Chromium supplementation enhances insulin ... - Wiley Online Library

ORIGINAL ARTICLE

doi: 10.1111/j.1463-1326.2008.00936.x

Chromium supplementation enhances insulin signalling in skeletal muscle of obese KK/HlJ diabetic mice W.-Y. Chen,1 C.-J. Chen,2 C.-H. Liu1 and F. C. Mao1 1

Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan

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Aim: Chromium is an essential nutrient required for glucose and lipid metabolism. Laboratory and clinical evidences indicate that chromium supplementation may improve insulin sensitivity by enhancing intracellular signalling. Considerable evidence suggests that serine phosphorylation of insulin receptor substrate 1 (IRS1) at 307 residue (IRS1-Ser307) inhibits insulin signalling and results in peripheral insulin resistance. Therefore, we investigated whether chromium-associated insulin action was mediated by modulation of IRS1-Ser307 phosphorylation. Methods: Male KK/HlJ mice (genetically obese and insulin resistant) were supplemented daily with chromium-containing milk powder or placebo for 7 weeks. In analysing functionally characterized insulin resistance, the changes of blood biochemicals, inflammatory factors and insulin signalling molecules in skeletal muscle were analysed. Results: Using KK mice model, we demonstrated that daily supplementation of trivalent chromium-containing milk powder reduced serum levels of glucose, insulin and triglycerides, and improved glucose and insulin tolerance. Mechanistic study showed that chromium supplementation activated postreceptor insulin signalling such as increasing IRS1, IRS1 tyrosine phosphorylation, p85a regulatory subunit of phosphatidylinositol 3-kinase and glucose transporter 4 expression, stimulating Akt activity, downregulating c-Jun N-terminal kinase (JNK) activity and decreasing IRS1 ubiquitinization and insulin resistance-associated IRS1 phosphorylation (IRS1-Ser307) in skeletal muscle. In addition, chromium supplementation attenuated pro-inflammatory cytokine expression in both blood circulation and skeletal muscle. Conclusion: Our data suggest that chromium-containing milk powder supplementation can provide a beneficial effect in diabetic subjects by enhancing insulin signalling in skeletal muscle. The improvement in insulin signalling by chromium was associated with the decreased IRS1-Ser307 phosphorylation, JNK activity and pro-inflammatory cytokine production. Keywords: chromium, inflammation, insulin resistance, IRS-ser307, JNK Received 29 February 2008; accepted 12 June 2008

Introduction Insulin resistance resulting in hyperglycaemia and compensatory hyperinsulinaemia dysregulates many physiological processes that contribute to life-threatening metabolic diseases and is a known risk factor for the development of cardiovascular disorders [1]. Type 2 diabetes is one of the world’s most important chronic diseases because of its increasing prevalence and

complications [2]. Insulin resistance has been recognized as a main pathogenic factor in the development of type 2 diabetes. Clinical studies have revealed that hypoglycaemic treatment with little effect on insulin resistance may not significantly reduce obesity and diabetes-associated complications [3,4]. Therefore, it is evident that insulin resistance is a critical therapeutic target for the control of diabetes-associated complications.

Correspondence: Frank Chiahung Mao, Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan. E-mail: [email protected] 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd

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Diabetes, Obesity and Metabolism, 11, 2009, 293–303

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Chromium supplementation in obese KK/HlJ diabetic mice

