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PubMed2002

GLUT4 protein is differently modulated during development of obesity in monosodium glutamate-treated mice.

The aim of the present study was to investigate the GLUT4 protein expression during the development of obesity in monosodium glutamate- (MSG) treated mice. Control (C) and neonatally MSG-treated 2-month-old (2-mo), 4-month-old (4-mo) and 7-month-old (7-mo) mice were analyzed. Anthropometric data, basal glycemia and insulinemia were measured; and the GLUT4 protein was assessed by Western blotting in white adipose tissue (WAT), skeletal muscle gastrocnemius (SM) and heart (H). Compared to age-matched C mice, the 2-mo and 4-mo MSG mice were already obese, but metabolically they showed increased or preserved whole-body insulin sensitivity, respectively. At these ages they showed unchanged total GLUT4 content in SM and H. However, in plasma membrane fraction from WAT, the MSG showed increased GLUT4 content at both 2- (by 60%) and 4-month (by 45%) of age. When the GLUT4 protein was expressed by unit of adipocyte surface area the protein amount was increased by 36 and 220% in 2-mo and 4-mo MSG mice, respectively. At 7 months of age, obesity was fully established in MSG mice, showing a strongly insulin resistant condition. Additionally, in the 7-mo MSG-mice the GLUT4 protein was reduced in SM (by 40%), H (by 28%), PM and M fractions of WAT (by approximately 70%), and PM expressed by unit of adipocyte surface area (by 92%). The data demonstrate that early, during the accelerated development of obesity in MSG-treated mice, the GLUT4 content was increased in WAT, and that may play a key role in the development of obesity. Later on, when obesity is fully established, the GLUT4 protein was reduced in SM, heart and WAT, and that may be involved in the insulin resistance present in this condition.

Adipocytes↗
PubMed1998

Effect of growth hormone on the translocation of GLUT4 and its relation to insulin-like and anti-insulin action.

To elucidate the effect of growth hormone (GH) on the insulin signal transduction pathway leading to the translocation of glucose transporter-4 (GLUT4), we constructed Chinese hamster ovary cells that overexpressed GH receptor and GLUT4. Treatment with GH triggered GLUT4 translocation, and this translocation was completely inhibited by wortmannin. GH-induced GLUT4 translocation reached a maximum level after 30 min, and then gradually decreased and returned to the basal level after 2 h. Tyrosine phosphorylation of JAK2 also became maximal after 30 min and then gradually decreased. In contrast, GLUT4 translocation remained unchanged for 2 h after insulin treatment, and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) also remained constant for up to 2 h. Chronic GH treatment had almost no effect on insulin-stimulated Akt kinase activation and GLUT4 translocation. These results suggest that GH and insulin translocate GLUT4 in a similar manner, at least in part, and the difference in translocation depends on the difference in the tyrosine phosphorylation of JAK2 and IRS-1. The anti-insulin action of GH after chronic GH treatment does not appear to be mainly due to the inhibition of GLUT4 translocation.

Androstadienes↗
PubMed2002

Expression of insulin-responsive glucose transporter GLUT4 mRNA in the rat brain and spinal cord: an in situ hybridization study.

Following a previous immunocytochemical study of GLUT4 in the rat brain and spinal cord (J. Comp. Neurol. 399 (1998) 492), we now report the distribution and cellular expression of GLUT4 mRNA in the CNS using reverse transcription-polymerase chain reaction and non-radioactive in situ hybridization (ISH). The former technique demonstrated the expression of GLUT4 in the different regions examined while ISH with a specific riboprobe allowed the anatomical localization of GLUT4 mRNA. A strong hybridization signal was detected in the piriform and entorhinal cortices and in the pyramidal cell layer of the hippocampal CA1-CA3 areas. Numerous moderately labeled cells were additionally observed in the dentate gyrus granular layer, subiculum and most neocortical areas, as well as in different nuclei of the limbic and motor systems. In contrast, positive cell groups were scarce in the hypothalamus. In the hindbrain, a strong expression of GLUT4 mRNA was observed in the large cell bodies of the red nucleus and cerebellar Purkinje cell layer. Moreover, different groups of moderately labeled cells were found in the deep cerebellar and medullary motor nuclei, in various reticular fields and in the ventral horn of the spinal cord. The present results of ISH mostly agree with the immunocytochemical data reported by our group, although the immunoreactive cells were generally less numerous. However, the fact that a high expression of GLUT4 mRNA was observed in cell bodies of the piriform lobe, hippocampus and substantia nigra, whereas the immunoreactivity for GLUT4 was low in these regions, suggests the existence of post-transcriptional regulation of GLUT4 expression which may depend on the physiological conditions of the animals.

