The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.

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Insulin signalling and the regulation of glucose and lipid metabolism

Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.

Rho GTPases in cell biology.

The RAS oncogenes were identified almost 20 years ago. Since then, we have learnt that they are members of a large family of small GTPases that bind GTP and hydrolyse it to GDP. This is then exchanged for GTP and the cycle is repeated. The switching between these two states regulates a wide range of cellular processes. A branch of the RAS family--the RHO proteins--is also involved in cancer, but what is the role of these proteins and would they make good therapeutic targets?

RHO-GTPases and cancer.

The GLUT4 Glucose Transporter

https://www.uv.es/elanuza/Dinamica/Rho%20GTPasas.pdf

Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking.

The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface. Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation. We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP. On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin. Here we show that phosphorylated Cbl recruits the CrkII-C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.

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Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10

Since the discovery of insulin roughly 80 yr ago, much has been learned about how target cells receive, interpret, and respond to this peptide hormone. For example, we now know that insulin activates the tyrosine kinase activity of its cell surface receptor, thereby triggering intracellular signaling cascades that regulate many cellular processes. With respect to glucose homeostasis, these include the function of insulin to suppress hepatic glucose production and to increase glucose uptake in muscle and adipose tissues, the latter resulting from the translocation of the glucose transporter 4 (GLUT4) to the cell surface membrane. Although simple in broad outline, elucidating the molecular intricacies of these receptor-signaling pathways and membrane-trafficking processes continues to challenge the creative ingenuity of scientists, and many questions remain unresolved, or even perhaps unasked. The identification and functional characterization of specific molecules required for both insulin signaling and GLUT4 vesicle trafficking remain key issues in our pursuit of developing specific therapeutic agents to treat and/or prevent this debilitating disease process. To this end, the combined efforts of numerous research groups employing a range of experimental approaches has led to a clearer molecular picture of how insulin regulates the membrane trafficking of GLUT4.

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Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes.

Glucose is cleared from the bloodstream by a family of facilitative transporters (GLUTs), which catalyze the transport of glucose down its concentration gradient and into cells of target tissues, primarily striated muscle and adipose. Currently, there are five established functional facilitative glucose transporter isoforms (GLUT1-4 and GLUTX1), with GLUT5 being a fructose transporter. GLUT1 is ubiquitously expressed with particularly high levels in human erythrocytes and in the endothelial cells lining the blood vessels of the brain. GLUT3 is expressed primarily in neurons and, together, GLUT1 and GLUT3 allow glucose to cross the blood-brain barrier and enter neurons. GLUT2 is a low-affinity (high Km) glucose transporter present in liver, intestine, kidney, and pancreatic beta cells. This transporter functions as part of the glucose sensor system in beta cells and in the basolateral transport of intestinal epithelial cells that absorb glucose from the diet. A new facilitative glucose transporter protein, GLUTX1, has been identified and appears to be important in early blastocyst development. The GLUT4 isoform is the major insulin-responsive transporter that is predominantly restricted to striated muscle and adipose tissue. In contrast to the other GLUT isoforms, which are primarily localized to the cell surface membrane, GLUT4 transporter proteins are sequestered into specialized storage vesicles that remain within the cell's interior under basal conditions. As postprandial glucose levels rise, the subsequent increase in circulating insulin activates intracellular signaling cascades that ultimately result in the translocation of the GLUT4 storage compartments to the plasma membrane. Importantly, this process is readily reversible such that when circulating insulin levels decline, GLUT4 transporters are removed from the plasma membrane by endocytosis and are recycled back to their intracellular storage compartments. Therefore, by establishing an internal membrane compartment as the default localization for the GLUT4 transporters, insulin-responsive tissues are poised to respond rapidly and efficiently to fluctuations in circulating insulin levels. Unfortunately, the complexity of these regulatory processes provides numerous potential targets that may be defective and eventually result in peripheral tissue insulin resistance and possibly diabetes. As such, understanding the molecular details of GLUT4 expression, GLUT4 vesicle compartment biogenesis, GLUT4 sequestration, vesicle trafficking, and fusion with the plasma membrane has become a major focus for many laboratories. This chapter will focus on recently elucidated insulin signal transduction pathways and GLUT4 vesicle trafficking components that are necessary for insulin-stimulated glucose uptake and GLUT4 translocation in adipocytes.

/pdf/intracellular-organization-of-insulin-signaling-and-glut4-54huyn5fjr.pdf

Intracellular organization of insulin signaling and GLUT4 translocation.

http://fisica.uc.pt/data/20062007/apontamentos/apnt_1202_5.pdf

Bridging the GAP between insulin signaling and GLUT4 translocation

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

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Robert T. Watson

Papers

Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10

Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes.

Intracellular organization of insulin signaling and GLUT4 translocation.

Bridging the GAP between insulin signaling and GLUT4 translocation

Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.