Calpain-10 gene and protein expression in human skeletal muscle: effect of acute lipid-induced insulin resistance and type 2 diabetes

OBJECTIVE: Our objective was to investigate the effect of lipid-induced insulin resistance and type 2 diabetes on skeletal muscle calpain-10 mRNA and protein levels. RESEARCH DESIGN AND METHODS: In the first part of this study, 10 healthy subjects underwent hyperinsulinemic euglycemic (4.5 mmol/liter) clamps for 6 h with iv infusion of either saline or a 20% Intralipid emulsion (Fresenius Kabi AG, Bad Homburg, Germany). Skeletal muscle biopsies were taken before and after 3- and 6-h insulin infusion and analyzed for calpain-10 mRNA and protein expression. In the second part of the study, muscle samples obtained after an overnight fast in 10 long-standing, sedentary type 2 diabetes patients, 10 sedentary, weight-matched, normoglycemic controls, and 10 age-matched, endurance-trained cyclists were analyzed for calpain-10 mRNA and protein content. RESULTS: Intralipid infusion in healthy subjects reduced whole body glucose disposal by approximately 50% (P<0.001). Calpain-10 mRNA (P=0.01) but not protein content was reduced after 6-h insulin infusion in both the saline and Intralipid emulsion trials. Skeletal muscle calpain-10 mRNA and protein content did not differ between the type 2 diabetes patients and normoglycemic controls, but there was a strong trend for total calpain-10 protein to be greater in the endurance-trained athletes (P=0.06). CONCLUSIONS: These data indicate that skeletal muscle calpain-10 expression is not modified by insulin resistance per se and suggest that hyperinsulinemia and exercise training may modulate human skeletal muscle calpain-10 expression.     

Molecular mechanisms of skeletal muscle insulin resistance in type 2 diabetes

Type 2 (non-insulin-dependent) diabetes mellitus afflicts millions of people worldwide and is one of the main causes of morbidity and mortality. Current therapeutic agents to treat Type 2 diabetes are insufficient and thus, newer approaches are desperately needed. Type 2 diabetes is manifested by progressive metabolic impairments in tissues such as skeletal muscle, adipose tissue and liver, such that these tissues become less responsive to insulin. Skeletal muscle is quantitatively the most important tissue involved in maintaining glucose homeostasis under insulin-stimulated conditions, and is a major site of insulin resistance in Type 2 diabetic patients. At the cellular level, glucose transport into skeletal muscle is the rate-limiting step for whole body glucose uptake and a primary site of insulin resistance in Type 2 diabetes. Thus, skeletal muscle is a key insulin target tissue that harbours intrinsic defects that impinges upon whole body glucose homeostasis. Here, we review the current knowledge of signalling events that regulate glucose transport in human skeletal muscle.     

Impairment of insulin-induced vasodilation is associated with muscle insulin resistance in type 2 diabetes

To clarify the association between the actions of insulin on the vascular wall and on the muscles in diabetes, we evaluated insulin-mediated vasodilation and muscle glucose uptake simultaneously using the euglycemic hyperinsulinemic glucose clamp technique and the calculation of total peripheral vascular resistance (TPR) from arterial pulse wave analysis in 19 Japanese patients with type 2 diabetes who had no signs of atherosclerosis. During the clamp study, the plasma norepinephrine (NE) level and plasma renin activity (PRA) increased without showing any significant correlation to the glucose infusion rate (GIR); a marker of muscle insulin sensitivity, and no changes of other plasma vasoactive hormone levels were observed. TPR decreased over time during the clamp study. The decrease of TPR from baseline was 0.88 +/- 0.02 at 1 h (mean +/- S.E.M., P < 0.01) and 0.79 +/- 0.03 at 2 h (P < 0.01), and the relative change in TPR from baseline was negatively correlated with GIR (r = -0.48 at 1 and 2 h; both P < 0.05). Our results suggest that there is also insulin resistance in the vascular wall, and this phenomenon may be associated with muscle insulin resistance in type 2 diabetes.     

