Glucose entry into muscle cells is precisely regulated by insulin, through recruitment of GLUT4 (glucose transporter-4) to the membrane of muscle and fat cells. Work done over more than two decades has contributed to mapping the insulin signalling and GLUT4 vesicle trafficking events underpinning this response. In spite of this intensive scientific research, there are outstanding questions that continue to challenge us today. The present review summarizes the knowledge in the field, with emphasis on the latest breakthroughs in insulin signalling at the level of AS160 (Akt substrate of 160 kDa), TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) and their target Rab proteins; in vesicle trafficking at the level of vesicle mobilization, tethering, docking and fusion with the membrane; and in the participation of the cytoskeleton to achieve optimal temporal and spatial location of insulin-derived signals and GLUT4 vesicles.

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.

Insulin enhances plasmalemmal-directed traffic of glucose transporter-4 (GLUT4), but it is unknown whether insulin regulates GLUT4 traffic through endosomal compartments. In L6 myoblasts expressing Myctagged GLUT4, insulin markedly stimulated the rate of GLUT4myc recycling. In myoblasts stimulated with insulin to maximize surface GLUT4myc levels, we followed the rates of surface-labeled GLUT4myc endocytosis and chased its intracellular distribution in space and time using confocal immunofluorescence microscopy. Surface-labeled GLUT4myc internalized rapidly (t1 ⁄2 3 min), reaching the early endosome by 2 min and the transferrin receptor-rich, perinuclear recycling endosome by 20 min. Upon re-addition of insulin, the t1 ⁄2 of GLUT4 disappearance from the plasma membrane was unchanged (3 min), but strikingly, GLUT4myc reached the recycling endosome by 10 and left by 20 min. This effect of insulin was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002 or by transiently transfected dominant-negative phosphatidylinositol 3-kinase and protein kinase B mutants. In contrast, insulin did not alter the rate of arrival of rhodamine-labeled transferrin at the recycling endosome. These results reveal a heretofore unknown effect of insulin to accelerate interendosomal travel rates of GLUT4 and identify the recycling endosome as an obligatory stage in insulindependent GLUT4 recycling.

The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored. Here we make use of L6 myoblasts stably expressing GLUT4 with an exofacially directed Myc-tag (GLUT4myc) to determine the exocytic and endocytic rates of the transporter. Insulin caused a rapid (t1 ⁄2 ‫؍‬ 4 min) gain, whereas hyperosmolarity (0.45 M sucrose) caused a slow (t1 ⁄2 ‫؍‬ 20 min) gain in surface GLUT4myc molecules. With prior insulin stimulation followed by addition of hypertonic sucrose, the increase in surface GLUT4myc was partly additive. Unlike the effect of insulin, the GLUT4myc gain caused by hyperosmolarity was insensitive to wortmannin or to tetanus toxin cleavage of VAMP2 and VAMP3. Disappearance of GLUT4myc from the cell surface was rapid (t1 ⁄2 ‫؍‬ 1.5 min). Insulin had no effect on the initial rate of GLUT4myc internalization. In contrast, hyperosmolarity almost completely abolished GLUT4myc internalization. Surface GLUT4myc accumulation in response to hyperosmolarity was only partially blocked by inhibition of tyrosine kinases with erbstatin analog (erbstatin A) and genistein. However, neither inhibitor interfered with the ability of hyperosmolarity to block GLUT4myc internalization. We propose that hyperosmolarity increases surface GLUT4myc by preventing GLUT4 endocytosis and stimulating its exocytosis via a pathway independent of phosphatidylinositol 3-kinase activity and of VAMP2 or VAMP3. A tetanus toxin-insensitive v-SNARE such as TI-VAMP detected in these cells, might mediate membrane fusion of the hyperosmolarity-sensitive pool. The glucose transporter 4 (GLUT4)1 is the predominant glucose transporter of muscle and adipose cells. In untreated adipocytes, GLUT4 recycles constitutively between the plasma membrane and intracellular loci (1, 2), with the steady-state distribution favoring the latter. Morphological and biochemical studies have detected GLUT4 in distinct but inter-related intracellular pools, including sorting endosomes, TGN, recycling endosomes, and specialized GLUT4 exocytic vesicles (3-6). GLUT4 endocytosis occurs via clathrin-coated vesicles, assisted by the GTPase dynamin. Thus, inhibition of clathrin-coated vesicle formation via K ϩ depletion (7), interference with dynamin-amphiphysin pairing (8), or expression of GTPase-deficient dynamin (9, 10), all prevent GLUT4 internalization in adipocytes. Little is known about the traffic of this transporter in muscle cells, despite the fact that muscle represents the largest in vivo site of glucose utilization.Insulin shifts the subcellular distribution of GLUT4 resulting in a new steady state where a large fraction of GLUT4 resides at the plasma membrane of skeletal muscle (11-13), primary adipose cells (14, 15), L6 muscle cells in culture (16), and 3T3-L1 adipocytes (1). Studies in adipocytes indicate that this shift occurs primarily through the stimulation of GLUT4 exocytosis (1, 2), but whether or not insulin inhibits GLUT4 endocytosis is still debatable (1, 2, 17, 18). The contribution of exocytic and endocytic pa...

