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Chromium picolinate attenuates hyperglycemia-induced oxidative stress in streptozotocin-induced diabetic rats
Journal of Trace Elements in Medicine and Biology 27 (2013) 117–121
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Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
Clinical studies
Chromium picolinate attenuates hyperglycemia-induced oxidative stress in streptozotocin-induced diabetic rats Bhuvaneshwari Sundaram, Aanchal Aggarwal, Rajat Sandhir ∗ Department of Biochemistry, Panjab University, Chandigarh 160014, India
a r t i c l e
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Article history: Received 19 June 2012 Accepted 6 September 2012 Keywords: Antioxidants Chromium picolinate Hyperglycemia Oxidative stress Liver
a b s t r a c t Chromium picolinate is advocated as an anti-diabetic agent for impaired glycemic control. It is a transition metal that exists in various oxidation states and may thereby act as a pro-oxidant. The present study has been designed to examine the effect of chromium picolinate supplementation on hyperglycemiainduced oxidative stress. Diabetes was induced in male Wistar rats by a single intraperitoneal injection of streptozotocin (50 mg/kg body weight) and chromium was administered orally as chromium picolinate (1 mg/kg body weight) daily for a period of four weeks after the induction of diabetes. As is characteristic of diabetic condition, hyperglycemia was associated with an increase in oxidative stress in liver in terms of increased lipid peroxidation and decreased glutathione levels. The activity of antioxidant enzymes like superoxide dismutase, catalase and glutathione reductase were significantly reduced in liver of diabetic animals. Levels of ␣-tocopherol and ascorbic acid were found to be considerably lower in plasma of diabetic rats. Chromium picolinate administration on the other hand was found to have beneficial effect in normalizing glucose levels, lipid peroxidation and antioxidant status. The results from the present study demonstrate potential of chromium picolinate to attenuate hyperglycemia-induced oxidative stress in experimental diabetes. © 2012 Elsevier GmbH. All rights reserved.
Introduction Diabetes mellitus is a chronic metabolic disorder of carbohydrate and lipid metabolism leading to various complications [1–4]. Oxidative stress as a consequence of imbalance between radicalgenerating and radical-scavenging system plays an important role in the pathogenesis of diabetic complications [5]. Diabetes is closely associated with unbalanced trace elements that stem from chronic uncontrolled hyperglycemia [6]. Out of all the trace elements studied, chromium appears to be the most promising element in the therapeutics of diabetes. Chromium plays beneficial role in diabetes by facilitating insulin signaling thereby improving insulin sensitivity [7]. Deficiency of chromium has been associated with impaired glucose tolerance, fasting hyperglycemia, glucosuria, increased body fat, dyslipidemia and impaired fertility [8]. We have previously observed that CrP exerts anti-diabetic effect through regulation of carbohydrate and lipid metabolism [9,10]. It is a transition element that exists in several oxidation states and may increase oxidative stress [11]. Cr (III) and Cr (VI) species been shown to act as a pro-oxidant by increasing ROS production that induces DNA damage [12]. Contrarily, Cr
∗ Corresponding author. Tel.: +91 172 2534131/38. E-mail address: [email protected] (R. Sandhir). 0946-672X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jtemb.2012.09.002
(III) has also been shown to improve cellular antioxidant capacity in both animal and cell culture models of diabetes [13–15]. Furthermore, organic complexes of Cr (III) have been found to be less toxic as compared to inorganic salts [16]. Chromium dinicocysteinate, an organic Cr (III) formulation has hypoglycemic effects and lowers oxidative stress [17]. Hence, it is important to monitor the antioxidant status in diabetic rats supplemented with Cr (III) to recognize the precise influence of chromium picolinate, an organic complex on antioxidant defence system in diabetes. Therefore, the present study was planned with the aim to evaluate the effect of chromium picolinate on oxidative stress induced by hyperglycemia in liver of diabetic rats. Materials and methods Materials Streptozotocin (STZ), 2,4,6-tripyridyl-S-triazine (TPTZ) was obtained from Sigma Chemical Company, St. Louis, MO, USA. 5,5 -Dithiobis-(2-nitrobenzoic acid) (DTNB), nitroblue tetrazolium (NBT), dinitrophenyl hydrazine (DNPH) were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Picolinic acid and chromium chloride were obtained from Himedia Laboratories Pvt. Ltd., Mumbai, India. All other chemicals used were of analytical grade.
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Synthesis of chromium picolinate
Glutathione (GSH) levels
CrP was synthesized according to the method described by Stearns [18]. CrP synthesized was characterized to be a mononuclear complex.
GSH was estimated by reduction of DTNB to form 2-nitro-5mercaptobenzoic acid with absorption maxima at 412 nm [22]. Concentration of GSH was calculated by comparing with the standards and the results were expressed as mol GSH/mg protein.
