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PubMed2001

Effect of oral creatine supplementation on human muscle GLUT4 protein content after immobilization.

The purpose of this study was to investigate the effect of oral creatine supplementation on muscle GLUT4 protein content and total creatine and glycogen content during muscle disuse and subsequent training. A double-blind placebo-controlled trial was performed with 22 young healthy volunteers. The right leg of each subject was immobilized using a cast for 2 weeks, after which subjects participated in a 10-week heavy resistance training program involving the knee-extensor muscles (three sessions per week). Half of the subjects received creatine monohydrate supplements (20 g daily during the immobilization period and 15 and 5 g daily during the first 3 and the last 7 weeks of rehabilitation training, respectively), whereas the other 11 subjects ingested placebo (maltodextrine). Muscle GLUT4 protein content and glycogen and total creatine concentrations were assayed in needle biopsy samples from the vastus lateralis muscle before and after immobilization and after 3 and 10 weeks of training. Immobilization decreased GLUT4 in the placebo group (-20%, P < 0.05), but not in the creatine group (+9% NS). Glycogen and total creatine were unchanged in both groups during the immobilization period. In the placebo group, during training, GLUT4 was normalized, and glycogen and total creatine were stable. Conversely, in the creatine group, GLUT4 increased by approximately 40% (P < 0.05) during rehabilitation. Muscle glycogen and total creatine levels were higher in the creatine group after 3 weeks of rehabilitation (P < 0.05), but not after 10 weeks of rehabilitation. We concluded that 1) oral creatine supplementation offsets the decline in muscle GLUT4 protein content that occurs during immobilization, and 2) oral creatine supplementation increases GLUT4 protein content during subsequent rehabilitation training in healthy subjects.

Administration, Oral↗
PubMed2001

GLUT4 is reduced in slow muscle fibers of type 2 diabetic patients: is insulin resistance in type 2 diabetes a slow, type 1 fiber disease?

To gain further insight into the mechanisms underlying muscle insulin resistance, the influence of obesity and type 2 diabetes on GLUT4 immunoreactivity in slow and fast skeletal muscle fibers was studied. Through a newly developed, very sensitive method using immunohistochemistry combined with morphometry, GLUT4 density was found to be significantly higher in slow compared with fast fibers in biopsy specimens from lean and obese subjects. In contrast, in type 2 diabetic subjects, GLUT4 density was significantly lower in slow compared with fast fibers. GLUT4 density in slow fibers from diabetic patients was reduced by 9% compared with the weight-matched obese subjects and by 18% compared with the lean control group. The slow-fiber fraction was reduced to 86% in the obese subjects and to 75% in the diabetic subjects compared with the control group. Estimated GLUT4 contribution from slow fibers was reduced to 77% in the obese subjects and to 61% in type 2 diabetic patients compared with the control subjects. We propose that a reduction in the fraction of slow-twitch fibers, combined with a reduction in GLUT4 expression in slow fibers, may reduce the insulin-sensitive GLUT4 pool in type 2 diabetes and thus contribute to skeletal muscle insulin resistance.

Adult↗
PubMed2002

Need for GLUT4 activation to reach maximum effect of insulin-mediated glucose uptake in brown adipocytes isolated from GLUT4myc-expressing mice.

There is a need to understand whether the amount of GLUT4 at the cell surface determines the extent of glucose uptake in response to insulin. Thus, we created a heterozygous mouse expressing modest levels of myc-tagged GLUT4 (GLUT4myc) in insulin-sensitive tissues under the control of the human GLUT4 promoter. Insulin stimulated 2-deoxyglucose uptake 6.5-fold in isolated brown adipocytes. GLUT1 did not contribute to the insulin response. The stimulation by insulin was completely blocked by wortmannin and partly (55 +/- 2%) by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Insulin increased surface exposure of GLUT4myc twofold (determined by fluorescent or enzyme-linked myc immunodetection in intact adipocytes). Such increase was completely blocked by wortmannin but insensitive to SB203580. Insulin increased the kinase activity of the p38 MAPK beta-isoform 1.9-fold without affecting p38-alpha. In summary, the GLUT4myc mouse is a promising model for measuring GLUT4 translocation in intact primary cells. It affords direct comparison between GLUT4 translocation and glucose uptake in similar cell preparations, allowing one to study the regulation of GLUT4 activity. Using this animal model, we found that stimulation of glucose uptake into brown adipocytes involves both GLUT4 translocation and activation.