Insulin increases glucose uptake into muscle by enhancing the surface recycling of GLUT4 transporters. In myoblasts, insulin signals bifurcate downstream of phosphatidylinositol 3-kinase into separate Akt and Rac/actin arms. Akt-mediated Rab-GAP AS160 phosphorylation and Rac/actin are required for net insulin gain of GLUT4, but the specific steps (vesicle recruitment, docking or fusion) regulated by Rac, actin dynamics, and AS160 target Rab8A are unknown. In L6 myoblasts expressing GLUT4myc, blocking vesicle fusion by tetanus toxin cleavage of VAMP2 impeded GLUT4myc membrane insertion without diminishing its build-up at the cell periphery. Conversely, actin disruption by dominant negative Rac or Latrunculin B abolished insulin-induced surface and submembrane GLUT4myc accumulation. Expression of non-phosphorylatable AS160 (AS160-4P) abrogated membrane insertion of GLUT4myc and partially reduced its cortical build-up, an effect magnified by selective Rab8A knockdown. We propose that insulin-induced actin dynamics participates in GLUT4myc vesicle retention beneath the membrane, whereas AS160 phosphorylation is essential for GLUT4myc vesicle-membrane docking/fusion and also contributes to GLUT4myc cortical availability through Rab8A.

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Background: Veno-arterial extracorporeal life support (VA-ECLS) is widely used to treat refractory cardiogenic shock. However, increased left ventricular (LV) afterload in VA-ECLS can worsen pulmonary congestion and compromise myocardial recovery. Our objectives were to explore the efficacy, safety, and optimal timing of adjunctive LV venting strategies. Methods: A systematic search was performed on Medline, EMBASE, PubMed, CDSR, CCRCT, CINAHL, ClinicalTrials.Gov, and WHO ICTRP from inception until January 2019 for all relevant studies, including LV venting. Data were analyzed for mortality and weaning from VA-ECLS on the basis of timing of LV venting, along with adverse complications. Results: A total of 7995 patients were included from 62 observational studies, wherein 3458 patients had LV venting during VA-ECLS. LV venting significantly improved weaning from VA-ECLS (odds ratio, 0.62 [95% CI, 0.47–0.83]; P =0.001) and reduced short-term (30 day; risk ratio [RR], 0.86 [95% CI, 0.77–0.96]; P =0.008) but not in-hospital (RR, 0.92 [95% CI, 0.83–1.01] P =0.09) or long-term (6 months; RR, 0.96 [95% CI, 0.90–1.03]; P =0.27) mortality. Early (<12 hours; RR, 0.86 [95% CI, 0.75–0.99]; P =0.03) but not late (≥12 hours; RR, 0.99 [95% CI, 0.71–1.38]; P =0.93) LV venting significantly reduced short-term mortality. Patients with LV venting spent more time on VA-ECLS (3.6 versus 2.8 days, P <0.001), and mechanical ventilation (7.1 versus 4.6 days, P =0.013). With the exception of hemolysis (RR, 2.18 [95% CI, 1.58–3.01]; P <0.00001), overall adverse events did not differ. Conclusions: LV venting, especially if done early (<12 hours), appears to be associated with an increased success of weaning and reduced short-term mortality. Future studies are required to delineate the importance of any or early LV venting adjuncts on mortality and morbidity outcomes.

Insulin increases glucose uptake into muscle via glucose transporter-4 (GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol (PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed the following: (a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. (b) Insulin caused greater immunological availability of the C terminus than myc epitopes, suggesting that C terminus unmasking had occurred. Delivering phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) to intact cells significantly increased lawnassociated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate (PI3P) increased the detection of C terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion, and C terminus unmasking, through distinct phosphoinositides. PI(3,4,5)P 3 causes arrival and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.Insulin promotes the uptake of glucose into muscle and fat tissues through a rapid gain in surface-bound glucose transporters (1-3). The muscle-and fat-specific glucose transporter GLUT4 1 cycles continuously between the plasma membrane and intracellular stores, with the steady-state distribution largely favoring the latter. Insulin changes this steady-state resulting in a net gain in surface GLUT4 (4 -6) largely as a result of enhancing the exocytic rate of GLUT4 cycling (7,8). Of significance, insulin resistance and diabetes are accompanied by defective GLUT4 gain at the plasma membrane of muscle and fat cells (9 -11).It is well established that signaling from class IA phosphatidylinositol (PI) 3-kinase is required for the insulin-dependent net gain in surface GLUT4 (12-15), but the specific step(s) in GLUT4 cycling that are regulated are not elucidated. We have recently shown that class IA PI 3-kinase is required for the insulin-dependent acceleration of GLUT4 transit through the recycling endosome (16). A second input of class IA PI 3-kinase in muscle cells is the spatial-temporal actin remodeling and its possible contribution to segregating specific signaling molecules (17, 18). However, it is not known whether fusion of insulin-sensitive GLUT4 vesicles with the plasma membrane is a regulated step, nor w...