The maintenance of blood glucose homeostasis is a complex process involving insulin secretion and insulin metabolic responsiveness. Peripheral skeletal muscle is particularly important for the action of insulin because skeletal muscle is normally responsible for over 75% of all insulin-mediated glucose disposal [5]. Normally, ligand and receptor engagement initiates insulinmediated glucose uptake. Insulin binds to the insulin receptor (IR) on the sarcolemma of skeletal muscle, increases IR tyrosine kinase activity and phosphorylates insulin receptor substrates (IRS). Tyrosine phosphorylated IRS1 recruits and activates phosphatidylinositol 3-kinase (PI3-k), which increases serine phosphorylation of downstream protein kinase B/Akt. The activation of Akt facilitates translocation of glucose transporter 4 (Glut 4) to the sarcolemma to facilitate glucose entry into the cell [6]. Therefore, maintaining proper responses of the IRS–PI3-k–Akt pathway is crucial for normal metabolic signalling of insulin in skeletal muscle. Insulin resistance is characterized mainly by the poor responsiveness of tissues to insulin. Generally, insulin resistance is a consequence of dysregulated insulin signalling that arises from defects at many levels such as decreases in IR concentration and kinase activity, IRS concentration and tyrosine phosphorylation, PI3-k/Akt activity and glucose transporter translocation or increases in protein or lipid phosphatase activity [7]. Covalent modification of the IRS proteins by serine phosphorylation is another mechanism resulting in insulin resistance. Phosphorylation of IRS1 at serine residue (312 in humans and 307 in rats) is an emerging inhibitory target for impaired insulin signalling [8]. Phosphorylation of Ser307 inhibits insulin-stimulated tyrosine phosphorylation and activation of IRS1. Several intracellular protein kinases contribute to this site of phosphorylation, such as Akt and c-Jun N-terminal kinase (JNK) [9,10]. Because this modification plays an important role in interference with insulin signalling, the regulation of Ser307 phosphorylation of IRS1 is critical to reveal a molecular basis for insulin resistance. Chromium is an essential nutrient required for optimal insulin activity and normal carbohydrate and lipid metabolism [11]. Accumulating evidence shows that tissue chromium levels of subjects with diabetes are lower than those of normal control subjects, and a correlation exists between low circulating levels of chromium and the incidence of type 2 diabetes [12–14]. It has been proposed that chromium may increase the number of IRs, enhance receptor binding and enhance intracellular signalling leading to the improvement of insulin resistance [15–18]. However, it is not clear whether the inhibitory Ser307 phosphorylation of IRS1 plays a role in the determination

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of chromium-mediated improvement in insulin resistance. In this study, we investigated whether chromium improved skeletal muscle insulin signalling in obese KK/ HlJ diabetic mice, and if that proved to be the case, tried to determine the potential upstream regulators involved.

Methods Chromium-containing Milk Powder Milk powder containing 10 p.p.m. trivalent chromium was provided by Maxluck Biotechnology Corporation, Taipei, Taiwan [19].

Animals and Diets Male KK/HlJ mice, polygenic models of type 2 diabetes, which exhibit a multigenic syndrome of moderate obesity, hyperinsulinaemia and hyperglycaemia [20,21], were maintained on a 12-h light/12-h dark cycle with free access to regular mouse chow (5008 Rodent LabDiet; PMI Nutrition International, St Louis, MO, USA) and water. At 10 weeks of age, the KK/HlJ mice were switched to feed with high-fat chow (high-fat Rodent TestDiet; PMI Nutrition International; 67% of calories provided by fat) for the enhancement of obesity and hyperlipidaemia. After 4 weeks of induction, obese mice (body weight (BW) between 37 and 38 g) were selected and were randomly divided into two groups. The chromium group (n ¼ 12) was supplemented with trivalent chromium formulated with milk powder (80 mg/kg BW/day) for 7 weeks and the other group (n ¼ 12) was supplemented with placebo milk powder for the same time period. The caloric content in regular chow, high-fat chow and chromiumcontaining milk powder is 4.15, 7.10 and 4 kcal/g respectively. In all experiments, the authors adhered to the Guidelines for the Care and Use of Laboratory Animals, as recommended by the Taiwan government.

Chromium Analysis Collected tissue samples were digested by adding 65% nitric acid and heated at 65 °C for 1 h [22]. After digestion, the concentration of chromium in each sample was analysed by graphite furnace atomic absorption spectrophotometry (Hitachi Z-2000 series polarized Zeeman atomic absorption spectrophotometer).

Glucose Tolerance Test [23] Before chromium supplementation and after 5 weeks of supplementation, the obese mice were given the glucose # 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd

