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PubMed1993

Suppression of GLUT4 expression in skeletal muscle of rats that are obese from high fat feeding but not from high carbohydrate feeding or genetic obesity.

In this study we determined whether alterations in the expression of GLUT4, the major insulin-regulatable glucose transporter, in skeletal muscle could explain the insulin-resistant glucose uptake characteristic of both dietary-induced and genetic obesity. GLUT1 expression was measured for comparison. To assess glucose transporter protein levels in dietary-induced obesity, postnuclear membranes were prepared from hindlimb muscle of Sprague-Dawley rats fed chow ad libitum (control), a high calorie/carbohydrate diet, or a high fat (80%) diet for 7 weeks. Immunoblotting revealed that GLUT4 protein levels decreased 34% in high fat-fed rats, but were unaltered in high calorie/carbohydrate-fed obese rats compared to control values. GLUT4 mRNA per DNA decreased 47% in muscle of high fat-fed rats compared to that in control or high calorie-fed rats; GLUT1 mRNA was reduced 31%, and actin mRNA tended to decrease (29%). To assess GLUT4 and GLUT1 expression in genetic obesity, similar studies were carried out in 5- and 20-week-old lean and obese Zucker rats as well as in 20-week-old obese Zucker rats 36 h after streptozotocin injection to lower insulin levels. GLUT4 protein and mRNA levels were unaltered in hindlimb muscle of obese Zucker rats at either age or in the acutely diabetic state, whereas GLUT1 protein and mRNA levels decreased 40-45%. Comparison of these results with recent data in adipocytes demonstrates tissue-specific regulation of expression of GLUT4 and GLUT1. Thus, obesity due to high fat feeding, but not that due to high calorie/carbohydrate feeding or genetics, is associated with pretranslational suppression of GLUT4 expression in skeletal muscle. In at least some forms of obesity, the level of GLUT4 expression in muscle appears to be only one factor in, or may even be unrelated to, the degree of insulin-responsive glucose transport in vivo.

Actins↗
PubMed1997

The small guanosine triphosphate-binding protein Rab4 is involved in insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-L1 cells.

Insulin's stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter GLUT4 to the plasma membrane. Small GTP-binding proteins have been implicated in the regulation of vesicular traffic. We studied the effects of microinjection of wild-type Rab4 glutathione S-transferase fusion protein (WT Rab4), a GTP-binding defective mutant (Rab4 N121I), a guanosine triphosphatase-defective mutant (Rab4 Q67L), and a Rab4 antibody on insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. Microinjection of Rab4 N121I and Rab4 antibodies had no effect on basal GLUT4 staining, but inhibited insulin-induced GLUT4 translocation by 50% compared with that in control IgG-injected cells. WT Rab4 and Rab4 Q67L microinjection had no effect on either basal or insulin-induced GLUT4 translocation. Premixing and coinjection of the Rab4 antibody with WT Rab4 almost completely abolished its inhibitory effect on insulin-induced GLUT4 translocation. In contrast, microinjection of an antibody directed against the highly conserved region of Rab3 proteins had no effect on insulin-induced GLUT4. These results point to a direct role of Rab4 in insulin-induced GLUT4 translocation, and that this effect is dependent on nucleotide binding to the protein. We also studied the effect of microinjection of the same proteins on insulin-induced actin filament rearrangement (membrane ruffling) in the same cell line. Microinjection of Rab4 N121I and Rab4 antibodies inhibited insulin-induced membrane ruffling by 40%, whereas WT Rab4 or a Rab3 antibody injection had no effect on cytoskeletal rearrangement. In summary, 1) Rab4 is a necessary component of the insulin/GLUT4 translocation signaling pathway; 2) the function of Rab4 in this pathway requires GTP binding; 3) Rab4 also participates in the process of insulin-induced membrane ruffling; and 4) Rab3 proteins do not seem to be involved in these processes.

3T3 Cells↗
PubMed1999

The insulin-sensitive glucose transporter (GLUT4) is involved in early bone growth in control and diabetic mice, but is regulated through the insulin-like growth factor I receptor.

