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PubMed1996

Comparison of isoproterenol with BRL37344 in activation of beta 3-adrenoceptors to inhibit the uptake of [14C]deoxy-D-glucose and translocation of glucose transporter (GLUT4) to membrane fraction in rat adipocytes.

In an attempt to understand the role of beta 3-adrenoceptors in the regulation of glucose uptake, the effect of isoproterenol was compared with BRL37344 in isolated white adipocytes of the rat using [14C]deoxy-D-glucose as an indicator. In the presence of BRL37344, the specific agonist of beta 3-adrenoceptors, spontaneous uptake of [14C]deoxy-D-glucose (glucose uptake) into adipocytes was markedly attenuated. Similar concentration-dependent inhibition of glucose uptake was also observed in the samples treated with isoproterenol, an agonist for all kinds of beta-adrenoceptors. Action of BRL37344 was blocked by propranolol at concentrations sufficient to abolish the activity of isoproterenol. Pindolol reversed BRL37344-induced inhibition more effectively than propranolol. Moreover, unlike the action of isoproterenol, BRL37344 did not modify the insulin-stimulated glucose uptake. Translocation of glucose transporter (GLUT4) from cytosol to membrane stimulated with insulin was reduced by isoproterenol but not by BRL37344. Combination with the findings that isoproterenol prolonged the time for insulin to reach maximum stimulation of glucose uptake, leads to the conclusion that delay of insulin action by isoproterenol can be considered as one of the mechanisms for this inhibition. The results obtained suggest that BRL37344 decreased the spontaneous uptake of glucose via an activation of beta 3-adrenoceptors while the insulin stimulated glucose uptake was inhibited by isoproterenol but not by BRL37344.

Adipocytes↗
PubMed1994

Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.

Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.

3T3 Cells↗
PubMed1996

Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells.

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (approximately 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti(-)[insulin receptor substrate 1 (IRS1)] and anti-alpha-p85 antibodies show the same level of insulin-stimulated activity, approximately 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of alpha-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

3T3 Cells↗
PubMed1997

Glucose transport and glucose transporter GLUT4 are regulated by product(s) of intermediary metabolism in cardiomyocytes.

Alternative substrates of energy metabolism are thought to contribute to the impairment of heart and muscle glucose utilization in insulin-resistant states. We have investigated the acute effects of substrates in isolated rat cardiomyocytes. Exposure to lactate, pyruvate, propionate, acetate, palmitate, beta-hydroxybutyrate or alpha-oxoglutarate led to the depression of glucose transport by up to 50%, with lactate, pyruvate and propionate being the most potent agents. The percentage inhibition was greater in cardiomyocytes in which glucose transport was stimulated with the alpha-adrenergic agonist phenylephrine or with a submaximal insulin concentration than in basal or fully insulin-stimulated cells. Cardiomyocytes from fasted or diabetic rats displayed a similar sensitivity to substrates as did cells from control animals. On the other hand, the amination product of pyruvate (alanine), as well as valine and the aminotransferase inhibitors cycloserine and amino-oxyacetate, stimulated glucose transport about 2-fold. In addition, the effect of pyruvate was counteracted by cycloserine. Since reversible transamination reactions are known to affect the pool size of the citrate cycle, the influence of substrates, amino acids and aminotransferase inhibitors on citrate, malate and glutamate content was examined. A significant negative correlation was found between alterations in glucose transport and the levels of citrate (P < 0.01) or malate (P < 0.01), and there was a positive correlation between glucose transport and glutamate levels (P < 0.05). In contrast, there was no correlation with changes in [1-(14)C]pyruvate oxidation or in glucose-6-phosphate levels. Finally, pyruvate decreased the abundance of GLUT4 glucose transporters at the surface of phenylephrine- or insulin-stimulated cells by 34% and 27 % respectively, as determined by using the selective photoaffinity label [3H]ATB-BMPA [[3H]2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-man nos-4-yloxy)propyl-2-amine]. In conclusion, cardiomyocyte glucose transport is subject to counter-regulation by alternative substrates. The glucose transport system appears to be controlled by (a) compound(s) of intermediary metabolism (other than glucose 6-phosphate), but in a different way than pyruvate dehydrogenase. Transport inhibition eventually occurs via a decrease in the amount of glucose transporters in the plasma membrane.

