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PubMed2001

Identification of discrete classes of endosome-derived small vesicles as a major cellular pool for recycling membrane proteins.

Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5'-[gamma-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface.

Aminopeptidases↗
PubMed1996

Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.

3T3 Cells↗
PubMed2003

Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation.

Phosphatidylinositol-3-phosphate (PtdIns-3-P) is considered as a lipid constitutively present on endosomes; it does not seem to have a dynamic role in signalling. In contrast, phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) plays a crucial role in different signalling pathways including translocation of the glucose transporter protein GLUT4 to the plasma membrane upon insulin receptor activation. GLUT4 translocation requires activation of two distinct pathways involving phosphatidylinositol 3-kinase (PI 3-K) and the small GTP-binding protein TC10, respectively. The contribution of each pathway remains to be elucidated. Here we show that insulin specifically induces the formation of PtdIns-3-P in insulin- responsive cells. The insulin-mediated formation of PtdIns-3-P occurs through the activation of TC10 at the lipid rafts subdomain of the plasma membrane. Exogenous PtdIns-3-P induces the plasma membrane translocation of both overexpressed and endogenous GLUT4. These data indicate that PtdIns-3-P is specifically produced downstream from insulin-mediated activation of TC10 to promote the plasma membrane translocation of GLUT4. These results give a new insight into the intracellular role of PtdIns-3-P and shed light on some aspects of insulin signalling so far not completely understood.

3T3 Cells↗
PubMed2004

Glucose transporter isoform 4 is expressed in the syncytiotrophoblast of first trimester human placenta.

BACKGROUND: Placental glucose transport mechanisms in early pregnancy are poorly understood. The aims of this study were to investigate the expression of glucose transporter (GLUT) isoforms 1, 3 and 4 in first trimester villous tissue, to assess the effects of insulin on glucose uptake and compare them with term. METHODS: The expression of GLUT isoforms was investigated using immunohistochemistry, Western blot and reverse transcription (RT)-PCR in trophoblast tissue from terminations at 6-13 weeks gestation and term. The effects of insulin (300 ng/ml, 1 h) on glucose uptake were studied in villous fragments. RESULTS: In the first trimester, GLUT1 and GLUT3 were present in the microvillous membrane and the cytotrophoblast, and GLUT4 in perinuclear membranes in the cytosol of the syncytiotrophoblast (ST). GLUT4 protein (48 kDa) and mRNA were identified in trophoblast homogenates. Whereas GLUT1 was expressed abundantly in term placenta, the expression of GLUT3 and 4 was markedly lower at term compared with first trimester. Insulin increased glucose uptake by 182% (n=6, P<0.05) in first trimester fragments, but not in term fragments. CONCLUSIONS: The insulin-regulatable GLUT4 is expressed in the cytosol of first trimester ST compatible with a role for GLUT4 in placental glucose transport in early pregnancy. The placental expression pattern of GLUT isoforms in early pregnancy is distinct from that later in pregnancy.

Blotting, Western↗
PubMed1994

Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures.

Glucose transporters are membrane-embedded proteins that mediate the uptake of glucose from the surrounding medium into the cell. Glucose is the main fuel for most cells, and its uptake is rate-limiting for glucose utilization. For this reason, it is expected that glucose transport is tightly regulated. Whereas rapid regulation of glucose transporters by hormones has been known for some time, the regulation of glucose transporters by substrate availability (i.e., by glucose itself) is less well understood. This question has been approached by scientists from two angles: one, by measuring the consequence of diabetic states (in which there is surplus of glucose availability) on the expression of glucose transporter genes, and another one, by measuring the effect of glucose availability and glucose deprivation in cell cultures on glucose transporter gene expression. The results from both camps are unfortunately not coincident, due in part to the coexistence of other variables in the diabetic animals, and to the lack of ideal cell cultures. In spite of these caveats, the profuse literature on both approaches propelled us to find commonalities within each approach. This review concludes that in animal studies, one isoform of glucose transporters, the GLUT4 type, is down-regulated by high levels of circulating glucose in muscle but not in fat cells. This down-regulation of the protein is independent of regulation of transcription. In contrast, in fat cells, high glucose levels depress GLUT4 mRNA levels. In cell culture studies, high glucose levels lead to lower expression of the GLUT1 transporter isoform relative to glucose-deprived cultures. Glucose levels do not affect the amount of GLUT4 transporter isoform. The down-regulation of the GLUT1 transporter protein is caused by pre- and post-transcriptional mechanisms, the prevalence of each being cell-type specific. No glucose-responsive elements have been identified on either the GLUT1 or GLUT4 genes, and no information is available on the glucose metabolites that mediate the response of glucose transporter gene expression to glucose availability.

