Biol Trace Elem Res (2017) 180:246–254 DOI 10.1007/s12011-017-1014-2

Hypoglycemic and Hypolipidemic Effects of Leucine, Zinc, and Chromium, Alone and in Combination, in Rats with Type 2 Diabetes Hassan Sadri 1 & Negar Nowroozi Larki 2,3 & Saeed Kolahian 2,3

Received: 31 January 2017 / Accepted: 4 April 2017 / Published online: 13 April 2017 # Springer Science+Business Media New York 2017

Abstract For the increasing development of diabetes, dietary habits and using appropriate supplements can play important roles in the treatment or reduction of risk for this disease. The objective of this study was to investigate the effects of leucine (Leu), zinc (Zn), and chromium (Cr) supplementation, alone or in combination, in rats with type 2 diabetes (T2D). Seventyseven adult male Wistar rats were randomly assigned in 11 groups, using nutritional supplements and insulin (INS) or glibenclamide (GLC). Supplementing Leu significantly reduced blood glucose, triglycerides (TG), nonesterified fatty acids (NEFA), low-density lipoprotein (LDL), and increased high-density lipoprotein (HDL) concentrations compared to vehicle-treated T2D animals, and those improvements were associated with reduced area under the 2-h blood glucose response curve (AUC). Supplementation of T2D animals with Zn improved serum lipid profile as well as blood glucose concentrations but was not comparable with the INS, GLC, and Leu groups. Supplementary Cr did not improve blood glucose and AUC in T2D rats, whereas it reduced serum TG and LDL and increased HDL concentrations. In conclusion, supplementation of diabetic rats with Leu was more effective in improving blood glucose and consequently decreasing glucose AUC than other nutritional supplements. Supplementary

* Hassan Sadri [email protected]

1

Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz 516616471, Iran

2

Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology and ICePhA, University of Tuebingen, 72074 Tuebingen, Germany

3

Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz 516616471, Iran

Zn and Cr only improved serum lipid profile. The combination of the nutritional supplements did not improve blood glucose level. Nevertheless, supplementation with Leu-Zn, Leu-Cr, Zn-Cr, and Leu-Zn-Cr led to an improved response in serum lipid profile over each supplement given alone. Keywords Leucine . Zinc . Chromium . Type 2 diabetes

Introduction Type 2 diabetes mellitus, characterized by hyperglycemia, is one of the most common metabolic disorders with prevalence of 8.3% of adults in the world [1]. Decreases in insulin secretion, defects in glucose uptake in peripheral tissues including skeletal muscle and adipose tissue, and increase in glucose production in the liver, primarily due to insulin resistance, are the main characters of diabetes mellitus [2]. Current antidiabetic medications such as metformin and sulphonylureas are associated with many side effects and may be ineffective in long-term therapy due to declining function of the pancreatic β-cell in diabetic patients [3–6]. Nowadays, many clinical and nutritional studies have focused on lifestyle modifications, in particular, diet pattern that could potentially reduce the risk of developing metabolic disorders including type 2 diabetes [7]. Consequently, there is an increasing interest in the role of nutritional supplements with potential hypoglycemic and hypolipidemic activities in the treatment or reduction of risk for diabetes. Leucine (Leu), an essential branchedchain amino acid (BCAA), is not only as a building block for protein synthesis but also is a well-known potent stimulus of insulin secretion through serving as metabolic fuel as well as allosteric activator of glutamate dehydrogenase (a key enzyme controlling the oxidation of glutamate) in the pancreatic β-cell [8, 9]. Zinc (Zn), the second most abundant trace

