Glucose Transport In Rat Adipocytes Research Articles

Fractionation analysis of the subcellular distribution of GLUT-4 in 3T3-L1 adipocytes.

AbstractIn 1980, two groups simultaneously provided evidence of the existence of an intracellular pool of glucose transporters in rat adipocytes (1,2). We now know that facilitative glucose uptake occurs through a family of highly related integral membrane proteins that share significant sequence similarity. Of the established glucose transporter isoforms, GLUT-4 is highly expressed in adipose tissue and striated muscle (3). In the basal state, GLUT-4 cycles slowly between the plasma membrane and one or more intracellular compartments, with the vast majority of the transporter residing in vesicular compartments within the cell interior (4–6). Activation of the insulin receptor triggers a large increase in the rate of GLUT-4 vesicle exocytosis and a smaller decrease in the rate of internalization by endocytosis (7–10). The stimulation of exocytosis by insulin is probably the major step for GLUT-4 translocation because complete inhibition of GLUT-4 endocytosis only modestly increases plasma membrane-associated GLUT-4 protein without affecting the extent of insulin-stimulated GLUT-4 translocation (11–13). In contrast to GLUT-4, GLUT-1 is an intracellular and plasma membrane localized in the basal state and displays a modest insulin-stimulated redistribution to the plasma membrane. Thus, the overall insulin-dependent shift in the cellular dynamics of GLUT-4 vesicle trafficking results in a net increase of GLUT-4 on the cell surface, thereby increasing the rate of glucose uptake.KeywordsSucrose CushionPyridoxine HydrochlorideVesicular CompartmentAdipocyte DevelopmentJA20 RotorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Long-term effect of dietary overload lithium on the glucose metabolism in broiler chickens

Lithium, like insulin, activates glycogen synthase and stimulates glucose transport in rat adipocytes. To investigate the effect of dietary overload lithium on glucose metabolism in broiler chickens, one-day-old chicks were fed a basal diet supplemented with 0 (control) or 100mg lithium/kg (overload lithium) for 35days. Compared to controls, glucose disappearance rates were lower (p=0.035) 15–120min after glucose gavage, and blood glucose concentrations were lower (p=0.038) 30min after insulin injection in overload lithium broilers. Overload lithium decreased (p<0.05) glycogen and glucose-6-phosphate concentrations in liver, but increased (p<0.05) their concentrations in pectoralis major. Overload lithium increased (p<0.05) mRNA expression of glucose transporter (GLUT) 3 and GLUT9 in liver, and GLUT1, GLUT3, GLUT8, and GLUT9 in pectoralis major, but decreased (p<0.05) cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in liver and mitochondrial PEPCK in pectoralis major. These results suggest that dietary overload lithium decreases glucose tolerance and gluconeogenesis, but increases insulin sensitivity and glucose transport in broiler chickens.

Curcumin is a direct inhibitor of glucose transport in adipocytes

Curcumin has been reported to inhibit insulin signaling and translocation of GLUT4 to the cell surface in 3T3-L1 adipocytes. We have investigated the effect of curcumin on insulin signaling in primary rat adipocytes. Curcumin (20μM) inhibited both basal and insulin-stimulated glucose transport (2-deoxyglucose uptake), but had no effect on insulin inhibition of lipolysis. Dose–response experiments demonstrated that curcumin (0–100μM) inhibited basal and insulin-stimulated glucose transport, but even at the highest concentration tested did not affect lipolysis. Inhibition was equal in cells that had been pre-incubated with curcumin and in cells to which curcumin was added immediately before the glucose transport assay. Similarly, time-course experiments revealed that the inhibitory effect of curcumin was evident at the earliest time point tested (30s). Thus it is unlikely that inhibition of insulin signaling or of translocation of GLUT4 to the cell surface is involved in the inhibitory effect of curcumin. Curcumin did not affect the stimulatory action of insulin on phosphorylation of Akt at serine 473. We conclude that curcumin is a direct inhibitor of glucose transporters in rat adipocytes.