Children with uncontrolled type I (insulin-dependent) diabetes mellitus are characterized by a slow growth rate, which improves upon adequate therapy. While skeletal growth is an energy-consuming process involving high glucose utilization, the role of glucose transporters (GLUT) and their regulation in the bone formation process are not yet fully understood. Thus, we studied both in vivo and in vitro early endochondral bone formation in control and streptozotocin-induced young diabetic mice. Using in situ hybridization and immunohistochemistry techniques, we demonstrated the novel existence of the insulin-sensitive glucose transporter (GLUT4), as well as GLUT1, in juvenile-derived murine mandibular condyles and in the humeral growth plate-two models for endochondral bone formation. Insulin-like growth factor (IGF) I receptors (IGF-I-R), but not insulin receptors (IR), were shown to have cellular distribution similar to GLUT4, being more abundant in mature chondrocytes. Further, in the skeletal growth centers of streptozotocin-induced diabetic mice, GLUT4, IGF-I, and IGF-I and insulin receptor levels, but not GLUT1 were markedly reduced. The decrease in GLUT4 and in IGF-I and insulin receptors was associated with severe histological changes in the mandibular condyles and humeral growth plate. Insulin therapy restored IR levels to normalcy, whereas IGF-I-R and GLUT4 levels were only partially recovered. Thus, GLUT4 and IGF-I-R have a potential role in early bone growth in mice. Further, during early bone growth GLUT4 may be regulated through the IGF-I receptor rather than via the insulin receptor. We propose that skeletal growth retardation in type I diabetes may be associated with reduced expression of the GLUT4 and IGF-I receptor in the bone growth center.

Animals↗
PubMed2005

Akt activation is required at a late stage of insulin-induced GLUT4 translocation to the plasma membrane.

Insulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortex-this process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PM-this process may be the major Akt-dependent step in the insulin regulation of glucose transport.

3T3 Cells↗
PubMed1999

Association of AP1 adaptor complexes with GLUT4 vesicles.

Nycodenz gradients have been used to examine the in vitro effects of GTP-(gamma)-S on adaptor complex association with GLUT4 vesicles. On addition of GTP-(gamma)-S, GLUT4 fractionates as a heavier population of vesicles, which we suggest is due to a budding or coating reaction. Under these conditions there is an increase in co-sedimentation of GLUT4 with AP1, but not with AP3. Western blotting of proteins associated with isolated GLUT4 vesicles shows the presence of high levels of AP1 and some AP3 but very little AP2 adaptor complexes. Cell free, in vitro association of the AP1 complex with GLUT4 vesicles is increased approximately 4-fold by the addition of GTP-(gamma)-S and an ATP regenerating system. Following GTP-(gamma)-S treatment in vitro, ARF is also recruited to GLUT4 vesicles, and the temperature dependence of ARF recruitment closely parallels that of AP1. The recruitment of both AP1 and ARF are partially blocked by brefeldin A. These data demonstrate that the coating of GLUT4 vesicles can be studied in isolated cell-free fractions. Furthermore, at least two distinct adaptor complexes can associate with the GLUT4 vesicles and it is likely that these adaptors are involved in mediating distinct intracellular sorting events at the level of TGN and endosomes.

Adaptor Protein Complex 1↗
PubMed2000

Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts.

Insulin-responsive trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well established. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole body glucose homeostasis. GLUT4 is expressed in a very limited number of tissues, most highly in adipose and muscle, while IRAP is expressed in many tissues. IRAP's physiological role in any of the tissues in which it is expressed, however, is unknown. The fact that IRAP, which traffics by the same insulin-regulated pathway as GLUT4, is expressed in 'non-insulin responsive' tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway. The existence of an insulin-responsive pathway in other cell types would allow regulation of IRAP activity at the plasma membrane as a potentially important physiological function of insulin. To address this question we use reporter molecules for both GLUT4 and IRAP trafficking to measure insulin-stimulated translocation in undifferentiated cells by quantitative fluorescence microscopy. One reporter (vpTR), a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor, has been previously characterized. The other is a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP. By comparing these reporters to the transferrin receptor, a marker for general endocytic trafficking, we demonstrate the existence of a specialized, insulin-regulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4. The magnitude of translocation in these undifferentiated cells (approximately threefold) is similar to that reported for the translocation of GLUT4 in muscle cells. Thus, undifferentiated cells have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important.