Calpain 3 gene expression in skeletal muscle is associated with body fat content and measures of insulin resistance

OBJECTIVE: To investigate whether skeletal muscle gene expression of calpain 3 is related to obesity and insulin resistance. DESIGN: Cross-sectional studies in 27 non-diabetic human subjects and in Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes. MEASUREMENTS: Expression of CAPN3 in skeletal muscle was measured using Taqman fluorogenic PCR. In the human subjects, body composition was assessed by DEXA and insulin sensitivity was measured by euglycemic-hyperinsulinemic clamp. In Psammomys obesus, body composition was determined by carcass analysis, and substrate oxidation rates, physical activity and energy expenditure were measured by whole-body indirect calorimetry. RESULTS: In human subjects, calpain 3 gene expression was negatively correlated with total (P = 0.022) and central abdominal fat mass (P = 0.034), and with blood glucose concentration in non-obese subjects (P = 0.017). In Psammomys obesus, calpain 3 gene expression was negatively correlated with circulating glucose (P = 0.013) and insulin (P = 0.034), and with body fat mass (P = 0.049). Indirect calorimetry revealed associations between calpain 3 gene expression and carbohydrate oxidation (P = 0.009) and energy expenditure (P = 0.013). CONCLUSION/INTERPRETATION: Lower levels of expression of calpain 3 in skeletal muscle were associated with reduced carbohydrate oxidation and elevated circulating glucose and insulin concentrations, and also with increased body fat and in particular abdominal fat. Therefore, reduced expression of calpain 3 in both humans and Psammomys obesus was associated with phenotypes related to obesity and insulin resistance.     

Extracellular superoxide dismutase gene polymorphism is associated with insulin resistance and the susceptibility to type 2 diabetes

Oxidative stress has been implicated in pancreatic beta-cell damage, insulin resistance and vascular function in diabetic patients and the dysfunction of antioxidant enzymes may be associated with the pathogenesis of diabetes. Extracellular superoxide dismutase (EC-SOD) is found in the extracellular matrix of tissues and the major scavenger of superoxide radical. To investigate the role of genetic variability for the pathogenesis of type 2 diabetes, we scanned the protein coding exon and flanking introns of EC-SOD gene for mutation in Japanese type 2 diabetic patients. We identified two missense mutations, Ala40Thr (GCG-->ACG) and Arg213Gly (CGG-->GGG), and a silent mutation, Leu53Leu (CTG-->TTG). For one of these variants, the Ala40Thr polymorphism, the frequency of Thr allele and the number of subjects with Thr allele (Ala/Thr+Thr/Thr) were higher in type 2 diabetic patients (n=205) than those in non-diabetic subjects (n=220) (33.2% versus 24.1%, p=0.003 and 55.6% versus 42.7%, p=0.008, respectively). The patients with Thr allele also showed earlier age at diagnosis of diabetes (42.2+/-7.8 years versus 44.4+/-6.9 years, p=0.037) and higher prevalence of hypertension (53.5% versus 38.5%, p=0.032) than those without the allele. Insulin sensitivity, furthermore, was evaluated in 71 type 2 diabetic patients with short insulin tolerance test (SITT). The patients with Thr allele showed lower insulin sensitivity (Kitt value of SITT) than those without the allele (1.78+/-0.78%/min versus 2.33+/-1.02%/min, p=0.012), although no significant differences in other clinical and biochemical characteristics were observed between two groups. These results suggest that the genetic variant of EC-SOD gene is associated with insulin resistance and the susceptibility to type 2 diabetes.     