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tolerance test by intraperitoneal glucose injection (0.5 g/ kg BW) after an overnight fast. Blood samples were collected from tail veins before and 30, 60, 120 and 180 min after glucose injection. Blood glucose was measured by glucose oxidase reaction (blood glucose monitoring system, model One Touch II; LifeScan, Milpitas, CA, USA). Insulin Tolerance Test After 6 weeks of supplementation, obese mice were given the insulin tolerance test by intraperitoneal insulin injection (1 U/kg BW) after an overnight fast. Blood glucose was measured at 0, 30 and 60 min after the insulin injection. Biochemical Analyses After the experiments, blood samples were collected from overnight fasting animals. Blood glucose was measured by glucose oxidase reaction. Serum triglycerides and cholesterols were measured by clinical chemistry analysers (Hitachi Autoanalyzer 7070 and Roche I 800). The levels of insulin, hs-CRP, TNF-a and IL-6 were determined with enzyme immunosorbent assay (ELISA) kits (insulin: Crystal Chem Inc., Chicago, IL; hs-CRP: Kamiya Biomedical Company, Seattle, WA, USA; TNF-a and IL-6: R&D Systems, Minneapolis, MN, USA), following the procedures provided by the respective manufacturers. Homeostasis Model Assessment Index Insulin sensitivity was assessed from fasting insulin and glucose levels and by the previously validated homeostasis model assessment (HOMA) insulin resistance index (HOMA-IR) [24] as follows: HOMA-IR ¼ fasting insulin (mU/ml) fasting glucose (mmol/l)/22.5. The function of pancreatic b-cell was assessed by HOMA index (HOMA-B) [24]: [fasting insulin (mU/L) 20/fasting glucose (mmol/l) 3.5]. Tissue Preparation, Immunoprecipitation and Western Blot Analysis After the experiments, the animals were anaesthetized, perfused with cold normal saline and sacrificed. Both gastrocnemius muscles were removed, frozen in liquid nitrogen and kept at 80 °C until processing. For protein extraction, tissues were placed in ice-cold protein lysis buffer (1% Triton X-100; 50 mM Tris–HCl, pH 7.6; 150 mM NaCl) and 1% protease inhibitor cocktail. After homogenization on ice, the tissue debris were pelleted by centrifugation at 17 000 g for 15 min at 4 °C. The protein concentrations in supernatants were determined by the 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd


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Bradford assay (Bio-Rad, Richmond, CA, USA). For immunoprecipitation, protein A-agarose beads were washed with radioimmunoprecipitation (RIPA) buffer [0.15 mM NaCl, 10 mM phosphate, 1% Nonidat P-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulphate (SDS)] and then incubated with antibody against IRS1 (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. After removal of the unbound antibodies, protein extracts (1000 mg) were added with gentle shaking and incubated for an additional 4 h at room temperature. Immunoprecipitates were washed once with RIPA buffer and then three times with buffer containing 150 mM NaCl, 50 mM Tris–HCl (pH 7.6) and 0.1 mM EDTA. Finally, the immunoprecipitates were subjected to western blot with antibodies against IRS1-Ser307 (1 : 500; Santa Cruz Biotechnology), phosphotyrosine (1 : 1000; BD Transduction Laboratories), ubiquitin (1 : 1000; Santa Cruz Biotechnology) and IRS1 (1 : 500; Santa Cruz Biotechnology). For western blot, equal amounts of protein (100 mg) were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were first incubated with 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h at room temperature to reduce non-specific binding. Membranes were washed with PBS containing 0.1% Tween-20 (PBST), and then incubated for 1 h at room temperature with the indicated antibodies including TNF-a (1 : 1000; Santa Cruz Biotechnology), IL6 (1 : 1000; R&D Systems), IR (1 : 100; Abcam, Cambridge, MA, USA), PI3-k p85a (1 : 500; Santa Cruz Biotechnology), Akt (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), Akt-Ser473 (1 : 1000; Cell Signaling Technology), JNK-Thr183/Tyr185 (1 : 1000; R&D Systems), JNK (1 : 1000; R&D Systems), Glut 4 (1 : 5000; Abcam) and b-actin (1 : 2000; Santa Cruz Biotechnology). After the membranes were washed again with PBST buffer, a 1 : 10 000 dilution of horseradish peroxidase-labelled immunoglobulin G was added at room temperature for 1 h. The blots were developed using ECL Western blotting reagents and quantified by optical densitometry (Image Master ID; Pharmacia Biotech, Upsalla, Sweden) of developed autoradiographs. The intensity of each signal was corrected by the values obtained from the immunodetection of b-actin, and the relative protein intensity was expressed as folds of the content in the placebo group.

Isolation of RNA and Reverse Transcriptase-polymerase Chain Reaction (RT–PCR) The isolation of RNA, synthesis of cDNA, and PCR were carried out as previously reported [25]. DNA fragments of

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specific genes and internal controls were co-amplified in one tube. The PCR reaction was carried out in the following conditions: one cycle of 94 °C for 3 min, 30 cycles of (94 °C for 50 s, 61 °C for 40 s and 72 °C for 45 s) and then 72 °C for 5 min. The amplified DNA fragments were resolved by 1.5% agarose gel electrophoresis and stained with ethidium bromide. The intensity of each signal was determined by a computer image analysis system (IS1000; Alpha Innotech Corporation San Leandro, CA, USA). The primer sets used in this study were 5¢-CCCGTTCGGTGCCAAATAGCCGT and 5¢-CCTTGAGTGTCTGCGCGAATCACTTTG for IRS1; 5¢-TCCTGTGGCATCCATGAAACT and 5¢-GGAGCAATGATCTTGATCTTC for b-actin.

