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Effect of Chromium Niacinate and Chromium Picolinate Supplementation on Lipid Peroxidation, TNF-α, IL-6, CRP, Glycated Hemoglobin, Triglycerides and Cholesterol Levels in blood of Streptozotocin-treated Diabetic Rats

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Источник: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568689/

Association of plasma chromium with metabolic syndrome among Chinese adults: a case-control study

  • Sijing Chen1,2,
  • Li Zhou1,2,
  • Qianqian Guo1,2,
  • Can Fang1,2,
  • Mengke Wang1,2,
  • Xiaobo Peng1,2,
  • Jiawei Yin1,2,
  • Shuzhen Li1,2,
  • Yalun Zhu1,2,
  • Wei Yang1,2,
  • Yan Zhang3,
  • Zhilei Shan1,2,
  • Xiaoyi Chen4 &
  • Liegang Liu1,2

Nutrition Journalvolume 19, Article number: 107 (2020) Cite this article

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Abstract

Backgroud

Chromium has been suggested playing a role in alleviating diabetes, insulin resistance and lipid anomalies, but the effect on metabolic syndrome (MetS) in humans remains controversial.

Methods

We conducted a matched case-control study in a Chinese population, involving 2141 MetS cases and 2141 healthy controls, which were 1:1 matched by age (±2 years) and sex. Plasma chromium was measured by inductively coupled plasma mass spectrometry.

Results

Plasma chromium levels were lower in MetS group than in control group (mean: 4.36 μg/L and 4.66 μg/L, respectively, P < 0.001), and progressively decreased with the number of MetS components (P for trend < 0.001). After adjustment for potential confounding factors, the odds ratios (95% confidence intervals) for MetS across increasing quartiles of plasma chromium levels were 1 (reference), 0.84 (0.67–1.05), 0.76 (0.61–0.95), and 0.62 (0.49–0.78), respectively (P for trend < 0.001). For the components of MetS (high waist circumference, high triglycerides and high blood glucose), the odds ratios (95% confidence intervals) of the highest quartiles were 0.77 (0.61–0.95), 0.67 (0.55–0.80), and 0.53 (0.44–0.64), respectively (P for trend < 0.05).

Conclusions

Our results indicated that plasma chromium levels were inversely associated with MetS in Chinese adults. The association may be explained by the relations between plasma chromium levels and high waist circumference, and the triglycerides and blood glucose levels.

Peer Review reports

Introduction

Metabolic syndrome (MetS), known as a constellation of metabolic abnormalities, which includes abdominal obesity, high triglycerides, low high-density lipoprotein (HDL) cholesterol, high blood pressure, and elevated fasting blood glucose, is now both a public health and a clinical problem. MetS is epidemic all over the world and its incidence has been rising year-on-year [1]. Recent data indicated that about 33.9% of the adults in Mainland China had MetS [2]. In addition, MetS has been realized a major contributor to the epidemic of cardiovascular disease and type 2 diabetes mellitus [3], and it may increase the risk of mortality [4].

Chromium is an essential trace element, which has been suggested playing a potential role in alleviating diabetes, insulin resistance and lipid anomalies. The beneficial mechanism has been investigated in experimental studies [5,6,7,8,9]. However, the epidemiological evidence of the protective effect of chromium on MetS is very limited, and has inconsistent conclusion so far. A prospective study suggested an inverse association between chromium and incidence of MetS in American young adults, and the inverse association was mainly explained by its relation to blood lipids [10]. There was another case-control study suggesting an association between low chromium levels and increased risk of nonfatal myocardial infarction [11]. Besides, our previous study found that plasma chromium concentrations were inversely associated with type 2 diabetes mellitus and pre-diabetes mellitus [12]. Yet some studies did not support the inverse relationship between chromium and MetS [13, 14]. So far, clinical trials evaluating chromium supplementation on glucose and lipid profiles have yielded conflicting results [15,16,17,18].

Accordingly, in this matched case-control study, we aimed to examine the association of plasma chromium levels with MetS along with its components in a large Chinese population.

Methods

Study population

The present study was a case-control study conducted in Wuhan, China, during the period of March 2013 to December 2017. The study population consisted of 2141 MetS cases and 2141 healthy controls, which were 1:1 matched by age (±2 years) and sex. All participants were aged 18 years or older, consecutively recruited from the general population undergoing a routine health checkup in the Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology. Patients with clinical significant neurological, endocrinological or other systemic diseases, as well as acute illness and chronic inflammatory or infective diseases were excluded from the study. All the participants enrolled were of Chinese Han ethnicity. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Tongji Medical College.

Definition of MetS

The definition of MetS was based on the harmonized definition for MetS in 2009 [19]. To be considered as a case of MetS, participants had to meet at least three of the following criteria: 1. Abdominal obesity: waist circumference ≥ 85 cm in men and ≥ 80 cm in women; 2. Hypertriglyceridemia: ≥ 150 mg/dL; 3. Low levels of HDL cholesterol: < 40 mg/dL in men and < 50 mg/dL in women; 4. High blood pressure: ≥ 130/85 mmHg and/or use of antihypertensive medication; 5. High fasting glucose: ≥ 100 mg/dL and/or current use of antidiabetic medication and/or self-reported history of diabetes. The controls had zero to two components of MetS which were mentioned above.

Data collection

Demographics, health status, and lifestyle data were obtained from the questionnaires, including sex, age, education level, history of disease (diabetes, hypertension and hyperlipemia), family history of diabetes, physical activity, current smoking status, and current alcohol drinking status. Education level was classified as none or elementary school, middle school, and high school or college. Physical activity was classified as at least once/week or no. Current smoking status was classified as yes (at least one cigarette per day over the previous 6 months) or no. Current alcohol drinking status was classified as yes (drink alcohol beverage more than once a week over the previous 6 months) or no. Anthropometric data including height (m), mass (kg), waist circumference and blood pressure were measured with standardized techniques by trained and certified technicians. BMI (body mass index) was calculated as mass divided by the square of height (kg/m2). Waist circumference was obtained at the mid-point between the lowest rib and the iliac crest to the nearest 0.1 cm, after inhalation and exhalation. Hip circumference was measured at the outermost points of the greater trochanters. The ratio of waist-to-hip circumference was used as an index of fat distribution. Blood pressure was measured at rest in the seated position using a standardized automated sphygmomanometer after 5 min of rest, and repeated in both arms.

Laboratory measurements

Blood samples were collected in all participants after an overnight fast of at least 10 h. Details of measurement of fasting plasma glucose, fasting plasma insulin, total cholesterol, triglyceride, HDL cholesterol, low-density lipoprotein (LDL) cholesterol and calculation of homeostasis model assessment of insulin resistance (HOMA-IR) and HOMA of β-cell function (HOMA-β) have been described previously [20]. Plasma malonaldehyde (MDA) was measured with an MDA assay kit (Jiancheng, Inc., Nanjing, China).

Measurement of plasma chromium concentrations

Plasma chromium concentrations were measured in the Ministry of Education Key Laboratory of Environment and Health and School of Public Health at Tongji Medical College of Huazhong University of Science and Technology, using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7700 Series, Tokyo, Japan). Plasma samples were stored at − 80 °C. The case and control specimens were measured randomly in the daily measurement, with laboratory personnel blinded to the case–control status. For quality assurance, metals in standard reference materials were measured once in every 20 samples using certified reference material. The certified concentrations of human plasma controls (ClinChek no. 8883 and 8884) were 3.56 ± 0.89 μg/L and 11.1 ± 2.22 μg/L, respectively. The limit of detection (LOD) for chromium was 0.01 μg/L, and concentrations of plasma chromium levels below the LOD (0.7%) were imputed at LOD/√2. Quality control was performed (1 out of 20 samples), and the inter-assay and intra-assay coefficients of variation were < 10 and < 8%, respectively.

Statistical analysis

Descriptive statistics were calculated for all demographic and clinical characteristics of the study subjects, and summarized as numbers (percentages) for categorical data, mean ± standard deviations (SDs) for normally distributed data, and medians (interquartile ranges) for non-normally distributed data. Comparisons between MetS and controls were performed by t test or Mann-Whitney U test for continuous variables, and chi-square tests for categorical variables. In addition, subjects were divided into 6 groups according to their possession of 0, 1, 2, 3, 4 or 5 components of MetS. Multiple imputation based on 5 replications and a fully conditional specification method in SPSS was used to account for missing data.

For calculation of the odds ratio (OR) for MetS, plasma chromium concentration was categorized in quartiles according to the control group: category 1, < 3.27 μg/L; category 2, 3.28–4.46 μg/L; category 3, 4.47–5.87 μg/L, and category 4, > 5.88 μg/L. Conditional logistic regression was used to assess the association of MetS with plasma chromium concentrations. The ORs and 95% confidence intervals (CIs) of MetS were calculated between the quartiles of chromium using the lowest quartile as the reference category, and also by per 1 μg/L chromium as continuous variable. We considered three models with progressive degrees of adjustment: model 1 adjusted for age; model 2 additionally adjusted for education, current smoking status, current alcohol drinking status, physical activity and family history of diabetes; and model 3 further adjusted for BMI. Tests of linear trend across increasing chromium quartiles were conducted by assigning the median value to each quartile and treating it as a continuous variable. Furthermore, the ORs of the MetS components including high waist circumference, high triglycerides, low HDL cholesterol, high blood pressure, and high blood glucose were calculated using binary logistic regression.

To evaluate the consistency of the association between chromium and MetS by participant characteristics, additional analyses were run, stratifying age (< 50, ≥50), sex, BMI (< 24, ≥24), physical activity, current smoking status, and current drinking alcohol status. The interactions between these stratification variables and plasma chromium were tested by adding multiplicative terms into the multivariate logistic regression models; the likelihood ratio tests were conducted to examine the interactions.

Statistical analyses were performed with SPSS for Windows, version 24.0 (SPSS Inc., Chicago, Illinois). P values reported are two tailed, and values below 0.05 were considered statistically significant.

Results

Anthropometric and metabolic characteristics of the 2141 MetS and 2141 controls are reported in Table 1. Compared with control subjects, the individuals with MetS had higher prevalence of family history of diabetes and lower rate of smoking and activity (P < 0.05). As expected, we observed higher levels of BMI, waist circumference, hip circumference, waist-to-hip circumference ratio, systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting plasma glucose, fasting plasma insulin, HOMA-IR, triglycerides, total cholesterol, LDL cholesterol and lower levels of HDL cholesterol in MetS than in the controls (P < 0.001). MetS group had higher MDA levels than the control group (P < 0.001).

Full size table

Plasma chromium concentrations were significantly decreased in the individuals with MetS compared with controls (mean: 4.36 μg/L in MetS, and 4.66 μg/L in controls, P < 0.001). For the 5 components of MetS, participants with high triglycerides and high blood glucose had significant lower levels of plasma chromium (P < 0.001). Furthermore, plasma chromium levels progressively decreased with the number of MetS components (P for trend < 0.001) (Table 2).

Full size table

Significant inverse associations between the levels of plasma chromium concentration and MetS were observed, and multiple adjusted models showed similar results (Table 3). After overall multivariable adjustment of age, education, current smoking status, current alcohol drinking status, physical activity, family history of diabetes, and BMI, the ORs (95% CIs) for MetS from the lowest to the highest quartiles were 1 (reference), 0.84 (0.67–1.05), 0.76 (0.61–0.95), and 0.62 (0.49–0.78), respectively (P for trend < 0.001). When plasma chromium concentration was considered as a continuous variable, the overall OR (95% CI) of having MetS was 0.95 (0.92–0.98) per 1 μg/L increment of chromium concentration.

Full size table

The associations of plasma chromium concentrations with each component of MetS were examined afterwards. Similar inverse associations were observed in high waist circumference, high triglycerides and high blood glucose, and the full adjusted ORs (95% CIs) of the highest quartiles were 0.77 (0.61–0.95), 0.67 (0.55–0.80), and 0.53 (0.44–0.64), respectively (P for trend < 0.05) (Table 4). As for low HDL cholesterol, significant associations were observed in model 1, but not in model 2 and 3. Association of plasma chromium concentrations with high blood pressure was not found in this study (Table 4).

Full size table

In stratified analysis (Table 5), ORs (95% CIs) of the highest quartiles of all subgroups decreased significantly, indicating the robust association. No interaction was recognized between age, sex, BMI, physical activity, smoking, drinking alcohol and chromium (P for interaction > 0.05).

Full size table

Discussion

In this matched case-control study, we found that plasma chromium concentrations were inversely associated with the prevalence of MetS among Chinese adults. The inverse association was mainly explained by the relations between plasma chromium concentrations and waist circumference, the triglycerides and blood glucose levels. The associations were not appreciably changed by multivariate adjustment, and were consistent in the stratified analyses.

Chromium coming from foods varies and is usually very low [21]. Dietary intake of chromium from Asian diets ranged from 59.9 to 224 μg per day [22]. It is difficult in estimating dietary chromium due to its wide variability and low content in food sources, so a sensitive and reliable biomarker for chromium intake is required in epidemiological studies. Plasma chromium is considered a reliable objective biomarker for chromium exposure [23]. Previous studies reporting plasma chromium concentrations in large populations were sparse. Currently, there is no international acceptable value or range for the plasma chromium concentration in the general population. The mean concentration of plasma chromium in our population was 4.51 ± 2.24 μg/L, higher than the previously published studies, which varied from 0.2 to 0.86 μg/L [24,25,26]. A possible explanation for it may be higher contamination for the population. As some studies indicated that chromium exposure may come from industrial pollution like coal and oil combustion, the metal fabrication industry and the leather tanning sector, and China had a dramatic increase of anthropogenic chromium emissions from 1990 to 2009 [27].

There existed few high-quality evidence focused on the relationship between chromium and MetS at present. Limited epidemiological study yielded controversial results. A 23-year follow-up study including 3648 American adults indicated that toenail chromium levels were inversely and longitudinally associated with incidence of MetS [10]. However, another cross-sectional study conducted in Korea did not support the association between toenail chromium concentrations and MetS and its components [13].

In our study, significant associations between chromium concentrations and waist circumference, triglycerides and blood glucose levels were noticed. These associations might explain the latent mechanism involved in the relationship of chromium and MetS.

The association of plasma chromium with high blood glucose was the strongest among the components of MetS in this study. Although the pathogenesis of MetS remains unclear, recent interest has focused on the possible involvement of insulin resistance as a linking factor [28]. Coincident with this, our previous study has elaborated the inverse association between plasma chromium concentrations and type 2 diabetes mellitus and pre-diabetes mellitus in a case-control study [12]. In addition, evidences in animal and in vitro studies supported the association as well. A lot of studies demonstrated that chromium may up-regulate insulin-stimulated insulin signal transduction by a variety of mechanisms [5, 6, 8, 29,30,31]. However, it is worth concerning the causality of chromium status and high blood glucose. On one hand, the low levels of chromium might result in the diminution of insulin signal transduction, and further aggravate the development of insulin resistance. On the other hand, chromium lost and excreted from human body increased with aging and was related to the diabetes [32]. Large losses of chromium over more than 2 years’ diabetes duration may change the chromium homeostasis [33]. Further studies are warranted to investigate the causality of chromium status and high blood glucose.

Moreover, the effects of chromium on obesity and dyslipidemia has also been studied. The animal studies indicated that chromium might reduce the weight of obese rats and lipids levels as well [5, 9, 34, 35]. However, clinical trials were inconclusive with regard to weight control and lipid metabolism improvement. Although some studies claimed beneficial effects of chromium supplementation [36, 37], systematic reviews found it inadequate to inform firm decisions about the efficacy of chromium supplements on weight loss or lipid metabolism in overweight or obese adults because of the low-quality evidence [15, 18, 38].

The strengths of our study included the matched case-control study design, the large number of participants and objectively measured plasma chromium levels. In addition, chromium levels in plasma were measured using the state-of-the-art ICP-MS method. A few limitations need to be considered. First, the case-control nature of our study does not allow us to infer any causality and address temporal relationship between plasma chromium and MetS. Second, we could not differentiate trivalent chromium from hexavalent chromium in plasma measurement. Trivalent chromium is suggested to be beneficial and hexavalent chromium is toxic to human health [39]. Thus, the combination of these two forms may attenuate the association that may exist between trivalent chromium and MetS. Third, the classification of current smoking and alcohol drinking status and physical activity was not detailed enough. Additionally, the lack of information on the other unknown or unmeasured factors might also confound our results. Finally, the generalizability of our findings may be limited since all participants were of Chinese Han ethnicity. However, a homogenous ethnic background may reduce residual confounding from unmeasured genetic and cultural variability.

Conclusions

Our study demonstrated an inverse association between plasma chromium levels and MetS in a Chinese population. The association was mainly accounted for the relations between plasma chromium levels and high waist circumference, and the triglycerides and blood glucose levels. Further studies are warranted to confirm our findings in prospective cohorts and to elucidate the potential mechanisms underlying the relationship between chromium and MetS, as well as MetS components.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

Confidence interval

Diastolic blood pressure

Malonaldehyde

High-density lipoprotein

Homeostasis model assessment of β-cell function

Homeostasis model assessment of insulin resistance

Limit of detection

Low-density lipoprotein

Metabolic syndrome

Odds ratio

Systolic blood pressure

Standard deviation

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Acknowledgements

We gratefully acknowledge the nurses of the Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, for their diligent work on collecting blood samples.

Funding

This study was supported by the National Natural Science Foundation of China (81803239); the Major International (Regional) Joint Research Project (81820108027); the Fundamental Research Funds for the Central Universities (2019kfyXMBZ050); and the Angel Nutrition Research Fund.

Author information

Affiliations

  1. Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China

    Sijing Chen, Li Zhou, Qianqian Guo, Can Fang, Mengke Wang, Xiaobo Peng, Jiawei Yin, Shuzhen Li, Yalun Zhu, Wei Yang, Zhilei Shan & Liegang Liu

  2. Ministry of Education Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China

    Sijing Chen, Li Zhou, Qianqian Guo, Can Fang, Mengke Wang, Xiaobo Peng, Jiawei Yin, Shuzhen Li, Yalun Zhu, Wei Yang, Zhilei Shan & Liegang Liu

  3. The Hubei Provincial Key Laboratory of Yeast Function, Yichang, 443003, Hubei, China

    Yan Zhang

  4. Department of Nutrition and Food Hygiene, School of Public Health, Guangzhou Medical University, Guangzhou, 511436, China

    Xiaoyi Chen

Contributions

SC, LZ, ZS, XC, LL designed the study; QG, CF, MW, XP, SL, YZ acquired the data; SC, LZ, JY analyzed and interpreted the data; SC drafted the article; WY, YZ, ZS, XC, LL substantively revised it. All authors have approved the final version of the article.

Corresponding authors

Correspondence to Xiaoyi Chen or Liegang Liu.

Ethics declarations

Ethics approval and consent to participate

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Tongji Medical College.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

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Supplementary information

Additional file 1: Table S1

STROBE-nut: An extension of the STROBE statement for nutritional epidemiology.

Additional file 2: Table S2

The Strengthening the Reporting Observational studies in Epidemiology – Molecular Epidemiology (STROBE-ME) Reporting Recommendations: Extended from STROBE statement.

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Opinion controversy to chromium picolinate therapy’s safety and efficacy: ignoring ‘anecdotes’ of case reports or recognising individual risks and new guidelines urgency to introduce innovation by predictive diagnostics? Academic research paper on "Clinical medicine"

The

Journal

REVIEW

Open Access

Opinion controversy to chromium picolinate therapy's safety and efficacy: ignoring 'anecdotes' of case reports or recognising individual risks and new guidelines urgency to introduce innovation by predictive diagnostics?

Olga Golubnitschaja* and Kristina Yeghiazaryan

Abstract

Due to the important physiologic function of trivalent chromium in glucose/insulin homeostasis, some commercial organisations promote Cr3+ supplements in maintaining proper carbohydrate and lipid metabolism; regulation of reducing carbohydrate carvings and appetite; prevention of insulin resistance and glucose intolerance; regulation of body composition, including reducing fat mass and increasing lean body mass; optimal body building for athletes; losing weight; treatment of atypical depression as an antidepressant; and prevention of obesity and type 2 diabetes mellitus. On one hand, case reports are commented as 'nonevidence-based anecdotes'. On the other hand, a number of independent studies warn against adverse health outcomes assigned to chromium picolinate (CrPic) dietary application. This review analyses opinion controversies, demonstrates highly individual reactions towards CrPic dietary supplements and highlights risks when the dietary supplements are used freely as therapeutic agents, without application of advanced diagnostic tools to predict individual outcomes.

Keywords: Patient records, Individual profiles, Subcellular imaging patterns, Therapy response, Pre/diabetes care, New guidelines, Predictive preventive personalised medicine, Dietary supplements, Healthcare, Integrative medicine

Review

Changing long-held beliefs is never easy

As a consequence of the accumulating clinical data and knowledge about the epidemiology and pathological mechanisms of the most frequent causes of morbidity and mortality, we are currently reconsidering our view of the origins and progression of cardiovascular, oncologic and neurodegenerative diseases. The majority of these pathologies are of chronic nature: they progress from precursor lesions over one or even several decades of life; therefore, it is often too late for effective therapeutic intervention. An excellent example is the epidemic scale of type 2 diabetes mellitus witnessed in the European Union. In most industrialised countries and countries with large populations, the permanently growing cohort of diabetics

* Correspondence: [email protected]

Department of Radiology, Rheinische Friedrich-Wilhelms-University of Bonn,

Sigmund-Freud-Str. 25, Bonn 53105, Germany

Bio Med Central

creates a serious healthcare problem and a dramatic health economic burden. The estimate for diabetes prevalence in the years 2025-2030 is half a billion patients worldwide (see Figure 1).

Moreover, the contemporary onset of the dominant type 2 diabetes was already observed in subpopulations of teenagers [3]. Severe complications secondary to early onset of diabetes mellitus, such as retinopathy, nephropathy, silent ischaemia, dementia and cancer (Figure 2), soon may lead to collapsing healthcare systems [4].

Optimistic versus pessimistic prognosis in future developments of the healthcare sector depends much on diagnostic, preventive and treatment approaches which diabetes care will preferably adopt in the near future. Without innovation in healthcare, diabetes-related complications may cause such a dramatic level of burden that any performance of personalised medicine will not be feasible from an economical point of view. In contrast, effective

© 2012 Golubnitschaja and Yeghiazaryan; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestrictec use, distribution, and reproduction in any medium, provided the original work is properly cited.

.1 460

1980 1985 1990 1995 2000 2005 2010 2015 2020 2025 2030 2035 2040

Figure 1 Worldwide prognosis of the epidemic scale of diabetes. Single asterisks denote estimations as published around the year 2000; double asterisks denote stepwise worsening prognosis (from black circles to black diamond) as published in 2003-2008; current prognoses are marked in red colour (adapted from [1,2]).

utilisation of advanced early/predictive diagnostics and targeted prevention could enable rapidly ageing populations in Europe and elsewhere to be economically effective for the society. Therefore, it is timely to consider the integrative medical approaches to advance pre/diabetes care utilising the innovation by predictive diagnostics as the basis for concomitant targeted prevention, patient profiling as the basis for individualised treatment algorithms and medical approaches tailored to the patient [5-8].

The message is that new guidelines should create a robust juristic and economic platform for advanced medical services utilising the cost-effective models of risk assessment followed by tailored treatments focussed on the precursor stages of chronic disease [9].

Potential role of biologically active chromium in diabetes prevention

Chromium is an essential trace element in human physiology. The physiologic value of chromium compounds

depends on the oxidative states of chromium ions that vary from bivalent to hexavalent ones. The best investigated forms are trivalent and hexavalent chromium compounds. The latter is frequently used in a variety of industrial technologies. Chronic exposure to it has been shown to be responsible for a spectrum of severe health problems affecting the skin and inner organs [10]. Investigations of its carcinogenic effects are in progress at molecular and cellular levels [11,12]. In contrast, triva-lent chromium is considered physiologically safe and is an essential trace metal functionally involved in several metabolic pathways in human beings as published elsewhere. The recommended daily allowance of trivalent chromium corresponds to the range of 50-200 ^g [13]. Dietary deficiency of chromium leads to disturbances in carbohydrate metabolisms; increases risk of glucose intolerance, insulin resistance, hyperglycaemia and predisposes to obesity and type 2 diabetes mellitus [14,15].

Controversial benefits by Cr3+ nutrient supplement in foods

Biologically valuable forms of chromium are compounds of trivalent chromium bound to low molecular weight organic complexes such as oligopeptides. This recognition led to the development of a commercial branch with Cr3+ nutrient supplements which suppose to promote or even improve health status.

What are the currently available 'pro' versus 'contra' arguments to approve/disprove potential benefits of Cr3+ nutrient supplement?

Chromium is an essential metal, but only traces of triva-lent chromium are utilised in the human body for glucose conversion by insulin-driven reactions in carbohydrate metabolic pathways. Furthermore, naturally available food sources used in well-balanced diets are capable to cover the requirement of trivalent chromium completely: fruits, vegetables, meat, fish, grains, brewer's yeast, etc. [16]. Cr deficiency cases are extremely rare, and evidence-based biochemical justification for artificial source needed

I microcirculation disturbances retinopathy

nephropathy

vasculopathy, atherosclerosis, valvular calcification & degeneration shortened re-operation time of implanted bioprosthesis,

cardiomyopathy, silent myocardial infarcts, sudden death polyneuropathy, neurodegenerative diseases cancerogenesis

extensive necrosis, "diabetic foot"

Figure 2 Severe complications developed secondary to diabetes: from 'upstream' (up) to 'downstream' (bottom) in the cascade of pathologic processes (adapted from [4]).

additionally to standard foods has not been provided in the literature. Nevertheless, due to the important physiologic function of trivalent chromium in maintaining proper carbohydrate and lipid metabolism and glucose/ insulin homeostasis, some commercial organisations promote Cr3+ nutrient supplements in

• regulation of reducing carbohydrate carvings and appetite

• prevention of insulin resistance and glucose intolerance

• regulation of body composition, including reducing fat mass and increasing lean body mass

• optimal body building for athletes

• losing weight

• treatment of atypical depression as an antidepressant

• prevention of obesity and type 2 diabetes mellitus.

The widely spread chemical compound used as a trivalent chromium nutrient supplement is chromium picolinate (CrPic). CrPic has become a very popular nutritional supplement for treating type 2 diabetic, obese and diabetes-predisposed individuals [17]. However, over a decade of human studies with CrPic indicate that the supplement has not demonstrated effects on the body composition of healthy individuals, even when taken in combination with an exercise training programme [18]. Also, a potential weight loss by CrPic therapy has not been confirmed [19].

After the evidence-based search into the issue, the US Food and Drug Administration concluded that the 'relationship between CrPic intake and insulin resistance is highly uncertain' [20]. Furthermore, some later studies have concluded that 'CrPic does not improve key features of metabolic syndrome in obese nondiabetic adults' [21]. Nevertheless, it is reported that CrPic generates sales for more than US$100 million annually [22]. The CrPic supplements are freely available in numerous forms including chewing gums, pills, sports drinks and nutrition bars [23]. CrPic is commonly supplied in the range of 200-500 ^g as a daily dose [13]. A big advantage of CrPic is its high bioavailability due to increased absorbing capacity in human tissues compared with dietary chromium [24]. However, the mechanism of action of CrPic at molecular level is not completely understood. In 1999, Speetjens et al. reported that CrPic cleaves DNA molecules by a radical mechanism: in the presence of oxygen from air and biologically available reductants, CrPic generates hydroxyl radicals damaging DNA molecules [25]. Further, organisms exposed to CrPic demonstrate the tendency to accumulate intracellular Cr3+ ions that may lead to long-term genotoxic effects by formation of covalent bonds to DNA [26]. Taken together, cytotoxic, genotoxic and mutagenic effects as well as activity damaging to the mitochondria and induction of

apoptosis have been reported for CrPic-using mammalian cell cultures, Drosophila and animal models [27-30]. Potential toxicity of CrPic has raised concerns about the safety of CrPic-related nutrient supplements.

Animal experiments to model treatment effects of CrPic on human beings

To simulate treatment effects of CrPic, a spectrum of models has been used. To simulate type 2 diabetes melli-tus (DM), a well-acknowledged animal model of db/db mice was utilised in a recent project [31]. The experimental design is summarised in Figure 3.