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element in the body, is an important component for insulin biosynthesis through stabilizing insulin hexamers and their pancreatic storage [10–12]. Zinc also appears to have an insulinaemic effects on target tissues, probably through increasing the phosphorylation of the insulin receptor substrate-1 [13] and of the protein kinase B, a key enzyme in insulin signaling pathway [14]. On the other hand, chromium (Cr), biologically active as a component of the oligopeptide low-molecular-weight-chromium binding peptide (also known as chromoduline), is thought to improve insulin sensitivity by binding to the insulin receptor on target cells and potentiating the insulin response through stimulating the tyrosine kinase activity of the insulin-activated insulin receptor [15, 16]. The beneficial effects of dietary Leu [17–20], Zn [11, 21, 22], and Cr [23–25] to modulate glucose metabolism and insulin sensitivity were tested in some laboratory animal and human studies. However, to our knowledge, there is no study performed to address the effects of a combination of Leu, Zn, and Cr on glucose metabolism. We hypothesized that a combination of Leu, Zn, and Cr supplements, with potential hypoglycemic and hypolipidemic effects will improve blood glucose control and lipid profile over each supplement given alone. Therefore, the objective of the current study was to characterize the effects of Leu, Zn, and Cr supplementation, alone or in combination, on glycemic status and lipid profile in rats with type 2 diabetes.

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treated with combination of Zn and Cr (Zn-Cr); and (11) T2D treated with combination of Leu, Zn, and Cr (Leu-Zn-Cr). All animal protocols of the experiment were performed in accordance with the guidelines for the Care and Use of Laboratory Animals as adopted by the Ethics Committee of the Faculty of Veterinary Medicine of University of Tabriz, Iran (Permit Number: A-12-30511). Type 2 Diabetes Induction The rats were allocated into two dietary regimens of either normal chow diet in nondiabetic control (CTR) group consisting (as a percentage of total kcal) of 12% fat, 60% carbohydrate, and 28% protein (Javaneh Khorasan Co., Mashhad, Iran) or high-fat diet (HFD) in diabetic groups consisting of 40% fat, 42% carbohydrate, and 18% protein (Javaneh Khorasan Co., Mashhad, Iran) for a period of 2 weeks. Subsequently, animals were fasted overnight, and the rats on the HFD received a low-dose intraperitoneal injection of streptozotocin [(STZ); 35 mg/kg; Sigma-Aldrich, Inc., St. Louis, Mo., USA] [26]. After administration of STZ, the animals had free access to food and water. Animals in both the STZ-injected and CTR animals continued to receive their original diets throughout the period of study. The normal chow diet contained cornstarch, sucrose, casein, soybean oil, mineral mix, vitamin mix, DL-methionine, and choline chloride. This diet contained Zn and Cr at 22.8 and 0.48 mg/kg, respectively. Diabetes Confirmation

Materials and Methods Animals and Experimental Groups Seventy-seven adult male Wistar rats (8 weeks old), weighting 150–200 g, were used in this study. The rats in each group were housed separately in single cages in a temperaturecontrolled room (22 ± 2 °C) with a 12:12-h light:dark cycle. They were fed ad libitum with a commercially available food pellet diet (normal diet) and had free access to water during the acclimation period (10 days). The blood glucose concentrations were analyzed in all animals in samples taken from the tail veins using a blood glucose monitor (Glucose monitor, Canada). All animals showed blood glucose concentrations of 80 ± 5 mg/dL. Afterwards, the rats were randomly categorized into the following 11 groups, each including seven animals: (1) nondiabetic (as a negative control; CTR); (2) nontreated diabetic (as a positive control; T2D); (3) T2D treated with neutral protamine Hagedorn (NPH) insulin (INS); (4) T2D treated with glibenclamide (GLC); (5) T2D treated with Leu; 6) T2D treated with Zn; (7) T2D treated with Cr; (8) T2D treated with combination of Leu and Zn (Leu-Zn); (9) T2D treated with combination of Leu and Cr (Leu-Cr); (10) T2D