3-Hydroxybutyric Acid↗
PubMed1997

The effect of in vivo thyroxine treatment on insulin receptors, glucose transport and GLUT4 in rat adipocytes.

Daily i.p. administration of thyroxine (T4; 50 micrograms/100 g BW) to adult male rats for one week doubled the thyroxinemia and induced significant augmentation of glycemia and insulinemia. Hyperthyroxinemia was also manifested by significant elevation of liver malic enzyme activity and by the presence of smaller fat cells. Adipocytes of T4 treated rats displayed significantly lower density of insulin receptors, decreased insulin stimulatory effect on glucose transport and less glucose transporter (GLUT4) protein in plasma membranes after insulin stimulation as those of controls. These results indicate a deleterious effect of hyperthyroxinemia on insulin controlled glucose metabolism in fat cells.

Adipocytes↗
PubMed2002

The TC10-interacting protein CIP4/2 is required for insulin-stimulated Glut4 translocation in 3T3L1 adipocytes.

The GTPase TC10 plays a critical role in insulin-stimulated glucose transport. We report here the identification of the TC10-interacting protein CIP4/2 (Cdc42-interacting protein 4/2) as an effector in this pathway. CIP4/2 localizes to an intracellular compartment under basal conditions and translocates to the plasma membrane on insulin stimulation. Overexpression of constitutively active TC10 brings CIP4/2 to the plasma membrane, whereas overexpression of an inhibitory form of TC10 blocks the translocation of CIP4/2 produced by insulin. Overexpression of mutant forms of CIP4/2 containing an N-terminal deletion or with diminished TC10 binding inhibits insulin-stimulated Glut4 translocation. These data suggest that CIP4/2 may play an important role in insulin-stimulated glucose transport as a downstream effector of TC10.

3T3 Cells↗
PubMed2002

An increase in the myocardial PCr/ATP ratio in GLUT4 null mice.

ATP and creatine phosphate (PCr) are prime myocardial high-energy phosphates. Their relative concentrations are conserved among mammalian species and across a range of physiologic cardiac workloads. The cardiac PCr/ATP ratio is decreased with several pathologic conditions, such as ischemia and heart failure, but there are no reports of an increase in the cardiac PCr/ATP ratio in any species or with interventions. We studied the in vivo energetics in transgenic mice lacking expression of the glucose transport protein GLUT4 (G4N) and observed a significant 60% increase in the myocardial PCr/ATP ratio in G4N that was confirmed in three different experimental settings including intact animals. The higher PCr/ATP in G4N is cardiac-specific and is due to higher total cardiac creatine (CR) concentrations in G4N than in wild-type (WT). However, [ATP], [ADP], and -DG(-ATP) did not differ between the strains. Expression of the creatine transport protein (CreaT) that is responsible for creatine uptake in myocytes was preserved in G4N cardiac tissue. These observations demonstrate, for the first time to our knowledge, that G4N manifest a unique increase in the cardiac PCr/ATP ratio, which suggests a novel genetic strategy for increasing myocardial creatine levels.

Adenosine Diphosphate↗
PubMed1998

Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide.

The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C2-ceramide inhibited insulin-stimulated glucose transport by approximately 50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C2-ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C2-ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C2-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C2-ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide's inhibition of Akt. These studies demonstrate ceramide's capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis.

3T3 Cells↗
PubMed2002

APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes.