Animals and induction of diabetes Superoxide dismutase Male albino rats (Wistar strain) weighing between 160 and 180 g were used as an animal model. The animals were obtained from the Central Animal House of Panjab University and were housed in polypropylene cages. The animals were fed standard commercial pellet diet (Hindustan Lever Ltd., India) and water ad libitum. The animals were kept in good hygienic conditions in the departmental animal house. All institutional guidelines were followed for use and care of animals. Experimental design The rats were randomly divided into four groups of 6 animals each and were treated as described below: Control: Animals received citrate buffer intraperitoneally and isotonic saline, orally. Control + CrP: Animals were administered chromium picolinate orally (1 mg/kg body weight) daily for a period of four weeks. Diabetic: Animals received single injection of STZ (50 mg/kg body weight) intraperitoneally and were also given isotonic saline, orally for the duration of the study. Diabetic + CrP: Diabetic animals were administered chromium orally as chromium picolinate (1 mg/kg body weight) daily for a period of four weeks after the induction of diabetes. CrP was administrated at a dose of 1 mg/kg body weight as dose lower than this has been reported to be partially effective in lowering glucose levels [19]. It has been observed that supplementation at this and at higher doses has no significant toxic effect [20].
Superoxide dismutase (SOD) activity was assayed in liver homogenate by following the reduction of NBT at 560 nm [23]. The results were expressed as units/mg protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by 50%. Catalase Activity of catalase was assayed by monitoring the breakdown of H2 O2 to H2 O and O2 at 240 nm [24]. Activity of the enzyme was expressed as moles of H2 O2 decomposed/min/mg protein. Glutathione reductase Glutathione reductase (GR) was assayed by monitoring the conversion of oxidized glutathione to its reduced form coupled with oxidation of NADPH measured at 340 nm [25]. Activity of the enzyme was expressed as nmol of NADPH oxidized/min/mg protein. ˛-Tocopherol levels Plasma ␣-tocopherol was estimated by reduction of ferric ions to ferrous ions which bind to TPTZ to produce purple color with absorption maxima at 600 nm [26]. The concentration of ␣tocopherol was calculated by comparing with the standards and the results were expressed as g/mL.
Collection and preparation of sample Ascorbic acid levels The rats were anesthetized and blood was collected and centrifuged at 3000 × g for 10 min. The plasma was separated and used for estimation of glucose, ␣-tocopherol, ascorbic acid, aspartate aminotransferase and alanine aminotransferase activity. Liver homogenate was prepared in 5 volumes of 0.1 M Tris–HCl buffer (pH 7.4) containing 0.25 M sucrose and used for the assay of lipid peroxidation and glutathione levels. The homogenate was centrifuged at 2000 × g for 10 min at 4 ◦ C to remove cell debris and the supernatant thus obtained, used for the assay of superoxide dismutase, catalase and glutathione reductase activity. Biochemical estimations Plasma glucose levels Glucose was estimated in plasma by the glucose oxidase–peroxidase method using a commercially available kit (Reckon, Gujarat, India). The results were expressed as mM. Lipid peroxidation Lipid peroxidation was measured by monitoring the formation of thiobarbituric acid (TBA) reactive metabolites of lipids such as malondialdehyde (MDA). TBA–MDA chromophore with a maxima at 532 nm was measured and the results were expressed as nmol MDA/mg protein [21].
Plasma ascorbic acid was estimated by measuring the formation of hydrazone with 2,4-dinitrophenyl hydrazine (DNPH) in the presence of sulfuric acid having absorption maxima at 520 nm [27]. Concentration of ascorbic acid was calculated by comparing with the standard and the results were expressed as g/mL. Alanine aminotransferase (ALT) ALT activity in plasma was assayed by measuring the oxidation of NADH coupled to conversion of alanine and oxoglutarate to pyruvate and glutamate in the presence of lactate dehydrogenase at 340 nm. Results were expressed as units/L [28]. Aspartate aminotransferase (AST) AST activity in plasma was assayed by measuring the oxidation of NADH coupled to conversion of aspartate and oxoglutarate to oxaloacetate and glutamate in the presence of malate dehydrogenase at 340 nm and the results were expressed as units/L [28]. Protein content The concentration of protein was estimated using bovine serum albumin as standard [29].
B. Sundaram et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 117–121
20
3
*
*
16 14 12 10 8
*†
6 4
Lipid Peroxidation (nmol MDA/mg protein)
18
Plasma Glucose (mM)
119
2
2.5 2
*†
1.5 1 0.5
0
0
Control
Control+CrP
Diabetic
Diabetic+CrP
Control
Control+CrP
Diabetic
Diabetic+CrP
Fig. 1. Effect of chromium picolinate on plasma glucose levels of control and diabetic rats. Values are mean ± S.D. from 6 animals/group. *p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
Fig. 2. Effect of chromium picolinate administration on lipid peroxidation in liver of control and diabetic rats. Values are mean ± S.D. from 6 animals/group. *p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
Statistical analysis
Table 2 Effect of chromium picolinate administration on the levels of ␣-tocopherol and ascorbic acid in plasma of control and diabetic rats.