Subcellular imaging insights into CrPic therapy Quantitative subcellular imaging by 'comet assay' analysis

The single-cell gel electrophoresis assay is a simple and effective method for evaluating DNA damage in cells. It is based on the ability of denatured, cleaved DNA fragments to migrate out of the cell under the influence of an electric field, whereas undamaged DNA remains within the confines of the nucleoid and migrates slower. The assessment of DNA damage is done via the evaluation of the DNA 'comet' tail shape and migration pattern. The cells are immobilised in a bed of low-melting point agarose, on a Trevigen CometSlide™ (Gaithersburg, MD, USA). After cell lysis, samples are treated with alkali to unwind and denature the DNA and hydrolyse sites of damage. After performing electrophoresis, staining with a fluorescent DNA-intercalating dye (SYBR® Green I; Trevigen Inc., Gaithersburg, MD, USA) is done, and the sample is visualised by epifluorescence microscopy. The alkaline electrophoresis is very sensitive and allows for the detection of small amounts of damage.

In our experiments, DNA damage was designated to four classes based on the visual aspect considering the extent of DNA migration as published earlier [32]. Comets with a bright head and almost no tail were classified as class I, indicating minimal DNA damage, whereas comets with no visible head and a long diffuse tail were classified as class IV, revealing complete DNA fragmentation. Comets with intermediate characteristics were assigned to classes II and III based on the ratio R = T/r, in which T represents the comet's tail length and r is the radius of the comet's head. The characteristic value of R for class I is 1 and that for class IV is ^ (r =0). Comets with R values ranging between 1< R >3 were designated as class II. The comet classification is demonstrated in Figure 4A. Figure 4B demonstrates comet patterns typical for experimental group 1 (left) with the lowest level of comets of class IV (apoptosis) and experimental groups 5-7 (right) with the highest level of apoptosis.

Animal model

Type 2 DM mice without treatment

Group 3

Type 2 DM mice CrPic supplement: 5 mg/kg Duration: 6 weeks-6 months of age

Group 4

Type 2 DM mice CrPic supplement: 10 mg/kg Duration: 6 weeks-6 months of age

Group 5

Type 2 DM mice CrPic supplement: 100 mg/kg Duration: 6 weeks-6 months of age

Group 6

Type 2 DM mice CrPic supplement: 100 mg/kg Duration: 3-6 months of age

Group 7

Type 2 DM mice CrPic supplement: 250 mg/kg Duration: 3-6 months of age

Figure 3 Experimental design to model CrPic treatment effects. Animals were arranged in eight groups. Group 1 represented the healthy control. Group 2 was the modelmice for type 2 DM. Groups 3-7 were diabetes modelmice treated under individualalgorithms: 5 mg/kg CrPic from 6 weeks to 6 months of age (group 3), 10 mg/kg CrPic from 6 weeks to 6 months of age (group 4), 100 mg/kg CrPic from 6 weeks to 6 months of age (group 5), 100 mg/kg CrPic from 3 to 6 months of age (group 6), and 250 mg/kg CrPic from 3 to 6 months of age (group 7). Kidney tissue samples were collected and stored at -80 °C tillthe analysis described in the below section was performed.

Statistical analysis reveals increased heterogeneity of individual reactions towards the highest doses of CrPic

Statistical analysis of comets in classes I (intact DNA) and IV (apoptosis) is demonstrated in Figure 5A, B, respectively. The red frame marks the experimental groups with the highest rates of statistical deviations towards the mean value. The highest heterogeneity is obvious for groups 5, 6 and 7, i.e. the experimental groups with the highest doses of CrPic applied as treatment.

DNA damage in untreated diabetic group is significantly higher compared to healthy control group

Quantitative subcellular imaging by comet assay analysis revealed significantly higher DNA damage in the untreated diabetic group compared to healthy controls (see

Figure 6) that is well in agreement with data published and reviewed for diabetic patients [4,8].

Individuality within the untreated diabetic group

Subcellular imaging generally revealed group-specific patterns when diabetic animals were compared with controls, although higher heterogeneity was demonstrated within the untreated diabetic group: in some animals, intact DNA (class I comets) was monitored, whereas only comet classes with damaged DNA were monitored for others (see Figure 7). This heterogeneity clearly demonstrates an individualised reaction of organisms towards diabetic condition. This is a very important observation that corresponds well with the clinical picture of diabetes [8]. Hence, it is conclusive that the

animal model used is suitable for issue-related studies to simulate the medical condition of diabetes and investigate individualised therapeutic effects.

Highly individual reaction of diabetic animals towards CrPic dietary supplements

Despite group-specific patterns, the subcellular imaging by the comet assay indicated that each animal within a

group responded individually to the identical dosage of CrPic administered and the treatment duration. Within the diabetic groups, experimental animals demonstrated highly individual comet patterns with respect to single dosages and treatment algorithms. This observation is important in regard of the highly individual response to the treatment algorithms applied. The paper concludes that there are individual reactions of diabetic animals towards doses and duration of CrPic treatment. This observation

Group (¿roup

Figure 5 Statistical analyses for the comet types (A) I and (B) IV (see 'comet classification' in Figure 4A) in groups of comparison as described in Figure 3 ('experimental design'). Analyses were carried out using SPSS 17.0 software (SPSS, Chicago, IL, USA) by the application of univariable variance analysis with Bonferroni. The red box marks the groups with the highest deviation towards the mean value, i.e. the greatest heterogeneity within corresponding groups. The individualvalues which go overboard by statisticalcalculations are marked with black circles (for the standard deviation in groups 4 and 6) and asterisk (for the mean value in group 4).

A 10-, 9 8 7 6 5 4

Figure 6 Comparative analysis in the experimental groups. (A)

The diagram presents the mean values of the individual experimentalgroup for the comets of class I (intact chromosomal DNA): axe X, group numbering (1-7); axe Y, corresponding mean values (% of comets). The highest and lowest rates correspond tc controlgroup 1 and untreated diabetic group 2, respectively. (B) The diagram presents the mean values of the individualexperimental group for the ratio of comets with damaged DNA (classes III and IV) to the comets with undamaged DNA (classes I and II). Similarly to the diagram in (A), the highest and lowest rates of DNA damage correspond to controlgroup 1 and untreated diabetic group 2, respectively. The green lines mark the levelcorresponding to the mean value of the controlgroup; in contrast, the red lines mark the levelcorresponding to the mean value of the untreated diabetic group.

can explain discrepancies found in the literature concerning harmful effects of CrPic therapy [13,24-28].

The authors interpreted that CrPic treatment effects are unpredictable for the patient cohort as a whole, due to highly individual reactions towards therapy. Individuals should be treated personally on the basis of individual results going by predictive diagnostics and therapy monitoring.

Group-pecific comparative quantification of apoptosis under CrPic treatments

All diabetic groups demonstrate apoptotic rates which are significantly higher compared to the control group

(see Figure 8). The highest rates of apoptosis were monitored in the diabetic groups treated with the highest doses of CrPic, namely in the groups 5, 6 and 7.

Assumed and registered adverse health outcomes by CrPic treatments

Taken the above observations together, the authors conclude possible risks for individual long-term effects when CrPic is freely used as a therapeutic nutritional modality agent without application of advanced diagnostic tools to predict individual outcomes.

In experimental models

In terms of potential treatments of gestational diabetes, there is very little information about the safety of CrPic applied as a dietary supplement in pregnant women. However, experiments performed with CrPic administered to pregnant mice resulted in skeletal birth defects in the developing fetus [33].

Comparative cytotoxic and genotoxic studies of triva-lent chromium demonstrated that the compound's safety depends on the ligand bound to the chromium ion [34]. Thereby, CrPic produces significantly more oxidative stress and DNA damage compared, for example, to niacin-bound chromium(III). The implicated toxicity of CrPic may result in renal impairment; severe biochemical, histological and morphological changes in the eye; skin blisters and pustules; anaemia; hemolysis; tissue oedema; liver dysfunction; neuronal cell injury; impaired cognitive, perceptual and motor activity; enhanced production of hydroxyl radicals; chromosomal aberration; depletion of antioxidant enzymes; oxidative stress and DNA damage [23,29,34-38]. Increased apoptotic effects by CrPic were demonstrated by the authors of this study for kidney and for circulating leukocytes elsewhere [17,39]. Potential carcinogenic effects are assumed [40,41].

In humans

A growing body of case reports warns against adverse health outcomes assigned to CrPic dietary application, whereas by others, they are interpreted as 'anecdotal reports' [42]. Hence, case reports have described acute kidney failure, liver damage and anaemia by taking high dosage of CrPic as a dietary supplement [43,44]. Adverse cutaneous reactions to CrPic supplements have also been described [45].

Furthermore, there are some concerns that CrPic may affect the levels of neurotransmitters leading to potential risks for patients treated for depression, bipolar disorder and schizophrenia [18]. In a broader sense, CrPic supplements are hormone-related and may influence hormone secretion through their function in the endocrine/ metabolic system [46]. Chromium supplements taken

CONTROL

untreated DIABETIC group

treated DIABETIC groups

Figure 7 Diagram demonstrates individual reactions towards CrPic treatments in the groups of comparison (1-7, group numbering is marked in the middle). Individuallevels of intact DNA (axe X, individualnumbering; axe Y, comet class I in %) are highly heterogeneous ir diabetic groups compared to controlgroup 1. The highest levelof heterogeneity is evident in groups 5, 6 and 7 with the highest doses of CrPic supplements (see 'experimentaldesign' in Figure 3) The turquoise lines mark the levelcorresponding to the mean value of the controlgroup; in contrast, the red arrows mark the levelcorresponding to the mean of the untreated diabetic group.

90 80 70 60 50 40 30 20 10 0

Figure 8 Diagram presents mean values of individual experimental groups for the comets of class IV (apoptosis): axe X, group numbering (1-7); axe Y, corresponding mean values (% of comets). The lowest rates correspond to controlgroup 1 (green line); the highest rates correspond to diabetic groups treated with high doses of CrPic, namely experimentalgroups 5, 6 and 7. The red line marks the levelcorresponding to the mean value of the untreated diabetic group.

together with medications that block the formation of prostaglandins, such as ibuprofen, indomethacin, na-proxen and aspirin, may increase the absorption of chromium in the body followed by unpredictable consequences such as long-term genotoxic effects caused by formation of covalent bonds to DNA molecules [26]. Some additive medication effects may be expected if CrPic therapy is combined with diabetes treatments, causing blood glucose levels to dip too low.

Here, the authors are wishing to stress the point that according to our analysis, the above listed adverse health effects are not expected to happen to everybody. They may occur individually with a severity grade depending on individual predispositions but can be effectively avoided by application of advanced diagnostic tools for the therapy monitoring and to predict individual outcomes. Further, the authors appeal to react effectively towards the case reports demonstrating adverse health outcomes by artificial dietary supplements often regarded as harmless by the public and lay media.

Conclusions and recommendations

Artificial supplements for diabetes prevention: hype or hope?

Definitely, CrPic therapy is solely one example of several therapy forms which currently are applied 'across-the-board' in diabetes care. The administration of artificial supplements is an attempt to prevent or at least to postpone the onset of type 2 diabetes in the group of risk and with individuals who are unwilling to make prudent changes in their diets and sedentary habits. Should this approach be considered as the hype or the hope? Considering type 2 diabetes as a multifactorial disease, our

answer is that the above question is rather of rhetoric nature.

High efficacy of a balanced diet, an individually optimised lifestyle and personalised treatment regiments can hardly be substituted by a limited number of single supplements to cover all the multifactorial risks such as the upward trends of population ageing, environmental risk factors, urbanisation, additive effects of diverse stress factors, incorrectly chosen lifestyle including unfavourable nutritional habits, increasing prevalence of obesity, low physical activity, etc.

Although ageing is the well-acknowledged factor contributing to the disease's development, there are completely new epidemiologic factors characteristic of the twenty-first century that speed up the disease's progression particularly in the youth and in the young adults. Hence, it has been demonstrated that the prognosed DM rate progression will be inversely increasing with age (younger age = higher progression), and the youngest group of 20-39-year-old people will be delivering the highest rates of diabetic progression which will double the diabetes mellitus cohort of this age group by the year 2030 compared to 2010 [47]. This is a completely new situation and a very big challenge for most societies around the globe, requiring special competencies of several groups of professionals as well as innovative approaches in healthcare and health economy.

The population at-risk for diabetes is huge and increasing in a pandemic scale. One of the reasons might be the failed attempt to prevent the disease by the application of artificial supplements and drugs with hardly recognised individual risks. Consequently, a multimodal approach of integrative medicine by predictive diagnostics, targeted prevention and individually created treatment algorithms is highly desirable.

As discussed and reviewed earlier, more individualised treatments are desirable in effective protection against diabetic retinopathy and polyneuropathy, diabetes-related cardiovascular complications and cancer [8]. Further, field-related research is needed to establish simplified non-invasive diagnostic approaches for routine medical practice which would allow for an accurate prediction of individualised therapy risks and outcomes. A promising technological platform has been recently created using the detection of circulating nucleic acids in blood plasma [48] and clinical proteomics of body fluids [49].

Targeted measures require a creation of new guidelines that are essential to regulate (renoprotective) therapy approaches and the application of more individualised therapeutic modalities for advanced diabetes care. These measures should provide a legitimate regulation for well-timed predictive diagnostics, an effective prevention and the creation of individualised treatment algorithms in pre/diabetes [50].

Competing interests

The authors declare that they have no competing interests. Authors' contributions

KY participated in the design of the study and coordinated the performance of the experimentalpart. OG performed the design of the project and its coordination and created the concept of the manuscript. Both authors read and approved the final manuscript.

Acknowledgements

The project has been granted by NIH ('Is chromium-picolinate renoprotective in diabetes?', Nationallnstitutes of Health, study no. 1R21 AT003012-01A1). The authors thank Prof. Dr. M. Mozaffari (Georgia Health Sciences University, USA) for providing tissue samples in the project. For the statisticalanalysis and assistance in preparation of the originalpaper [31], the authors thank V. Peeva, M.Sc. and A. Shenoy, M.Sc., University of Bonn, Germany.

Received: 11 July 2012 Accepted: 17 August 2012 Published: 7 October 2012

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THE POSSIBLE PROTECTIVE EFFECTS OF CYMBOPOGON (LEMONGRASS) DECOCTION ON CHROMIUM PICOLINATE INDUCED PULMONARY ALVEOLAR CHANGES IN ADULT MALE ALBINO RATS.

THE POSSIBLE PROTECTIVE EFFECTS OF CYMBOPOGON (LEMONGRASS) DECOCTION ON CHROMIUM PICOLINATE INDUCED PULMONARY ALVEOLAR CHANGES IN ADULT MALE ALBINO RATS. Fardous S Karawya , El Sayed Aly Mohamed Metwally Department of Histology and Cell Biology, Department of Anatomy, Faculty of Medicine, Alexandria University. ABSTRACT Introduction: Chromium is a naturally – occurring heavy metal found in several forms (hexavalent Cr VI and trivalent Cr III). Copper, Manganese, Selenium and Chromium are all trace elements which are important in human diet. Any of these elements may have pernicious effects if taken in quantity or if the usual mechanisms of elimination are impaired Over exposure to chromium can occur in welders and other workers in the metallurgical industry, persons taking chromium – containing dietary supplements, patients who have received metallic surgical implants and individuals who ingest chromium salts in drinking water. The interest in herbal therapies and its expansive involvement in the health sector are not surprising. The herb Cymbopogon (Lemongrass), known as halfa-baris is highly reputed in folkloric medicine as an effective diuretic, renal or abdominal antispasmodic agent and for relieving bronchial asthma. Aim of the work: assessment of the possible protective effects of Lemongrass decoction on Chromium picolinate induced pulmonary alveolar changes of male albino rats. Material & Methods: the animals were divided into 3 groups. Group I (control group) which were further subdivided into 2 equal subgroups .Group II(experimental group ) Chromium picolinate (Cr(pic)3 ) was dissolved in water and administered at a concentration of 5 mg/kg/day in drinking water ad libitum for one month. Group III (protected group) received Chromium picolinate by the same route and duration concomitant with Lemongrass decoction 1%. Results: administration of Chromium picolinate induced alteration in the pulmonary alveoli as evident by proliferation and abnormal vacuolation of type II pneumocyte, numerous foamy macrophages, intra-alveolar cellular debris and exfoliated cells, thickening of inter-alveolar septa due to mononuclear cellular infiltration, congestion of the blood vessels associated with increased collagen deposition. Concomitant administration of lemongrass ameliorates most of these changes. Conclusion: Chromium picolinate is toxic to pulmonary alveolar epithelium and concomitant administration of Lemongrass effectively protects lung tissues. Key words: Slimming drugs- Chromium picolinate –Lemongrass- alveoli- Albino rats. INTRODUCTION Chromium (Cr III, VI) is an “essential trace element” and is a widely used industrial chemical, extensively used in paints, metal finishes, and steel including stainless steel manufacturing, chrome and wood treatment. Cr III compounds are used as micronutrients and nutritional supplements and have been demonstrated to exhibit a significant number of health benefits in animals and humans (Mirasol, 2000; Baselt 2008; and Anderson 2000). One form in particular, chromium picolinate, is popular because it is one of the more easily absorbed forms. Sales of chromium picolinate as a nutritional supplement were second only to calcium in the year 2000 (Ben Best, 2000). It is marketed as a weight loss aid for dieters and an ergogenic (muscle- building) aid for bodybuilders and athletes and also used for depression and polycystic ovary syndrome (PCOS) (Alemany et al., 2003; Wayne et al., 1999; and Urmila and Goyal, 2003). It works together with insulin to metabolize carbohydrates, so it is used for improving blood sugar control in people with prediabetes, type 1 and type 2 diabetes. Despite widespread use of Chromium picolinate, significant controversy still exists regarding the effect of Chromium supplementation on parameters assessing human health (William et al., 2004). Respiratory and dermal toxicity of chromium is well documented. Workers exposed to chromium develop nasal irritation, nasal ulcer, hypersensitivity reaction and chrome holes of the skin (Baselt, 2008). The inhalation of chromium compounds has been shown to be associated with the development of cancer in workers in the chromate industry (Langard, 1982). It also damages the genetic material and causes oxidative stress and DNA damage (David et al., 2008; Stout et al., 2009; and Dayan and paine, 2001). Applying medicamental herb approach has emerged on using medicines with natural and especially herbal origin (Khayatnouri et al., 2011). The herb Cymbopogon (Lemongrass) known as halfa-baris, has many health benefits and healing properties. The primary chemical component in lemongrass is citral which has strong anti- microbial and anti-fungal properties, so it prevents and cures bacterial infections in the colon, stomach, urinary tract and respiratory system. Its leaves and stems are high in folic acid and essential vitamins such as pantothenic acid (vitamin B5), pyridoxine (vitamin B-6) and thiamin (vitamin B-1). It also contains many anti-oxidant minerals and vitamins such as vitamin C, vitamin A, potassium, zinc, calcium, iron, manganese, copper, and magnesium. Lemongrass tea can act as a diuretic and is highly effective in flushing toxins and waste out of the body; improving the function of many different organs including the liver, spleen and kidneys. It can help you lose weight by shedding unnecessary water along with the impurities. Many people use lemongrass as a calmative agent; to help them deal with anxiety and nervousness (Sayed and Elserwy, 2011; Adeneye and Agbaje, 2007; Chandrasekhar and Joshi, 2006; Cheel et al., 2005; Figueirinha et al., 2010; Gayathri et al., 2011; Komorowski et al., 2008). The present study was designed to explore the possible protective effects of lemongrass on Chromium picolinate induced alveolar changes in male albino rats. MATERIAL & METHODS Preparation of lemongrass decoction 1% by addition of 2 gms dried leaves and flowering tops to 200 ml water and boiling (Adeneye and Agbaje, 2007; and Gayathri et al., 2011). Forty healthy adult male albino rats of 3 months age 150-200 gm were acclimated for a week. The animal procedures were performed in accordance with Guidelines for Ethical Conduct in the Care and Use of Animals, maintained at room temperature of 25 ± 2°C with 12-hour dark-light cycle. Animals were fed with standard rodent diet. There was no water and light restriction throughout the experimental period. They were divided into 3 groups:- Group I (Control group): 20 rats were further subdivided into two equal subgroups 10 animals each. Subgroup Ia given physiological saline and subgroup Ib given Lemongrass decoction 1% orally by gavage for one month. Group II (experimental group): 10 rats received Chromium picolinate dissolved in water and administered at a concentration of 5 mg/kg/day of Cr (pic) 3, in the drinking water ad libitum for one month orally by gavage. The chromium intake in this study is similar to other studies utilizing rodents (Komorowski et al., 2008; Jain et al., 2007 and Mozaffari et al., 2005). Group III (Protected group): 10 rats received the same dose of Chromium piclonate as in group II concomitant with Lemongrass decoction 1% for one month orally by gavage. At the end of the experimental period, the animals of all groups were sacrificed by decapitation under ether anaesthesia. Lungs were dissected out. Parts of the specimens were preserved in formal saline for preparation of paraffin blocks. Sections were cut 5µm thickness and were subjected to the following staining procedures: 1. Haematoxylin & Eosin stain for routine histological examination (Carletons et al., 1980). 2. Gomori" s Trichrome stain for demonstration of collagen fibers (Carletons et al., 1980). 3. Some of the specimens were immediately removed after sacrification and immediately fixed in 3% phosphate buffered glutaralehyde (Ph 7.4) for 2 hours at 4ºC, and further processed for examination and photography of the ultrastructure by Joel -100 CX transmission electron microscope Faculty of Science Alex university (Trevor and Graham, 1996; Girgis et al., 1988). RESULTS I- Light Microscopic Results: H&E stained sections of the control group ( subgroup Ia,b) showed lung alveoli lined with type I and type II pneumocytes. Type I pneumocytes are flattened cells with flattened nuclei, type II pneumocytes are rounded cells with rounded nuclei. The alveoli are separated by very thin interalveolar septa (Fig 1a, b). Compared with the control group chromium picolinate treated rats for one month (group II) showed collapsed alveoli with proliferation and vacuolation of type II pneumocytes, numerous foamy alveolar macrophages, intra-alveolar cellular debris and thickening of inter-alveolar septa with cellular infiltration and acidophilic vacuolated material (Fig 2 a, b). Concomitant administration of Lemongrass with Chromium picolinate (group III) revealed evident reduction of all alveolar changes except for mild thickening of inter-alveolar septa by cellular infiltration associated with mild congestion of blood vessels (Fig 3 a,b,c, d ). Gomori"s Trichrome stained section of the control group showed normal distribution of collagen fibers (Fig 4 a,b). Collagen fibers increased between the alveoli of chromium picolinate treated rats (Fig 4 c), while lung tissue of the protected group revealed more or less the normal pattern of collagen distribution (Fig 4 d,e) . II- Ultrastructural Results: The lungs of the control group (subgroup Ia,b) revealed, patent alveoli with thin walls lined with two types of cells, the flattened pneumocyte type I (Fig 5 a) and large cuboidal pneumocyte type II with its characteristic apical microvillous border and lamellar bodies. The inter-alveolar septa appeared thin containing few cells and fibers (Fig 5 b). Electron micrographs of Chromium picolinate treated rat lung for one month showed alteration in the alveolar architecture. The alveoli appeared collapsed and lined by either destructed type I and Type II pneumocyte (Fig 6 a,b), or may be evidently proliferated and abnormally vacuolated type II pneumocyte, (Fig 7 a, b, c, d) numerous foamy macrophages with vacuolated cytoplasm and many lysosomes (Fig 8 a,b, c,d ). Desquamated cells and cellular debris were also seen within the lumen of the alveoli (Fig 9 a,b,c ). The inter-alveolar septa were thickened due to mononuclear cellular infiltration, congested blood vessels (Fig 10 a,b), presence of hyaline material (Fig 11a,b) and increased collagen deposition (Fig12 a,b). The lung tissues of the protected group revealed considerable degree of preservation of the alveolar architecture. Most of the alveoli were inflated and lined with more or less normal type I and II pneumocytes (Fig13 a,b,c). Few septal cells associated with less collagen fibers and less vacuolated macrophages were also noted (Fig14 a,b,c). DISCUSSION Obesity is increasing at an alarming rate, so it possesses serious health hazards and its treatment is often disappointing. One of the key goals for enhancing weight loss is to increase the sensitivity of the cells throughout the body to insulin. Micronutrient is proposed as a global approach for the obese person (Choes et al., 2007; Bloniarz and Zareba, 2007). The trace mineral chromium is an essential nutrient involved in the regulation of carbohydrate, lipid and protein metabolism via an enhancement of insulin action. Chromium plays a key role in cellular sensitivity to insulin. It has lately gained a great deal of lay attention as an aid to weight loss. Also chromium picolinate was reported to ergogenic properties related to body composition changes in young men. (Friedman, 2000; Korc, 2004; Chaturvedi, 2007; Mehta and Farmer, 2007 and Dandona et al., 2005) Chromium picolinate (Cr (pic) 3), contains trivalent chromium. It is widely used because of claims that it exerts antidiabetic and weight-reduction effects (Anderson, 2000; Trumbo and Ellwood, 2006 and Vincent, 2003). On the other hand, other studies have raised concerns regarding the safety of Cr (pic) 3. It is a very stable hydrophobic molecule which allows it to be readily absorbed from the digestive tract and readily cross membranes. But it appears that once inside the cells, trivalent chromium (Cr3+) picolinate can be reduced to divalent chromium (Cr2+) which can participate in the Fenton reaction in the presence of hydrogen peroxide to generate hydroxyl radicals which cause DNA damage (Stearns et al., 1995; Speetjens et al., 1999; Bagchi et al., 2002 and Coryell and Stearns 2006). Animal studies have shown chromium (VI) to cause respiratory toxicity in the form of tumors, ulcerations, bronchitis and pneumonia or renal toxicity because the kidney serves not only as its major route of elimination but also accumulates chromium (Lamson and Plaza, 2002; Hepburn and Vincent, 2002, 2003; and Beaumont et al., 2008). In view of the previously mentioned facts, it was quite essential to investigate the histological effects of oral chromium picolinate on the pulmonary alveolar cells and the possible protective effect of lemongrass. In the present study chromium picolinate (group II) induced diffuse changes in the alveolar architecture as evident by collapsed alveoli, very thick interalvelolar septum due to mononuclear cellular infiltration, congested blood vessels, numerous foamy macrophage, acidophilic hyaline material, extravasated erythrocytes, desquamated cells in the alveoli associated with increased collagen fibers. Chromate and their reduction products cause many types of DNA damage. Some of these types of DNA damage can be decreased by antioxidants vitamins C and E which suggests that oxidative damage might have a role in causing these types of DNA damage within the cell (De Flora, 2000; Bagchi, 2000; Sugden et al., 2001). These previous studies provided important mechanistic data into chromium – induced toxicity. Macrophage accumulation and neutrophil infiltration in the present study add our understanding of chromium induced pulmonary toxicity. We suggest that chromium induced expression of many cytokines and chemokines may play an important role in the activation of alveolar macrophages. Activated macrophages secrete proinflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor α. The macrophage-derived inflammatory cytokines have two major effects: (1) expression of adhesion molecules on endothelial cells for extravasation of monocytes and lymphocytes; and (2) stimulation of targeted migration of mononuclear cells to the area of inflammation (chemotaxis). Thus, additional mononuclear phagocytes are recruited to the tissue from the intravascular space. The inflammation can be seen as thickening of interalveolar septa. Septum thickening leads to alveoloar collapse. In the present study, chromium exposure induced lung injury as well as a chronic inflammatory response. The predominant immune cells in the lung airways and tissue were neutrophils and lymphoid cells, respectively. We hypothesize that chromium will induce an inflammatory microenvironment in the lung that will promote proliferation and selection of growth-altered cells. These findings are apparent in group II. Chronic inflammation is involved in the pathogenesis of many cancers, including those of the lung (Coussens and Werb, 2002; Lin and Karin, 2007). The presence of neutrophils and macrophages in the lung after chromium exposure is consistent with welding fume studies in which a significant increase in neutrophils and macrophages was also detected in the lung of exposed rodents (Antonini et al., 2007; Solano-Lopez et al., 2006; Taylor et al., 2003 and Zeidler-Erdely et al., 2008) Neutrophils initiate the debridement of damaged tissue, phagocytose any pathogens, and amplify the inflammatory response through production of cytokines (Coussens and Werb, 2002; Eming et al., 2007). A major function of macrophages is to continue phagocytosis at sites of tissue injury (Freeman et al., 2007). To this end, neutrophils and macrophages release highly active substances, including reactive oxygen species (ROS) and reactive nitrogen species that may promote a microenvironment that directly damages DNA or interferes with the mechanisms of DNA repair (Azad et al., 2008; Federico et al., 2007). In the chromium exposure, these reactive species may further exacerbate DNA damage in surviving and/or proliferating epithelial cells and thus promote initiating events in chromium carcinogenesis (O’Brien et al., 2003). Macrophages also produce cytokines and growth factors in order to .stimulate cell proliferation and angiogenesis (Eming et al., 2007) We also observed that chromium exposure resulted in oxidative stress, thus chromium may promote inflammation, cell survival, and repair of the airways after lung injury. In keeping with this hypothesis, we observed proliferative epithelial cells, which is consistent with promoting cell survival in an environment of genotoxic chromium induced injury and inflammation. In the present study pneumocytes type II was the most altered cell. The increase in the number and size of type II pneumocytes which were noticed in the present study might be due to its role to replace the type I pneumocytes. When alveolar epithelium is exposed to toxic agent, particularly if there is extensive destruction of the sensitive type I pneumocytes, type II pneumocytes increase in size and number being precursor stem cells for type I pneumocytes (Stevens and Lowe, 1997) Also type II pneumocytes had deformed surfactant material. The maintenance of the alveoli depends on the presence of a surface tension lowering substance known as pulmonary surfactant. It consists of 90% phospholipids and 10% proteins. Pulmonary surfactant is made and secreted by pneumocyte type II cells in whose cytoplasm it is stored in the form of lamellated bodies. Abnormalities of phospholipid metabolism most often manifest themselves by the accumulation of phospholipids in various tissues of the body. These accumulations may then interfere with cellular functions and lead to the establishment of acute or chronic disease states. The accumulation of phospholipids in the lung has been referred to, pulmonary alveolar proteinosis (PAP). The alveoli in PAP are filled with proteinaceous material, which has been analyzed extensively and determined to be normal surfactant composed of lipids and surfactant-associated proteins. Evidence exists of a defect in the homeostatic mechanism of either the production of surfactant or the clearance by alveolar macrophages. A clear relationship has been demonstrated between PAP and impaired macrophage maturation or function, which might account for the high association with malignancies and unusual infections (Griese et al., 2010; Cummings et al., 2012; Suzuki, 2010; Carey and Trapnell, 2010). The present study revealed accumulation of phospholipid in the macrophages (foam cell) and in the cytoplasm of type II pneumocytes. In the lung because of its unique architecture, accumulation of phospholipids may occur both intracellularly and extracellularly. Intracellular accumulation of phospholipids can interfere with cellular functions resulting in impaired or abnormal pulmonary responsiveness in a variety of situations. The cytoplasmic accumulation of phospholipids in alveolar macrophages has been shown to impair its phagocytic activity. Impaired phagocytic activity of macrophages may lead to decreased resistance of the lung to infection (Carraway, 2000). Extracellular accumulation of phospholipids in the alveoli interferes with gas exchange causing respiratory insufficiency. In addition, alveolar clearance of toxic substances has been shown to be severely impaired in the lungs of rats with phospholipidosis ( Presneill, 2004 ). The extravasation of erythrocytes was most possibly the sequel of endothelial cell damage in the alveolar capillaries. Also extravascular localization of leukocytes implies acute vascular injury which is a consistent feature of injury caused by most pulmonary toxicants (Cotran, 1987). Exudation of leukocytes (mostly neutrophils into the alveolar lumen of exposed animals was most probably attributed to increased permeability of the alveolar capillaries. Besides, the toxic material was found to stimulate macrophages and pneumocytes to release chemoattractants for neutrophils. Also the infiltrating inflammatory cells may account for the damaging of the alveolar and interstitial pulmonary structures through the lytic effect of their enzymes. On the other hand light microscopic examination of the Trichrome stained sections of the experimental group showed that the amount of collagen fibers increased. The agents that induced phospholipidosis produce lung fibrosis. Pulmonary fibrosis is the end – stage of a group of chronic diseases. A complex set of tissue reactions must occur for the formation and accumulation of fibrous connective tissue that defines pulmonary fibrosis. The pathogenesis of pulmonary fibrosis begins as an inflammatory response to injury when immune cells are excessively or inappropriately activated. These immune cells include macrophages and neutrophils that release toxic mediators, compromising epithelial integrity and promoting tissue injury. The normal repair process involves the recruitment and activation of mesenchymal cells resulting in extracellular matrix deposition, re-epithelialization and restoration of normal lung architecture (Hamdy et al., 2012; Kliment and Oury, 2010). Also previous researches reported that reactive oxygen species promotes the development of inflammation and increases activity at sites of inflammation and induces the proliferation of the fibroblast leading to severe pulmonary fibrosis (Shi et al., 2014; Kliment and Oury, 2010). Moreover the results of the present study revealed destruction of type I and type II pneumocytes. Excessive and persistent formation of ROS from inflammatory cells (i.e., macrophages and neutrophils) is considered the hallmark of genotoxicity. The overall genotoxic response will depend on the effectiveness and efficiency of intra- and extracellular antioxidant defense systems, DNA repair systems, and processes leading to apoptotic and necrotic processes in those cells carrying premutagenic lesions (Starosta and Griese, 2006; Trapani et al., 2003 and Stout et al., 2009). The lemongrass group (group III) in the present study showed thin interalveolar septa, few mononuclear cellular infiltration, few extravasated RBCs and decreased collagen fibers. In agreement with these findings previous studies reported that lemongrass constitutes an important source of antioxidants, also contains minerals that function as co – factors in the antioxidant enzymes (Arhoghro et al., 2010; Figueirinha et al., 2010; Omotode, 2009 and Tiwari et al., 2010). Conclusion Over viewing our results we find that exposure to chromium induces chronic inflammation and injury in the lung. Furthermore, this chromium induced injury and inflammation was associated with epithelial cell proliferation. Taken together, we suggest that these early disease processes promote a microenvironment that may participate in the initiation and promotion of neoplastic cells and contribute over time to chromium carcinogenesis on the other hand lemongrass produced sufficient protection against these damage . Fig 1 a,b: photomicrograph of the control rat lung showing, normal architecture of the alveoli (A) separated by very thin interalveolar septa. The alveoli are lined by flat type I pneumocyte (↑) and rounded type II pneumocyte (↑↑). H&E stain Mic Mag a X100-bX 400. Fig 2 a,b : photomicrograph of the rat lung group II showing, collapsed alveoli (A) separated by very thick interalveolar septa. Note mononuclear cellular infiltration, congestion of the blood vessels (V), acidophilic hyaline material (*) numerous foamy macrophages (↑) and extravasated RBCs (R). H&E stain Mic Mag a X100-bX 400. Fig 3 a,b, c,d : photomicrograph of the rat lung group III showing, preserved lung architecture except very mild increase in thickness of interalveolar septa, some acidophilic hyaline material (*), exfoliated cells (E ) and congested blood vessels (V). H&E stain Mic Mag. a ,bX100-c,dX 400. Fig 4 a,b,c,d, e : photomicrograph of the rat lung showing a, b - normal distribution of collagen fibers in the control group. c- group II showing thick interaveolar septa, congestion of the blood vessels and increase in collagen fibers. d, e - group III showing very mild increase in interalveolar septa associated with mild increase in collagen fibers. Gomori"s Trichrome stain Mic Mag X 200. Fig 5 a,b : electron micrograph of the control rat lung showing, open alveoli (A) lined by flat nucleus of type I pneumocyte ( P1) and type II pneumocyte (P2) with characteristic lamellated structure (↑) and apical microvilli (mv). Mic Mag a X4000-b X3000. Fig 6 a,b : electron micrograph of rat lung group II showing collapsed alveoli (A), destruction( ↑) and abnormal nucleus of type I pneumocyte ( P1 ). Destruction of type II pneumocyte (↑↑) filled with empty lamellated structure (P2), thick interalveolar septa, hyaline material (*) and extravasated RBCs (R). Mic Mag a X 5000-b X 3000. Fig 7 a,b,c,d : electron micrograph of rat lung group II showing collapsed alveoli (A ) separated by thick interalveolar septa. Note numerous type II pneumocyte filled with empty lamellated structure (P2), alveolar macrophage (↑) and congested blood vessels (V). Mic Mag a X 1500-b X 1000- c X 2500- d X 2000. Fig 8 a,b,c,d : electron micrograph of rat lung group II showing, numerous alveolar macrophages ( foam cell ) filled with many vacuoles (↑). Note extravasated RBCs (R) and hyaline material (*). Mic Mag a,b,d X 3000- c X 1500. Fig 9 a, b, c: electron micrograph of rat lung group II showing, interalveolar septa contain desquamated cell and cellular debris (↑↑), alveolar macrophages (↑). Note type II pneumocyte containing empty lamellated structure (P2), congested blood vessels (V) and hyaline material (*). Mic Mag a , c X 1000-b X 1500. Fig 10 a, b: electron micrograph of rat lung group II showing, thick interalveolar septa due to mononuclear cellular infiltration (↑) and congestion of the blood vessels (V). Note type II pneumocyte filled with empty lamellated structure (P2) and cellular debris (↑↑). Mic Mag a X 1500- b X 2000. Fig 11 a, b: electron micrograph of rat lung group II showing, thick interalveolar septa, hyaline material (*), congested blood vessels (V), increased collagen fiber (↑) and desquamated cell (↑↑). Mic Mag a X 2500-b X 3000. Fig 12 a, b: electron micrograph of rat lung group II showing, abnormal type II pneumocyte with irregular nuclei ( P 2 ), marked increase of collagen fibers (↑) and extravasated RBCs (R). Mic Mag a X 4000- b X 3000. Fig 13 a, b, c: electron micrograph of rat lung group III showing, more or less normal architecture of the alveoli lined by type I pneumocyte (P 1) and type II pneumocyte (P 2) with apical microvilli (mv) and lamellated structure (↑). Note presence of extravasated RBCs (R), few collagen fibers (↑↑) and some hyaline material (*). Mic Mag a,b X 3000- c X 4000. Fig 14 a, b, c: electron micrograph of rat lung group III showing, type II pneumocyte with apical microvilli (mv) and lamellated structure (P2) and alveolar macrophage with less vacuoles(↑). Note mild increase in collagen fibers (C) and hyaline material (*). Mic Mag a X 3000- b X 2500- c X 4000. REFFERENCES 1- Adeneye AA. And Agbaje EO. 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Effect of chromium piclonate on histopathological alteration in STZ and neonatal STZ diabetic rats. j. Cell Mol. Med. 2003; 7(3) 322-9. 69- Vincent JB: The potential value and toxicity of chromium picolinate as a nutritional supplement, weight loss agent and muscle development agent. Sports Med 2003; 33:213-30. 70- Wayne WC, Lyndon JJ, Stephanie LD, Deanna CC, Richard A A and William J E. Effects of resistance training and chromium piclonate on body composition and skeletal muscle in older men. Journal of Applied physiology 1999; 86(1); 29-39. 71- William T, Cefalu MD and Frank B. Role of chromium in human health and in diabetes. Diabetes Care 2004; 27(11): 2741-51. 72- Zeidler-Erdely PC, Kashon ML, Battelli LA, Young SH, Erdely A, Roberts JR, et al. Pulmonary inflammation and tumor induction in lung tumor susceptible A/J and resistant C57BL/6J mice exposed to welding fume. Part Fibre Toxicol. 2008; 5:12.