Adipocytes↗
PubMed2000

Gi-mediated translocation of GLUT4 is independent of p85/p110alpha and p110gamma phosphoinositide 3-kinases but might involve the activation of Akt kinase.

Activation of phosphoinositide 3-kinase (PI-3K) is essential for insulin-stimulated translocation of GLUT4 and glucose transport in insulin target tissues. A novel p110gamma PI-3K was reported to be activated by G(i)-coupled receptors via Gbetagamma subunits. We asked whether the stimulation of G(i)-coupled receptors would trigger GLUT4 translocation and glucose uptake by the activation of Gbetagamma-dependent p110gamma PI-3K. We find that this translocation and glucose uptake can be induced by the ligand stimulation of G(i)-coupled alpha(2A) adrenergic receptor and fMet-Leu-Phe receptor in cells stably expressing these receptors. The noradrenaline ('noradrenaline')- and fMet-Leu-Phe-stimulated GLUT4 translocations were abolished by pretreatment with pertussis toxin. Pretreatment with wortmannin or genistein also inhibited the G(i)-mediated GLUT4 translocation. On ligand stimulation of these two kinds of G(i)-coupled receptor, although there was a slight increase in PtdIns(3,4,5)P(3) production, activation of either the p85/p110alpha PI-3K or Gbetagamma-dependent p110gamma PI-3K was not observed even in Chinese hamster ovary cells stably overexpressing exogenous p101/p110gamma. The G(i)-mediated GLUT4 translocation was accompanied by activation of the serine-threonine kinase Akt; the inhibitory effects of pertussis toxin, wortmannin and genistein on G(i)-mediated GLUT4 translocation paralleled their inhibitory effects on Akt activation. In contrast, the activation of some other G(i)-coupled receptors, such as prostaglandin EP3alpha receptor and platelet-activating factor receptor, did not cause either pertussis-toxin-sensitive translocation of GLUT4myc or activation of Akt kinase. These results indicate that the ligand stimulation of some G(i)-coupled receptors triggers GLUT4 translocation that occurs independently of p85/p110alpha-type and p110gamma-type PI-3Ks but might involve the activation of Akt kinase.

Adrenergic alpha-Agonists↗
PubMed2000

Overexpression of wild-type Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and affected GSK3 beta regulation, but did not promote insulin-stimulated GLUT4 translocation or glucose transport in L6 myotubes.

We have developed a simple, direct and sensitive method to detect GLUT4 on the cell surface. Using this system, we found that PI3-kinase plays a key role in the signaling pathway of insulin-stimulated GLUT4 translocation. One of the down stream effectors of PI3-kinase is serine-threonine kinase Akt (protein kinase B, RAK-PK), but the involvement of Akt in insulin-stimulated GLUT4 translocation is controversial. To investigate whether Akt1 regulates insulin-stimulated GLUT4 translocation and glucose uptake in L6 myotubes, we established L6 myotubes stably expressing c-myc epitope-tagged GLUT4 (GLUT4myc) and mouse wild type (WT) Akt1. We found that overexpression of WT Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and increased the basal activity of GSK3 beta, but did not promote insulin-stimulated GLUT4 translocation or glucose uptake. These data supported the result that Akt is not a main signaling molecule to transmit the signal of insulin-stimulated GLUT4 translocation or glucose uptake from insulin-activated PI3-kinase.

Amino Acid Sequence↗
PubMed2001

[Effect of alcohol extract of Cornus officinalis Sieb. et Zucc on GLUT4 expression in skeletal muscle in type 2 (non-insulin-dependent) diabetic mellitus rats].

OBJECTIVE: Based on its effects of decreasing postprandial plasma glucose and increasing insulin level in non-insulin-dependent diabetic mellitus (NIDDM) rats, we studied the effects of Alcohol extract of Cornus officinalis Sieb. et Zucc on the GLUT4 expression in NIDDM model rats. METHOD: The rat model of NIDDM was made. The animals were divided into three groups(six for each group): group I: control; group II: NIDDM model; group III: NIDDM model + Cornus officinalis Sieb. et Zucc. The drug was given orally to animals one time a day, uninterrupted for a month. The GLUT4 mRNA and its protein expression in skeletal muscle were observed with Northern blot and Western blot method, respectively. RESULT: The GLUT4 mRNA expression in skeletal muscle was decreased remarkably in NIDDM rats (P < 0.01, compared to control group). Alcohol extract of Cornus officinalis Sieb. et Zucc increased GLUT4 mRNA expression in NIDDM rats(P < 0.01), compared to NIDDM model group). The result of GLUT4 protein expression was similar to GLUT4 mRNA expression. CONCLUSION: This experiment demonstrated that alcohol extract of Cornus officinalis Sieb. et Zucc can increase GLUT4 mRNA and its protein expression in NIDDM rats through promoting proliferation of islet and increasing postprandial secretion of insulin and therefore accelerate glucose transport.

