Lack of inducible nitric oxide synthase does not prevent aging-associated insulin resistance

Inducible nitric oxide synthase (iNOS) is involved in obesity-induced insulin resistance. Since aging is accompanied by increased iNOS expression, the effect of iNOS gene deletion on aging-associated insulin resistance was investigated in 7-month-old (adult) and 22-month-old (old) iNOS knockout and wild-type mice using the hyperinsulinemic–euglycemic clamp. While body weight and fat mass were increased, muscle mass was reduced with aging in wild-type mice. However, body composition was not changed with aging in iNOS knockout mice due to increased locomotor activity. NO metabolites in plasma, and protein levels of iNOS and nitrotyrosine in skeletal muscle increased with aging in wild-type mice. Deletion of iNOS gene attenuated NO metabolites and nitrotyrosine with aging in iNOS knockout mice. Glucose uptake in whole body and skeletal muscle was reduced with aging in both wild-type and iNOS knockout mice and there was no difference between two groups. Plasma level of tumor necrosis factor-α and gene expression of proinflammatory cytokines in peripheral tissues were increased with aging in both groups, and that was more heightened in iNOS knockout mice. These results suggest that lack of iNOS does not prevent aging-associated insulin resistance in mice and heightened production of proinflammatory cytokines may be involved.     

Glycogen synthase kinase 3 inhibition improves insulin-stimulated glucose metabolism but not hypertension in high-fat-fed C57BL/6J mice

Aims/hypothesis In the current study, the effect of a highly specific peptide inhibitor of glycogen synthase kinase 3 (GSK3) (L803-mts) on glucose metabolism and BP was examined in a high-fat (HF) fed mouse model of diabetes. Methods C57/BL6J mice were placed on an HF diet for 3 months and treated with L803-mts for 20 days, following which glucose metabolism was examined by euglycaemic–hyperinsulinaemic clamp studies. BP and heart rate were measured by radio-telemetry. Results The HF mice were obese, with impaired glucose tolerance and high plasma insulin and leptin levels. L803-mts treatment significantly reduced the insulin levels and doubled the glucose infusion rate required to maintain a euglycaemic condition in the HF+L803-mts group compared with the HF group. Insulin failed to suppress the endogenous glucose production rate in the HF group while decreasing it by 75% in the HF+L803-mts group, accompanied by increased liver glycogen synthase activity and net hepatic glycogen synthesis. GSK3 inhibition also reduced peripheral insulin resistance. Plasma glucose disappearance rate increased by 60% in the HF+L803-mts group compared with the HF group. In addition, glucose uptake in heart and gastrocnemius muscle was markedly improved. Although mean arterial pressure increased following the HF diet, it did not change significantly during the 12 days of L803-mts treatment. Conclusions/interpretation These studies demonstrate that GSK3 inhibition improved hepatic and peripheral insulin resistance in a mouse model of HF-induced diabetes, but it failed to have an effect on BP. GSK3 may represent an important therapeutic target for insulin resistance.     

T cell protein tyrosine phosphatase (TCPTP) deficiency in muscle does not alter insulin signalling and glucose homeostasis in mice

Aims/hypothesis  

Insulin activates insulin receptor protein tyrosine kinase and downstream phosphatidylinositol-3-kinase (PI3K)/Akt signalling in muscle to promote glucose uptake. The insulin receptor can serve as a substrate for the protein tyrosine phosphatase (PTP) 1B and T cell protein tyrosine phosphatase (TCPTP), which share a striking 74% sequence identity in their catalytic domains. PTP1B is a validated therapeutic target for the alleviation of insulin resistance in type 2 diabetes. PTP1B dephosphorylates the insulin receptor in liver and muscle to regulate glucose homeostasis, whereas TCPTP regulates insulin receptor signalling and gluconeogenesis in the liver. In this study we assessed for the first time the role of TCPTP in the regulation of insulin receptor signalling in muscle.     

