Translocation In Skeletal Muscle Research Articles

Munc18c is not rate-limiting for glucose and long-chain fatty acid uptake in the heart

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)- and SNARE-associated proteins have not yet been assessed in regulation of cardiac glucose uptake, nor in the regulation of long-chain fatty acid (LCFA) uptake in any tissue. Munc18c is a SNARE-associated protein that regulates GLUT4 translocation in skeletal muscle and adipose tissue. Using cardiomyocytes from Munc18c(-/+) mice (with 56% reduction of Munc18c protein expression), we investigated whether this syntaxin4-associated protein is involved in regulation of cardiac substrate uptake. Basal, insulin- and oligomycin (a 5' AMP-activated protein kinase-activating agent)-stimulated glucose and LCFA uptake were not altered significantly in Munc18c(-/+) cardiomyocytes compared to wild-type cells. We conclude, therefore, that Munc18c is not rate-limiting for cardiac substrate uptake, neither under basal conditions nor when maximally stimulated metabolically.

Epigallocatechin gallate promotes GLUT4 translocation in skeletal muscle

In this study, we investigated whether epigallocatechin gallate (EGCg) affects glucose uptake activity and the translocation of insulin-sensitive glucose transporter (GLUT) 4 in skeletal muscle. A single oral administration of EGCg at 75 mg/kg body weight promoted GLUT4 translocation in skeletal muscle of rats. EGCg significantly increased glucose uptake accompanying GLUT4 translocation in L6 myotubes at 1 nM. The translocation of GLUT4 was also observed both in skeletal muscle of mice and rats ex vivo and in insulin-resistant L6 myotubes. Wortmannin, an inhibitor of phosphatidylinositol 3′-kinase, inhibited both EGCg- and insulin-increased glucose uptakes, while genistein, an inhibitor of tyrosine kinase, failed to inhibit the EGCg-increased uptake. Therefore, EGCg may improve hyperglycemia by promoting GLUT4 translocation in skeletal muscle with partially different mechanism from insulin.

Contractile C2C12myotube model for studying exercise-inducible responses in skeletal muscle

Adequate exercise leads to a vast variety of physiological changes in skeletal muscle as well as other tissues/organs and is also responsible for maintaining healthy muscle displaying enhanced insulin-responsive glucose uptake via GLUT4 translocation. We generated highly developed contractile C(2)C(12) myotubes by manipulating intracellular Ca(2+) transients with electric pulse stimulation (EPS) that is endowed with properties similar to those of in vivo skeletal muscle in terms of 1) excitation-induced contractile activity as a result of de novo sarcomere formation, 2) activation of both the AMP kinase and stress-activated MAP kinase cascades, and 3) improved insulin responsiveness as assessed by GLUT4 recycling. Tbc1d1, a Rab-GAP implicated in exercise-induced GLUT4 translocation in skeletal muscle, also appeared to be phosphorylated on Ser(231) after EPS-induced contraction. In addition, a switch in myosin heavy-chain (MHC) expression from "fast type" to "slow type" was observed in the C(2)C(12) myotubes endowed with EPS-induced repetitive contractility. Taking advantage of these highly developed contractile C(2)C(12) myotubes, we identified myotube-derived factors responsive to EPS-evoked contraction, including the CXC chemokines CXCL1/KC and CXCL5/LIX, as well as IL-6, previously reported to be upregulated in contracting muscles in vivo. Importantly, animal treadmill experiments revealed that exercise significantly increased systemic levels of CXCL1/KC, perhaps derived from contracting muscle. Taken together, these results confirm that we have established a specialized muscle cell culture model allowing contraction-inducible cellular responses to be explored. Utilizing this model, we identified contraction-inducible myokines potentially linked to the metabolic alterations, immune responses, and angiogenesis induced by exercise.