All values were expressed as mean ± S.D. of six animals per group and differences between means were analyzed by one way analysis of variance (ANOVA). The intergroup differences were determined by Student’s t test with p < 0.05 considered significant. Results Effect of CrP on plasma glucose levels Fig. 1 depicts the plasma glucose levels in control and diabetic rats supplemented with chromium. Diabetic animals had more than two fold increase in plasma glucose levels as compared to the controls. However, CrP administration to the diabetic rats attenuated hyperglycemia observed in the animals, while no significant change was observed in the control animals. Effect of CrP on lipid peroxidation As shown in Fig. 2 there was about two-fold increase in lipid peroxidation in liver of diabetic rats as compared to the controls. CrP administration significantly lowered lipid peroxidation in the diabetic animals while no effect was observed in the control animals. Effect of CrP on GSH levels and antioxidant enzymes As shown in Table 1, GSH levels were significantly lowered by 61.9% in the diabetic animals as compared to controls. Also, there was a decline in activity of antioxidant enzymes like SOD, catalase and GR by 51%, 33% and 27% respectively as compared to the controls. GSH levels and activity of antioxidant enzymes were significantly restored in the CrP treated diabetic animals. However,
Groups
␣-Tocopherol (g/mL)
Control Control + CrP Diabetic Diabetic + CrP
9.8 9.6 5.7 8.6
± ± ± ±
0.8 0.4 0.6* 0.4* , †
Ascorbic acid (g/mL) 13.0 12.4 7.2 9.8
± ± ± ±
0.8 0.9 0.3* 0.3* , †
Values are mean ± S.D. from 6 animals in each group. * p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
CrP supplementation did not alter the activities of these enzymes in the controls. Effect of CrP on plasma antioxidants Table 2 depicts the levels of antioxidants (␣-tocopherol and ascorbic acid) in various groups. A significant decrease in levels of ␣-tocopherol and ascorbic acid were observed in the diabetic animals as compared to the controls. CrP administration to the diabetic animals was found to increase the antioxidant levels significantly as compared to controls. However, chromium administration to the control rats did not produce any significant change. These results suggest that the antioxidant capacity in plasma was depleted in the diabetic animals and CrP treatment for four weeks significantly restored this capacity. Effect of CrP on transaminases Table 3 shows that the activity of ALT and AST in plasma of diabetic rats. It was observed that there was a significant increase in the activity of these enzymes in the diabetic rats as compared to
Table 1 Effect of chromium picolinate administration on the activities of superoxide dismutase, catalase, glutathione reductase and total glutathione levels in liver of control and diabetic rats. Groups
GSH (mol/mg protein)
Control Control + CrP Diabetic Diabetic + CrP
41.51 40.58 16.41 36.31
± ± ± ±
2.08 4.07 1.56* 2.94* , †
GR (nmol of NADPH oxidized/min/mg protein) 104.62 106.39 76.43 85.91
Values are mean ± S.D. from 6 animals in each group. * p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
± ± ± ±
10.16 8.85 7.17* 6.12* , †
SOD (Units/mg protein) 14.61 14.80 7.10 23.69
± ± ± ±
1.33 1.63 0.60* 1.07* , †
Catalase (mol of H2 O2 decomposed/min/mg protein) 82.21 80.27 55.42 84.07
± ± ± ±
0.56 0.87 1.42* 0.57* , †
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B. Sundaram et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 117–121
Table 3 Effect of chromium picolinate administration on the activities of alanine aminotransferase and aspartate aminotransferase in plasma of control and diabetic rats. Groups
ALT (Units/L)
Control Control + CrP Diabetic Diabetic + CrP
30.62 29.41 49.42 39.83
± ± ± ±
AST (Units/L) 0.96 1.85 0.80* 1.23* , †
82.75 82.09 99.34 95.29
± ± ± ±
5.68 7.29 2.99* 2.14* , †
Values are mean ± S.D. from 6 animals in each group. * p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
the controls. CrP supplementation normalized the activity of these enzymes in diabetic rats while there was no significant change observed in control animals suggesting that CrP is not hepatotoxic. Discussion Trivalent chromium is an essential nutrient required for glucose and lipid metabolism [30,31]. Chromium deficiency has been associated with diabetic-like condition [32,33]. The results from our study show that administration of CrP at a dose of 1 mg/kg body weight for four weeks was able to normalize plasma glucose levels in diabetic animals. Abdourahman and Edwards [33] observed that supplementation of CrP at a dose of 1 and 10 mg/kg/day to diabetic rats for thirty-two weeks showed improvement in glucose tolerance and insulin sensitivity [33]. Recent clinical studies suggested that doses of 200 g/kg, 400 g/kg body weight of CrP can improve blood indices for diabetics but is not sufficient to reverse all glucose abnormalities [30,34]. Another clinical trial suggested that a dose of 1 mg/kg body weight daily for four months had pronounced effect in lowering glucose and glycated hemoglobin levels without any toxic effects [19]. Reference dose of chromium for humans has been calculated as 1 mg/kg body weight [35]. Chromium has been shown to exert anti-diabetic action through several