Three days after the STZ injection and an overnight fast, the presence of diabetes was verified by blood glucose concentrations above 250 mg/dL, as determined in samples taken from the tail veins by using a blood glucose monitor (Glucose monitor®, Canada). Treatment with Nutritional Supplements and Sampling Treatment with nutritional supplements was started from the next day after confirmation of the induction of diabetes and continued up to 4 weeks. Leu (AllMax, USA) 15 g/L [8], Zn (Zinc sulfate, Alhavi, Iran) 10 mg/L [27], or Cr (Picolinate chromium, Swanson, USA) 5 mg/L [28] were added to the drinking water of the diabetic rats. In addition, a subset of the diabetic rats received NPH insulin (an intermediate-acting insulin; Isophane Lansulin®, Exir, Iran) in a dose of 2 U/day subcutaneously or glibenclamide (glyburide®, Iran Najo, Iran) at 20 mg/L throughout the treatment period. Daily food intake was recorded throughout the trial, and the average daily food consumption per rat for each group was subsequently determined. At the end of each week, fasting blood glucose concentrations were determined using a blood glucose

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monitor (Glucose monitor®, Canada), and individual body weights (BW) were recorded. The animals in all of the groups were killed by exsanguination at the end of experimental protocol after an overnight fast (12 h), and whole blood was collected by cardiac puncture, and serum was obtained after centrifugation at 1000×g for 10 min. Different tissue samples were collected as part of another project and stored frozen for future analysis. Blood glucose concentrations were measured using a blood glucose monitor (Glucose monitor®, Canada). Blood serum was analyzed for the concentrations of triglycerides (TG), nonesterified fatty acids (NEFA), low-density lipoprotein (LDL), highdensity lipoprotein (HDL), and cholesterol by a certified laboratory for clinical studies (Veterinary Diagnostic Lab, DPACo., Tabriz, Iran). The serum concentrations of insulin were determined using a standard commercial ELISA kit (BioMark Technologies Inc., Canada).

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Results Body Weight, Feed Intake, and Blood Glucose Body weight, feed intake, and blood glucose concentrations (recorded during the treatment period) of the experimental groups are presented in Table 1. A tendency was observed for a greater BW gain (p = 0.08) and consequently a greater final BW (p = 0.07) in the GLC group compared with the T2D group; there was no difference among the other groups. Feed intake was comparable among the groups, except that the CTR group had a greater (p < 0.0001) feed intake than the other rats. As expected, blood glucose concentrations were lower in the CTR as compared to the other groups (p < 0.0001). Blood glucose concentrations in the INS, GLC, Leu, and Leu-Zn animals were significantly lower as compared with the T2D animals (p < 0.01). Interestingly, the improvement in the blood glucose concentrations of the animals treated with Leu was comparable with those of INS and GLC.

Oral Glucose Tolerance Test Oral glucose tolerance test (OGTT) was performed in the week 4 of the experiment after an overnight fast (12 h). Rats were orally (by gavage) dosed with a 20% glucose solution (2 g/kg BW), and blood glucose concentrations were subsequently determined at 0 (just prior to oral glucose dosing), 30, 60, and 120 min after administration of glucose, in samples taken from the tail veins by using a blood glucose monitor (Glucose monitor®, Canada). The area under curve (AUC) was calculated from concentration-time curves by using linear trapezoidal method. The AUC of the curves of each experimental group were compared and tested for significance to the nontreated diabetic group (T2D), to represent glucose uptake by the tissues. Animals were not anesthetized for this procedure.

Statistical Analysis Statistical analysis of the data was performed using the MIXED procedure of SAS software (version 9.3; SAS Institute Inc., Cary, NC). The model included treatment as the fixed effects and rat as the random effect. Initial BW was included in the statistical model as a covariate for analysis of covariance applied to their corresponding measurements during the experimental period. The Tukey-Kramer adjustment was used for correction of the multiple comparisons. The data were presented as the means ± SEM. The results are presented in Table 1 as the least squares means and standard errors of the means. The threshold of significance was set at p < 0.05; trends were declared at 0.05 < p < 0.10.