APS is a Cbl-binding protein that is tyrosine phosphorylated by the insulin receptor kinase. Insulin-stimulated phosphorylation of tyrosine 618 in APS is necessary for its association with c-Cbl and the subsequent tyrosine phosphorylation of Cbl by the insulin receptor in both 3T3-L1 adipocytes and CHO-IR cells. When overexpressed in these cells, wild-type APS but not an APS/Y(618)F mutant facilitated the tyrosine phosphorylation of coexpressed Cbl and its association with Crk upon insulin stimulation. APS-facilitated phosphorylation occurred on tyrosines 371, 700, and 774 in the Cbl protein. APS also interacted directly with the c-Cbl-associated protein (CAP) and colocalized with the protein in cells. The association was dependent on the SH3 domains of CAP and was independent of insulin treatment. Overexpression of the APS/Y(618)F mutant in 3T3-L1 adipocytes blocked the insulin-stimulated tyrosine phosphorylation of endogenous Cbl and binding to Crk. Moreover, the translocation of GLUT4 from intracellular vesicles to the plasma membrane was also inhibited by overexpression of the APS/Y(618)F mutant. These data suggest that APS serves as an adapter protein linking the CAP/Cbl pathway to the insulin receptor and, further, that APS-facilitated Cbl tyrosine phosphorylation catalyzed by the insulin receptor is a crucial event in the stimulation of glucose transport by insulin.

3T3 Cells↗
PubMed2003

SKIP negatively regulates insulin-induced GLUT4 translocation and membrane ruffle formation.

Skeletal muscle and kidney enriched inositol phosphatase (SKIP) is an inositol polyphosphate 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to downregulate intracellular levels. In this study, we show that SKIP inhibits phosphoinositide 3-kinase signaling in insulin-stimulated CHO cells. Ectopic expression of SKIP did not inhibit insulin-induced PI(3,4,5)P3 generation but did rapidly decrease insulin-induced intracellular PI(3,4,5)P3 levels compared with those in control cells. Further, insulin-induced phosphorylation of some downstream targets such as Akt and p70 S6 kinase was markedly inhibited by the ectopic expression of SKIP, whereas phosphorylation of mitogen-activated protein kinase was not. In contrast, downregulation of intracellular SKIP levels by antisense oligonucleotides dramatically enhanced Akt (protein kinase B) phosphorylation in response to insulin, suggesting that endogenous SKIP downregulates insulin signaling. SKIP also markedly inhibited GLUT4 translocation and membrane ruffle formation. We conclude that SKIP preferentially regulates glucose transport and actin cytoskeletal rearrangement among a variety of PI(3,4,5)P3 downstream events.

Amino Acid Sequence↗
PubMed2005

Muscle-specific deletion of the Glut4 glucose transporter alters multiple regulatory steps in glycogen metabolism.

Mice with muscle-specific knockout of the Glut4 glucose transporter (muscle-G4KO) are insulin resistant and mildly diabetic. Here we show that despite markedly reduced glucose transport in muscle, muscle glycogen content in the fasted state is increased. We sought to determine the mechanism(s). Basal glycogen synthase activity is increased by 34% and glycogen phosphorylase activity is decreased by 17% (P < 0.05) in muscle of muscle-G4KO mice. Contraction-induced glycogen breakdown is normal. The increased glycogen synthase activity occurs in spite of decreased signaling through the insulin receptor substrate 1 (IRS-1)-phosphoinositide (PI) 3-kinase-Akt pathway and increased glycogen synthase kinase 3beta (GSK3beta) activity in the basal state. Hexokinase II is increased, leading to an approximately twofold increase in glucose-6-phosphate levels. In addition, the levels of two scaffolding proteins that are glycogen-targeting subunits of protein phosphatase 1 (PP1), the muscle-specific regulatory subunit (RGL) and the protein targeting to glycogen (PTG), are strikingly increased by 3.2- to 4.2-fold in muscle of muscle-G4KO mice compared to wild-type mice. The catalytic activity of PP1, which dephosphorylates and activates glycogen synthase, is also increased. This dominates over the GSK3 effects, since glycogen synthase phosphorylation on the GSK3-regulated site is decreased. Thus, the markedly reduced glucose transport in muscle results in increased glycogen synthase activity due to increased hexokinase II, glucose-6-phosphate, and RGL and PTG levels and enhanced PP1 activity. This, combined with decreased glycogen phosphorylase activity, results in increased glycogen content in muscle in the fasted state when glucose transport is reduced.