Animals↗
PubMed2006

Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC).

The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein-protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulin-dependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas components of the ubiquitin-proteasome degradation system were generally reduced. Proteins participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related proteins, alpha-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 +/- 0.5-fold by SILAC and 2.9 +/- 0.8-fold by immunoblotting. Insulin caused GLUT4 and alpha-actinin-4 co-localization as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably alpha-actinin-4, associate with the transporter, potentially to facilitate its routing to the plasma membrane.

Actinin↗
PubMed1997

GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes.

GLUT4, the insulin-responsive glucose transporter, plays an important role in postprandial glucose disposal. Altered GLUT4 activity is suggested to be one of the factors responsible for decreased glucose uptake in muscle and adipose tissue in obesity and diabetes. To assess the effect of GLUT4 expression on whole-body glucose homeostasis, we disrupted the murine GLUT4 gene by homologous recombination. Male mice heterozygous for the mutation (GLUT4 +/-) exhibited a decrease in GLUT4 expression in adipose tissue and skeletal muscle. This decrease in GLUT4 expression did not result in obesity but led to increased serum glucose and insulin, reduced muscle glucose uptake, hypertension, and diabetic histopathologies in the heart and liver similar to those of humans with non-insulin-dependent diabetes mellitus (NIDDM). The male GLUT4 +/- mice represent a good model for studying the development of NIDDM without the complications associated with obesity.

Animals↗
PubMed2001

GLUT4 vesicle trafficking in rat adipocytes after ethanol feeding: regulation by heterotrimeric G-proteins.

Long-term ethanol consumption decreases insulin-stimulated glucose uptake in isolated rat adipocytes. Here we investigate the mechanisms for this decrease. Male Wistar rats were fed for 4 weeks with a liquid diet containing 35% of the calories from ethanol and compared with pair-fed controls. Stimulation of 3-O-methylglucose transport in isolated adipocytes by insulin was decreased by 70% after ethanol feeding. However, stimulation by insulin of the tyrosine phosphorylation of the p85 subunit of phosphoinositide 3-kinase and the phosphorylation of Akt were not affected by ethanol feeding. GLUT4 was mobilized from intracellular light microsomes in response to insulin in both pair-fed and ethanol-fed rats, resulting in 4.3-fold and 3.3-fold increases in GLUT4 associated with plasma membrane in pair-fed and ethanol-fed rats respectively. Surface-accessible GLUT4, assessed by a trypsin cleavage assay or cell-surface labelling with bis-mannose photolabel, was increased 2.3-fold and 1.6-fold respectively, in pair-fed rats after treatment with insulin. In contrast, insulin did not increase surface-accessible GLUT4 in ethanol-fed rats. Treatment of adipocytes with R-phenylisopropyladenosine, an adenosine A1 receptor agonist, increased the transport of 3-O-methylglucose and trypsin-accessible GLUT4, in adipocytes from both pair-fed and ethanol-fed rats. These results demonstrate that whereas the insulin-mediated signalling and translocation of GLUT4 to the plasma membrane is maintained after ethanol feeding, the final fusion of GLUT4 vesicles to the plasma membrane is disrupted, preventing the stimulation of glucose uptake by insulin. Fusion of GLUT4 with the plasma membrane can be stimulated by the activation of adenosine A1 receptors.

3-O-Methylglucose↗
PubMed1996

Lipid mediators of insulin resistance: ceramide signalling down-regulates GLUT4 gene transcription in 3T3-L1 adipocytes.

We have previously demonstrated that chronic exposure of 3T3-L1 adipocytes to tumour necrosis factor-alpha (TNF) resulted in a marked decrease (approximately 90%) in cellular GLUT4 (insulin-responsive glucose transporter) mRNA content as a result of a decreased transcription rate of the GLUT4 gene (approximately 75%) and a reduced half-life of its mRNA (9 to 4.5 h). Investigation of the signalling mechanism responsible for this regulation demonstrated that in the 3T3-L1 adipocytes, sphingomyelin levels decreased to 50% of control levels within 40 min of exposure to TNF, consistent with activation of a sphingomyelinase. In the same manner as with TNF, treatment of the adipocytes with 1-3 microM C6-ceramide, a membrane-permeable analogue of ceramide, decreased GLUT4 mRNA content by approximately 60%. Subsequent investigations revealed that transcription of the GLUT4 gene was reduced by approximately 65% in response to C6-ceramide, demonstrating that the decrease in mRNA content is mediated by a reduction in the transcription of the genc. No effect on GLUT4 mRNA stability was observed after exposure of the adipocytes to C6-ceramide. These observations are interesting in light of our previous data demonstrating that TNF affects both GLUT4 transcription and mRNA stability in the 3T3-L1 adipocytes. In conclusion, the effect of ceramide on GLUT4 gene expression is at the level of transcription, suggesting that another pathway controls mRNA stability. These data establish that ceramide-initiated signal transduction pathways exist within the adipocyte, and provide a potential mechanism for control of GLUT4 gene expression.