Animals↗
PubMed2002

Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1.

Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria.

Adaptation, Physiological↗
PubMed2006

Insulin-regulated aminopeptidase marks an antigen-stimulated recycling compartment in mast cells.

Insulin-regulated aminopeptidase (IRAP) is a marker for insulin-sensitive recycling compartments of fat and muscle cells that contain the glucose transporter isoform GLUT4. Unlike GLUT4, IRAP is expressed in many other cell types. Thus, it is a potential marker for regulated recycling compartments that are analogous to GLUT4 vesicles. In bone marrow-derived mast cells, IRAP is highly expressed and localizes to an intracellular compartment different from secretory granules. Using cell-surface biotinylation, we determined that IRAP underwent rapid redistribution to the plasma membrane on antigen/immunoglobulin E (IgE) stimulation and was re-internalized within 30 min. When granule exocytosis was inhibited, by removing extracellular calcium, adding the protein kinase C inhibitor bisindolylmaleimide or the phosphatidylinositol 3-kinase inhibitor wortmannin, IRAP redistribution was still detected in stimulated cells. However, the redistribution of IRAP required intracellular calcium. By immunofluorescence, IRAP significantly co-localized with the transferrin receptor (TfR), a marker for constitutively recycling endosomes. However, antigen/IgE stimulation did not increase TfR on the cell surface, indicating that IRAP and TfR may follow different pathways to the plasma membrane. In rat peritoneal mast cells, the distributions of IRAP and TfR overlapped to only a limited extent, indicating that overlap may decrease with cell differentiation. We propose that IRAP vesicles represent a second IgE-sensitive exocytotic compartment in mast cells, which is regulated differently from secretory granules, and that these vesicles may be similar to GLUT4 vesicles.

Aminopeptidases↗
PubMed2004

Creatine supplementation increases glucose oxidation and AMPK phosphorylation and reduces lactate production in L6 rat skeletal muscle cells.

Recent observations have suggested that creatine supplementation might have a beneficial effect on glucoregulation in skeletal muscle. However, conclusive studies on the direct effects of creatine on glucose uptake and metabolism are lacking. The objective of this study was to investigate the effects of creatine supplementation on basal and insulin-stimulated glucose transporter (GLUT4) translocation, glucose uptake, glycogen content, glycogen synthesis, lactate production, glucose oxidation and AMP-activated protein kinase (AMPK) phosphorylation in L6 rat skeletal muscle cells. Four treatment groups were studied: control, insulin (100 nM), creatine (0.5 mM) and creatine + insulin. After 48 h of creatine supplementation the creatine and phosphocreatine contents of L6 myoblasts increased by approximately 9.3- and approximately 5.1-fold, respectively, but the ATP content of the cells was not affected. Insulin significantly increased 2-deoxyglucose uptake ( approximately 1.9-fold), GLUT4 translocation ( approximately 1.8-fold), the incorporation of D-[U-(14)C]glucose into glycogen ( approximately 2.3-fold), lactate production ( approximately 1.5-fold) and (14)CO(2) production ( approximately 1.5-fold). Creatine neither altered the glycogen and GLUT4 contents of the cells nor the insulin-stimulated rates of 2-DG uptake, GLUT4 translocation, glycogen synthesis and glucose oxidation. However, creatine significantly reduced by approximately 42% the basal rate of lactate production and increased by approximately 40% the basal rate of (14)CO(2) production. This is in agreement with the approximately 35% increase in citrate synthase activity and also with the approximately 2-fold increase in the phosphorylation of both alpha-1 and alpha-2 isoforms of AMPK after creatine supplementation. We conclude that 48 h of creatine supplementation does not alter insulin-stimulated glucose uptake and glucose metabolism; however, it activates AMPK, shifts basal glucose metabolism towards oxidation and reduces lactate production in L6 rat skeletal muscle cells.