A High-fructose Diet Impairs Akt and PKCζ Phosphorylation and GLUT4 Translocation in Rat Skeletal Muscle

The molecular mechanism of insulin resistance induced by high-fructose feeding is not fully understood. The present study investigated the role of downstream signaling molecules of phosphatidylinositol 3-kinase (PI3K) in the insulin-stimulated skeletal muscle of high-fructose-fed rats. Rats were divided into chow-fed and fructose-fed groups. The results of the euglycemic clamp study (insulin infusion rates: 6 mU/kg BW/min) showed a significant decrease in the glucose infusion rate (GIR) and the metabolic clearance rate of glucose (MCR) in fructose-fed rats compared with chow-fed rats. In skeletal muscle removed immediately after the clamp procedure, high-fructose feeding did not alter protein levels of protein kinase B (PKB/Akt), protein kinase C zeta (PKCzeta), or glucose transporter 4 (GLUT4). However, insulin-stimulated phosphorylation of Akt and PKCzeta and GLUT4 translocation to the plasma membrane were reduced. Our findings suggest that insulin resistance in fructose-fed rats is associated with impaired Akt and PKCzeta activation and GLUT4 translocation in skeletal muscle.

Frontiers: Skeletal muscle sodium pump regulation: a translocation paradigm

The skeletal muscle sodium pump plays a major role in the removal of K(+) ions from the circulation postprandial, or after a physical activity bout, thereby preventing the development of hyperkalemia and fatigue. Insulin and muscle contractions stimulate Na(+)-K(+)-ATPase activity in skeletal muscle, at least partially via translocation of sodium pump units to the plasma membrane from intracellular stores. The molecular mechanism of this phenomenon is poorly understood. Due to the contradictory reports in the literature, the very existence of the translocation of Na(+)-K(+)-ATPase to the skeletal muscle cell surface is questionable. This review summarizes more than 30 years work on the skeletal muscle sodium pump translocation paradigm. Furthermore, the methodological caveats of major approaches to study the sodium pump translocation in skeletal muscle are discussed. An understanding of the molecular regulation of Na(+)-K(+)-ATPase in skeletal muscle will have important clinical implications for the understanding of the development of complications associated with the metabolic syndrome, such as cardiovascular diseases or increased muscle fatigue in diabetic patients.

Distinct Signals Regulate AS160 Phosphorylation in Response to Insulin, AICAR, and Contraction in Mouse Skeletal Muscle

Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by wortmannin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK alpha2-inactive (alpha2i) transgenic mice. AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK alpha2i transgenic mice. Combined AMPK alpha2 and Akt inhibition by wortmannin treatment of AMPK alpha2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK alpha2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.

Altered GLUT4 Translocation in Skeletal Muscle of 12/15-Lipoxygenase Knockout Mice

We have recently shown that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of actin microfilaments in rat cardiomyocytes, and that inhibition of 12-lipoxygenase abrogates insulin-stimulated GLUT4 translocation in these cells. In the present study, we used mice that were null for the leukocyte 12/15-lipoxygenase to explore the implications of this enzyme for insulin action under IN VIVO conditions. Insulin induced a profound reduction in blood glucose in both control and knockout mice. However, significantly higher serum insulin levels were observed in these animals. GLUT4 expression in heart and skeletal muscle was unaffected in KO mice. Insulin-regulated serine phosphorylation of Akt and GSK3alpha and GSK3beta was unaltered in heart and skeletal muscle of knockout mice, suggesting unaltered insulin signaling. Fractionation of hind limb muscles showed that insulin had induced a prominent translocation of GLUT4 to skeletal muscle plasma membranes in control mice. However, this response was largely reduced in knockout animals. Our data show that the lack of leukocyte 12/15-lipoxygenase does not lead to the development of an insulin-resistant phenotype. However, perturbation of GLUT4 translocation in skeletal muscle of knockout mice may indicate latent insulin resistance, and supports our hypothesis that eicosanoids are involved in insulin-mediated regulation of muscle glucose transport.