3T3 Cells↗
PubMed2004

Regulation of GLUT4 gene expression during exercise.

Glucose transport into muscle is important for the maintenance of normoglycemia. Thus, understanding mechanisms that regulate expression of GLUT4, the main glucose transporter in skeletal muscle, is important to identify targets for the treatment of diabetes. Exercise increases the expression of GLUT4 mRNA and protein, and we have been investigating the mechanisms involved. Transcription of the GLUT4 gene is transiently activated after an acute bout of exercise and GLUT4 protein can be increased as much as two- to threefold after a few days of repeated exercise bouts. Studies of the GLUT4 promoter have identified two sets of DNA sequences that are important for metabolic regulation and also for increased transcription of the gene in response to exercise. These DNA elements have been shown to bind the transcription factors myocyte enhancer factor 2 (MEF2) and GLUT4 enhancer factor (GEF). The mechanisms that activate these proteins remain one of the important areas of research in this field. Signals that link muscle contraction to the activation of transcription factors (MEF2, GEF) involved in increased expression of GLUT4 during exercise is another area needing further research. Two signals that show promise are changes in the energy charge (acting through AMP activated kinase [AMPK]) and changes in intracellular calcium (acting through calcineurin [a calcium-calmodulin activated phosphatase] and calcium-calmodulin activated kinase [CAMK]). There is good evidence that both increased AMPK activity and increased CAMK activity cause increased transcription of the GLUT4 gene. It remains to be demonstrated that exercise is acting through one or both of these mechanisms.

Animals↗
PubMed1997

Insulin sensitizer YM268 ameliorates insulin resistance by normalizing the decreased content of GLUT4 in adipose tissue of obese Zucker rats.

Genetically obese Zucker rats exhibit mild hyperglycaemia and hyperinsulinaemia suggesting the existence of peripheral insulin resistance. We have examined the effects of YM268, an analogue of thiazolidinedione, on the content and translocation of a glucose transporter (GLUT4) in epididymal adipose tissue in 11-week-old obese and lean Zucker rats. The administration of YM268 at a dose of 10 mg/kg for 2 weeks ameliorated hyperglycaemia, hyperinsulinaemia, and impaired glucose tolerance after glucose load in obese rats. The GLUT4 content per fat pad in obese rats was reduced to 36% of that in lean littermates. Obese rats treated with YM268 increased GLUT4 concentrations in their fat pads from a basal value of 36% up to 191% of the level in lean rats. Furthermore, in adipocytes prepared from obese rats, an increase in the ratio of GLUT4 in plasma membrane to the total amount of GLUT4 (PM-GLUT4 ratio) induced by the submaximal concentration of insulin (0.3 nmol/l) was significantly attenuated compared with that in lean rats. But the maximum effect of insulin (3 nmol/l) was not attenuated. Meanwhile, YM268 had no significant effect on the attenuated PM-GLUT4 ratio in response to insulin in obese rats. These data suggested that one of the mechanisms by which YM268 improved insulin resistance in obese Zucker rats was to normalize the decreased GLUT4 content in the adipose tissue.

Adipose Tissue↗
PubMed2001

GLUT4 protein expression in obese and lean 12-month-old rats: insights from different types of data analysis.

GLUT4 protein expression in white adipose tissue (WAT) and skeletal muscle (SM) was investigated in 2-month-old, 12-month-old spontaneously obese or 12-month-old calorie-restricted lean Wistar rats, by considering different parameters of analysis, such as tissue and body weight, and total protein yield of the tissue. In WAT, an approximately 70% decrease was observed in plasma membrane and microsomal GLUT4 protein, expressed as microg protein or g tissue, in both 12-month-old obese and 12-month-old lean rats compared to 2-month-old rats. However, when plasma membrane and microsomal GLUT4 tissue contents were expressed as g body weight, they were the same. In SM, GLUT4 protein content, expressed as microg protein, was similar in 2-month-old and 12-month-old obese rats, whereas it was reduced in 12-month-old obese rats, when expressed as g tissue or g body weight, which may play an important role in insulin resistance. Weight loss did not change the SM GLUT4 content. These results show that altered insulin sensitivity is accompanied by modulation of GLUT4 protein expression. However, the true role of WAT and SM GLUT4 contents in whole-body or tissue insulin sensitivity should be determined considering not only GLUT4 protein expression, but also the strong morphostructural changes in these tissues, which require different types of data analysis.