Adenosine Triphosphate↗
PubMed2003

Intracellular segregation of phosphatidylinositol-3,4,5-trisphosphate by insulin-dependent actin remodeling in L6 skeletal muscle cells.

Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular pool to the cell surface through a mechanism that is dependent on phosphatidylinositol (PI) 3-kinase (PI3-K) and cortical actin remodeling. Here we test the hypothesis that insulin-dependent actin filament remodeling determines the location of insulin signaling molecules. It has been shown previously that insulin treatment of L6 myotubes leads to a rapid rearrangement of actin filaments into submembrane structures where the p85 regulatory subunit of PI3-K and organelles containing GLUT4, VAMP2, and the insulin-regulated aminopeptidase (IRAP) colocalize. We now report that insulin receptor substrate-1 and the p110alpha catalytic subunit of PI3-K (but not p110beta) also colocalize with the actin structures. Akt-1 was also found in the remodeled actin structures, unlike another PI3-K effector, atypical protein kinase C lambda. Transiently transfected green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of general receptor for phosphoinositides-1 (GRP1) or Akt (ligands of phosphatidylinositol-3,4,5-trisphosphate [PI-3,4,5-P(3)]) migrated to the periphery of the live cells; in fixed cells, they were detected in the insulin-induced actin structures. These results suggest that PI-3,4,5-P(3) is generated on membranes located within the actin mesh. Actin remodeling and GLUT4 externalization were blocked in cells highly expressing GFP-PH-GRP1, suggesting that PI-3,4,5-P(3) is required for both phenomena. We propose that PI-3,4,5-P(3) leads to actin remodeling, which in turn segregates p85alpha and p110alpha, thus localizing PI-3,4,5-P(3) production on membranes trapped by the actin mesh. Insulin-stimulated actin remodeling may spatially coordinate the localized generation of PI-3,4,5-P(3) and recruitment of Akt, ultimately leading to GLUT4 insertion at the plasma membrane.

Actins↗
PubMed1995

Molecular actions of insulin on glucose transport.

Low basal glucose uptake by insulin-sensitive muscle and adipose cells reflects rapid endocytic retrieval of GLUT4 glucose transporters from the cell surface and their retention in intracellular membranes. Both GLUT4 endocytosis and its intracellular retention are governed by a dileucine motif in its COOH-terminal region. Acute stimulation of sugar uptake by insulin results from GLUT4 redistribution to the plasma membrane and may reflect disruption of dileucine motif function as well as enhanced bulk membrane exocytosis. Candidate signaling elements for these postulated actions of insulin are PI 3-kinase and p21ras, both acutely activated by the hormone. Recent work in our laboratory and others demonstrates the localization of PI 3-kinase to intracellular membranes via its docking to the insulin receptor substrate-1 (IRS-1). An important hypothesis for future testing is that 3' phosphoinositides generated in the endosomal tubulovesicular system in response to insulin cause budding or movements of GLUT4-containing membranes to the cell surface.

Animals↗
PubMed2002

Insulin-stimulated cytosol alkalinization facilitates optimal activation of glucose transport in cardiomyocytes.