Blood Insulin and Lipid Profile The serum insulin concentrations were highest in the INS, GLC, Zn, Cr, and Zn-Cr, followed by the T2D, Leu, and Leu-Zn-Cr and lowest in the CTR (p < 0.001; Fig. 1). The serum concentrations of TG in the treated groups were higher than that in the CTR group (p < 0.001), except in the case of Leu-Zn, Leu-Cr, and Leu-Zn-Cr that were comparable with that of CTR group (Fig. 2). Treatment with the nutritional supplement, but not with insulin and glibenclamide, led to a decrease in the serum TG concentrations in the diabetic animals (Fig. 2). The serum concentrations of NEFA showed almost similar changes as those of the serum TG among the groups (p < 0.001; Fig. 3). The serum concentrations of LDL in the Leu-Zn, Leu-Cr, Zn-Cr, and Leu-Zn-Cr animals were comparable with those of the CTR animals (Fig. 4). Treatment with insulin and glibenclamide of diabetic rats did not improve the serum LDL, whereas treatment with the nutritional supplements did (p < 0.05; Fig. 4). The serum concentrations of HDL in the experimental groups inversely changed as compared with those of the serum LDL (p < 0.001; Fig. 5). Treatment with the nutritional supplements, except with Cr, led to an increase in the serum HDL concentrations of the diabetic animals. Interestingly, supplementation of diabetic rats with combination of Leu, Cr, and Zn was associated with higher serum HDL concentrations than CTR animals (Fig. 5). The serum concentrations of cholesterol in the experimental groups showed almost the same changes as the serum LDL (p < 0.001; Fig. 6).

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Table 1 Body weight (BW), feed intake, and blood glucose (during supplementation period) of the experimental groups. Data are least squares means and standard error of the mean Treatments Item

CTR

T2D

INS

GLC

Leu

Zn

Cr

Leu-Zn Leu-Cr

Zn-Cr

Leu-Zn-Cr SEM P value

Initial BW (g)

143.6 140.8

162.9

169.1

163.6

154.3

130.4

151.6

141.1

137.4

168.1

6.27

–

BW gain (g)

75.8

75.3

90.3

73.7

75.0

66.1

81.3

73.1

67.6

85.2

6.98

0.08

7.52 1.03 30.7

0.07 <.0001 <.0001

52.4

Final BW (g) 224.9 201.8 228.1 244.0 226.3 226.1 213.5 232.0 222.2 216.1 238.7 11.3b 12.4b 10.9b 11.4b 11.1b Feed intake (g/d) 21.5a 12.3b 11.8b 13.4b 11.5b 12.1b Blood glucose (mg/dL) 99.2e 359.8a 190.7d 184.7d 178.1d 261.5bc 291.0abc 240.3cd 322.7ab 308.6abc 311.4abc CTR control, T2D type 2 diabetes, INS NPH insulin, GLC glibenclamide, Leu leucine, Zn zinc, Cr chromium a–e

Significant differences (p < 0.05) among treatments are indicated by different letters

OGTT and AUC

Discussion

The blood concentrations of glucose in the CTR, T2D, and diabetic rats treated with the nutritional supplements demonstrated significant changes after the oral glucose loading (p < 0.001; Fig. 7a, b). The animals in the T2D had a significant increase in the blood glucose concentrations throughout the total measurement period (120 min) as compared to the CTR animals (Fig. 2); additionally, it did not return to the initial value (0 min level) even at the end of the period tested. The AUC did not differ between T2D and diabetic rats treated with Zn, Cr, Leu-Cr, and Zn-Cr. However, treatments of diabetic rats with Leu and Leu-Zn resulted in a significant reduction in the AUC compared to the T2D group. Furthermore, Leu and Leu-Zn were as effective as insulin and glibenclamide in reducing glycaemia, leading to comparable outcomes (Fig. 7a, b).

The induction of type 2 diabetes used in our study would resemble the natural history and the metabolic characteristics of the human syndrome. Feeding rats with HFD for 2 weeks tend to induce insulin resistance in the absence of hyperglycemia, mimicking the human situation of prediabetes, including hyperinsulinemia [26, 29]. Following 2 weeks of HFD, treatment with STZ leads to a transition from an insulinresistant state to a state of type 2 diabetes [26, 29]. In this regard, the dose of STZ has indeed a significant impact on the phenotype of HFD-fed rats, and previous studies agree on the HFD and low-dose STZ rat as a suitable model of type 2 diabetes and for testing of anti-diabetic compounds [26, 29–32]. In the present study, drinking water intake was not recorded. Therefore, one limitation of our study is the absence of water intake data and subsequently actual intakes of the supplements. The absence of these data makes it difficult to determine the dose response relationship.

