Philip D. Miles

Phosphoinositide signalling links O -GlcNAc transferase to insulin resistance

Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes.

Improved insulin-sensitivity in mice heterozygous for PPAR-γ deficiency

The thiazolidinedione class of insulin-sensitizing, antidiabetic drugs interacts with peroxisome proliferator-activated receptor gamma (PPAR-gamma). To gain insight into the role of this nuclear receptor in insulin resistance and diabetes, we conducted metabolic studies in the PPAR-gamma gene knockout mouse model. Because homozygous PPAR-gamma-null mice die in development, we studied glucose metabolism in mice heterozygous for the mutation (PPAR-gamma(+/-) mice). We identified no statistically significant differences in body weight, basal glucose, insulin, or FFA levels between the wild-type (WT) and PPAR-gamma(+/-) groups. Nor was there a difference in glucose excursion between the groups of mice during oral glucose tolerance test, but insulin concentrations of the WT group were greater than those of the PPAR-gamma(+/-) group, and insulin-induced increase in glucose disposal rate was significantly increased in PPAR-gamma(+/-) mice. Likewise, the insulin-induced suppression of hepatic glucose production was significantly greater in the PPAR-gamma(+/-) mice than in the WT mice. Taken together, these results indicate that - counterintuitively - although pharmacological activation of PPAR-gamma improves insulin sensitivity, a similar effect is obtained by genetically reducing the expression levels of the receptor.

Metabolic Effects of Troglitazone on Fructose-Induced Insulin Resistance in the Rat

Troglitazone is a new orally active hypoglycemic agent that has been shown to reduce insulin resistance and hyperinsulinemia in both diabetic animal models and non-insulin-dependent diabetes mellitus (NIDDM) subjects. To determine whether this drug could prevent the development of fructose-induced insulin resistance and related abnormalities, we studied the effects of troglitazone on the insulin resistance induced by fructose feeding in rats. Normal male Sprague-Dawley rats were fed a high-fructose diet for 3 weeks with and without troglitazone as a food admixture (0.2%) or were fed normal chow to serve as a control group. In vivo insulin resistnace was measured by the euglycemic hyperinsulinemic clamp technique at two different insulin infusion rates, 29 (submaximal stimulation) and 290 (maximal stimulation) pmol.kg−1 · min−1. Fructose feeding markedly reduced submaximal glucose disposal rate (GDR) (113.8 ± 8.3 vs. 176.0 ± 5.6 µmol.kg−1 · min−1 P < 0.05) and maximal GDR (255.9 ± 5.6 vs. 313.6 ± 10.5 µmol.kg−1 · min−1 P < 0.05), reduced the suppressibility of submaximal hepatic glucose production (HGP; 45.5 ± 5.0 vs. 11.7 ± 5.0 µmol.kg−1 · min−1 P < 0.05), and resulted in hypertriglyceridemia and hypertension. Troglitazone treatment completely restored the GDR (submaximal 158.2 ± 5.6, maximal 305.3 ± 6.1 µmol.kg−1 · min−1) and submaximal HGP (9.4 ± 2.8 µmol.kg−1 · min−1) to control levels and also normalized the elevated plasma triglyceride concentration and systolic blood pressure levels in fructose-fed rats. These results suggest that troglitazone treatment could completely prevent the insulin resistance, hypertension, and hypertriglyceridemia induced by a diet high in fructose and that the drug might prove useful in the treatment and/or prevention of nonhyperglycemic insulin-resistant states as well as in the treatment of established NIDDM.

