GLUT4 Mobilization Research Articles

Interleukin-6 Treatment Results in GLUT4 Translocation and AMPK Phosphorylation in Neuronal SH-SY5Y Cells.

Interleukin-6 (IL-6) is a pleiotropic cytokine that can be released from the brain during prolonged exercise. In peripheral tissues, exercise induced IL-6 can result in GLUT4 translocation and increased glucose uptake through AMPK activation. GLUT4 is expressed in the brain and can be recruited to axonal plasma membranes with neuronal activity through AMPK activation. The aim of this study is to examine if IL-6 treatment: (1) results in AMPK activation in neuronal cells, (2) increases the activation of proteins involved in GLUT4 translocation, and (3) increases neuronal glucose uptake. Retinoic acid was used to differentiate SH-SY5Y neuronal cells. Treatment with 100 nM of insulin increased the phosphorylation of Akt and AS160 (p < 0.05). Treatment with 20 ng/mL of IL-6 resulted in the phosphorylation of STAT3 at Tyr705 (p ≤ 0.05) as well as AS160 (p < 0.05). Fluorescent Glut4GFP imaging revealed treatment with 20ng/mL of IL-6 resulted in a significant mobilization towards the plasma membrane after 5 min until 30 min. There was no difference in GLUT4 mobilization between the insulin and IL-6 treated groups. Importantly, IL-6 treatment increased glucose uptake. Our findings demonstrate that IL-6 and insulin can phosphorylate AS160 via different signaling pathways (AMPK and PI3K/Akt, respectively) and promote GLUT4 translocation towards the neuronal plasma membrane, resulting in increased neuronal glucose uptake in SH-SY5Y cells.

GLUT4 Mobilization Supports Energetic Demands of Active Synapses

The brain is highly sensitive to proper fuel availability as evidenced by the rapid decline in neuronal function during ischemic attacks and acute severe hypoglycemia. We previously showed that sustained presynaptic function requires activity-driven glycolysis. Here, we provide strong evidence that during action potential (AP) firing, nerve terminals rely on the glucose transporter GLUT4 as a glycolytic regulatory system to meet the activity-driven increase in energy demands. Activity at synapses triggers insertion of GLUT4 into the axonal plasma membrane driven by activation of the metabolic sensor AMP kinase. Furthermore, we show that genetic ablation of GLUT4 leads to an arrest of synaptic vesicle recycling during sustained AP firing, similar to what is observed during acute glucose deprivation. The reliance on this biochemical regulatory system for "exercising" synapses is reminiscent of that occurring in exercising muscle to sustain cellular function and identifies nerve terminals as critical sites of proper metabolic control.

Metabolic alterations derived from absence of Two-Pore Channel 1 at cardiac level.

Two-pore channels (TPCs or TPCNs) are novel voltage-gated ion channels that have been postulated to act as Ca2+ and/or Na+ channels expressed exclusively in acidic organelles such as endosomes and lysosomes. TPCNs participate in the regulation of diverse biological processes and recently have been proposed to be involved in the pathophysiology of metabolic disorders such as obesity, fatty liver disease and type 2 diabetes mellitus. Due to the importance of these pathologies in the development of cardiovascular diseases, we aimed to study the possible role of two-pore channel 1 (TPCN1) in the regulation of cardiac metabolism. To explore the cardiac function of TPCN1, we developed proteomic approaches as 2-DE-MALDI-MS and LC-MALDI-MS in the cardiac left ventricle of TPCN1 KO and WT mice, and found alterations in several proteins implicated in glucose and fatty acid metabolism in TPCN1 KO vs. WT mice. The results confirmed the altered expression of HFABP, a key fatty acid transport protein, and of enolase and PGK1, the key enzymes in the glycolytic process. Finally, in vitro experiments performed in neonatal rat cardiomyocytes, in which TPCN1 was silenced using siRNAs, confirmed that the downregulation of TPCN1 gene expression increased 2-deoxy-D-[3H]-glucose uptake and GLUT4 mobilization into cell peripherals in cardiac cells. Our results are the first to suggest a potential role for TPCNs in cardiac metabolism regulation.

SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

RAB10 is a regulator of insulin-stimulated translocation of the GLUT4 glucose transporter to the plasma membrane (PM) of adipocytes, which is essential for whole-body glucose homeostasis. We establish SEC16A as a novel RAB10 effector in this process. Colocalization of SEC16A with RAB10 is augmented by insulin stimulation, and SEC16A knockdown attenuates insulin-induced GLUT4 translocation, phenocopying RAB10 knockdown. We show that SEC16A and RAB10 promote insulin-stimulated mobilization of GLUT4 from a perinuclear recycling endosome/TGN compartment. We propose RAB10-SEC16A functions to accelerate formation of the vesicles that ferry GLUT4 to the PM during insulin stimulation. Because GLUT4 continually cycles between the PM and intracellular compartments, the maintenance of elevated cell-surface GLUT4 in the presence of insulin requires accelerated biogenesis of the specialized GLUT4 transport vesicles. The function of SEC16A in GLUT4 trafficking is independent of its previously characterized activity in ER exit site formation and therefore independent of canonical COPII-coated vesicle function. However, our data support a role for SEC23A, but not the other COPII components SEC13, SEC23B, and SEC31, in the insulin stimulation of GLUT4 trafficking, suggesting that vesicles derived from subcomplexes of COPII coat proteins have a role in the specialized trafficking of GLUT4.

Sustained AS160 and TBC1D1 phosphorylations in human skeletal muscle 30 min after a single bout of exercise.

phosphorylation of AS160 and TBC1D1 plays an important role for GLUT4 mobilization to the cell surface. The phosphorylation of AS160 and TBC1D1 in humans in response to acute exercise is not fully characterized. to study AS160 and TBC1D1 phosphorylation in human skeletal muscle after aerobic exercise followed by a hyperinsulinemic euglycemic clamp. eight healthy men were studied on two occasions: 1) in the resting state and 2) in the hours after a 1-h bout of ergometer cycling. A hyperinsulinemic euglycemic clamp was initiated 240 min after exercise and in a time-matched nonexercised control condition. We obtained muscle biopsies 30 min after exercise and in a time-matched nonexercised control condition (t = 30) and after 30 min of insulin stimulation (t = 270) and investigated site-specific phosphorylation of AS160 and TBC1D1. phosphorylation on AS160 and TBC1D1 was increased 30 min after the exercise bout, whereas phosphorylation of the putative upstream kinases, Akt and AMPK, was unchanged compared with resting control condition. Exercise augmented insulin-stimulated phosphorylation on AS160 at Ser(341) and Ser(704) 270 min after exercise. No additional exercise effects were observed on insulin-stimulated phosphorylation of Thr(642) and Ser(588) on AS160 or Ser(237) and Thr(596) on TBC1D1. AS160 and TBC1D1 phosphorylations were evident 30 min after exercise without simultaneously increased Akt and AMPK phosphorylation. Unlike TBC1D1, insulin-stimulated site-specific AS160 phosphorylation is modified by prior exercise, but these sites do not include Thr(642) and Ser(588). Together, these data provide new insights into phosphorylation of key regulators of glucose transport in human skeletal muscle.

Signal transduction meets vesicle traffic: the software and hardware of GLUT4 translocation

Skeletal muscle is the major tissue disposing of dietary glucose, a function regulated by insulin-elicited signals that impart mobilization of GLUT4 glucose transporters to the plasma membrane. This phenomenon, also central to adipocyte biology, has been the subject of intense and productive research for decades. We focus on muscle cell studies scrutinizing insulin signals and vesicle traffic in a spatiotemporal manner. Using the analogy of an integrated circuit to approach the intersection between signal transduction and vesicle mobilization, we identify signaling relays ("software") that engage structural/mechanical elements ("hardware") to enact the rapid mobilization and incorporation of GLUT4 into the cell surface. We emphasize how insulin signal transduction switches from tyrosine through lipid and serine phosphorylation down to activation of small G proteins of the Rab and Rho families, describe key negative regulation step of Rab GTPases through the GTPase-activating protein activity of the Akt substrate of 160 kDa (AS160), and focus on the mechanical effectors engaged by Rabs 8A and 10 (the molecular motor myosin Va), and the Rho GTPase Rac1 (actin filament branching and severing through Arp2/3 and cofilin). Finally, we illustrate how actin filaments interact with myosin 1c and α-Actinin4 to promote vesicle tethering as preamble to fusion with the membrane.