Animals↗
PubMed2004

Muscle insulin resistance amended with exercise training: role of GLUT4 expression.

PURPOSE: Muscle insulin resistance is characterized by the inability of a normal insulin concentration to produce a favorable rate of glucose uptake. The muscle of the obese Zucker rat is highly insulin resistant. The purpose of this review is to discuss the cellular defects associated with the muscle insulin resistance of the obese Zucker rat, as well as the mechanisms by which exercise training alleviates or compensates for these defects. Emphasis will be given to the importance of an increased GLUT4 expression on alleviating muscle insulin resistance. METHODS: A review of the relative research from my laboratory and the scientific literature was performed to obtain information on the muscle insulin resistance of the obese Zucker rat and its response to exercise training. RESULTS: The insulin resistance of the obese Zucker rat results from defects in the insulin signaling cascade, which limits translocation of the glucose transporter GLUT4 to the plasma membrane upon insulin binding to its receptor. Exercise training improves the muscle insulin resistance of obese Zucker rat but does not correct the defects in insulin signaling or GLUT4 translocation. The improvement in insulin resistance, i.e., glucose transport, is correlated with an increased expression of GLUT4 protein. Preventing GLUT4 overexpression during exercise training will inhibit the improvement in insulin-stimulated glucose transport. CONCLUSION: Exercise training does not correct but compensates for the defects in muscle insulin resistance by increasing expression of GLUT4. This increase in GLUT4 protein is essential for the improvement in muscle insulin resistance.

Animals↗
PubMed1991

Chronic treatment with insulin selectively down-regulates cell-surface GLUT4 glucose transporters in 3T3-L1 adipocytes.

A new method for photoaffinity labeling of glucose transporters has been used to compare the effects of glucose-starvation, acute-insulin, and chronic-insulin treatments on the cell-surface glucose transporters in 3T3-L1 adipocytes. Starvation alone increased the cell-surface levels of GLUT1 and GLUT4 by approximately 4- and approximately 2-fold, respectively. As shown by Calderhead, D, M., Kitagawa, K., Tanner, L.T., Holman, G.D., and Lienhard, G.E. (1990) J. Biol. Chem. 265, 13800-13808) acute-insulin treatment increased cell-surface GLUT1 and GLUT4 by approximately 5- and approximately 15-fold respectively. In contrast to this, chronic-insulin treatment gave a further 3-4-fold increase in both cell-surface and total cellular GLUT1, but availability of GLUT4 at the cell-surface was down-regulated to half the level found in the acute treatment but with no change in the total cellular level. This effect occurred in starved and non-starved cells and suggests that starvation, acute-insulin, and chronic-insulin treatments regulate glucose transporter availability through independent mechanisms. The down-regulation of GLUT4 reached a maximally reduced cell-surface level in 6 h while the rise in GLUT1 reached a maximum after 24-48 h. The rise in GLUT1 appeared to compensate for the decline in cell-surface GLUT4 as glucose transport activity was further increased during the long term treatment with insulin. The down-regulation of GLUT4 due to the chronic-insulin treatment is associated with a marked resistance of the cells to restimulate glucose transport and particularly to recruit further GLUT4 to the cell-surface following an additional insulin treatment. The defect appears to be in the signaling mechanism that is responsible for translocation.

Adipose Tissue↗
PubMed1993

The glucose transporter GluT4 and secretory carrier membrane proteins (SCAMPs) colocalize in rat adipocytes and partially segregate during insulin stimulation.

