There is little doubt that we are in the midst of a worldwide epidemic of diabetes. Over 20 million people in the US are thought to be afflicted, a fourth of whom are undiagnosed. Insulin resistance is recognized as a characteristic trait of the disease, defined by the inability to respond to normal circulating levels of insulin. The primary lesion in this state involves defects in the uptake and storage of glucose in muscle and fat cells. Targeting these defects holds the key to the development of new therapeutic approaches. However, understanding the specific lesions that cause insulin resistance will first require a better grasp of the cell biology of insulin action. To this end, we investigate the molecular events involved in the regulation of glucose uptake and storage by insulin, with special attention to mechanisms underlying the specificity of the actions of the hormone.
Insulin-stimulated glucose transport is mediated by the glucose transporter Glut4, which translocates from intracellular compartments to the plasma membrane of muscle and fat cells in response to insulin. Although the PI-3 kinase pathway is required for this action of insulin, we and others have shown that other signaling events are involved. We discovered a novel PI-3 kinase-independent pathway that is localized to discrete domains of the plasma membrane called lipid rafts. The signaling cascade is initiated by the phosphorylation of the protooncogene c-Cbl, recruited to the receptor by the adapter protein APS, and anchored to lipid raft microdomains by a multifunctional protein called CAP. Upon phosphorylation, Cbl in turn recruits the Crk/C3G complex, which activates the small GTPase TC10. CAP also interacts with and regulates cytoskeletal proteins involved in cell migration and growth. We are investigating the molecular dynamics by which these complexes form, their physiological roles in knockout mouse models, and how crosstalk from other signaling pathways might influence these events in cells.
Small GTPases function as “molecular switches” that cycle between inactive GDP-bound and active GTP-bound states. Many receptor tyrosine kinases utilize small GTPases to transduce, amplify, or convert signals to downstream effectors, particularly during regulation of vesicle trafficking. One such molecular switch is the Rho family G protein TC10. We have identified several effectors of TC10 that are involved in insulin-stimulated glucose transport. These include the adaptor proteins CIP4, TCGAP, Par6, and Exo70. These proteins are recruited to the plasma membrane upon the activation of TC10 by insulin, and in turn regulate different downstream processes, including protein kinases, lipid kinases and other G proteins, including those in the Rab family. We are investigating the molecular details underlying the regulation of TC10 activity, focusing on guanyl nucleotide exchange factors (GEF), guanyl nucleotide dissociation inhibitors (GDI) and GTPase-activating proteins (GAP) that are implicated in insulin action. We are also focusing on events downstream of TC10, and study the physiological role of TC10 with knockout mouse models.
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The exocyst is an octameric complex first identified in yeast as a tethering site for targeted exocytosis. We discovered that this complex plays a key role in the regulation of glucose transport, targeting Glut4 vesicles to sites of docking and fusion in adipocytes. The exocyst appears to be assembled in two subcomplexes, a target (t-) complex that comes together at the plasma membrane, and a vesicular (v-) complex that assembles at Glut4 vesicles. The assembly of each sub-complex appears to be controlled by distinct G proteins activated by a different process, and then unified prior to vesicle docking. The activation of TC10 recruits the t-exocyst to the plasma membrane, while activation of the G protein RalA recruits the v-exocyst, and then brings together the complex. We are investigating the order of exocyst assembly, its coordination with other transport machineries during Glut4 trafficking and additional regulatory mechanisms that influence the exocyst.
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Defects in the regulation of non-oxidative glucose metabolism by insulin may be among the primary lesions in insulin resistance. In peripheral tissues, insulin modulates glycogen accumulation through a coordinate increase in glucose transport and regulation of glycogen metabolizing enzymes, while in liver insulin blocks glucose output by inhibiting gluconeogenesis, promoting glycogenesis and blocking glycogenolysis. The principle mechanism by which hormones control glycogen metabolism is through regulation of glycogen synthase. Insulin activates glycogen synthase by several mechanisms, although the most prominent contribution involves promotion of the net dephosphorylation of the enzyme. We identified PTG as a protein phosphatase-scaffolding molecule that localizes exclusively to glycogen particles, and promotes the dephosphorylation of glycogen synthase and phosphorylase. We are investigating how hormones such as insulin control glycogen metabolism via the function of PTG and its related molecules.
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Obesity induces a state of chronic low grade inflammation that contributes to the development of metabolic syndrome and diabetes. The inflammatory changes in obesity include monocyte activation, increased circulating inflammatory factors, and inflammation in fat tissue. A key component of the inflammatory response to obesity is the infiltration of adipose tissue with macrophages. These adipose tissue macrophages (ATMs) are a source of inflammatory cytokines that alter adipocyte insulin sensitivity and are required for the development of diabetes in obese mice. We have performed detailed characterization of the properties of ATMs isolated from lean and obese mice using a model of high fat diet feeding. These studies have shown that obesity leads to significant alterations in the inflammatory properties of ATMs and have revealed mechanisms by which activated macrophages impair fat cell function. We are continuing to investigate the biology of ATMs in obesity to devise strategies that might prevent their activation and block adipose tissue inflammation. Additionally, we are studying the function of ATMs in knockout mice to identify the important inflammatory signalling pathways relevant in obesity and diabetes.
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Read about our laboratory's recent discoveries regarding macrophages and their role in insulin resistance here and in The Journal of Clinical Investigation, Diabetes and The American Journal of Physiology.
