Neil Ruderman - US grants

Vascular disease is the major cause of morbidity and mortality in diabetes. Two types have been defined--microvascular disease affecting predominantly the eye and kidney and macrovascular disease (atherosclerosis) affecting both peripheral and coronary arteries. To study the pathogenesis of these disorders, relevant functional, morphological and metabolic parameters will be compared in animal models with diabetes and in in vitro systems. Particular emphasis will be placed on studying the normal metabolism and function of the vascular wall and its components and delineating how they are altered in diabetes. These studies will utilize cultured endothelial and smooth muscle cells, isolated capillaries, and segments of large arteries from control and diabetic animals. Among the areas to be studied are cell motility and growth, the metabolism of fuels and connective tissue and prostaglandin synthesis. In addition the biological effects of insulin will be assessed as will the nature of the insulin receptor in endothelial and smooth muscle cells. We will also attempt to study the pathogenesis of macrovascular disease in the rabbit, and animal model in which diabetes has so far been shown to lessen the severity of atherosclerosis for reasons unknown. Several potential atherogenic factors will be assessed including circulating lipoproteins and platelets, the metabolism of lipids and liproteins by the arterial wall, and the severity of diabetes. Finally, one of the projects will evaluate a morphometric method for studying the natural history of diabetic microangiopathy. This work will be carried out in man and will utilize a unique population of identical twins, some concordant and some discordant for diabetes. The studies in this program should provide important insights into the pathogenesis of macrovascular and microvascular disease in diabetes. They will also, hopefully, provide a groundwork for devising approaches for preventing these complications.

It has long been known that after exercise glucose utilization and glycogen synthesis in skeletal muscle are enhanced. We have recently demonstrated that another post-exercise change in muscle is an altered response to insulin. Thus, using intact rats and a perfused hindquarter preparation, we found that the stimulation of glucose and AIB transport and glycogen synthesis by insulin were enhanced after voluntary exercise or electrical stimulation. In addition after intense exercise insulin increased 0-2 consumption, an effect it did not have in resting muscle. The overall objectives of this proposal are to examine this characterization of the post-exercise state and to explore the physiological relevance and possible mechanisms for the increase in insulin effect. Using the perfused rat hindquarter, the intact rat and man, we will carry out studies with the following specific aims: (1) To characterize further the effects of prior exercise on insulin action in muscle. The basis for the enhanced ability of insulin to stimulate glycogen synthesis in the hindquarter preparation will be examined, as will the effects of prior exercise on the ability of insulin to stimulate protein synthesis and activate pyruvate dehydrogenase and glycogen synthase; (2) To study possible mechanisms for the post-exercise increase in insulin sensitivity. Insulin binding and insulin-stimulated phosphorylation of the insulin receptor will be examined in muscle, as will the effect of exercise itself on receptor phosphorylation. (3) To determine whether prior exercise increases insulin sensitivity in vivo. Whole body and individual organ insulin sensitivity will be assessed in the conscious rat using an insulin-glucose clamp technique developed by one of the applicants. (4) To examine the basis for increased thermogenesis in the post-exercise state. Processes that could contribute to the insulin-induced increase in thermogenesis in the perfused hindquarter will be studied. In addition we will assess physiological determinants and possible mechanisms for the post-exercise increase in dietary-induced thermogenesis we have observed in humans. These studies should help delineate the array of metabolic alterations produced by insulin in the post-exercise state. They should also cast light on the mechanisms by which sensitivity and insulin-induced thermogenesis in muscle are enhanced by prior exercise.