Activation of liver X receptors promotes lipid accumulation but does not alter insulin action in human skeletal muscle cells

Aims/hypothesis The aim of this study was to investigate the effects of liver X receptor (LXR) activation on lipid metabolism and insulin action in human skeletal muscle cells prepared from control subjects and from patients with type 2 diabetes.Subjects and methods Cultured myotubes were obtained from muscle biopsies of 11 lean, healthy control subjects and ten patients with type 2 diabetes. The mRNA levels of LXR isoforms and lipogenic genes were estimated by RT-quantitative PCR, and the effects of LXR agonists on insulin action were evaluated by assays of protein kinase B serine 473 phosphorylation and glycogen synthesis.Results Both LXRα and LXRβ were expressed in human skeletal muscle and adipose tissue and there was no difference in their mRNA abundance in tissues from patients with type 2 diabetes compared with control subjects. In cultured muscle cells, LXR activation by T0901317 strongly increased expression of the genes encoding lipogenic enzymes, including sterol regulatory element binding protein 1c, fatty acid synthase and stearoyl-CoA desaturase 1, and also promoted triglyceride accumulation in the presence of a high glucose concentration. Importantly, these effects on lipid metabolism did not affect protein kinase B activation by insulin. Furthermore, LXR agonists did not modify insulin action in muscle cells from patients with type 2 diabetes.Conclusions/interpretation These data suggest that LXR agonists may lead to increased utilisation of lipids and glucose in muscle cells without affecting the mechanism of action of insulin. However, the long-term consequences of triglyceride accumulation in muscle should be evaluated before the development of effective LXR-based therapeutic agents.D. Cozzone, C. Debard and N. Dif contributed equally to this work.     

SOD1, but not SOD3, deficiency accelerates diabetic renal injury in C57BL/6-Ins2Akita diabetic mice

Superoxide dismutase (SOD) is a major defender against excessive superoxide generated under hyperglycemia. We have recently reported that renal SOD1 (cytosolic CuZn-SOD) and SOD3 (extracellular CuZn-SOD) isoenzymes are remarkably down-regulated in KK/Ta-Ins2^Akita diabetic mice, which exhibit progressive diabetic nephropathy (DN), but not in DN-resistant C57BL/6- Ins2^Akita (C57BL/6-Akita) diabetic mice. To determine the role of SOD1 and SOD3 in DN, we generated C57BL/6-Akita diabetic mice with deficiency of SOD1 and/or SOD3 and investigated their renal phenotype at the age of 20 weeks. Increased glomerular superoxide levels were observed in SOD1^?/?SOD3^+/+ and SOD1^?/?SOD3^?/? C57BL/6-Akita mice but not in SOD1^+/+SOD3^?/? C57BL/6-Akita mice. The SOD1^?/?SOD3^+/+ and SOD1^?/?SOD3^?/? C57BL/6-Akita mice exhibited higher glomerular filtration rate, increased urinary albumin levels, and advanced mesangial expansion as compared with SOD1^+/+SOD3^+/+ C57BL/6-Akita mice, yet the severity of DN did not differ between the SOD1^?/?SOD3^+/+ and SOD1^?/?SOD3^?/? C57BL/6-Akita groups. Increased renal mRNA expression of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), reduced glomerular nitric oxide (NO), and increased renal prostaglandin E2 (PGE2) production were noted in the SOD1^?/?SOD3^+/+ and SOD1^?/?SOD3^?/? C57BL/6-Akita mice. This finding indicates that such renal changes in fibrogenic cytokines, NO, and PGE2, possibly caused by superoxide excess, would contribute to the development of overt albuminuria by promoting mesangial expansion, endothelial dysfunction, and glomerular hyperfiltration. The present results demonstrate that deficiency of SOD1, but not SOD3, increases renal superoxide in the setting of diabetes and causes overt renal injury in nephropathy-resistant diabetic mice, and that SOD3 deficiency does not provide additive effects on the severity of DN in SOD1-deficient C57BL/6-Akita mice.     

Amelioration of insulin resistance but not hyperinsulinemia in obese mice overexpressing GLUT4 selectively in skeletal muscle