脂肪分化相关蛋白基因与2型糖尿病相关

目的通过单核苷酸多态性(SNP)的检测及基因分型来研究脂肪分化相关蛋白基因(ADRP)与2型糖尿病的相关性。方法使用PCR测序方法,对ADRP基因的启动子、外显子以及临近的内含子,分别在正常人群、2型糖尿病人群进行测序,确定该基因是否与华东地区汉族2型糖尿病患者相关。结果所测ADRP基因片段总长度5003个碱基,有23个SNP,高频、低频分别是5个、18个。启动子的一个SNP(P/-407)在2型糖尿病人群与正常人群的等位基因型、基因型差异均有显著性(P<0.05);两个错义突变Ser251Pro、Thr396Ala分别在两个2型糖尿病家系中传递。结论ADRP可能与华东地区汉族人2型糖尿病相关。     

Adiponectin, skeletal muscle adiponectin receptor expression and insulin resistance following dexamethasone

Objective Skeletal muscle is a major site of adiponectin action and of glucocorticoid‐induced insulin resistance. Little human data exist however, regarding the impact of exogenous glucocorticoids on adiponectin receptors in skeletal muscle. Design and patients Twelve subjects with type 2 diabetes and 12 controls underwent blood sampling and muscle biopsy of vastus lateralis before and after 4 days of 4 mg dexamethasone. Measurements (i) Total and high molecular weight (HMW) plasma adiponectin, glucose and insulin; (ii) Skeletal muscle adiponectin receptor AdipoR1 and AdipoR2 mRNA levels by quantitative real time RT‐PCR. Results Baseline total adiponectin (8·0 ± 0·89 vs. 12·5 ± 1·46 µg/ml, P = 0·013), HMW adiponectin (2·8 ± 0·44 vs. 5·9 ± 1·04 µg/ml, P = 0·014) and AdipoR2 mRNA levels (mean ΔC_T 14·71 ± 0·35 vs. 13·37 ± 0·28, P = 0·017) were significantly lower in diabetic subjects. After dexamethasone, AdipoR2 mRNA fell in the controls but there was no change in the diabetic group, while there was a significant increase in total (P = 0·002) and HMW adiponectin (P < 0·001) across both groups. Total and HMW plasma adiponectin correlated with clinical and biochemical measures of insulin sensitivity. However following dexamethasone which increased insulin resistance, the relationship between adiponectin and the biochemical measures was lost. Conclusions Plasma adiponectin and skeletal muscle AdipoR2 mRNA expression are reduced in subjects with diabetes; both are likely to contribute to the observed insulin resistance. Dexamethasone inhibits AdipoR2 mRNA expression in nondiabetic subjects, while there is a small rise in plasma adiponectin levels. The close relationship between plasma adiponectin and biochemical measures of insulin sensitivity is lost in the setting of glucocorticoid‐induced insulin resistance.     

Restoration of insulin secretion in pancreatic islets of protein-deficient rats by reduced expression of insulin receptor substrate (IRS)-1 and IRS-2

Autocrine and paracrine insulin signaling may participate in the fine control of insulin secretion. In the present study, tissue distribution and protein amounts of the insulin receptor and its major substrates, insulin receptor substrate (IRS)-1 and IRS-2, were evaluated in a model of impaired glucose-induced insulin secretion, the protein-deficient rat. Immunoblot and RT-PCR studies showed that the insulin receptor and IRS-2 expression are increased, whilst IRS-1 protein and mRNA contents are decreased in pancreatic islets of protein-deficient rats. Immunohistochemical studies revealed that the insulin receptor and IRS-1 and -2 are present in the great majority of islet cells; however, the greatest staining was localized at the periphery, suggesting a co-localization with non-insulin-secreting cells. Exogenous insulin stimulation of isolated islets promoted higher insulin receptor and IRS-1 and -2 tyrosine phosphorylation in islets from protein-deficient rats, as compared with controls. Moreover, insulin-induced IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activity are increased in islets of protein-deficient rats. The reduction of IRS-1 and IRS-2 protein expression in islets isolated from protein-deficient rats by the use of antisense IRS-1 or IRS-2 phosphorthioate-modified oligonucleotides partially restored glucose-induced insulin secretion. Thus, the impairment of insulin cell signaling through members of the IRS family of proteins in isolated rat pancreatic islets improves glucose-induced insulin secretion. The present data reinforced the role of insulin paracrine and autocrine signaling in the control of its own secretion.     

