Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.     

Protein kinase Czeta mediates insulin-induced glucose transport through actin remodeling in L6 muscle cells

Protein kinase C (PKC) zeta has been implicated in insulin-induced glucose uptake in skeletal muscle cell, although the underlying mechanism remains unknown. In this study, we investigated the effect of PKCzeta on actin remodeling and glucose transport in differentiated rat L6 muscle cells expressing myc-tagged glucose transporter 4 (GLUT4). On insulin stimulation, PKCzeta translocated from low-density microsomes to plasma membrane accompanied by increase in GLUT4 translocation and glucose uptake. Z-scan confocal microscopy revealed a spatial colocalization of relocated PKCzeta with the small GTPase Rac-1, actin, and GLUT4 after insulin stimulation. The insulin-mediated colocalization, PKCzeta distribution, GLUT4 translocation, and glucose uptake were inhibited by wortmannin and cell-permeable PKCzeta pseudosubstrate peptide. In stable transfected cells, overexpression of PKCzeta caused an insulin-like effect on actin remodeling accompanied by a 2.1-fold increase in GLUT4 translocation and 1.7-fold increase in glucose uptake in the absence of insulin. The effects of PKCzeta overexpression were abolished by cell-permeable PKCzeta pseudosubstrate peptide, but not wortmannin. Transient transfection of constitutively active Rac-1 recruited PKCzeta to new structures resembling actin remodeling, whereas dominant negative Rac-1 prevented the insulin-mediated PKCzeta translocation. Together, these results suggest that PKCzeta mediates insulin effect on glucose transport through actin remodeling in muscle cells.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.     

Mechanisms of insulin-dependent glucose transport into porcine and bovine skeletal muscle

Euglycemic, hyperinsulinemic clamp tests have shown that adult ruminants are less insulin-sensitive than monogastric omnivores. The present study was carried out to elucidate possible cellular mechanisms contributing to this impaired insulin sensitivity of ruminants. Western blotting was used to measure glucose transporters 1 and 4 (GLUT1, GLUT4) in oxidative (musculus masseter and diaphragm) and glycolytic (musculus longissimus dorsi and semitendinosus) skeletal muscle in the crude membranes of pigs and cows. Muscles were characterized biochemically. To determine insulin-stimulated 3-O-D-[(3)H]-methylglucose (3-O-MG) uptake and GLUT4 translocation, porcine and bovine musculus semitendinosus strips were removed by open muscle biopsy and incubated without and with 0.1 or 20 mIU insulin/ml. GLUT4 translocation was analyzed using subcellular fractionation techniques to isolate partially purified plasma membranes and cytoplasmic vesicles and using Western blotting. GLUT4 protein contents were significantly higher in oxidative than in glycolytic muscles in pigs and cows. GLUT1 protein contents were significantly higher in glycolytic than in oxidative muscles in bovines but not in porcines. The 3-O-MG uptake into musculus semitendinosus was similar in both species. Maximum insulin-induced GLUT4 translocation into musculus semitendinosus plasma membrane was significantly lower in bovines than in porcines. These results indicate that GLUT1 is the predominant glucose transporter in bovine glycolytic muscles and that a reinforced insulin-independent glucose uptake via GLUT1 may compensate for the impaired insulin-stimulated GLUT4 translocation, resulting in a similar 3-O-MG uptake in bovine and porcine musculus semitendinosus. These findings may explain at least in part the impaired in vivo insulin sensitivity of adult ruminants compared with that of omnivorous monogastric animals.     

Cdc42 is a Rho GTPase family member that can mediate insulin signaling to glucose transport in 3T3-L1 adipocytes

We investigated the role of cdc42, a Rho GTPase family member, in insulin-induced glucose transport in 3T3-L1 adipocytes. Microinjection of anti-cdc42 antibody or cdc42 siRNA led to decreased insulin-induced and constitutively active G(q) (CA-G(q); Q209L)-induced GLUT4 translocation. Adenovirus-mediated expression of constitutively active cdc42 (CA-cdc42; V12) stimulated 2-deoxyglucose uptake to 56% of the maximal insulin response, and this was blocked by treatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin, or LY294002. Both insulin and CA-G(q) expression caused an increase in cdc42 activity, showing that cdc42 is activated by insulin and is downstream of G alpha(q/11) in this activation pathway. Immunoprecipitation experiments showed that insulin enhanced a direct association of cdc42 and p85, and both insulin treatment and CA-cdc42 expression stimulated PI3-kinase activity in immunoprecipitates with anti-cdc42 antibody. Furthermore, the effects of insulin, CA-G(q), and CA-cdc42 on GLUT4 translocation or 2-deoxyglucose uptake were inhibited by microinjection of anti-protein kinase C lambda (PKC lambda) antibody or overexpression of a kinase-deficient PKC lambda construct. In summary, activated cdc42 can mediate 1) insulin-stimulated GLUT4 translocation and 2) glucose transport in a PI3-kinase-dependent manner. 3) Insulin treatment and constitutively active G(q) expression can enhance the cdc42 activity state as well as the association of cdc42 with activated PI3-kinase. 4) PKC lambda inhibition blocks CA-cdc42, CA-G(q), and insulin-stimulated GLUT4 translocation. Taken together, these data indicate that cdc42 can mediate insulin signaling to GLUT4 translocation and lies downstream of G alpha(q/11) and upstream of PI3-kinase and PKC lambda in this stimulatory pathway.     