Nesfatin-1 in Human and Murine Cardiomyocytes: Synthesis, Secretion, and Mobilization of GLUT-4

Nesfatin-1, a satiety-inducing peptide identified in hypothalamic regions that regulate energy balance, is an integral regulator of energy homeostasis and a putative glucose-dependent insulin coadjuvant. We investigated its production by human cardiomyocytes and its effects on glucose uptake, in the main cardiac glucose transporter GLUT-4 and in intracellular signaling. Quantitative RT-PCR, Western blots, confocal immunofluorescence microscopy, and ELISA of human and murine cardiomyocytes and/or cardiac tissue showed that cardiomyocytes can synthesize and secrete nesfatin-1. Confocal microscopy of cultured cardiomyocytes after GLUT-4 labeling showed that nesfatin-1 mobilizes this glucose transporter to cell peripherals. The rate of 2-deoxy-D-[(3)H]glucose incorporation demonstrated that nesfatin-1 induces glucose uptake by HL-1 cells and cultured cardiomyocytes. Nesfatin-1 induced dose- and time-dependent increases in the phosphorylation of ERK1/2, AKT, and AS160. In murine and human cardiac tissue, nesfatin-1 levels varied with diet and coronary health. In conclusion, human and murine cardiomyocytes can synthesize and secrete nesfatin-1, which is able to induce glucose uptake and the mobilization of the glucose transporter GLUT-4 in these cells. Nesfatin-1 cardiac levels are regulated by diet and coronary health.

Insulin Mobilizes Peripheral Endosomal GLUT4 Compartments to the Plasma Membrane

Insulin-stimulated GLUT4 redistribution to the plasma membrane (PM) plays essential roles in glucose homeostasis in mammals. Besides GLUT4 storage vesicles (GSVs) being released by Rab10 activation, endosomal GLUT4 compartments located at the cell periphery are also suggested to contribute to insulin-stimulated GLUT4 accumulation at the PM. Peripheral endosomal GLUT4 compartments are well suited to be studies with total internal reflection fluorescence (TIRF) microscopy. However, visualizing these GLUT4-positive structures after insulin is hindered by the fact that a large amount of GLUT4 is present on the PM after insulin stimulation, obscuring all intracellular GLUT4 compartments.In this study, a new GLUT4 probe, GLUT4-exoGFP, is developed. In combination with Bromophenol Blue (BPB), GLUT4-exoGFP can be used to specifically visualize intracellular GLUT4 compartments, without interference from GLUT4 at the PM. Employing this probe we studied the mobilization of GLUT4 from peripheral GLUT4 compartments. Our results indicate that Rab4A-positive endosomes serve as an important reservoir of GLUT4 molecules that are delivered to the PM after insulin stimulation.

Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of ∼20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min−1) and was decreased during the steady-state responses to insulin (0.18 min−1), AICAR (0.16 min−1), and A-769662 (0.24 min−1). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.