Adipose Tissue↗
PubMed2000

Use of a novel impermeable biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface GLUT4 content in skeletal muscle from type 2 diabetic patients.

Cell surface GLUT4 levels in skeletal muscle from nine type 2 diabetic subjects and nine healthy control subjects have been assessed by a new technique that involves the use of a biotinylated photo-affinity label. A profound impairment in GLUT4 translocation to the skeletal muscle cell surface in response to insulin was observed in type 2 diabetic patients. Levels of insulin-stimulated cell surface GLUT4 above basal in type 2 diabetic patients were only approximately 10% of those observed in healthy subjects. The magnitude of the defect in GLUT4 translocation in type 2 diabetic patients was greater than that observed for glucose transport activity, which was approximately 50% of that in healthy subjects. Reduced GLUT4 translocation is therefore a major contributor to the impaired glucose transport activity in skeletal muscle from type 2 diabetic subjects. When a marked impairment in GLUT4 translocation occurs, the contribution of other transporters to transport activity becomes apparent. In response to hypoxia, marked reductions in skeletal muscle cell surface GLUT4 levels were also observed in type 2 diabetic patients. Therefore, a defect in a common late stage in signal transduction and/or a direct impairment in the GLUT4 translocation process accounts for reduced glucose transport in type 2 diabetic patients.

Biological Transport↗
PubMed2005

Altered hepatic and muscle substrate utilization provoked by GLUT4 ablation.

Studies were conducted to explore altered substrate utilization and metabolism in GLUT4 null mice. Liver fatty acid synthase mRNA and fatty acid synthesis rates were dramatically increased in GLUT4 null mice compared with control mice and were supported by increased rates of the pentose phosphate pathway oxidative phase and sterol regulatory binding protein mRNA expression. Increased GLUT2 protein content, glucokinase mRNA, and glucose-6-phosphate in GLUT4 null mice may provide substrate for the enhanced fatty acid synthesis. Increased fatty acid synthesis, however, did not lead to hepatic triglyceride accumulation in GLUT4 null mice because of increased hepatic triglyceride secretion rates. GLUT4 null mice rapidly cleared orally administered olive oil, had reduced serum triglyceride concentrations in the fed and the fasted state, and increased skeletal muscle lipoprotein lipase when compared with controls. Oleate oxidation rates were increased in GLUT4 null skeletal muscle in association with mitochondrial hyperplasia/hypertrophy. This study demonstrated that GLUT4 null mice had increased hepatic glucose uptake and conversion into triglyceride for subsequent use by muscle. The ability of GLUT4 null mice to alter hepatic carbohydrate and lipid metabolism to provide proper nutrients for peripheral tissues may explain (in part) their ability to resist diabetes when fed a normal diet.

Animals↗
PubMed2000

Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.

Actin-based cytoskeletons have been implicated in insulin-stimulated glucose transport and translocation of the insulin-regulated glucose transporter, GLUT4, from the intracellular pool to the plasma membrane. However, most previous studies were done using adherent cell systems such as L6 myotubes and 3T3-L1 adipocytes, and very little information is available on the significance of the actin filaments to the insulin action in isolated adipocytes, a widely used experimental system. In the present study, we investigated the physiological role of actin filaments in the subcellular trafficking of GLUT4 in isolated rat adipocytes. We first compared the effects of two actin-disrupting reagents, latrunculin A and cytochalasin D, on the organization of the actin filaments as well as on the insulin action on glucose transport by laser confocal microscopy combined with biochemical analysis of the insulin action. Treatment of the cells with latrunculin A induced dose- and time-dependent disappearance of the filamentous actin, which correlated very well with inhibition of the insulin effect on glucose transport. Although cytochalasin D at 50 microM significantly inhibited insulin-stimulated glucose transport, it was not effective in disassembly of the actin filaments; rather, many intense punctate signals were observed in cytochalasin D-treated cells. In the actin-disrupted adipocytes treated with latrunculin A, insulin-induced GLUT4 translocation was inhibited completely. In addition, latrunculin A remarkably inhibited both insulin-induced glucose transport and GLUT4 translocation in the presense of D(k)-(62-85), a potent inhibitor of GLUT4 endocytosis, suggesting that intactness of the actin filaments was necessary for insulin-induced exocytosis of the GLUT4-containing vesicles. On the other hand, latrunculin A showed little inhibitory effect on either endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 or decay of the glucose transport activity after addition of wortmannin in insulin-stimulated cells. The results of our experiment show clearly that, in rat adipocytes, (i) latrunculin A may be a more suitable tool than cytochalasin D for disruption of actin filaments, and (ii) actin filaments play a crucial role in exocytotic recruitment of GLUT4 to the plasma membrane from the intracellular pool, but not in its endocytosis.

