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Objective: Chromium has gained popularity as a nutritional supplement for diabetic patients. This study evaluated the effect of chronic administration of a chromium complex of d-phenylalanine (Cr(d-phe)3) on glucose and insulin tolerance in obese mice. The study tested the hypothesis that Cr(d-phe)3 suppresses endoplasmic reticulum (ER) stress and insulin resistance in these animals.
Methods and Procedures: C57BL lean and ob/ob obese mice were randomly divided to orally receive vehicle or Cr(d-phe)3 (3.8 μg of elemental chromium/kg/day) for 6 months. Insulin sensitivity was evaluated by glucose and insulin tolerance tests. Protein levels of phosphorylated pancreatic ER kinase (PERK), α subunit of translation initiation factor 2 (eIF2α) and inositol-requiring enzyme-1 (IRE-1), p-c-Jun, and insulin receptor substrate-1 (IRS-1) phosphoserine-307 were assessed by western blotting. In vitro ER stress was induced by treating cultured muscle cells with thapsigargin in the presence or absence of Cr(d-phe)3.
Results: ob/ob mice showed poor glucose and insulin tolerance compared to the lean controls, which was attenuated by Cr(d-phe)3. Markers of insulin resistance (phospho-c-Jun and IRS-1 phosphoserine) and ER stress (p-PERK, p-IRE-1, p-eIF2α), which were elevated in ob/ob mice, were attenuated following Cr(d-phe)3 treatment. Chromium treatment was also associated with a reduction in liver triglyceride levels and lipid accumulation. In cultured myotubes, Cr(d-phe)3 attenuated ER stress induced by thapsigargin.
Discussion: Oral Cr(d-phe)3 treatment reduces glucose intolerance, insulin resistance, and hepatic ER stress in obese, insulin-resistant mice.
Insulin resistance, concomitant with diabetes, metabolic syndrome, obesity, and hypertension, is a major risk factor for the development and progression of cardiovascular diseases, the leading causes of mortality and morbidity (1). The currently accepted therapeutic regimens include pharmacological treatment (biguanides, thiazolidinediones), caloric restriction, and physical exercise, which are effective, although none is deemed the ultimate cure. In consequence, there has been a substantial demand for development of new agents targeting the insulin-signaling cascade to improve overall insulin sensitivity and long-term benefit in the management of diabetes, obesity, and insulin resistance. Mineral chromium, which is thought to play a key role in carbohydrate metabolism by potentiating the action of insulin, has drawn recent interest for its role in diabetes and obesity (2). Dietary deficiency of chromium is believed to be positively associated with the risk of diabetes and its complications (3). This is supported by findings from clinical trials that dietary chromium supplementation can lower blood-glucose levels and improve lipid profile in diabetic patients (reviewed in ref. 4).
Better bioavailability of low-molecular-weight organic chromium complexes, and the identification of the biologically active form of chromium as a complex with an oligopeptide, prompted the design and evaluation of low-molecular-weight organic chromium complexes as potential therapeutic agents in treating insulin in type 2 diabetes (5). In several animal and human studies, chromium complex of picolinic acid, the most popularly used dietary supplement, has been shown to modulate intracellular pathways of glucose metabolism and improve comorbidities associated with insulin resistance (6).
In a recent clinical trial, Martin and associates demonstrated an improvement in insulin sensitivity and glycated hemoglobin in subjects with type 2 diabetes who received chromium picolinate (7). Despite these documented evidence for the beneficial effects of chromium, two recent studies suggested that chromium treatment may not have any effect on insulin sensitivity or glycated hemoglobin levels in obese patients with poorly controlled diabetes (8,9). Based on its evaluations of the existing scientific evidence, the US Food and Drug Administration (FDA) has allowed the following qualified health claim: “Chromium picolinate may reduce the risk of insulin resistance, and therefore possibly may reduce the risk of type 2 diabetes. FDA concludes, however, that the existence of such a relationship between chromium picolinate and either insulin resistance or type 2 diabetes is highly uncertain.” To understand the role of chromium in treatment of diabetes, these discrepancies warrant further extensive studies.
Although the molecular mechanisms leading to insulin resistance remain elusive, emerging evidence suggests that endoplasmic reticulum (ER) stress may play a pivotal role in this phenomenon (10,11). Ozcan and colleagues showed elevated ER stress markers in adipocytes and liver cells of genetically obese and high-fat fed mice (12). ER stress causes activation of c-Jun N-terminal kinase (JNK) that phosphorylates, a serine residue (307) on insulin receptor substrate-1 (IRS-1), leading to the suppression of insulin signaling (12). Consequently, molecules that suppress ER stress can improve diabetes and associated comorbidities (13,14). Based on this background, it is pertinent to assess the potential effect of chromium on ER stress.