A molecular mathematical model of glucose mobilization and uptake

A new molecular mathematical model is developed by considering the kinetics of GLUT2, GLUT3, and GLUT4, the process of glucose mobilization by glycogen phosphorylase and glycogen synthase in liver, and the dynamics of the insulin signaling pathway. The new model can qualitatively reproduce the experimental glucose and insulin data. It also enables us to use the Bendixson criterion about the existence of periodic orbits of a two-dimensional dynamical system to mathematically predict that the oscillations of glucose and insulin are not caused by liver, instead they would be caused by the mechanism of insulin secretion from pancreatic β cells. Furthermore it enables us to conduct a parametric sensitivity analysis. The analysis shows that both glucose and insulin are most sensitive to the rate constant for conversion of PI(3, 4, 5)P 3 to PI(4, 5)P 2, the multiplicative factor modulating the rate constant for conversion of PI(3, 4, 5)P 3 to PI(4, 5)P 2, the multiplicative factor that modulates insulin receptor dephosphorylation rate, and the maximum velocity of GLUT4. Moreover, the sensitivity analysis predicts that an increase of the apparent velocity of GLUT4, a combination of elevated mobilization rate of GLUT4 to the plasma membrane and an extended duration of GLUT4 on the plasma membrane, will result in a decrease in the needs of plasma insulin. On the other hand, an increase of the GLUT4 internalization rate results in an elevated demand of insulin to stimulate the mobilization of GLUT4 from the intracellular store to the plasma membrane.

Insulin sensitivity and inhibition by forskolin, dipyridamole and pentobarbital of glucose transport in three L6 muscle cell lines

L6 skeletal muscle myoblasts stably overexpressing glucose transporter GLUT1 or GLUT4 with exofacial myc-epitope tags were characterized for their response to insulin. In clonally selected cultures, 2-deoxyglucose uptake into L6-GLUT1myc myoblasts and myotubes was linear within the time of study. In L6-GLUT1myc and L6-GLUT4myc myoblasts, 100 nmol/L insulin treatment increased the GLUT1 content of the plasma membrane by 1.58+/-0.01 fold and the GLUT4 content 1.96+/-0.11 fold, as well as the 2-deoxyglucose uptake 1.53+/-0.09 and 1.86+/-0.17 fold respectively, all by a wortmannin-inhibitable manner. The phosphorylation of Akt in these two cell lines was increased by insulin. L6-GLUT1myc myoblasts showed a dose-dependent stimulation of glucose uptake by insulin, with unaltered sensitivity and maximal responsiveness compared with wild type cells. By contrast, the improved insulin responsiveness and sensitivity of glucose uptake were observed in L6-GLUT4myc myoblasts. Earlier studies indicated that forskolin might affect insulin-stimulated GLUT4 translocation. A 65% decrease of insulin-stimulated 2-deoxyglucose uptake in GLUT4myc cells was not due to an effect on GLUT4 mobilization to the plasma membrane, but instead on direct inhibition of GLUT4. Forskolin and dipyridamole are more potent inhibitors of GLUT4 than GLUT1. Alternatively, pentobarbital inhibits GLUT1 more than GLUT4. The use of these inhibitors confirmed that the overexpressed GLUT1 or GLUT4 are the major functional glucose transporters in unstimulated and insulin-stimulated L6 myoblasts. Therefore, L6-GLUT1myc and L6-GLUT4myc cells provide a platform to screen compounds that may have differential effects on GLUT isoform activity or may influence GLUT isoform mobilization to the cell surface of muscle cells.

Chromium Activates Glucose Transporter 4 Trafficking and Enhances Insulin-Stimulated Glucose Transport in 3T3-L1 Adipocytes via a Cholesterol-Dependent Mechanism