Actins↗
PubMed1992

Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells.

L6 myoblasts spontaneously undergo differentiation and cell fusion into myotubes. These cells express both GLUT1 and GLUT4 glucose transporters, but their expression varies during myogenesis. We now report that the subcellular distribution and the protein processing by glycosylation of both glucose transporter isoforms also change during myogenesis. Crude plasma membrane and light microsome fractions were isolated from either myoblasts or myotubes and characterized by the presence of two functional proteins, the Na+/K(+)-ATPase and the dihydropyridine receptor (DHPR). Immunoreactive alpha 1 subunit of the Na+/K(+)-ATPase was faint in the crude plasma membrane fraction from myoblasts, but abundant in both membrane fractions from myotubes. In contrast, the alpha 1 subunit of the DHPR, which is expressed only in differentiated muscle, was detected in crude plasma membrane from myotubes but not from myoblasts. Therefore, crude plasma membrane fractions from myoblasts and myotubes contain cell surface markers, and the composition of these membranes appears to be developmentally regulated during myogenesis. GLUT1 protein was more abundant in the crude plasma membrane relative to the light microsome fraction prepared from either myoblasts or myotubes. The molecular size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the GLUT1 transporters in myotubes was smaller than that in myoblasts (Mr 47,000 and 53,000, respectively). GLUT4 protein (Mr 48,000) was barely detectable in the crude plasma membrane fraction and was almost absent in the light microsome fraction prepared from myoblasts. However, GLUT4 protein was abundant in myotubes and was predominantly located in the light microsome fraction. Treatment with endoglycosidase F reduced the molecular size of the transporters in all fractions to Mr 46,000 for GLUT1 and Mr 47,000 for GLUT4 proteins. In myotubes, acute insulin treatment increased the crude plasma membrane content of GLUT1 marginally and of GLUT4 markedly, with a concomitant decrease in the light microsomal fraction. These results indicate that: (a) the subcellular distribution of glucose transporters is regulated during myogenesis, GLUT4 being preferentially sorted to intracellular membranes; (b) both GLUT1 and GLUT4 transporters are processed by N-linked glycosylation to form the mature transporters in the course of myogenesis; and (c) insulin causes modest recruitment of GLUT1 transporters and marked recruitment of GLUT4 transporters, from light microsomes to plasma membranes in L6 myotubes.

Animals↗
PubMed1993

Domains that confer intracellular sequestration of the Glut4 glucose transporter in Xenopus oocytes.

The Glut4 glucose transporter is poorly functional compared with other glucose transporter isoforms when expressed in Xenopus oocytes. To investigate the molecular basis for this poor functionality, we compared the biosynthesis and targeting of Glut1 and Glut4 in oocytes after microinjection of the corresponding mRNAs. Both Glut1 and Glut4 were present as lower molecular weight endoglycosidase H-sensitive and higher molecular weight endoglycosidase H-resistant. Subcellular fractionation indicated that Glut1 was targeted to the plasma membrane with a 6.6-fold greater efficiency than was Glut4. Confocal immunofluorescence microscopy confirmed the relative enrichment of Glut1 in the plasma membrane and the efficient intracellular sequestration of Glut4. As in mammalian cells, the endoglycosidase H-resistant form of Glut4 was concentrated in low-density intracellular vesicles, whereas Glut1 was distributed in intracellular vesicles of higher average density. The structural basis for the differential localization of Glut1 and Glut4 was investigated by determining the plasma membrane content of a series of chimeric Glut1/Glut4 molecules. These data indicated that two distinct regions of Glut4, encompassing residues 24-132 and the COOH-terminal cytoplasmic tail, confer intracellular sequestration on the chimeric transporter molecules. At least part of the sequestration effect of the more N-terminal domain was due to the incomplete maturation of chimeras containing this region, resulting in the accumulation of lower molecular weight endoglycosidase H-sensitive and endoglycosidase H-resistant forms, whereas the COOH-terminal cytoplasmic tail conferred sequestration of fully glycosylated chimeras in a low-density intracellular membrane compartment.