mechanisms that include: (i) Increased GLUT4 trafficking at the plasma membrane thereby increasing insulin dependent glucose transport in adipocytes cultured under diabetic conditions [36]. (ii) Enhancing insulin binding, insulin receptor number and -cell sensitivity [7,39]. (iii) Regulating carbohydrate and lipid metabolism [9,10]. Hyperglycemia is known to accentuate oxidative stress in liver, kidneys and other organs [37,38]. MDA, an index of oxidative insult was significantly increased in liver of diabetic animals and CrP supplementation attenuated hyperglycemia-induced increase in MDA levels. Animal models and clinical studies have also demonstrated elevated lipid peroxidation in both type-1 and type-2 diabetes [39,40]. It has been observed that multi-mineral enriched yeast containing chromium by itself could lower lipid peroxidation in diabetes [41,42]. CrP might reduce lipid peroxidation by modulating glucose/insulin system [42]. The beneficial effect of chromium on lipid peroxidation in diabetic animals is most likely due to normalization of blood glucose levels as observed in our study. Some studies suggest that chromium increases production of ROS and lipid peroxidation under both in vitro and in vivo conditions [43]. A recent study by Jain et al. [17] compared the anti-diabetic effect of CrP and chromium dinicocysteinate (CDNC) and observed that CDNC was more effective than CrP in lowering glucose levels and modulating signal transduction pathways associated with vascular inflammation in diabetic animals. There were no differences in plasma chromium levels in animals administered with CrP and CNDC suggesting nearby equal absorption of both
compounds. Also, no signs of toxicity were observed in animals supplemented with CrP and CDNC. CDNC was more effective than CrP in improving hyperglycemia associated changes [17]. Further in a clinical study CDNC was observed to be more effective than CrP in type-2 diabetic patients in reducing blood levels of TNF␣, insulin and oxidative stress [44]. It appears that the beneficial effect of chromium might be attributed to its organic form as they are more bio-available and less toxic. GSH, an important molecule in regulation of redox state that protects cells from oxidative damage, was found to be altered in liver of diabetic animals. Administration of CrP to diabetic animals led to increase in GSH levels. Increase in ROS generation due to hyperglycemia contributes to lowered GSH thus lowering GSH/GSSG ratios [45]. GR uses NADPH generated by HMP shunt for the conversion of GSSG to GSH [46,47]. The activity of the HMP shunt is altered in diabetes which might affect NADPH concentration that may be responsible for lowering GSH levels in diabetic mice [47]. In our earlier study, increase in glucose-6-phosphate dehydrogenase activity (G6PD) was observed in the CrP supplemented diabetic rats [10]. It appears that increased NADPH levels as a result of increased G6PD activity might probably help in the restoration of the GSH levels in the liver of CrP treated diabetic animals. Our results suggest that activity of antioxidant enzymes comprising of SOD, catalase and GR in liver of the diabetic rats was significantly lower than those in the control group. These results suggest that the ability to scavenge or inactivate free radicals is weakened in the diabetic animals. Numerous studies affirmed lowering of hepatic antioxidant enzymes activity in diabetes mellitus [48–50]. However, they were restored in CrP treated diabetic animals. Refaie et al. [51] also found that activity of these antioxidant enzymes were normalized after CrP supplementation in liver of diabetic rats. Several clinical studies also demonstrated that chromium administration could improve SOD and catalase activity in plasma of diabetic subjects [42,52]. Thus, it is suggested that improved antioxidant status may be associated with improvement in insulin sensitivity which could be potentiated by chromium administration [42]. In the present study, diabetic animals had significantly lower levels of plasma ␣-tocopherol and ascorbic acid compared to control animals. CrP administration on the other hand, improved the levels of these antioxidants. It was suggested that antioxidant capacity was depleted in serum of diabetic subjects attributed to lowered blood vitamin C and vitamin E levels [53]. Also, low ␣tocopherol is a risk factor for onset of type 2 diabetes mellitus [54]. Decrease in ␣-tocopherol and ascorbic acid levels in diabetic animals compared with the control animals can be due to increased utilization of these antioxidants to detoxify increased free radicals production. Thus, this improvement may be attributed to decreased utilization of these antioxidants in CrP treated diabetic animals. Consistent with previous reports [34], there was no change in AST and ALT activities in CrP supplemented rats compared with placebo supplemented diabetic rats, which suggests that there is no hepatotoxicity. A recent report suggests that CrP supplementation lowers the blood levels of ALT and AST in diabetic rats [55]. The data in this study also indicated that AST and ALT activities in diabetic rats showed large increase and CrP supplemented animals could improve their levels to near control values indicating no liver toxicity.