Statistical Analysis The data are expressed as mean values standard deviation of the mean ( s.d.). In glucose tolerance test and insulin tolerance test experiments, statistical analysis was carried out using analysis of variance (ANOVA), followed by Bonferroni test to assess the statistical significance between placebo and chromium groups. Comparison of statistical significance between the placebo and chromium groups in other experiments was conducted using Student’s t-test. A level of p < 0.05 was considered statistically significant.

Results Effect of Chromium Supplementation on Animal Growth and Chromium Tissue Distribution High-fat diets have been shown to significantly promote the development of obesity and hyperglycaemia in insulin-resistant, obesity-prone KK/HlJ mice [26]. In our preliminary studies, high fat–fed KK/HIJ mice showed remarkable hyperglycaemia than regular chow-fed KK/ HIJ mice and high fat–fed C57BL/6JNarl mice (data not shown). Thus, we initiated this study using fat-fed KK/HIJ mice model. Daily supplementation of chromium-containing milk powder (80 mg/kg BW/day) for 7 weeks had little effects on body weight, epididymal fat weight and average food intake (table 1). At this supplemented dosage and protocol, the obtained results further showed that chromium supplementation effectively increased chromium level in serum, skeletal and fat. However, the distribution of chromium in urine and liver was not changed (table 2). These results indicate that this supplemented dosage and protocol could increase the absorption and elevate tissue levels of chromium.

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Table 1 Chromium supplementation had little effect on food intake and tissue weight in animals

Body weight (g) Food intake (g/day/mouse) Epididymal fat (g)

Placebo (n 5 12)

Chromium (n 5 12)

41.4 2.6 3.30 0.12 1.46 0.21

39.2 3.9 3.25 0.09 1.31 0.22

Body weight and epididymal fat weight were measured after chromium-containing or placebo milk powder supplementation for 7 weeks. During this period, the average food intake in animals was recorded. Values are expressed as mean s.d.

Effect of Chromium Supplementation on Glucose and Lipid Metabolism In our study, the fasting blood glucose was 115 16 mg/dl in normal diets and 162 19 mg/dl after high-fat diet treatment. Daily supplementation of chromium-containing milk powder for 7 weeks remarkably decreased the fasting blood glucose level in diabetic mice (table 3), indicating a hypoglycaemic effect. Regarding the glucose tolerance, figure 1 shows the blood glucose variations during the glucose tolerance test for the placebo and chromium groups. As shown in figure 1A, at the beginning of the supplementation, both groups exhibited similar glucose tolerance response. Remarkably, after chromium supplementation (figure 1B), the clearance of blood glucose after glucose injection was comparably effective. In addition to increased glucose clearance, chromium supplementation also lowered the plasma level of triglycerides (table 3). However, there was no significant difference in total cholesterol between these two groups (table 3). These results indicate that chromium-containing milk powder supplementation may improve glucose and lipid metabolism in type 2 diabetic mice. Effect of Chromium Supplementation on Insulin Sensitivity Analysis of fasting blood insulin revealed that the highfat diet increased the level from 0.35 0.28 to Table 2 Chromium supplementation increased tissue level of chromium in animals Placebo (n 5 12) Serum (ppb) Urine (ppb) Skeletal muscle (ppb) Fat (ppb) Liver (ppb)

0.21 8.82 34.0 23.9 73.2

0.056 7.24 8.7 15.1 25.9

Chromium (n 5 12) 0.32 7.18 61.9 47.9 56.8

0.178* 6.14 24.8** 19.2** 12.3

Levels of chromium in tissues were determined after chromium-containing or placebo milk powder supplementation for 7 weeks. Values are expressed as mean s.d. *p < 0.05 and **p < 0.001 vs. placebo group. # 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd

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Table 3 Chromium supplementation improved metabolic parameters and insulin sensitivity index Placebo (n 5 12) Blood glucose (mg/dl) Triglycerides (mg/dl) Total cholesterol (mg/dl) Insulin (ng/ml) HOMA-IR HOMA-B