Secretory carrier membrane proteins (SCAMPs) mark the recycling system for the insulin-responsive glucose transporter, GluT4, in rat adipocytes. Anti-GluT4 and anti-SCAMP antibodies each immunoadsorbed vesicles containing both antigens from a low density microsomal fraction that is enriched in both antigens. The immunoadsorbed vesicles also contain VAMPs (synaptobrevins), synaptic vesicle membrane proteins. All three antigens were colocalized in low density microsomal vesicles from both basal and insulin-stimulated adipocytes. The SCAMPs have the same electrophoretic mobility as a major polypeptides detected in GluT4 vesicles. During insulin stimulation, 40% each of GluT4 and VAMPs redistribute from low density microsomes to the plasma membrane fraction; however, < 10% of the SCAMPs redistribute. Immunocytochemical staining of adipose tissue shows almost complete coincidence of SCAMPs and GluT4 in the basal state and extensive redistribution of both antigens to the cell periphery during insulin stimulation. Segregation of antigens during stimulation is not as distinct as observed by fractionation, although there are regions at the cell border where the SCAMPs appear more concentrated than GluT4. These data suggest that during insulin stimulation, in contrast to the behaviour of GluT4, SCAMPs remain tightly associated with the recycling system.

Adipose Tissue↗
PubMed1993

Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells.

The two glucose transporter isoforms GLUT4 and GLUT1 present in 3T3-L1 cells were labeled in the insulin-stimulated and basal states with the impermeant bis-mannose photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine. The redistributions of these labeled transporters from the plasma membrane to the low density microsome membrane fraction were followed while cells were maintained at either insulin-stimulated or basal steady states. In both these steady states GLUT4 and GLUT1 were continuously recycled. Analysis of the time courses for tracer-tagged GLUT4 and GLUT1 redistribution showed that the endocytosis rate constants were only approximately 30% slower in the insulin-stimulated (0.08 and 0.093 min-1) compared with the basal (0.116 and 0.121 min-1) state. In the insulin-stimulated state, the rate constants for GLUT4 and GLUT1 exocytosis (0.086 and 0.096 min-1) were similar to those of endocytosis. In contrast, the exocytosis rate constants of GLUT4 and GLUT1 in the basal state were 0.01 and 0.035 min-1. We therefore conclude that the main effect of insulin is to increase GLUT4 and GLUT1 exocytosis rate constants by approximately 9- and 3-fold, respectively, and that the unique feature of the GLUT4 isoform is the very slow rate of exocytosis in the basal state.

3T3 Cells↗
PubMed1999

Cloning of an L-3-hydroxyacyl-CoA dehydrogenase that interacts with the GLUT4 C-terminus.

Evidence indicates that the carboxy-terminal cytoplasmic domain of glucose transporter 4 (GLUT4) is important for the regulation of GLUT4 in muscle and adipocytes. We cloned from a human skeletal muscle cDNA library a 34-kDa protein which interacts with GLUT4 C-terminal cytoplasmic domain in a two-hybrid system and also with GLUT4 C-terminus synthetic peptide in an in vitro binding assay. This protein, called YP10, showed a high degree (>90%) of sequence homology with l-3-hydroxyacyl-CoA dehydrogenase (HAD) and had a dehydrogenase activity similar to pig heart HAD, which was inhibited by GLUT4 C-terminus synthetic peptide. An antiserum raised against pig heart HAD also reacted with YP10. Western blot analysis using this antiserum revealed abundant immunoreactivity only in the mitochondria- and plasma membrane-enriched fractions of rat adipocytes. Northern blots revealed that YP10 mRNA is most abundant in skeletal and heart muscle. These findings suggest that YP10, a HAD isoform, interacts with GLUT4 at the plasma membrane and may play a role in cross-talk between glucose transport and fatty acid metabolism.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1993

Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase.

We examined the role of 185-kDa insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) in the signaling pathway of insulin-stimulated GLUT4 translocation. We had already developed a novel cell line to detect GLUT4 on the cell surface, directly and sensitively (Kanai, F., Nishioka, Y., Hayashi, H., Kamohara, S., Todaka, M., and Ebina, Y. (1993) J. Biol. Chem. 268, 14523-14526). We stably expressed a mutant insulin receptor in which Tyr972 was replaced with phenylalanine. Insulin-stimulated tyrosyl phosphorylation of IRS-1 and GLUT4 translocation were decreased in cells expressing the mutant receptor, as compared to findings in cells expressing the normal receptor. Wortmannin, an inhibitor of PI3-kinase, inhibits the insulin-stimulated PI3-kinase activity and GLUT4 translocation at 50 nM, but not the NaF-stimulated GLUT4 translocation. These results suggest that the tyrosine phosphorylation of IRS-1 and activation of PI3-kinase may be involved in the signaling pathway of the insulin-stimulated GLUT4 translocation.

Androstadienes↗
PubMed1997

Botulinum E toxin light chain does not cleave SNAP-23 and only partially impairs insulin stimulation of GLUT4 translocation in 3T3-L1 cells.