L-Proline as a modulator of ectodermal differentiation in ES cells. Focus on “l-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture

current research is focusing on defining the control of pluripotency and lineage determination of pluripotent cells, particularly of embryonic stem (ES) cells. ES cells are usually produced from the inner cell mass (ICM) of the blastocyst and are considered equivalent to ICM cells captured during a

Bradykinin Augments Insulin-Stimulated Glucose Transport in Rat Adipocytes via Endothelial Nitric Oxide Synthase–Mediated Inhibition of Jun NH2-Terminal Kinase

An increase in bradykinin has been suggested to contribute to the enhanced insulin sensitivity observed in the presence of ACE inhibitors. To investigate a potential direct, nonvascular effect on an insulin target tissue, the effect of bradykinin on glucose uptake and insulin signaling was studied in primary rat adipocytes. Whereas basal glucose uptake was not altered, bradykinin augmented insulin-stimulated glucose uptake twofold, which was blocked by HOE-140, a bradykinin B2 receptor antagonist. The bradykinin effect on glucose uptake was nitric oxide (NO) dependent, mimicked by NO donors and absent in adipocytes from endothelial NO synthase-/- mice. Investigation of insulin signaling revealed that bradykinin enhanced insulin receptor substrate-1 (IRS-1) Tyr phosphorylation, Akt/protein kinase B phosphorylation, and GLUT4 translocation. In contrast, insulin-stimulated extracellular signal-regulated kinase1/2 and Jun NH2-terminal kinase (JNK) activation were decreased in the presence of bradykinin, accompanied by decreased IRS-1 Ser307 phosphorylation. Furthermore, bradykinin did not enhance insulin action in the presence of the JNK inhibitor, SP-600125, or in adipocytes from JNK1-/- mice. These data indicate that bradykinin enhances insulin sensitivity in adipocytes via an NO-dependent pathway that acts by modulating the feedback inhibition of insulin signaling at the level of IRS-1.

Insulin Receptor Substrates-1 and -2 Are Both Depleted but via Different Mechanisms after Down-Regulation of Glucose Transport in Rat Adipocytes

Alterations in muscle and adipose tissue insulin receptor substrate (IRS)-1 and IRS-2 are associated with, and commonly believed to contribute to, development of insulin resistance. In this study, we investigated the mechanisms behind previously observed reductions in IRS levels due to high concentrations of glucose and insulin and their significance in the impairment of glucose uptake capacity in primary rat adipocytes. Semiquantitative RT-PCR analysis showed that insulin (10(4) microU/ml) alone or in combination with glucose (15 mm) markedly suppressed IRS-2 gene expression, whereas IRS-1 mRNA was unaffected by the culture conditions. The negative effect of a high glucose/high insulin setting on IRS-1 protein level was still exerted when protein synthesis was inhibited with cycloheximide. Impairment of glucose uptake capacity after treatment with high glucose and insulin was most pronounced after 3 h, whereas IRS-1 and IRS-2 protein levels were unaffected up to 6 h but were reduced after 16 h. Moreover, impaired glucose uptake capacity could only partially be reversed by subsequent incubation at physiological conditions. These novel results suggest that: 1) in a high glucose/high insulin setting depletion of IRS-1 and IRS-2 protein, respectively, occurs via different mechanisms, and IRS-2 gene expression is suppressed, whereas IRS-1 depletion is due to posttranslational mechanisms; 2) IRS-1 and IRS-2 protein depletion is a secondary event in the development of insulin resistance in this model of hyperglycemia/hyperinsulinemia; and 3) depletion of cellular IRS in adipose tissue may be a consequence rather than a cause of insulin resistance and hyperinsulinemia in type 2 diabetes.

