D. M. Ouwens

Cardiac dysfunction induced by high-fat diet is associated with altered myocardial insulin signalling in rats.

Aims/hypothesisDiabetic cardiomyopathy (DCM) is common in type 2 diabetes. In DCM, insulin resistance may alter cardiac substrate supply and utilisation leading to changes in myocardial metabolism and cardiac function. In rats, exposure to excessive alimentary fat, inducing a type 2 diabetic phenotype, may result in myocardial insulin resistance and cardiac functional changes resembling DCM.Materials and methodsRats received high-fat (HFD) or low-fat (LFD) diets for 7 weeks. Prior to killing, insulin or saline was injected i.p. Contractile function and insulin signalling were assessed in papillary muscles and ventricular lysates, respectively.ResultsFasting and post-load blood glucose levels were increased in HFD- vs LFD-rats (all p<0.02). Mean heart weight, but not body weight, was increased in HFD-rats (p<0.01). HFD-hearts showed structural changes and triglyceride accumulation. HFD-muscles developed higher baseline and maximum forces, but showed impaired recovery from higher workloads. Insulin-associated modulation of Ca2+-induced force augmentation was abolished in HFD-muscles. HFD reduced insulin-stimulated IRS1-associated phosphatidylinositol 3′-kinase activity and phosphorylation of protein kinase B, glycogen synthase kinase-3β, endothelial nitric oxide synthase, and forkhead transcription factors by 40–60% (all p<0.05). Insulin-mediated phosphorylation of phospholamban, a critical regulator of myocardial contractility, was decreased in HFD-hearts (p<0.05).Conclusions/interpretationHFD induced a hypertrophy-like cardiac phenotype, characterised by a higher basal contractile force, an impaired recovery from increased workloads and decreased insulin-mediated protection against Ca2+ overload. Cardiac dysfunction was associated with myocardial insulin resistance and phospholamban hypophosphorylation. Our data suggest that myocardial insulin resistance, resulting from exposure to excessive alimentary fat, may contribute to the pathogenesis of diabetes-related heart disease.

Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.

Expression of a dominant-negative Ras mutant does not affect stimulation of glucose uptake and glycogen synthesis by insulin.

SummaryIt has previously been shown that insulin-induced stimulation of glucose uptake and glycogen synthesis requires activation of phosphatidylinositol-3-kinase (PI3kinase). Insulin also induces formation of RasGTP in cells and various studies have yielded inconsistent data with respect to the contribution of signalling pathways activated by RasGTP, to insulin-stimulated glucose uptake and glycogen synthesis. We have examined the requirement of RasGTP-mediated signalling for these insulin responses by expression of a dominant negative mutant of Ras (RasN17) in cells by vaccinia virus mediated gene transfer. This Ras-mutant abrogates the signalling pathways mediated by endogenous RasGTP. Subsequently, the ability of insulin to stimulate 2-deoxyglucose uptake and glycogen was examined. We observed that expression of RasN17 in 3T3L1 adipocytes did not affect the stimulation of hexose uptake by insulin. Similarly, expression of RasN17 in A14 cells, an NIH 3T3-derived cell line with high expression of insulin receptors, did not affect insulin-induced stimulation of glycogen synthesis. In both cell lines, insulin-induced phosphorylation of Mapkinase (Erk1,2) was abrogated after expression of RasN17, demonstrating the functional interference by RasN17 with signalling mediated by endogenous RasGTP. Wortmannin, an inhibitor of PBkinase, abolished dose-dependently the insulin-induced stimulation of hexose uptake and glycogen synthesis without an effect on RasGTP levels in both cell types. We conclude that stimulation of glucose transport and glycogen synthesis by insulin occurs independently of RasGTP-mediated signalling.

Does dipeptidyl peptidase-4 inhibition prevent the diabetogenic effects of glucocorticoids in men with the metabolic syndrome? A randomized controlled trial

OBJECTIVE Anti-inflammatory glucocorticoid (GC) therapy often induces hyperglycemia due to insulin resistance and islet-cell dysfunction. Incretin-based therapies may preserve glucose tolerance and pancreatic islet-cell function. In this study, we hypothesized that concomitant administration of the dipeptidyl peptidase-4 inhibitor sitagliptin and prednisolone in men at high risk to develop type 2 diabetes could protect against the GC-induced diabetogenic effects. DESIGN AND METHODS Men with the metabolic syndrome but without diabetes received prednisolone 30  mg once daily plus sitagliptin 100  mg once daily (n=14), prednisolone (n=12) or sitagliptin alone (n=14) or placebo (n=12) for 14 days in a double-blind 2 × 2 randomized-controlled study. Glucose, insulin, C-peptide, and glucagon were measured in the fasted state and following a standardized mixed-meal test. β-cell function parameters were assessed both from a hyperglycemic-arginine clamp procedure and from the meal test. Insulin sensitivity (M-value) was measured by euglycemic clamp. RESULTS Prednisolone increased postprandial area under the curve (AUC)-glucose by 17% (P<0.001 vs placebo) and postprandial AUC-glucagon by 50% (P<0.001). Prednisolone reduced 1st and 2nd phase glucose-stimulated- and combined hyperglycemia-arginine-stimulated C-peptide secretion (all P ≤ 0.001). When sitagliptin was added, both clamp-measured β-cell function (P=NS for 1st and 2nd phase vs placebo) and postprandial hyperglucagonemia (P=NS vs placebo) remained unaffected. However, administration of sitagliptin could not prevent prednisolone-induced increment in postprandial glucose concentrations (P<0.001 vs placebo). M-value was not altered by any treatment. CONCLUSION Fourteen-day treatment with high-dose prednisolone impaired postprandial glucose metabolism in subjects with the metabolic syndrome. Concomitant treatment with sitagliptin improved various aspects of pancreatic islet-cell function, but did not prevent deterioration of glucose tolerance by GC treatment.

Dynamics of insulin signalling in liver during hyperinsulinemic euglycaemic clamp conditions in vivo and the effects of high-fat feeding in male mice

Abstract Insulin is an important regulator of hepatic carbohydrate, lipid, and protein metabolism, and the regulation of these processes by insulin is disturbed under conditions of insulin resistance and type 2 diabetes. Despite these alterations, the impact of insulin resistance on insulin signalling in the liver is not well defined. Variations in time and dose of insulin stimulation as well as plasma glucose levels may underlie this. The present study aimed at determining the dynamics of activation of hepatic insulin signalling in vivo at insulin concentrations resembling those achieved after a meal, and addressing the effects of high-fat feeding. An unexpected finding of this study was the biphasic activation pattern of the IRS-PI3K-PKB/Akt pathway. Our findings indicate that the first burst of activation contributes to regulation of glucose metabolism. The physiological function of the second peak is still unknown, but may involve regulation of protein synthesis. Finally, high-fat feeding caused hepatic insulin resistance, as illustrated by a reduced suppression of hepatic glucose production. A sustained increased phosphorylation of the serine/threonine kinases p70S6kinase and Jun N-terminal kinase in the absence of insulin may underlie the abrogated phosphorylation of the IRS proteins and their downstream targets.