162 206 147 1.76 16.2 158.1

19 39 13 0.62 6.0 83.3

Chromium (n 5 12) 132 173 145 1.30 9.6 172.4

16*** 49* 19 0.58* 4.0*** 105.6

HOMA, homeostasis model assessment; IR, insulin receptor.Levels of fasting blood glucose, triglycerides, total cholesterol, and insulin were determined after chromium-containing or placebo milk powder supplementation for 7 weeks. Insulin sensitivity index (HOMA-IR) and the function of pancreatic b-cell (HOMA-B) were calculated from fasting serum glucose and insulin values as described in Methods. Values are expressed as mean s.d. *p < 0.05 and ***p < 0.001 vs. placebo group.

1.76 0.62 ng/ml and chromium supplementation markedly decreased the insulin level (table 3), indicating a hypoinsulinaemic effect. The analysis of insulin tolerance was performed by measuring the dynamic

Fig. 1 Chromium supplementation improved glucose tolerance. Glucose tolerance test was performed on animals (A) before and (B) 5 weeks after chromium-containing milk powder supplementation. The level of blood glucose was measured at 0, 30, 60, 120 and 180 min after intraperitoneal glucose (0.5 g/kg BW) injection in both the placebo (n ¼ 12) and chromium (n ¼ 12) groups. Values are expressed as mean s.d. *p < 0.05 and **p < 0.01 vs. the placebo group. 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd

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change of blood glucose after insulin injection. The elimination of blood glucose was more efficient in chromium-supplemented animals as shown by glucose variations (figure 2A) and decreased percentage (figure 2B). Furthermore, the chromium supplementation significantly decreased the HOMA-IR in the chromium group when compared with the placebo group (table 3). In contrast, the function of pancreatic b-cell was not changed as evidenced by HOMA-B (table 3). These results indicate that chromium-containing milk powder supplementation may improve insulin sensitivity in type 2 diabetic mice.

Effect of Chromium Supplementation on Pro-inflammatory Cytokine Production Chronic inflammation and elevated expression of proinflammatory cytokines correlate well with several clinical disorders, including obesity and insulin resistance [27,28]. In our study, ELISA data illustrated the effect of

Fig. 2 Chromium supplementation improved insulin tolerance. Insulin tolerance test was performed on animals 6 weeks after chromium-containing milk powder supplementation. The level of blood glucose was measured at 0, 30 and 60 min after intraperitoneal insulin (1 U/kg BW) injection in both the placebo (n ¼ 12) and the chromium (n ¼ 12) groups (A). The relative glucose level at different time points was also expressed as a fold change from basal (0 min) (B). Values are expressed as mean s.d. *p < 0.5, **p < 0.01 and ***p < 0.001 vs. the placebo group.

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chromium supplementation on hs-CRP, TNF-a and IL-6 levels in the serum of diabetic mice. There was a significant reduction in hs-CRP, TNF-a and IL-6 levels in serum of the chromium group compared with that of the placebo group (figure 3). Skeletal muscles are critical target tissues for the development of insulin resistance [5]. In addition to reducing circulating levels of TNF-a and IL-6, chromium-containing milk powder also downregulated TNF-a and IL-6 expression in skeletal muscles, as demonstrated by the results of western blot analysis (figure 4). These findings reveal that chromium-containing milk powder supplementation can attenuate inflammatory response in diabetic mice by lowering pro-inflammatory cytokines in blood circulation and in skeletal muscles.

Fig. 4 Chromium supplementation reduced the expression of TNF-a and IL-6 in muscle. Cellular proteins were extracted from muscles in the placebo ( ; n ¼ 12) and chromium ( ; n ¼ 12) groups after 7 weeks of chromium-containing milk powder supplementation and subjected to western blot with antibodies against TNF-a, IL-6 and b-actin. Two representative samples from each group are shown (A). The relative protein intensity was expressed as folds of the content in the placebo group (B). Values are expressed as mean s.d. *p < 0.5 and **p < 0.01 vs. each sample from the placebo group.

Effect of Chromium Supplementation on Insulin Signalling in Skeletal Muscles

Fig. 3 Chromium supplementation reduced circulating pro-inflammatory cytokines. Serum samples were collected from the placebo ( ; n ¼ 12) and chromium ( ; n ¼ 12) groups after 7 weeks of chromium-containing milk powder supplementation. The samples were then subjected to measurement of hs-CRP, TNF-a and IL-6 with commercially available enzyme immunosorbent assay kits. Values are expressed as mean s.d. *p < 0.5, **p < 0.01 and ***p < 0.001 vs. the placebo group.