Fig. 1 Blood insulin concentrations in the experimental groups. Different superscript letters (a–e) indicate significant differences among the groups (p < 0.05). CTR control, T2D type 2 diabetes, INS NPH insulin, GLC glibenclamide, Leu leucine, Zn zinc, Cr chromium. Data for insulin were recently published by Kolahian et al. (49)

Fig. 2 Blood triglycerides concentrations in the experimental groups. Different superscript letters (a–e) indicate significant differences among the groups (p < 0.05). CTR control, T2D type 2 diabetes, INS NPH insulin, GLC glibenclamide, Leu leucine, Zn zinc, Cr chromium

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Fig. 3 Blood nonesterified fatty acid concentrations in the experimental groups. Different superscript letters (a–d) indicate significant differences among the groups (p < 0.05). CTR control, T2D type 2 diabetes, INS NPH insulin, GLC glibenclamide, Leu leucine, Zn zinc, Cr chromium

Fig. 5 Blood high-density lipoprotein concentrations in the experimental groups. Different superscript letters (a–d) indicate significant differences among the groups (p < 0.05). CTR control, T2D type 2 diabetes, INS NPH insulin, GLC glibenclamide, Leu leucine, Zn zinc, Cr chromium

In the current study, we sought to determine how Leu supplementation, alone or in combination with Zn and Cr, could impact on glycemic status and lipid profile in rats with type 2 diabetes. Leucine supplementation significantly reduced blood glucose concentrations compared to the nontreated diabetic animals (T2D) throughout the study period, even though Leu had no effect on BW or BW gain. The improvement in glycemic control was associated with a reduced area under the 2-h blood glucose response curve. Previous studies in human have also shown that supplying extra Leu through high protein diet can reduce glycaemia in patients with type 2 diabetes with no change in BW [33], but this was, at least in part, due to an improvement in insulin secretion [34]. Leucine is known to act as an insulin secretagogue, through its

mitochondrial oxidative decarboxylation as well as by allosterically activating glutamate dehydrogenase, a key enzyme controlling the oxidation of amino acid glutamate, in the pancreatic β-cell [8]. It has been proposed that stimulation of mitochondrial activity in the pancreatic β-cell depends on both the generation of acetyl-CoA and α-ketoglutarate [34], resulting in closing of ATP-sensitive K + channels. This leads to an increase in plasma membrane depolarization, influx of extracellular Ca2+, which then triggers exocytosis of insulin granules from pancreatic β-cells [8, 9]. In pancreatic β-cells, Leu also regulates β-cell proliferation, which is mediated through the mTOR signaling pathway [35]. However, in the current study, serum insulin concentrations in the Leu group were comparable with those of T2D group, reflecting that

Fig. 4 Blood low-density lipoprotein concentrations in the experimental groups. Different superscript letters (a–d) indicate significant differences among the groups (p < 0.05). CTR control, T2D type 2 diabetes, INS NPH insulin, GLC glibenclamide, Leu leucine, Zn zinc, Cr chromium

Fig. 6 Blood cholesterol concentrations in the experimental groups. Different superscript letters (a–d) indicate significant differences among the groups (p < 0.05). CTR control, T2D type 2 diabetes, INS NPH insulin, GLC glibenclamide, Leu leucine, Zn zinc, Cr chromium