Abnormalities in intracellular pH regulation have been proposed to be important in type 2 diabetes and the associated cardiomyopathy and hypertension. We have therefore investigated the dependence of insulin-stimulated glucose transport on cytosolic pH in cardiomyocytes. Insulin treatment of cardiomyocytes resulted in a marked alkalinization of the cytoplasm as measured using carboxy-semi-napthorhodofluor-1. The alkalinizing effect of insulin was blocked by treatment with either cariporide (which inhibits the Na+/H+ exchanger) or by bafilomycin A1 (which inhibits H+-ATPase activity). After treatments with cariporide or bafilomycin A1, insulin stimulation of insulin receptor and insulin receptor substrate-1 phosphorylation and Akt activity were normal. In contrast, glucose transport activity and the levels of functional GLUT4 at the plasma membrane (detected using an exofacial photolabel) were reduced by approximately 50%. Immunocytochemical analysis revealed that insulin treatment caused a translocation of the GLUT4 from perinuclear structures and increased its co-localization with cell surface syntaxin 4. However, neither cariporide nor bafilomycin A1 treatment reduced the translocation of immunodetectable GLUT4 to the sarcolemma region of the cell. It is therefore hypothesized that insulin-stimulated cytosol alkalinization facilitates the final stages of translocation and incorporation of fully functional GLUT4 at the surface-limiting membrane.

Animals↗
PubMed2005

Effects of chronic Akt activation on glucose uptake in the heart.

Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake (P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo (P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin.

Animals↗
PubMed1999

Contribution of alpha-adrenergic and beta-adrenergic stimulation to ischemia-induced glucose transporter (GLUT) 4 and GLUT1 translocation in the isolated perfused rat heart.

The intracellular signaling mechanism of the ischemia-stimulated glucose transporter (GLUT) translocation in the heart is not yet characterized. It has been suggested that catecholamines released during ischemia may be involved in this pathway. The purpose of this study was to evaluate the contribution of alpha-adrenoceptors and beta-adrenoceptors to ischemia-mediated GLUT4 and GLUT1 translocation in the isolated, Langendorff-perfused rat heart. Additionally, GLUT translocation was studied in response to catecholamine stimulation with phenylephrine (Phy) and isoproterenol (Iso). The results were compared with myocardial uptake of glucose analogue [18F]fluorodeoxyglucose (FDG). Subcellular analysis of GLUT4 and GLUT1 protein on plasma membrane vesicles (PM) and intracellular membrane vesicles (IM) using membrane preparation and immunoblotting revealed that alpha- and beta-receptor agonists stimulated GLUT4 translocation from IM to PM (2.5-fold for Phy and 2.1-fold for Iso, P<0.05 versus control), which was completely inhibited by phentolamine (Phe) and propranolol (Pro), respectively. Plasmalemmal GLUT1 moderately rose after Iso exposure, and this was prevented by Pro. In contrast, ischemia-stimulated GLUT4 translocation (2.2-fold, P<0.05 versus control) was only inhibited by alpha-adrenergic antagonist Phe but not by beta-adrenergic antagonist Pro. Similarly, Phe but not Pro inhibited ischemia-stimulated GLUT1 translocation. GLUT data were confirmed by FDG uptake monitored using bismuth germanate detectors. The catecholamine-stimulated FDG uptake (6.9-fold for Phy and 8.9-fold for Iso) was significantly inhibited by Phe and Pro; however, only Phe but not Pro significantly reduced the ischemia-induced 2.5-fold increase in FDG uptake (P<0.05 versus ischemia). This study suggests that alpha-adrenoceptor stimulation may play a role in the ischemia-mediated increase in glucose transporter trafficking leading to the stimulation of FDG uptake in the isolated, perfused rat heart, whereas beta-adrenergic activation does not participate in this signaling pathway.

Adrenergic alpha-Agonists↗
PubMedDate not supplied

Insulin resistance and the alterations of glucose transporter-4 in adipose cells from cachectic tumor-bearing rats.