Источник: http://www.academia.edu/16299719/THE_POSSIBLE_PROTECTIVE_EFFECTS_OF_CYMBOPOGON_LEMONGRASS_DECOCTION_ON_CHROMIUM_PICOLINATE_INDUCED_PULMONARY_ALVEOLAR_CHANGES_IN_ADULT_MALE_ALBINO_RATS

Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE−/− mice

Abstract

Increasing evidence suggests thrombospondin-1 (TSP-1), a potent proatherogenic matricellular protein, as a putative link between hyperglycemia and atherosclerotic complications in diabetes. We previously reported that the micronutrient chromium picolinate (CrP), with long-standing cardiovascular benefits, inhibits TSP-1 expression in glucose-stimulated human aortic smooth muscle cells in vitro. Here, we investigated the atheroprotective action of orally administered CrP in type 1 diabetic apolipoprotein E-deficient (ApoE−/−) mice and elucidated the role of TSP-1 in this process. CrP decreased lipid burden and neointimal thickness in aortic root lesions of hyperglycemic ApoE−/− mice; also, smooth muscle cell (SMC), macrophage and leukocyte abundance was prevented coupled with reduced cell proliferation. Attenuated lesion progression was accompanied with inhibition of hyperglycemia-induced TSP-1 expression and reduced protein O-glycosylation following CrP treatment; also, PCNA and vimentin (SMC synthetic marker) expression were reduced while SM-MHC (SMC contractile marker) levels were increased. To confirm a direct role of TSP-1 in diabetic atherosclerosis, hyperglycemic TSP-1−/−/ApoE−/− double knockout mice were compared with age-matched hyperglycemic ApoE−/− littermates. Lack of TSP-1 prevented lesion formation in hyperglycemic ApoE−/− mice, mimicking the atheroprotective phenotype of CrP-treated mice. These results suggest that therapeutic TSP-1 inhibition may have important atheroprotective potential in diabetic vascular disease.

Introduction

Vascular disease is the leading cause of increased morbidity and mortality in diabetes. Risks of atherosclerotic complications are enhanced two-to-four-fold in diabetic patients1,2, accounting for >80% of deaths and hospitalizations in these individuals. Clinical studies, including recent animal data3,4,5,6,7, indicate that elevated glucose levels, independent of hyperlipidemia, may have profound proatherogenic effects in diabetes. Previous epidemiological studies1,2 have revealed a strong association between cumulative glycemic exposure and intima-media thickness (IMT) of the carotid artery, an early marker of atherosclerosis. Together, these reports support the notion that hyperglycemia is an important risk factor for development of macrovascular complications in diabetes. However, mechanisms underlying hyperglycemia-induced atherosclerosis are incompletely understood.

Thrombospondin-1 (TSP-1), a potent proatherogenic and anti-angiogenic protein, belongs to a family of matricellular proteins controlling cell-cell and cell-matrix interactions8. Earlier studies have shown that TSP-1 expression is significantly enhanced in response to vascular injury and in atherosclerotic lesions, with augmented expression in vascular smooth muscle cells (VSMC)9,10,11. Growing literature indicates distinct cell-and tissue-specific effects of TSP-1; both in vivo and in vitro studies have revealed that TSP-1 stimulates VSMC proliferation12 while inducing endothelial cell (EC) apoptosis13. The TSP protein family has been previously linked to atherosclerotic vascular disease based on GENEQUEST studies demonstrating an association between specific single nucleotide polymorphisms in the TSP genes with coronary artery disease and myocardial infarction14. These findings, confirmed by multiple human studies, lend support to TSP-1 as an alternative pathway for development of atherosclerosis.

Diabetic patients and diabetic animal models have been reported to have elevated TSP-1 levels in the plasma and walls of the large blood vessels15,16. Earlier work demonstrated that high glucose in vitro, characteristic of a diabetic milieu, upregulates TSP-1 expression in cells of the large blood vessel (VSMC, EC, fibroblasts)16. We have further shown that hyperglycemia in vitro increases TSP-1 expression via a transcriptional mechanism in primary human aortic smooth muscle cell (HASMC) cultures17,18. Together, these findings implicate TSP-1 as a putative link between hyperglycemia and accelerated atherosclerotic complications in diabetes. However, the impact of therapeutic TSP-1 inhibition on diabetic atherogenesis remains unexplored.

We recently reported19 that chromium picolinate (CrP), the most bioavailable form of the mineral nutrient trivalent chromium (Cr3+) at pharmacological concentrations, inhibits TSP-1 expression in glucose-stimulated HASMC in vitro. In addition, we have found that TSP-1 inhibition was accompanied with attenuated HASMC proliferation in response to Cr3+ and this effect was specific for high glucose conditions19. Accumulating data have indicated optimal regulatory effects of Cr3+ on carbohydrate and lipid metabolism20,21. Unlike its hexavalent counterpart (Cr6+), Cr3+ is relatively stable, with minimal toxicity issues at doses allowable for dietary intake22,23. Numerous studies have also indicated favorable glycemic and cardiovascular effects of Cr3+ 24,25,26,27,28. Nevertheless, clinical significance of Cr3+ in health and disease has been challenged by a dearth of mechanistic understanding of Cr3+ action.

Diabetic patients have low circulating and tissue Cr3+ levels compared to non-diabetic individuals29. There is increasing evidence that inadequate Cr3+ intake may elevate blood glucose and lipid levels30. Clinical studies have revealed that CrP in combination with biotin reduces insulin resistance and lowers the plasma atherogenic index in a cohort of type 2 diabetic patients31. Previous studies have also demonstrated that in STZ-induced diabetic Sprague Dawley rats in vivo and glucose-stimulated cultured monocytes in vitro, different Cr3+ formulations reduced lipid peroxidation and pro-inflammatory cytokine secretion32,33,34. Moreover, in a hypercholesterolemic rabbit model of atherosclerosis, intramuscular administration of chromium chloride (CrCl3) lowered serum cholesterol levels and reduced the size of lipid deposits in coronary and aortic vasculature35. Despite a favorable response to Cr3+ in vascular disease, the precise effect and mechanisms of the nutraceutical CrP in large vessels in the setting of diabetic atherosclerosis has remained elusive.

The present study provides the first demonstration that orally administered CrP impedes development of atherosclerotic lesions in STZ-induced hyperglycemic ApoE−/− mice, a mouse model of combined atherosclerosis and type 1 diabetes. Our data suggests that inhibition of TSP-1 expression, possibly mediated via reduced protein O-glycosylation, and blockade of VSMC phenotypic switching in the large vessel are important atheroprotective mechanisms of CrP in vivo. Notably, we have shown that genetic deletion of TSP-1 protects ApoE−/− mice against hyperglycemia-induced atherosclerosis, mimicking the protective phenotype of CrP-treated diabetic atherosclerotic mice.

Results

Oral chromium picolinate has no effect on body weight, blood glucose and lipid profiles in hyperglycemic ApoE−/− mice

No significant differences in body weights were observed in STZ-induced hyperglycemic ApoE−/− mice treated with or without CrP compared with non-hyperglycemic ApoE−/− mice (Fig. 1a). As expected, non-fasted blood glucose levels increased 2–3-fold following STZ treatment in ApoE−/− mice vs. non-STZ-treated ApoE−/− (Fig. 1b); consistent with earlier reports, multiple low-dose STZ did not adversely affect animal well-being or survival. Interestingly, under conditions of experimental type 1 diabetes in ApoE−/− mice, CrP provided in drinking water had no effect on the non-fasted blood glucose levels in these mice. Although a slight reduction (~15%) in glucose levels was noted at 12 weeks of age following CrP administration, this effect was abolished at later time points and the animals continued to remain hyperglycemic attaining non-fasted blood glucose levels ≥250 mg/dl. Moreover, while hyperglycemia increased plasma total cholesterol and total triglyceride levels in STZ-ApoE−/− mice (~1.6-fold versus ApoE−/− control), there was no statistically significant effect of CrP administration on the lipid profiles in hyperglycemic ApoE−/− mice (Figs. 1c,d). Similar to these findings, CrP did not affect either body weight or total cholesterol levels in non-diabetic ApoE−/− control mice (data not shown).

Six weeks old male ApoE−/− mice were treated with 50 mg/Kg/day streptozotocin or sodium citrate buffer (vehicle control) i.p. for 5 consecutive days. This was followed by treatment with or without CrP (8 μg/Kg/day) in drinking water beginning at 8 weeks of age. (a) Body weight and (b) non-fasted blood glucose levels were measured every two weeks from 8–18 weeks of age. (c) Plasma total cholesterol and (d) plasma total triglyceride levels were measured at end point of the study (18 weeks). Results are expressed are mean ± SD (n = 10–17 mice per group); *p ≤ 0.05 vs. Control.

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Chromium picolinate impedes hyperglycemia-induced atherosclerosis in ApoE−/− mice

Atherosclerotic lesion development was examined in all treatment groups using aortic root morphometry. Lipid-filled lesions were significantly increased in STZ-induced hyperglycemic ApoE−/− mice compared with age-matched non-hyperglycemic ApoE−/− littermates, shown by oil red O (ORO) staining. In contrast, CrP treatment diminished lesion formation in hyperglycemic ApoE−/− mice (Fig. 2a). Quantification of ORO-positive staining revealed that while lesion burden was augmented in STZ-ApoE−/− mice (2.3-fold vs. Controls), CrP remarkably reduced lipid-filled lesions in hyperglycemic ApoE−/− (62.6% vs. STZ, Fig. 2b). Aortic root H & E staining showed reduced neointimal thickening in response to CrP (Fig. 2c). Specifically, while the intimal-medial thickness was increased by 66.7% in aortic roots of hyperglycemic ApoE−/− mice compared to non-hyperglycemic ApoE−/−, CrP-treated mice demonstrated decreased neointimal thickening (30% vs. STZ only, Fig. 2d). Aortic root morphometry using Masson-Trichrome (MT) staining showed no statistically significant differences in the lesion collagen content in hyperglycemic ApoE−/− mice vs. non-hyperglycemic ApoE−/−, although higher collagen amounts were noted in regions outside the aortic root vessel wall in STZ-treated ApoE−/− mice. Moreover, CrP treatment had no effect on collagen accumulation in the aortic root lesions of hyperglycemic ApoE−/− mice compared to STZ only animals (Fig. 2e,f). Interestingly, lesion formation remained unaffected by CrP in ApoE−/− control mice that were not subjected to STZ-induced hyperglycemia (Supplementary Fig. S1).

STZ-induced hyperglycemic ApoE−/− mice were treated with or without CrP (8 μg/Kg/day) from 8–18 wks of age. Aortic root morphometry was performed as described in Methods. All comparisons were made between Control (ApoE−/− no STZ), STZ (ApoE−/− with STZ) and STZ + CrP (ApoE−/− with STZ plus CrP). Shown are (a,b) representative images and summary data for quantification of ORO-positive staining (Control: n = 6; STZ: n = 7; STZ + CrP: n = 7), (c,d) representative H & E images and summary data for neointimal thickness (Control: n = 5; STZ: n = 7; STZ + CrP: n = 7), (e,f) representative images and summary data for quantification of MT staining (Control: n = 6; STZ: n = 8; STZ+CrP: n = 8). All microscopic images were captured at 4X magnification. Specific regions used for analyses are marked by dotted lines. Results are presented as fold of Control; all values are expressed as mean ± SD; *p ≤ 0.003 vs. Control, #p ≤ 0.026 vs. STZ.

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Atherosclerotic lesions were also monitored using a non-invasive auxiliary approach, High-frequency Ultrasound Imaging. Congruent to the aortic root morphometric data, both left ventricular outflow tract and transaortic arch diameters were reduced (~20%) in STZ-ApoE−/− vs. non-STZ-ApoE−/− mice. Notably, this reduction in vessel diameter was completely abrogated in CrP-treated mice (Supplementary Table S1). Furthermore, ultrasound imaging of carotid vessels revealed significant reduction in vessel diameter in hyperglycemic ApoE−/− mice (~25% vs. Control); in contrast, this decrease in vessel diameter was completely obliterated in STZ-ApoE−/− mice treated with CrP (Figs. 3a–c). Further histological experiments were conducted to verify whether the reduction in vessel diameter in STZ-ApoE−/− mice was related to formation of atherosclerotic lesions in the carotid vasculature. Specifically, H & E staining of carotid vessel tissue sections derived from STZ-induced hyperglycemic ApoE−/− mice revealed distinct lesions in the vessel wall resulting in luminal obstruction (Supplemental Fig. S2a). Importantly, a strong negative correlation (-0.91) was noted between the carotid vessel internal diameter and % luminal obstruction of the carotid vessels (Supplemental Fig. S2b), validating the ultrasound assessments of lesion development in the carotid vasculature (Fig. 3). Taken together, these data demonstrate an atheroprotective effect of oral CrP in hyperglycemic ApoE−/− mice.

(a) Shown are representative ultrasound images of left and right common carotid artery; yellow arrows indicate neointimal thickening of vessel wall resulting in reduction in vessel diameter in STZ mice. Shown are the summary data for (b), left and (c), right carotid artery diameters. LCCA- left common carotid artery; LECA- left external carotid artery; RCCA- right common carotid artery; RECA- right external carotid artery. Results are expressed as mean ± SD (n = 4–7 mice/group) *p ≤ 0.0001 vs. Control; +p ≤ 0.0001 vs. STZ.

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Chromium picolinate prevents hyperglycemia-induced increase in cellular proliferation and inflammatory lesion burden in STZ-treated ApoE−/− mice

Next, we characterized lesion cellularity in response to CrP in vivo. Smooth muscle cell abundance and cell proliferation was assessed using α-SMA and PCNA immunohistochemistry, respectively. STZ-induced hyperglycemia increased α-SMA (1.6-fold) and PCNA staining (2.0-fold) in ApoE−/− mice, indicating enhanced smooth muscle cell content and cell proliferation. On the contrary, there was a significant decrease in α-SMA and PCNA positive staining in aortic roots of STZ-ApoE−/− mice that received CrP (~40–48% vs. STZ only, Fig. 4); as expected, α-SMA immunostaining demonstrated positive medial staining of smooth muscle cells in control mice whereas increased α-SMA staining was limited to the neointimal layer in the aortic root of STZ mice (Supplemental Fig. S3). The PCNA staining patterns were confirmed using a second proliferation marker, Ki67. As shown in Supplemental Fig. S4a,b, Ki67 expression was increased (2.2-fold) in aortic root lesions of STZ-induced hyperglycemic ApoE−/− mice compared to ApoE−/− control mice, recapitulating the PCNA immunostaining results (Fig. 4c). Moreover, CrP treatment reduced (54%) Ki67 expression similar to its effect on PCNA expression in hyperglycemic ApoE−/− mice compared to STZ only ApoE−/− (Supplemental Fig. S4a,b). Notably, double immunofluorescence experiments using α-SMA and PCNA antibodies demonstrated increased PCNA-positive smooth muscle cell content in aortic root lesions of STZ mice vs. controls. In contrast, increased smooth muscle cell proliferation was prevented in aortic root sections of STZ + CrP mice, depicted by a lack of PCNA-positive α-SMA staining (Fig. 4a, merge). Next, immunohistochemistry demonstrated enhanced macrophage and leukocyte infiltration into aortic root lesions of hyperglycemic ApoE−/− mice while the lesion burden of inflammatory cells was reduced in CrP-treated hyperglycemic mice (Figs. 5a–c). Specifically, immunostaining quantifications revealed > 2.4-fold increase in CD68 and CD45 expression in aortic root lesions of STZ-ApoE−/− mice compared with ApoE−/− controls. In contrast, oral CrP decreased both CD68 and CD45 expression in aortic roots of hyperglycemic ApoE−/− mice (54–64% vs. STZ only, Figs. 5b–d) indicative of attenuated macrophage and leukocyte accumulation, respectively. In each case, the specificity of the immunofluorescence staining was confirmed in parallel aortic root sections incubated in the absence of the corresponding primary antibody (Supplementary Fig. S5). Together, these results demonstrate that CrP in vivo prevents hyperglycemia-induced smooth muscle cell and inflammatory cell abundance as well as inhibits smooth muscle cell proliferation in diabetic atherosclerotic mice, resembling a state of reduced plaque development.

Shown are (a) representative images for PCNA and α-SMA co-staining (10x magnification). Regions used for immunostaining quantifications are outlined by dotted lines; arrows indicate PCNA-positive smooth muscle cells. (b,c) Summary data for quantification of α-SMA-and PCNA-positive staining. All results are presented as fold of Control and values are expressed as mean ± SD (Control: n = 6; STZ: n = 7; STZ+CrP: n = 6); *p < 0.0001 vs. Control, #p < 0.0001 vs. STZ.