Hypoglycemic and Hypolipidemic Effects

Fig. 7 Oral glucose tolerance test (a: upper panel) and are under the 2-h blood glucose response curve (b: lower panel) in the experimental groups. Glycaemia in initial (0) and in 30, 60, and 120 min after gavage of 2 g/kg body weight of glucose. Data are expressed in mean ± SEM. Different superscript letters (a–f) indicate significant differences among groups (p < 0.05). CTR control, T2D type 2 diabetes, INS NPH insulin, GLC glibenclamide, Leu leucine, Zn zinc, Cr chromium

there was no apparent insulin response to the Leu supplementation. Thus, the unchanged serum insulin concentrations, together with the lower blood glucose concentrations and improved glucose tolerance test are suggestive of an improvement of insulin sensitivity in this group. The beneficial effects of dietary Leu to improve insulin sensitivity and glucose tolerance were also documented in mice [18, 20]. Thus, metabolic benefits of Leu supplementation to modulate glucose metabolism are likely mediated via insulin-dependent and insulin-independent pathways; in the case of the latter, probably through modulating glucose transporter 4 (GLUT4) translocation to the sarcolemma [17, 19]. However, in contrast to the beneficial effects of Leu in the improvement of glucose homeostasis described here, others have proposed that

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elevation in the blood concentrations of BCAA including Leu, may contribute to the development of insulin resistance and type 2 diabetes [36–38]. However, it is more probable that increased plasma BCAA is due to reduced expression and activity of BCAA catabolic enzyme expression in obese and diabetic conditions [39–41], and thus, the elevated circulating levels of BCAA is likely a consequence rather than a cause of insulin resistance [42]. We have further shown that the metabolic benefits of Leu supplementation in T2D rats were associated with reduced serum TG, NEFA, and LDL, and increased serum HDL. Increasing Leu intake was also associated with a significant decrease in plasma total and LDL cholesterol levels in mice with ad libitum consumption of HFD [20]. The cholesterol-reducing effect of Leu might be mediated through mTOR and/or other potential mediators [20] that need further investigations to be fully understood. In the current study, Zn supplementation improved blood glucose concentrations in the animals treated with Zn as compared with the T2D animals but was not comparable with the INS, GLC, and Leu groups. The potential beneficial effects of Zn supplementation to improve the symptoms of diabetes have been investigated in both animal models and in diabetic patients. Zinc supplementation attenuated fasting hyperglycaemia and hyperinsulinaemia in obese ob/ob mice [43], improved polydipsia and high HDL cholesterol levels in STZ-induced diabetic rats [44], and ameliorated the diabetic phenotype in type 2 diabetes patients [45]. In addition, meta-analysis of studies examining the effects of Zn supplementation in human subjects has shown that Zn causes a significant reduction in blood glucose concentration [21, 22]. Zinc is highly concentrated in pancreatic β-cells and plays important roles in systemic glycemic control through its effects on insulin biosynthesis, processing, and storage as well as by inhibiting hepatic clearance of insulin, resulting in elevated circulating insulin levels [10–12]. Furthermore, Zn may support the actions of insulin on target tissues, probably through increasing the phosphorylation of the insulin receptor substrate-1 [13] and of the protein kinase B, a key enzyme in insulin signaling pathway [14]. In addition, in the present study, supplementation of diabetic rats with Zn was associated with improvement in serum lipid profile including TG, LDL, and HDL: insulin-like effect of Zn to increase rate of lipogenesis was already demonstrated in rat adipocytes treated with high Zn concentrations [46]. Zinc and Cu can exhibit important interactions and competitive inhibition in transport and bioavailability. The site of the interaction between Zn and Cu is thought to occur in the intestine because of competing for binding sites on an absorption-enhancing protein or a transporter [47, 48]. In addition, high amounts of Zn may result in competition with Cu for the Cu2+ binding site on CuZnsuperoxide dismutase, thus, causing Cu2+ to be unable to transfer electrons [49]. Therefore, the potential interaction between Zn and Cu should be taken into consideration, especially when these elements are used together.