The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis

Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [(32)P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of glucose stimulation in MIN6 beta cells. Tyrosine phosphorylation of Munc18c was also detected in 3T3L1 adipocytes and increased with insulin stimulation, suggesting that this may be a conserved mechanism. Syntaxin 4 binding to Munc18c decreased as Munc18c phosphorylation levels increased in pervanadate-treated cells, suggesting that phosphorylation dissociates the Munc18c-Syntaxin 4 complex. Munc18c phosphorylation was localized to the N-terminal 255 residues. Mutagenesis of one residue in this region, Y219F, significantly increased the affinity of Munc18c for Syntaxin 4, whereas mutation of three other candidate sites was without effect. Moreover, Munc18c-Y219F expression in MIN6 cells functionally inhibited glucose-stimulated SNARE complex formation and insulin granule exocytosis. These data support a novel and conserved mechanism for the dissociation of Munc18c-Syntaxin 4 complexes in a stimulus-dependent manner to facilitate the increase in Syntaxin 4-VAMP2 association and to promote vesicle/granule fusion.

Regulation of glucose transporters by insulin and extracellular glucose in C2C12myotubes

It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C(2)C(12) myocytes expressing exofacial-Myc-GLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C(2)C(12) myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation ( approximately 3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C(2)C(12) myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C(2)C(12) myotubes can be achieved through surprisingly acute glucose-dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C(2)C(12) myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.

Inhibition of calpain results in impaired contraction-stimulated GLUT4 translocation in skeletal muscle

It was previously found that transgenic mice that overexpress the calpain inhibitor calpastatin (CsTg) have an approximately 3-fold increase in GLUT4 protein in their skeletal muscles. Despite the increase in GLUT4, which appears to be due to inhibition of its proteolysis by calpain, insulin-stimulated glucose transport is not increased in CsTg muscles. PKB (Akt) protein level is reduced approximately 60% in CsTg muscles, suggesting a possible mechanism for the relative insulin resistance. Muscle contractions stimulate glucose transport by a mechanism that is independent of insulin signaling. The purpose of this study was to test the hypothesis that the threefold increase in GLUT4 in CsTg would result in a large increase in contraction-stimulated glucose transport. CAMKII and AMPK mediate steps in the contraction-stimulated pathway. The protein levels of AMPK and CAMKII were increased three- to fourfold in CsTg muscles, suggesting that these proteins are also calpain substrates. Despite the large increases in GLUT4, AMPK, and CAMKII, contraction-stimulated GLUT4 translocation and glucose transport were not increased above wild-type values. These findings suggest that inhibition of calpain results in impairment of a step in the GLUT4 translocation process downstream of the insulin- and contraction-signaling pathways. They also provide evidence that CAMKII and AMPK are calpain substrates.

Effect of astragalus polysaccharide on insulin resistance in KKAy mice: alteration in PKB/GLUT4 signal transduction

Astragalus polysaccharide (APS) has been shown to improve glycemic control by increasing insulin sensitivity in the skeletal muscle of type 2 diabetic rats. To investigate whether the effect of APS on glucose disposal is associated with the altered insulin signaling, APS (700mg/kg/day) or vehicle was administered to 12-week-old KKAy and C57BL/6J mice for 8 weeks. KKAy mice developed hyperglycemia, hyperinsulinemia, obesity and impaired oral glucose tolerance, which were alleviated by treatment with APS. While skeletal muscle protein kinase B (PKB) level from KKAy and C57BL/6J mice were similar, insulin stimulated serine473 phosphorylation of PKB protein content was 56% lower in KKAy than that in C57BL/6J mice. APS treatment increased serine473 phosphorylation of PKB in KKAy mice (1.4 fold) compared to control, which was associated with a 1.3 fold increase in plasma membrane glucose transporter 4 (GLUT-4) protein content. In contrast, APS have no effect on C57BL/6J mice. These results indicate that defect in insulin-stimulated PKB serine473 phosphorylation and plasma membrane GLUT-4 translocation in skeletal muscle may play important roles in the development of insulin resistance in KKAy mice. The glucoregulatory effects of APS were dependent of an elevated insulin-stimulated PKB serine473 phosphorylation and plasma membrane GLUT4 protein expression in KKAy mice. (This study is supported by National Natural Sciences Foundation of China grant 30370673)

Syntaxin 4 transgenic mice exhibit enhanced insulin-mediated glucose uptake in skeletal muscle.