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Shown are representative images and summary data for quantification of (a,b) CD68-positive staining and (c,d) CD45-positive staining. All images were captured at 15X magnification. Histology of the immunofluorescence images are shown in the corresponding H & E-stained images (indicated by the black box). Specific regions of the immunofluorescence images used for data analyses are marked by dotted lines. Results are presented as fold of Control and all values are expressed are mean ± SD (for CD45, Control: n = 6; STZ: n = 6; STZ+CrP: n = 5; for CD68, Control: n = 5; STZ: n = 5; STZ+CrP: n = 5); *p ≤ 0.0008 vs. Control; #p ≤ 0.0018 vs. STZ.

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Chromium picolinate in vivo inhibits TSP-1 expression, decreases protein O-glycosylation and blocks VSMC phenotypic switching in aortic vessels of STZ-induced hyperglycemic ApoE−/− mice

We reported earlier that both CrCl3 and CrP in vitro inhibit TSP-1 expression in glucose-stimulated HASMC primary cultures19. To investigate whether the atheroprotective action of CrP observed in the current study correlates with TSP-1 expression in vivo, aortic tissue lysates prepared from all treatment groups were subjected to immunoblotting. As shown in Fig. 6a, TSP-1 expression was increased in aortic vessels of hyperglycemic ApoE−/− mice (2.45-fold vs. Control). Concomitant to enhanced TSP-1 expression, hyperglycemia augmented protein O-GlcNAc levels in the aortic vasculature of STZ-ApoE−/− mice. Specifically, immunoblotting of aortic lysates revealed elevated O-GlcNAc levels on several proteins ranging from approximately 100–260 kDa and 45–60 kDa in STZ-ApoE−/− mice (Fig. 6b). This increase in protein O-GlcNAcylation was also accompanied with enhanced OGT expression (3.2-fold, Fig. 6c), a major regulator of O-GlcNAcylation. In contrast, TSP-1 expression was inhibited in aortic vessels of hyperglycemic ApoE−/− treated with CrP (76.7% vs. STZ, Fig. 6a). Moreover, downregulation of TSP-1 expression was accompanied with reduced O-GlcNAc protein modification and attenuated OGT expression in hyperglycemic ApoE−/− mice subjected to CrP treatment (~55% vs. STZ, Fig. 6b,c). Notably, while STZ-induced hyperglycemia augmented cell proliferation marker PCNA expression in ApoE−/− mice (3.6-fold vs. Control), CrP administration significantly inhibited PCNA expression (80% vs. STZ, Fig. 6d). Consistent with effects on cell proliferation, hyperglycemic ApoE−/− mice showed enhanced vimentin expression together with reduced SM-MHC expression in the aortic vasculature. Specifically, densitometry of immunoblots revealed 2-fold increase in vimentin expression and 56.5% decrease in SM-MHC expression in aortic lysates of STZ-ApoE−/− mice compared with ApoE−/− Controls. On the contrary, CrP significantly inhibited vimentin expression (62.5%) while increasing SM-MHC expression (2.18-fold) in hyperglycemic ApoE−/− mice (Figs. 6e–g). Together, these data demonstrate an important link between attenuated lesion formation, downregulation of TSP-1 expression and reduced protein O-GlcNAcylation in CrP- treated hyperglycemic ApoE−/− mice.

Shown are representative immunoblots depicting (a) TSP-1, (b) O-GlcNAc, (c) OGT, (d) PCNA, (e) SM-MHC expression (upper panel) and vimentin expression (middle panel). Membranes were probed with anti-β-actin used as loading control. In each case, graphs represent summary data for densitometric quantification of immunoblots (at least 3 mice per group). For c and e, lane images show proteins detected on a single immunoblot; however, lanes were rearranged for clarity of presentation; the corresponding original uncropped blots are presented in Supplementary Fig. S7. All results are presented as fold of Control; values are expressed as means ± SD; *p ≤ 0.05 vs. Control; #p ≤ 0.05 vs. STZ.

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TSP-1 deficiency prevents development of atherosclerotic lesions in STZ-induced hyperglycemic ApoE−/− mice

To investigate a direct role of TSP-1 in hyperglycemia-induced atherosclerosis, we generated TSP-1−/−/ApoE−/− double knockout mice that were subjected to STZ-induced hyperglycemia, as described in Methods; these mice were compared with age-matched STZ-induced hyperglycemic ApoE−/− littermates, both genotypes being at 18 weeks of age. Similar to STZ-treated ApoE−/−, TSP-1−/−/ApoE−/− dKO mice developed significant hyperglycemia upon STZ treatment achieving non-fasted blood glucose levels ≥ 250 mg/dl. However, under these conditions of experimental diabetes, no statistically significant differences were observed in the body weights, plasma total cholesterol and total triglyceride levels between the mice genotypes (Supplementary Table S2). Aortic root morphometry showed remarkable decrease in lipid content (Fig. 7a) and neointimal thickening (Fig. 7c) in hyperglycemic TSP-1−/−/ApoE−/− mice compared with age-matched hyperglycemic ApoE−/− littermates. Specifically, morphometric quantification depicted reduced lesion burden (2.9-fold) and neointimal thickness (1.7-fold) in aortic roots of STZ-treated TSP-1−/−/ApoE−/− mice (vs. STZ-ApoE−/−, Fig. 7b–d). Similar to CrP-treated mice, there was no difference in the collagen content of lesions in either genotypes in response to hyperglycemia (Fig. 7e,f). Furthermore, cellular characterization of lesions revealed attenuated PCNA and α-SMA expression in hyperglycemic TSP-1−/−/ApoE−/− dKO mice (vs. hyperglycemic ApoE−/−, Fig. 8a–c); while STZ-TSP-1−/−/ApoE−/− mice showed positive medial SMA staining, enhanced α-SMA staining was limited to the neointimal surface of the aortic root in STZ-ApoE−/− mice. Importantly, double immunofluorescence staining showed lower abundance of PCNA-positive smooth muscle cells in aortic root lesions of STZ-TSP-1−/−/ApoE−/−, depicting attenuated smooth muscle cell proliferation (Fig. 8a, merge), compared to STZ-ApoE−/− mice. Further, both CD68 and CD45 expression levels were ameliorated in hyperglycemic TSP-1−/−/ApoE−/− dKO mice (vs. STZ-ApoE−/−, Figs. 8d–g). Specific immunostaining quantifications showed marked reduction in PCNA expression (2.1-fold), further confirmed by Ki67 immunostaining (Supplemental Fig. S4c,d); moreover, α-SMA, CD68 and CD45 expression profiles were attenuated in aortic root lesions of STZ-induced hyperglycemic TSP-1−/−/ApoE−/− mice (1.6-, 2.1- and 2.6-fold, respectively vs. age-matched STZ-ApoE−/− littermates), indicating attenuated cell proliferation, lower smooth muscle cell abundance and reduced macrophage as well as leukocyte lesion invasion. Collectively, these data clearly demonstrate that TSP-1 deletion protects ApoE−/− mice against hyperglycemia-induced atherosclerosis.

Age-matched male ApoE−/− and TSP-1−/−/ApoE−/− dKO mice were subjected to STZ-induced hyperglycemia; aortic root morphometry was performed as described in Methods. Shown are (a,b) representative ORO images and summary data for quantification of ORO positive staining (STZ-ApoE: n = 7; STZ-TSP-1/ApoE: n = 5); (c,d) representative H & E images and summary data for neointimal thickness (STZ-ApoE: n = 7; STZ-TSP-1/ApoE: n = 5); (e,f) representative images and summary data for quantification of MT positive staining (STZ-ApoE: n = 8; STZ-TSP-1/ApoE: n = 5). Specific regions used for analyses are indicated by dotted lines. Results are presented as fold of ApoE−/− Control; all values are expressed as mean ± SD; *p ≤ 0.0013 vs. STZ-ApoE−/−.

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Shown are representative images for (a) PCNA and α-SMA co-staining (10X magnification), (b,c) quantification data for PCNA and α-SMA staining (STZ-ApoE−/−: n = 7; STZ-TSP-1/ApoE: n = 6), (d) CD68 staining (15X magnification) depicting macrophage content and (e) CD45 staining (15X magnification) depicting leukocyte abundance and (f,g) summary data for quantification of CD68 (STZ-ApoE: n = 5; STZ-TSP-1/ApoE: n = 5) and CD45 (STZ-ApoE: n = 6; STZ-TSP-1/ApoE: n = 5) positive staining. Regions used for analysis are marked via dotted lines; arrows indicate PCNA-positive smooth muscle cells. Histology of the immunofluorescence images are shown in the corresponding H & E-stained images (indicated by black box). Results are presented as fold of ApoE−/− Control. All values are expressed are mean ± SD; *p ≤ 0.0019 vs. STZ-ApoE−/−.

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Discussion

The present study provides the first demonstration for an atheroprotective effect of the nutraceutical CrP in a mouse model of diabetic atherosclerosis. We further show that attenuated lesion progression is accompanied with inhibition of TSP-1 expression in response to CrP in vivo. Notably, the current work delineates a direct role of TSP-1 in hyperglycemia-driven atherosclerosis.

Clinical data on the cardiovascular benefits of Cr3+ in diabetes have been agnostic36,37, confounded by the question of whether the effects are mediated via glycemic regulation. Contrary to few earlier reports26,34,38, orally administered CrP did not have any effect on circulating glucose levels in hyperglycemic ApoE−/− mice in the current study. While such discrepancy could be attributed to differences in species (rat vs. mice), Cr3+ formulation and dose as well as animal models (wild-type vs. ApoE−/−) utilized, it is important to note that the reduced susceptibility to atherosclerosis in CrP-treated mice was not related to lower lipid profiles in these animals; indeed, plasma total cholesterol and total triglyceride levels were not significantly different between hyperglycemic ApoE−/− mice treated with and without CrP. A possible explanation for such species-dependent differential glycemic response may relate to generation of a metabolite specific to rats, capable of modulating glucose metabolism. As such, our findings suggest an alternate mechanism of atheroprotection by Cr3+, independent of glucose and lipid control.

Previous epidemiological data and animal studies using rabbits have indicated a correlation between Cr3+ intake, incidence of coronary artery disease and serum lipid deposition31,35,39. Both in vitro and in vivo studies have implicated that Cr3+ may lower risks of vascular inflammation under conditions of elevated glucose levels32,33,34. In line with these reports, the present study has revealed that oral CrP significantly impedes lesion formation and reduces neointimal thickening in the aortic sinus of type 1 diabetic atherosclerotic mice. These morphometric results were also confirmed by ultrasound imaging of the aortic and carotid vasculature. Previous studies have indicated a correlation between conventional ORO staining methods and Ultrasound Imaging approaches for vascular lesion detection in mice40. Our data lend additional support to ultrasound imaging as a useful non-invasive methodology for progressive atherosclerotic lesion monitoring in mice. Reduced lesion severity in response to CrP was further illustrated by cellular lesion characterization. Morphometric quantification of aortic root lesions revealed that CrP prevents plaque development in hyperglycemic ApoE−/− mice, depicted by the profound decrease in hyperglycemia-induced proliferative smooth muscle cell abundance as well as macrophage and leukocyte infiltration.

Atherosclerosis is a multifactorial progressive disease comprising of a series of cellular and molecular events, triggering enhanced inflammatory response in the vessel wall. Internalization of atherogenic lipoproteins by monocyte-derived macrophages and activation of inflammatory pathways are important pathogenic mediators of atherosclerosis. This, in turn, may initiate release of a repertoire of growth factors and cytokines promoting VSMC activation, a key step responsible for initiation and progression of atherosclerotic lesions41. Diabetic patients are predisposed to aberrant VSMC activation42, characterized by enhanced migratory, proliferative and synthetic SMC phenotype. Previous work from our laboratory and others16,17,19 have shown that hyperglycemia, mimicking diabetes, upregulates the proatherogenic protein TSP-1 expression. TSP-1, a secreted glycoprotein, is expressed by many different cell types including endothelial cells, SMC and fibroblasts16, and has been widely implicated in atherosclerotic vascular disease14,43. The multidomain structure of TSP-1 has been attributed to the cell-and tissue-specific mechanisms of TSP-1 upregulation observed under glucose stimulation17,44,45. Earlier reports, including ours, have demonstrated that intracellular protein O-GlcNAcylation, a dynamic posttranslational protein modification, plays a pivotal role in the transcriptional upregulation of TSP-1 in VSMC and endothelial cells17,18,44. More recently, we have shown that both CrCl3 and CrP, regardless of its anionic ligand, reduces protein O-GlcNAcylation in primary HASMC cultures; notably reduced O-GlcNAcylation was accompanied with downregulation of TSP-1 expression in glucose-stimulated cells treated with Cr3+19. The current data that inhibition of TSP-1 expression occurs concomitant to attenuated OGT and protein O-GlcNAc expression support the overall concept that altered O-GlcNAcylation of nucleocytoplasmic proteins modulates downregulation of TSP-1 expression by CrP in diabetic macrovessels.

Growing evidence indicates that TSP-1 stimulates VSMC migration and proliferation, contributing to neointimal hyperplasia46. We have previously shown that high glucose-induced TSP-1 expression leads to abnormally enhanced cell proliferation in HASMC cultures. Specifically, anti-TSP-1 antibody and TSP-1-targeted siRNA blocked glucose-stimulated HASMC proliferation in vitro17. More recently, we demonstrated that pharmacological concentrations of CrCl3 and CrP inhibit TSP-1 expression and attenuate HASMC proliferation in glucose-stimulated cells19. Our current findings that CrP in vivo abrogates TSP-1 expression together with lowered SMC content and reduced cell proliferation fits well with the role of TSP-1 on VSMC activation. Interestingly, as opposed to Cr3+’s inhibitory effects on glucose-induced proliferation of aortic SMC cultures isolated from wild-type mice, we have found that the anti-proliferative effects of Cr3+ were completely obliterated in glucose-stimulated aortic SMCs derived from TSP-1 transgenic mice, constitutively overexpressing TSP-1 in the arterial SMC (Supplemental Fig. S6); these data extend support to the idea that the anti-proliferative effects of Cr3+ are specific for TSP-1.

In a healthy vessel, SMCs typically localize within medial layers of the arterial wall expressing a plethora of proteins and signaling mediators that modulate its contractile functions. However, exposure to proatherogenic stimuli such as hyperglycemia provokes VSMC phenotypic transition from a quiescent ‘contractile’ state to the proliferative ‘synthetic’ phase47, capable of increased lipid uptake and production of extracellular matrix proteins. Our results showing enhanced SM-MHC (SMC contractile marker) expression together with decreased vimentin (SMC synthetic marker) expression in CrP-treated mice suggest a possible regulatory role of Cr3 + on VSMC de-differentiation. Cogent to earlier reports, our data suggest that reduced protein O-GlcNAcylation by CrP may block TSP-1 upregulation in the large vessel impeding enhanced VSMC proliferation and atherosclerotic lesion formation in diabetes. Given that diabetic ApoE−/− mice have elevated cholesterol levels, one might argue that inhibition of atherosclerosis by CrP may be due to changes in the hyperlipidemic status of these animals. However, it is worth noting that the lipid levels remained unaffected by CrP in the current study despite attenuated lesion burden. Future studies are currently underway to determine the conceptual link between O-GlcNAc signaling and VSMC de-differentiation in diabetic atherosclerosis.

Finally, the present study provides the first evidence for a direct role of TSP-1 in hyperglycemia-induced atherosclerosis. Specifically, TSP-1 deficiency diminished both lesion severity and cellularity in STZ-induced type 1 diabetic ApoE−/− mice. Earlier reports have shown that ApoE−/− and TSP-1−/−/ApoE−/− dKO mice developed comparable aortic root lesion area and plaque burden following 24 weeks of normocholesterolemic diet48. Also, TSP-1−/−/ApoE−/− dKO mice at 36 weeks of age manifested increased collagen accumulation and inflammatory cell invasion into lesions triggering enhanced necrotic core formation compared to age-matched ApoE−/− littermates; these results have suggested a potential role of TSP-1 in macrophage-mediated phagocytosis and plaque maturation48. As opposed to these earlier findings, we have found reduced macrophage and leukocyte abundance in the aortic root lesions of diabetic TSP-1−/−/ApoE−/− dKO mice compared with age-matched diabetic ApoE−/− littermates. Importantly, lack of TSP-1 protected ApoE−/− mice against hyperglycemia-induced atherosclerosis. Moreover, we have shown that hyperglycemic ApoE−/− mice with TSP-1 deficiency mimicked the protective phenotype of CrP-treated diabetic atherosclerotic mice, which may have significant clinical implications.

Interestingly, congruent with earlier reports48, no significant difference in lipid-filled lesions was noted between ‘non-diabetic’ ApoE−/− and age-matched ‘non-diabetic’ TSP-1−/−/ApoE−/− littermate mice (data not shown). These results clearly highlight a role of TSP-1 that may be specific for hyperglycemia-driven atherosclerosis, with a significant bearing upon diabetic vascular disease. Our data also concur with the counterbalancing effects of TSP-1 on early vs late stage lesions49. Reassuringly, the current findings are in agreement with the previously reported involvement of TSP-1 in endothelium activation and infiltration of monocyte-derived macrophages contributing to foam cell formation48, and suggest differential patterns of vascular inflammatory burden triggered by hyperglycemia. Overall, the present study prompts us to speculate that inhibition of TSP-1-mediated VSMC phenotypic transition may represent an underlying mechanism of atheroprotection by CrP in diabetes. Although TSP-1 is well known to affect endothelial cell functions, it however remains unclear at this point how CrP affects endothelial cells; additional studies are needed to determine cell-specific effects of CrP on TSP-1 regulation. Furthermore, the impact of VSMC-specific TSP-1 deletion on diabetic atherogenesis warrants future investigation.

In summary, the present study provides strong evidence for an atheroprotective effect of orally administered CrP in a mouse model of type 1 diabetic atherosclerosis. We have also shown that the anti-atherogenic effect of CrP is accompanied with TSP-1 inhibition and reduced VSMC phenotypic transition. Notably, our data underscore TSP-1 as an important driving force for hyperglycemia-induced atherosclerosis. Taken together, the present study suggests key atheroprotective potential of therapeutic TSP-1 inhibition in diabetic macrovascular complications.

Methods

Mouse models

All animal procedures were approved by the Institutional Animal Care and Use Committee at Northeast Ohio Medical University in accordance with the NIH guidelines for the Care and Use of Laboratory Animals. Breeder pairs for ApoE−/− mice (stock # 002052) and TSP-1−/− mice (stock # 006141), congenic with C57BL/6 J mice, were originally purchased from The Jackson Laboratories (Bar Harbor, ME, USA) and the mice colonies were expanded and maintained in our animal facility. TSP-1−/−/ApoE−/− double knockout (dKO) mice were generated by intercrossing TSP-1−/− mice with ApoE−/− mice, obtained from our in-house breeding colonies. The first generation of offsprings (F1) for TSP-1 and ApoE allele were genotyped and identified as male and female double heterozygous mice; these double heterozygous mice were then bred leading to the second generation of offsprings (F2). From F2, mice identified as TSP-1+/−/ApoE−/− were further intercrossed leading to the generation of the double homozygous TSP-1−/−/ApoE−/− mice. ApoE−/− and TSP-1−/− genotypes were confirmed by PCR according to established protocols provided by Jackson Laboratories. All animals were housed in a pathogen-free environment and kept on 12:12 h light/dark cycle. All mice were weaned at 4 weeks of age and provided access to regular chow diet ad libitum until 18 weeks of age.

Induction of hyperglycemia and chromium picolinate administration

Upon weaning, animals were randomly assigned to three groups: ApoE−/−, no STZ (Control); ApoE−/−, with STZ (STZ) and ApoE−/−, with STZ plus CrP (STZ + CrP). Hyperglycemia was induced in six-week old male ApoE−/− mice using a multiple low-dose STZ (Sigma) regimen, as described previously50. Briefly, age-matched ApoE−/− littermate mice were injected intraperitoneally with either STZ (50 mg/Kg/day) or sodium citrate buffer (vehicle control) for 5 consecutive days. Ten days after the first STZ injection, blood samples collected by lateral tail incision were used for glucose estimation using a one-touch glucometer. Mice with non-fasted blood glucose levels >250 mg/dl were identified as hyperglycemic. A subset of these hyperglycemic ApoE−/− mice received chromium picolinate (8 μg Cr3+ /Kg/day) provided in drinking water starting at 8 weeks of age. This particular dose of CrP was chosen based on previous rodent studies23,51. Additionally, this dose approximately equates to an equivalent dose of 560 μg Cr3+ for a 70-kg adult human, representative of commercially available CrP supplements22,52. Fresh drinking water ± CrP (Nutrition 21, Purchase, NY) was prepared weekly and CrP concentration was adjusted based on the changes in animal weight. Body weight and non-fasted blood glucose levels were monitored every two weeks; animals were harvested at 18 weeks of age.

In a parallel study, six-week old male TSP-1−/−/ApoE−/− dKO mice and age-matched ApoE−/− littermates were subjected to STZ-induced hyperglycemia, as described above. Mice with non-fasted blood glucose levels >250 mg/dl were identified as hyperglycemic. Both genotypes were maintained on regular chow diet ad libitum until 18 weeks of age.

Plasma Lipid Analyses

After an overnight fasting, subsets of mice sacrificed at endpoint were used for estimation of plasma total cholesterol and total triglyceride levels using standard enzymatic kits (Thermo Fisher, Waltham, MA).

Aortic Root Morphometry

Mice were euthanized using 200 mg/Kg sodium pentobarbital injected intraperitoneally, perfused with PBS followed by formalin, and the heart, ascending aorta including aortic arch and carotid tissue were isolated. Aortic root sections (8–10 micron thickness) of formalin-fixed, OCT-embedded frozen hearts were cut at the point where the aortic valve leaflets were first visible. Care was taken to ensure that serial sections were collected from regions of the aortic root representing about 100–150 microns following the valve leaflet. Additional care was exercised to ensure that all measurements were taken within similar regions of the aortic root among all treatment groups for quantification and comparison. Sections were concurrently stained with 0.5% w/v Oil red O (ORO), hematoxylin and eosin (H & E) and Masson-trichrome (MT) to assess atherosclerotic lesions, intima-media thickness (IMT) and collagen content respectively, as reported earlier53. For ORO and MT staining, sections were counterstained with hematoxylin. All sections were mounted with DPX mounting media, observed using Olympus BX40 microscope and images were captured using 4X magnification. For quantitative morphometry, at least 5 animals per treatment group with an average of 20 tissue sections per group were analyzed using Image J software as previously described54. Analysis of collagen content was based on the positive MT staining per plaque area, which included both lipid and non-lipid regions; however, lumen area, valve leaflets, vessel walls and regions outside the vessel walls were excluded in these quantifications. Specifically, lesion area was selected using a magnetic lasso tool in Adobe Photoshop; this was copied and pasted into a new image file which was subsequently used for measuring the MT-stained region using Image J software. Line tracings were drawn to mark the luminal perimeter, the inner perimeter and the outer perimeter of the aortic root or carotid vessel cross-sectional image and the corresponding area were determined. Neointimal thickness was determined by subtracting the aortic root luminal perimeter from the aortic root outer perimeter. Percent luminal obstruction of the carotid vessel was calculated as follows: [(Area enclosed by inner perimeter - Area enclosed by luminal perimeter) x100]/Area enclosed by inner perimeter. All image quantifications were performed by team members blinded to the identity of all sections.

En-face atherosclerotic lesion assay

Mice were euthanized, perfused with PBS followed by formalin, and the heart, ascending aorta including aortic arch and carotid tissue were removed under a dissecting microscope. The entire aorta from the heart, including right and left common carotid arteries, extending 10–20 mm after iliac bifurcations were processed for ‘en-face’ quantitative atherosclerotic lesion assay. Briefly, aortic and carotid vessels were dissected free of fat and adventitial tissue, opened longitudinally and stained with 0.05% freshly-made Oil red O (ORO) solution. Each stained aortae was then digitally scanned and the percentage of the aorta covered by ORO-positive lipid-filled lesions was determined using Adobe Photoshop, as reported earlier53.

High-frequency Ultrasound Imaging

Vascular lesions were measured non-invasively by High-frequency Ultrasound Imaging using the Vevo 770 High-resolution Imaging System (VisualSonics, Inc. Toronto, Canada), as previously reported55. Briefly, both internal and external diameters of left and right common carotids were measured using B-mode (2-dimensional) images. In addition, left ventricular outflow tract (LVOT) and transverse aortic arch diameters were measured.

Immunohistochemistry

Aortic root sections from each animal were subjected to immunohistochemistry using anti-PCNA (Abcam, Cambridge, MA), anti-αSMA (Sigma, St. Louis, MO), anti-CD68 (Bioss, Woburn, MA), anti-CD45 (Bioss, Woburn, MA) and anti-Ki67 (Abcam, Cambridge, MA) antibodies. Briefly, tissue sections were incubated in ice-cold acetone (5–10 mins) and blocked with 5% donkey or goat serum (90 mins) at room temperature. Following an overnight incubation with primary antibodies (anti-PCNA-1:200; anti-α-SMA-1:200; anti-CD68–1:50; anti-CD45–1:150; anti-Ki67-1:100) at 4 °C, sections were incubated with Alexa Fluor 488 goat anti-mouse (for α-SMA) or Alexa Flour 594 donkey anti-rabbit IgG secondary antibodies (1:1000 or 1:500) and mounted on DAPI-containing mounting media (Vectashield, Vector Laboratories). For co-staining experiments, consecutive slides from serial sections were sequentially stained first with PCNA antibody followed by α-SMA antibody. To control for non-specific staining, identical sections were incubated in the absence of the corresponding primary antibodies, where no background staining was noted. Sections were observed using Olympus fluorescence IX71 microscope (10X or 15X magnification) and images were digitally captured using a set of identical parameters across all sections, specific for each antibody. For immunohistochemistry quantifications, lesion area within the aortic root was outlined, rest of the image cropped away and specific positive staining within lesions was quantified. For each individual treatment group, at least 5 mice with an average of 20 tissue sections per group were utilized for all quantifications. All immunostaining images were quantified in a blinded randomized manner using the Image J software. Results are expressed as fold of control for positive staining.

Immunoblotting

Aortic tissue lysates were prepared in SDS lysis buffer, as described earlier56 and protein content was determined using BCA protein assay. Equal amounts of proteins (35 μg) were resolved on 8% SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed using anti-TSP-1 (1:500-1:1000, Neomarkers, Freemont, CA), anti-O-GlcNAc (RL2, 1:1000; Abcam, Cambridge, MA), anti-OGT (1:1000, Cell Signaling, Danvers MA), anti-PCNA (1:300, Abcam, Cambridge, MA), anti-SM-MHC (1:2000, Proteintech, Rosemont, IL) and anti-vimentin (1:1000, Cell Signaling, Danvers, MA) antibodies. Membranes were stripped and re-probed with anti-β-actin used as a loading control; equal protein loading of samples was also confirmed by staining the membranes with Ponceau S. All immunoblot images were captured using Protein Simple and densitometric analyses was performed using the Image J software.

Primary Cultures of Mouse Aortic Smooth Muscle Cell

Primary cultures of mouse aortic smooth muscle cells (aSMC) isolated from TSP-1-transgenic mice, constitutively overexpressing TSP-1 in the arterial SMCs of the aortic vessel, and wild-type mice were kindly provided as a gift by Dr. Olga Stenina Adognravi (Cleveland Clinic, Cleveland, OH). Cells were maintained in complete DMEM/F12 media supplemented with 10% FBS and 1% antibiotics/antimycotic solution. aSMC primary cultures between passages 3–6 were used in all experiments; the contractile phenotype of aSMC was confirmed by α-SMA staining.

Cell Proliferation Assay

About 5000–7000 mouse aortic smooth muscle cells were plated on 96-well tissue-culture plates in complete DMEM/F12 medium containing 10% FBS. After allowing for an overnight growth, the cells were placed in low glucose (5.5 mM) serum-free DMEM media and further incubated with or without 20 mM glucose in the presence or absence of 100 nM chromium chloride (CrCl3) for 72 hours. Cell proliferation was measured at endpoint using the WST-1 cell proliferation reagent (Cayman Chemicals), as reported earlier19. Data are represented as % of Control (wild type); all values are expressed as mean ± SD from four independent experiments.