The present study evaluated the impact of chronic treatment with a chromium complex of the amino acid d-phenylalanine (Cr(d-phe)3) (15) on glucose tolerance and insulin sensitivity in obese mice with type 2 diabetes. The study also assessed the effect of chronic chromium therapy on plasma and liver lipid levels. Further, in an attempt to understand the potential mechanisms involved, the effect of chromium treatment on hepatic ER stress and cellular markers of insulin resistance was investigated.
Methods and Procedures
All chemicals, unless stated otherwise, were obtained from Sigma Chemical (St. Louis, MO). The Micro BCA protein assay kit was purchased from Pierce Chemical (Rockford, IL).
Synthesis of Cr(d-phe)3
Cr(d-phe)3 was synthesized and characterized as described previously (15). Briefly, aqueous solutions of CrCl3·6H2O (2.6 g, 10 mmol in 50 ml water) and d-phenylalanine (4.8 g, 30 mmol in 50 ml water) were mixed at 80 °C and refluxed for 4 h. The homogeneous green reaction mixture was freeze-dried. The greenish-violet solid obtained was washed with acetone and dried in an air oven.
Animal treatment protocol
The experimental procedures described in this study were approved by the University of Wyoming (Laramie, WY) Animal Use and Care Committee. In brief, homozygous B6.V-lep<ob>/J male mice were obtained from the Jackson Laboratory (Bar Harbor, ME) at 5 weeks of age and were housed within the School of Pharmacy Animal Facility at the University of Wyoming with free access to food and water. The ob/ob obese (leptin deficient) mice and age-matched wild-type C57BL/6J male mice were randomly divided to receive either vehicle (H2O) or Cr(d-phe)3, provided in drinking water for 6 months starting at 2 months of age. On the basis of calculated water intake, Cr(d-phe)3 was administered to provide a dose of ∼45 μg/kg/day (corresponding to ∼3.8 μg of elemental chromium/kg/day).
Plasma leptin, insulin, and lipids measurement
Plasma leptin and insulin levels were measured by radioimmunoassay and mouse insulin enzyme-linked immunoassay using commercially available kits per manufacturer's specifications (Linco Research, St. Charles, MO). Serum triglyceride and cholesterol levels were measured using an assay kit from Diagnostic Chemicals, Oxford, CT.
Glucose and insulin tolerance tests
At the end of the treatment schedule, mice were subjected to the intraperitoneal glucose tolerance test (GTT) as described previously (16). Briefly, the mice were fasted overnight (∼12 h), and glucose challenge was initiated with intraperitoneal injection of glucose (1 g/kg). Glucose levels were determined in blood drops obtained by clipping the tail of the mice immediately before glucose challenge, as well as at 15-, 60-, and 120- min intervals. Serum glucose levels were determined using ACCU-CHEK Advantage Glucose Analyzer (Roche Diagnostics, IN). An insulin tolerance test was performed at the end of 5 months of treatment. Mice (without overnight starving) were given intraperitoneal injections of insulin (5 U/kg). Blood-glucose levels were determined by the tail-clip method at different time points as described previously (17).
Liver triglyceride assay
Liver triglyceride levels were determined as described previously (18). Briefly, 0.2 g liver tissue from each animal was homogenized with 0.62 ml of chloroform/methanol (1/2 by volume). After vortexing for 15 min, 0.2 ml of chloroform and 0.2 ml of water were added sequentially, mixed, and centrifuged briefly at 3,000 rpm to separate the phases. The lower phase was transferred to another tube, and the residue was mixed with 0.31 ml chloroform for the second-step vortex and centrifugation. The lower phase obtained by the centrifugation was mixed with the first chloroform phase in the same tube. After evaporation with nitrogen gas at 55 °C, the lipid extract was dissolved in 0.3 ml 2-propanol. Triglyceride levels were measured spectrophotometrically (Spectra Max 190 Microplate Spectrophotometer; Molecular Devices, Sunnyvale, CA). The liver sections were stained with hematoxylin and eosin.