Animals↗
PubMed2006

Loss of cortical actin filaments in insulin-resistant skeletal muscle cells impairs GLUT4 vesicle trafficking and glucose transport.

Study has demonstrated an essential role of cortical filamentous actin (F-actin) in insulin-regulated glucose uptake by skeletal muscle. Here, we tested whether perturbations in F-actin contributed to impaired insulin responsiveness provoked by hyperinsulinemia. In L6 myotubes stably expressing GLUT4 that carries an exofacial myc-epitope tag, acute insulin stimulation (20 min, 100 nM) increased GLUT4myc translocation and glucose uptake by approximately 2-fold. In contrast, a hyperinsulinemic state, induced by inclusion of 5 nM insulin in the medium for 12 h decreased the ability of insulin to stimulate these processes. Defects in insulin signaling did not readily account for the observed disruption. In contrast, hyperinsulinemia reduced cortical F-actin. This occurred concomitant with a loss of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), a lipid involved in cytoskeletal regulation. Restoration of plasma membrane PIP(2) in hyperinsulinemic cells restored F-actin and insulin responsiveness. Consistent with these in vitro observations suggesting that the hyperinsulinemic state negatively affects cortical F-actin structure, epitrochlearis skeletal muscle from insulin-resistant hyperinsulinemic Zucker fatty rats displayed a similar loss of F-actin structure compared with that in muscle from lean insulin-sensitive littermates. We propose that a component of insulin-induced insulin resistance in skeletal muscle involves defects in PIP(2)/F-actin structure essential for insulin-regulated glucose transport.

Actin Cytoskeleton↗
PubMed2006

A role of lipin in human obesity and insulin resistance: relation to adipocyte glucose transport and GLUT4 expression.

The mouse lipin gene, Lpin1, is important for adipose tissue development and is a candidate gene for insulin resistance. Here, we investigate the adipose tissue expression levels of the human LPIN1 gene in relation to various clinical variables as well as adipocyte function. LPIN1 gene expression was induced at an early step in human preadipocyte differentiation in parallel with peroxisome proliferator-activated receptor gamma. Lipin mRNA levels were higher in fat cells than in adipose tissue segments but showed no difference between subcutaneous and omental depots. Moreover, LPIN1 expression levels were reduced in obesity, improved following weight reduction in obese subjects, and were downregulated in women with the metabolic syndrome. With respect to adipocyte function, adipose LPIN1 gene expression was strongly associated with both basal and insulin-mediated subcutaneous adipocyte glucose transport as well as mRNA levels of glucose transporter 4 (GLUT4). We show that body fat accumulation is a major regulator of human adipose LPIN1 expression and suggest a role of LPIN1 in human preadipocyte as well as mature adipocyte function.

Adipocytes↗
PubMed2006

Peroxisome proliferator-activated receptor-alpha-null mice have increased white adipose tissue glucose utilization, GLUT4, and fat mass: Role in liver and brain.

Activation of the peroxisome proliferator-activated receptor (PPAR)-alpha increases lipid catabolism and lowers the concentration of circulating lipid, but its role in the control of glucose metabolism is not as clearly established. Here we compared PPARalpha knockout mice with wild type and confirmed that the former developed hypoglycemia during fasting. This was associated with only a slight increase in insulin sensitivity but a dramatic increase in whole-body and adipose tissue glucose use rates in the fasting state. The white sc and visceral fat depots were larger due to an increase in the size and number of adipocytes, and their level of GLUT4 expression was higher and no longer regulated by the fed-to-fast transition. To evaluate whether these adipocyte deregulations were secondary to the absence of PPARalpha from liver, we reexpresssed this transcription factor in the liver of knockout mice using recombinant adenoviruses. Whereas more than 90% of the hepatocytes were infected and PPARalpha expression was restored to normal levels, the whole-body glucose use rate remained elevated. Next, to evaluate whether brain PPARalpha could affect glucose homeostasis, we activated brain PPARalpha in wild-type mice by infusing WY14643 into the lateral ventricle and showed that whole-body glucose use was reduced. Hence, our data show that PPARalpha is involved in the regulation of glucose homeostasis, insulin sensitivity, fat accumulation, and adipose tissue glucose use by a mechanism that does not require PPARalpha expression in the liver. By contrast, activation of PPARalpha in the brain stimulates peripheral glucose use. This suggests that the alteration in adipocyte glucose metabolism in the knockout mice may result from the absence of PPARalpha in the brain.

