Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.

Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actinrelated protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-⌬WA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-⌬WA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.Insulin stimulates glucose uptake by skeletal muscle and adipose tissues primarily through regulation of the subcellular distribution of the glucose transporter 4 (GLUT4) 1 (1, 2). In response to insulin, a fraction of GLUT4 present in intracellular membranes is redistributed to the plasma membrane, resulting in an increase of GLUT4 on the cell surface and enhanced glucose uptake by these cells. This effect of insulin is important in maintaining glucose homeostasis in humans, and impaired insulin action can contribute to the pathogenesis of type 2 diabetes (3). The precise mechanism by which insulin directs exocytosis of GLUT4-containing membrane vesicles remains obscure. However, it is established that activation of the insulin receptor tyrosine kinase catalyzes tyrosine phosphorylation of insulin receptor substrate proteins that bind to Srchomology 2 domain-containing molecules, including the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (4, 5). This results in activation of the p110 catalytic subunit of the kinase, which then phosphorylates cellular polyphosphoinositides at the D-3 position, forming signaling molecules such as phosphatidylinositol 3,4,5-trisphosphate. Multiple studies using various pharmacologic inhibitors, overexpression of constitutively active or dominant negative mutants, and microinjection of blocking antibodies have suggested a necessary role of the p85/p110-type PI 3-kinase in insulin-stimulated GLUT4 translocation and glucose transport (1, 2, 6 -8).On the other hand, several lines of evidence suggest the requirement of PI 3-kinase-independent pathway(s) in G...

Glucose homeostasis is controlled in part by regulation of glucose uptake into muscle and adipose tissue. Intracellular membrane vesicles containing the GLUT4 glucose transporter move towards the cell cortex in response to insulin and then fuse with the plasma membrane. Here we show that the fusion step is retarded by the inhibition of phosphatidylinositol (PI) 3-kinase. Treatment of insulin-stimulated 3T3-L1 adipocytes with the PI 3-kinase inhibitor LY294002 causes the accumulation of GLUT4-containing vesicles just beneath the cell surface. This accumulation of GLUT4-containing vesicles near the plasma membrane prior to fusion requires an intact cytoskeletal network and the unconventional myosin motor Myo1c. Remarkably, enhanced Myo1c expression under these conditions causes extensive membrane ruffling and overrides the block in membrane fusion caused by LY294002, restoring the display of GLUT4 on the cell exterior. Ultrafast microscopic analysis revealed that insulin treatment leads to the mobilization of GLUT4-containing vesicles to these regions of Myo1c-induced membrane ruffles. Thus, localized membrane remodeling driven by the Myo1c motor appears to facilitate the fusion of exocytic GLUT4-containing vesicles with the adipocyte plasma membrane.

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.

Chronic oxidative stress results in decreased responsiveness to insulin, eventually leading to diabetes and cardiovascular disease. Activation of the JNK signaling pathway can mediate many of the effects of stress on insulin resistance through inhibitory phosphorylation of insulin receptor substrate 1. By contrast, exercise, which acutely increases oxidative stress in the muscle, improves insulin sensitivity and glucose tolerance in patients with Type 2 diabetes. To elucidate the mechanism underlying the contrasting effects of acute versus chronic oxidative stress on insulin sensitivity, we used a cellular model of insulin-resistant muscle to induce either chronic or acute oxidative stress and investigate their effects on insulin and JNK signaling. Chronic oxidative stress resulted in increased levels of phosphorylated (activated) JNK in the cytoplasm, whereas acute oxidative stress led to redistribution of JNK-specific phosphatase MKP7 from the nucleus into the cytoplasm, reduction in cytoplasmic phospho-JNK, and a concurrent accumulation of phospho-JNK in the nucleus. Acute oxidative stress restored normal insulin sensitivity and glucose uptake in insulin-resistant muscle cells, and this effect was dependent on MKP7. We propose that the contrasting effects of acute and chronic stress on insulin sensitivity are driven by changes in subcellular distribution of MKP7 and activated JNK.Chronic oxidative stress is one of the major sources of metabolic abnormalities associated with Type 2 diabetes (1-3). High glucose and fatty acid levels lead to increased production of reactive oxygen species (ROS), 2 which can cause insulin resistance in peripheral metabolic tissues. This leads to decreased glucose uptake in muscle and adipose tissue, and eventually, pancreatic ␤ cell failure, glucose intolerance, and frank diabetes (4 -8).The mechanistic link between increased ROS levels and insulin resistance is activation of several signaling pathways, primarily mitogen-activated protein kinases (MAPK) pathways. JNK (Jun N-terminal kinases) are MAP kinases activated by cellular stresses, including oxidative stress, and play a role in apoptosis and survival, stress resistance, and immune response (9). Upstream signaling leading to JNK activation involves stress-induced MAPK kinases MEKK4 and MEKK7, as well as scaffold protein JIP (JNK-interacting protein) (10). Activation of JNK leads to dimerization followed by translocation into the nucleus, where it can phosphorylate its downstream target c-Jun, leading to activation of stress response and apoptotic pathways. JNKs are specifically dephosphorylated and inactivated by MAP kinase phosphatase 7 (MKP7), which also acts as a shuttle protein and was proposed to be involved in JNK nucleocytoplasmic translocation (11).Obesity increases JNK activation in muscle and adipose tissue in mice. Genetic ablation or pharmacological inhibition of JNK results in marked improvement of insulin sensitivity in mouse models of diet-induced obesity and insulin resistance (6, 12, 13). Mechanistically, J...

The identification of highly potent and orally bioavailable GPR39 agonists is reported. Compound 1, found in a phenotypic screening campaign, was transformed into compound 2 with good activity on both the rat and human GPR39 receptor. This compound was further optimized to improve ligand efficiency and pharmacokinetic properties to yield GPR39 agonists for the potential oral treatment of type 2 diabetes. Thus, compound 3 is the first potent GPR39 agonist (EC 50 s ≤ 1 nM for human and rat receptor) that is orally bioavailable in mice and robustly induced acute GLP-1 levels.

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and transGolgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNAbased gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.Blood glucose homeostasis is maintained by insulin in part through glucose disposal into muscle and fat tissues. This is achieved by means of a redistribution of the facilitative glucose transporter GLUT4 from an internal sequestration compartment to the cell surface where it facilitates glucose uptake into these cells. In the absence of insulin GLUT4 recycles very slowly from internal membranes to the plasma membrane, whereas insulin markedly increases the exocytic rate of these GLUT4-containing vesicles. This results in a net increase of GLUT4 at the plasma membrane. This regulated trafficking pathway for GLUT4 appears to be distinct from that of many other membrane proteins such as the transferrin receptor or the GLUT1 glucose transporter protein.Insulin stimulation of adipocytes increases the abundance of these latter proteins at the cell surface by ϳ2-fold, whereas the amount of GLUT4 is increased between 10 -20-fold (1, 2). Other insulin-regulated exocytic pathways likely operate in adipocytes as well, such as a secretory mechanism for peptides like Acrp30/adiponectin. Like transferrin receptor recycling to the cell surface, Acrp30 secretion is only modestly stimulated by insulin (3). This large difference in insulin sensitivity indicates that the molecular mechanisms of GLUT4 sequestration and trafficking have unique regulatory elements. To understand insulin action on glucose transport it will be necessary to elucidate the nature of GLUT4 sequestration in intracellular membranes, the transit of GLUT4-containing intracellular membranes toward the cell surface, and the fusion of these membranes with the plasma membrane.Although the trafficking of the transferrin receptor and GLUT4 differ in their response to insulin stimulation, it appears that ϳ50% of the ...