TNF-α-Induced Insulin Resistance In Vivo and Its Prevention by Troglitazone

Tumor necrosis factor (TNF)-α may play a role in the insulin resistance of obesity and NIDDM. Troglitazone is a new orally active hypoglycemic agent that has been shown to ameliorate insulin resistance and hyperinsu-linemia in both diabetic animal models and NIDDM subjects. To determine whether this drug could prevent the development of TNF-α-induced insulin resistance, glucose turnover was assessed in rats infused with cytokine and pretreated with troglitazone. Normal male Sprague-Dawley rats were fed normal powdered food with or without troglitazone as a food admixture (0.2%). After ∼10 days, rats were infused with TNF-a for 4–5 days, producing a plasma concentration of 632 ± 30 pg/ml. In vivo insulin action was measured by the euglycemic-hyperinsulinemic clamp technique at a sub-maximal (24 μmol · kg−1 · min−1) and maximal insulin infusion rate (240 μmol · kg−1 · min−1). TNF-α infusion resulted in a pronounced reduction in submaximal insulin-stimulated glucose disposal rate (GDR) (97 ± 10 vs. 141 ± 4 μmol · kg−1 · min−1 P < 0.05), maximal GDR (175 ± 8 vs. 267 ± 6 μmol · kg−1 · min−1 P < 0.01), and in insulin receptor-tyrosine kinase activity (IR-TKA) (248 ± 39 vs. 406 ± 32 pmol ATP/pmol IR, P < 0.05). It also led to a marked increase in basal insulin (90 ± 24 vs. 48 ± 6 pmol/1, P < 0.05) and free fatty acid (FFA) concentration (2.56 ± 0.76 vs. 0.87 ± 0.13 mmol/1, p < 0.01). Troglitazone treatment completely prevented the TNF-α-induced decline in submaximal GDR (133 ± 16 vs. 141 ± 4 umol · kg−1 · min−1, NS) and maximal GDR (271 ± 19 vs. 267 ± 6 μmol · kg1 · min1, NS). The hyperlipidemia was partially corrected by troglitazone (1.53 ± 0.28 vs. 0.87 ± 0.13 mmol/1, P < 0.05), while IR-TKA and insulin concentration remained unaffected by the drug. Troglitazone restores insulin action possibly by lowering the FFA concentration of the blood and/or by stimulating glucose uptake at an intracellular point distal to insulin receptor autophosphorylation in muscle. If TNF-α plays a role in the development of the obe-sity/NIDDM syndrome, troglitazone may prove useful in its treatment.

12/15-Lipoxygenase Is Required for the Early Onset of High Fat Diet-Induced Adipose Tissue Inflammation and Insulin Resistance in Mice

Background Recent understanding that insulin resistance is an inflammatory condition necessitates searching for genes that regulate inflammation in insulin sensitive tissues. 12/15-lipoxygenase (12/15LO) regulates the expression of proinflammatory cytokines and chemokines and is implicated in the early development of diet-induced atherosclerosis. Thus, we tested the hypothesis that 12/15LO is involved in the onset of high fat diet (HFD)-induced insulin resistance. Methodology/Principal Findings Cells over-expressing 12/15LO secreted two potent chemokines, MCP-1 and osteopontin, implicated in the development of insulin resistance. We assessed adipose tissue inflammation and whole body insulin resistance in wild type (WT) and 12/15LO knockout (KO) mice after 2–4 weeks on HFD. In adipose tissue from WT mice, HFD resulted in recruitment of CD11b+, F4/80+ macrophages and elevated protein levels of the inflammatory markers IL-1β, IL-6, IL-10, IL-12, IFNγ, Cxcl1 and TNFα. Remarkably, adipose tissue from HFD-fed 12/15LO KO mice was not infiltrated by macrophages and did not display any increase in the inflammatory markers compared to adipose tissue from normal chow-fed mice. WT mice developed severe whole body (hepatic and skeletal muscle) insulin resistance after HFD, as measured by hyperinsulinemic euglycemic clamp. In contrast, 12/15LO KO mice exhibited no HFD-induced change in insulin-stimulated glucose disposal rate or hepatic glucose output during clamp studies. Insulin-stimulated Akt phosphorylation in muscle tissue from HFD-fed mice was significantly greater in 12/15LO KO mice than in WT mice. Conclusions These results demonstrate that 12/15LO mediates early stages of adipose tissue inflammation and whole body insulin resistance induced by high fat feeding.

