- Email: [email protected]
Protein tyrosine phosphatase 1B is impaired in skeletal muscle of diabetic psammomys obesus
S156
Poster Session I
still unclear whether this signaling system is contributed to insulin resistance in the skeletal muscle in vivo. First, to investigate insulin signaling system that regulates glycogen synthesis and glucose transport in the skeletal muscle, male Balblc mice starved for 24 hours were administrated insulin (2U/kg) through portal veins under pentobarbital anesthesia. At various times, soleus muscles were taken and frozen immediately in liquid nitrogen, and then used to measure PI 3-kinase, Akt, GSK-3 and glycogen synthase activities. In the mouse skeletal muscle, PI 3-kinase and Akt activities were stimulated as early as 3 mitt after insulin administration, and maximum stimulation was seen at 5 min (proximately ‘I-fold and 4-fold, respectively). PI 3-kinase activity was decreased to 3-fold at 15 min. whereas almost no reduction was seen in Akt activity. Glycogen synthase activity in the skeletal muscle was activated 5 min after insulin injection (1.2-fold), and maximum activation was observed at 15 min (1.7-fold). At 3 min after insulin administration, a decrease in GSK-3 activity was seen in mouse skeletal muscle, and the maximum inhibition of GSK3 in response to insulin was 15 min after administration (proximately 60% of the control). Next, to evaluate whether insulin resistance is associated with impaired insulin signaling in the skeletal muscle in viva, TNFo or glucosamine was used to induce insulin resintance, and then insulin-stimulated PI 3-kinase activity was measured. Proximately 20% decrease in PI 3-kinase activity was observed 6hr after TNF-o administration (100 pg/kg). Four hours continuous infusion of glucosamine (30 ~mobkg/min) also resulted in 30% reduction of PI 3-kinase activity in the muscle. In this study we have shown that administration of insulin to mice from portal vein elicits GSK inhibition and PI 3-kinase, Akt, glycogen synthase activation. We have also demonstrated that insulin signaling is impared under insulin resistance. These data suggest that measurement of these kinase activities after transportal insulin injection would be useful to evaluate insulin resistance status in the muscle in viva.
P623 IncreasedInsulin Secretion Fails To Compensate for Insulin Resistance in Mice with Impaired Glucose Disposal under High-Fat Diet GIOVANNI PACINI ’ , Bo Ah& 2. ’ Bioengineering, LADSEB-CNR, Padua, Italy; 2 Medicine, Lund University, MalmBSweden
Aim. Insulin sensitivity is reduced by high-fat diet in mice. This study aimed to evaluate if increased insulin secretion may compensate for the reduced insulin sensitivity. Methods. From the age of 4 weeks high-fat diet (58% fat on energy basis) was given to C57BIJ6.l mice (I-IF) whereas ordinary chow was given to controls (CT). After 6-10 months, glucose (1 gikg) was injected i.v. in anesthetized mice (alone in 24 CT and in 24 HF (regular test), and with an additional insulin injection of 1 U/kg at 10 min (insulin test) in 12 CT and in 12 HF). Samples were taken over 50 min for analysis of insulin and glucose with the minimal model of glucose disappearance that gives the insulin sensitivity index (Sr). Insulin secretion was represented by the insulin area (AUC) and glucose disposal by the slope of glucose disappearance from 5 to 50 min (I&). The total insulin effect was the unitless disposition index (DI=AUC x St). Results. HF exhibited higher body weight (41f2 vs. 26f0.4 g), higher basal glucose (10.4f0.2 vs. 7.9f0.2 mM) and higher basal insulin (702f84 vs. 348f36 PM), all PcO.05. In both experiments HF had a markedly reduced Ko (l.lfO.l vs. 1.8f0.18 min.’ with the regular test and 3.6f0.2 vs. 5.0f0.2% min.’ with the insulin test) and St (0.38f0.09 vs. 0.90f0.15 lOA min.‘/PM and 0.13f0.02 vs. 1.02f0.25 lo4 min.‘/PM) all P~O.002. In the regular test AUC was 50f8 nM mitt vs. 23f2 nM min in CT, P