The effects of gold-thioglucose (GTG) treatment were examined in mice overexpressing GLUT4 selectively in skeletal muscle (MLC-GLUT4 mice) and in age-matched controls. Groups of MLC-GLUT4 and control mice were injected with GTG or saline at 5 weeks of age. At 12 weeks following the injections, GTG-treated control mice exhibited a 35% increase in body weight versus saline-treated controls. Similarly, a 30% increase in body weight was observed in GTG-treated MLC-GLUT4 mice compared with saline-treated MLC-GLUT4 mice 12 weeks after the injections. In saline-treated lean MLC-GLUT4 and control mice, intraperitoneal injection of insulin decreased blood glucose in 1 hour by 63% and 38%, respectively. Insulin also decreased blood glucose by 40% in GTG-treated obese MLC-GLUT4 mice after 1 hour. However, insulin did not reduce blood glucose levels in GTG-treated obese control mice. The ability of insulin to clear blood glucose in GTG-treated obese MLC-GLUT4 mice is associated with increased skeletal muscle GLUT4 content and white adipose tissue (WAT) GLUT4 content as compared with GTG-treated obese controls. However, fasting blood glucose levels in GTG-treated obese MLC-GLUT4 and control mice were elevated by approximately 30% compared with saline-treated groups. Lastly, although GTG-treated obese MLC-GLUT4 mice exhibited improved glucose clearance in response to insulin, they nevertheless remained as hyperinsulinemic as GTG-treated obese control mice. These results suggest that genetic overexpression of GLUT4 in skeletal muscle may ameliorate the development of insulin resistance associated with obesity but cannot restore normal glucose and insulin levels.     

Diabetes induces endothelial dysfunction but does not increase neointimal formation in high-fat diet fed C57BL/6J mice

Studies of diabetic vascular disease have traditionally used murine models of type 1 diabetes and genetic models of type 2 diabetes. Because the majority of patients with type 2 diabetes have diet induced obesity, we sought to study the effect of diabetes on arterial disease in a mouse model of diet induced obesity/diabetes. C57Bl/6 mice fed a high-fat diet for 9 weeks developed type 2 diabetes characterized by elevated body weight, hyperglycemia, and hyperinsulinemia. Arteries from diabetic mice exhibited a marked decrease in endothelium-dependent vasodilation, a modest decrease in endothelium independent vasodilation, and an increase in sensitivity to adrenergic vasoconstricting agents. Insulin stimulated protein kinase B (akt) and endothelial nitric oxide synthase (eNOS) phosphorylation were preserved in arteries from diabetic mice; however, eNOS protein dimers were markedly diminished. Arterial nitrotyrosine staining indicated that increased levels of peroxynitrite contributed to eNOS dimer disruption in the diabetic mice. The abnormal vasomotion was not an acute response to the high-fat diet, as short term high-fat diet feeding had no effect on endothelium dependent dilation. A trend toward smaller neointimal lesions was noted in high-fat diet fed mice after femoral artery wire denudation injury. In summary, disrupted eNOS dimer formation rather than impaired insulin mediated eNOS phosphorylation contributed to the endothelial dysfunction in diet induced obese/diabetic mice. The lack of an increase in neointimal formation indicates that additional diabetes associated parameters (such as hyperlipidemia and atherosclerotic vascular disease) may need to be present to increase neointimal formation in this model.     

Endothelial nitric oxide synthase haplotypes are related to blood pressure elevation, but not to resistance to antihypertensive drug therapy

OBJECTIVES: Most hypertensive patients require two or more drugs to control arterial blood pressure effectively. Although endothelial nitric oxide synthase (eNOS) haplotypes have been associated with hypertension, it is unknown whether eNOS genotypes/haplotypes are associated with resistance to antihypertensive therapy. METHODS: We studied the distribution of three eNOS genetic polymorphisms: single nucleotide polymorphisms in the promoter region (T(-786)C), and in exon 7 (Glu298Asp), and a variable number of tandem repeats in intron 4 (b/a). Genotypes were determined for 111 normotensive controls (NT), 116 hypertensive individuals who were well controlled (HT), and 100 hypertensive individuals who were resistant to conventional antihypertensive therapy (RHT). We also compared the distribution of eNOS haplotypes in the three groups of subjects. RESULTS: No differences were found in genotype or allele distribution among the three groups (all P > 0.05). Conversely, the 'C Glu b' haplotype was more commonly found in the NT than in the HT or RHT groups (21 versus 8 and 7%, respectively; both P < 0.00625). In addition, the 'C Asp b' haplotype was more commonly found in the HT or RHT groups than in the NT group (22 and 20%, respectively, versus 8%; both P < 0.00625). The distribution of eNOS haplotypes was not significantly different in the HT and RHT groups (P > 0.05). CONCLUSIONS: Whereas our findings suggest a protective effect for the 'C Glu b' haplotype against hypertension and that the 'C Asp b' haplotype increases the susceptibility to hypertension, our results suggest that eNOS haplotypes are not associated with resistance to antihypertensive therapy.     

