G_α11 Signaling through ARF6 Regulates F-Actin Mobilization and GLUT4 Glucose Transporter Translocation to the Plasma Membrane

Abstract

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G ␣ q or G ␣ 11 proteins. Here we report that murine G ␣ 11 is most abundant in fat and that expression of the constitutively active form of G ␣ 11 [G ␣ 11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G ␣ 11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G ␣ 11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G ␣ 11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.

EHD2 and the Novel EH Domain Binding Protein EHBP1 Couple Endocytosis to the Actin Cytoskeleton

“…After immunogold labeling, the cell preparations were fixed and processed as described (20). For immunogold labeling of EHD2, GLUT4, and actin at plasma membrane, lawns from 3T3-L1 adipocytes were generated as described (23). Immediately after preparation, membrane lawns were fixed on coverslips with 3.7% formaldehyde and labeled with primary antibodies, followed by 6-and 12-nm gold-tagged secondary antibodies.…”

“…The EHBP1 protein was detected by immunoblotting with affinity-purified anti-EHBP1 antibody. For Northern blot analysis, total RNA from 3T3-L1 fibroblasts, differentiated adipocytes, and different mouse tissues was isolated as described (20,23). 10 g of total RNA was resolved on a 1.2% denaturing gel and was transferred to a Nytran membrane.…”

“…Cell Culture and Transfection of Differentiated 3T3-L1 Adipocytes-3T3-L1 fibroblasts were grown to confluency and differentiated as described previously (23). Differentiated 3T3-L1 adipocytes were transfected by electroporation as described (24).…”

A Phosphatidylinositol 3-Kinase-independent Insulin Signaling Pathway to N-WASP/Arp2/3/F-actin Required for GLUT4 Glucose Transporter Recycling

“…Furthermore, interleukin-4 and anti-integrin antibodies do not apparently stimulate glucose transport in adipocytes, although these agonists activate PI 3-kinase (10,11). Other studies suggest that both endothelin-1 and physical exercise stimulate glucose uptake independent of PI 3-kinase activation in cultured adipocytes and muscle, respectively (12)(13)(14)(15). Recent reports indicate that insulin stimulation of GLUT4 translocation requires activation of the small GTPase TC10 through a PI 3-kinase-independent pathway involving the docking of adaptor protein CAP and tyrosine phosphorylation of c-Cbl in adipocytes (16,17).…”

“…In searching for such PI 3-kinase-independent signaling elements that may be downstream of TC10, we focused on recent findings from our laboratory (13,18,19) and others (20 -26) linking the microtubule-and actin-based cytoskeleton to recycling of GLUT4-containing vesicles. In particular, insulin elicits actin filament (F-actin) formation (13, 18 -24, 27, 28), and reagents that cause actin depolymerization inhibit insulin-induced GLUT4 translocation and glucose uptake (18 -24).…”