Conclusion The study clearly demonstrates that there was a significant increase in oxidative stress in diabetic animals and CrP treatment for four weeks restored antioxidant profile, without any
B. Sundaram et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 117–121
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Clinical studies
Chromium picolinate attenuates hyperglycemia-induced oxidative stress in streptozotocin-induced diabetic rats Bhuvaneshwari Sundaram, Aanchal Aggarwal, Rajat Sandhir ∗ Department of Biochemistry, Panjab University, Chandigarh 160014, India
a r t i c l e
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Article history: Received 19 June 2012 Accepted 6 September 2012 Keywords: Antioxidants Chromium picolinate Hyperglycemia Oxidative stress Liver
a b s t r a c t Chromium picolinate is advocated as an anti-diabetic agent for impaired glycemic control. It is a transition metal that exists in various oxidation states and may thereby act as a pro-oxidant. The present study has been designed to examine the effect of chromium picolinate supplementation on hyperglycemiainduced oxidative stress. Diabetes was induced in male Wistar rats by a single intraperitoneal injection of streptozotocin (50 mg/kg body weight) and chromium was administered orally as chromium picolinate (1 mg/kg body weight) daily for a period of four weeks after the induction of diabetes. As is characteristic of diabetic condition, hyperglycemia was associated with an increase in oxidative stress in liver in terms of increased lipid peroxidation and decreased glutathione levels. The activity of antioxidant enzymes like superoxide dismutase, catalase and glutathione reductase were significantly reduced in liver of diabetic animals. Levels of ␣-tocopherol and ascorbic acid were found to be considerably lower in plasma of diabetic rats. Chromium picolinate administration on the other hand was found to have beneficial effect in normalizing glucose levels, lipid peroxidation and antioxidant status. The results from the present study demonstrate potential of chromium picolinate to attenuate hyperglycemia-induced oxidative stress in experimental diabetes. © 2012 Elsevier GmbH. All rights reserved.
Introduction Diabetes mellitus is a chronic metabolic disorder of carbohydrate and lipid metabolism leading to various complications [1–4]. Oxidative stress as a consequence of imbalance between radicalgenerating and radical-scavenging system plays an important role in the pathogenesis of diabetic complications [5]. Diabetes is closely associated with unbalanced trace elements that stem from chronic uncontrolled hyperglycemia [6]. Out of all the trace elements studied, chromium appears to be the most promising element in the therapeutics of diabetes. Chromium plays beneficial role in diabetes by facilitating insulin signaling thereby improving insulin sensitivity [7]. Deficiency of chromium has been associated with impaired glucose tolerance, fasting hyperglycemia, glucosuria, increased body fat, dyslipidemia and impaired fertility [8]. We have previously observed that CrP exerts anti-diabetic effect through regulation of carbohydrate and lipid metabolism [9,10]. It is a transition element that exists in several oxidation states and may increase oxidative stress [11]. Cr (III) and Cr (VI) species been shown to act as a pro-oxidant by increasing ROS production that induces DNA damage [12]. Contrarily, Cr
∗ Corresponding author. Tel.: +91 172 2534131/38. E-mail address: [email protected] (R. Sandhir). 0946-672X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jtemb.2012.09.002
(III) has also been shown to improve cellular antioxidant capacity in both animal and cell culture models of diabetes [13–15]. Furthermore, organic complexes of Cr (III) have been found to be less toxic as compared to inorganic salts [16]. Chromium dinicocysteinate, an organic Cr (III) formulation has hypoglycemic effects and lowers oxidative stress [17]. Hence, it is important to monitor the antioxidant status in diabetic rats supplemented with Cr (III) to recognize the precise influence of chromium picolinate, an organic complex on antioxidant defence system in diabetes. Therefore, the present study was planned with the aim to evaluate the effect of chromium picolinate on oxidative stress induced by hyperglycemia in liver of diabetic rats. Materials and methods Materials Streptozotocin (STZ), 2,4,6-tripyridyl-S-triazine (TPTZ) was obtained from Sigma Chemical Company, St. Louis, MO, USA. 5,5 -Dithiobis-(2-nitrobenzoic acid) (DTNB), nitroblue tetrazolium (NBT), dinitrophenyl hydrazine (DNPH) were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Picolinic acid and chromium chloride were obtained from Himedia Laboratories Pvt. Ltd., Mumbai, India. All other chemicals used were of analytical grade.
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Synthesis of chromium picolinate
Glutathione (GSH) levels
CrP was synthesized according to the method described by Stearns [18]. CrP synthesized was characterized to be a mononuclear complex.
GSH was estimated by reduction of DTNB to form 2-nitro-5mercaptobenzoic acid with absorption maxima at 412 nm [22]. Concentration of GSH was calculated by comparing with the standards and the results were expressed as mol GSH/mg protein.