The stimulation of glucose uptake into fat and muscle by insulin results predominantly from the translocation of the glucose transporter, GLUT4, from an intracellular vesicle pool to the cell surface. Homologues of several key proteins known to be involved in the process of synaptic vesicle fusion have been identified on GLUT4 vesicles, including VAMP2 and cellubrevin. Syntaxin 4, SNAP-23 and/or SNAP-25 are also implicated in this process. Bacterial toxins that specifically cleave these proteins have been utilised to assess their involvement in cell function. We aimed to distinguish which of the SNAP isoforms are specifically involved in GLUT4 translocation. Here we show that both human (h) and mouse (m) SNAP-23, unlike SNAP-25, are not substrates for Botulinum E toxin light chain (BoNT/E). Furthermore, we demonstrate that microinjection of differentiated 3T3-L1 cells with BoNT/E inhibited insulin stimulation of GLUT4 translocation only slightly, 27%, whereas tetanus toxin light chain, that cleaves VAMP2, inhibited insulin stimulation of GLUT4 translocation by 80%. These studies therefore do not support a major role for SNAP-25 in insulin stimulation of GLUT4 translocation and place SNAP-23 as a prime candidate for a role in this process.

3T3 Cells↗
PubMed1998

Separation of IRS-1 and PI3-kinase from GLUT4 vesicles in rat skeletal muscle.

In fat and muscle tissues, insulin stimulates cellular glucose uptake by initiating a phosphorylation cascade which ultimately results in the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular storage pool(s) to the plasma membrane in fat and to t-tubules in skeletal muscle. Insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) are known to be involved in cellular responses to insulin such as GLUT4 translocation, but the biochemical mechanism(s) connecting IRS-1 and PI3-kinase to GLUT4-containing intracellular membranes remains unclear. Here, in control and insulin-stimulated rat skeletal muscle, the intracellular localization of these two proteins was compared to that of GLUT4 using subcellular fractionation by sucrose velocity gradients followed by immunoblotting. Our data show that insulin-sensitive GLUT4-containing vesicles are present in fractions 1 through 10, whereas IRS-1 and PI3-kinase are found in fractions 16 through 24. These results indicate that in intracellular fractions derived from skeletal muscle, IRS-1 and PI3-kinase are excluded from membranes harboring GLUT4.

Animals↗
PubMed2005

Reduced insulin-stimulated GLUT4 bioavailability in stroke-prone spontaneously hypertensive rats.

AIMS/HYPOTHESIS: Insulin-stimulated glucose transport is impaired in a genetic model of hypertension, the stroke-prone spontaneously hypertensive rat (SHRSP), yet the molecular mechanisms that underlie this defect in the animals remain unclear. METHODS: We examined the effects of insulin on the trafficking of the insulin-responsive glucose transporter GLUT4 to the plasma membrane in isolated adipocytes from SHRSP and normotensive control Wistar-Kyoto (WKY) rats. RESULTS: Treatment of isolated adipocytes with insulin resulted in trafficking of GLUT4 to the plasma membrane. There was no significant difference in the magnitude of insulin-stimulated GLUT4 trafficking from intracellular membranes to the plasma membrane between strains. In contrast, we demonstrated that there is a significant reduction in GLUT4 accessible to the glucose photolabel Bio-LC-ATB-BGPA at the plasma membrane of SHRSP adipocytes compared with control rats. CONCLUSIONS/INTERPRETATION: We propose that a large proportion of GLUT4 translocated to the plasma membrane in response to insulin is not able to bind substrate and catalyse transport in the SHRSP. Therefore, there is a reduction in bioavailable GLUT4 in SHRSP animals that is likely to account, at least in part, for the reduced insulin-stimulated glucose uptake.

Adipocytes↗
PubMed2005

Protein kinase-zeta interacts with munc18c: role in GLUT4 trafficking.