P624 Positive Regulation of Insulin Receptor Signaling by tbe GrblO Adapter Protein BARBARA GIOVANNONE, Gabriele Tiller, Robert J. Smith. Metabolism Section, Joslin Diabetes Center; Boston, MA, United States of America GrblO is an adapter protein that binds with high affinity to the tyrosine kinase and C-terminal regions of the insulin receptor r?Jsubunit via two tandem protein interaction domains. GrblO is postulated to link other proteins to the insulin receptor and to function as a regulator of insulin signaling. Studies published to date have provided conflicting evidence for stimulatory or inhibitory actions of GrblO on insulin-stimulated mitogenesis and other downstream insulin responses. Data on the effects of GrblO on insulin receptor tyrosine phosphorylation have not been reported, and there is limited information on its effects on the phosphorylation of insulin receptor substrates. Therefore, the objective of this study was to determine insulin receptor, IRS-l, and She tyrosine phosphorylation in insulin responsive cells following either a modest increase in the abundance of the full-length GrblO protein, or the introduction of high levels of a truncated C-terminal fragment of GrblO, which should compete for binding of the native protein to insulin receptors. A 2-fold increase in full-length GrblO induced by transient transfection of R/IR mouse fibroblasts augmented insulin receptor tyrosine phosphorylation (40% increase 2 min after insulin stimulation, P=O.O29, and 170% increase 10 min after insulin, P=O.O5). A much higher level of expression of truncated GrblO had no effect on receptor phosphorylation. The tyrosine phosphorylation of IRS-l and She were unaffected by full-length GrblO overexpression, whereas the truncated GrblO construct resulted in a 40% decrease in IRS-l (P=O.O6) and a 20% decrease in She tyrosine phosphorylation (P~O.005) 10 min after insulin. These findings support the conclusion that the intact GrblO protein can augment insulin receptor phosphorylation. The greater effect of GrblO at 10 min vs 2 min after insulin stimulation is consistent with a potential role of GrblO in delaying receptor dephosphorylation. Inhibition of IRS-l and She phosphorylation by high levels of truncated GrblO further supports a role for the native, full-length adapter protein in augmenting insulin receptor signaling. Studies in a related cell line have shown inhibition of ligand-induced receptor internalization by increased expression of either full-length or truncated GrblO. These findings suggest that GrblO may increase insulin receptor phosphorylation and regulate insulin signaling by modulating the localization or tralhcking of insulin receptors.
P625 Protein Tyrosine Phosphatase 1B Is Impaired in Skeletal Muscle of Diabetic Psammomys Obesus YUKIO IKEDA’, Ehud Ziv’, Eleazar Shafrir2, Luitgard Mosthaf ‘. ’ Department of Molecular Signaling, Hagedom Research Institute, Gentofte, Denmark; 2 Diabetes Research Unit, Haa’assah University Hospital, Jerusalem, Israel Psammomys obesus (sand rat) is an animal model of nutritionally induced insulin resistance and type 2 diabetes. To examine whether protein tyrosine phosphatase (PTPase) is involved in the susceptibility to insulin resistance or diabetes, we measured PTPase expression and the enzyme activity towards the insulin receptor in skeletal muscle and liver of diabetic animals from a diabetes prone (DP) line, prediabetic (normoinsulinemic, normoglycemic) animals from a DP line and control animals from a diabetes resistant (DR) line. The expression level of protein tyrosine phosphatase 1B (PTP 1B) in the skeletal muscle was increased by 83% in the diabetic animals compared with the control animals. However, PTP 1B activity in the particulate fraction was decreased to 60% in diabetic animals (60 f 11 arbitrary units) when compared with the control animals (100 * 12 arbitrary units) or with the prediabetic animals (100 f 10 arbitrary units). In addition, PTP 1B activity, as well as total PTPase activity, was
Track 4. Basic Research
inversely correlated to serum glucose concentrations (r = -0.434, p < 0.02) and insulin levels (r = -0.408, p < 0.05). These findings suggest that PTP 1B is not involved in the genetic susceptibility to insulin resistance in these animals but is secondarily impaired by some factors in the diabetic milieu. The overexpression of PTP 1B in skeletal muscle may represent a compensatory mechanism against the impairment of the enzyme.