Duality in the Mastoparan Action on Glucose Transport in Rat Adipocytes

Mastoparan, a tetradecapeptide purified from wasp venom, has been shown to stimulate glucose transport in rat adipocytes although the mechanism of its action has remained undefined. Here, we characterized the action of mastoparan on glucose transport in rat adipocytes. Mastoparan at a concentration of 20 microM or more caused a dose-dependent release of lactate dehydrogenase (LDH) from the cells, which closely correlated with its stimulatory effect on glucose uptake. The mastoparan-induced glucose uptake was inhibited neither by deprivation of ATP with KCN nor by addition of phloretin, a direct inhibitor of glucose transporter, suggesting that the ability of mastoparan to stimulate glucose uptake did not derive from activation of the glucose transport system (i.e. translocation or activation of GLUT4 and/or GLUT1). On the other hand, mastoparan at a lower concentration (15 microM or below), which showed an insignificant effect on LDH release, potentiated the insulin action on glucose transport and Akt phosphorylation in the presence of adenosine deaminase. The effect of mastoparan was not additive to that of phenylisopropyladenosine and was completely abolished by pretreatment of adipocytes with pertussis toxin (1 microg/ml for 2 hours). Thus, the present study disclosed duality in the action of mastoparan on glucose uptake in rat adipocytes. At a concentration of 15 microM or less, it enhances the insulin action on glucose transport by a pertussis toxin-sensitive Gi protein-dependent mechanism. At higher concentrations, however, mastoparan increases non-specific permeability of the plasma membrane, which causes LDH release as well as glucose uptake not mediated through glucose transporter.

Histamine does not induce an amine oxidase-dependent adipogenic effect in murine preadipocytes.

Mice deficient in H3-receptors exhibit increased food intake, body weight, and adiposity [1]. Also intracerebroventricular administration of histamine suppresses appetite [2]. Chronic administration of H3-receptor antagonists reduces food intake and body weight gain in rodents. Although changes in release of neurotransmitters which regulate appetite may explain observed changes in food consumption and subsequent alterations of fat deposition, there may be a direct action of histamine on adipose cells. Previously we reported that histamine stimulates glucose transport in rat adipocytes, in presence of vanadate [3]. Also histamine has a dual influence on lipid mobilization: it stimulates lipolysis when H1receptors and amine oxidases are blocked [4] or inhibits glycerol release when incubated at high concentration with mature adipocytes [5]. Here, we studied whether histamine was able to influence adipose differentiation of cultured preadipocytes. Since amine oxidation by semicarbazide-sensitive amine oxidase (SSAO) has been shown to mediate adipogenic effect of benzylamine [6] or methylamine [7] in 3T3-F442A cells, we also examined whether histamine was subjected to oxidative deamination in these murine cells.

Benzylamine exhibits insulin-like effects on glucose disposal, glucose transport, and fat cell lipolysis in rabbits and diabetic mice.

Benzylamine, a substrate of semicarbazide-sensitive amine oxidase (SSAO), stimulates glucose transport in rat adipocytes and improves glucose disposal in diabetic rats only in the presence of vanadate. These effects have been described to result from a synergism between the hydrogen peroxide formed during amine oxidation and vanadate, via the generation of pervanadate, a powerful insulin mimicker. However, it has also been reported that benzylamine alone can stimulate glucose uptake and inhibit lipolysis in human fat cells. In this work, we therefore investigated whether benzylamine on its own was able to induce both in vivo and in vitro insulin-like responses in animal models other than rat. In rabbits, the i.v. infusion of 7 micromol/kg benzylamine before a glucose tolerance test resulted in a net reduction of the hyperglycemic response without a change in insulin secretion. Benzylamine also improved glucose tolerance and reduced lipid mobilization in hyperglycemic/obese mice. In vitro, 0.1 mM benzylamine stimulated glucose transport and inhibited lipolysis in mouse and rabbit adipocytes. These effects were blocked by previous treatments with semicarbazide, a SSAO inhibitor. Levels of benzylamine oxidation were more elevated in mouse than in rabbit adipose tissues, whereas the reverse was observed for skeletal muscles. Finally, benzylamine was unable to stimulate insulin secretion by isolated pancreatic islets from both species and SSAO activity was hardly detectable in pancreas. Together, our results bring evidence that benzylamine on its own can improve glucose tolerance in rabbit and mouse, likely by stimulating glucose uptake via amine oxidase activation in insulin-sensitive tissues.

Tyramine stimulates glucose uptake in insulin-sensitive tissues in vitro and in vivo via its oxidation by amine oxidases.