3T3 Cells↗
PubMed2006

Muscle GLUT4 regulation by estrogen receptors ERbeta and ERalpha.

Estrogen is known to influence glucose homeostasis with dominant effects in the liver, but the role of estrogen receptors in muscle glucose metabolism is unknown. In the present study, we investigated the expression of the two estrogen receptors, ERalpha and ERbeta, and their influence on regulation of the glucose transporter, GLUT4, and its associated structural protein, caveolin-1, in mouse gastrocnemius muscle. Immunohistochemical analysis revealed that ERalpha and ERbeta are coexpressed in the nuclei of most muscle cells, and that their levels were not affected by absence of estradiol [in aromatase-knockout (ArKO) mice]. GLUT4 expression on the muscle cell membrane was not affected by loss of ERbeta but was extremely reduced in ERalpha(-/-) mice and elevated in ArKO mice. RT-PCR confirmed a parallel reduction in GLUT4 mRNA levels in ERalpha(-/-) mice. Upon treatment of ArKO mice with the ERbeta agonist 2,3-bis(4-hydroxyphenyl)propionitrile, GLUT4 expression was reduced. By immunofluorescence and Western blotting, caveolin-1 expression was higher in ArKO mice and lower in ERbeta(-/-) and ERalpha(-/-) mice than in WT littermates. GLUT4 and caveolin-1 were colocalized in WT and ArKO mice but not in ERbeta(-/-) and ERalpha(-/-) mice. These results reveal that ERalpha is a positive regulator of GLUT4 expression, whereas ERbeta has a suppressive role. Both ERbeta and ERalpha are necessary for optimal caveolin-1 expression. Taken together, these results indicate that colocalization of caveolin-1 and GLUT4 is not an absolute requirement for muscle glucose metabolism but that reduction in GLUT4 could be contributing to the insulin resistance observed in ERalpha(-/-) mice.

Animals↗
PubMed2001

Insulin can regulate GLUT4 internalization by signaling to Rab5 and the motor protein dynein.

Insulin stimulates glucose transport by promoting translocation of the insulin-sensitive glucose transporter isoform 4 (GLUT4) from an intracellular compartment to the cell surface. This movement is accomplished by stimulation of GLUT4 exocytosis as well as inhibition of endocytosis. However, the molecular mechanisms for these effects remain unclear. In this study, we found that the GTP-binding protein Rab5 physically associated with the motor protein dynein in immunoprecipitants from both untransfected cells and cells transfected with GFP-Rab5 constructs. Microinjection of anti-Rab5 or anti-dynein antibody into 3T3-L1 adipocytes increased the basal level of surface GLUT4, did not change the insulin-stimulated surface GLUT4 level, and inhibited GLUT4 internalization after the removal of insulin. Photoaffinity labeling of Rab5 with [gamma-(32)P]GTP-azidoanilide showed that insulin inhibited Rab5-GTP loading. By using microtubule-capture assays, we found that insulin also caused a significant decrease in the binding of dynein to microtubules. Furthermore, pretreatment of cells with the PI3-kinase inhibitor LY294002 inhibited the effects of insulin on both Rab5-GTP loading and dynein binding to microtubules. In conclusion, these data indicate that insulin signaling inhibits Rab5 activity and the interaction of dynein with microtubules in a PI3-kinase-dependent manner, and that these effects may inhibit the rate of GLUT4 internalization. As such, our results present a previously uncharacterized insulin-signaling pathway involving Rab5, the motor protein dynein, and the cytoskeleton to regulate directional GLUT4 movement, facilitating GLUT4 distribution to the cell surface.

3T3 Cells↗
PubMed1991

Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP.