Common variation in oxidative phosphorylation genes is not a major cause of insulin resistance or type 2 diabetes

Aims/hypothesis

There is substantial evidence that mitochondrial dysfunction is linked to insulin resistance and is present in several tissues relevant to the pathogenesis of type 2 diabetes. Here, we examined whether common variation in genes involved in oxidative phosphorylation (OxPhos) contributes to type 2 diabetes susceptibility or influences diabetes-related metabolic traits.

Methods

OxPhos gene variants (n?=?10) that had been nominally associated (p?n?=?10,108) were selected for follow-up in 3,599 type 2 diabetic and 4,956 glucose-tolerant Danish individuals. A meta-analysis of these variants was performed in 11,729 type 2 diabetic patients and 43,943 non-diabetic individuals. The impact on OGTT-derived metabolic traits was evaluated in 5,869 treatment-naive individuals from the Danish Inter99 study.

Results

The minor alleles of COX10 rs9915302 (p?=?0.02) and COX5B rs1466100 (p?=?0.005) showed nominal association with type 2 diabetes in our Danish cohort. However, in the meta-analysis, none of the investigated variants showed a robust association with type 2 diabetes after correction for multiple testing. Among the alleles potentially associated with type 2 diabetes, none negatively influenced surrogate markers of insulin sensitivity in non-diabetic participants, while the minor alleles of UQCRC1 rs2228561 and COX10 rs10521253 showed a weak (p?p?Conclusions/interpretation We cannot rule out the possibility that common variants in or near OxPhos genes may influence beta cell function in non-diabetic individuals. However, our quantitative trait studies and a sufficiently large meta-analysis indicate that common variation in proximity to the examined OxPhos genes is not a major cause of insulin resistance or type 2 diabetes.     

Increased insulin sensitivity and upregulation of insulin receptor, insulin receptor substrate (IRS)-1 and IRS-2 in liver of Ames dwarf mice

In the present study we have used hypopituitary Ames dwarf mice, which lack GH, prolactin and TSH, to investigate the consequences of the deficiency of these hormones on glucose homeostasis and on the initial components of the insulin signal transduction pathway in the liver. Ames dwarf mice displayed hypersensitivity to insulin since they maintained lower fasting glucose concentrations (73% of control values), had significantly reduced amounts of insulin (58% of control values), and exhibited an increased hypoglycemic response to exogenous insulin. Probably as a result of reduced insulin production, Ames dwarf mice displayed intolerance to glucose. The insulin-stimulated phosphorylation of the insulin receptor (IR) tended to be increased in the liver of Ames dwarf mice, while IR receptor protein content was increased by 38%. Insulin-stimulated phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 was increased by 61 and 72% respectively, while IRS-1 and IRS-2 protein levels were increased by 76 and 95%. The insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase with IRS-1 was increased by 28%, but unaltered with IRS-2. Interestingly, while the insulin-stimulated phosphotyrosine-derived PI 3-kinase activity was not changed, insulin-stimulated protein kinase B activation was increased by 41% in this tissue. These alterations may account for the insulin hypersensitivity exhibited by these animals. The present findings in long-lived Ames dwarf mice add to the evidence that insulin signaling is importantly related to the regulation of aging and life span.     

Exercise increases skeletal muscle GLUT4 gene expression in patients with type 2 diabetes

The aim of the study was to determine the effect of a single bout of exercise on GLUT4 gene expression in muscle of patients with type 2 diabetes (T2D) and control subjects, matched for age and body mass index. Nine patients with T2D and nine control subjects performed 60 min of cycling exercise at ~55% peak power (W(max) ). Skeletal muscle biopsies were obtained at baseline, immediately post and 3-h post exercise. GLUT4 mRNA expression increased (p < 0.05) to a similar extent immediately post exercise in control (~60%) and T2D (~66%) subjects, and remained elevated (p < 0.05) 3-h post exercise with no differences between groups. Similarly, p-AMP-activated protein kinase, p38 mitogen-activated kinase and proliferator-activated receptor gamma co-activator-alpha mRNA expression were increased (p < 0.05) post exercise, and were not different between the groups. In conclusion, a single bout of exercise increased skeletal muscle GLUT4 mRNA expression in patients with T2D to a similar extent as in control subjects.     