Insulin-stimulated translocation of GLUT4 vesicles from an intracellular compartment to the plasma membrane in 3T3L1 adipocytes is mediated through a syntaxin 4 (Syn4)- and Munc18c-dependent mechanism. To investigate the impact of increasing Syn4 protein abundance on glucose homeostasis in vivo, we engineered tetracycline-repressible transgenic mice to overexpress Syn4 by fivefold in skeletal muscle and pancreas and threefold in adipose tissue. Increases in Syn4 caused increases in Munc18c protein, indicating that Syn4 regulates Munc18c expression in vivo. An important finding was that female Syn4 transgenic mice exhibited an increased rate of glucose clearance during glucose tolerance tests that was repressible by the administration of tetracycline. Insulin-stimulated glucose uptake in skeletal muscle was increased by twofold in Syn4 transgenic mice compared with wild-type mice as assessed by hyperinsulinemic-euglycemic clamp analysis, consistent with a twofold increase in insulin-stimulated GLUT4 translocation in skeletal muscle. Hepatic insulin action was unaffected. Moreover, insulin content and glucose-stimulated insulin secretion by islets isolated from Syn4 transgenic mice did not differ from that of wild-type mice. In sum, these data suggest that increasing the number of Syn4-Munc18c "fusion sites" at the plasma membrane of skeletal muscle increases the amount of GLUT4 available to increase the overall rate of insulin-mediated glucose uptake in vivo.

Anti‐obesity actions of green tea: Possible involvements in modulation of the glucose uptake system and suppression of the adipogenesis‐related transcription factors

To investigate mechanisms of the anti-obesity actions of green tea in vivo, rats were given green tea instead of drinking water for 3 weeks. It was confirmed that green tea reduced adipose tissue weight without any change in body weight, other tissue weights, and food and water intakes. Green tea also significantly reduced the plasma levels of cholesterols and free fatty acids. Certain catechins existed in the plasma at 0.24 microM under our experimental conditions, though most of them existed as conjugated forms. For mechanisms of the anti-obesity actions, green tea significantly reduced glucose uptake accompanied by a decrease in translocation of glucose transporter 4 (GLUT4) in adipose tissue, while it significantly stimulated the glucose uptake with GLUT4 translocation in skeletal muscle. Moreover, green tea suppressed the expression of peroxisome proliferator-activated receptor gamma and the activation of sterol regulatory element binding protein-1 in adipose tissue. In conclusion, green tea modulates the glucose uptake system in adipose tissue and skeletal muscle and suppresses the expression and/or activation of adipogenesis-related transcription factors, as the possible mechanisms of its anti-obesity actions.

Impairment of Insulin-induced GLUT4 Translocation in Skeletal Muscle of KK-Ay Mouse, a Genetic Animal Model of Type 2 Diabetes

In skeletal muscle, insulin-induced glucose transport is known to start from the translocation of glucose transporter (GLUT) 4 from intracellular pool to plasma membrane. KK-Ay mouse, a genetic animal model of type 2 diabetes, develops moderate degrees of obesity and diabetes that are especially apparent in animals of more than 11 week old. Defect in insulin-induced GLUT4 translocation may also be involved in the geneses of type 2 diabetes mellitus. We examined the alteration of insulin-induced GLUT4 translocation in the skeletal muscle. After isolation of low density microsomal membrane and plasma membrane in skeletal muscle, insulin-induced translocation of GLUT4 in KK-Ay mouse were examined by Western blotting method. The insulin-induced translocation of GLUT4 from low density microsomal membranes to plasma membrane was significantly reduced in skeletal muscle of KK-Ay mice when compared with that in the ddY mice. These results support a view that the reduction of insulin-induced GLUT4 translocation in skeletal muscle may be involved in the deferioration of insulin sensitivity in diabetic KK-Ay mice.