Evidence suggests that chromium supplementation may alleviate symptoms associated with diabetes, such as high blood glucose and lipid abnormalities, yet a molecular mechanism remains unclear. Here, we report that trivalent chromium in the chloride (CrCl3) or picolinate (CrPic) salt forms mobilize the glucose transporter, GLUT4, to the plasma membrane in 3T3-L1 adipocytes. Concomitant with an increase in GLUT4 at the plasma membrane, insulin-stimulated glucose transport was enhanced by chromium treatment. In contrast, the chromium-mobilized pool of transporters was not active in the absence of insulin. Microscopic analysis of an exofacially Myc-tagged enhanced green fluorescent protein-GLUT4 construct revealed that the chromium-induced accumulation of GLUT4-containing vesicles occurred adjacent to the inner cell surface membrane. With insulin these transporters physically incorporated into the plasma membrane. Regulation of GLUT4 translocation by chromium did not involve known insulin signaling proteins such as the insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, and Akt. Consistent with a reported effect of chromium on increasing membrane fluidity, we found that chromium treatment decreased plasma membrane cholesterol. Interestingly, cholesterol add-back to the plasma membrane prevented the beneficial effect of chromium on both GLUT4 mobilization and insulin-stimulated glucose transport. Furthermore, chromium action was absent in methyl-beta-cyclodextrin-pretreated cells already displaying reduced plasma membrane cholesterol and increased GLUT4 translocation. Together, these data reveal a novel mechanism by which chromium may enhance GLUT4 trafficking and insulin-stimulated glucose transport. Moreover, these findings at the level of the cell are consistent with in vivo observations of improved glucose tolerance and decreased circulating cholesterol levels after chromium supplementation.

Minireview: Recent Developments in the Regulation of Glucose Transporter-4 Traffic: New Signals, Locations, and Partners

Glucose transporter (GLUT) 4 is the major glucose transporter of muscle and adipose cells, exquisitely regulated by insulin through posttranslational events. Twenty years after the seminal observations that GLUT4 levels rapidly rise at the plasma membrane (PM) and drop in endomembranes in response to an acute insulin challenge, we are still mapping the intracellular traffic of the transporter and the regulatory events that insulin unleashes. Newly synthesized GLUT4 enters an insulin-responsive compartment aided by GGA2 (an Arf-binding protein). In cultured adipocytes and myocytes, GLUT4 concentrates in a perinuclear pole through participation of microtubules and the EHD1 Eps15 homology domain-containing protein 1. In the absence of stimuli, GLUT4 distributes between recycling endosomes and the insulin-responsive compartment. A handful of proteins that bind to GLUT4 appear to regulate its half-life (e.g. Ubc9) and tethering within endomembranes (e.g. TUG). Insulin-derived signals promote not only GLUT4 mobilization toward the PM but also its traffic between endosomal compartments and internalization from the PM. Class IA phosphatidylinositol (PI) 3-kinase plays a pivotal role at several steps of GLUT4 mobilization. The PI 3-kinase --> atypical PKC and --> Akt/PKB --> AS160 signaling cascades are major regulators of GLUT4 exocytosis aided by small GTPases. At the cell periphery, GLUT4-containing vesicles tether, dock, and fuse with the PM assisted by the exocyst complex followed by engagement of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex [with vesicle-associated membrane protein (VAMP)2 as the vesicular (v)-SNARE and soluble NSF-attachment protein (SNAP)23 and syntaxin4 as target (t)-SNAREs] regulated by the accessory proteins Munc18c, Synip and Tomosyn. Vesicle tethering and fusion are regulated by insulin through input from class IA PI 3-kinase.

Modulation of the Actin Cytoskeleton via Gelsolin Regulates Vacuolar H+-ATPase Recycling