Western blot analysis for markers of ER stress and insulin resistance
The liver of each mouse was rapidly removed and homogenized in a lysis buffer containing 20 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail. Samples were then sonicated for 15 s and centrifuged at 12,000g for 20 min at 4 °C. The protein concentration of the supernatant was assessed using the Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts (50 μg protein/lane) of protein and prestained molecular weight marker (Gibco-BRL, Gaithersburg, MD) were loaded onto 7% sodium dodecyl sulfate-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad) before being separated and transferred to nitrocellulose membranes (0.2 μm pore size, Bio-Rad). Membranes were incubated for 1 h in a blocking solution containing 5% nonfat milk in Tris-buffered saline, washed in Tris-buffered saline and incubated overnight at 4 °C with antiphospho-PERK (1:250), antiphospho-IRE, anti-IRE (1:1,000), anti-eIF2α (1:500) and antiphospho- eIF2α (1:500), anti phospho-IRS-1 (1:1,000), anti-IRS-1 (1:1,000), antiphospho-c-Jun (1:1,000), and antitotal JNK (1:1,000) antibody. Rabbit polyclonal anti-p-IRE and anti-IRE antibodies were kindly provided by Fumihiko Urano, University of Massachusetts. All other antibodies were obtained from Cell Signaling (Beverly, MA). After incubation with the primary antibody, blots were incubated with an antirabbit IgG HRP-linked antibody at a dilution of 1:5,000 for 1 h at room temperature. Immunoreactive bands were detected using the Super Signal West Dura Extended Duration Substrate (Pierce, Milwaukee, WI). The intensity of bands was measured with a scanning densitometer (Model GS-800; Bio-Rad), coupled with Bio-Rad PC analysis software.
Immunoprecipitation was performed as described previously (19). Briefly, cell lysates from the liver (1 mg of total protein) were prepared in 1 ml radioimmunoprecipitation assay buffer in 1.5-ml microtubes. Anti-IRS-1 antibody was added (0.6 μg) to each sample and incubated overnight at 4 °C. The immunoprecipitates were captured on Protein A/G Sepharose beads (Pierce, Rockford, IL) by incubating the beads for 2 h at 4 °C. The beads were washed three times with cold radioimmunoprecipitation assay buffer, followed by a final wash with phosphate-buffered saline. The beads were then suspended in 2× Laemmli buffer and heated at 95 °C for 5 min, and the supernatant was used for western blotting.
Cell culture and treatment
The skeletal muscle cell line C2C12 from adult mouse legs (American Type Culture Collection) was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin under a humidified atmosphere of 5% CO2 in air and maintained at low confluence. To induce differentiation, the culture medium was changed to 1% fetal bovine serum instead of 10% fetal bovine serum when cells reached 70–80% confluence. Once differentiated into myotubes, the cells were rendered quiescent by incubating them with serum-free medium for 24 h. During the last 6 h of quiescence, the myotubes were treated with thapsigargin (1 μmol/l) in the presence or absence of Cr(d-phe)3. At the end of the treatment period, the cells were lysed with radioimmunoprecipitation assay buffer and western blotted with antiphospho α subunit of translation initiation factor 2 (eIF2α) as described above. The complete experiment, including differentiation of myotubes, was repeated three times.
Data are expressed as mean ± s.e.m. and statistically evaluated using a 2-way ANOVA using the Scheffe F-test for post hoc analysis (Jandel Scientific, San Rafael, CA). A “P” value of <0.05 was considered to be statistically significant.
General features of lean and ob/ob mice treated with Cr(d-phe)3
As expected, ob/ob mice had a significantly higher body, heart, liver, and kidney mass compared to age-matched lean controls. Chronic treatment with Cr(d-phe)3 was not associated with changes in body mass (Table 1).
Table 1. General features of lean and ob/ob mice with or without a 6-month treatment of Cr(d-phe)3 at 45 μg/kg/day (3.8 μg of elemental chromium/kg/day) for 6 months
Effect of Cr(d-phe)3 treatment on plasma leptin, insulin, and lipids levels
Plasma leptin levels were undetectable in ob/ob mice, and treatment with Cr(d-phe)3 did not alter leptin levels in these animals (Figure 1a), although a small increase in plasma leptin levels was observed in lean mice treated with Cr(d-phe)3 (Figure 1a). Blood levels of insulin, triglyceride, and cholesterol were significantly elevated in obese mice compared to the lean control, suggesting the presence of insulin resistance and hyperlipidemia. Chronic treatment with Cr(d-phe)3 did not alter the basal serum levels of insulin, triglyceride, or cholesterol (Figure 1b–d).