Tyramine and benzylamine have been described as stimulators of glucose transport in adipocytes. This effect is dependent on amine oxidation by monoamine oxidase (MAO) or semicarbazide-sensitive amine oxidase (SSAO) and on the subsequent hydrogen peroxide formation as already demonstrated by blockade with oxidase inhibitors or antioxidants and potentiation with vanadate. In this work, we extended these observations to skeletal muscle and cardiac myocytes using in vitro and in vivo approaches. Tissue distribution studies showed that substantial extrahepatic peripheral MAO activities exist in kidney and gut, but also in insulin-sensitive tissues: heart, adipose tissue, and skeletal muscles. SSAO activity is also widely distributed and present at a lower level than MAO, except in fat depots where both oxidases were equally involved in tyramine oxidation. When tested in vitro at millimolar doses, tyramine caused a large stimulation of glucose transport in rat adipocytes and in skeletal and cardiac muscles. In vivo administration of tyramine (4 mg/kg i.p.) lowered the hyperglycemic responses to a glucose challenge in control and in streptozotocin-treated rats. This positive effect on glucose disposal was obtained without vanadate and was abolished by SSAO and MAO inhibitors. Tyramine increased hexose uptake in vivo in insulin-sensitive tissues, whereas it induced only transient effects on plasma insulin or cardiovascular parameters. In conclusion, activation of the amine oxidases present in insulin-sensitive tissues induces insulin-like effects, readily detectable in vitro, and increasing peripheral glucose utilization in vivo.

Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H2O2 formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or β-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H(2)O(2) formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or beta-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

Effects of myricetin on glycemia and glycogen metabolism in diabetic rats

In our previous study, we found that myricetin, a naturally occurring bioflavonoid, was able to stimulate glucose transport in rat adipocytes and enhance insulin-stimulated lipogenesis. We report here that after 2 days of treatment with myricetin (3 mg/12 h), hyperglycemia in diabetic rats was reduced by 50% and the hypertriglyceridemia that is often associated with diabetes was normalised. Treatment with myricetin increased hepatic glycogen and glucose-6-phosphate content. It increased hepatic glycogen synthase I activity without having any effect on total glycogen synthase nor phosphorylase a activity. It lowered phosphorylase a activity in the muscle. Thus, the hypoglycemic effect of myricetin is likely to be due to its effect on glycogen metabolism. There was no indication of serious hepatotoxicity with myricetin treatment and therefore, myricetin could be of therapeutic potential in diabetes.

Okadaic Acid Activates Atypical Protein Kinase C (ζ/λ) in Rat and 3T3/L1 Adipocytes: AN APPARENT REQUIREMENT FOR ACTIVATION OF GLUT4 TRANSLOCATION AND GLUCOSE TRANSPORT

Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is known to provoke insulin-like effects on GLUT4 translocation and glucose transport, but the underlying mechanism is obscure. Presently, we found in both rat adipocytes and 3T3/L1 adipocytes that okadaic acid provoked partial insulin-like increases in glucose transport, which were inhibited by phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin and LY294002, and inhibitors of atypical protein kinase C (PKC) isoforms, zeta and lambda. Moreover, in both cell types, okadaic acid provoked increases in the activity of immunoprecipitable PKC-zeta/lambda by a PI 3-kinase-dependent mechanism. In keeping with apparent PI 3-kinase dependence of stimulatory effects of okadaic acid on glucose transport and PKC-zeta/lambda activity, okadaic acid provoked insulin-like increases in membrane PI 3-kinase activity in rat adipocytes; the mechanism for PI 3-kinase activation was uncertain, however, because it was not apparent in phosphotyrosine immunoprecipitates. Of further note, okadaic acid provoked partial insulin-like increases in the translocation of hemagglutinin antigen-tagged GLUT4 to the plasma membrane in transiently transfected rat adipocytes, and these stimulatory effects on hemagglutinin antigen-tagged GLUT4 translocation were inhibited by co-expression of kinase-inactive forms of PKC-zeta and PKC-lambda but not by a double mutant (T308A, S473A), activation-resistant form of protein kinase B. Our findings suggest that, as with insulin, PI 3-kinase-dependent atypical PKCs, zeta and lambda, are required for okadaic acid-induced increases in GLUT4 translocation and glucose transport in rat adipocytes and 3T3/L1 adipocytes.

Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ε) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

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

Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

Atypical protein kinase (PK)C isoforms, ζ and λ, have been reported to be activated by insulin via phosphoinositide 3-kinase, and have been suggested to be required for insulin-stimulated glucose transport. Here, we have examined the effects of transiently expressed wild-type (WT), constitutively active (Constit) and kinase-inactive (KI) forms of atypical PKCs, ζ and λ, on haemagglutinin antigen (HAA)-tagged glucose transporter 4 (GLUT4) translocation in rat adipocytes, and compared these effects with each other and with those of comparable forms of conventional (α, β) and novel (δ, ε) PKCs, which have also been proposed to be required for insulin-stimulated glucose transport. KI-PKC-ζ evoked consistent, sizeable (overall mean of 65%) inhibitory effects on insulin-stimulated, but not basal or guanosine-5´-[γ-thio]triphosphate-stimulated, HAA-GLUT4 translocation; moreover, inhibitory effects of KI-PKC-ζ were largely reversed by co-transfection of WT-PKC-ζ. Like KI-PKC-ζ, KI-PKC-λ inhibited insulin-stimulated HAA-GLUT4 translocation by approx. 40–60%, and the combination of KI-PKC-ζ and KI-PKC-λ caused nearly complete (85%) inhibition. Of particular interest is the fact that inhibitory effects of KI forms of PKC-ζ and PKC-λ were largely reversed by the opposite WT forms, i.e. PKC-λ and PKC-ζ respectively. In contrast with KI forms of atypical PKCs, KI forms of PKC-α, PKC-β2, PKC-δ and PKC-ε had little or no effect on insulin-stimulated HAA-GLUT4 translocation. Concerning the question of sufficiency, overexpression of WT-PKC-ζ enhanced insulin effects on HAA-GLUT4 translocation, whereas WT forms of PKC-α, PKC-β2, PKC-δ and PKC-ε did not affect GLUT4 translocation; furthermore, Constit PKC-ζ evoked increases in HAA-GLUT4 translocation approaching those of insulin, but Constit forms of PKC-α and PKC-β2 were without effect. Our findings suggest that, among PKCs, the atypical PKCs, ζ and λ, appear to be specifically, but interchangeably, required for insulin effects on HAA-GLUT4 translocation.

Stimulation of IRS-1-associated Phosphatidylinositol 3-Kinase and Akt/Protein Kinase B but Not Glucose Transport by β1-Integrin Signaling in Rat Adipocytes

The signal transduction pathway by which insulin stimulates glucose transport is not understood, but a role for complexes of insulin receptor substrate (IRS) proteins and phosphatidylinositol (PI) 3-kinase as well as for Akt/protein kinase B (PKB) has been proposed. Here, we present evidence suggesting that formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation are insufficient to stimulate glucose transport in rat adipocytes. Cross-linking of beta1-integrin on the surface of rat adipocytes by anti-beta1-integrin antibody and fibronectin was found to cause greater IRS-1 tyrosine phosphorylation, IRS-1-associated PI 3-kinase activity, and Akt/PKB activation, detected by anti-serine 473 antibody, than did 1 nM insulin. Clustering of beta1-integrin also significantly potentiated stimulation of insulin receptor and IRS-1 tyrosine phosphorylation, IRS-associated PI 3-kinase activity, and Akt/PKB activation caused by submaximal concentrations of insulin. In contrast, beta1-integrin clustering caused neither a change in deoxyglucose transport nor an effect on the ability of insulin to stimulate deoxyglucose uptake at any concentration along the entire dose-response relationship range. The data suggest that (i) beta1-integrins can engage tyrosine kinase signaling pathways in isolated fat cells, potentially regulating fat cell functions and (ii) either formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation is not necessary for regulation of glucose transport in fat cells or an additional signaling pathway is required.