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.     

Current understanding of glucose transporter 4 expression and functional mechanisms

Glucose is used aerobically and anaerobically to generate energy for cells. Glucose transporters (GLUTs) are transmembrane proteins that transport glucose across the cell membrane. Insulin promotes glucose utilization in part through promoting glucose entry into the skeletal and adipose tissues. This has been thought to be achieved through insulin-induced GLUT4 translocation from intracellular compartments to the cell membrane, which increases the overall rate of glucose flux into a cell. The insulin-induced GLUT4 translocation has been investigated extensively. Recently, significant progress has been made in our understanding of GLUT4 expression and translocation. Here, we summarized the methods and reagents used to determine the expression levels of Slc2a4 mRNA and GLUT4 protein, and GLUT4 translocation in the skeletal muscle, adipose tissues, heart and brain. Overall, a variety of methods such real-time polymerase chain reaction, immunohistochemistry, fluorescence microscopy, fusion proteins, stable cell line and transgenic animals have been used to answer particular questions related to GLUT4 system and insulin action. It seems that insulin-induced GLUT4 translocation can be observed in the heart and brain in addition to the skeletal muscle and adipocytes. Hormones other than insulin can induce GLUT4 translocation. Clearly, more studies of GLUT4 are warranted in the future to advance of our understanding of glucose homeostasis.     

Tissue-specific alterations of glucose transport and molecular mechanisms of intertissue communication in obesity and type 2 diabetes.

Insulin resistance plays a major role in the pathogenesis of type 2 diabetes. Insulin regulates blood glucose levels primarily by promoting glucose uptake from the blood into multiple tissues and by suppressing glucose production from the liver. The glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in muscle and adipose tissue. Decreased GLUT4 expression in adipose tissue is a common feature of many insulin resistant states. GLUT4 expression is preserved in skeletal muscle in many insulin resistant states. However, functional defects in the intracellular trafficking and plasma membrane translocation of GLUT4 result in impaired insulin-stimulated glucose uptake in muscle. Tissue-specific genetic knockout of GLUT4 expression in adipose tissue or muscle of mice has provided new insights into the pathogenesis of insulin resistance. We recently determined that the expression of serum retinol binding protein (RBP4) is induced in adipose tissue as a consequence of decreased GLUT4 expression. We found that RBP4 is elevated in the serum of insulin resistant humans and mice. Furthermore, we found that increasing serum RBP4 levels by transgenic overexpression or by injection of purified RBP4 protein into normal mice causes insulin resistance. Therefore, RBP4 appears to play an important role in mediating adipose tissue communication with other insulin target tissues in insulin resistant states.     

Dimethyl sulfoxide enhances GLUT4 translocation through a reduction in GLUT4 endocytosis in insulin-stimulated 3T3-L1 adipocytes

Insulin increases muscle and fat cell glucose uptake by inducing the translocation of glucose transporter GLUT4 from intracellular compartments to the plasma membrane. Here, we have demonstrated that in 3T3-L1 adipocytes, DMSO at concentrations higher than 7.5% augmented cell surface GLUT4 levels in the absence and presence of insulin, but that at lower concentrations, DMSO only enhanced GLUT4 levels in insulin-stimulated cells. At a 5% concentration, DMSO also increased cell surface levels of the transferrin receptor and GLUT1. Glucose uptake experiments indicated that while DMSO enhanced cell surface glucose transporter levels, it also inhibited glucose transporter activity. Our studies further demonstrated that DMSO did not sensitize the adipocytes for insulin and that its effect on GLUT4 was readily reversible (t1/2∼12 min) and maintained in insulin-resistant adipocytes. An enhancement of insulin-induced GLUT4 translocation was not observed in 3T3-L1 preadipocytes and L6 myotubes, indicating cell specificity. DMSO did not enhance insulin signaling nor exocytosis of GLUT4 vesicles, but inhibited GLUT4 internalization. While other chemical chaperones (glycerol and 4-phenyl butyric acid) also acutely enhanced insulin-induced GLUT4 translocation, these effects were not mediated via changes in GLUT4 endocytosis. We conclude that DMSO is the first molecule to be described that instantaneously enhances insulin-induced increases in cell surface GLUT4 levels in adipocytes, at least in part through a reduction in GLUT4 endocytosis.     