Adiponectin receptor gene expression in human skeletal muscle cells is not regulated by fibrates and thiazolidinediones

BACKGROUND: Thiazolidinediones as PPARgamma agonists and fibrates as PPARalpha agonists improve insulin sensitivity in insulin-responsive tissues. Recent data show an induction of adiponectin receptor 2 (AdipoR2) by PPARalpha and PPARgamma agonists in human macrophages. OBJECTIVE: In this study, we examined the effects of thiazolidinediones and fibrates on the expression of adiponectin receptors in human skeletal muscle cells, an important cell type in the context of insulin resistance. RESULTS AND METHODS: In vitro differentiated human myotubes treated with troglitazone or rosiglitazone (20 h) showed no significant changes in AdipoR1 and AdipoR2 mRNA expression. PPARgamma activation was controlled by determination of PPARgamma mRNA induction. Likewise, differentiated myotubes treated with Wy-14,643 or fenofibrate (20 h) revealed no significant regulation of AdipoR1 and AdipoR2 mRNA. PPARalpha activation was assessed by measuring PDHK4 mRNA expression. CONCLUSION: Induction of AdipoR gene expression in human skeletal muscle cells is not involved in the insulin-sensitizing effects of thiazolidinediones or fibrates.     

Retinol-binding protein 4 is associated with insulin resistance and body fat distribution in nonobese subjects without type 2 diabetes

BACKGROUND: Adipose tissue is responsible for releasing various adipokines that have been related to insulin resistance. Understanding the relationship of these adipokines to insulin resistance may foster the development of new treatments for diabetes. OBJECTIVES: The primary objective of this study was to determine whether an association between retinol-binding protein 4 (RBP4) and insulin resistance exists in nonobese individuals without a family history or diagnosis of diabetes. The secondary objective was to determine by a dual energy x-ray absorptiometry scan which adipose tissue depot most closely relates to RBP4 levels. DESIGN: Cross-sectional analysis of 92 study participants ranging in age from 20 to 83 yr was performed. The range of body mass index (BMI) was from 18 to 30 kg/m(2). Exclusion criteria were a BMI greater than 30 kg/m(2), family history of diabetes, or a diagnosis of diabetes. Insulin sensitivity was determined by a hyperinsulinemic euglycemic clamp. Body fat was measured by dual energy x-ray absorptiometry scan. RESULTS: RBP4 values were lower in females (35.8 +/- 1.7 microg/ml) compared with males (39.9 +/- 1.4 microg/ml; P = 0.06). RBP4 levels were found to correlate negatively with insulin sensitivity (r = -0.32; P = 0.002) and positively with age (r = 0.38; P < 0.001). RBP4 levels did not correlate with BMI (r = -0.13; P = 0.22), trunk fat (r = 0.16; P = 0.22), or percent body fat (r = 0.07; P = 0.65). However, RBP4 levels did correlate with percent trunk fat (r = 0.36; P = 0.001). CONCLUSION: These findings indicate a relationship between RBP4, insulin sensitivity, and percent trunk fat in individuals who may not have features of insulin resistance.     

Serum visfatin is associated with type 2 diabetes mellitus independent of insulin resistance and obesity

Objective

The aim of this study was to evaluate the association of serum visfatin, adiponectin and leptin with 2 diabetes mellitus (T2DM) in the context of the role of obesity or insulin resistance, which is not well understood.

Methods

A total of 76 newly-diagnosed T2DM patients and 76 healthy control subjects, matched for age, body mass index (BMI) and sex ratio, were enrolled. Anthropometric parameters, glycemic and lipid profile, insulin resistance (measured by homeostasis model assessment of insulin resistance index [HOMA-IR]), leptin, adiponectin, and visfatin were assessed.