Effect of Cr(d-phe)3 treatment on glucose and insulin tolerance tests
Whole-body insulin sensitivity was assessed by performing GTTs and insulin tolerance tests in both lean and obese mice chronically treated with Cr(d-phe)3 (Figure 2a,b). The basal fasting blood-glucose level in the lean mice was 93.2 ± 3.1 mg/dl and that for the obese mice was 141 ± 8.9 mg/dl, indicating overt hyperglycemia in the obese mice. Following glucose challenge in the lean animals, blood-glucose levels peaked at 15 min and returned to near-normal levels within 120 min. In contrast, ob/ob mice exhibited severe hyperglycemia upon administration of glucose and exhibited impaired glucose tolerance as evidenced by the high postchallenge blood glucose levels even at 120 min. Chronic ingestion of Cr(d-phe)3 significantly improved glucose tolerance in ob/ob mice, with glucose disposal curves showing significantly lower blood levels of glucose at 15, 60, and 120 min. Treatment with Cr(d-phe)3 did not alter the glucose disposal rate in lean mice. In both obese and lean groups, chronic treatment with Cr(d-phe)3 had no apparent effect on the basal levels of fasting blood glucose.
Obese animals subjected to insulin challenge showed marked reduction in postchallenge blood-glucose levels (Figure 2b). The blood-glucose levels in obese mice treated with chromium were significantly lower at 15 and 60 min compared to the glucose levels at the corresponding time points in the obese mice receiving the vehicle. Surprisingly, in contrast to established reports, lean mice subjected to insulin challenge did not exhibit a precipitous fall in blood-glucose levels. Not having starved the animals prior to the test may have contributed to this discrepancy. Treatment with Cr(d-phe)3 did not alter the glucose disposal rates in lean animals following insulin challenge.
Effect of Cr(d-phe)3 treatment on indicators of hepatic ER stress
Insulin resistance and obesity are associated with an increase in ER stress (12). To test whether chromium complexes improve glucose tolerance and insulin sensitivity via reducing ER stress, ER stress indicators such as pancreatic ER kinase (PERK), inositol-requiring enzyme-1 (IRE-1), and eIF2α were assessed in hepatic tissues of obese and lean mice treated with or without Cr(d-phe)3. Consistent with previous reports (12,14), levels of phosphorylated PERK (Thr980), eIF2α, and IRE-1 were significantly higher in obese mice compared to lean controls (Figure 3). This obesity-associated increase in p-PERK, p-IRE, and eIF2α was reduced in Cr(d-phe)3-treated obese mice. In the lean group, chronic therapy with Cr(d-phe)3 did not alter the protein levels of ER stress indicators as seen by unchanged levels of IRE-1 protein (Figure 3).
Effect of Cr(d-phe)3 treatment on markers of insulin resistance
Insulin resistance and obesity are associated with activation of c-Jun-N-terminal kinase (JNK) and subsequent JNK-mediated serine phosphorylation of IRS-1 at Ser-307 that negatively regulates insulin signaling (20). Accordingly, the levels of phospho-c-Jun and IRS-1 phospho-serine-307 were elevated in the livers of obese mice compared to lean control. Treatment with Cr (d-phe)3 suppressed tyrosine phosphorylation of c-Jun and serine phosphorylation of IRS-1 in livers of ob/ob mice (Figure 4).
Effect of Cr(d-phe)3 treatment on liver triglyceride content and morphology in ob/ob mice
Obesity and insulin resistance has been associated with alterations in liver lipid metabolism (21). Analysis of hepatic triglyceride content showed a significant increase in liver triglycerides levels in the ob/ob mice compared to lean controls (Figure 5a). Cr(d-phe)3-treated obese animals had a significant reduction in hepatic triglyceride levels compared to vehicle control. Histological examination of the ob/ob mouse livers revealed an increase in the number and size of intracellular vacuoles characteristic of lipid droplets (Figure 5b). In contrast, the number of vacuoles was significantly lower in the chromium-treated animals, indicating partial reversal of obesity-induced fat accumulation in liver by chromium treatment.
Effect of Cr(d-phe)3 on thapsigargin-induced ER stress in cultured myotubes
Lysates from myotubes treated with thapsigargin (an agent that promotes ER stress by depletion of calcium stores) caused a significant increase in phosphorylated e-IF2α, an early marker of ER stress, which was inhibited by treating the cells with Cr(d-phe)3 (Figure 6). The inhibition was evident even at 10 nmol/l concentrations of Cr(d-phe)3. Chromium treatment did not alter the total protein levels of eIF2α.