Effect of insulin and contraction up on glucose transport in skeletal muscle

The major glucose transporter protein expressed in skeletal muscle is GLUT4. Both muscle contraction and insulin induce translocation of GLUT4 from the intracellular pool to the plasma membrane. The intracellular pathways that lead to contraction- and insulin-stimulated GLUT4 translocation seem to be different, allowing the attainment of a maximal effect when acting together. Insulin utilizes a phosphatidylinositol 3-kinase-dependent mechanism, whereas the exercise signal may be initiated by calcium release from the sarcoplasmic reticulum or from autocrine- or paracrine-mediated activation of glucose transport. During exercise skeletal muscle utilizes more glucose than when at rest. However, endurance training leads to decreased glucose utilization during sub-maximal exercise, in spite of a large increase in the total GLUT4 content associated with training. The mechanisms involved in this reduction have not been totally elucidated, but appear to cause the decrease of the amount of GLUT4 translocated to the plasma membrane by altering the exercise-induced enhancement of glucose transport capacity. On the other hand, the effect of resistance training is controversial. Recent studies, however, demonstrated the improvement in insulin sensitivity correlated with increasing muscle mass. New studies should be designed to define the molecular basis for these important adaptations to skeletal muscle. Since during exercise the muscle may utilize insulin-independent mechanisms to increase glucose uptake, the mechanisms involved should provide important knowledge to the understanding and managing peripheral insulin resistance.     

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.     

Insulin-stimulated glucose uptake involves the transition of glucose transporters to a caveolae-rich fraction within the plasma membrane: implications for type II diabetes.

BACKGROUND: Adipose and muscle tissues express an insulin-sensitive glucose transporter (GLUT4). This transporter has been shown to translocate from intracellular stores to the plasma membrane following insulin stimulation. The molecular mechanisms signalling this event and the details of the translocation pathway remain unknown. In type II diabetes, the cellular transport of glucose in response to insulin is impaired, partly explaining why blood-glucose levels in patients are not lowered by insulin as in normal individuals. MATERIALS AND METHODS: Isolated rat epididymal adipocytes were stimulated with insulin and subjected to subcellular fractionation and to measurement of glucose uptake. A caveolae-rich fraction was isolated from the plasma membranes after detergent solubilization and ultracentrifugal floatation in a sucrose gradient. Presence of GLUT4 and caveolin was determined by immunoblotting after SDS-PAGE. RESULTS: In freshly isolated adipocytes, insulin induced a rapid translocation of GLUT4 to the plasma membrane fraction, which was followed by a slower transition of the transporter into a detergent resistant caveolae-rich region of the plasma membrane. The insulin-stimulated appearance of transporters in the caveolae-rich fraction occurred in parallel with enhanced glucose uptake by cells. Treatment with isoproterenol plus adenosine deaminase rapidly inhibited insulin-stimulated glucose transport by 40%, and at the same time GLUT4 disappeared from the caveolae-rich fraction and from plasma membranes as a whole. CONCLUSIONS: Insulin stimulates glucose uptake in adipocytes by rapidly translocating GLUT4 from intracellular stores to the plasma membrane. This is followed by a slower transition of GLUT4 to the caveolae-rich regions of the plasma membrane, where glucose transport appears to take place. These results have implications for an understanding of the defect in glucose transport involved in type II diabetes.     

A high-fructose diet impairs Akt and PKCzeta 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.     

Insulin-stimulated glucose transport in muscle. Evidence for a protein-kinase-C-dependent component which is unaltered in insulin-resistant mice.

The aim of our work was to investigate a possible role of protein kinase C (PKC) in insulin-stimulated glucose uptake in mouse skeletal muscle, and to search for a defect in PKC activation in insulin resistance found in obesity. In isolated soleus muscle of lean mice, insulin (100 nM) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (1 microM) acutely stimulated glucose uptake 3- and 2-fold respectively. The effects of insulin and TPA were not additive. When PKC activity was down-regulated by long-term (24 h) TPA pretreatment, before measurement of glucose transport, the TPA effect was abolished, but in addition insulin-stimulated glucose transport returned to basal values. Furthermore, polymyxin B, which inhibits PKC in muscle extracts, prevented insulin-stimulated glucose uptake in muscle. In muscle of obese insulin-resistant mice, glucose uptake evoked by insulin was decreased, whereas the TPA effect, expressed as a fold increase, was unaltered. Thus both agents stimulated glucose transport to the same extent. Furthermore, no difference was observed when PKC activation by TPA was measured in muscle from lean and obese mice. These results suggest that: (1) PKC is involved in the insulin effect on glucose transport in muscle; (2) PKC activation explains only part of the insulin stimulation of glucose transport; (3) the defect in insulin response in obese mice does not appear to be due to an alteration in the PKC-dependent component of glucose transport. We propose that insulin stimulation of glucose uptake occurs by a sequential two-step mechanism, with first translocation of transporters to the plasma membrane, which is PKC dependent, and second, activation of the glucose transporters. In obesity only the activation step was decreased, whereas the translocation step was unaltered.     