Animals and induction of diabetes Superoxide dismutase Male albino rats (Wistar strain) weighing between 160 and 180 g were used as an animal model. The animals were obtained from the Central Animal House of Panjab University and were housed in polypropylene cages. The animals were fed standard commercial pellet diet (Hindustan Lever Ltd., India) and water ad libitum. The animals were kept in good hygienic conditions in the departmental animal house. All institutional guidelines were followed for use and care of animals. Experimental design The rats were randomly divided into four groups of 6 animals each and were treated as described below: Control: Animals received citrate buffer intraperitoneally and isotonic saline, orally. Control + CrP: Animals were administered chromium picolinate orally (1 mg/kg body weight) daily for a period of four weeks. Diabetic: Animals received single injection of STZ (50 mg/kg body weight) intraperitoneally and were also given isotonic saline, orally for the duration of the study. Diabetic + CrP: Diabetic animals were administered chromium orally as chromium picolinate (1 mg/kg body weight) daily for a period of four weeks after the induction of diabetes. CrP was administrated at a dose of 1 mg/kg body weight as dose lower than this has been reported to be partially effective in lowering glucose levels [19]. It has been observed that supplementation at this and at higher doses has no significant toxic effect [20].
Superoxide dismutase (SOD) activity was assayed in liver homogenate by following the reduction of NBT at 560 nm [23]. The results were expressed as units/mg protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by 50%. Catalase Activity of catalase was assayed by monitoring the breakdown of H2 O2 to H2 O and O2 at 240 nm [24]. Activity of the enzyme was expressed as moles of H2 O2 decomposed/min/mg protein. Glutathione reductase Glutathione reductase (GR) was assayed by monitoring the conversion of oxidized glutathione to its reduced form coupled with oxidation of NADPH measured at 340 nm [25]. Activity of the enzyme was expressed as nmol of NADPH oxidized/min/mg protein. ˛-Tocopherol levels Plasma ␣-tocopherol was estimated by reduction of ferric ions to ferrous ions which bind to TPTZ to produce purple color with absorption maxima at 600 nm [26]. The concentration of ␣tocopherol was calculated by comparing with the standards and the results were expressed as g/mL.
Collection and preparation of sample Ascorbic acid levels The rats were anesthetized and blood was collected and centrifuged at 3000 × g for 10 min. The plasma was separated and used for estimation of glucose, ␣-tocopherol, ascorbic acid, aspartate aminotransferase and alanine aminotransferase activity. Liver homogenate was prepared in 5 volumes of 0.1 M Tris–HCl buffer (pH 7.4) containing 0.25 M sucrose and used for the assay of lipid peroxidation and glutathione levels. The homogenate was centrifuged at 2000 × g for 10 min at 4 ◦ C to remove cell debris and the supernatant thus obtained, used for the assay of superoxide dismutase, catalase and glutathione reductase activity. Biochemical estimations Plasma glucose levels Glucose was estimated in plasma by the glucose oxidase–peroxidase method using a commercially available kit (Reckon, Gujarat, India). The results were expressed as mM. Lipid peroxidation Lipid peroxidation was measured by monitoring the formation of thiobarbituric acid (TBA) reactive metabolites of lipids such as malondialdehyde (MDA). TBA–MDA chromophore with a maxima at 532 nm was measured and the results were expressed as nmol MDA/mg protein [21].
Plasma ascorbic acid was estimated by measuring the formation of hydrazone with 2,4-dinitrophenyl hydrazine (DNPH) in the presence of sulfuric acid having absorption maxima at 520 nm [27]. Concentration of ascorbic acid was calculated by comparing with the standard and the results were expressed as g/mL. Alanine aminotransferase (ALT) ALT activity in plasma was assayed by measuring the oxidation of NADH coupled to conversion of alanine and oxoglutarate to pyruvate and glutamate in the presence of lactate dehydrogenase at 340 nm. Results were expressed as units/L [28]. Aspartate aminotransferase (AST) AST activity in plasma was assayed by measuring the oxidation of NADH coupled to conversion of aspartate and oxoglutarate to oxaloacetate and glutamate in the presence of malate dehydrogenase at 340 nm and the results were expressed as units/L [28]. Protein content The concentration of protein was estimated using bovine serum albumin as standard [29].
B. Sundaram et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 117–121
20
3
*
*
16 14 12 10 8
*†
6 4
Lipid Peroxidation (nmol MDA/mg protein)
18
Plasma Glucose (mM)
119
2
2.5 2
*†
1.5 1 0.5
0
0
Control
Control+CrP
Diabetic
Diabetic+CrP
Control
Control+CrP
Diabetic
Diabetic+CrP
Fig. 1. Effect of chromium picolinate on plasma glucose levels of control and diabetic rats. Values are mean ± S.D. from 6 animals/group. *p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
Fig. 2. Effect of chromium picolinate administration on lipid peroxidation in liver of control and diabetic rats. Values are mean ± S.D. from 6 animals/group. *p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
Statistical analysis
Table 2 Effect of chromium picolinate administration on the levels of ␣-tocopherol and ascorbic acid in plasma of control and diabetic rats.