Impairment of insulin-stimulated GLUT4 translocation in skeletal muscle and adipose tissue in the Tsumura Suzuki obese diabetic mouse: a new genetic animal model of type 2 diabetes.

In skeletal muscle and adipocytes, insulin-stimulated glucose transport has been known to occur through the translocation of glucose transporter (GLUT) 4 from the intracellular pool to the plasma membrane. The Tsumura Suzuki obese diabetic (TSOD) mouse, a new genetic animal model of type 2 diabetes, develops moderate degrees of obesity and diabetes that are especially apparent in animals more than 11 weeks old. A defect in insulin stimulation of GLUT4 translocation also contributes to the characteristics of type 2 diabetes. To characterize this mouse further, we examined the alteration in insulin-stimulated GLUT4 translocation in the skeletal muscle and adipose tissue. For glucose and insulin tolerance tests, the mice were given glucose or insulin and blood samples were collected. After isolation of low-density microsomal membrane and plasma membrane from skeletal muscle and adipose tissue, insulin-stimulated translocation of GLUT4 in these TSOD mice was examined by Western blot. TSOD mice showed a significant increase in blood glucose after the glucose load, and exhibited a significantly attenuated decrease in blood glucose concentrations after administration of insulin, compared with that in control Tsumura Suzuki non-obese (TSNO) mice. The insulin-stimulated translocation of GLUT4 from low-density microsomal membranes to plasma membrane was significantly reduced in both skeletal muscle and adipose tissue of TSOD mice. These results indicate that the reduced insulin sensitivity in diabetic TSOD mice is presumably due, at least in part, to the impaired GLUT4 translocation by insulin in both skeletal muscle and adipocytes.

GLUT-4 translocation in skeletal muscle studied with a cell-free assay: involvement of phospholipase D

GLUT-4-containing membranes immunoprecipitated from insulin-stimulated rat skeletal muscle produce the phospholipase D (PLD) product phosphatidic acid. In vitro stimulation of PLD in crude membrane with ammonium sulfate (5 mM) resulted in transfer of GLUT-4 (3.0-fold vs. control) as well as transferrin receptor proteins from large to small membrane structures. The in vitro GLUT-4 transfer could be blocked by neomycin (a PLD inhibitor), and neomycin also reduced insulin-stimulated glucose transport in intact incubated soleus muscles. Furthermore, protein kinase B(beta) (PKB(beta)) was found to associate with the GLUT-4 protein and was transferred to small vesicles in response to ammonium sulfate in vitro. Finally, addition of cytosolic proteins, prepared from basal skeletal muscle, and GTP nucleotides to an enriched GLUT-4 membrane fraction resulted in in vitro transfer of GLUT-4 to small membranes (6.8-fold vs. unstimulated control). The cytosol and nucleotide-induced GLUT-4 transfer could be blocked by neomycin and N-ethylmaleimide. In conclusion, we have developed a cell-free assay that demonstrates in vitro GLUT-4 transfer. This transfer may suggest release of GLUT-4-containing vesicles from donor GLUT-4 membranes involving PLD activity and binding of PKB(beta) to GLUT-4.

Defective insulin-induced GLUT4 translocation in skeletal muscle of high fat-fed rats is associated with alterations in both Akt/protein kinase B and atypical protein kinase C (zeta/lambda) activities.