BACKGROUND: Insulin resistance may play an important role in cancer cachexia; however, its mechanisms remain to be clarified. METHODS: Cellular mechanisms of insulin resistance in tumor-bearing rats (TBR) were investigated in isolated adipose cells by measuring 3-O-[14C]methyl glucose transport activity and glucose transporter-4 (GLUT4) protein in low-density microsomes at a basal state and in the plasma membrane at an insulin-stimulated state. RESULTS: The insulin-stimulated glucose transport activity in adipose cells from TBR was significantly lower than that of control rats (CTR) (0.51 +/- 0.25 and 2.27 +/- 0.11 fmol/cell/min, respectively). The amount of GLUT4 in low-density microsomes at a basal state and in plasma membrane at an insulin-stimulated state was less in TBR than in CTR. CONCLUSIONS: These data suggest that the insulin resistance seen in the adipose cells of these tumor-bearing rats was caused in part by both a decreased amount of GLUT4 protein in a basal state and a decreased translocation of GLUT4 in response to insulin stimulation.

Adipocytes↗
PubMed2005

Endofacial competitive inhibition of glucose transporter-4 intrinsic activity by the mitogen-activated protein kinase inhibitor SB203580.

The translocation of glucose transporter-4 (GLUT4) to the cell surface is a complex multistep process that involves movement of GLUT4 vesicles from a reservoir compartment, and docking and fusion of the vesicles with the plasma membrane. It has recently been proposed that a p38 mitogen-activated protein kinase (MAPK)-dependent step may lead to intrinsic activation of the transporters exposed at the cell surface. In contrast to data obtained in muscle and adipocyte cell lines, we found that no insulin activation of p38 MAPK occurred in rat adipose cells. However, the p38 MAPK inhibitor SB203580 consistently inhibited transport activity after preincubation with the adipose cells. These apparently contradictory findings led us to hypothesize that the inhibitor may have a direct effect on the transport catalytic activity of GLUT4 that was independent of inhibition of the kinase. Kinetic analysis of 3-O-methyl-d-glucose transport activity revealed that SB203580 was a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but was a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport. This pattern of inhibition of GLUT4 was also observed with cytochalasin B. The pattern of inhibition is consistent with interaction at the endofacial surface, but not the exofacial surface of the transporter. Occupation of the endofacial substrate site reduces maximum velocity under zero-trans conditions, because return of the substrate site to the outside is blocked, and no substrate is present inside to displace the inhibitor. Under equilibrium-exchange conditions, internal substrate competitively displaces the inhibitor, and the transport K(m) is increased.

Adipocytes↗
PubMed1993

The antidiabetic agent pioglitazone increases expression of glucose transporters in 3T3-F442A cells by increasing messenger ribonucleic acid transcript stability.

Whereas adipocytes normally play an important role as a major site for systemic energy homeostasis, adipocyte function is markedly altered in disorders such as diabetes. In this study, we investigated the effect of pioglitazone, a novel antidiabetic agent known to lower plasma glucose in animal models of diabetes mellitus, on expression of glucose transporters GLUT1 and GLUT4 in 3T3-F442A cells. Treatment of confluent 3T3-F442A preadipocyte cultures for 7 days with pioglitazone (1 microM) and insulin (1 microgram/ml) resulted in nearly 100% differentiation of cells to lipid-accumulating adipocytes, and such adipocytes showed a markedly increased capacity for glucose uptake. Analysis of messenger RNA transcripts encoding GLUT1 and GLUT4 glucose transporters over the 7-day differentiation period indicated time-dependent increases in abundance of each type that were maximal at more than 5-fold with the combined presence of insulin and pioglitazone. In accord, GLUT1 and GLUT4 protein levels also increased to maximal levels of 10-fold and 7-fold, respectively, over those in undifferentiated preadipocytes. Increased messenger RNA half-lives from 2.2 to greater than 24 h for GLUT1 and from 1.2 to greater than 24 h for GLUT4 correlated with this induced adipocyte differentiation. Taken together, these findings indicated that pioglitazone markedly enhanced expression of cellular glucose transporters, and the mechanism for this action was mainly stabilization of transporter messenger RNA transcripts. Such increased expression of glucose transporters in adipocytes establishes the cells in a state active for glucose uptake, thus ultimately facilitating storage and metabolism as well.