All values were expressed as mean ± S.D. of six animals per group and differences between means were analyzed by one way analysis of variance (ANOVA). The intergroup differences were determined by Student’s t test with p < 0.05 considered significant. Results Effect of CrP on plasma glucose levels Fig. 1 depicts the plasma glucose levels in control and diabetic rats supplemented with chromium. Diabetic animals had more than two fold increase in plasma glucose levels as compared to the controls. However, CrP administration to the diabetic rats attenuated hyperglycemia observed in the animals, while no significant change was observed in the control animals. Effect of CrP on lipid peroxidation As shown in Fig. 2 there was about two-fold increase in lipid peroxidation in liver of diabetic rats as compared to the controls. CrP administration significantly lowered lipid peroxidation in the diabetic animals while no effect was observed in the control animals. Effect of CrP on GSH levels and antioxidant enzymes As shown in Table 1, GSH levels were significantly lowered by 61.9% in the diabetic animals as compared to controls. Also, there was a decline in activity of antioxidant enzymes like SOD, catalase and GR by 51%, 33% and 27% respectively as compared to the controls. GSH levels and activity of antioxidant enzymes were significantly restored in the CrP treated diabetic animals. However,
Groups
␣-Tocopherol (g/mL)
Control Control + CrP Diabetic Diabetic + CrP
9.8 9.6 5.7 8.6
± ± ± ±
0.8 0.4 0.6* 0.4* , †
Ascorbic acid (g/mL) 13.0 12.4 7.2 9.8
± ± ± ±
0.8 0.9 0.3* 0.3* , †
Values are mean ± S.D. from 6 animals in each group. * p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
CrP supplementation did not alter the activities of these enzymes in the controls. Effect of CrP on plasma antioxidants Table 2 depicts the levels of antioxidants (␣-tocopherol and ascorbic acid) in various groups. A significant decrease in levels of ␣-tocopherol and ascorbic acid were observed in the diabetic animals as compared to the controls. CrP administration to the diabetic animals was found to increase the antioxidant levels significantly as compared to controls. However, chromium administration to the control rats did not produce any significant change. These results suggest that the antioxidant capacity in plasma was depleted in the diabetic animals and CrP treatment for four weeks significantly restored this capacity. Effect of CrP on transaminases Table 3 shows that the activity of ALT and AST in plasma of diabetic rats. It was observed that there was a significant increase in the activity of these enzymes in the diabetic rats as compared to
Table 1 Effect of chromium picolinate administration on the activities of superoxide dismutase, catalase, glutathione reductase and total glutathione levels in liver of control and diabetic rats. Groups
GSH (mol/mg protein)
Control Control + CrP Diabetic Diabetic + CrP
41.51 40.58 16.41 36.31
± ± ± ±
2.08 4.07 1.56* 2.94* , †
GR (nmol of NADPH oxidized/min/mg protein) 104.62 106.39 76.43 85.91
Values are mean ± S.D. from 6 animals in each group. * p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
± ± ± ±
10.16 8.85 7.17* 6.12* , †
SOD (Units/mg protein) 14.61 14.80 7.10 23.69
± ± ± ±
1.33 1.63 0.60* 1.07* , †
Catalase (mol of H2 O2 decomposed/min/mg protein) 82.21 80.27 55.42 84.07
± ± ± ±
0.56 0.87 1.42* 0.57* , †
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B. Sundaram et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 117–121
Table 3 Effect of chromium picolinate administration on the activities of alanine aminotransferase and aspartate aminotransferase in plasma of control and diabetic rats. Groups
ALT (Units/L)
Control Control + CrP Diabetic Diabetic + CrP
30.62 29.41 49.42 39.83
± ± ± ±
AST (Units/L) 0.96 1.85 0.80* 1.23* , †
82.75 82.09 99.34 95.29
± ± ± ±
5.68 7.29 2.99* 2.14* , †
Values are mean ± S.D. from 6 animals in each group. * p < 0.05 compared with control animals. † p < 0.05 compared with diabetic animals.