Tumor necrosis factor alpha receptor 1 deficiency in hepatocytes does not protect from non-alcoholic steatohepatitis,but attenuates insulin resistance in mice

BACKGROUND End-stage liver disease caused by non-alcoholic steatohepatitis(NASH) is the second leading indication for liver transplantation. To date, only moderately effective pharmacotherapies exist to treat NASH. Understanding the pathogenesis of NASH is therefore crucial for the development of new therapies. The inflammatory cytokine tumor necrosis factor alpha(TNF-α) is important for the progression of liver disease. TNF signaling via TNF receptor 1(TNFR1) has been hypothesized to be important for the development of NASH and hepatocellular carcinoma in whole-body knockout animal models.AIM To investigate the role of TNFR1 signaling in hepatocytes for steatohepatitis development in a mouse model of diet-induced NASH.METHODS NASH was induced by a western-style fast-food diet in mice deficient for TNFR1 in hepatocytes(TNFR1~(ΔHEP)) and their wild-type littermates(TNFR1~(fl/fl)). Glucose tolerance was assessed after 18 wk and insulin resistance after 19 wk of feeding. After 20 wk mice were assessed for features of NASH and the metabolic syndrome such as liver weight, liver steatosis, liver fibrosis and markers of liver inflammation.RESULTS Obesity, liver injury, inflammation, steatosis and fibrosis was not different between TNFR1~(ΔHEP) and TNFR1~(fl/fl) mice. However, Tnfr1 deficiency in hepatocytes protected against glucose intolerance and insulin resistance.CONCLUSION Our results indicate that deficiency of TNFR1 signaling in hepatocytes does not protect from diet-induced NASH. However, improved insulin resistance in this model strengthens the role of the liver in glucose homeostasis.     

Long-standing hyperglycemia in C57BL/6J mice does not affect retinal glutathione levels or endothelial/pericyte ratio in retinal capillaries

Free radicals have been suggested to play a role in the development of diabetic retinopathy. The aim of the present study was to examine whether the metabolic perturbations caused by high-fat feeding of two strains of mice, the C57BL6/J mice and the NMRI mice, interfere with one of the free radical enzyme defense systems in the retina, i. e., glutathione (GSH), and whether morphological changes occur in the retinal vessels. C57BL/6J mice and NMRI mice were fed a high-fat diet (55%) for 18 months. High-fat fed mice of both strains developed overweight, hyperinsulinemia, and hyperlipidemia. In addition, the high-fat fed C57BL/6J mice also developed sustained hyperglycemia for at least 15 months. The C57BL/6J mice had lower retinal GSH levels than the NMRI mice, both when given a normal diet (29.6+/-1.2 vs. 37.1+/-1.4 nmol/mg protein; p<0.01) and when given a high-fat diet (27.0+/-1.6 vs. 34.7+/-2.6 nmol/mg protein; p<0.05). Despite the long-standing hyperglycemia, hyperinsulinemia and hyperlipidemia in the C57BL/6J mice, high-fat feeding did not cause any changes in the retinal tissue levels of GSH (27.0+/-1.6 vs. 29. 6+/-1.2 nmol/mg protein) or cysteine (7.61+/-0.63 vs. 6.80+/-0.59 nmol/mg protein). Similarly, high-fat feeding did not affect retinal GSH or cysteine levels in NMRI mice. No light microscopical retinal vessel changes were seen, either in C57BL/6J or in NMRI mice. The study therefore shows that long-standing metabolic perturbations induced by dietary obesity do not induce signs of retinopathy in two different strains of mice. Further studies are needed to explore whether this is explained by increased expression of protecting systems making these strains of mice resistant to effects of oxidative stress.     