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To elicit the potential mechanisms underlying chromiummediated improvement in insulin resistance, we analysed the alteration of intracellular insulin signalling molecules in skeletal muscles. Representative blots of immunoprecipitation/western blot and western blot analysis of IR, P-IRS1-Ser307, P-IRS1-Tyr, IRS1-Ub, IRS1, PI3-k p85a, P-Akt-Ser473, Akt, P-JNK, JNK and Glut 4 are shown in figure 5A. There was no difference in the IR protein content between the placebo and the chromium groups (figure 5A). Significantly, there was an increased expression of IRS1 tyrosine phosphorylation (P-IRS1-Tyr), IRS1, PI3-k p85a, Akt phosphorylation (P-Akt-Ser) and Glut 4 in diabetic mice supplemented with chromiumcontaining milk powder when compared with the placebo group (figure 5A). However, chromium supplementation decreased JNK phosphorylation, IRS1 phosphorylation in 307 serine residue and IRS1 ubiquitinization (figure 5A). RNA analysis revealed that the transcriptional level of IRS1 was not changed by chromium supplementation # 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd

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Fig. 5 Chromium supplementation enhanced insulin signalling in muscles. Cellular proteins were extracted from muscles in the placebo ( ; n ¼ 12) and chromium ( ; n ¼ 12) groups after 7 weeks of chromium-containing milk powder supplementation and subjected to western blot with antibodies against insulin receptor (IR), PI3-k p85a, Akt-Ser473 (P-Akt-Ser), Akt, c-Jun N-terminal kinase (JNK)-Thr183/Tyr185 (P-JNK), JNK, Glut 4 and b-actin. Some samples were first immunoprecipitated with anti-insulin receptor substrate 1 (IRS1) antibody followed by western blot with antibodies against IRS1-Ser307 (P-IRS1-Ser307), phosphotyrosine (P-IRS1-Tyr), ubiquitin (IRS1-Ub) and IRS1. Two representative samples from each group and statistical graph are shown (A). Cellular RNAs were extracted from muscles in the placebo ( ; n ¼ 12) and chromium ( ; n ¼ 12) groups after 7 weeks of chromium-containing milk powder supplementation and subjected to RT–PCR for the detection of IRS1 and b-actin mRNA. Two representative samples from each group and statistical graph are shown (B). The relative intensity was expressed as folds of the content in the placebo group. Values are expressed as mean s.d. *p < 0.5, **p < 0.01 and ***p < 0.001 vs. each sample from the placebo group.

(figure 5B). These results indicate that chromiumcontaining milk powder supplementation may enhance postreceptor insulin signalling through activating 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd

positive regulators such as IRS-1, PI3-k p85a, Akt and Glut 4, and/or inactivating negative regulators such as JNK and IRS1-Ser307 in skeletal muscles of KK/H1J mice.

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Discussion In this study, using a KK mice model, we demonstrated the hypoglycaemic and hypoinsulinaemic effects of trivalent chromium in type 2 diabetic animals through a formulation of chromium-containing milk powder (figures 1 and 2, table 3). The results from biochemical study showed that chromium supplementation activated postreceptor insulin signalling such as increasing IRS1 and Glut 4 expression, stimulating PI3-k and Akt activity, downregulating JNK activity and decreasing insulin resistance-associated IRS1 phosphorylation in skeletal muscles (figure 5). In addition, chromium supplementation attenuated pro-inflammatory cytokine expression in both blood circulation and skeletal muscles (figures 3 and 4). Our findings suggest that chromium enhances skeletal muscle cellular insulin signalling, at least in part, by involving immune suppression and finally results in the improvement of insulin resistance. Chromium supplementation facilitates normal protein, fat and carbohydrate metabolism and is widely used by the public in many countries. In this study, we supplemented the diet of obese KK/HlJ diabetic mice with chromium-containing milk powder for 7 weeks. This supplementation did not change animal growth (table 1) but significantly elevated tissue levels of chromium (table 2). The results revealed that chromium supplementation significantly reduced the levels of blood glucose, plasma insulin and triglycerides (table 3) and improved glucose, insulin tolerance (figures 1 and 2) and HOMA-IR (table 3) in type 2 diabetic mice. Similar results demonstrating the ability of chromium to improve glucose and insulin tolerance and to reduce blood glucose and lipids have been reported previously [15,29]. However, chromium supplementation had little effect on reduction of serum cholesterol (table 3). Therefore, our findings imply that the improvement of insulin resistance by chromium-containing milk powder is more relevant than lipid profiles. A human study also demonstrated similar results and conclusions. Daily supplementation of chromium-containing milk powder improved insulin resistance but had little effect on metabolic lipid parameters in type 2 diabetes mellitus patients [19]. Insulin stimulation of IRS1–PI3-k–Akt is required for glucose transporter translocation and is a classic insulinsignalling cascade [6]. Normally, skeletal muscle is the major tissue in glucose metabolism and impaired insulin metabolic signalling in this tissue is crucial for development of systemic insulin resistance [30,31]. In chromium-supplemented animals, elevated expressions of IRS1, activity-associated IRS1 tyrosine phosphorylation,