P626 Redistribution of Signaling Target for Insulin-Mimetic CijNTER MiiLLER, Stefan Deutschlond GmbH Disease Main, Germany
Proteins from Caveolae as Potential Compounds Welte, Susanne Wied. Aventis Pharma Group Metabolic Diseases, Frankfurt am
Phosphoinositolglycans (PIG) representing the polar glycan head group of the glycosylphosphatidylinositol (GPI) membrane anchor of GPI-modified plasma membrane proteins (GPI proteins) have been demonstrated to exert potent insulin-mimetic metabolic and signaling activity in isolated adipose and muscle cells. In rat adipocytes this activity depends on the presence of a 115-kDa polypeptide residing in caveolae of the plasma membrane where it may interact with GPI roteins as the natural ligands. Purpose of the Study: We investigated whether caveolar localization of certain inactive signaling molecules could provide a compartmental basis for their regulated activation by PIG and explain cross-talk with insulin signaling. Methods: It has been shown that caveolin, a principle component of caveolae membranes, functionally supresses the activity of dual acylated Src-family tyrosine kinases (e.g. p~59~Y”) through the so-called caveolin-scaffolding domain. Results: PIG (0.5 PM) as well as caveolin scaffolding domain peptide (25 PM, introduced into cells by electroporation) induced tyrosine phosphorylation of the insulin receptor substrates 1, 2 and 3 (IRS) independent of the insulin receptor kinase thereby eliciting potent stimulation of glucose transport and metabolism in isolated rat adipocytes accompanied by up to 7.5- and 3.7-fold, respectively, activation of ~~59~““. In correlation to p~59~Y” activation by PIG, several GPI proteins (e.g. Gcel) were significantly deprived from tbe caveolae relative to the resident proteins caveolin and the 115-kDa receptor. Conclusion: The dissociation of GPl proteins from the 115-kDa receptor protein by PIG structures may abolish the inhibitory effect of caveolin on p~59~fl via causing their dissociation and subsequent movement of pp59LY” into non-caveolar areas of the plasma membrane, where tyrosine phosphorylationof IRS and thereby initiation of metabolic insulin signaling occurs. Thus, redistribution of GPI proteins from caveolae may represent a novel target for insulin signal transduction therapy.
P627 The Acute and Chronic Stimulatory Effects of Endothelin-1 on Glucose lkansport Are Mediated by Distinct Pathways in 3T3-Ll AdipoCytes KENICHI ISHIBASHI, Takeshi Imamura, Prem M. Sharma, Satoshi Ugi, Jerrold M. Olefsky. Endocrinology and Metabolism, University of California LA Jolla, CA, United States of America We have recently shown that pretreatment with Endothelin-1 (ET-l) for 20 min stimulates GLUT4 translocation in a PI3-kinase dependent manner in 3T3-Ll adipocytes (JBC. 274.33691-5). This study presents another pathway by which ET-l potentiates glucose transport in 3T3-Ll adipocytes. ET-l treatment (10 nM) exhibits a biphasic effect on 2-deoxyglucose (2-DOG) uptake, with a rapid increase up to -2.5 fold within 20 min, followed by a transient decline at 1 h, reaching a maximal effect (-4 fold) at approximately 6 h and recovering almost to basal levels after 24 h. Insulin stimulation (3 ng/ml) leads to a monophasic effect on 2-DOG uptake, with a maximum increase at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-l induced glucose uptake both at 20 min and 6 h, while the ETB receptor
s157
antagonist, BQ 788, was without effect. Interestingly, ET-l stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-l involve distinct mechanisms. GLUT4 translocation assay showed that 20 min. but not 6 h, exposure to ET-l led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min. exposure to ET-l increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-l induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the Gai inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-l induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-Ll adipocytes.
P628 Relationship of Insulin Activation of Protein Kinase B in Human Muscle Cells with Myogeuic Diffenmtiation but Not Glucose Uptake or Glycogen Synthesis JAN KRUETZPBLDT, Christiana Kausch, Hans U. Haering, Michael Stumvoll. Medizinische Klinik n! Universitaet Tuebingen, Tuebingen, Germany Aim: Activation of protein kinase B (PKB) has been implicated in myogenic differentiation and insulin‘s metabolic effects in animal muscle cell lines. To evaluate analogous functions of PKB-activation in human muscle cells we compared cultured myotubes (MT) with high and low insulin-stimulated phosphorylation of serine 473 in PKB (SeP73). Methods: Myotubes derived from muscle biopsies from 20 healthy volunteers were screened for insulin-stimulated Se#73-phosphorylation by immunoblotting. Cell lines with high (H, 2.73+0.22relU, n=3) and low (L, 0.86f0.02relU. n=3; ~~0.05) insulin-induced Ser473-phosphorylation were selected and compared for muscle-specific markers, insulin signaling and action. Results: H exhibited thicker and longer multinucleated MT compared to L. Creatine kinase activity was higher in H (2.90f0.23 vs 0.62fO.O2relU, ~~0.05) as was expression of a-sarcomeric actin (2.35f0.06 vs 1.08f0.34relU, pcO.05). Insulin activated insulin receptor kinase (2.07f0.27 vs 1.78fO.l3relU, p=O.38) and IRS-l-associatedphosphatidylinositol 3-kinase (PI3K. 2.02f0.22 vs 1.76&0.27relU, p&51) were not different between H and L. In addition, insulin-stimulated glucose uptake (1.38f0.11 vs 1.47f0.08-fold basal, p=O.53) and glycogen synthesis (2.1 lf0.21 vs 2.05f0.18-fold basal, p&86) were not different. Reduced insulin-stimulated Ser“73-phosphorylation in L was not compensated for by protein kinase C-dependent mechanisms, since also in tbe presence of 20wM PKC inhibitor RO 31-8220 (RO) insulin-stimulated glucose uptake was not different between H and L (1.2540.07 vs 1.34f0.07-fold basal, p=O.40), while RO significantly reduced basal glucose uptake by 45% and 52%. respectively. Conclusions: These results suggest a potential regulatory role of PKB in myogenic differentiation of human muscle cells. Such a regulation seems not to involve PI3K. Maximal insulin activation of PKB is not a prerequisite for maximal insulin-stimulated glucose uptake or glycogen synthesis.