Animals↗
PubMed1994

Identification and isolation of glycoproteins that translocate to the cell surface from GLUT4-enriched vesicles in an insulin-dependent fashion.

It has previously been shown that in fat cells, the intracellular reservoir of the glucose transporter isoform GLUT4 consists of a membrane vesicle population highly enriched in this transporter (approximately 15% of the protein content) that has a relatively simple protein pattern as revealed by SDS-polyacrylamide gel electrophoresis and silver staining (Zorzano, A., Wilkinson, W., Kotliar, N., Thoidis, G., Wadzinski, B. E., Ruoho, A. E., and Pilch, P. F. (1989) J. Biol. Chem. 264, 12358-12363). Upon exposure of adipocytes to insulin, the cell-surface (plasma membrane) content of GLUT4 is dramatically enhanced, and this transporter appears to continually cycle from intracellular vesicles to the plasma membrane. To identify other proteins that may recycle in a similar fashion to GLUT4 and that may participate in the insulin-dependent alteration in the cellular location of GLUT4 and other membrane proteins, we performed immunoadsorption of GLUT4-enriched vesicles together with biotinylation of membranes using a cell-impermeant analog of biotin. We find that immunoadsorbed GLUT4-containing vesicles can be fractionated into Triton X-100-soluble and -insoluble fractions. The first includes three major glycoprotein components with molecular masses of 110, 160, and 230 kDa and a few minor polypeptides with lower molecular masses. A Triton X-100-resistant fraction consists of GLUT4 and an approximately 25-kDa protein. All three major Triton-soluble proteins (110, 160, and 230 kDa) isolated from the immunoimmobilized vesicles on wheat germ agglutinin-agarose are strongly biotinylated in an insulin-dependent fashion, i.e. they cycle to and from the cell surface in an apparently identical manner to GLUT4. Sequence analysis of two tryptic fragments from p160 reveals that it is a novel protein containing sequence with no homology to known proteins.

Adipocytes↗
PubMed1993

Glycemia and exercise training alter glucose transport and GLUT4 in the Zucker rat.

The purpose of this study was to determine the effects of chronic hyperglycemia and/or exercise training on the muscle concentration of the insulin-responsive glucose transporter protein, GLUT4, and on maximally insulin-stimulated hindlimb muscle glucose transport. Five-wk-old lean and obese Zucker rats were randomly assigned to sham-operated control (CTL) or 90% pancreatectomized (PX) groups. Obese-PX animals were further randomized into sedentary or exercise trained groups (15-wk treadmill running for 2 h.d-1, 5 d.wk-1, 15% grade, at 15-18 m.min-1). Muscle GLUT4 protein content and maximally insulin-stimulated glucose transport were determined in gastrocnemius, plantaris, and soleus muscles. At 20 wk, lean-PX displayed mild fasting hyperglycemia but normal insulin levels. Obese-PX rats had insulin levels similar to lean-CTL rats but had severe hyperglycemia. Hyperglycemia in lean-PX was associated with a 28% decrease in maximal glucose transport and a 65% decrease in muscle GLUT4 (P < 0.05) compared with lean-CTL. In obese-PX, maximal glucose transport was not affected, but muscle GLUT4 was reduced by 62% (P < 0.05) compared to obese-CTL. Exercise training obese-PX reduced hyperglycemia, increased maximal glucose transport by 45%, and increased muscle GLUT4 by > 2-fold (P < 0.05) compared with obese-CTL. Thus, hyperglycemia associated with PX may be an important factor in the reduction of muscle GLUT4 levels in lean and obese rats. The reduced GLUT4 was accompanied by reduced maximal glucose transport in lean but not obese rats. Exercise training reduced hyperglycemia, normalized glucose transport, and increased muscle GLUT4 in obese-PX.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1999

Effects of transiently expressed atypical (zeta, lambda), conventional (alpha, beta) and novel (delta, epsilon) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda.