3T3 Cells↗
PubMed1993

In vivo metformin treatment ameliorates insulin resistance: evidence for potentiation of insulin-induced translocation and increased functional activity of glucose transporters in obese (fa/fa) Zucker rat adipocytes.

To examine the cellular mechanism of the antihyperglycemic action of in vivo metformin (M) we used an animal model of severe insulin resistance, the genetically obese (fa/fa) Zucker rat. The animals were treated with or without M (250 mg/kg.day) which was supplied with the drinking water. Three weeks of in vivo M-treatment had no effect on body weight and several blood lipid parameters, but markedly reduced plasma insulin levels by 45% (-M: 2932 +/- 166 vs. +M: 1614 +/- 85 pmol/liter, P < 0.01); plasma glucose was slightly but significantly decreased by 8.3% (-M: 7.2 +/- 0.2 vs. +M: 6.6 +/- 0.16 mmol/liter, P < 0.05). Adipocytes were isolated and incubated with or without insulin. In vivo M-treatment had no effect on basal 3-O-methylglucose uptake. In contrast, in vivo M-treatment increased insulin-stimulated glucose transport by 2.6 +/- 0.6-fold (P < 0.01). Measurement of cell surface insulin receptors revealed no effect of M on neither specific [125I]insulin binding nor on insulin receptor kinase activity. Insulin-mediated translocation of both GLUT1 and GLUT4 glucose transporters was enhanced by in vivo M-treatment, GLUT1 by 26.1%, GLUT4 by 30.5%. To fully account for the M-induced increment of insulin-stimulated glucose transport (2.6-fold), these data suggest that M increased the functional activity of glucose transporters. We conclude that amelioration of insulin resistance in (fa/fa) Zucker rats after 3 weeks of in vivo M-treatment is associated with 1) a marked reduction of in vivo hyperinsulinemia, 2) an increase of insulin-stimulated glucose transport in adipocytes; 3) this increase of insulin-stimulated glucose transport is accompanied with both a potentiation of insulin-induced translocation of GLUT1 and GLUT4 glucose transporters from an intracellular pool to the plasma membrane as well as increased functional activity of plasma membrane glucose transporters. 4) This M-effect seems to be independent of de novo glucose transporter synthesis, since total cellular GLUT1 and GLUT4 glucose transporter number were uneffected by M. 5) These results strongly suggest a direct action of M at the level of glucose transport, since neither tracer insulin binding nor insulin receptor kinase activity were significantly altered by M.

3-O-Methylglucose↗
PubMed1996

Insulin induces a change in Rab5 subcellular localization in adipocytes independently of phosphatidylinositol 3-kinase activation.

We investigated whether Rab5, a small guanosine triphosphatase that regulates early endocytic transport in different cell types is involved in the insulin-regulated endocytic pathways in adipocytes. Rab5 was detected in freshly isolated adipocytes and 3T3-L1 adipocytes, but its expression level was not markedly increased with adipocyte differentiation. After subcellular fractionation of adipocytes incubated in the absence of insulin, Rab5 was found to be abundant in plasma membrane and cytosol, but was also present in high and low density microsomes. This subcellular distribution was compatible with a role in early endocytosis. When cells were incubated with insulin, the concentration of Rab5 decreased by about 50% in the internal compartments. In contrast to Rab4, which also leaves the low density microsomes in response to insulin, Rab5 was not found in Glut4-containing vesicles purified by immunoadsorption on antibodies to Glut4. When adipocytes were treated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, the effect of insulin on Rab5 movement was not affected, whereas the insulin-induced movements of Rab4 and Glut4 were abolished. In parallel, wortmannin inhibited the increase in horseradish peroxidase uptake induced by insulin, an index of fluid phase endocytosis, but did not prevent the endocytosis of the glucose transporters. As a whole, our results suggest that Rab5 is not involved in insulin-stimulated Glut4 exocytosis. These results are compatible with the postulated role of Rab5 in the endocytotic pathway, at a step that does not require phosphatidyl-inositol 3-kinase activation.

3T3 Cells↗