The overall objectives of this project are twofold: first to examine the mechanisms responsible for alterations in cerebral microvascular hexose transport and fuel metabolism we have observed in the diabetic rat; and second to examine the relationship between these alterations and the regulation of myo- inositol transport, NaKATPase activity and polyol metabolism. The proposed studies will utilize intact rats, isolated microvessels and glomeruli and cultured microvascular cells. The specific aims are as follows: 1). To determine the factors and mechanisms responsible for the downregulation of hexose transport across the blood-brain barrier (BBB) of diabetic rats. Hexose transport in vivo will be related to alterations in hexose transporter number, structure and (mRNA) in microvessels isolated from diabetic and other rats in which plasma insulin or glucose are chronically altered. 2). To examine the mechanism(s) by which hyperglycemia downregulates hexose (3-methylglucose) transport in cerebral microvascular endothelium (CMEC). The role of glucose metabolism and protein synthesis will be evaluated. Many of the parameters studied will be the same as those in Aim 1, thereby enabling us to compare the mechanism(s) by which hexose transport is downregulated in vivo in the diabetic rat and in vitro in CMEC grown in a glucose-enriched medium. 3). To determine the basis for upregulation of B-hydroxybutyrate transport across the BBB in diabetes and its relation to altered hexose transport. 4). To determine the functional consequences of altered transport and fuel metabolism in microvessels of diabetic rats. The ability to maintain myo-inositol and glutathione content, NaK ATPase activity and AIB transport will be assessed in microvessels from at least two sites and in isolated glomeruli. The effect of diabetes on O2 consumption and fuel metabolism in these tissues will also be examined. 5). To determine the effect of hyperglycemia on myo-inositol transport, polyol metabolism and NaK ATPase in CMEC and pericytes. These studies should provide fundamental information about the early metabolic and functional alterations produced by diabetes in the microvasculature and the mechanisms by which they occur. They will hopefully provide a groundwork for understanding the dysregulation of signal transduction in the microvasculature and for developing metabolically-based therapies to prevent the development of microvascular disease.

ABSTRACT A novel phosphatidylinositol-3 kinase (PI3K) that appears to play a key role in cell transformation and growth has recently been described by Cantley and co-workers. Over the past 6 months, the principal investigator, while on sabbatical in Cantley's laboratory, has demonstrated that insulin dramatically increases the activity of this enzyme in CHO cells transfected with human insulin receptors (CHO-HIR). The proposed studies will examine the basis for this effect, the physiological relevance of PI3K will be explored. The specific aims are (1) To complete the characterization of the time-course and dose response to insulin of PI3K in CHO-HIR, both in vitro (anti-P-tyr and anti-insulin receptor antibody immunoprecipitates) and in intact cells. (2) To compare the response of CHO-HIR to those of CHO cells that are deficient in insulin receptors or have been transfected with mutant receptors that do not express tyrosine kinase. (3) To evaluate the relation of PI3K activation to acute and long- term metabolic effects of insulin. (4) To test the hypothesis that activation of its intrinsic tyrosine kinase causes the insulin receptor to tissues and determine whether it is altered by ageing or nutritional and hormonal perturbations. These studies should provide important information about the physiological relevance of PI3K and its regulation by insulin. Based on our preliminary data, we also anticipate they will provide new insights into the mechanism of signal transduction by insulin receptor kinase.

Skeletal muscle is a site of insulin resistance after denervation and in pathological states characterized by hyperinsulinemia, hyperglycemia and/or persistent elevations of plasma free fatty acid (FFA). This proposal will examine the hypothesis that insulin resistance in these situations involves alterations in a diacylglycerol-protein kinase C (DAG-PKC) signalling system. Based on preliminary data with an incubated soleus muscle preparation, we are proposing a model in which increases in DAG in these conditions (1) occur in a specific pool, (2) are predominantly due to DAG synthesis de novo and (3) are associated with an increase in PKC, and ultimately with alterations In DAG-PKC signalling that result in insulin resistance. The proposed studies will both test this paradigm and explore the biological role of the DAG-PKC signalling system in insulin action. Using incubated and perfused muscle preparations, we will carry out studies with the following aims: 1. To determine the mechanisms for the increase in DAG synthesis in insulin+glucose-stimulated and denervated soleus muscles. 2. To characterize the interrelationships between changes in DAG mass and synthesis and PKC activity. 3. To characterize the temporal relations between changes in DAG-PKC signalling and the development of insulin resistance. 4. To determine whether alterations in DAG-PKC affect insulin-mediated gene expression. 5. To compare DAG-PKC signalling in the soleus , a predominantly slow-twitch red muscle, and the extensor digitorum longus (EDL), a muscle composed mainly of white fibers. 6. To evaluate the effect of prior exercise, a maneuver that increases insulin sensitivity in muscle, on DAG-PKC signalling. These studies should provide novel information about the linkage between fuel-metabolism and signal transduction in skeletal muscle. They should also yield new insights into the role of DAG-PKC signalling in the pathogenesis of insulin resistance.