The major findings from the present study are that chronic treatment with Cr(d-phe)3 improves glucose tolerance and insulin resistance in an obese mouse model of type 2 diabetes. Furthermore, the data presented here suggest that chromium suppresses markers of insulin resistance such as serine phosphorylation of IRS-1, tyrosine phosphorylation of JNK, and hepatic ER stress in obese animals. Chromium treatment also reduced triglyceride levels and lipid accumulation in the liver of obese animals without altering the total body mass of these animals. In cultured myocytes, chromium inhibited thapsigargin-induced ER stress. Taken together, these results demonstrate that chronic chromium therapy may have beneficial effects in the treatment of diabetes and insulin resistance associated with obesity.
The beneficial effects of chromium complexes in animal models of diabetes and insulin resistance have been previously reported. Using an insulin-resistant, type 2 diabetic rat model (JCR:LA-corpulent rat), Cefalu and co-workers have demonstrated that supplementation of chromium picolinate at a dose of ∼10 μg chromium/kg/day for 12 weeks improves glucose disposal rates and enhances insulin-stimulated phosphorylation of IRS-1 and PI-3K activity in skeletal muscle. (The paper erroneously reported the chromium dose as 18 μg/kg/day) (22). Their data suggest that chromium picolinate improves insulin action by augmenting insulin signal transduction, which is in accordance with the results of the current study. Clodfelder and co-workers have shown that oral treatment with much higher doses of chromium (1,000 μg Cr/kg) given as a trinuclear chromium propionate complex causes a reduction in plasma insulin, glycated hemoglobin, and plasma lipids in Zucker obese rats (model for early stages of type 2 diabetes) and Zucker diabetic fatty rats (a model for type 2 diabetes). Despite the high dose of chromium used in these experiments, the animals failed to show any changes in glucose levels following a GTT, although there was a reduction in post-GTT plasma insulin levels (23). In a rat model of dexamethasone-induced diabetes, Kim and co-workers have shown that higher doses of chromium picolinate (30 mg/kg/day corresponding to 3.7 mg/kg/day of elemental chromium) cause a significant decrease in plasma glucose levels following an insulin tolerance test (24). In accordance with the data from Clodfelder and co-workers (23), treatment with chromium did not lower blood-glucose levels following GTT although insulin levels were significantly reduced (24). It difficult to compare these studies with the present study owing to the different sources and doses of chromium used and differences in the animal species and model employed. However, taken together, these results help to further substantiate the claim that chromium and its complexes may have beneficial effects in the treatment or control of diabetes and insulin resistance.
Previous studies (17,22,23) have demonstrated that treatment with chromium may reduce serum lipid levels in diabetic conditions. However, the present study fails to confirm any lipid-lowering effect from chromium treatment in obese mice. This difference may be attributed to the low dose of chromium (∼3.8 μg/kg/day of elemental chromium) used in the current study. Although chromium treatment failed to reduce serum lipid levels in this study, it significantly reduced liver triglyceride levels and liver lipid accumulation. The disparity between the serum and liver lipid levels suggests that chromium may be acting at the level of lipid transporters either enhancing the transport of lipids from the liver or preventing active reuptake of lipids by the liver. Recent studies show that chromium attenuates ABCA1, a protein that regulates cholesterol efflux, suggesting a role for chromium in lipid transport (25).
Agents that reduce ER stress have been shown to enhance insulin signaling and ameliorate glucose intolerance (14). ER stress is thought to suppress insulin receptor signaling through the hyper-activation of c-Jun N-terminal kinase (JNK) (26). Insulin signaling is negatively regulated by JNK through serine phosphorylation of residue 307 within IRS-1 in liver and adipose tissues (27). The phosphorylation of IRS-1 Ser307 prevents association of its protein tyrosine binding with insulin receptor β-subunit, thereby preventing IRS-1 binding to the receptor and insulin-dependent activation of PI3-kinase (20). Additionally, Ser307 phosphorylation of IRS-1 accelerates its ubiquitin-proteosome-mediated degradation (28). Attenuation of ER stress, JNK activation, and Ser307 phosphorylation of IRS-1 may represent a novel mechanism by which chromium imparts its beneficial effects in improving insulin sensitivity.
In summary, the present study shows that chronic administration of low-molecular chromium complexes improves glucose tolerance and ER stress in obese type 2 diabetic mice. Chromium treatment also attenuates hepatic triglyceride levels and lipid accumulation. These effects of chromium may be mediated by attenuation of obesity and insulin resistance stimulated ER stress and consequent reduction in JNK activation. Thus, the ability of chromium complexes to alleviate ER stress and improve glucose tolerance provides further support for the use of chromium in treatment or prophylaxis of diabetes and related diseases.
This work was supported by the American Diabetes Association Junior Faculty Award to N.S. We thank Ms Virginia L. Cole for editing the manuscript.