The cellular mechanism by which high-fat feeding induces skeletal muscle insulin resistance was investigated in the present study. Insulin-stimulated glucose transport was impaired ( approximately 40-60%) in muscles of high fat-fed rats. Muscle GLUT4 expression was significantly lower in these animals ( approximately 40%, P < 0.05) but only in type IIa-enriched muscle. Insulin stimulated the translocation of GLUT4 to both the plasma membrane and the transverse (T)-tubules in chow-fed rats. In marked contrast, GLUT4 translocation was completely abrogated in the muscle of insulin-stimulated high fat-fed rats. High-fat feeding markedly decreased insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity but not insulin-induced tyrosine phosphorylation of the insulin receptor and IRS proteins in muscle. Impairment of PI 3-kinase function was associated with defective Akt/protein kinase B kinase activity (-40%, P < 0.01) in insulin-stimulated muscle of high fat-fed rats, despite unaltered phosphorylation (Ser473/Thr308) of the enzyme. Interestingly, basal activity of atypical protein kinase C (aPKC) was elevated in muscle of high fat-fed rats compared with chow-fed controls. Whereas insulin induced a twofold increase in aPKC kinase activity in the muscle of chow-fed rats, the hormone failed to further increase the kinase activity in high fat-fed rat muscle. In conclusion, it was found that GLUT4 translocation to both the plasma membrane and the T-tubules is impaired in the muscle of high fat-fed rats. We identified PI 3-kinase as the first step of the insulin signaling pathway to be impaired by high-fat feeding, and this was associated with alterations in both Akt and aPKC kinase activities.

Syntaxin 4 heterozygous knockout mice develop muscle insulin resistance.

To investigate the physiological function of syntaxin 4 in the regulation of GLUT4 vesicle trafficking, we used homologous recombination to generate syntaxin 4-knockout mice. Homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, whereas heterozygous knockout mice, Syn4(+/-), had normal viability with no significant impairment in growth, development, or reproduction. However, the Syn4(+/-) mice manifested impaired glucose tolerance with a 50% reduction in whole-body glucose uptake. This defect was attributed to a 50% reduction in skeletal muscle glucose transport determined by 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp procedures. In parallel, insulin-stimulated GLUT4 translocation in skeletal muscle was also significantly reduced in these mice. In contrast, Syn4(+/-) mice displayed normal insulin-stimulated glucose uptake and metabolism in adipose tissue and liver. Together, these data demonstrate that syntaxin 4 plays a critical physiological role in insulin-stimulated glucose uptake in skeletal muscle. Furthermore, reduction in syntaxin 4 protein levels in this tissue can account for the impairment in whole-body insulin-stimulated glucose metabolism in this animal model.

Munc18c Regulates Insulin-stimulated GLUT4 Translocation to the Transverse Tubules in Skeletal Muscle

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin- and exercise-stimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.

5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle.

It has previously been reported that exercise causes an increase in glucose uptake in skeletal muscle and also an increase in 5' AMP-activated protein kinase (AMPK) activity. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA-riboside), an analog of adenosine, is taken up into cells and phosphorylated to form AICA-riboside monophosphate (ZMP), which can also activate AMPK. This study was designed to determine whether the increase in glucose uptake observed with AMPK activation by AICA-riboside is due to GLUT4 translocation from an intracellular location to the plasma membranes, similar to that seen in response to contraction. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine erythrocytes, 8 mmol/l glucose, and +/-2 mmol/AICA-riboside or +/-60 nmol/l insulin. Perfusion medium containing AICA-riboside was found to significantly increase AMPK activity, glucose uptake, and GLUT4 translocation in skeletal muscle above basal levels. Insulin-perfused muscles showed significant increases in glucose uptake and GLUT4 translocation, but AMPK activation was not significantly changed from basal levels. These results provide evidence that the increased glucose uptake observed with AMPK activation by AICA-riboside in perfused rat hindlimb muscles is due to an increase in the translocation of GLUT4 to surface membranes.