AIMS/HYPOTHESIS: Insulin-stimulated glucose transport requires a signalling cascade through kinases protein kinase (PK) Czeta/lambda and PKB that leads to movement of GLUT4 vesicles to the plasma membrane. The aim of this study was to identify missing links between the upstream insulin-regulated kinases and the GLUT4 vesicle trafficking system. MATERIALS AND METHODS: A yeast two-hybrid screen was conducted, using as bait full-length mouse munc18c, a protein known to be part of the GLUT4 vesicle trafficking machinery. RESULTS: The yeast two-hybrid screen identified PKCzeta as a novel interactor with munc18c. Glutathione S transferase (GST) pull-downs with GST-tagged munc18c constructs confirmed the interaction, mapped a key region of munc18c that binds PKCzeta to residues 295-338 and showed that the N-terminal region of PKCzeta was required for the interaction. Endogenous munc18c was shown to associate with endogenous PKCzeta in vivo in various cell types. Importantly, insulin stimulation increased the association by approximately three-fold. Moreover, disruption of PKCzeta binding to munc18c by deletion of residues 295-338 of munc18c or deletion of the N-terminal region of PKCzeta markedly inhibited the ability of insulin to stimulate glucose uptake or GLUT4 translocation. CONCLUSIONS/INTERPRETATION: We have identified a physiological interaction between munc18c and PKCzeta that is insulin-regulated. This establishes a link between a kinase (PKCzeta) involved in the insulin signalling cascade and a known component of the GLUT4 vesicle trafficking pathway (munc18c). The results indicate that PKCzeta regulates munc18c and suggest a model whereby insulin triggers the docking of PKCzeta to munc18c, resulting in enhanced GLUT4 translocation to the plasma membrane.

Animals↗
PubMed1992

Subcellular distribution and activity of glucose transporter isoforms GLUT1 and GLUT4 transiently expressed in COS-7 cells.

In adipose and muscle cells, the glucose transporter isoform GLUT4 is mainly located in an intracellular, vesicular compartment from which it is translocated to the plasma membrane in response to insulin. In order to test the hypothesis that this preferential targeting of a glucose transporter to an intracellular storage site is conferred only by its primary sequence, we compared the subcellular distribution of the fat/muscle glucose transporter GLUT4 with that of the erythrocyte/brain-type glucose transporter GLUT1 after transient expression in COS-7 cells. Full-length cDNA was ligated into the expression vector pCMV that is driven by the cytomegalovirus promoter, and introduced into COS cells by the DEAE-dextran method. Cells were homogenized and fractionated by differential centrifugation to yield plasma membranes and a Golgi-enriched fraction of intracellular membranes (low-density microsomes). In these membrane fractions, the abundance of glucose transporters was assessed by immunoblotting with specific antibodies against GLUT1 and GLUT4, and their transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Uptake rates of 2-deoxyglucose assayed in parallel samples were higher in cells expressing GLUT1 or GLUT4 as compared with control cells (transfection of pCMV without transporter cDNA). Reconstituted glucose transport activity in plasma membranes was about 5-fold higher after expression of GLUT1 and GLUT4 as compared with control cells. The relative amount of GLUT4 in the low-density microsomes as detected by reconstitution and immunoblotting exceeded that of the GLUT1, but was much lower than that observed in typical insulin-sensitive cells, e.g., rat fat cells or 3T3-L1 adipocytes. These data indicate that COS-7 cells transfected with glucose transporter cDNA express the active transport proteins and can be used for functional studies.

Amino Acid Sequence↗
PubMed2004

Ischemic preconditioning activates AMPK in a PKC-dependent manner and induces GLUT4 up-regulation in the late phase of cardioprotection.

OBJECTIVE: The aim of this study was to determine the role of AMP-activated protein kinase (AMPK) and its link to protein kinase C (PKC) in the late phase of cardioprotection afforded by ischemic preconditioning (PC) against myocardial stunning. METHODS AND RESULTS: Rabbits were instrumented with a balloon occluder around a coronary artery and with a Doppler sensor to monitor the thickening fraction (TF). Conscious rabbits underwent five cycles of 5-min ischemia/5-min reperfusion (I/R) on 2 consecutive days (days 1 and 2). Reduction of TF after I/R was significantly less and recovery of TF was faster on day 2, indicating a late PC effect. PC provoked translocation of PKC- from the cytosol to the membrane and significantly increased AMPK activity by 100% immediately after PC. The mRNA level of GLUT4, a glucose transporter, was elevated by 150% at 3 h after PC, and the total protein level of GLUT4 was increased by 107% at 24 h after PC. The level of sarcolemmal GLUT4 protein after I/R on day 2 was 41% higher than its level after I/R on day 1. AMPK activation and up-regulation of GLUT4 by PC were abrogated by pre-treatment with PKC inhibitors. CONCLUSION: PC activated AMPK and up-regulated GLUT4 expression in a PKC-dependent manner. This GLUT4 up-regulation at 24 h after PC may contribute to attenuation of myocardial stunning.

AMP-Activated Protein Kinases↗