the controls. CrP supplementation normalized the activity of these enzymes in diabetic rats while there was no significant change observed in control animals suggesting that CrP is not hepatotoxic. Discussion Trivalent chromium is an essential nutrient required for glucose and lipid metabolism [30,31]. Chromium deficiency has been associated with diabetic-like condition [32,33]. The results from our study show that administration of CrP at a dose of 1 mg/kg body weight for four weeks was able to normalize plasma glucose levels in diabetic animals. Abdourahman and Edwards [33] observed that supplementation of CrP at a dose of 1 and 10 mg/kg/day to diabetic rats for thirty-two weeks showed improvement in glucose tolerance and insulin sensitivity [33]. Recent clinical studies suggested that doses of 200 g/kg, 400 g/kg body weight of CrP can improve blood indices for diabetics but is not sufficient to reverse all glucose abnormalities [30,34]. Another clinical trial suggested that a dose of 1 mg/kg body weight daily for four months had pronounced effect in lowering glucose and glycated hemoglobin levels without any toxic effects [19]. Reference dose of chromium for humans has been calculated as 1 mg/kg body weight [35]. Chromium has been shown to exert anti-diabetic action through several mechanisms that include: (i) Increased GLUT4 trafficking at the plasma membrane thereby increasing insulin dependent glucose transport in adipocytes cultured under diabetic conditions [36]. (ii) Enhancing insulin binding, insulin receptor number and -cell sensitivity [7,39]. (iii) Regulating carbohydrate and lipid metabolism [9,10]. Hyperglycemia is known to accentuate oxidative stress in liver, kidneys and other organs [37,38]. MDA, an index of oxidative insult was significantly increased in liver of diabetic animals and CrP supplementation attenuated hyperglycemia-induced increase in MDA levels. Animal models and clinical studies have also demonstrated elevated lipid peroxidation in both type-1 and type-2 diabetes [39,40]. It has been observed that multi-mineral enriched yeast containing chromium by itself could lower lipid peroxidation in diabetes [41,42]. CrP might reduce lipid peroxidation by modulating glucose/insulin system [42]. The beneficial effect of chromium on lipid peroxidation in diabetic animals is most likely due to normalization of blood glucose levels as observed in our study. Some studies suggest that chromium increases production of ROS and lipid peroxidation under both in vitro and in vivo conditions [43]. A recent study by Jain et al. [17] compared the anti-diabetic effect of CrP and chromium dinicocysteinate (CDNC) and observed that CDNC was more effective than CrP in lowering glucose levels and modulating signal transduction pathways associated with vascular inflammation in diabetic animals. There were no differences in plasma chromium levels in animals administered with CrP and CNDC suggesting nearby equal absorption of both
compounds. Also, no signs of toxicity were observed in animals supplemented with CrP and CDNC. CDNC was more effective than CrP in improving hyperglycemia associated changes [17]. Further in a clinical study CDNC was observed to be more effective than CrP in type-2 diabetic patients in reducing blood levels of TNF␣, insulin and oxidative stress [44]. It appears that the beneficial effect of chromium might be attributed to its organic form as they are more bio-available and less toxic. GSH, an important molecule in regulation of redox state that protects cells from oxidative damage, was found to be altered in liver of diabetic animals. Administration of CrP to diabetic animals led to increase in GSH levels. Increase in ROS generation due to hyperglycemia contributes to lowered GSH thus lowering GSH/GSSG ratios [45]. GR uses NADPH generated by HMP shunt for the conversion of GSSG to GSH [46,47]. The activity of the HMP shunt is altered in diabetes which might affect NADPH concentration that may be responsible for lowering GSH levels in diabetic mice [47]. In our earlier study, increase in glucose-6-phosphate dehydrogenase activity (G6PD) was observed in the CrP supplemented diabetic rats [10]. It appears that increased NADPH levels as a result of increased G6PD activity might probably help in the restoration of the GSH levels in the liver of CrP treated diabetic animals. Our results suggest that activity of antioxidant enzymes comprising of SOD, catalase and GR in liver of the diabetic rats was significantly lower than those in the control group. These results suggest that the ability to scavenge or inactivate free radicals is weakened in the diabetic animals. Numerous studies affirmed lowering of hepatic antioxidant enzymes activity in diabetes mellitus [48–50]. However, they were restored in CrP treated diabetic animals. Refaie et al. [51] also found that activity of these antioxidant enzymes were normalized after CrP supplementation in liver of diabetic rats. Several clinical studies also demonstrated that chromium administration could improve SOD and catalase activity in plasma of diabetic subjects [42,52]. Thus, it is suggested that improved antioxidant status may be associated with improvement in insulin sensitivity which could be potentiated by chromium administration [42]. In the present study, diabetic animals had significantly lower levels of plasma ␣-tocopherol and ascorbic acid compared to control animals. CrP administration on the other hand, improved the levels of these antioxidants. It was suggested that antioxidant capacity was depleted in serum of diabetic subjects attributed to lowered blood vitamin C and vitamin E levels [53]. Also, low ␣tocopherol is a risk factor for onset of type 2 diabetes mellitus [54]. Decrease in ␣-tocopherol and ascorbic acid levels in diabetic animals compared with the control animals can be due to increased utilization of these antioxidants to detoxify increased free radicals production. Thus, this improvement may be attributed to decreased utilization of these antioxidants in CrP treated diabetic animals. Consistent with previous reports [34], there was no change in AST and ALT activities in CrP supplemented rats compared with placebo supplemented diabetic rats, which suggests that there is no hepatotoxicity. A recent report suggests that CrP supplementation lowers the blood levels of ALT and AST in diabetic rats [55]. The data in this study also indicated that AST and ALT activities in diabetic rats showed large increase and CrP supplemented animals could improve their levels to near control values indicating no liver toxicity.
Conclusion The study clearly demonstrates that there was a significant increase in oxidative stress in diabetic animals and CrP treatment for four weeks restored antioxidant profile, without any
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