Statistical Analyses

For all morphometric and immunohistochemistry quantifications, at least 5 mice per treatment group were utilized with an average of 20 tissue sections per treatment group for each measurement. Sections derived from identical regions of the aortic root following the valve leaflet were used in all treatment groups for quantifications and comparisons. Please note, immunohistochemistry and morphometric data collected from all animals were included in our quantifications. All images were quantified by team members blinded to the identity of the treatment groups, in order to minimize bias and intentional exclusion of animals from the study. Differences in group sizes for some measurements were due to lack of additional tissue sections from the corresponding mice. For immunoblotting, aortic tissue lysates prepared from at least 3 mice per group were utilized. Image J software was used for densitometry of immunoblots and positive staining quantification. For indicated immunoblots, lane images show proteins detected on a single blot; however, lanes were rearranged for clarity of presentation. All data are presented as fold of control; values are expressed as mean ± SD, to depict variability of data. For comparison between two treatment groups, statistical analysis was done using unpaired Student’s t-test. For comparison between three groups, one-way analysis of variance (ANOVA) was used. Differences between mean values were considered statistically significant at P ≤ 0.05.

Additional Information

How to cite this article: Ganguly, R. et al. Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE−/− mice. Sci. Rep.7, 45279; doi: 10.1038/srep45279 (2017).

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    CAS

Источник: https://www.nature.com/articles/srep45279

Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE−/− mice

Abstract

Increasing evidence suggests thrombospondin-1 (TSP-1), a potent proatherogenic matricellular protein, as a putative link between hyperglycemia and atherosclerotic complications in diabetes. We previously reported that the micronutrient chromium picolinate (CrP), with long-standing cardiovascular benefits, inhibits TSP-1 expression in glucose-stimulated human aortic smooth muscle cells in vitro. Here, we investigated the atheroprotective action of orally administered CrP in type 1 diabetic apolipoprotein E-deficient (ApoE−/−) mice and elucidated the role of TSP-1 in this process. CrP decreased lipid burden and neointimal thickness in aortic root lesions of hyperglycemic ApoE−/− mice; also, smooth muscle cell (SMC), macrophage and leukocyte abundance was prevented coupled with reduced cell proliferation. Attenuated lesion progression was accompanied with inhibition of hyperglycemia-induced TSP-1 expression and reduced protein O-glycosylation following CrP treatment; also, PCNA and vimentin (SMC synthetic marker) expression were reduced while SM-MHC (SMC contractile marker) levels were increased. To confirm a direct role of TSP-1 in diabetic atherosclerosis, hyperglycemic TSP-1−/−/ApoE−/− double knockout mice were compared with age-matched hyperglycemic ApoE−/− littermates. Lack of TSP-1 prevented lesion formation in hyperglycemic ApoE−/− mice, mimicking the atheroprotective phenotype of CrP-treated mice. These results suggest that therapeutic TSP-1 inhibition may have important atheroprotective potential in diabetic vascular disease.

Introduction

Vascular disease is the leading cause of increased morbidity and mortality in diabetes. Risks of atherosclerotic complications are enhanced two-to-four-fold in diabetic patients1,2, accounting for >80% of deaths and hospitalizations in these individuals. Clinical studies, including recent animal data3,4,5,6,7, indicate that elevated glucose levels, independent of hyperlipidemia, may have profound proatherogenic effects in diabetes. Previous epidemiological studies1,2 have revealed a strong association between cumulative glycemic exposure and intima-media thickness (IMT) of the carotid artery, an early marker of atherosclerosis. Together, these reports support the notion that hyperglycemia is an important risk factor for development of macrovascular complications in diabetes. However, mechanisms underlying hyperglycemia-induced atherosclerosis are incompletely understood.

Thrombospondin-1 (TSP-1), a potent proatherogenic and anti-angiogenic protein, belongs to a family of matricellular proteins controlling cell-cell and cell-matrix interactions8. Earlier studies have shown that TSP-1 expression is significantly enhanced in response to vascular injury and in atherosclerotic lesions, with augmented expression in vascular smooth muscle cells (VSMC)9,10,11. Growing literature indicates distinct cell-and tissue-specific effects of TSP-1; both in vivo and in vitro studies have revealed that TSP-1 stimulates VSMC proliferation12 while inducing endothelial cell (EC) apoptosis13. The TSP protein family has been previously linked to atherosclerotic vascular disease based on GENEQUEST studies demonstrating an association between specific single nucleotide polymorphisms in the TSP genes with coronary artery disease and myocardial infarction14. These findings, confirmed by multiple human studies, lend support to TSP-1 as an alternative pathway for development of atherosclerosis.

Diabetic patients and diabetic animal models have been reported to have elevated TSP-1 levels in the plasma and walls of the large blood vessels15,16. Earlier work demonstrated that high glucose in vitro, characteristic of a diabetic milieu, upregulates TSP-1 expression in cells of the large blood vessel (VSMC, EC, fibroblasts)16. We have further shown that hyperglycemia in vitro increases TSP-1 expression via a transcriptional mechanism in primary human aortic smooth muscle cell (HASMC) cultures17,18. Together, these findings implicate TSP-1 as a putative link between hyperglycemia and accelerated atherosclerotic complications in diabetes. However, the impact of therapeutic TSP-1 inhibition on diabetic atherogenesis remains unexplored.

We recently reported19 that chromium picolinate (CrP), the most bioavailable form of the mineral nutrient trivalent chromium (Cr3+) at pharmacological concentrations, inhibits TSP-1 expression in glucose-stimulated HASMC in vitro. In addition, we have found that TSP-1 inhibition was accompanied with attenuated HASMC proliferation in response to Cr3+ and this effect was specific for high glucose conditions19. Accumulating data have indicated optimal regulatory effects of Cr3+ on carbohydrate and lipid metabolism20,21. Unlike its hexavalent counterpart (Cr6+), Cr3+ is relatively stable, with minimal toxicity issues at doses allowable for dietary intake22,23. Numerous studies have also indicated favorable glycemic and cardiovascular effects of Cr3+ 24,25,26,27,28. Nevertheless, clinical significance of Cr3+ in health and disease has been challenged by a dearth of mechanistic understanding of Cr3+ action.

Diabetic patients have low circulating and tissue Cr3+ levels compared to non-diabetic individuals29. There is increasing evidence that inadequate Cr3+ intake may elevate blood glucose and lipid levels30. Clinical studies have revealed that CrP in combination with biotin reduces insulin resistance and lowers the plasma atherogenic index in a cohort of type 2 diabetic patients31. Previous studies have also demonstrated that in STZ-induced diabetic Sprague Dawley rats in vivo and glucose-stimulated cultured monocytes in vitro, different Cr3+ formulations reduced lipid peroxidation and pro-inflammatory cytokine secretion32,33,34. Moreover, in a hypercholesterolemic rabbit model of atherosclerosis, intramuscular administration of chromium chloride (CrCl3) lowered serum cholesterol levels and reduced the size of lipid deposits in coronary and aortic vasculature35. Despite a favorable response to Cr3+ in vascular disease, the precise effect and mechanisms of the nutraceutical CrP in large vessels in the setting of diabetic atherosclerosis has remained elusive.

The present study provides the first demonstration that orally administered CrP impedes development of atherosclerotic lesions in STZ-induced hyperglycemic ApoE−/− mice, a mouse model of combined atherosclerosis and type 1 diabetes. Our data suggests that inhibition of TSP-1 expression, possibly mediated via reduced protein O-glycosylation, and blockade of VSMC phenotypic switching in the large vessel are important atheroprotective mechanisms of CrP in vivo. Notably, we have shown that genetic deletion of TSP-1 protects ApoE−/− mice against hyperglycemia-induced atherosclerosis, mimicking the protective phenotype of CrP-treated diabetic atherosclerotic mice.

Results

Oral chromium picolinate has no effect on body weight, blood glucose and lipid profiles in hyperglycemic ApoE−/− mice

No significant differences in body weights were observed in STZ-induced hyperglycemic ApoE−/− mice treated with or without CrP compared with non-hyperglycemic ApoE−/− mice (Fig. 1a). As expected, non-fasted blood glucose levels increased 2–3-fold following STZ treatment in ApoE−/− mice vs. non-STZ-treated ApoE−/− (Fig. 1b); consistent with earlier reports, multiple low-dose STZ did not adversely affect animal well-being or survival. Interestingly, under conditions of experimental type 1 diabetes in ApoE−/− mice, CrP provided in drinking water had no effect on the non-fasted blood glucose levels in these mice. Although a slight reduction (~15%) in glucose levels was noted at 12 weeks of age following CrP administration, this effect was abolished at later time points and the animals continued to remain hyperglycemic attaining non-fasted blood glucose levels ≥250 mg/dl. Moreover, while hyperglycemia increased plasma total cholesterol and total triglyceride levels in STZ-ApoE−/− mice (~1.6-fold versus ApoE−/− control), there was no statistically significant effect of CrP administration on the lipid profiles in hyperglycemic ApoE−/− mice (Figs. 1c,d). Similar to these findings, CrP did not affect either body weight or total cholesterol levels in non-diabetic ApoE−/− control mice (data not shown).

Six weeks old male ApoE−/− mice were treated with 50 mg/Kg/day streptozotocin or sodium citrate buffer (vehicle control) i.p. for 5 consecutive days. This was followed by treatment with or without CrP (8 μg/Kg/day) in drinking water beginning at 8 weeks of age. (a) Body weight and (b) non-fasted blood glucose levels were measured every two weeks from 8–18 weeks of age. (c) Plasma total cholesterol and (d) plasma total triglyceride levels were measured at end point of the study (18 weeks). Results are expressed are mean ± SD (n = 10–17 mice per group); *p ≤ 0.05 vs. Control.

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Chromium picolinate impedes hyperglycemia-induced atherosclerosis in ApoE−/− mice

Atherosclerotic lesion development was examined in all treatment groups using aortic root morphometry. Lipid-filled lesions were significantly increased in STZ-induced hyperglycemic ApoE−/− mice compared with age-matched non-hyperglycemic ApoE−/− littermates, shown by oil red O (ORO) staining. In contrast, CrP treatment diminished lesion formation in hyperglycemic ApoE−/− mice (Fig. 2a). Quantification of ORO-positive staining revealed that while lesion burden was augmented in STZ-ApoE−/− mice (2.3-fold vs. Controls), CrP remarkably reduced lipid-filled lesions in hyperglycemic ApoE−/− (62.6% vs. STZ, Fig. 2b). Aortic root H & E staining showed reduced neointimal thickening in response to CrP (Fig. 2c). Specifically, while the intimal-medial thickness was increased by 66.7% in aortic roots of hyperglycemic ApoE−/− mice compared to non-hyperglycemic ApoE−/−, CrP-treated mice demonstrated decreased neointimal thickening (30% vs. STZ only, Fig. 2d). Aortic root morphometry using Masson-Trichrome (MT) staining showed no statistically significant differences in the lesion collagen content in hyperglycemic ApoE−/− mice vs. non-hyperglycemic ApoE−/−, although higher collagen amounts were noted in regions outside the aortic root vessel wall in STZ-treated ApoE−/− mice. Moreover, CrP treatment had no effect on collagen accumulation in the aortic root lesions of hyperglycemic ApoE−/− mice compared to STZ only animals (Fig. 2e,f). Interestingly, lesion formation remained unaffected by CrP in ApoE−/− control mice that were not subjected to STZ-induced hyperglycemia (Supplementary Fig. S1).

STZ-induced hyperglycemic ApoE−/− mice were treated with or without CrP (8 μg/Kg/day) from 8–18 wks of age. Aortic root morphometry was performed as described in Methods. All comparisons were made between Control (ApoE−/− no STZ), STZ (ApoE−/− with STZ) and STZ + CrP (ApoE−/− with STZ plus CrP). Shown are (a,b) representative images and summary data for quantification of ORO-positive staining (Control: n = 6; STZ: n = 7; STZ + CrP: n = 7), (c,d) representative H & E images and summary data for neointimal thickness (Control: n = 5; STZ: n = 7; STZ + CrP: n = 7), (e,f) representative images and summary data for quantification of MT staining (Control: n = 6; STZ: n = 8; STZ+CrP: n = 8). All microscopic images were captured at 4X magnification. Specific regions used for analyses are marked by dotted lines. Results are presented as fold of Control; all values are expressed as mean ± SD; *p ≤ 0.003 vs. Control, #p ≤ 0.026 vs. STZ.

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Atherosclerotic lesions were also monitored using a non-invasive auxiliary approach, High-frequency Ultrasound Imaging. Congruent to the aortic root morphometric data, both left ventricular outflow tract and transaortic arch diameters were reduced (~20%) in STZ-ApoE−/− vs. non-STZ-ApoE−/− mice. Notably, this reduction in vessel diameter was completely abrogated in CrP-treated mice (Supplementary Table S1). Furthermore, ultrasound imaging of carotid vessels revealed significant reduction in vessel diameter in hyperglycemic ApoE−/− mice (~25% vs. Control); in contrast, this decrease in vessel diameter was completely obliterated in STZ-ApoE−/− mice treated with CrP (Figs. 3a–c). Further histological experiments were conducted to verify whether the reduction in vessel diameter in STZ-ApoE−/− mice was related to formation of atherosclerotic lesions in the carotid vasculature. Specifically, H & E staining of carotid vessel tissue sections derived from STZ-induced hyperglycemic ApoE−/− mice revealed distinct lesions in the vessel wall resulting in luminal obstruction (Supplemental Fig. S2a). Importantly, a strong negative correlation (-0.91) was noted between the carotid vessel internal diameter and % luminal obstruction of the carotid vessels (Supplemental Fig. S2b), validating the ultrasound assessments of lesion development in the carotid vasculature (Fig. 3). Taken together, these data demonstrate an atheroprotective effect of oral CrP in hyperglycemic ApoE−/− mice.

(a) Shown are representative ultrasound images of left and right common carotid artery; yellow arrows indicate neointimal thickening of vessel wall resulting in reduction in vessel diameter in STZ mice. Shown are the summary data for (b), left and (c), right carotid artery diameters. LCCA- left common carotid artery; LECA- left external carotid artery; RCCA- right common carotid artery; RECA- right external carotid artery. Results are expressed as mean ± SD (n = 4–7 mice/group) *p ≤ 0.0001 vs. Control; +p ≤ 0.0001 vs. STZ.

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Chromium picolinate prevents hyperglycemia-induced increase in cellular proliferation and inflammatory lesion burden in STZ-treated ApoE−/− mice

Next, we characterized lesion cellularity in response to CrP in vivo. Smooth muscle cell abundance and cell proliferation was assessed using α-SMA and PCNA immunohistochemistry, respectively. STZ-induced hyperglycemia increased α-SMA (1.6-fold) and PCNA staining (2.0-fold) in ApoE−/− mice, indicating enhanced smooth muscle cell content and cell Windows Movie Maker 2021 Crack 9.2.0.6 For Windows Download [Latest]. On the contrary, there was a significant decrease in α-SMA and PCNA positive staining in aortic roots of STZ-ApoE−/− mice that received CrP (~40–48% vs. STZ only, Fig. 4); as expected, α-SMA immunostaining demonstrated positive medial staining of smooth muscle cells in control mice whereas increased α-SMA staining was limited to the neointimal layer in the aortic root of STZ mice (Supplemental Fig. S3). The PCNA staining patterns were confirmed using a second proliferation marker, Ki67. As shown in Supplemental Fig. S4a,b, Ki67 expression was increased (2.2-fold) in aortic root lesions of STZ-induced hyperglycemic ApoE−/− mice compared to ApoE−/− control mice, recapitulating the PCNA immunostaining results (Fig. 4c). Moreover, CrP treatment reduced (54%) Ki67 expression similar to its effect on PCNA expression in hyperglycemic ApoE−/− mice compared to STZ only ApoE−/− (Supplemental Fig. S4a,b). Notably, double immunofluorescence experiments using α-SMA and PCNA antibodies demonstrated increased PCNA-positive smooth muscle cell content in aortic root lesions of STZ mice vs. controls. In contrast, increased smooth muscle cell proliferation was prevented in aortic root sections of STZ + CrP mice, depicted by a lack of PCNA-positive α-SMA staining (Fig. 4a, merge). Next, immunohistochemistry demonstrated enhanced macrophage and leukocyte infiltration into aortic root lesions of hyperglycemic ApoE−/− mice while the lesion burden of inflammatory cells was reduced in CrP-treated hyperglycemic mice (Figs. 5a–c). Specifically, immunostaining quantifications revealed > 2.4-fold increase in CD68 and CD45 expression in aortic root lesions of STZ-ApoE−/− mice compared with ApoE−/− controls. In contrast, oral CrP decreased both CD68 and CD45 expression in aortic roots of hyperglycemic ApoE−/− mice (54–64% vs. STZ only, Figs. 5b–d) indicative of attenuated macrophage and leukocyte accumulation, respectively. In each case, the specificity of the immunofluorescence staining was confirmed in parallel aortic root sections incubated in the absence of the corresponding primary antibody (Supplementary Fig. S5). Together, these results demonstrate that CrP in vivo prevents hyperglycemia-induced smooth muscle cell and inflammatory cell abundance as well as inhibits smooth muscle cell proliferation in diabetic atherosclerotic mice, resembling a state of reduced plaque development.

Shown are (a) representative images for PCNA and α-SMA co-staining (10x magnification). Regions used for immunostaining quantifications are outlined by dotted lines; arrows indicate PCNA-positive smooth muscle cells. (b,c) Summary data for quantification of α-SMA-and PCNA-positive staining. All results are presented as fold of Control and values are expressed as mean ± SD (Control: n = 6; STZ: n = 7; STZ+CrP: n = 6); *p < 0.0001 vs. Control, #p < 0.0001 vs. STZ.

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Shown are representative images and summary data for quantification of (a,b) CD68-positive staining and (c,d) CD45-positive staining. All images were captured at 15X magnification. Histology of the immunofluorescence images are shown in the corresponding H & E-stained images (indicated by the black box). Specific regions of the immunofluorescence images used for data analyses are marked by dotted lines. Results are presented as fold of Control and all values are expressed are mean ± SD (for CD45, Control: n = 6; STZ: n = 6; STZ+CrP: n = 5; for CD68, Control: n = 5; STZ: n = 5; STZ+CrP: n = 5); *p ≤ 0.0008 vs. Control; #p ≤ 0.0018 vs. STZ.

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Chromium picolinate in vivo inhibits TSP-1 expression, decreases protein O-glycosylation and blocks VSMC phenotypic switching in aortic vessels of STZ-induced hyperglycemic ApoE−/− mice

We reported earlier that both CrCl3 and CrP in vitro inhibit TSP-1 expression in glucose-stimulated HASMC primary cultures19. To investigate whether the atheroprotective action of CrP observed in the current study correlates with TSP-1 expression in vivo, aortic tissue lysates prepared from all treatment groups were subjected to immunoblotting. As shown in Fig. 6a, TSP-1 expression was increased in aortic vessels of hyperglycemic ApoE−/− mice (2.45-fold vs. Control). Concomitant to enhanced TSP-1 expression, hyperglycemia augmented protein O-GlcNAc levels in the aortic vasculature of STZ-ApoE−/− mice. Specifically, immunoblotting of aortic lysates revealed elevated O-GlcNAc levels on several proteins ranging from approximately 100–260 kDa and 45–60 kDa in STZ-ApoE−/− mice (Fig. 6b). This increase in protein O-GlcNAcylation was also accompanied with enhanced OGT expression (3.2-fold, Fig. 6c), a major regulator of O-GlcNAcylation. In contrast, TSP-1 expression was inhibited in aortic vessels of hyperglycemic ApoE−/− treated with CrP (76.7% vs. STZ, Fig. 6a). Moreover, downregulation of TSP-1 expression was accompanied with reduced O-GlcNAc protein modification and attenuated OGT expression in hyperglycemic ApoE−/− mice subjected to CrP treatment (~55% vs. STZ, Fig. 6b,c). Notably, while STZ-induced hyperglycemia augmented cell proliferation marker PCNA expression in ApoE−/− mice (3.6-fold vs. Control), CrP administration significantly inhibited PCNA expression (80% vs. STZ, Fig. 6d). Consistent with effects on cell proliferation, hyperglycemic ApoE−/− mice showed enhanced vimentin expression together with reduced SM-MHC expression in the aortic vasculature. Specifically, densitometry of immunoblots revealed 2-fold increase in vimentin expression and 56.5% decrease in SM-MHC expression in aortic lysates of STZ-ApoE−/− mice compared with ApoE−/− Controls. On the contrary, CrP significantly inhibited vimentin expression (62.5%) while increasing SM-MHC expression (2.18-fold) in hyperglycemic ApoE−/− mice (Figs. 6e–g). Together, these data demonstrate an important link between attenuated lesion formation, downregulation of TSP-1 expression and reduced protein O-GlcNAcylation in CrP- treated hyperglycemic ApoE−/− mice.

Shown are representative immunoblots depicting (a) TSP-1, (b) O-GlcNAc, (c) OGT, (d) PCNA, (e) SM-MHC expression (upper panel) and vimentin expression (middle panel). Membranes were probed with anti-β-actin used as loading control. In each case, graphs represent summary data for densitometric quantification of immunoblots (at least 3 mice per group). For c and e, lane images show proteins detected on a single immunoblot; however, lanes were rearranged for clarity of presentation; the corresponding original uncropped blots are presented in Supplementary Fig. S7. All results are presented as fold of Control; values are expressed as means ± SD; *p ≤ 0.05 vs. Control; #p ≤ 0.05 vs. STZ.

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TSP-1 deficiency prevents development of atherosclerotic lesions in STZ-induced hyperglycemic ApoE−/− mice

To investigate a direct role of TSP-1 in hyperglycemia-induced atherosclerosis, we generated TSP-1−/−/ApoE−/− double knockout mice that were subjected to STZ-induced hyperglycemia, as described in Methods; these mice were compared with age-matched STZ-induced hyperglycemic ApoE−/− littermates, both genotypes being at 18 weeks of age. Similar to STZ-treated ApoE−/−, TSP-1−/−/ApoE−/− dKO mice developed significant hyperglycemia upon STZ treatment achieving non-fasted blood glucose levels ≥ 250 mg/dl. However, under these conditions of experimental diabetes, no statistically significant differences were observed in the body weights, plasma total cholesterol and total triglyceride levels between the mice genotypes (Supplementary Table S2). Aortic root morphometry showed remarkable decrease in lipid content (Fig. 7a) and neointimal thickening (Fig. 7c) in hyperglycemic TSP-1−/−/ApoE−/− mice compared with age-matched hyperglycemic ApoE−/− littermates. Specifically, morphometric quantification depicted reduced lesion burden (2.9-fold) and neointimal thickness (1.7-fold) in aortic roots of STZ-treated TSP-1−/−/ApoE−/− mice (vs. STZ-ApoE−/−, Fig. 7b–d). Similar to CrP-treated mice, there was no difference in the collagen content of lesions in either genotypes in response to hyperglycemia (Fig. 7e,f). Furthermore, cellular characterization of lesions revealed attenuated PCNA and α-SMA expression in hyperglycemic TSP-1−/−/ApoE−/− dKO mice (vs. hyperglycemic ApoE−/−, Fig. 8a–c); while STZ-TSP-1−/−/ApoE−/− mice showed positive medial SMA staining, enhanced α-SMA staining was limited to the neointimal surface of the aortic root in STZ-ApoE−/− mice. Importantly, double immunofluorescence staining showed lower abundance of PCNA-positive smooth muscle cells in aortic root lesions of STZ-TSP-1−/−/ApoE−/−, depicting attenuated smooth muscle cell proliferation (Fig. 8a, merge), compared to STZ-ApoE−/− mice. Further, both CD68 and CD45 expression levels were ameliorated in hyperglycemic TSP-1−/−/ApoE−/− dKO mice (vs. STZ-ApoE−/−, Figs. 8d–g). Specific immunostaining quantifications showed marked reduction in PCNA expression (2.1-fold), further confirmed by Ki67 immunostaining (Supplemental Fig. S4c,d); moreover, α-SMA, CD68 and CD45 expression profiles were attenuated in aortic root lesions of STZ-induced hyperglycemic TSP-1−/−/ApoE−/− mice (1.6- 2.1- and 2.6-fold, respectively vs. age-matched STZ-ApoE−/− littermates), indicating attenuated cell proliferation, lower smooth muscle cell abundance and reduced macrophage as well as leukocyte lesion invasion. Collectively, these data clearly demonstrate that TSP-1 deletion protects ApoE−/− mice against hyperglycemia-induced atherosclerosis.

Age-matched male ApoE−/− and TSP-1−/−/ApoE−/− dKO mice were subjected to STZ-induced hyperglycemia; aortic root morphometry was performed as described in Methods. Shown are (a,b) representative ORO images and summary data for quantification of ORO positive staining (STZ-ApoE: n = 7; STZ-TSP-1/ApoE: n = 5); (c,d) representative H & E images and summary data for neointimal thickness (STZ-ApoE: n = 7; STZ-TSP-1/ApoE: n = 5); (e,f) representative images and summary data for quantification of MT positive staining (STZ-ApoE: n = 8; STZ-TSP-1/ApoE: n = 5). Specific regions used for analyses are indicated by dotted lines. Results are presented as fold of ApoE−/− Control; all values are expressed as mean ± SD; *p ≤ 0.0013 vs. STZ-ApoE−/−.

Full size image

Shown are representative images for (a) PCNA and α-SMA co-staining (10X magnification), (b,c) quantification data for PCNA and α-SMA staining (STZ-ApoE−/−: n = 7; STZ-TSP-1/ApoE: n = 6), (d) CD68 staining (15X magnification) depicting macrophage content and (e) CD45 staining (15X magnification) depicting leukocyte abundance and (f,g) summary data for quantification of CD68 (STZ-ApoE: n = 5; STZ-TSP-1/ApoE: n = 5) and CD45 (STZ-ApoE: n = 6; STZ-TSP-1/ApoE: n = 5) positive staining. Regions used for analysis are marked via dotted lines; arrows indicate PCNA-positive smooth muscle cells. Histology of the immunofluorescence images are shown in the corresponding H & E-stained images (indicated by black box). Results are presented as fold of ApoE−/− Control. All values are expressed are mean ± SD; *p ≤ 0.0019 vs. STZ-ApoE−/−.

Full size image

Discussion

The present study provides the first demonstration for an atheroprotective effect of the nutraceutical CrP in a mouse model of diabetic atherosclerosis. We further show that attenuated lesion progression is accompanied with inhibition of TSP-1 expression in response to CrP in vivo. Notably, the current work delineates a direct role of TSP-1 in hyperglycemia-driven atherosclerosis.

Clinical data on the cardiovascular benefits of Cr3+ in diabetes have been agnostic36,37, confounded by the question of whether the effects are mediated via glycemic regulation. Contrary to few earlier reports26,34,38, orally administered CrP did not have any effect on circulating glucose levels in hyperglycemic ApoE−/− mice in the current study. While such discrepancy could be attributed to differences in species (rat vs. mice), Cr3+ formulation and dose as well as animal models (wild-type vs. ApoE−/−) utilized, it is important to note that the reduced susceptibility to atherosclerosis in CrP-treated mice was not related to lower lipid profiles in these animals; indeed, plasma total cholesterol and total triglyceride levels were not significantly different between hyperglycemic ApoE−/− mice treated with and without CrP. A possible explanation for such species-dependent differential glycemic response may relate to generation of a metabolite specific to rats, capable of modulating glucose metabolism. As such, our findings suggest an alternate mechanism of atheroprotection by Cr3+, independent of glucose and lipid control.

Previous epidemiological data and animal studies using rabbits have indicated a correlation between Cr3+ intake, incidence of coronary artery disease and serum lipid deposition31,35,39. Both in vitro and in vivo studies have implicated that Cr3+ may lower risks of vascular inflammation under conditions of elevated glucose levels32,33,34. In line with these reports, the present study has revealed that oral CrP significantly impedes lesion formation and reduces neointimal thickening in the aortic sinus of type 1 diabetic atherosclerotic mice. These morphometric results were also confirmed by ultrasound imaging of the aortic and carotid vasculature. Previous studies have indicated a correlation between conventional ORO staining methods and Ultrasound Imaging approaches for vascular lesion detection in mice40. Our data lend additional support to ultrasound imaging as a useful non-invasive methodology for progressive atherosclerotic lesion monitoring in mice. Reduced lesion severity in response to CrP was further illustrated by cellular lesion characterization. Morphometric quantification of aortic root lesions revealed that CrP prevents plaque development in hyperglycemic ApoE−/− mice, depicted by the profound decrease in hyperglycemia-induced proliferative smooth muscle cell abundance as well as macrophage and leukocyte infiltration.