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p85a regulatory subunit of PI3-k, Akt serine phosphorylation and Glut 4 were detected in skeletal muscle (figure 5A). These findings suggest that this IRS1–PI3k–Akt cascade is one of the potential intervention targets for chromium-mediated improvement of insulin resistance. Generally, insulin resistance is a consequence of dysregulated insulin signalling that arises from various sources. Increasing IR expression and receptor binding is one of the proposed action mechanisms of chromium treatment. Chromium supplementation might increase a chromium-containing oligopeptide present in insulin-sensitive cells that binds to the IR, markedly increasing the activity of insulin-stimulated tyrosine kinase and phosphorylation of IRS1 and Glut 4 [15–18]. In our study, western blot data revealed that there was no difference in IR expression between the placebo and chromium groups in skeletal muscle (figure 5A). That is, chromium might act at postreceptor downstream insulin signalling. There are a number of mechanisms that may lead to an impairment of the insulin-signalling pathway downstream of ligand and receptor engagement. Considerable evidence is largely consistent with the hypothesis that serine phosphorylation of the IR or IRS1 proteins inhibits insulin signalling. More than 100 potential serine phosphorylation sites exist in IRS1. Among them, serine 307 residue (Ser312 in human IRS1) is located next to the phosphotyrosine-binding domain in IRS1 and its phosphorylation inhibits the activation of IRS1 and might target IRS1 for ubiquitin/proteasome-mediated degradation, causing insulin resistance [8]. This insulin resistance-associated inhibitory IRS1-Ser307 phosphorylation was markedly attenuated by chromium (figure 5A). The decreased IRS1-Ser307 phosphorylation might also stabilize IRS1 protein (figure 5A). This hypothesis was supported by the findings that chromium supplementation decreased IRS1 ubiquitinization and increased IRS1 tyrosine phosphorylation (figure 5A). However, the level of IRS1 mRNA was not changed by chromium (figure 5B). That is, the relief of the IRS1-Ser307-negative regulator is one of the intervention targets for chromium’s effect. IRS1-Ser307 is phosphorylated through several mechanisms, including insulin-stimulated kinases or stress-activated kinases like JNK. JNK activity is abnormally elevated in obesity and insulin resistance [9]. It has been reported that activation of JNK induces serine 307 phosphorylation of IRS1 leading to a decrease in the insulin-stimulated PI3-k activity [32]. Our data showed a decrease in JNK activity in parallel with the reduction of IRS1-Ser307 phosphorylation (figure 5A), suggesting that the downregulation of JNK activity may have an important role in enhancing the # 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd

