GLUCOSAMINE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Type 2 diabetes mellitus (T2DM) is a global epidemic with grave health consequences. Insulin resistance is a characteristic feature of most patients with T2DM and is consequently a principal target for intervention. Peroxisome Proliferator-Activated Receptors (PPARs), a subfamily of nuclear receptors, are important regulators of glucose and lipid homeostasis. Since therapy with pharmacologic PPAR-g agonists improves insulin sensitivity and glycemic control in T2DM, while PPAR-a agonists lower VLDL production, combined stimulation of both isoforms is becoming a preferred approach. PPARs are lipid-activated transcription factors of the nuclear hormone receptor superfamily that regulate the expression of genes controlling lipid and glucose metabolism. The PPAR-a isoform, which is expressed abundantly in liver, intestine and renal cortex and to a lesser extent in skeletal muscle, governs lipid catabolism and homeostasis in liver. This isoform is activated by the fibrate class of hypolipidemic agents, as well as by a variety of fatty acids and eicosanoids. PPAR-g, expressed mainly in adipose tissue and immune cells, appears to play an important role in adipocyte differentiation and lipogenesis. Activation of PPAR-g by the thiazolidinedione class of antidiabetic drugs improves glucose homeostasis and insulin action in T2DM. Furthermore, it has been recently reported that thiazolidinedione administration increases plasma levels of Adiponectin in rodents and humans, apparently due to increased gene expression in adipocytes. Since this protein is believed to improve insulin action, this provides an interesting additional explanation to explain the benefit in whole-body glucose metabolism. Indeed, adiponectin administration has been shown to lower hepatic glucose production in isolated hepatocytes and in conscious mice, suggesting enhanced hepatic sensitivity to insulin. Furthermore, it has recently been determined that measuring the ratio of the two circulating forms of adiponectin is probably of greater physiologic consequence than total adiponectin levels. Indeed, since the high molecular weight (HMW) form is biologically active while the low molecular weight (LMW) form appears to have dominant negative properties, the ratio of HMW/total adiponectin levels is the measure which correlates best with potency of its in vivo effects on insulin sensitivity. Additional beneficial effects on diabetic dyslipidemia have been observed with PPAR-g agonists. This may be at least in part attributable to improved insulin action. However, since pioglitazone also has some PPAR-a agonist activity, this may contribute to the beneficial effects of this agent. To date, published data on lipid ameliorating effects of pioglitazone has also been limited to studies of more chronic duration. High-throughput gene expression profiling in insulin resistant Zucker rats revealed that improved insulin action with a PPAR-g agonist was associated with >3-fold increases in the expression of many genes in fat and muscle, particularly genes regulating lipogenesis (eg. fatty acid synthase), differentiation (eg.ADD1/SREBP1c) and fatty acid binding. This data was confirmed by RTQ-PCR and correlated with previous observations in other rodent models. Additionally, PPAR-a stimulation affects the expression of target genes CD36/FAT7 and peroxisomal acyl Co-A oxidase in muscle. It remains to be determined whether expression of these 'signature' genes can be altered by PPAR agonists when administered in vivo in humans. Recently it was shown that human obesity and aging are both characterized by increased macrophage infiltration into adipose tissue. Thus, adipose tissue depots contain macrophages and macrophage precursors, whose tissue-specific functions remain to be fully delineated. Indeed, macrophages serve important immune and scavenger functions, are the primary mediators of the innate immune response, and are important participants in adaptive immunity. Adipose tissue produces several proinflammatory, procoagulant, and acute-phase molecules in direct proportion to the degree of adiposity. Among these molecules, TNF-a, IL-6, PAI-1, NO, factor VII, and MCP-1 have all been implicated in the development of adverse pathophysiological phenotypes associated with obesity. The strong relationship between adipose tissue macrophage content and indicators of adiposity suggests a source for the increased adipose tissue production of proinflammatory molecules and acute phase proteins associated with obesity. There is recent evidence that the largest class of genes significantly upregulated in obesity consists of macrophage and inflammatory genes in white adipose tissue. Furthermore, macrophage accumulation in adipose tissue is tightly correlated with increasing adipocyte size. Since the latter is strongly correlated with insulin action, this suggests that macrophage infiltration is likely to be associated with insulin resistance. Since adipose tissue macrophage content was shown to increase with age, this might be another factor contributing to insulin resistance in aging. Indeed, increased macrophage infiltration into fat is likely to enhance the adipose tissue production of these proinflammatory and acute-phase molecules and thereby contribute to the pathophysiological consequences of obesity. Of note, PPAR-gamma receptors have been shown to negatively regulate macrophage activation. Pioglitazone, while predominantly a PPAR-g agonist, does have some affinity for PPAR-a receptors and consequently offers theoretical advantages over other available thiazolidenediones. While thiazolidenediones are known to have acute effects on insulin action within hours in animals, their acute administration has never been studied in humans. We hypothesize that pioglitazone has acute effects on both hepatic and peripheral insulin action and on plasma lipid profiles in humans with T2DM. To further confirm that beneficial metabolic effects are due to activation of specific PPAR receptors, we will also examine the effect of pioglitazone on the expression of PPAR-regulated genes. This study, using state-of-the-art techniques of in vivo physiology and molecular biology, should provide novel insights about potential acute metabolic effects of thiazolidinediones in humans. Additionally, these studies would address for the first time in humans whether pioglitazone's beneficial effects on insulin action in skeletal muscle and adipose tissue might be at least in part due to effects on gene expression. SPECIFIC AIMS We will study 15 insulin resistant, T2DM subjects after oral administration of either placebo or pioglitazone. We will compare the following parameters under all experimental conditions: 1. Whole body in vivo glucose turnover during 6 hour euglycemic (90 mg/dl), stepped hyperinsulinemic clamp studies using isotope techniques 2. Circulating levels of free fatty acids (FFA), adiponectin, and other 'adipokines' (adipose-derived cytokines and acute phase reactants) 3. Gene expression, by real-time quantitative polymerase chain reaction (RTQ-PCR), in adipose tissue and in skeletal muscle. Adipose tissue will be separated into adipocyte, stromal cell and macrophage fractions. 4. Adipose macrophage content, by immunohistochemistry and Florescence Activated Cell Sorting (FACS) analysis 5. We will thereby determine whether acute administration of pioglitazone can impact insulin action and lipid metabolism in insulin resistant human T2DM subjects, and whether these metabolic effects might be mediated by altered expression of genes known to be regulated by PPAR-g agonists.