Atherosclerosis is a multifactorial progressive disease comprising of a series of cellular and molecular events, triggering enhanced inflammatory response in the vessel wall. Internalization of atherogenic lipoproteins by monocyte-derived macrophages and activation of inflammatory pathways are important pathogenic mediators of atherosclerosis. This, in turn, may initiate release of a repertoire of growth factors and cytokines promoting VSMC activation, a key step responsible for initiation and progression of atherosclerotic lesions41. Diabetic patients are predisposed to aberrant VSMC activation42, characterized by enhanced migratory, proliferative and synthetic SMC phenotype. Previous work from our laboratory and others16,17,19 have shown that hyperglycemia, mimicking diabetes, upregulates the proatherogenic protein TSP-1 expression. TSP-1, a secreted glycoprotein, is expressed by many different cell types including endothelial cells, SMC and fibroblasts16, and has been widely implicated in atherosclerotic vascular disease14,43. The multidomain structure of TSP-1 has been attributed to the cell-and tissue-specific mechanisms of TSP-1 upregulation observed under glucose stimulation17,44,45. Earlier reports, including ours, have demonstrated that intracellular protein O-GlcNAcylation, a dynamic posttranslational protein modification, plays a pivotal role in the transcriptional upregulation of TSP-1 in VSMC and endothelial cells17,18,44. More recently, we have shown that both CrCl3 and CrP, regardless of its anionic ligand, reduces protein O-GlcNAcylation in primary HASMC cultures; notably reduced O-GlcNAcylation was accompanied with downregulation of TSP-1 expression in glucose-stimulated cells treated with Cr3+19. The current data that inhibition of TSP-1 expression occurs concomitant to attenuated OGT and protein O-GlcNAc expression support the overall concept that altered O-GlcNAcylation of nucleocytoplasmic proteins modulates downregulation of TSP-1 expression by CrP in diabetic macrovessels.

Growing evidence indicates that TSP-1 stimulates VSMC migration and proliferation, contributing to neointimal hyperplasia46. We have previously shown that high glucose-induced TSP-1 expression leads to abnormally enhanced cell proliferation in HASMC cultures. Specifically, anti-TSP-1 antibody and TSP-1-targeted siRNA blocked glucose-stimulated HASMC proliferation in vitro17. More recently, we demonstrated that pharmacological concentrations of CrCl3 and CrP inhibit TSP-1 expression and attenuate HASMC proliferation in glucose-stimulated cells19. Our current findings that CrP in vivo abrogates TSP-1 expression together with lowered SMC content and reduced cell proliferation fits well with the role of TSP-1 on VSMC activation. Interestingly, as opposed to Cr3+’s inhibitory effects on glucose-induced proliferation of aortic SMC cultures isolated from wild-type mice, we have found that the anti-proliferative effects of Cr3+ were completely obliterated in glucose-stimulated aortic SMCs derived from TSP-1 transgenic mice, constitutively overexpressing TSP-1 in the arterial SMC (Supplemental Fig. S6); these data extend support to the idea that the anti-proliferative effects of Cr3+ are specific for TSP-1.

In a healthy vessel, SMCs typically localize within medial layers of the arterial wall expressing a plethora of proteins and signaling mediators that modulate its contractile functions. However, exposure to proatherogenic stimuli such as hyperglycemia provokes VSMC phenotypic transition from a quiescent ‘contractile’ state to the proliferative ‘synthetic’ phase47, capable of increased lipid uptake and production of extracellular matrix proteins. Our results showing enhanced SM-MHC (SMC contractile marker) expression together with decreased vimentin (SMC synthetic marker) expression in CrP-treated mice suggest a possible regulatory role of Cr3 + on VSMC de-differentiation. Cogent to earlier reports, our data suggest that reduced protein O-GlcNAcylation by CrP may block TSP-1 upregulation in the large vessel impeding enhanced VSMC proliferation and atherosclerotic lesion formation in diabetes. Given that diabetic ApoE−/− mice have elevated cholesterol levels, one might argue that inhibition of atherosclerosis by CrP may be due to changes in the hyperlipidemic status of these animals. However, it is worth noting that the lipid levels remained unaffected by CrP in the current study despite attenuated lesion burden. Future studies are currently underway to determine the conceptual link between O-GlcNAc signaling and VSMC de-differentiation in diabetic atherosclerosis.

Finally, the present study provides the first evidence for a direct role of TSP-1 in hyperglycemia-induced atherosclerosis. Specifically, TSP-1 deficiency diminished both lesion severity and cellularity in STZ-induced type 1 diabetic ApoE−/− mice. Earlier reports have shown that ApoE−/− and TSP-1−/−/ApoE−/− dKO mice developed comparable aortic root lesion area and plaque burden following 24 weeks of normocholesterolemic diet48. Also, TSP-1−/−/ApoE−/− dKO mice at 36 weeks of age manifested increased collagen accumulation and inflammatory cell invasion into lesions triggering enhanced necrotic core formation compared to age-matched ApoE−/− littermates; these results have suggested a potential role of TSP-1 in macrophage-mediated phagocytosis and plaque maturation48. As opposed to these earlier findings, we have found reduced macrophage and leukocyte abundance in the aortic root lesions of diabetic TSP-1−/−/ApoE−/− dKO mice compared with age-matched diabetic ApoE−/− littermates. Importantly, lack of TSP-1 protected ApoE−/− mice against hyperglycemia-induced atherosclerosis. Moreover, we have shown that hyperglycemic ApoE−/− mice with TSP-1 deficiency mimicked the protective phenotype of CrP-treated diabetic atherosclerotic mice, which may have significant clinical implications.

Interestingly, congruent with earlier reports48, no significant difference in lipid-filled lesions was noted between ‘non-diabetic’ ApoE−/− and age-matched ‘non-diabetic’ TSP-1−/−/ApoE−/− littermate mice (data not shown). These results clearly highlight a role of TSP-1 that may be specific for hyperglycemia-driven atherosclerosis, with a significant bearing upon diabetic vascular disease. Our data also concur with the counterbalancing effects of TSP-1 on early vs late stage lesions49. Reassuringly, the current findings are in agreement with the previously reported involvement of TSP-1 in endothelium activation and infiltration of monocyte-derived macrophages contributing to foam cell formation48, and suggest differential patterns of vascular inflammatory burden triggered by hyperglycemia. Overall, the present study prompts us to speculate that inhibition of TSP-1-mediated VSMC phenotypic transition may represent an underlying mechanism of atheroprotection by CrP in diabetes. Although TSP-1 is well known to affect endothelial cell functions, it however remains unclear at this point how CrP affects endothelial cells; additional studies are needed to determine cell-specific effects of CrP on TSP-1 regulation. Furthermore, the impact of VSMC-specific TSP-1 deletion on diabetic atherogenesis warrants future investigation.

In summary, the present study provides strong evidence for an atheroprotective effect of orally administered CrP in a mouse model of type 1 diabetic atherosclerosis. We have also shown that the anti-atherogenic effect of CrP is accompanied with TSP-1 inhibition and reduced VSMC phenotypic transition. Notably, our data underscore TSP-1 as an important driving force for hyperglycemia-induced atherosclerosis. Taken together, the present study suggests key atheroprotective potential of therapeutic TSP-1 inhibition in diabetic macrovascular complications.

Methods

Mouse models

All animal procedures were approved by the Institutional Animal Care and Use Committee at Northeast Ohio Medical University in accordance with the NIH guidelines for the Care and Use of Laboratory Animals. Breeder pairs for ApoE−/− mice (stock # 002052) and TSP-1−/− mice (stock # 006141), congenic with C57BL/6 J mice, were originally purchased from The Jackson Laboratories (Bar Harbor, ME, USA) and the mice colonies were expanded and maintained in our animal facility. TSP-1−/−/ApoE−/− double knockout (dKO) mice were generated by intercrossing TSP-1−/− mice with ApoE−/− mice, obtained from our in-house breeding colonies. The first generation of offsprings (F1) for TSP-1 and ApoE allele were genotyped and identified as male and female double heterozygous mice; these double heterozygous mice were then bred leading to the second generation of offsprings (F2). From F2, mice identified as TSP-1+/−/ApoE−/− were further intercrossed leading to the generation of the double homozygous TSP-1−/−/ApoE−/− mice. ApoE−/− and TSP-1−/− genotypes were confirmed by PCR according to established protocols provided by Jackson Laboratories. All animals were housed in a pathogen-free environment and kept on 12:12 h light/dark cycle. All mice were weaned at 4 weeks of age and provided access to regular chow diet ad libitum until 18 weeks of age.

Induction of hyperglycemia and chromium picolinate administration

Upon weaning, animals were randomly assigned to three groups: ApoE−/−, no STZ (Control); ApoE−/−, with STZ (STZ) and ApoE−/−, with STZ plus CrP (STZ + CrP). Hyperglycemia was induced in six-week old male ApoE−/− mice using a multiple low-dose STZ (Sigma) regimen, as described previously50. Briefly, age-matched ApoE−/− littermate mice were injected intraperitoneally with either STZ (50 mg/Kg/day) or sodium citrate buffer (vehicle control) for 5 consecutive days. Ten days after the first STZ injection, blood samples collected by lateral tail incision were used for glucose estimation using a one-touch glucometer. Mice with non-fasted blood glucose levels >250 mg/dl were identified as hyperglycemic. A subset of these hyperglycemic ApoE−/− mice received chromium picolinate (8 μg Cr3+ /Kg/day) provided in drinking water starting at 8 weeks of age. This particular dose of CrP was chosen based on previous rodent studies23,51. Additionally, this dose approximately equates to an equivalent dose of 560 μg Cr3+ for a 70-kg adult human, representative of commercially available CrP supplements22,52. Fresh drinking water ± CrP (Nutrition 21, Purchase, NY) was prepared weekly and CrP concentration was adjusted based on the changes in animal weight. Body weight and non-fasted blood glucose levels were monitored every two weeks; animals were harvested at 18 weeks of age.

In a parallel study, six-week old male TSP-1−/−/ApoE−/− dKO mice and age-matched ApoE−/− littermates were subjected to STZ-induced hyperglycemia, as described above. Mice with non-fasted blood glucose levels >250 mg/dl were identified as hyperglycemic. Both genotypes were maintained on regular chow diet ad libitum until 18 weeks of age.

Plasma Lipid Analyses

After an overnight fasting, subsets of mice sacrificed at endpoint were used for estimation of plasma total cholesterol and total triglyceride levels using standard enzymatic kits (Thermo Fisher, Waltham, MA).

Aortic Root Morphometry

Mice were euthanized using 200 mg/Kg sodium pentobarbital injected intraperitoneally, perfused with PBS followed by formalin, and the heart, ascending aorta including aortic arch and carotid tissue were isolated. Aortic root sections (8–10 micron thickness) of formalin-fixed, OCT-embedded frozen hearts were cut at the point where the aortic valve leaflets were first visible. Care was taken to ensure that serial sections were collected from regions of the aortic root representing about 100–150 microns following the valve leaflet. Additional care was exercised to ensure that all measurements were taken within similar regions of the aortic root among all treatment groups for quantification and comparison. Sections were concurrently stained with 0.5% w/v Oil red O (ORO), hematoxylin and eosin (H & E) and Masson-trichrome (MT) to assess atherosclerotic lesions, intima-media thickness (IMT) and collagen content respectively, as reported earlier53. For ORO and MT staining, sections were counterstained with hematoxylin. All sections were mounted with DPX mounting media, observed using Olympus BX40 microscope and images were captured using 4X magnification. For quantitative morphometry, at least 5 animals per treatment group with an average of 20 tissue sections per group were analyzed using Image J software as previously described54. Analysis of collagen content was based on the positive MT staining per plaque area, which included both lipid and non-lipid regions; however, lumen area, valve leaflets, vessel walls and regions outside the vessel walls were excluded in these quantifications. Specifically, lesion area was selected using a magnetic lasso tool in Adobe Photoshop; this was copied and pasted into a new image file which was subsequently used for measuring the MT-stained region using Image J software. Line tracings were drawn to mark the luminal perimeter, the inner perimeter and the outer perimeter of the aortic root or carotid vessel cross-sectional image and the corresponding area were determined. Neointimal thickness was determined by subtracting the aortic root luminal perimeter from the aortic root outer perimeter. Percent luminal obstruction of the carotid vessel was calculated as follows: [(Area enclosed by inner perimeter - Area enclosed by luminal perimeter) x100]/Area enclosed by inner perimeter. All image quantifications were performed by team members blinded to the identity of all sections.

En-face atherosclerotic lesion assay

Mice were euthanized, perfused with PBS followed by formalin, and the heart, ascending aorta including aortic arch and carotid tissue were removed under a dissecting microscope. The entire aorta from the heart, including right and left common carotid arteries, extending 10–20 mm after iliac bifurcations were processed for ‘en-face’ quantitative atherosclerotic lesion assay. Briefly, aortic and carotid vessels were dissected free of fat and adventitial tissue, opened longitudinally and stained with 0.05% freshly-made Oil red O (ORO) solution. Each stained aortae was then digitally scanned and the percentage of the aorta covered by ORO-positive lipid-filled lesions was determined using Adobe Photoshop, as reported earlier53.

High-frequency Ultrasound Imaging

Vascular lesions were measured non-invasively by High-frequency Ultrasound Imaging using the Vevo 770 High-resolution Imaging System (VisualSonics, Inc. Toronto, Canada), as previously reported55. Briefly, both internal and external diameters of left and right common carotids were measured using B-mode (2-dimensional) images. In addition, left ventricular outflow tract (LVOT) and transverse aortic arch diameters were measured.

Immunohistochemistry

Aortic root sections from each animal were subjected to immunohistochemistry using anti-PCNA (Abcam, Cambridge, MA), anti-αSMA (Sigma, St. Louis, MO), anti-CD68 (Bioss, Woburn, MA), anti-CD45 (Bioss, Woburn, MA) and anti-Ki67 (Abcam, Cambridge, MA) antibodies. Briefly, tissue sections were incubated in ice-cold acetone (5–10 mins) and blocked with 5% donkey or goat serum (90 mins) at room temperature. Following an overnight incubation with primary antibodies (anti-PCNA-1:200; anti-α-SMA-1:200; anti-CD68–1:50; anti-CD45–1:150; anti-Ki67-1:100) at 4 °C, sections were incubated with Alexa Fluor 488 goat anti-mouse (for α-SMA) or Alexa Flour 594 donkey anti-rabbit IgG secondary antibodies (1:1000 or 1:500) and mounted on DAPI-containing mounting media (Vectashield, Vector Laboratories). For co-staining experiments, consecutive slides from serial sections were sequentially stained first with PCNA antibody followed by α-SMA antibody. To control for non-specific staining, identical sections were incubated in the absence of the corresponding primary antibodies, where no background staining was noted. Sections were observed using Olympus fluorescence IX71 microscope (10X or 15X magnification) and images were digitally captured using a set of identical parameters across all sections, specific for each antibody. For immunohistochemistry quantifications, lesion area within the aortic root was outlined, rest of the image cropped away and specific positive staining within lesions was quantified. TemplateToaster 6.0.0.11708 Full Crack Download Free Is Here each individual treatment group, at least 5 mice with an average of 20 tissue sections per group were utilized for all quantifications. All immunostaining images were quantified in a blinded randomized manner using the Image J software. Results are expressed as fold of control for positive staining.

Immunoblotting

Aortic tissue lysates were prepared in SDS lysis buffer, as described earlier56 and protein content was determined using BCA protein assay. Equal amounts of proteins (35 μg) were resolved on 8% SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed using anti-TSP-1 (1:500-1:1000, Neomarkers, Freemont, CA), anti-O-GlcNAc (RL2, 1:1000; Abcam, Cambridge, MA), anti-OGT (1:1000, Cell Signaling, Danvers MA), anti-PCNA (1:300, Abcam, Cambridge, MA), anti-SM-MHC (1:2000, Proteintech, Rosemont, IL) and anti-vimentin (1:1000, Cell Signaling, Danvers, MA) antibodies. Membranes were stripped and re-probed with anti-β-actin used as a loading control; equal protein loading of samples was also confirmed by staining the membranes with Ponceau S. All immunoblot images were captured using Protein Simple and densitometric analyses was performed using the Image J software.

Primary Cultures of Mouse Aortic Smooth Muscle Cell

Primary cultures of mouse aortic smooth muscle cells (aSMC) isolated from TSP-1-transgenic mice, constitutively overexpressing TSP-1 in the arterial SMCs of the aortic vessel, and wild-type mice were kindly provided as a gift by Dr. Olga Stenina Adognravi (Cleveland Clinic, Cleveland, OH). Cells were maintained in complete DMEM/F12 media supplemented with 10% FBS and 1% antibiotics/antimycotic solution. aSMC primary cultures between passages 3–6 were used in all experiments; the contractile phenotype of aSMC was confirmed by α-SMA staining.

Cell Proliferation Assay

About 5000–7000 mouse aortic smooth muscle cells were plated on 96-well tissue-culture plates in complete DMEM/F12 medium containing 10% FBS. After allowing for an overnight growth, the cells were placed in low glucose (5.5 mM) serum-free DMEM media and further incubated with or without 20 mM glucose in the presence or absence of 100 nM chromium chloride (CrCl3) for 72 hours. Cell proliferation was measured at endpoint using the WST-1 cell proliferation reagent (Cayman Chemicals), as reported earlier19. Data are represented as % of Control (wild type); all values are expressed as mean ± SD from four independent experiments.

Statistical Analyses

For all morphometric and immunohistochemistry quantifications, at least 5 mice per treatment group were utilized with an average of 20 tissue sections per treatment group for each measurement. Sections derived from identical regions of the aortic root following the valve leaflet were used in all treatment groups for quantifications and comparisons. Please note, immunohistochemistry and morphometric data collected from all animals were included in our quantifications. All images were quantified by team members blinded to the identity of the treatment groups, in order to minimize bias and intentional exclusion of animals from the study. Differences in group sizes for some measurements were due to lack of additional tissue sections from the corresponding mice. For immunoblotting, aortic tissue lysates prepared from at least 3 mice per group were utilized. Image J software was used for densitometry of immunoblots and positive staining quantification. For indicated immunoblots, lane images show proteins detected on a single blot; however, lanes were rearranged for clarity of presentation. All data are presented as fold of control; values are expressed as mean ± SD, to depict variability of data. For comparison between two treatment groups, statistical analysis was done using unpaired Student’s t-test. For comparison between three groups, one-way analysis of variance (ANOVA) was used. Differences between mean values were considered statistically significant at P ≤ 0.05.

Additional Information

How to cite this article: Ganguly, R. et al. Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE−/− mice. Sci. Rep.7, 45279; doi: 10.1038/srep45279 (2017).

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    CAS

Источник: https://www.nature.com/articles/srep45279

THE POSSIBLE PROTECTIVE EFFECTS OF CYMBOPOGON (LEMONGRASS) DECOCTION ON CHROMIUM PICOLINATE INDUCED PULMONARY ALVEOLAR CHANGES IN ADULT MALE ALBINO RATS.