The role of the actin cytoskeleton in regulating membrane protein trafficking is complex and depends on the cell type and protein being examined. Using the epididymis as a model system in which luminal acidification is crucial for sperm maturation and storage, we now report that modulation of the actin cytoskeleton by the calcium-activated actin-capping and -severing protein gelsolin plays a key role in regulating vacuolar H(+)-ATPase (V-ATPase) recycling. Epididymal clear cells contain abundant V-ATPase in their apical pole, and an increase in their cell-surface V-ATPase expression correlates with an increase in luminal proton secretion. We have shown that apical membrane accumulation of V-ATPase is triggered by an elevation in cAMP following activation of bicarbonate-regulated soluble adenylyl cyclase in response to alkaline luminal pH (Pastor-Soler, N., Beaulieu, V., Litvin, T. N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L. R., and Breton, S. (2003) J. Biol. Chem. 278, 49523-49529). Here, we show that clear cells express high levels of gelsolin, indicating a potential role in the functional activity of these cells. When jasplakinolide was used to overcome the severing action of gelsolin by polymerizing actin, complete inhibition of the alkaline pH- and cAMP-induced apical membrane accumulation of V-ATPase was observed. Conversely, when gelsolin-mediated actin filament elongation was inhibited using a 10-residue peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate-binding region (phosphoinositide-binding domain 2) of gelsolin, significant V-ATPase apical membrane mobilization was induced, even at acidic luminal pH. In contrast, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) and the phospholipase C inhibitor U-73122 inhibited the alkaline pH-induced V-ATPase apical accumulation. Thus, maintenance of the actin cytoskeleton in a depolymerized state by gelsolin facilitates calcium-dependent apical accumulation of V-ATPase in response to luminal pH alkalinization. Gelsolin is present in other cell types that express the V-ATPase in their plasma membrane and recycling vesicles, including kidney intercalated cells and osteoclasts. Therefore, modulation of the actin cortex by this severing and capping protein may represent a common mechanism by which these cells regulate their rate of proton secretion.

Dipyridamole alters cardiac substrate preference by inducing translocation of FAT/CD36, but not that of GLUT4.

In cardiac myocytes, uptake rates of glucose and long-chain fatty acids (FA) are regulated by translocation of GLUT4 and FA translocase (FAT)/CD36, respectively, from intracellular stores to the sarcolemma. Insulin and contractions are two major physiological stimuli able to induce translocation of both transporters and therefore enhance the uptake of both substrates. Interestingly, the cardiovascular drug dipyridamole was able to enhance FA uptake but had no effect on glucose uptake. The selective stimulatory effect of dipyridamole on FA uptake was unrelated to its effects on phosphodiesterase inhibition and on nucleoside transport inhibition. However, dipyridamole-stimulated FA uptake was abolished in the presence of sulfo-N-succinimidylpalmitate, which indicated that FAT/CD36 is involved in the uptake process. Furthermore, the effect was additive to that of insulin but not to that of the AMP-elevating agent oligomycin, indicating that dipyridamole stimulates FAT/CD36-mediated FA uptake by activating the AMP-activated protein kinase (AMPK) signaling pathway. Dipyridamole, however, neither influenced the intracellular AMP content nor induced activation of AMPK. Finally, dipyridamole was able to induce FAT/CD36 translocation from intracellular storage sites to the sarcolemma but had no effect on the subcellular distribution of GLUT4. It is concluded that beyond AMP-activated protein kinase the contraction-induced and AMPK-mediated signal branches off into separate mobilization of GLUT4 and of FAT/CD36, and that dipyridamole activates a yet unidentified target in the FAT/CD36 mobilizing branch.

Glucosamine-induced insulin resistance is coupled to O-linked glycosylation of Munc18c

Evidence suggests that glucosamine inhibits distal components regulating insulin-stimulated GLUT4 translocation to the plasma membrane. Here we assessed whether key membrane docking and fusion events were targeted. Consistent with a plasma membrane-localized effect, 3T3-L1 adipocytes exposed to glucosamine displayed an increase in cell-surface O-linked glycosylation and a simultaneously impaired mobilization of GLUT4 by insulin. Analysis of syntaxin 4 and SNAP23, plasma membrane-localized target receptor proteins (t-SNAREs) for the GLUT4 vesicle, showed that they were not cell-surface targets of O-linked glycosylation. However, the syntaxin 4 binding protein, Munc18c, was targeted by O-linked glycosylation. This occurred concomitantly with a block in insulin-stimulated association of syntaxin 4 with its cognate GLUT4 vesicle receptor protein (v-SNARE), VAMP2. In conclusion, our data suggest that the mechanism by which glucosamine inhibits insulin-stimulated GLUT4 translocation involves modification of Munc18c.

Closing in on the cause of insulin resistance and type 2 diabetes.

Closing in on the cause of insulin resistance and type 2 diabetes.