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postreceptor insulin signalling in skeletal muscle of diabetic mice when supplemented with chromiumcontaining milk powder. JNK is a member of the mitogen-activated protein kinase (MAP) family and can be activated by several stress factors, including pro-inflammatory cytokines such as TNF-a and IL-6 [9,33,34]. Accumulating evidence indicates that low-grade chronic inflammation plays a fundamental role in the development of insulin resistance and inflammatory cytokines such as TNF-a and IL-6 likely contribute to the link between inflammation and skeletal muscle insulin resistance [5,28,35–37]. TNF-a is a pleiotropic cytokine and is mainly produced by macrophages and also by many other cells, including skeletal muscle cells. TNF-a is the first cytokine recognized to have a direct role in promoting insulin resistance [38]. Increased levels of TNF-a have been noted in skeletal muscle tissue and cultured skeletal muscle cells from humans and animals with insulin resistance and/ or diabetes [39,40]. TNF-a reduces insulin-stimulated receptor tyrosine kinase activity at low concentrations and can also decrease the expression of IR, IRS1 and Glut 4 at higher concentrations as well as increase the phosphorylation of serine 307 in IRS1 [41]. This relative increase in serine-to-tyrosine phosphorylation may lead to increased ubiquitinization/proteosomal degradation of IRS1 or decreased ability of IRS1 to engage the p85a subunit of PI3-k, thus impairing insulin signalling. Circulating IL-6 levels are also increased in insulin-resistant states such as obesity, impaired glucose tolerance and type 2 diabetes [37,42–44]. In vivo acute IL-6 treatment in mice reduces insulin-stimulated skeletal muscle glucose uptake associated with defects in IRS1/PI3-k activity and increases in fatty acyl-CoA levels in skeletal muscle [45]. Moreover, IL-6 induces rapid recruitment of IRS1 to the IL-6 receptor complex in cultured skeletal muscle cells and induces a rapid and transient IRS1 serine phosphorylation resulting in increased IRS1 ubiquitinization in skeletal muscle tissue [46]. Thus, TNF-a and IL-6 play important roles in insulin resistance and the inflammation process through their multiple actions. In our study, we found that the well-documented proinflammatory markers associated with type 2 diabetes such as TNF-a, IL-6 and hs-CRP were lowered in the chromium-supplemented group. This reduction of proinflammatory cytokine production occurred in systemic blood circulation (figure 3) and in skeletal muscle (figure 4). Together with the previously reported immune suppressive effect of chromium [47], these findings reveal that the downregulation of pro-inflammatory cytokine expression by chromium is one of the potential mechanisms that may attenuate IRS1-Ser307-mediated 2008 The Authors Journal Compilation # 2008 Blackwell Publishing Ltd


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inhibition of insulin signalling in skeletal muscle. Hence, we suggest that the increase in the IRS1–PI3-k–Akt cascade in skeletal muscle by chromium may be linked to the immune suppressive effect by reducing TNF-a and IL-6 expression, and in turn by inhibiting the JNK activity and IRS1-Ser307 phosphorylation. Trivalent chromium is an essential element required for normal glucose and lipid metabolism, and insufficient dietary chromium is associated with maturity-onset diabetes and/or cardiovascular disease [48,49]. Thus, chromium supplement is widely used as an alternative remedy for type 2 diabetes mellitus. In this study, animals daily obtained 3.69 mg chromium/animal and 6.89 mg chromium/animal from chow and supplementation in the placebo and the chromium groups respectively. Our experimental results showed the absorption and elevation of chromium in body tissues (table 2) and further demonstrated its beneficial effect on insulin signalling (figure 5). These improvement effects were also observed in other animal conditions such as regular chow-fed KK/HIJ mice and high fat–fed C57BL/6JNarl mice (data not shown). Unfortunately, the specific pharmacological dosage of chromium was not defined by this study. A recent clinical study showed that chromium-containing milk powder reduced hyperglycaemia and hyperinsulinaemia in type 2 diabetic patients [19]. In parallel with those clinical observations, our findings suggested that appropriate elevation in chromium tissue levels could have beneficial effect on diabetic subjects. Serine phosphorylation of IRS1 at 307 residue contributes to peripheral insulin resistance by interfering with the insulin-stimulated IRS1–PI3-k–Akt signalling cascade. In summary, our data suggest that chromium supplementation can provide a beneficial effect in diabetic subjects by enhancing insulin signalling in skeletal muscle. The improvement in insulin signalling by chromium was associated with decreased IRS1-Ser307 phosphorylation, JNK activity and pro-inflammatory cytokine production. These results suggest that chromium-containing milk powder supplementation might improve insulin resistance through enhancing postreceptor insulin signalling in skeletal muscle involving attenuation of insulin resistance-associated IRS1-Ser307 phosphorylation, at least in part, resulting from inhibition of TNF-a and IL-6-related JNK activation.

Acknowledgements This study was supported by NSC95-2313-B-005-039 from National Science Council, TCVGH-NCHU-957602 from Taichung Veterans General Hospital and National Chung Hsing University, and the Ministry of Education

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(Taiwan) under the ATU plan. We also express our gratitude to Maxluck Biotechnology Corporation, Taipei, Taiwan, for financial support.

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