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The role of Cr in glucose metabolism has been suggested several years ago by Schwarz and Mertz (1959) and may improve insulin sensitivity at the cellular level [50]. Chromium is biologically active as a component of the oligopeptide apo-low-molecular-weight-chromium binding peptide (also known as apo-chromoduline) which is part of an insulin-signaling pathway. The Fe-transport protein transferrin was shown as the molecular agent responsible for maintaining chromic ion levels in the circulation and for transporting Cr from mobile pools to target tissues in an insulin-responsive manner [51, 52]. Thus, at high concentrations of either Cr or Fe, such as hemochromatotic diabetic condition (an iron overload disorder), they may act as antagonist, probably due to shared binding site on transferring [53–55]. The holo-chromodulin, Cr-loaded form of apochromodulin, binds to the insulin receptor and potentiates the insulin response through stimulating the tyrosine kinase activity of the insulin-activated insulin receptor [15, 56]. This may lead to eightfold difference in insulin receptor activation as demonstrated in the experiments using rat adipocyte cells with equal serum insulin concentrations with or without Cr [15]. In the present study, supplementary Cr did not improve blood glucose, and area under the 2-h blood glucose response curve in T2D rats. However, Cr supplementation led to an improvement in serum lipid profile including TG, LDL, and HDL in the T2D rats. Krol and Krejpcio (2011) reported that supplementary Cr had no effect on fasting glucose concentrations, whereas it improved insulin sensitivity and reduced serum total and LDL cholesterol, and TG concentrations in HFD-fed and STZ-injected rats [57]. In studies examining HFD and STZ, reduced blood cholesterol and TG concentrations appear to be a consistent effect of Cr supplementation, whereas changes in fasting glucose and insulin levels and body mass are not consistent. This may reflect that the origin of the diabetes may influence potential beneficial effects of Cr supplementation [16]. Furthermore, studies regarding effects of Cr feeding on glucose metabolism and insulin resistance in human have yielded contrasting results [24, 25, 58, 59]. A recent improved meta-analysis of clinical studies since 2007 showed no significant effect of Cr supplements on fasting glucose [58]. In addition, in a review of published studies [59] and consequently the American Diabetes Association [60] concluded that there is no sufficient evidence supporting the use of supplemental Cr to improve glycemic control in patients suffering with type 2 diabetes. With regard to the improved insulin sensitivity in the peripheral tissues of the rat models of diabetes after Cr administration, the lack of effects in human studies may be due to the fact that humans receive a comparably smaller dose than the rodent models [16]. In the present study, the combination of different nutritional supplements did not show any significant positive effects on blood glucose and area under the 2-h blood glucose response curve. However, supplementation with Leu-Zn, Leu-

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Cr, Zn-Cr, and Leu-Zn-Cr elicited improved response in terms of serum lipid profile over each supplement given alone. The etiology and pathophysiology of the biological effects of diabetes mellitus, especially with respect to cell damage, are mainly attributed to oxidative stress and its related complications. Results reported about the oxidative status of the animals as part of this experiment [61] demonstrated a significant redox imbalance in the T2D animals compared to the CTR animals. In this experiment, the animals in the T2D exhibited higher serum myeloperoxidase, glutathione peroxidase, and superoxide dismutase activities compared to the CTR animals [61]. It is likely that the increased serum enzymatic antioxidant activities found in this experiment [61] occurred as a compensatory response for opposing the increased oxidative stress associated with induced diabetes. Interestingly, supplementation with Leu-Zn, Leu-Cr, Zn-Cr, and Leu-Zn-Cr resulted in a greater reduction in serum enzymatic antioxidant activities compared to each supplement alone, probably due to improved lipid profile and attenuated fat-mediated oxidative stress in these groups.

Conclusion In conclusion, supplementation with Leu was more effective in improving blood glucose, and consequently decreasing glucose AUC compared with other nutritional supplements. In addition, Leu supplementation reduced serum TG, NEFA, and LDL and increased serum HDL. Supplementation with Zn improved blood glucose concentrations though was not comparable with those of INS, GLC, and Leu. Supplementation of diabetic rats with Cr did not improve blood glucose control, but it was associated with improvement in serum lipid profile. Our data does not provide evidence that combination of Leu, Zn, and Cr has a beneficial effect on blood glucose and glucose AUC, whereas supplementation with Leu-Zn, Leu-Cr, Zn-Cr, and Leu-Zn-Cr led to an improvement in the serum lipid profile over each supplement given alone. Acknowledgments This study was financially supported by the Research Council of University of Tabriz. The authors also express their appreciation to A. Sadeghi for his help in conducting this experiment.

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