High levels of whole-body energy expenditure are associated with a lower coupling of skeletal muscle mitochondria in C57Bl/6 mice

Considerable variation in energy expenditure is observed in C57Bl/6 mice on a high-fat diet. Because muscle tissue is a major determinant of whole-body energy expenditure, we set out to determine the variation in energy expenditure and its possible association with skeletal muscle mitochondrial function upon high-fat diet intervention. Metabolic cages using indirect calorimetry were used to assess whole-body energy metabolism in C57Bl/6 male mice during the first 3 days of high-fat diet intervention. Mice were grouped in a negative or positive residual nocturnal energy expenditure group after correction of total nocturnal energy expenditure for body mass by residual analysis. The positive residual energy expenditure group was characterized by higher uncorrected total nocturnal energy expenditure and food intake. On day 7, mitochondria were isolated from the skeletal muscle of the hind limb. Mitochondrial density was determined by mitochondrial protein content and did not differ between the positive and negative residual energy expenditure groups. Using high-resolution respirometry, mitochondrial oxidative function was assessed using various substrates. Mitochondria from the positive residual energy expenditure group were characterized by a lower adenosine diphosphate-stimulated respiration and lower respiratory control rates using palmitoyl-coenzyme A as substrate. These results indicate that reduced mitochondrial coupling is associated with positive residual energy expenditure and high rates of total energy expenditure in vivo.     

Failure of dietary quercetin to alter the temporal progression of insulin resistance among tissues of C57BL/6J mice during the development of diet-induced obesity

Aims/hypotheses  High-fat diets produce obesity and glucose intolerance by promoting the development of insulin resistance in peripheral tissues and liver. The present studies sought to identify the initial site(s) where insulin resistance develops using a moderately high-fat diet and to assess whether the bioflavonoid, quercetin, ameliorates progression of this sequence. Methods  Four cohorts of male C57BL/6J mice were placed on diets formulated to be low-fat (10% of energy from fat), high-fat (45% of energy from fat) or high-fat plus 1.2% quercetin (wt/wt). After 3 and 8 weeks, cohorts were evaluated using euglycaemic–hyperinsulinaemic clamps, metabolomic analysis of fatty acylcarnitines and acute in vitro assessments of insulin signalling among tissues. Results  After 3 and 8 weeks, the high-fat diet produced whole-body insulin resistance without altering insulin-dependent glucose uptake in peripheral tissues. The primary defect was impaired suppression of hepatic glucose production by insulin at both times. Quercetin initially exacerbated the effect of high-fat diet by further increasing hepatic insulin resistance, but by 8 weeks insulin resistance and hepatic responsiveness to insulin were similarly compromised in both high-fat groups. The high-fat diet, irrespective of quercetin, increased short-chain fatty acylcarnitines in liver but not in muscle, while reciprocally reducing hepatic long-chain fatty acylcarnitines and increasing them in muscle. Conclusions/interpretation  Failure of insulin to suppress hepatic glucose output is the initial defect that accounts for the insulin resistance that develops after short-term consumption of a high-fat (45% of energy) diet. Hepatic insulin resistance is associated with accumulation of short- and medium-, but not long-chain fatty acylcarnitines. Dietary quercetin does not ameliorate the progression of this sequence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Hyperglycaemia normalises insulin action on glucose metabolism but not the impaired activation of AKT and glycogen synthase in the skeletal muscle of patients with type 2 diabetes

Aims/hypothesis  

In type 2 diabetes, reduced insulin-stimulated glucose disposal, primarily glycogen synthesis, is associated with defective insulin activation of glycogen synthase (GS) in skeletal muscle. Hyperglycaemia may compensate for these defects, but to what extent it involves improved insulin signalling to glycogen synthesis remains to be clarified.     

Exposure to the common food additive carrageenan leads to glucose intolerance, insulin resistance and inhibition of insulin signalling in HepG2 cells and C57BL/6J mice

Aims/hypothesis  

The aim of this study was to determine the impact of the common food additive carrageenan (E407) on glucose tolerance, insulin sensitivity and insulin signalling in a mouse model and human hepatic cells, since carrageenan is known to cause inflammation through interaction with toll-like receptor (TLR)4, which is associated with inflammation in diabetes.     

Increasing skeletal muscle fatty acid transport protein 1 (FATP1) targets fatty acids to oxidation and does not predispose mice to diet-induced insulin resistance

Aims/hypothesis  

We examined in skeletal muscle (1) whether fatty acid transport protein (FATP) 1 channels long-chain fatty acid (LCFA) to specific metabolic fates in rats; and (2) whether FATP1-mediated increases in LCFA uptake exacerbate the development of diet-induced insulin resistance in mice. We also examined whether FATP1 is altered in insulin-resistant obese Zucker rats.