Atypical protein kinase (PK)C isoforms, zeta and lambda, have been reported to be activated by insulin via phosphoinositide 3-kinase, and have been suggested to be required for insulin-stimulated glucose transport. Here, we have examined the effects of transiently expressed wild-type (WT), constitutively active (Constit) and kinase-inactive (KI) forms of atypical PKCs, zeta and lambda, on haemagglutinin antigen (HAA)-tagged glucose transporter 4 (GLUT4) translocation in rat adipocytes, and compared these effects with each other and with those of comparable forms of conventional (alpha, beta) and novel (delta, epsilon) PKCs, which have also been proposed to be required for insulin-stimulated glucose transport. KI-PKC-zeta evoked consistent, sizeable (overall mean of 65%) inhibitory effects on insulin-stimulated, but not basal or guanosine-5'-[gamma-thio]triphosphate-stimulated, HAA-GLUT4 translocation; moreover, inhibitory effects of KI-PKC-zeta were largely reversed by co-transfection of WT-PKC-zeta. Like KI-PKC-zeta, KI-PKC-lambda inhibited insulin-stimulated HAA-GLUT4 translocation by approx. 40-60%, and the combination of KI-PKC-zeta and KI-PKC-lambda caused nearly complete (85%) inhibition. Of particular interest is the fact that inhibitory effects of KI forms of PKC-zeta and PKC-lambda were largely reversed by the opposite WT forms, i.e. PKC-lambda and PKC-zeta respectively. In contrast with KI forms of atypical PKCs, KI forms of PKC-alpha, PKC-beta2, PKC-delta and PKC-epsilon had little or no effect on insulin-stimulated HAA-GLUT4 translocation. Concerning the question of sufficiency, overexpression of WT-PKC-zeta enhanced insulin effects on HAA-GLUT4 translocation, whereas WT forms of PKC-alpha, PKC-beta2, PKC-delta and PKC-epsilon did not affect GLUT4 translocation; furthermore, Constit PKC-zeta evoked increases in HAA-GLUT4 translocation approaching those of insulin, but Constit forms of PKC-alpha and PKC-beta2 were without effect. Our findings suggest that, among PKCs, the atypical PKCs, zeta and lambda, appear to be specifically, but interchangeably, required for insulin effects on HAA-GLUT4 translocation.

Adipose Tissue↗
PubMed1999

Metabolism of d-glucose and its pentaacetate ester in muscles and pancreatic islets of GLUT4 null mice.

The metabolism of d-glucose and its pentaacetate ester was investigated in GLUT4 null mice and control C57Bl6/CBA mice. The incorporation of d-[U-(14)C]glucose (1.7 mM) into glycogen of diaphragm, soleus, and extensor digitorium longus muscles averaged, in the GLUT4 null mice, only 34 +/- 7% of the mean corresponding control values. The utilization of d-[5-(3)H]glucose and conversion of d-[U-(14)C]glucose to (14)CO(2) and radioactive acidic metabolites or amino acids were little affected, however, in the muscles from GLUT4 null mice. Likewise, under steady-state conditions, the intracellular pool of 6-deoxy-6-iodo-d-glucose (10 microM) was not significantly different in muscles from GLUT4 null and control mice. The incorporation of d-[U-(14)C]glucose pentaacetate (1.7 mM) into glycogen and utilization of d-[5-(3)H]glucose pentaacetate were also not significantly different in muscles from GLUT4 null and control animals. They were about 10-30 times lower than the corresponding values found with the unesterified hexose. In pancreatic islets, however, the metabolism of d-glucose pentaacetate was not lower than that of unesterified d-glucose. Moreover, the utilization of d-[5-(3)H]glucose and catabolism of d-[U-(14)C]glucose were significantly higher in the islets from GLUT4 null mice than in those from control animals. These findings indicate that the defect of d-glucose metabolism in GLUT4 null mice occurs in muscles but not in pancreatic islets, affects preferentially glycogen synthesis rather than glycolysis, and can be bypassed by using the pentaacetate ester of the hexose. The present data also reveal a striking difference between muscles and islets when comparing the metabolism of d-glucose to that of its ester.

Animals↗