Mobilization of GLUT-4 from intracellular vesicles by insulin and K(+) depolarization in cultured H9c2 myotubes.

The insulin-responsive glucose transporter, GLUT-4, moves from an intracellular compartment to the cell surface in response to insulin and/or muscle contraction. Treatment of H9c2 myotubes with insulin significantly increased uptake of 2-deoxyglucose. Depolarization of the myotubes by increasing extracellular [K(+)], which mimics the initial phases of excitation-contraction coupling, also increased 2-deoxyglucose uptake. The K(+)- but not insulin-evoked increase was blocked by dantrolene, an inhibitor of Ca(2+) release from the sarcoplasmic reticulum. In contrast, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked insulin- but not K(+)-stimulated 2-deoxyglucose uptake. Increased glucose uptake in response to insulin or K(+) depolarization was associated with increased GLUT-4 in plasma membranes and depletion of a population of small intracellular GLUT-4-containing vesicles. Similarly, in H9c2 cells transfected with c-myc-tagged GLUT-4, translocation of c-myc GLUT-4 to the cell surface was increased after stimulation with insulin or K(+) depolarization. Taken together, these data demonstrate that insulin and K(+) depolarization increase glucose uptake by recruiting GLUT-4 from intracellular vesicles to the plasma membrane of H9c2 myotubes via distinct signaling mechanisms.

Insulin-induced Glut4 recruitment in the fatty Zucker rat heart is not associated with changes in Glut4 content in the intracellular membrane.

Impaired cardiac glucose metabolism and glucose transport have been shown in the insulin resistant fatty Zucker rat. The aim of the present study was to examine the translocation of the insulin-sensitive glucose transporter (Glut4) in the heart of the fatty Zucker rat under in vivo conditions. Insulin was injected into both lean (FA/?) and fatty (fa/fa) Zucker rats via the tail vein. The time course of cardiac Glut4 translocation was studied by determining the subcellular distribution of Glut4 using a newly developed ELISA quantitation method. Insulin (10 U/kg) caused a 30% and 37% increase in plasma membrane Glut4 content at 20 min after injection in lean and fatty rats respectively. The plasma membrane Glut4 contents in the basal and insulin-stimulated states were significantly lower in the fatty rat when compared to the lean control. The dose effect of insulin (2.5-10 U/kg) on Glut4 mobilization to the plasma membrane was similar in both phenotypes. The time course of Glut4 mobilization to the plasma membrane (5-30 min), which was similar in both lean and fatty Zucker rats, showed that maximal translocation was reached at 5 min post insulin injection and persisted throughout the remaining 25 min. However, in fatty Zucker rats, Glut4 content in the intracellular membrane remained unchanged at all insulin doses and all time points studied. Collectively, these results show that Glut4 recruitment to the plasma membrane is responsive to insulin in the fatty Zucker rat heart and that the maximal response was similar to that in lean Zucker rats. However, the recruitment of Glut4 to the plasma membrane was not associated with changes in the intracellular membrane Glut4 content.

Differential Modification of Activities of the High-Affinity and Low-Affinity Insulin Receptors of 3T3-L1 Fibroblasts by Phosphonolipidsin Vivo

The low-affinity and high-affinity forms of the insulin receptor respond differently to modifications of cellular phospholipid content in mouse 3T3-L1 fibroblastsin vivo.When cells are cultured with 2-aminoethylphosphonate the resulting phosphonolipid, which has previously been demonstrated to prevent the insulin-induced differentiation of the fibroblasts into adipocytes [J. D. Smithet al., Biochem. Arch.8,339–344 (1992)] results in alterations in both the affinity for insulin and receptor number of the low-affinity receptor while leaving the high-affinity receptor unaffected. That this phospholipid modification induces a specific change in the cellular insulin effect is demonstrated by the lack of alteration in the mobilization of GLUT-4 and glucose transport in the lipid modified cells. The results suggest that this specific cellular phospholipid modification will be useful in dissecting the specific functions of the two forms of the mammalian insulin receptor.