Kinetics of insulin action in vivo : identification of rate-limiting steps

To examine the kinetic steps in insulins in vivo action, we have assessed the temporal relationship between arterial insulin, interstitial insulin, glucose disposal rate (GDR), and insulin receptor kinase (IRK) activity in muscle and between portal insulin, hepatic glucose production (HGP), and IRK activity in liver. Interstitial insulin, as measured by lymph-insulin concentration (muscle only), and IRK activity were used as independent methods to determine the arrival of insulin at its tissue site of action. Euglycemic clamps were conducted in seven mongrel dogs and consisted of an activation phase with a venous insulin infusion (7.2 nmol · kg−1 · min−1,100 min) and a deactivation phase. Liver and muscle biopsies were taken to assess IRK activity. Arterial, portal, and lymph insulin rose to 636 ± 12, 558 ± 18, and 402 ± 24 pmol/1, respectively. GDR increased from 13.9 ± 0.6 to 41.7 ± 2.8, and HGP declined from 14.4 ± 0.6 to 1.1 ± 0.6 μmol · kg−1 · min−1. Muscle and liver IRK activity increased significantly from 5.9 ± 0.9 to 14.6 ± 0.6 and 5.5 ± 0.7 to 23.7 ± 1.9 fmol P/fmol insulin receptor (IR), respectively. The time to half-maximum response (t½a) for stimulation of GDR (19.8 ± 4.8 min) and suppression of HGP (21.5 ± 3.7 min) were similar. The t½a for stimulation of GDR, muscle IRK, and rise in lymph insulin were not significantly different from one another and were all markedly greater than that for the approach to steady state of arterial insulin (2.3 ± 1.2 min, P < 0.01). The t½a for portal insulin (1.8 ± 0.8 min) was less than that for activation of liver IRK (11.3 ± 4.3, P < 0.05), which in turn was less than that for suppression of HGP (21.5 ± 3.7 min, P < 0.05). In skeletal muscle, the delay in insulin-stimulated GDR occurs before IR binding and is due to the time required for plasma insulin to gain access to the interstitial compartment. In liver, however, identification of the site(s) of delay in insulins effects to suppress HGP is dependent on whether insulin acts directly or indirectly on the liver. If its action is direct, there are two separate sites of delay: a prereceptor and a postreceptor delay. If, however, insulins effect on suppressing HGP is indirect, then a single extrahepatic site of delay is likely, which represents the time-limiting step of insulins ability to stimulate GDR and suppress HGP. Then, the locus of the site involves transendothelial passage of insulin to the interstitial space.

Mechanisms of insulin resistance in experimental hyperinsulinemic dogs.

This study was undertaken to characterize the insulin resistance and the mechanism thereof caused by chronic hyperinsulinemia produced in dogs by surgically diverting the veins of the pancreas from the portal vein to the vena cava. Pancreatic venous diversion (PVD, n = 8) caused a sustained increase in arterial insulin and decrease in portal insulin concentration compared with the control group (n = 6). Hyperinsulinemic euglycemic clamps were conducted 4 wk after surgery. The increase in the glucose disposal rate (GDR) was significantly less in the PVD group (39.0+/-5.0 vs. 27.9+/-3.2 micromol/kg/min, P < 0.01) compared with the control group, but the suppression of hepatic glucose production by insulin was similar for both groups. Muscle insulin receptor tyrosine kinase activity (IR-TKA) increased from 6.2+/-0.4 to 20.3+/-2.7 in the control group, but from 5.8+/-0.5 to only 12.7+/-1.7 fmol P/fmol IR in the PVD group (P < 0.01). With respect to the periphery, the time to half-maximum response (t1/2a) for arterial insulin was the same for both groups, whereas the t1/2a for lymph insulin (30+/-3 vs. 40+/-4 min, P < 0.05) and GDR (29+/-3 vs. 66+/-10 min, P < 0.01) were greater for the PVD group. Chronic hyperinsulinemia led to marked peripheral insulin resistance characterized by decreased insulin-stimulated GDR, and impaired activation of GDR kinetics due, in part, to reduced IR-TKA. Transendothelial insulin transport was impeded and was responsible for one third of the kinetic defect in insulin-resistant animals, while slower intracellular mechanisms of GDR were responsible for the remaining two thirds.