Glucose uptake by adipose tissue is mediated by two glucose transporters: GLUT4, which is most abundant, and GLUT1. While GLUT1 is expressed in many tissues, GLUT4 is unique to tissues that exhibit insulin-stimulated glucose uptake (heart and skeletal muscle and adipose tissue). In the diabetic state and during starvation, insulin-stimulated glucose uptake and GLUT4 expression are decreased in tissue adipocytes. Using 3T3-L1 adipocytes in culture, we investigated the possibility that these effects are mediated by elevated cellular cAMP. When 3T3-L1 adipocytes were treated for 16 hr with forskolin or 8-Br-cAMP, GLUT4 mRNA and protein were decreased by approximately 70%, while expression of GLUT1 mRNA and protein was increased 3-fold. These changes were accompanied by an increased basal rate of 2-deoxyglucose uptake and a loss of acute responsiveness of hexose uptake to insulin. The magnitude of GLUT4 mRNA depletion/GLUT1 mRNA accumulation was dependent upon the concentration of 8-Br-cAMP. The decrease of GLUT4 mRNA caused by 8-Br-cAMP was the result of a decreased transcription rate, while the half-life of the message was unaffected. The increase in GLUT1 mRNA caused by 8-Br-cAMP was the result of both transient transcriptional activation and mRNA stabilization. We suggest that down-regulation of GLUT4 mRNA in adipose tissue in the diabetic state and during starvation is the result of repression of transcription of the GLUT4 gene caused by cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Gq-coupled receptors transmit the signal for GLUT4 translocation via an insulin-independent pathway.

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces the translocation of glucose transporter type 4 (GLUT4) from an intracellular pool to the cell surface and increases glucose uptake in adipocytes. The GTP-binding protein(s) responsible for the translocation has remained to be identified. Using a sensitive and quantitative method to assess the translocation of c-MYC epitope-tagged GLUT4, we obtained evidence that the activation of receptor-coupled Gq (neither Gi nor Gs) triggered GLUT4 translocation in cells, independently of insulin signaling pathway(s). Platelet-activating factor (PAF) induced GLUT4 translocation in the cells expressing the Gi- and Gq-coupled PAF receptor, but the translocation was induced even after pretreatment with wortmannin, an islet-activating protein and phorbol 12, 13-dibutyrate. Norepinephrine triggered GLUT4 translocation in cells expressing the Gq-coupled alpha1-adrenergic receptor, but prostaglandin E2 did not cause GLUT4 translocation in cells expressing the Gs-coupled EP4 receptor or the Gi-coupled EP3alpha receptor. The norepinephrine-stimulated GLUT4 translocation and glucose uptake via Gq may possibly contribute to the fuel supply required for thermogenesis in brown adipocytes and for the enhanced contractility in cardiomyocytes, both of which have an abundant endogenous GLUT4.

3T3 Cells↗
PubMed1998

Association of acyl-CoA synthetase-1 with GLUT4-containing vesicles.

GLUT4, the glucose transporter present in insulin-sensitive tissues, resides in intracellular vesicular structures and translocates to the cell surface in response to insulin. In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from rat adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 antibody. We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein band, long chain acyl-CoA synthetase-1, specifically present in immunoadsorbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimmune serum. Acyl-CoA synthetase activity detected in GLUT4-enriched vesicles prepared by gradient centrifugation from insulin-treated adipocytes was decreased to about the same extent as GLUT4 protein. Additionally, immunoadsorbed GLUT4 vesicles were found to catalyze palmitoylation of proteins when incubated with labeled palmitate, a pathway that requires palmitate esterification with CoA. These data indicate that the insulin-sensitive membrane compartment that sequesters GLUT4 in fat cells contains long chain acyl-CoA synthetase-1 and its product fatty acyl-CoA, shown previously to be required for budding and fusion in membrane trafficking processes.

Acyl Coenzyme A↗
PubMed1999

Aldolase mediates the association of F-actin with the insulin-responsive glucose transporter GLUT4.