Adipocytes↗
PubMed1995

Action of metformin on glucose transport and glucose transporter GLUT1 and GLUT4 in heart muscle cells from healthy and diabetic rats.

The effects of the antidiabetic drug metformin on glucose transport were investigated in freshly isolated heart muscle cells from healthy and streptozotocin-diabetic rats. In vivo treatment of diabetic rats with metformin failed to affect the basal and insulin-stimulated rate of glucose transport measured in isolated cells. In vitro exposure to therapeutic concentrations (< or = 10(-4) M) of metformin did not influence glucose transport, even upon incubation times up to 5 h or in the presence of high glucose (20 nM). In contrast, higher metformin concentrations produced an 8- to 12-fold increase in glucose uptake (with a lag of 90 min, and a maximum at 180 min and approximately 5 mM). In the presence of submaximal insulin concentrations (< or = 3.10(-10) M), the effects of metformin (5 mM) and of insulin were more than additive, whereas, at saturating insulin concentrations (10(-8) M), partial additivity was observed. Like insulin, metformin caused an approximately 1.6-fold increase in the content of both glucose transporter isoforms GLUT1 and GLUT4 in the plasma membrane of cardiac myocytes, with a corresponding decrease in an intracellular membrane fraction. cAMP-elevating treatments depressed the metformin-, but not the insulin-dependent glucose uptake, by 20-30%. In myocytes from diabetic rats, the rate of metformin-activated glucose transport was similar to that of cells from control animals, whereas basal and insulin-stimulated transport were substantially diminished. Finally, metformin (5 mM) induced a slight depression of oxygen consumption and energy metabolism of myocytes (as determined by measuring their level of energy-rich phosphates) comparable to the effects of hypoxia in rat hearts. In conclusion, these data do not provide evidence in favor of the hypothesis that glucose uptake by muscle tissue represents the site of metformin's therapeutic action in vivo. On the other hand, the large, insulin-independent effect of metformin at high concentrations (approximately mM) in vitro may be related to the action of hypoxia and occurs through a redistribution of glucose carriers from an intracellular locus to the plasma membrane. The mechanism (or signal) involved in metformin's action is likely to differ from that triggered by insulin and is not impaired in the diabetic state.

Animals↗
PubMed2003

An insulin-related peptide expressed in 3T3L1 adipocytes is localized in GLUT4 vesicles and secreted in response to exogenous insulin, which augments the insulin-stimulated glucose uptake.

If an adipocyte is programmed to secrete insulin, then the insulin released may amplify the insulin action by an autocrine manner. To examine this hypothesis in vitro, we investigated the effects of expressing the preproinsulin gene in 3T3L1 adipocytes on (pro)insulin release and glucose uptake. The human preproinsulin gene was transferred into 3T3L1 adipocytes by infecting the cells with recombinant adenovirus Adex1CA human preproinsulin. Immunocytochemical studies showed that (pro)insulin is associated with vesicular structures that colocalize with GLUT4 vesicles but not with GLUT1 vesicles. We then examined insulin-induced proinsulin release from 3T3L1 adipocytes expressing the insulin gene. The exogenously administered insulin stimulated proinsulin release from these cells in a dose-dependent manner. HPLC determination revealed the existence of mature human insulin in these cells, which suggested the release of mature insulin into the medium. Further we monitored the (pro)insulin release from these cells with confocal laser microscopy using the expression of a fusion protein between insulin and green fluorescent protein (GFP). Time-lapse confocal laser-scanning microscopy revealed that the total number of vesicles containing insulin-GFP was decreased by the addition of 10(-7) M insulin within 1 minute. Finally, we examined the insulin-stimulated glucose uptake by these cells. The data showed that insulin-stimulated glucose uptake increased to about 150% of that of control cells in response to exogenously administered insulin, indicating that the insulin released augmented the insulin-stimulated glucose uptake in an autocrine manner. Thus, the data support our hypothesis, indicating that we could construct the insulin-regulated insulin release system in adipocytes by introducing the preproinsulin gene.