P629 GLUT4 Gene Expression in Insulin-Resistant Obese Rats Involves Post-Ihmscriptional Poly-A ‘Igil mRNA Modulation PATRICIA M. SERAPHIM, Thiago G.R. Assis, Claudia B. Volpato, Maria T. Nunes, Ubiratan E Machado. Physiology and Biophysics, Institute of Biomedical Sciences. University of &io Paulo, Srio Paula, Srio Paula, Brazil Recent reports have demonstrated that spontaneously obese12 month-old Wistar rats have insulin resistance which is accompanied by tissue specific
Poster Session I
still unclear whether this signaling system is contributed to insulin resistance in the skeletal muscle in vivo. First, to investigate insulin signaling system that regulates glycogen synthesis and glucose transport in the skeletal muscle, male Balblc mice starved for 24 hours were administrated insulin (2U/kg) through portal veins under pentobarbital anesthesia. At various times, soleus muscles were taken and frozen immediately in liquid nitrogen, and then used to measure PI 3-kinase, Akt, GSK-3 and glycogen synthase activities. In the mouse skeletal muscle, PI 3-kinase and Akt activities were stimulated as early as 3 mitt after insulin administration, and maximum stimulation was seen at 5 min (proximately ‘I-fold and 4-fold, respectively). PI 3-kinase activity was decreased to 3-fold at 15 min. whereas almost no reduction was seen in Akt activity. Glycogen synthase activity in the skeletal muscle was activated 5 min after insulin injection (1.2-fold), and maximum activation was observed at 15 min (1.7-fold). At 3 min after insulin administration, a decrease in GSK-3 activity was seen in mouse skeletal muscle, and the maximum inhibition of GSK3 in response to insulin was 15 min after administration (proximately 60% of the control). Next, to evaluate whether insulin resistance is associated with impaired insulin signaling in the skeletal muscle in viva, TNFo or glucosamine was used to induce insulin resintance, and then insulin-stimulated PI 3-kinase activity was measured. Proximately 20% decrease in PI 3-kinase activity was observed 6hr after TNF-o administration (100 pg/kg). Four hours continuous infusion of glucosamine (30 ~mobkg/min) also resulted in 30% reduction of PI 3-kinase activity in the muscle. In this study we have shown that administration of insulin to mice from portal vein elicits GSK inhibition and PI 3-kinase, Akt, glycogen synthase activation. We have also demonstrated that insulin signaling is impared under insulin resistance. These data suggest that measurement of these kinase activities after transportal insulin injection would be useful to evaluate insulin resistance status in the muscle in viva.