THE POSSIBLE PROTECTIVE EFFECTS OF CYMBOPOGON (LEMONGRASS) DECOCTION ON CHROMIUM PICOLINATE INDUCED PULMONARY ALVEOLAR CHANGES IN ADULT MALE ALBINO RATS. Fardous S KarawyaEl Sayed Aly Mohamed Metwally Department of Histology and Cell Biology, Department of Anatomy, avast premier 2019 crack - Crack Key For U Faculty of Medicine, Alexandria University. ABSTRACT Introduction: Chromium is a naturally – occurring heavy metal found in several forms (hexavalent Cr VI and trivalent Cr III). Copper, Manganese, Selenium and Chromium are all trace elements which are important in human diet. Any of these elements may have pernicious effects if taken in quantity or if the usual mechanisms of elimination are impaired Over exposure to chromium can occur in welders and other workers in the metallurgical industry, persons taking chromium – containing dietary supplements, patients who have received metallic surgical implants and individuals who ingest chromium salts in drinking water. The interest in herbal therapies and its expansive involvement in the health sector are not surprising. The herb Cymbopogon (Lemongrass), known as halfa-baris is highly reputed in folkloric medicine as an effective diuretic, renal or abdominal antispasmodic agent and for relieving bronchial asthma. Aim of the work: assessment of the possible protective effects of Lemongrass decoction on Chromium picolinate induced pulmonary alveolar changes of male albino rats. Material & Methods: the animals were divided into 3 groups. Group I (control group) which were further subdivided into 2 equal subgroups .Group II(experimental group ) Chromium picolinate (Cr(pic)3 ) was dissolved in water and administered at a concentration of 5 mg/kg/day in drinking water ad libitum for one month. Group III (protected group) received Chromium picolinate by the same route and duration concomitant with Lemongrass decoction 1%. Results: administration of Chromium picolinate induced alteration in the pulmonary alveoli as evident by proliferation and abnormal vacuolation of type II pneumocyte, numerous foamy macrophages, intra-alveolar cellular debris and exfoliated cells, thickening of inter-alveolar septa due to mononuclear cellular infiltration, congestion of the blood vessels associated with increased collagen deposition. Concomitant administration of lemongrass ameliorates most of these changes. Conclusion: Chromium picolinate is toxic to pulmonary alveolar epithelium and concomitant administration of Lemongrass effectively protects lung tissues. Key words: Slimming drugs- Chromium picolinate –Lemongrass- alveoli- Albino rats. INTRODUCTION Chromium (Cr III, VI) is an “essential trace element” and is a widely used industrial chemical, extensively used in paints, metal finishes, and steel including stainless steel manufacturing, chrome and wood treatment. Cr III compounds are used as micronutrients and nutritional supplements and have been demonstrated to exhibit a significant number of health benefits in animals and humans (Mirasol, 2000; Baselt 2008; and Anderson 2000). One form in particular, chromium picolinate, is popular because it is one of the more easily absorbed forms. Sales of chromium picolinate as a nutritional supplement were second only to calcium in the year 2000 (Ben Best, 2000). It is marketed as a weight loss aid for dieters and an ergogenic (muscle- building) aid for bodybuilders and athletes and also used for depression and polycystic ovary syndrome (PCOS) (Alemany et al., 2003; Wayne et al., 1999; and Urmila and Goyal, 2003). It works together with insulin to metabolize carbohydrates, so it is used for improving blood sugar control in people with prediabetes, type 1 and type 2 diabetes. Despite widespread use of Chromium picolinate, significant controversy still exists regarding the effect of Chromium supplementation on parameters assessing human health (William et al., 2004). Respiratory and dermal toxicity of chromium is well documented. Workers exposed to chromium develop nasal irritation, nasal ulcer, hypersensitivity reaction and chrome holes of the skin (Baselt, 2008). The inhalation of chromium compounds has been shown to be associated with the development of cancer in workers in the chromate industry (Langard, 1982). It also damages the genetic material and causes oxidative stress and DNA damage (David et al., 2008; Stout et al., 2009; and Dayan and paine, 2001). Applying medicamental herb approach has emerged on using medicines with natural and especially herbal origin (Khayatnouri et al., 2011). The herb Cymbopogon (Lemongrass) known as halfa-baris, has many health benefits and healing properties. The primary chemical component in lemongrass is citral which has strong anti- microbial and anti-fungal properties, so it prevents and cures bacterial infections in the colon, stomach, urinary tract and respiratory system. Its leaves and stems are high in folic acid and essential vitamins such as pantothenic acid (vitamin B5), pyridoxine (vitamin B-6) and thiamin (vitamin B-1). It also contains many anti-oxidant minerals and vitamins such as vitamin C, vitamin A, potassium, zinc, calcium, iron, manganese, copper, and magnesium. Lemongrass tea can act as a diuretic and is highly effective in flushing toxins and waste out of the body; improving the function of many different organs including the liver, spleen and kidneys. It can help you lose weight by shedding unnecessary water along with the impurities. Many people use lemongrass as a calmative agent; to help them deal with anxiety and nervousness (Sayed and Elserwy, 2011; Adeneye and Agbaje, 2007; Chandrasekhar and Joshi, 2006; Cheel et al., 2005; Figueirinha et al., 2010; Gayathri et al., 2011; Komorowski et al., 2008). The present study was designed to explore the possible protective effects of lemongrass on Chromium picolinate induced alveolar changes in male albino rats. MATERIAL & METHODS Preparation of lemongrass decoction 1% by addition of 2 gms dried leaves and flowering tops to 200 ml water and boiling (Adeneye and Agbaje, 2007; and Gayathri et al., 2011). Forty healthy adult male albino rats of 3 months age 150-200 gm were acclimated for a week. The animal procedures were performed in accordance with Guidelines for Ethical Conduct in the Care and Use of Animals, maintained at room temperature of 25 ± 2°C with 12-hour dark-light cycle. Animals were fed with standard rodent diet. There was no water and light restriction throughout the experimental period. They were divided into 3 groups:- Group I (Control group): 20 rats were further subdivided into two equal subgroups 10 animals each. Subgroup Ia given physiological saline and subgroup Ib given Lemongrass decoction 1% orally by gavage for one month. Group II (experimental group): 10 rats received Chromium picolinate dissolved in water and administered at a concentration of 5 mg/kg/day of Cr (pic) 3, in the drinking water ad libitum for one month orally by gavage. The chromium intake in this study is similar to other studies utilizing rodents (Komorowski et al., 2008; Jain et al., 2007 and Mozaffari et al., 2005). Group III (Protected group): 10 rats received the same dose of Chromium piclonate as in group II concomitant with Lemongrass decoction 1% for one month orally by gavage. At the end of the experimental period, the animals of all groups were sacrificed by decapitation under ether anaesthesia. Lungs were dissected out. Parts of the specimens were preserved in formal saline for preparation of paraffin blocks. Sections were cut 5µm thickness and were subjected to the following staining procedures: 1. Haematoxylin & Eosin stain for routine histological examination (Carletons et al., 1980). 2. Gomori" s Trichrome stain for demonstration of collagen fibers (Carletons et al., 1980). 3. Some of the specimens were immediately removed after sacrification and immediately fixed in 3% phosphate buffered glutaralehyde (Ph 7.4) for 2 hours at 4ºC, and further processed for examination and photography of the ultrastructure by Joel -100 CX transmission electron microscope Faculty of Science Alex university (Trevor and Graham, 1996; Girgis et al., 1988). RESULTS I- Light Microscopic Results: H&E stained sections of the control group ( subgroup Ia,b) showed lung alveoli lined with type I and type II pneumocytes. Type I pneumocytes are flattened cells with flattened nuclei, type II pneumocytes are rounded cells with rounded nuclei. The alveoli are separated by very thin interalveolar septa (Fig 1a, b). Compared with the control group chromium picolinate treated rats for one month (group II) showed collapsed alveoli with proliferation and vacuolation of type II pneumocytes, numerous foamy alveolar macrophages, intra-alveolar cellular debris and thickening of inter-alveolar septa with cellular infiltration and acidophilic vacuolated material (Fig 2 a, b). Concomitant administration of Lemongrass with Chromium picolinate (group III) revealed evident reduction of all alveolar changes except for mild thickening of inter-alveolar septa by cellular infiltration associated with mild congestion of blood vessels (Fig 3 a,b,c, d ). Gomori"s Trichrome stained section of the control group showed normal distribution of collagen fibers (Fig 4 a,b). Collagen fibers increased between the alveoli of chromium picolinate treated rats (Fig 4 c), while lung tissue of the protected group revealed more or less the normal pattern of collagen distribution (Fig 4 d,e). II- Ultrastructural Results: The lungs of the control group (subgroup Ia,b) revealed, patent alveoli with thin walls lined with two types of cells, the flattened pneumocyte type I (Fig 5 a) and large cuboidal pneumocyte type II with its characteristic apical microvillous border and lamellar bodies. The inter-alveolar septa appeared thin containing few cells and fibers (Fig 5 b). Electron micrographs of Chromium picolinate treated rat lung for one month showed alteration in the alveolar architecture. The alveoli appeared collapsed and lined by either destructed type I and Type II pneumocyte (Fig 6 a,b), or may be evidently proliferated and abnormally vacuolated type II pneumocyte, (Fig 7 a, b, c, d) numerous foamy macrophages with vacuolated cytoplasm and many lysosomes (Fig 8 a,b, c,d ). Desquamated cells and cellular debris were also seen within the lumen of the alveoli (Fig 9 a,b,c ). The inter-alveolar septa were thickened due to mononuclear cellular infiltration, congested blood vessels (Fig 10 a,b), presence of hyaline material (Fig 11a,b) and increased collagen deposition (Fig12 a,b). The lung tissues of the protected group revealed considerable degree of preservation of the alveolar architecture. Most of the alveoli were inflated and lined with more or less normal type I and II pneumocytes (Fig13 a,b,c). Few septal cells associated with less collagen fibers and less vacuolated macrophages were also noted (Fig14 a,b,c). DISCUSSION Obesity is increasing at an alarming rate, so it possesses serious health hazards and its treatment is often disappointing. One of the key goals for enhancing weight loss is to increase the sensitivity of the cells throughout the body to insulin. Micronutrient is proposed as a global approach for the obese person (Choes et al., 2007; Bloniarz and Zareba, 2007). The trace mineral chromium is an essential nutrient involved in the regulation of carbohydrate, lipid and protein metabolism via an enhancement of insulin action. Chromium plays a key role in cellular sensitivity to insulin. It has lately gained a great deal of lay attention as an aid to weight loss. Also chromium picolinate was reported to ergogenic properties related to body composition changes in young men. (Friedman, 2000; Korc, 2004; Chaturvedi, 2007; Mehta and Farmer, 2007 and Dandona et al., 2005) Chromium picolinate (Cr (pic) 3), contains trivalent chromium. It is widely used because of claims that it exerts antidiabetic and weight-reduction effects (Anderson, 2000; Trumbo and Ellwood, 2006 and Vincent, 2003). On the other hand, other studies have raised concerns regarding the safety of Cr (pic) 3. It is a very stable hydrophobic molecule which allows it to be readily absorbed from the digestive tract and readily cross membranes. But it appears that once inside the cells, trivalent chromium (Cr3+) picolinate can be reduced to divalent chromium (Cr2+) which can participate in the Fenton reaction in the presence of hydrogen peroxide to generate hydroxyl radicals which cause DNA damage (Stearns et al., 1995; Speetjens et al., 1999; Bagchi et al., 2002 and Coryell and Stearns 2006). Animal studies have shown chromium (VI) to cause respiratory toxicity in the form of tumors, ulcerations, bronchitis and pneumonia or renal toxicity because the kidney serves not only as its major route of elimination but also accumulates chromium (Lamson and Plaza, 2002; Hepburn and Vincent, 2002, 2003; and Beaumont et al., 2008). In view of the previously mentioned facts, it was quite essential to investigate the histological effects of oral chromium picolinate on the pulmonary alveolar cells and the possible protective effect of lemongrass. In the present study chromium picolinate (group II) induced diffuse changes in the alveolar architecture as evident by collapsed alveoli, very thick interalvelolar septum due to mononuclear cellular infiltration, congested blood vessels, numerous foamy macrophage, acidophilic hyaline material, extravasated erythrocytes, desquamated cells in the alveoli associated with increased collagen fibers. Chromate and their reduction products cause many types of DNA damage. Some of these types of DNA damage can be decreased by antioxidants vitamins C and E which suggests that oxidative damage might have a role in causing these types of DNA damage within the cell (De Flora, 2000; Bagchi, 2000; Sugden et al., 2001). These previous studies provided important mechanistic data into chromium – induced toxicity. Macrophage accumulation and neutrophil infiltration in the present study add our understanding of chromium induced pulmonary toxicity. We suggest that chromium induced expression of many cytokines and chemokines may play an important role in the activation of alveolar macrophages. Activated macrophages secrete proinflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor α. The macrophage-derived inflammatory cytokines have two major effects: (1) expression of adhesion molecules on endothelial cells for extravasation of monocytes and lymphocytes; and (2) stimulation of targeted migration of mononuclear cells to the area of inflammation (chemotaxis). Thus, additional mononuclear phagocytes are recruited to the tissue from the intravascular space. The inflammation can be seen hma pro vpn license key 2019 android - Activators Patch thickening of interalveolar septa. Septum thickening leads to alveoloar collapse. In the present study, chromium exposure induced lung injury as well as a chronic inflammatory response. The predominant immune cells in the lung airways and tissue were neutrophils and lymphoid cells, respectively. We hypothesize that chromium will induce an inflammatory microenvironment in the lung that will promote proliferation and selection of growth-altered cells. These findings are apparent in group II. Chronic inflammation is involved in the pathogenesis of many cancers, including those of the lung (Coussens and Werb, 2002; Lin and Karin, 2007). The presence of neutrophils and macrophages in the lung after chromium exposure is consistent with welding fume studies in which a significant increase in neutrophils and macrophages was also detected in the lung of exposed rodents (Antonini et al., 2007; Solano-Lopez et al., 2006; Taylor et al., 2003 and Zeidler-Erdely et chromium picolinate - Crack Key For U al., 2008) Neutrophils initiate the debridement of damaged tissue, phagocytose any pathogens, and amplify the inflammatory response through production of cytokines (Coussens and Werb, 2002; Eming et al., 2007). A major function of macrophages is to continue phagocytosis at sites of tissue injury (Freeman et al., 2007). To this end, neutrophils and macrophages release highly active substances, including reactive oxygen species (ROS) and reactive nitrogen species that may promote a microenvironment that directly damages DNA or interferes with the mechanisms of DNA repair (Azad et al., 2008; Federico et al., 2007). In the chromium exposure, these reactive species may further exacerbate DNA damage in surviving and/or proliferating epithelial cells and thus promote initiating events in chromium carcinogenesis (O’Brien et al., 2003). Macrophages also produce cytokines and growth factors in order to .stimulate cell proliferation and angiogenesis (Eming et al., 2007) We also observed that chromium exposure resulted in oxidative stress, thus chromium may promote inflammation, cell survival, and repair of the airways after lung injury. In keeping with this hypothesis, we observed proliferative epithelial cells, which is consistent with promoting cell survival in an chromium picolinate - Crack Key For U of genotoxic chromium induced injury and inflammation. In the present study pneumocytes type II was the most altered cell. The increase in the number and size of type II pneumocytes which were noticed in the present study might be due to its role to replace the type I pneumocytes. When alveolar epithelium is exposed to toxic agent, particularly if there is extensive destruction of the sensitive type I pneumocytes, type II pneumocytes increase in size and number being precursor stem cells for type I pneumocytes (Stevens and Lowe, 1997) Also type II pneumocytes had deformed surfactant material. The maintenance of the alveoli depends on the presence of a surface tension lowering substance known as pulmonary surfactant. It consists of 90% phospholipids and 10% proteins. Pulmonary surfactant is made and secreted by pneumocyte type II cells in whose cytoplasm it is stored in the form of lamellated bodies. Abnormalities of phospholipid metabolism most often manifest themselves by the accumulation of phospholipids in various tissues of the body. These accumulations may then interfere with cellular functions and lead to the establishment of acute or chronic disease states. The accumulation of phospholipids in the lung has been referred to, pulmonary alveolar proteinosis (PAP). The alveoli in PAP are filled with proteinaceous material, which has been analyzed extensively and determined to be normal surfactant composed of lipids and surfactant-associated proteins. Evidence exists of a defect in the homeostatic mechanism of either the production of surfactant or the clearance by alveolar macrophages. A clear relationship has been demonstrated between PAP and impaired macrophage maturation or function, which might account for the high association with malignancies and unusual infections (Griese et al., 2010; Cummings et al., 2012; Suzuki, 2010; Carey and Trapnell, 2010). The present study revealed accumulation of phospholipid in the macrophages (foam cell) and in the cytoplasm of type II pneumocytes. In the lung because of its unique architecture, accumulation of phospholipids may occur both intracellularly and extracellularly. Intracellular accumulation of phospholipids can interfere with cellular functions resulting in impaired or abnormal pulmonary responsiveness in a variety of situations. The cytoplasmic accumulation of phospholipids in alveolar macrophages has been shown to impair its phagocytic activity. Impaired phagocytic activity of macrophages may lead to decreased resistance of the lung to infection (Carraway, 2000). Extracellular accumulation of phospholipids in the alveoli interferes with gas exchange causing respiratory insufficiency. In addition, alveolar clearance of toxic substances has been shown to be severely impaired in the lungs of rats with phospholipidosis ( Presneill, 2004 ). The extravasation of erythrocytes was most possibly the sequel of endothelial cell damage in the alveolar capillaries. Also extravascular localization of leukocytes implies acute vascular injury which is a consistent feature of injury caused by most pulmonary toxicants (Cotran, 1987). Exudation of leukocytes (mostly neutrophils into the alveolar lumen of exposed animals was most probably attributed to increased permeability of the alveolar capillaries. Besides, the toxic material was found to stimulate macrophages and pneumocytes to release chemoattractants for neutrophils. Also the infiltrating inflammatory cells may account for the damaging of the alveolar and interstitial pulmonary structures through the lytic effect of their enzymes. On the other hand light microscopic examination of the Trichrome stained sections of the experimental group showed that the amount of collagen fibers increased. The agents that induced phospholipidosis produce lung fibrosis. Pulmonary fibrosis is the end – stage of a group of chronic diseases. A complex set of tissue reactions must occur for the formation and accumulation of fibrous connective tissue that defines pulmonary fibrosis. The pathogenesis of pulmonary fibrosis begins as an inflammatory response to injury when immune cells are excessively or inappropriately activated. These immune cells include macrophages and neutrophils that release toxic mediators, compromising epithelial integrity and promoting tissue injury. The normal repair process involves the recruitment and activation of mesenchymal cells resulting in extracellular matrix deposition, re-epithelialization and restoration of normal lung architecture (Hamdy et al., 2012; Kliment and Oury, 2010). Also previous researches reported that reactive oxygen species promotes the development of inflammation and increases activity at sites of inflammation and induces the proliferation of the fibroblast leading to severe pulmonary fibrosis (Shi et al., 2014; Kliment and Oury, 2010). Moreover the results of the present study revealed destruction of type I and type II pneumocytes. Excessive and persistent formation of ROS from inflammatory cells (i.e., macrophages and neutrophils) is considered the hallmark of genotoxicity. The overall genotoxic response will depend on the effectiveness and efficiency of intra- and extracellular antioxidant defense systems, DNA repair systems, and processes leading to apoptotic and necrotic processes in those cells carrying premutagenic lesions (Starosta and Griese, 2006; Trapani et al., 2003 and Stout et al., 2009). The lemongrass group (group III) in the present study showed thin interalveolar septa, few mononuclear cellular infiltration, few extravasated RBCs and decreased collagen fibers. In agreement with these findings previous studies reported that lemongrass constitutes an important source of antioxidants, also contains minerals that function as co – factors in the antioxidant enzymes (Arhoghro et al., 2010; Figueirinha et al., 2010; Omotode, 2009 and Tiwari et al., 2010). Conclusion Over viewing our results we find that exposure to chromium induces chronic inflammation and injury in the lung. Furthermore, this chromium induced injury and inflammation was associated with epithelial cell proliferation. Taken together, we suggest that these early disease processes promote a microenvironment that may participate in the initiation and promotion of neoplastic cells and contribute over time to chromium carcinogenesis on the other hand lemongrass produced sufficient protection against these damage. Fig 1 a,b: photomicrograph of the control rat lung showing, normal architecture of the alveoli (A) separated by very thin interalveolar septa. The alveoli are lined by flat type I pneumocyte (↑) and rounded type II pneumocyte (↑↑). H&E stain Mic Mag a X100-bX 400. Fig 2 a,b : photomicrograph of the rat lung group II showing, collapsed alveoli (A) separated by very thick interalveolar septa. Note mononuclear cellular infiltration, congestion of the blood vessels (V), acidophilic hyaline material (*) numerous foamy macrophages (↑) and extravasated RBCs (R). H&E stain Mic Mag a X100-bX 400. Fig 3 a,b, c,d : photomicrograph of the rat lung group III showing, preserved lung architecture except very mild increase in thickness of interalveolar septa, some acidophilic hyaline material (*), exfoliated cells (E ) and congested blood vessels (V). H&E stain Mic Mag. a ,bX100-c,dX 400. Fig 4 a,b,c,d, e : photomicrograph of the rat lung showing a, b - normal distribution of collagen fibers in the control group. c- group II showing thick interaveolar septa, congestion of the blood vessels and increase in collagen fibers. d, e - group III showing very mild increase in interalveolar septa associated with mild increase Save2pc Ultimate For Windows collagen fibers. Gomori"s Trichrome stain Mic Mag X 200. Fig 5 a,b : electron micrograph of the control rat lung showing, open alveoli (A) lined by flat nucleus of type I pneumocyte ( P1) and type II pneumocyte (P2) with characteristic lamellated structure (↑) and apical microvilli (mv). Mic Mag a X4000-b X3000. Fig 6 a,b : electron micrograph of rat lung group II showing collapsed alveoli (A), destruction( ↑) and abnormal nucleus of type I pneumocyte ( P1 ). Destruction of type II pneumocyte (↑↑) filled with empty lamellated structure (P2), thick interalveolar septa, hyaline material (*) and extravasated RBCs (R). Mic Mag a X 5000-b X 3000. Fig 7 a,b,c,d : electron micrograph of rat lung group II showing collapsed alveoli (A ) separated by thick interalveolar septa. Note numerous type II pneumocyte filled with empty lamellated structure (P2), alveolar macrophage (↑) and congested blood vessels (V). Mic Mag a X 1500-b X 1000- c X 2500- d X 2000. Fig 8 a,b,c,d : electron micrograph of rat lung group II showing, numerous alveolar macrophages ( foam cell ) filled with many vacuoles (↑). Note extravasated RBCs (R) and hyaline material (*). Mic Mag a,b,d X 3000- c X 1500. Fig 9 a, b, c: electron micrograph of rat lung group II showing, interalveolar septa contain desquamated cell and cellular debris (↑↑), alveolar macrophages (↑). Note type II pneumocyte containing empty lamellated structure (P2), congested blood vessels (V) and hyaline material (*). Mic Mag ac X 1000-b X 1500. Fig 10 a, b: electron micrograph of rat lung group II showing, thick interalveolar septa due to mononuclear cellular infiltration (↑) and congestion of the blood vessels (V). Note type II pneumocyte filled with empty lamellated structure (P2) and cellular debris (↑↑). Mic Mag a X 1500- b X 2000. Fig 11 a, b: electron micrograph of rat lung group II showing, thick interalveolar septa, hyaline material (*), congested blood vessels (V), increased collagen fiber (↑) and desquamated cell (↑↑). Mic Mag a X 2500-b X 3000. Fig 12 a, b: electron micrograph of rat lung group II showing, abnormal type II pneumocyte with irregular nuclei ( P 2 ), marked increase of collagen fibers (↑) and extravasated RBCs (R). Mic Mag a X 4000- b X 3000. Fig 13 a, b, c: electron micrograph of rat lung group III showing, more or less normal architecture of the alveoli lined by type I pneumocyte (P 1) and type II pneumocyte (P 2) with apical microvilli (mv) and lamellated structure (↑). Note presence of extravasated RBCs (R), few collagen fibers (↑↑) and some hyaline material (*). Mic Mag a,b X 3000- c X 4000. Fig 14 a, b, c: electron micrograph of rat lung group III showing, type II pneumocyte with apical microvilli (mv) and lamellated structure (P2) and alveolar macrophage with less vacuoles(↑). Note mild increase in collagen fibers (C) and hyaline material (*). Mic Mag a X 3000- b X 2500- c X 4000. REFFERENCES 1- Adeneye AA. And Agbaje EO. 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Effect of chromium piclonate on histopathological alteration in STZ and neonatal STZ diabetic rats. j. Cell Mol. Med. 2003; 7(3) 322-9. 69- Vincent JB: The potential value and toxicity of chromium picolinate as a nutritional supplement, weight loss agent and muscle development agent. Sports Med 2003; 33:213-30. 70- Wayne WC, Lyndon JJ, Stephanie LD, Deanna CC, Richard A A and William J E. Effects of resistance training and chromium piclonate on body composition and skeletal muscle in older men. Journal of Applied physiology 1999; 86(1); 29-39. 71- William T, Cefalu MD and Frank B. Role of chromium in human health and in diabetes. Diabetes Care 2004; 27(11): 2741-51. 72- Zeidler-Erdely PC, Kashon ML, Battelli LA, Young SH, Erdely A, Roberts JR, et al. Pulmonary inflammation and tumor induction in lung tumor susceptible A/J and resistant C57BL/6J mice exposed to welding fume. Part Fibre Toxicol. 2008; 5:12.

Источник: http://www.academia.edu/16299719/THE_POSSIBLE_PROTECTIVE_EFFECTS_OF_CYMBOPOGON_LEMONGRASS_DECOCTION_ON_CHROMIUM_PICOLINATE_INDUCED_PULMONARY_ALVEOLAR_CHANGES_IN_ADULT_MALE_ALBINO_RATS

Association of plasma chromium with metabolic syndrome among Chinese adults: a case-control study

  • Sijing Chen1,2,
  • Li Zhou1,2,
  • Qianqian Guo1,2,
  • Can Fang1,2,
  • Mengke Wang1,2,
  • Xiaobo Peng1,2,
  • Jiawei Yin1,2,
  • Shuzhen Li1,2,
  • Yalun Zhu1,2,
  • Wei Yang1,2,
  • Yan Zhang3,
  • Zhilei Shan1,2,
  • Xiaoyi Chen4 &
  • Liegang Liu1,2

Nutrition Journalvolume 19, Article number: 107 (2020) Cite this article

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Abstract

Backgroud

Chromium has been suggested playing a role in alleviating diabetes, insulin resistance and lipid anomalies, but the effect on metabolic syndrome (MetS) in humans remains controversial.

Methods

We conducted a matched case-control study in a Chinese population, involving 2141 MetS cases and 2141 healthy controls, which were 1:1 matched by age (±2 years) and sex. Plasma chromium was measured by inductively coupled plasma mass spectrometry.

Results

Plasma chromium levels were lower in MetS group than in control group (mean: 4.36 μg/L and 4.66 μg/L, respectively, P < 0.001), and progressively decreased with the number of MetS components (P for trend < 0.001). After adjustment for potential confounding factors, the odds ratios (95% confidence intervals) for MetS across increasing quartiles of plasma chromium levels were 1 (reference), 0.84 (0.67–1.05), 0.76 (0.61–0.95), and 0.62 (0.49–0.78), respectively (P for trend < 0.001). For the components of MetS (high waist circumference, high triglycerides and high blood glucose), the odds ratios (95% confidence intervals) of the highest quartiles were 0.77 (0.61–0.95), 0.67 (0.55–0.80), and 0.53 (0.44–0.64), respectively (P for trend < 0.05).

Conclusions

Our results indicated that plasma chromium levels were inversely associated with MetS in Chinese adults. The association may be explained by the relations between plasma chromium levels and high waist circumference, and the triglycerides and blood glucose levels.

Peer Review reports

Introduction

Metabolic syndrome (MetS), known as a constellation of metabolic abnormalities, which includes abdominal obesity, high triglycerides, low high-density lipoprotein (HDL) cholesterol, high blood pressure, and elevated fasting blood glucose, is now both a public health and a clinical problem. MetS is epidemic all over the world and its incidence has been rising year-on-year [1]. Recent data indicated that about 33.9% of the adults in Mainland China had MetS [2]. In addition, MetS has been realized a major contributor to the epidemic of cardiovascular disease and type 2 diabetes mellitus [3], and it may increase the risk of mortality [4].

Chromium is an essential trace element, which has been suggested playing a potential role in alleviating diabetes, insulin resistance and lipid anomalies. The beneficial mechanism has been investigated in experimental studies [5,6,7,8,9]. However, the epidemiological evidence of the protective effect of chromium on MetS is very limited, and has inconsistent conclusion so far. A prospective study suggested an inverse association between chromium and incidence of MetS in American young adults, and the inverse association was mainly explained by its relation to blood lipids [10]. There was another case-control study suggesting an association between low chromium levels and increased risk of nonfatal myocardial infarction [11]. Besides, our previous study found that plasma chromium concentrations were inversely associated with type 2 diabetes mellitus and pre-diabetes mellitus [12]. Yet some studies did not support the inverse relationship between chromium and MetS [13, 14]. So far, clinical trials evaluating chromium supplementation on glucose and lipid profiles have yielded conflicting results [15,16,17,18].

Accordingly, in this matched case-control study, we aimed to examine the association of plasma chromium levels with MetS along with its components in a large Chinese population.

Methods

Study population

The present study was a case-control study conducted in Wuhan, China, during the period of March 2013 to December 2017. The study population consisted of 2141 MetS cases and 2141 healthy controls, which were 1:1 matched by age (±2 years) and sex. All participants were aged 18 years or older, consecutively recruited from the general population undergoing a routine health checkup in the Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology. Patients with clinical significant neurological, endocrinological or other systemic diseases, as well as acute illness and chronic inflammatory or infective diseases were excluded from the study. All the participants enrolled were of Chinese Han ethnicity. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Tongji Medical College.

Definition of MetS

The definition of MetS was based on the harmonized definition for MetS in 2009 [19]. To be considered as a case of MetS, participants had to meet at least three of the following criteria: 1. Abdominal obesity: waist circumference ≥ 85 cm in men and ≥ 80 cm in women; 2. Hypertriglyceridemia: ≥ 150 mg/dL; 3. Low levels of HDL cholesterol: < 40 mg/dL in men and < 50 mg/dL in women; 4. High blood pressure: ≥ 130/85 mmHg and/or use of antihypertensive medication; 5. High fasting glucose: ≥ 100 mg/dL and/or current use of antidiabetic medication and/or self-reported history of diabetes. The controls had zero to two components of MetS which were mentioned above.

Data collection

Demographics, health status, and lifestyle data were obtained from the questionnaires, including sex, age, education level, history of disease (diabetes, hypertension and hyperlipemia), family history of diabetes, physical activity, current smoking status, and current alcohol drinking status. Education level was classified as none or elementary school, middle school, and high school or college. Physical activity was classified as at least once/week or no. Current smoking status was classified as yes (at least one cigarette per day over the previous 6 months) or no. Current alcohol drinking status was classified as yes (drink alcohol beverage more than once a week over the previous 6 months) or no. Anthropometric data including height (m), mass (kg), waist circumference and blood pressure were measured with standardized techniques by trained and certified technicians. BMI (body mass index) was calculated as mass divided by the square of height (kg/m2). Waist circumference was obtained at the mid-point between the lowest rib and the iliac crest to the nearest 0.1 cm, after inhalation and exhalation. Hip circumference was measured at the outermost Zenmate 7.7.0.0 Crack+ Keygen Code 2021 - Activators Patch of the greater trochanters. The ratio of waist-to-hip circumference was used as an index of fat distribution. Blood pressure was measured at rest in the seated position using a standardized automated sphygmomanometer after 5 min of rest, and repeated in both arms.

Laboratory measurements

Blood samples were collected in all participants after an overnight fast of at least 10 h. Details of measurement of fasting plasma glucose, fasting plasma insulin, total cholesterol, triglyceride, HDL cholesterol, low-density lipoprotein (LDL) cholesterol and calculation of homeostasis model assessment of insulin resistance (HOMA-IR) and HOMA of β-cell function (HOMA-β) have been described previously [20]. Plasma malonaldehyde (MDA) was measured with an MDA assay kit (Jiancheng, Inc., Nanjing, China).

Measurement of plasma chromium concentrations

Plasma chromium concentrations were measured in the Ministry of Education Key Laboratory of Environment and Banner maker software - Free Activators and School of Public Health at Tongji Medical College of Huazhong University of Science and Technology, using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7700 Series, Tokyo, Japan). Plasma samples were stored at − 80 °C. The case and control specimens were measured randomly in the daily measurement, with laboratory personnel blinded to the case–control status. For quality assurance, metals in standard reference materials were measured once in every 20 samples using certified reference material. The certified concentrations of human plasma controls (ClinChek no. 8883 and 8884) were 3.56 ± 0.89 μg/L and 11.1 ± 2.22 μg/L, respectively. The chromium picolinate - Crack Key For U of detection (LOD) for chromium was 0.01 μg/L, and concentrations of plasma chromium levels below the LOD (0.7%) were imputed at LOD/√2. Quality control was performed (1 out of 20 samples), and the inter-assay and intra-assay coefficients of variation were < 10 and < 8%, respectively.

Statistical analysis

Descriptive statistics were calculated for all demographic and clinical characteristics of the study subjects, and summarized as numbers (percentages) for categorical data, mean ± standard deviations (SDs) for normally distributed data, and medians (interquartile ranges) for non-normally distributed data. Comparisons between MetS and controls were performed by t test or Mann-Whitney U test for continuous variables, and chi-square tests for categorical variables. In addition, subjects were divided into 6 groups according to their possession of 0, 1, 2, 3, 4 or 5 components of MetS. Multiple imputation based on 5 replications and a fully conditional specification method in SPSS was used to account for missing data.

For calculation of the odds ratio (OR) for MetS, plasma chromium concentration was categorized in quartiles according to the control group: category 1, < 3.27 μg/L; category 2, 3.28–4.46 μg/L; category 3, 4.47–5.87 μg/L, and category 4, > 5.88 μg/L. Conditional logistic regression was used to assess the association of MetS with plasma chromium concentrations. The ORs and 95% confidence intervals (CIs) of MetS were calculated between the quartiles of chromium using the lowest quartile as the reference category, and also by per 1 μg/L chromium as continuous variable. We considered three models with progressive degrees of adjustment: model 1 adjusted for age; model 2 additionally adjusted for education, current smoking status, current alcohol drinking status, physical activity and family history of diabetes; and model 3 further adjusted for BMI. Tests of linear trend across increasing chromium quartiles were conducted by assigning the median value to each chromium picolinate - Crack Key For U and treating it as a continuous variable. Furthermore, the ORs of the MetS components including high waist circumference, high triglycerides, low HDL cholesterol, high blood pressure, and high blood glucose were calculated using binary logistic regression.

To evaluate the consistency of the association between chromium and MetS by participant characteristics, additional analyses were run, stratifying age (< 50, ≥50), sex, BMI (< 24, ≥24), physical activity, current smoking status, and current drinking alcohol status. The interactions between these stratification variables and plasma chromium were tested by adding multiplicative terms into the multivariate logistic regression models; the likelihood ratio tests were conducted to examine the interactions.

Statistical analyses were performed with SPSS for Windows, version 24.0 (SPSS Inc., Chicago, Illinois). P values reported are two tailed, and values below 0.05 were considered statistically significant.

Results

Anthropometric and metabolic characteristics of the 2141 MetS and 2141 controls are reported in Table 1. Compared with control subjects, the individuals with MetS had higher prevalence of family history of diabetes and lower rate of smoking and activity (P < 0.05). As expected, we observed higher levels of BMI, waist circumference, hip circumference, waist-to-hip circumference ratio, systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting plasma glucose, fasting plasma insulin, HOMA-IR, triglycerides, total cholesterol, LDL cholesterol and lower levels of HDL cholesterol in MetS than in the controls (P < 0.001). MetS group had higher MDA levels than the control group (P < 0.001).

Full size table

Plasma chromium concentrations were significantly decreased in the individuals with MetS compared with controls (mean: 4.36 μg/L in MetS, and 4.66 μg/L in controls, P < 0.001). For the 5 components of MetS, participants with high triglycerides and high blood glucose had significant lower levels of plasma chromium (P < 0.001). Furthermore, plasma chromium levels progressively decreased with the number of MetS components (P for trend < 0.001) (Table 2).

Full size table

Significant inverse associations between the levels of plasma chromium concentration and MetS were observed, and multiple adjusted models showed similar results (Table 3). After overall multivariable adjustment of age, education, current smoking status, current alcohol drinking status, physical activity, family history of diabetes, and BMI, the ORs (95% CIs) for MetS from the lowest to the highest quartiles were 1 (reference), 0.84 (0.67–1.05), 0.76 (0.61–0.95), and 360 Total Security 10.8.0.1357 Crack + Serial Keygen [Premium] Download (0.49–0.78), respectively (P for trend < 0.001). When plasma chromium concentration was considered as a continuous variable, the overall OR (95% CI) of having MetS was 0.95 (0.92–0.98) per 1 μg/L increment of chromium concentration.

Full size table

The associations of plasma chromium concentrations with each component of MetS were examined afterwards. Similar inverse associations were observed in high waist circumference, high triglycerides and high blood glucose, and the full adjusted ORs (95% CIs) of the highest quartiles were 0.77 (0.61–0.95), 0.67 (0.55–0.80), and 0.53 (0.44–0.64), respectively (P for trend < 0.05) (Table 4). As for low HDL cholesterol, significant associations were observed in model 1, but not in model 2 and 3. Association of plasma chromium concentrations with high blood pressure was not found in this study (Table 4).