To identify potential proteins interacting with the insulin-responsive glucose transporter (GLUT4), we generated fusion proteins of glutathione S-transferase (GST) and the final 30 amino acids from GLUT4 (GST-G4) or GLUT1 (GST-G1). Incubation of these carboxyl-terminal fusion proteins with adipocyte cell extracts revealed a specific interaction of GLUT4 with fructose 1, 6-bisphosphate aldolase. In the presence of aldolase, GST-G4 but not GST-G1 was able to co-pellet with filamentous (F)-actin. This interaction was prevented by incubation with the aldolase substrates, fructose 1,6-bisphosphate or glyceraldehyde 3-phosphate. Immunofluorescence confocal microscopy demonstrated a significant co-localization of aldolase and GLUT4 in intact 3T3L1 adipocytes, which decreased following insulin stimulation. Introduction into permeabilized 3T3L1 adipocytes of fructose 1,6-bisphosphate or the metabolic inhibitor 2-deoxyglucose, two agents that disrupt the interaction between aldolase and actin, inhibited insulin-stimulated GLUT4 exocytosis without affecting GLUT4 endocytosis. Furthermore, microinjection of an aldolase-specific antibody also inhibited insulin-stimulated GLUT4 translocation. These data suggest that aldolase functions as a scaffolding protein for GLUT4 and that glucose metabolism may provide a negative feedback signal for the regulation of glucose transport by insulin.

3T3 Cells↗
PubMed1999

Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin.

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble N-ethyl-maleimide-sensitive factor attachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.

3T3 Cells↗
PubMed2000

Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes.

Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.

Adipocytes↗
PubMed2000

Cellugyrin is a marker for a distinct population of intracellular Glut4-containing vesicles.

Although Glut4 traffic is routinely described as translocation from an "intracellular storage pool" to the plasma membrane, it has been long realized that Glut4 travels through at least two functionally distinct intracellular membrane compartments on the way to and from the cell surface. Biochemical separation and systematic studies of the individual Glut4-containing compartments have been limited by the lack of appropriate reagents. We have prepared a monoclonal antibody against a novel component protein of Glut4 vesicles and have identified this protein as cellugyrin, a ubiquitously expressed homologue of a major synaptic vesicle protein, synaptogyrin. By means of sucrose gradient centrifugation, immunoadsorption, and confocal microscopy, we have shown that virtually all cellugyrin is co-localized with Glut4 in the same vesicles. However, unlike Glut4, cellugyrin is not re-distributed to the plasma membrane in response to insulin stimulation, and at least 40-50% of the total population of Glut4 vesicles do not contain this protein. We suggest that cellugyrin represents a specific marker of a functionally distinct population of Glut4 vesicles that permanently maintains its intracellular localization and is not recruited to the plasma membrane by insulin.

Adipocytes↗
PubMed2002

Insulin and isoproterenol have opposing roles in the maintenance of cytosol pH and optimal fusion of GLUT4 vesicles with the plasma membrane.

Insulin treatment of rat adipocytes increases both cytoplasmic alkalinity and glucose transport activity. Both processes are blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. Isoproterenol pre-treatment reverses the alkalinizing effects of insulin and leads to attenuation of insulin-stimulated glucose transport activity and exposure of GLUT4 to photolabeling reagents at the cell surface. These effects of isoproterenol are mimicked by acid loading and are reversed by cell-alkalinizing conditions. However, neither isoproterenol nor acid loading alters the total level of GLUT4 at the plasma membrane as revealed by Western blotting of plasma membrane fractions or immunodetection of GLUT4 in plasma membrane lawns. GLUT4 is therefore occluded from participation in glucose transport catalysis by a pH-sensitive process. To examine the kinetics of trafficking that lead to these changes in cell surface GLUT4 occlusion, we have utilized a new biotinylated photolabel, GP15. This reagent has a 70-atom spacer between the biotin and the photolabeling diazirine group, and this allows quenching of the surface signal of biotinylated GLUT4 by extracellular avidin. The rates of GLUT4 internalization are only slightly altered by isoproterenol or acidification, mainly due to reduced recycling over long internalization times. By contrast, insulin stimulation of GLUT4 exocytosis is slowed by isoproterenol or acidification pre-treatments. Biphasic time courses are evident, with an initial burst of exposure at the cell surface followed by a slow phase. It is hypothesized that the burst kinetics are a consequence of a two-phase fusion reaction that is rapid in the presence of insulin but slowed by cytosol acidification.

Adipocytes↗
PubMed2001

Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport.

Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myc-tagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC(50) of 3 nm. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC(50) = 43 nm). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC(50) = 8 and 10 microm, respectively). The sensitivity of insulin-stimulated activation of PKC zeta/lambda, Akt1, Akt2, and Akt3 to wortmannin (IC(50) = 24, 30, 35, and 60 nm, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulin-dependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC(50) of 7 nm. Insulin-dependent p38 alpha and p38 beta MAPK activities were also markedly reduced by wortmannin (IC(50) = 6 and 2 nm, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (Delta p85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKC zeta/lambda, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.

Androstadienes↗