Pharmacological characterization of AMP 579, a novel adenosine A1/A2 receptor agonist and cardioprotective

AMP 579 1S-[1α,2β,3β,4α(S*)]-4-[7-[[1 -[(3-chloro-2-thienyl)methyl]propylamino]-3H-imidazo[4,5-b]pyridin-3-yl]-N-ethyl-2,3-dihydroxy cyclopentanecarboxamide) is a novel cardioprotective adenosine agonist with the following order of affinity at adenosine receptors: A 1 > A 2A > A 3 . Agonism at A 1 receptors was demonstrated in vitro in three different systems: 1) inhibition of lipolysis in rat and human isolated adipocytes, 2) restoration of insulin-dependent glucose transport in rat adipocytes, and 3) reduction of heart rate in spontaneously beating rat right atria. Agonism at A 2A receptors was reflected in vasorelaxation of porcine coronary arterial rings (IC 50 = 0.3 μM); in comparison, agonism at A 2B receptors was ∼100-fold weaker, as reflected in relaxation of guinea pig aorta (IC 50 = 28 μM). When given iv to conscious Sprague-Dawley (SD) rats, AMP 579 dose-dependently lowered free fatty acids (FFA), heart rate (HR), and mean arterial pressure (MAP), but was 25-fold more potent at reducing FFA than at decreasing HR and MAP. In anesthetized rats undergoing myocardial ischemia-reperfusion injury, AMP 579 (3 μg/kg + 0.3 μg/kg/min iv and 10 μg/kg + 1 μg/ kg/min iv) was able to reduce infarct size by 55% and 63%, respectively, compared to control animals, when given 10 min prior to and throughout the first hour of reperfusion. These cardioprotective doses of AMP 579 caused no significant change in blood pressure or coronary blood flow. In summary, AMP 579 is a novel adenosine A 1 /A 2A receptor agonist which causes long-lasting reductions in FFA in vivo and has cardioprotective effects in a rat model of myocardial ischemia-reperfusion injury at doses which have minimal hemodynamic effects. Thus, AMP 579 has significant potential for the therapy of acute myocardial infarction.

Stimulatory effect of lithium on glucose transport in rat adipocytes is not mediated by elevation of IP1.

Lithium has been shown to increase glucose uptake in skeletal muscle and adipose tissues. The therapeutic effect of lithium on bipolar disease is thought to be mediated by its inhibitory effect on myo-inositol-1-monophosphatase (IMPase). We tested the hypothesis that the stimulatory effect of lithium on glucose uptake results from inhibition of IMPase and the resultant accumulation of inositol monophosphates (IP1) by comparing the effects of lithium and a selective IMPase inhibitor, L-690,488, on isolated rat adipocytes. Insulin produced a concentration-dependent stimulation of 2-deoxy-D-[14C]glucose (2-DG) transport (10 microU/ml caused half-maximal activation). Acute exposure to lithium stimulated basal glucose transport activity in a concentration-dependent manner, with a threefold stimulation at 30 mM lithium. Lithium also potentiated insulin-stimulated 2-DG transport. Lithium produced a concomitant increase in IP1 accumulation. In contrast, L-690,488 increased IP1 to levels comparable to those of lithium without stimulatory effects on 2-DG transport. These results demonstrate that stimulatory effects of lithium on glucose transport are not mediated by the inhibition of IMPase and subsequent accumulation of IP1 in rat adipocytes.

Comparative Effects of GTPγS and Insulin on the Activation of Rho, Phosphatidylinositol 3-Kinase, and Protein Kinase N in Rat Adipocytes: RELATIONSHIP TO GLUCOSE TRANSPORT

Electroporation of rat adipocytes with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPgammaS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPgammaS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPgammaS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPgammaS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-zeta (PKC-zeta) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-zeta, GTPgammaS activated PKN but not PKC-zeta. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPgammaS on HA-GLUT4 translocation. Our findings suggest that (a) GTPgammaS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-zeta may contribute to increases in glucose transport during insulin, but not GTPgammaS, action.