Metabolic effects of troglitazone on fat-induced insulin resistance in the rat.

Troglitazone is a new orally active hypoglycemic agent that has been shown to ameliorate insulin resistance and hyperinsulinemia in both diabetic animal models and non-insulin-dependent diabetes mellitus (NIDDM) subjects. To determine whether this drug could prevent the development of diet-induced insulin resistance and related abnormalities, we studied its effect on insulin resistance induced by high-fat feeding in rats. Normal male Sprague-Dawley rats were fed a high-fat diet for 3 weeks with and without troglitazone as a food mixture (0.2%) or were fed normal chow. In vivo insulin action was measured using a euglycemic-hyperinsulinemic clamp at two different insulin infusion rates, 4 (submaximal stimulation) and 40 (maximal stimulation) mU/kg/min. Fat feeding markedly reduced the submaximal glucose disposal rate ([GDR], 26.4 +/- 1.3 v 37.5 +/- 1.4 mg/kg/min, P < .01) and maximal GDR (55.9 +/- 1.3 v 64.5 +/- 1.3 mg/kg/min, P < 0.5), reduced the suppressibility of submaximal hepatic glucose production ([HGP], 3.2 +/- 0.9 v 1.5 +/- 0.5 mg/kg/min, P < .05), and resulted in hyperlipidemia. Troglitazone treatment did not affect any of these parameters. Insulin resistance induced by fat feeding is the first experimental model in which troglitazone failed to correct or partially correct the insulin resistance.

Islet Amyloid Polypeptide (Amylin) Increases the Renal Excretion of Calcium in the Conscious Dog

Islet amyloid polypeptide (IAPP) is a member of the calcitonin/CGRP family and has been isolated from the β-cell of pancreatic islets. Recent evidence suggests that this peptide may be involved in calcium metabolism in that its administration resulted in lowering of serum calcium levels. To determine the mechanism of IAPP-induced hypocalcemia, the peptide was infused at 50 pmol/min/kg for 90 minutes in conscious male mongrel dogs. Infusion of the peptide resulted in a modest decline in the total serum calcium concentration (10.4±0.2 to 9.4±0.2 mg/dl; P<0.05) and a concomitant increase in urinary calcium excretion (3.6±0.6 to 6.9±2.0 mg/dl; P<0.01). Based on an extracellular volume of 7 liter in a 28 kg dog, the total decrement in calcium due to IAPP was 41.3±2.4 mg, whereas the total increase in urinary calcium was 3.2±0.7 mg. There were no detectable changes in calcitonin. We conclude that IAPP lowers serum calcium and increases the renal excretion of calcium independently of calcitonin. However, the calciuria can only account for a small component of the hypocalcemic effect and therefore, an additional calcium lowering effect of IAPP exits.

T-cadherin (Cdh13) in association with pancreatic β-cell granules contributes to second phase insulin secretion

Glucose homeostasis depends on adequate control of insulin secretion. We report the association of the cell-adhesion and adiponectin (APN)-binding glycoprotein T-cadherin (Cdh13) with insulin granules in mouse and human β-cells. Immunohistochemistry and electron microscopy of islets in situ and targeting of RFP-tagged T-cadherin to GFP-labeled insulin granules in isolated β-cells demonstrate this unusual location. Analyses of T-cadherin-deficient (Tcad-KO) mice show normal islet architecture and insulin content. However, T-cadherin is required for sufficient insulin release in vitro and in vivo. Primary islets from Tcad-KO mice were defective in glucose-induced but not KCl-mediated insulin secretion. In vivo, second phase insulin release in T-cad-KO mice during a hyperglycemic clamp was impaired while acute first phase release was unaffected. Tcad-KO mice showed progressive glucose intolerance by 5 mo of age without concomitant changes in peripheral insulin sensitivity. Our analyses detected no association of APN with T-cadherin on β-cell granules although colocalization was observed on the pancreatic vasculature. These data identify T-cadherin as a novel component of insulin granules and suggest that T-cadherin contributes to the regulation of insulin secretion independently of direct interactions with APN.