The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin

Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62982/1/nature01533.pd

Inflammation produces catecholamine resistance in obesity via activation of PDE3B by the protein kinases IKKε and TBK1.

Obesity produces a chronic inflammatory state involving the NFκB pathway, resulting in persistent elevation of the noncanonical IκB kinases IKKε and TBK1. In this study, we report that these kinases attenuate β-adrenergic signaling in white adipose tissue. Treatment of 3T3-L1 adipocytes with specific inhibitors of these kinases restored β-adrenergic signaling and lipolysis attenuated by TNFα and Poly (I:C). Conversely, overexpression of the kinases reduced induction of Ucp1, lipolysis, cAMP levels, and phosphorylation of hormone sensitive lipase in response to isoproterenol or forskolin. Noncanonical IKKs reduce catecholamine sensitivity by phosphorylating and activating the major adipocyte phosphodiesterase PDE3B. In vivo inhibition of these kinases by treatment of obese mice with the drug amlexanox reversed obesity-induced catecholamine resistance, and restored PKA signaling in response to injection of a β-3 adrenergic agonist. These studies suggest that by reducing production of cAMP in adipocytes, IKKε and TBK1 may contribute to the repression of energy expenditure during obesity. DOI: http://dx.doi.org/10.7554/eLife.01119.001

Lipid raft microdomain compartalization of TC10 is required for insulin signalling 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

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

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(1-6). 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(7). 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(8,9). On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin(10). 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62864/1/410944a0.pd

Signaling pathways involved in insulin -stimulated glucose transport.

Insulin is a key hormone secreted from pancreatic beta cells that is essential for glucose homeostasis. The hormone rapidly stimulates glucose uptake into muscle and adipocytes, via the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. Although the cellular dynamics of GLUT4 trafficking are well studied, our understanding of the signaling pathways linking the insulin receptor to GLUT4 translocation remains incomplete. While tyrosine phosphorylation of the IRS family of insulin receptor substrates and the subsequent activation of the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) is required, numerous studies indicate that it is not sufficient for this action of insulin. This thesis describes a new PI 3-kinase independent pathway that is specifically activated in response to insulin, downstream of the CAP/Cbl axis, which is also essential for the stimulation of GLUT4 translocation by insulin. Upon tyrosine phosphorylation in response to insulin, Cbl translocates with CAP to a lipid raft subdomain in the plasma membrane, due to the interaction of CAP with flotillin. Phospho-Cbl then binds to CrkII, in the process recruiting the Crk-associated protein C3G into lipid rafts. The Rho GTPase TC10 in lipid rafts is in turn activated by C3G due to its close proximity. This thesis also characterizes the mouse homologue of TC10 (TC10alpha) and another TC10-like Rho GTPase (TC10beta) cloned from 3T3-L1 adipocytes. Surprisingly, both TC10alpha and TC10beta reside in 3T3-L1 adipocytes, and expression of tagged forms of the proteins revealed their localization in caveolin-enriched lipid rafts. In addition, both GTPases can be activated by insulin. A novel Synip-interacting Rho GAP protein (TCGAP) was isolated from a yeast two-hybrid screen. This TCGAP specifically interacts with the WW domain of Synip, the Syntaxin 4 interacting protein that may regulate v- and t-SNARE interactions that are crucial to GLUT4 vesicle docking and fusion. The constitutive association between TCGAP and Synip facilitates the dissociation of Synip from Syntaxin 4 in response to insulin. In addition to the specific binding to Synip, TCGAP also interacts with Rho GTPases in a GTP-dependent manner through its Rho GAP domain. The substrate specificity of the Rho GAP activity of TCGAP correlates well with its in vitro binding affinity toward Rho GTPases. Elucidation of the events downstream of TC10 activation and inactivation may help further the understanding of the signaling pathways critical to insulin action.Ph.D.Biological SciencesCellular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127355/2/3029313.pd

Compartmentalization of the Exocyst Complex in Lipid Rafts Controls Glut4 Vesicle Tethering

Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8. Knockdowns of these proteins block insulin-stimulated glucose uptake. Moreover, their targeting to lipid rafts is required for glucose uptake and Glut4 docking at the plasma membrane. The assembly of this complex also requires the PDZ domain protein SAP97, a member of the MAGUKs family, which binds to Sec8 upon its translocation to the lipid raft. Exocyst assembly at lipid rafts sets up targeting sites for Glut4 vesicles, which transiently associate with these microdomains upon stimulation of cells with insulin. These results suggest that the TC10/exocyst complex/SAP97 axis plays an important role in the tethering of Glut4 vesicles to the plasma membrane in adipocytes

TCGAP, a multidomain Rho GTPase-activating protein involved in insulin-stimulated glucose transport

International audienceInsulin stimulates glucose uptake in fat and muscle cells via the translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the cell surface. The signaling pathways linking the insulin receptor to GLUT4 translocation in adipocytes involve activation of the Rho family GTPases TC10alpha and beta. We report here the identification of TCGAP, a potential effector for Rho family GTPases. TCGAP consists of N-terminal PX and SH3 domains, a central Rho GAP domain and multiple proline-rich regions in the C-terminus. TCGAP specifically interacts with cdc42 and TC10beta through its GAP domain. Although it has GAP activity in vitro, TCGAP is not active as a GAP in intact cells. TCGAP translocates to the plasma membrane in response to insulin in adipocytes. The N-terminal PX domain interacts specifically with phos phatidylinositol-(4,5)-bisphosphate. Overexpression of the full-length and C-terminal fragments of TCGAP inhibits insulin-stimulated glucose uptake and GLUT4 translocation. Thus, TCGAP may act as a downstream effector of TC10 in the regulation of insulin-stimulated glucose transport

TCGAP, a multidomain Rho GTPase‐activating protein involved in insulin‐stimulated glucose transport

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102135/1/emboj7595173-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102135/2/emboj7595173.pd