Results

On the contrary to adiponectin, serum leptin and visfatin levels were higher in T2DM patients compared with controls (10.07 ± 4.5, 15.87 ± 16.4, and 5.49 ± 2.4 vs. 12.22 ± 4.9 μg/ml, 8.5 ± 7.8 ng/ml and 3.58 ± 2.2 ng/ml, respectively, P < 0.01). Waist circumference and BMI were correlated with leptin and adiponectin but not with visfatin. Leptin, adiponectin and visfatin all were associated with T2DM following adjusting for obesity measures. After controlling for HOMA-IR, visfatin remained as an independent predictor of T2DM (odds ratio = 1.32, P < 0.05). In a multiple regression analysis to determine visfatin only triglycerides and fasting glucose remained in the model (P < 0.05).

Conclusion

Elevation of visfatin in T2DM is independent of obesity and insulin resistance and is mainly determined by fasting glucose and triglycerides.     

Retinol-binding protein 4 is associated with components of the metabolic syndrome, but not with insulin resistance, in men with type 2 diabetes or coronary artery disease

Aims/hypothesis Retinol-binding protein 4 (RBP4) has recently been reported to be associated with insulin resistance and the metabolic syndrome. This study tested the hypothesis that RBP4 is a marker of insulin resistance and the metabolic syndrome in patients with type 2 diabetes or coronary artery disease (CAD) or in non-diabetic control subjects without CAD. Methods Serum RBP4 was measured in 365 men (126 with type 2 diabetes, 143 with CAD and 96 control subjects) and correlated with the homeostasis model assessment of insulin resistance index (HOMA-IR), components of the metabolic syndrome and lipoprotein metabolism. RBP4 was detected by ELISA and validated by quantitative Western blotting. Results RBP4 concentrations detected by ELISA were shown to be strongly associated with the results gained in quantitative Western blots. There were no associations of RBP4 with HOMA-IR or HbA_1c in any of the groups studied. In patients with type 2 diabetes there were significant positive correlations of RBP4 with total cholesterol, LDL-cholesterol, VLDL-cholesterol, plasma triacylglycerol and hepatic lipase activity. In patients with CAD, there were significant associations of RBP4 with VLDL-cholesterol, plasma triacylglycerol and hepatic lipase activity, while non-diabetic control subjects without CAD showed positive correlations of RBP4 with VLDL-cholesterol and plasma triacylglycerol. Conclusions/interpretation RBP4 does not seem to be a valuable marker for identification of the metabolic syndrome or insulin resistance in male patients with type 2 diabetes or CAD. Independent associations of RBP4 with pro-atherogenic lipoproteins and enzymes of lipoprotein metabolism indicate a possible role of RBP4 in lipid metabolism.     

Decline in skeletal muscle mass is associated with cognitive decline in type 2 diabetes mellitus

AimsTo examine the longitudinal association between skeletal muscle mass (SMM) loss and cognitive decline over time in type 2 diabetes mellitus (T2DM).MethodsWe conducted a prospective cohort study of 453 patients from SMART2D cohort with follow-up intervals of 1.6 to 6.4 years. Baseline and follow-up measurements included bio-impedance analysis (BIA) measure of skeletal muscle mass index (SMI) and Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) measure of cognitive function. We examined the association between annual rate of SMI and RBANS scores using linear regression, adjusting for demographics, education, depression, clinical co-variables and presence of apolipoprotein E4 (APOE) _?4 allele.ResultsThe mean age of participants was 60.3 ± 7.4 years. Compared to patients with Tertile 1 SMI change, the group with greater SMI decline (Tertile 3 SMI change) experienced 0.30 decline in RBANS total score (95%CI ?0.57 to ?0.03; p = 0.030) in the adjusted analysis. RBANS scores for subdomains in immediate memory and visuo-spatial/construction were lower in Tertile 3 SMI change group with corresponding coefficients ?0.54 (95%CI ?1.01 to ?0.06; p = 0.026), and ?0.71 (95%CI ?1.30 to ?0.12; p = 0.019) respectively.ConclusionIn patients with T2DM, BIA measure of muscle mass loss over time was independently associated with cognitive decline globally and in the domains of memory and visuo-spatial/construction.