Bridging the GAP between insulin signaling and GLUT4 translocation

Upon binding and activating its cell-surface receptor, insulin triggers signaling cascades that regulate many cellular processes. Regarding glucose homeostasis, insulin suppresses hepatic glucose production and increases glucose transport into muscle and adipose tissues. At the cellular level, glucose uptake results from the insulin-stimulated translocation of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Although the signaling molecules that function proximal to the activated insulin receptor have been well characterized, it is not known how the distal insulin-signaling cascade interfaces with and mobilizes GLUT4-containing compartments. Recently, several candidate signaling molecules, including AS160, PIKfyve and synip, have been identified that might provide functional links between the insulin signaling cascade and GLUT4 compartments. Future work will focus on delineating the precise GLUT4 trafficking steps regulated by these molecules.     

Cross-talk mechanisms in the development of insulin resistance of skeletal muscle cells palmitate rather than tumour necrosis factor inhibits insulin-dependent protein kinase B (PKB)/Akt stimulation and glucose uptake.

Insulin resistance in skeletal muscle is one of the earliest symptoms associated with non-insulin-dependent diabetes mellitus (NIDDM). Tumour necrosis factor (TNF) and nonesterified fatty acids have been proposed to be crucial factors in the development of the insulin-resistant state. We here show that, although TNF downregulated insulin-induced insulin receptor (IR) and IR substrate (IRS)-1 phosphorylation as well as phosphoinositide 3-kinase (PI3-kinase) activity in pmi28 myotubes, this was, unlike in adipocytes, not sufficient to affect insulin-induced glucose transport. Rather, TNF increased membrane expression of GLUT1 and glucose transport in these muscle cells. In contrast, the nonesterified fatty acid palmitate inhibited insulin-induced signalling cascades not only at the level of IR and IRS-1 phosphorylation, but also at the level protein kinase B (PKB/Akt), which is thought to be directly involved in the insulin-induced translocation of GLUT4, and inhibited insulin-induced glucose uptake. Palmitate also abrogated TNF-dependent enhancement of basal glucose uptake, suggesting that palmitate has the capacity to render muscle cells resistant not only to insulin but also to TNF with respect to glucose transport by GLUT4 and GLUT1, respectively. Our data illustrate the complexity of the mechanisms governing insulin resistance of skeletal muscle, questioning the role of TNF as a direct inhibitor of glucose homoeostasis in this tissue and shedding new light on an as yet unrecognized multifunctional role for the predominant nonesterified fatty acid palmitate in this process.     

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.     

Separation of insulin signaling into distinct GLUT4 translocation and activation steps

GLUT4 (glucose transporter 4) plays a pivotal role in insulin-induced glucose uptake to maintain normal blood glucose levels. Here, we report that a cell-permeable phosphoinositide-binding peptide induced GLUT4 translocation to the plasma membrane without inhibiting IRAP (insulin-responsive aminopeptidase) endocytosis. However, unlike insulin treatment, the peptide treatment did not increase glucose uptake in 3T3-L1 adipocytes, indicating that GLUT4 translocation and activation are separate events. GLUT4 activation can occur at the plasma membrane, since insulin was able to increase glucose uptake with a shorter time lag when inactive GLUT4 was first translocated to the plasma membrane by pretreating the cells with this peptide. Inhibition of phosphatidylinositol (PI) 3-kinase activity failed to inhibit GLUT4 translocation by the peptide but did inhibit glucose uptake when insulin was added following peptide treatment. Insulin, but not the peptide, stimulated GLUT1 translocation. Surprisingly, the peptide pretreatment inhibited insulin-induced GLUT1 translocation, suggesting that the peptide treatment has both a stimulatory effect on GLUT4 translocation and an inhibitory effect on insulin-induced GLUT1 translocation. These results suggest that GLUT4 requires translocation to the plasma membrane, as well as activation at the plasma membrane, to initiate glucose uptake, and both of these steps normally require PI 3-kinase activation.