3T3 Cells↗
PubMed2004

Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles.

Insulin-stimulated glucose uptake involves the recruitment of the glucose transporter 4 isoform (GLUT4) from an intracellular location to the plasma membrane of fat and muscle cells. Although the activation of the PI3-kinase/protein kinase B (PKB) pathway is central to this effect of insulin, the key substrates for PKB that are involved require identification. Here we report that serine318 on the FYVE domain-containing PtdIns3P 5-kinase (PIKfyve) is a novel substrate for PKB, and show that phosphorylation stimulates the PtdIns3P 5-kinase activity of the enzyme. We also demonstrate that PIKfyve is phosphorylated on serine318 in intact cells in response to insulin, in a PI3-kinase-dependent manner, and that PIKfyve colocalises with a highly motile subpopulation of insulin-regulated aminopeptidase (IRAP)/GLUT4 vesicles. Finally, we demonstrate that overexpression of a PIKfyve[S318A] mutant in 3T3-L1 adipocytes enhances insulin-stimulated IRAP/GLUT4 vesicle translocation to the plasma membrane suggesting a role for PKB-dependent phosphorylation of PIKfyve in insulin-regulated IRAP/GLUT4 trafficking. The phosphorylation and activation of PIKfyve by PKB provides a novel signalling paradigm that may link plasma membrane-localised PtdIns(3,4,5)P3 signals via a protein kinase cascade to regulated PtdIns(3,5)P2 production, and thereby to the control of trafficking of other membrane cargos.

3T3-L1 Cells↗
PubMed1995

Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling.

(+/-)-5-([4-[2-Methyl-2(pyridylamino)ethoxy]phenyl]methyl) 2,4-thiazolidinedione (BRL 49653) is a new potent antidiabetic agent that improves insulin sensitivity in animal models of NIDDM. In C57BL/6 obese (ob/ob) mice, BRL 49653, included in the diet for 8 days, improved glucose tolerance. The half-maximal effective dose was 3 mumol/kg diet, which is equivalent to approximately 0.1 mg/kg body wt. Improvements in glucose tolerance were accompanied by significant reductions in circulating triacylglycerol, nonesterified fatty acids, and insulin. The insulin receptor number of epididymal white adipocytes prepared from obese mice treated with BRL 49653 (30 mumol/kg diet) for 14 days was increased twofold. The affinity of the receptor for insulin was unchanged. In the absence of added insulin, the rates of glucose transport in adipocytes from untreated and BRL 49653-treated obese mice were similar. Insulin (73 nmol/l) produced only a 1.5-fold increase in glucose transport in adipocytes from control obese mice, whereas after BRL 49653 treatment, insulin stimulated glucose transport 2.8-fold. BRL 49653 did not alter the sensitivity of glucose transport to insulin. The increase in insulin responsiveness was accompanied by a 2.5-fold increase in the total tissue content of the glucose transporter GLUT4. Glucose transport in adipocytes from lean littermates was not altered by BRL 49653. To establish the contribution of changes in glucose transporter trafficking to the BRL 49653-mediated increase in insulin action, the cell-impermeant bis-mannose photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos++ +-4-yloxy) -2-[2-3H]-propylamine was used to measure adipocyte cell-surface-associated glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipocytes↗