Full size table

In stratified analysis (Table 5), ORs (95% CIs) of the highest quartiles of all subgroups decreased significantly, indicating the robust association. No interaction was recognized between age, sex, BMI, physical activity, smoking, drinking alcohol and chromium (P for interaction > 0.05).

Full size table

Discussion

In this matched case-control study, we found that plasma chromium concentrations were inversely associated with the prevalence of MetS among Chinese adults. The inverse association was mainly explained by the relations between plasma chromium concentrations and waist circumference, the triglycerides and blood glucose levels. The associations were not appreciably changed by multivariate adjustment, and were consistent in the stratified analyses.

Chromium coming from foods varies and is usually very low [21]. Dietary intake of chromium from Asian diets ranged from 59.9 to 224 μg per day [22]. It is difficult in estimating dietary chromium due to its wide variability and low content in food sources, so a sensitive and reliable biomarker for chromium intake is required in epidemiological studies. Plasma chromium is considered a reliable objective biomarker for chromium exposure [23]. Previous studies reporting plasma chromium concentrations in large populations were sparse. Currently, there is no international acceptable value or range for the plasma chromium concentration in the general population. The mean concentration of plasma chromium in our population was 4.51 ± 2.24 μg/L, higher than the previously published studies, which varied from 0.2 to 0.86 μg/L [24,25,26]. A possible explanation for it may be higher contamination for the population. As some studies indicated that chromium exposure may come from industrial pollution like coal and oil combustion, the metal fabrication industry and the leather tanning sector, and China had a dramatic increase of anthropogenic chromium emissions from 1990 to 2009 [27].

There existed few high-quality evidence focused on the relationship between chromium and MetS at present. Limited epidemiological study yielded controversial results. A 23-year follow-up study including 3648 American adults indicated that toenail chromium levels were inversely and longitudinally associated with incidence of MetS [10]. However, another cross-sectional study conducted in Korea did not support the association between toenail chromium concentrations and MetS and its components [13].

In our study, significant associations between chromium concentrations and waist circumference, triglycerides and blood glucose levels were noticed. These associations might explain the latent mechanism involved in the relationship of chromium and MetS.

The association of plasma chromium with high blood glucose was the strongest among the components of MetS in this study. Although the pathogenesis of MetS remains unclear, recent interest has focused on the possible involvement of insulin resistance as a linking factor [28]. Coincident with this, our previous study has elaborated the inverse association between plasma chromium concentrations and type 2 diabetes mellitus and pre-diabetes mellitus in a case-control study [12]. In addition, evidences in animal and in vitro studies supported the association as well. A lot of studies demonstrated that chromium may up-regulate insulin-stimulated free download malwarebytes signal transduction by a variety of mechanisms [5, 6, 8, 29,30,31]. However, it is worth concerning the causality of chromium status and high blood glucose. On one hand, the low levels of chromium might result in the diminution of insulin signal transduction, and further aggravate the development of insulin resistance. On the other hand, chromium lost and excreted from human body increased with aging and was related to the diabetes [32]. Large losses of chromium over more than 2 years’ diabetes duration may change the chromium homeostasis [33]. Further studies are warranted to investigate the causality of chromium status and high blood glucose.

Moreover, the effects of chromium on obesity and dyslipidemia has also been studied. The animal studies indicated that chromium might reduce the weight of obese rats and lipids levels as well [5, 9, 34, 35]. However, clinical trials were inconclusive with regard to weight control and lipid metabolism improvement. Although some studies claimed beneficial effects of chromium supplementation [36, 37], systematic reviews found it inadequate to inform firm decisions about the efficacy of chromium supplements on weight loss or lipid metabolism in overweight or obese adults because of the low-quality evidence [15, 18, 38].

The strengths of our study included the matched case-control study design, the large number of participants and objectively measured plasma chromium levels. In addition, chromium levels in plasma were measured using the state-of-the-art ICP-MS method. A few limitations need to be considered. First, the case-control nature of our study does not allow us to infer any causality and address temporal relationship between plasma chromium and MetS. Second, we could not differentiate trivalent chromium from hexavalent chromium in plasma measurement. Trivalent chromium is suggested to be beneficial and hexavalent chromium is toxic to human health [39]. Thus, the combination of these two forms may attenuate the association that may exist between trivalent chromium and MetS. Third, the classification of current smoking and alcohol drinking status and physical activity was not detailed enough. Additionally, the lack of information on the other unknown or unmeasured factors might also confound our results. Finally, the generalizability of our findings may be limited since all participants were of Chinese Han ethnicity. However, a homogenous ethnic background may reduce residual confounding from unmeasured genetic and cultural variability.

Conclusions

Our study demonstrated an inverse association between plasma chromium levels and MetS in a Chinese population. The association was mainly accounted for the relations between plasma chromium levels and high waist circumference, and the triglycerides and blood glucose levels. Further studies are warranted to confirm our findings in prospective cohorts and to elucidate the potential mechanisms underlying the relationship between chromium and MetS, as well as MetS components.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

Confidence interval

Diastolic blood pressure

Malonaldehyde

High-density lipoprotein

Homeostasis model assessment of β-cell function

Homeostasis model assessment of insulin resistance

Limit of detection

Low-density lipoprotein

Metabolic syndrome

Odds ratio

Systolic blood pressure

Standard deviation

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Acknowledgements

We gratefully acknowledge the nurses of the Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, for their diligent work on collecting blood samples.

Funding

This study was supported by the National Natural Science Foundation of China (81803239); the Major International (Regional) Joint Research Project (81820108027); the Fundamental Research Funds for the Central Universities (2019kfyXMBZ050); and the Angel Nutrition Research Fund.

Author information

Affiliations

  1. Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China

    Sijing Chen, Li Zhou, Qianqian Guo, Can Fang, Mengke Wang, Xiaobo Peng, Jiawei Yin, Shuzhen Li, Yalun Zhu, Wei Yang, Zhilei Shan & Liegang Liu

  2. Ministry of Education Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China

    Sijing Chen, Li Zhou, Qianqian Guo, Can Fang, Mengke Wang, Xiaobo Peng, Jiawei Yin, Shuzhen Li, Yalun Zhu, Wei Yang, Zhilei Shan & Liegang Liu

  3. The Hubei Provincial Key Laboratory of Yeast Function, Yichang, 443003, Hubei, China

    Yan Zhang

  4. Department of Nutrition and Food Hygiene, School of Public Health, Guangzhou Medical University, Guangzhou, 511436, China

    Xiaoyi Chen

Contributions

SC, LZ, ZS, XC, LL designed the study; QG, CF, MW, XP, SL, YZ acquired the data; SC, LZ, JY analyzed and interpreted the data; SC drafted the article; WY, YZ, ZS, XC, LL substantively revised it. All authors have approved the final version of the article.

Corresponding authors

Correspondence to Xiaoyi Chen or Liegang Liu.

Ethics declarations

Ethics approval and consent to participate

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Tongji Medical College.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1: Table S1

STROBE-nut: An extension of the STROBE statement for nutritional epidemiology.

Additional file 2: Table S2

The Strengthening the Reporting Observational studies in Epidemiology – Molecular Epidemiology (STROBE-ME) Reporting Recommendations: Extended from STROBE statement.

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Источник: https://nutritionj.biomedcentral.com/articles/10.1186/s12937-020-00625-w

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Источник: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568689/
Record InformationVersion2.0Creation Date2009-06-19 21:58:47 UTCUpdate Date2014-12-24 20:23:54 UTCAccession NumberT3D1445IdentificationCommon NameChromium(III) picolinateClassSmall MoleculeDescriptionChromium(III) picolinate is a chemical compound of chromium. It is found in nutritional supplements to prevent or treat chromium deficiency. Chromium is a chemical element which has the symbol Cr and atomic number 24. It is found naturally occuring in rocks, animals, plants, and soil, and is usually mined as chromite ore. Chromium is most toxic in its +6 oxidation state (chromium(VI)) due to its greater ability to enter cells and higher redox potential. Trivalent chromium (chromium(III)) however, is biologically necessary for sugar and lipid metabolism in humans. (6)Compound Type
  • Aromatic Hydrocarbon
  • Chromium Compound
  • Ester
  • Household Toxin
  • Organic Compound
  • Organometallic
  • Pollutant
  • Synthetic Compound
Chemical Structure
Thumb
Synonyms
Synonym
2-Pyridinecarboxylic acid, chromium salt
Chromium 2-pyridinecarboxylate
Chromium picolinate
Chromium tripicolinate
Chromium(III) picolinic acid
Chromium(III) trispicolinat
Picolinic acid, chromium salt
Tris(2-pyridinecarboxylato-N(1),O(2))chromium
Chemical FormulaC18H12CrN3O6Average Molecular Mass418.301 g/molMonoisotopic Mass418.013 g/molCAS Registry Number14639-25-9IUPAC Namebis(pyridine-2-carbonyloxy)chromio pyridine-2-carboxylateTraditional Namebis(pyridine-2-carbonyloxy)chromio pyridine-2-carboxylateSMILESO=C(O[Cr](OC(=O)C1=CC=CC=N1)OC(=O)C1=CC=CC=N1)C1=CC=CC=N1InChI IdentifierInChI=1S/3C6H5NO2.Cr/c3*8-6(9)5-3-1-2-4-7-5;/h3*1-4H,(H,8,9);/q;;;+3/p-3InChI KeyInChIKey=CBDQOLKNTOMMTL-UHFFFAOYSA-KChemical TaxonomyDescription belongs to the class of organic compounds known as pyridinecarboxylic acids. Pyridinecarboxylic acids are compounds containing a pyridine ring bearing a carboxylic acid group.KingdomOrganic compounds Super ClassOrganoheterocyclic compounds ClassPyridines and derivatives Sub ClassPyridinecarboxylic acids and derivatives Direct ParentPyridinecarboxylic acids Alternative ParentsSubstituents
  • Pyridine carboxylic acid
  • Heteroaromatic compound
  • Carboxylic acid salt
  • Carboxylic acid derivative
  • Carboxylic acid
  • Monocarboxylic acid or derivatives
  • Organic transition metal salt
  • Azacycle
  • Organic nitrogen compound
  • Hydrocarbon derivative
  • Organic salt
  • Organic zwitterion
  • Organic oxide
  • Organooxygen compound
  • Organonitrogen compound
  • Organic chromium salt
  • Organopnictogen compound
  • Organic oxygen compound
  • Aromatic heteromonocyclic compound
Molecular FrameworkNot AvailableExternal DescriptorsBiological PropertiesStatusDetected and Not QuantifiedOriginExogenousCellular LocationsBiofluid LocationsNot AvailableTissue LocationsNot AvailablePathwaysNot AvailableApplicationsNot AvailableBiological RolesNot AvailableChemical RolesNot AvailablePhysical PropertiesStateSolidAppearanceWhite powder.Experimental Properties
PropertyValue
Melting PointNot Available
Boiling PointNot Available
SolubilityNot Available
LogPNot Available
Predicted PropertiesSpectraSpectra
Spectrum TypeDescriptionSplash KeyView
Predicted LC-MS/MSPredicted LC-MS/MS Spectrum - 10V, Positivesplash10-014i-0200900000-e6ad4bf13bc058964867JSpectraViewer
Predicted LC-MS/MSPredicted LC-MS/MS Spectrum - 20V, Positivesplash10-0aor-0940600000-a19373e40b1728b369e5JSpectraViewer
Predicted LC-MS/MSPredicted LC-MS/MS Spectrum - 40V, Positivesplash10-0pb9-9800000000-a806f52e639d9e4303ecJSpectraViewer
Predicted LC-MS/MSPredicted LC-MS/MS Spectrum - 10V, Negativesplash10-014i-0001900000-5f269f42947a703295b1JSpectraViewer
Predicted LC-MS/MSPredicted LC-MS/MS Spectrum - 20V, Negativesplash10-01b9-3602900000-5dfd974eae5e34f080d5JSpectraViewer
Predicted LC-MS/MSPredicted LC-MS/MS Spectrum - 40V, Negativesplash10-0fb9-9100000000-18b04917f863d1b32717JSpectraViewer
Toxicity ProfileRoute of ExposureOral (5) ; inhalation (5) ; dermal (5)Mechanism of ToxicityTrivalent chromium may also form complexes with peptides, proteins, and DNA, resulting in DNA-protein crosslinks, DNA strand breaks, DNA-DNA interstrand crosslinks, chromium-DNA adducts, chromosomal aberrations and alterations in cellular signaling pathways. It has been shown to induce carcinogenesis by overstimulating cellular regulatory pathways and increasing peroxide levels by activating certain mitogen-activated protein kinases. It can also cause transcriptional repression by cross-linking histone deacetylase 1-DNA methyltransferase 1 complexes to CYP1A1 promoter chromatin, inhibiting histone modification. Chromium may increase its own toxicity by modifying metal regulatory transcription factor 1, causing the inhibition of zinc-induced metallothionein transcription. (1, 5, 2, 3, 4)MetabolismChromium is absorbed from oral, inhalation, or dermal exposure and distributes to nearly all tissues, with the highest concentrations found in kidney and liver. Bone is also a major storage site and may contribute to long-term retention. Hexavalent chromium's similarity to sulfate and chromate allow it to be transported into cells via sulfate transport mechanisms. Inside the cell, hexavalent chromium is reduced first to pentavalent chromium, then to trivalent chromium by many substances including ascorbate, glutathione, and nicotinamide adenine dinucleotide. Chromium is almost entirely excreted with the urine. (1, 5)Toxicity ValuesNot AvailableLethal DoseNot AvailableCarcinogenicity (IARC Classification)3, not classifiable as to its carcinogenicity to humans. (7)Uses/SourcesChromium(III) picolinate is found in nutritional supplements to prevent or treat chromium deficiency.Minimum Risk LevelNot AvailableHealth EffectsChromium in its trivalent state is not very toxic. It may be oxidized to hexavalent chromium, a known carcinogen. Hexavalent chromium has also been shown to affect reproduction and development. (1)SymptomsChromium in its trivalent state is not very toxic, but it may be oxidized to hexavalent chromium. Breathing hexavalent chromium can cause irritation to the lining of the nose, nose ulcers, runny nose, and breathing problems, such as asthma, cough, shortness of breath, or wheezing. Ingestion of hexavalent chromium causes irritation and ulcers in the stomach and small intestine, as well as anemia. Skin contact can cause skin ulcers. (5)TreatmentThere is no know antidote for chromium poisoning. Exposure is usually handled with symptomatic treatment. (5) Normal ConcentrationsNot AvailableAbnormal ConcentrationsNot AvailableExternal LinksDrugBank IDNot AvailableHMDB IDNot AvailablePubChem Compound ID151932 ChEMBL IDNot AvailableChemSpider ID133913 KEGG IDNot AvailableUniProt IDNot AvailableOMIM IDChEBI ID50369 BioCyc IDNot AvailableCTD IDC079689 Stitch IDChromium(III) picolinate PDB IDNot AvailableACToR IDNot AvailableWikipedia LinkNot AvailableReferencesSynthesis ReferenceNot AvailableMSDST3D1445.pdfGeneral References
  1. Salnikow K, Zhitkovich A: Genetic and epigenetic mechanisms in metal carcinogenesis and cocarcinogenesis: nickel, arsenic, and chromium. Chem Res Toxicol. 2008 Jan;21(1):28-44. Epub 2007 Oct 30. [17970581 ]
  2. Kim G, Yurkow EJ: Chromium induces a persistent activation of mitogen-activated protein kinases by a redox-sensitive mechanism in H4 rat hepatoma cells. Cancer Res. 1996 May 1;56(9):2045-51. [8616849 ]
  3. Schnekenburger M, Talaska G, Puga A: Chromium cross-links histone deacetylase 1-DNA methyltransferase 1 complexes to chromatin, inhibiting histone-remodeling marks critical for transcriptional activation. Mol Cell Biol. 2007 Oct;27(20):7089-101. Epub 2007 Aug 6. [17682057 ]
  4. Kimura T: [Molecular mechanism involved in chromium(VI) toxicity]. Yakugaku Zasshi. 2007 Dec;127(12):1957-65. [18057785 ]
  5. ATSDR - Agency for Toxic Substances and Disease Registry (2008). Toxicological profile for chromium. U.S. Public Health Service in collaboration with U.S. Environmental Protection Agency (EPA). [Link]
  6. Wikipedia. Chromium. Last Updated 5 March 2009. [Link]
  7. International Agency for Research on Cancer (2014). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. [Link]
Gene RegulationUp-Regulated GenesNot AvailableDown-Regulated GenesNot Available
Источник: http://www.t3db.ca/toxins/T3D1445

Chromium

Overview

Chromium is a mineral. It is called an "essential trace element" because very small amounts of chromium are necessary for human health. There are two forms of chromium: trivalent chromium and hexavalent chromium. The first is found in foods and supplements and is safe for humans. The second is a known toxin that can cause skin problems and lung cancer.

Chromium is used for chromium deficiency. It is also used for diabetes, high cholesterol, a hormonal disorder that causes enlarged ovaries with cysts (polycystic ovary syndrome or PCOS), and many other conditions, but there is no good scientific evidence to support most of these uses.

Classification

Is a Form of:

Mineral

Primary Functions:

Chromium deficiency

Also Known As:

Acétate de Chrome, Atomic Number 24, Chlorure Chromique

How Does It Work?

Chromium might help keep blood sugar levels normal by improving the way our bodies use insulin.

Uses

  • Chromium deficiency.Taking chromium by mouth is effective for preventing chromium deficiency.
  • Diabetes. Taking chromium picolinate may lower fasting blood sugar and insulin levels in some people with type 2 diabetes. Chromium picolinate might also decrease weight gain in people taking a class of antidiabetes medications called sulfonylureas. Higher chromium doses might work better and faster than lower doses. Higher doses might also lower the level of certain blood fats (cholesterol and triglycerides) in some people with diabetes. Early research shows that chromium picolinate might have the same benefits in people with type 1 diabetes, people who have diabetes as a result of steroid treatment, and people with diabetes that develops during pregnancy. But chromium might not help everyone. Some researchers think that chromium supplements only benefit people with low chromium levels or malnutrition. Most people with diabetes don't have low chromium levels. Chromium might also help prevent diabetes. But research is limited.
  • High levels of cholesterol or other fats (lipids) in the blood (hyperlipidemia). Some research shows that taking 15-200 mcg of chromium daily for 6-12 weeks lowers low-density lipoprotein (LDL or "bad") cholesterol and total cholesterol levels in people with slightly high or high cholesterol levels. Other research suggests that taking chromium for 7-16 months lowers triglycerides and LDL and increases high-density lipoprotein (HDL or "good") cholesterol. Also, taking chromium alone or along with other supplements seems to reduce levels of blood fats in people with high blood fat levels. However, there is some evidence that taking chromium daily for 10 weeks does not improve cholesterol levels in postmenopausal women.

Recommended Dosing

The following doses have been studied in scientific research:

ADULTS
BY MOUTH:

  • General: The safe and tolerable upper intake levels of chromium are not known. However, daily adequate intake (AI) levels for chromium have been established: men 14 to 50 years, 35 mcg; men 51 and older, 30 mcg; women 19 to 50 years, 25 mcg; women 51 and older, 20 mcg; pregnant women 14 to 18 years, 29 mcg; 19 to 50 years, 30 mcg; breast-feeding women 14 to 18 years, 44 mcg; 19 to 50 years, 45 mcg.
  • For diabetes: In people with type 2 diabetes, 200-1000 mcg of chromium taken daily in single or divided doses has been used. Also, a specific combination product providing chromium 600 mcg plus biotin 2 mg daily (Diachrome by Nutrition 21) has been used for up to 3 months. In addition, 1000 mcg of chromium (as chromium yeast) together with 1000 mg of vitamin C and 800 IU of vitamin E daily for 6 months has been used. In people with gestational diabetes, 4-8 mcg/kg of chromium picolinate daily for 8 weeks has been used. In people with high blood sugar due to use of corticosteroid medication, 400 mcg of chromium once daily or 200 mcg three times daily has been used.
  • For high levels of cholesterol or other fats (lipids) in the blood (hyperlipidemia): 50-250 mcg of chromium as chromium chloride or chromium picolinate, or brewer's yeast containing 15-48 mcg of chromium, has been used 5-7 days weekly for up to 16 months. 200 mcg of chromium polynicotinate along with 100 mg of grape seed extract, taken twice daily for 2 months, has been used. One to two capsules of a specific supplement (Colenon) containing 240 mg of chitosan, 55 mg of Garcinia cambogia extract, and 19 mg of chromium taken daily for 4 weeks has been used.

CHILDREN
BY MOUTH:

  • General: The safe and tolerable upper intake levels of chromium in children are not known. However, daily adequate intake (AI) levels for chromium have been established: Infants 0 to 6 months, 0.2 mcg; 7 to 12 months, 5.5 mcg; children 1 to 3 years, 11 mcg; 4 to 8 years, 15 mcg; boys 9 to 13 years, 25 mcg; boys 14-18 years. 35 mcg; girls 9 to 13 years, 21 mcg; 14 to 18 years, 24 mcg.
  • For high levels of cholesterol or other fats (lipids) in the blood (hyperlipidemia): 400-600 mcg of chromium polynicotinate and 1000-1500 mg of glucomannan has been used twice daily for 8 weeks.

Chromium Supplements Frequently Asked Questions

What does chromium do to your body?

Chromium is a mineral that humans require in trace amounts. Chromium is known to enhance the action of insulin and also appears to be directly involved in carbohydrate, fat, and protein metabolism. Chromium stores in the body may be reduced under several conditions.

Are chromium supplements safe?

Cautions about supplements

Chromium deficiency is rare, and studies have not yet confirmed the benefits of taking supplements, so it is best to obtain chromium through food. However, large doses of chromium in supplement form can cause stomach problems, low blood sugar, and kidney or liver damage.

Does chromium help with weight loss?

So there are claims that chromium supplements can lower your appetite, help you burn more calories, cut your body fat, and boost your muscle mass. But a review of 24 studies that checked the effects of 200-1,000 micrograms of chromium a day found that there aren't any significant benefits.

Does chromium have side effects?

Chromium has been used safely in a small number of studies using doses of 200-1000 mcg daily for up to 2 years. Some people experience side effects such as skin irritation, headaches, dizziness, nausea, mood changes, impaired thinking, judgment, and coordination.

Does chromium burn belly fat?

Chromium is a mineral that enhances insulin, a hormone that's important for turning food into energy. Your body also needs it to store carbohydrates, fats, and proteins. So there are claims that chromium supplements can lower your appetite, help you burn more calories, cut your body fat, and boost your muscle mass.

What are the benefits of cinnamon and chromium?

Four-month treatment with a dietary supplement containing cinnamon, chromium and carnosine decreased FPG and increased fat-free mass in overweight or obese pre-diabetic subjects. These beneficial effects might open up new avenues in the prevention of diabetes.

Can chromium cause depression?

For example, one study showed that chromium may affect symptoms such as increased appetite and eating, carbohydrate cravings, and diurnal mood variation, a type of depression in which symptoms are worse in the morning but improve as the day goes on.

Where is chromium found in food?

Some of the best sources of chromium are broccoli, liver and brewer's yeast. Potatoes, whole grains, seafood, and meats also contain chromium.

Should I take chromium?

When taken by mouth: Chromium is LIKELY SAFE for most adults in medicinal amounts, short-term. Up to 1000 mcg per day of chromium has been used safely for up to 6 months. When taken by mouth in these doses for longer periods of time, chromium is POSSIBLY SAFE for most adults.

Does chromium make you poop?

Benefits and risks of chromium supplements

Chromium picolinate is a popular supplement often marketed to those wanting to build muscle or lose weight. Some of those taking the supplement also experienced side effects, including watery stool, vertigo, headaches, and hives.

What is the best chromium to take for weight loss?

Doses of up to 1,000 μg/day of chromium picolinate were used in these studies. Overall, this research found that chromium picolinate produced very small amounts of weight loss (2.4 pounds or 1.1 kg) after 12 to 16 weeks in overweight or obese adults.

Is chromium bad for your liver?

There have been no reported cases of chromium poisoning due to food intake, so the IOM has not fixed a maximum intake level. However, large doses of chromium in supplement form can cause stomach problems, low blood sugar, and kidney or liver damage.

Does chromium help blood sugar?

Chromium picolinate, specifically, has been shown to reduce insulin resistance and to help reduce the risk of cardiovascular disease and type 2 diabetes. Dietary chromium is poorly absorbed. Supplements containing 200-1,000 mcg chromium as chromium picolinate a day have been found to improve blood glucose control.

What's the difference between chromium and chromium picolinate?

However, chromium picolinate is an alternate form of chromium that is absorbed better. For this reason, this type is commonly found in dietary supplements. Chromium picolinate is the mineral chromium attached to three molecules of picolinic acid.

What are the signs of chromium deficiency?

Signs and symptoms

The claimed symptoms of chromium deficiency caused by long-term total parenteral nutrition are severely impaired glucose tolerance, weight loss, peripheral neuropathy and confusion.

Does chromium cause anxiety?

Behavioral or psychiatric conditions such as depression, anxiety, or schizophrenia: Chromium might affect brain chemistry and might make behavioral or psychiatric conditions worse. If you have one of these conditions, be careful when using chromium supplements. Pay attention to any changes in how you feel.

Which form of chromium is toxic?

Trivalent chromium, or chromium(III), is the form of chromium that is essential to human health. Hexavalent chromium, or chromium(VI), is an unequivocally toxic form.

How much chromium should I take?

In the United States, the recommended dietary reference intake (DRI) of chromium is 35 μg/day for adult men and 25 μg/day for adult women (20). After the age of 50, the recommended intake decreases slightly to 30 μg/day for men and 20 μg/day for women.

Do eggs have chromium?

Chromium is commonly found in egg yolk, whole grains, high-bran cereals, green beans, broccoli, nuts, and brewer's yeast.

What is chromium in food?

Processed meats, whole-grain products, high-bran cereals, green beans, broccoli, nuts, and egg yolk are good sources of chromium. Foods high in simple sugars, such as sucrose and fructose, are usually low in chromium and may actually promote chromium excretion (4).

How do you get chromium in your diet?

Foods that are good sources of chromium include:

  • Vegetables such as broccoli, potatoes, and green beans.
  • Whole-grain products.
  • Beef and poultry.
  • Fruits, including apples and bananas; grape juice.
  • Milk and dairy products.

Clinical Studies

  • ^ ab Parsons A, et al. A proof of concept randomised placebo controlled factorial trial to examine the efficacy of St John's wort for smoking cessation and chromium to prevent weight gain on smoking cessation. Drug Alcohol Depend. (2009)
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  • ^ abHexavalent Chromium.
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  • ^ Jain SK, et al. Effect of chromium dinicocysteinate supplementation on circulating levels of insulin, TNF-α, oxidative stress, and insulin resistance in type 2 diabetic subjects: randomized, double-blind, placebo-controlled study. Mol Nutr Food Res. (2012)
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  • ^ abc Davis CM, Vincent JB. Chromium oligopeptide activates insulin receptor tyrosine kinase activity. Biochemistry. (1997)
  • ^ ab Yamamoto A, Wada O, Manabe S. Evidence that chromium is an essential factor for biological activity of low-molecular-weight, chromium-binding substance. Biochem Biophys Res Commun. (1989)
  • ^ ab Myers MG Jr, White MF. The new elements of insulin signaling. Insulin receptor substrate-1 and proteins with SH2 domains. Diabetes. (1993)
  • ^ Vincent JB. Recent advances in the nutritional biochemistry of trivalent chromium. Proc Nutr Soc. (2004)
  • ^ Vincent JB. Chromium: celebrating 50 years as an essential element. Dalton Trans. (2010)
  • ^ Rutter GA, Da Silva Xavier G, Leclerc I. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem J. (2003)
  • ^ Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev. (2011)
  • ^ Zhao P, et al. A newly synthetic chromium complex-chromium (D-phenylalanine)3 activates AMP-activated protein kinase and stimulates glucose transport. Biochem Pharmacol. (2009)
  • ^ ab Anderson RA, Kozlovsky AS. Chromium intake, absorption and excretion of subjects consuming self-selected diets. Am J Clin Nutr. (1985)
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