P623 IncreasedInsulin Secretion Fails To Compensate for Insulin Resistance in Mice with Impaired Glucose Disposal under High-Fat Diet GIOVANNI PACINI ’ , Bo Ah& 2. ’ Bioengineering, LADSEB-CNR, Padua, Italy; 2 Medicine, Lund University, MalmBSweden
Aim. Insulin sensitivity is reduced by high-fat diet in mice. This study aimed to evaluate if increased insulin secretion may compensate for the reduced insulin sensitivity. Methods. From the age of 4 weeks high-fat diet (58% fat on energy basis) was given to C57BIJ6.l mice (I-IF) whereas ordinary chow was given to controls (CT). After 6-10 months, glucose (1 gikg) was injected i.v. in anesthetized mice (alone in 24 CT and in 24 HF (regular test), and with an additional insulin injection of 1 U/kg at 10 min (insulin test) in 12 CT and in 12 HF). Samples were taken over 50 min for analysis of insulin and glucose with the minimal model of glucose disappearance that gives the insulin sensitivity index (Sr). Insulin secretion was represented by the insulin area (AUC) and glucose disposal by the slope of glucose disappearance from 5 to 50 min (I&). The total insulin effect was the unitless disposition index (DI=AUC x St). Results. HF exhibited higher body weight (41f2 vs. 26f0.4 g), higher basal glucose (10.4f0.2 vs. 7.9f0.2 mM) and higher basal insulin (702f84 vs. 348f36 PM), all PcO.05. In both experiments HF had a markedly reduced Ko (l.lfO.l vs. 1.8f0.18 min.’ with the regular test and 3.6f0.2 vs. 5.0f0.2% min.’ with the insulin test) and St (0.38f0.09 vs. 0.90f0.15 lOA min.‘/PM and 0.13f0.02 vs. 1.02f0.25 lo4 min.‘/PM) all P~O.002. In the regular test AUC was 50f8 nM mitt vs. 23f2 nM min in CT, P
P624 Positive Regulation of Insulin Receptor Signaling by tbe GrblO Adapter Protein BARBARA GIOVANNONE, Gabriele Tiller, Robert J. Smith. Metabolism Section, Joslin Diabetes Center; Boston, MA, United States of America GrblO is an adapter protein that binds with high affinity to the tyrosine kinase and C-terminal regions of the insulin receptor r?Jsubunit via two tandem protein interaction domains. GrblO is postulated to link other proteins to the insulin receptor and to function as a regulator of insulin signaling. Studies published to date have provided conflicting evidence for stimulatory or inhibitory actions of GrblO on insulin-stimulated mitogenesis and other downstream insulin responses. Data on the effects of GrblO on insulin receptor tyrosine phosphorylation have not been reported, and there is limited information on its effects on the phosphorylation of insulin receptor substrates. Therefore, the objective of this study was to determine insulin receptor, IRS-l, and She tyrosine phosphorylation in insulin responsive cells following either a modest increase in the abundance of the full-length GrblO protein, or the introduction of high levels of a truncated C-terminal fragment of GrblO, which should compete for binding of the native protein to insulin receptors. A 2-fold increase in full-length GrblO induced by transient transfection of R/IR mouse fibroblasts augmented insulin receptor tyrosine phosphorylation (40% increase 2 min after insulin stimulation, P=O.O29, and 170% increase 10 min after insulin, P=O.O5). A much higher level of expression of truncated GrblO had no effect on receptor phosphorylation. The tyrosine phosphorylation of IRS-l and She were unaffected by full-length GrblO overexpression, whereas the truncated GrblO construct resulted in a 40% decrease in IRS-l (P=O.O6) and a 20% decrease in She tyrosine phosphorylation (P~O.005) 10 min after insulin. These findings support the conclusion that the intact GrblO protein can augment insulin receptor phosphorylation. The greater effect of GrblO at 10 min vs 2 min after insulin stimulation is consistent with a potential role of GrblO in delaying receptor dephosphorylation. Inhibition of IRS-l and She phosphorylation by high levels of truncated GrblO further supports a role for the native, full-length adapter protein in augmenting insulin receptor signaling. Studies in a related cell line have shown inhibition of ligand-induced receptor internalization by increased expression of either full-length or truncated GrblO. These findings suggest that GrblO may increase insulin receptor phosphorylation and regulate insulin signaling by modulating the localization or tralhcking of insulin receptors.
P625 Protein Tyrosine Phosphatase 1B Is Impaired in Skeletal Muscle of Diabetic Psammomys Obesus YUKIO IKEDA’, Ehud Ziv’, Eleazar Shafrir2, Luitgard Mosthaf ‘. ’ Department of Molecular Signaling, Hagedom Research Institute, Gentofte, Denmark; 2 Diabetes Research Unit, Haa’assah University Hospital, Jerusalem, Israel Psammomys obesus (sand rat) is an animal model of nutritionally induced insulin resistance and type 2 diabetes. To examine whether protein tyrosine phosphatase (PTPase) is involved in the susceptibility to insulin resistance or diabetes, we measured PTPase expression and the enzyme activity towards the insulin receptor in skeletal muscle and liver of diabetic animals from a diabetes prone (DP) line, prediabetic (normoinsulinemic, normoglycemic) animals from a DP line and control animals from a diabetes resistant (DR) line. The expression level of protein tyrosine phosphatase 1B (PTP 1B) in the skeletal muscle was increased by 83% in the diabetic animals compared with the control animals. However, PTP 1B activity in the particulate fraction was decreased to 60% in diabetic animals (60 f 11 arbitrary units) when compared with the control animals (100 * 12 arbitrary units) or with the prediabetic animals (100 f 10 arbitrary units). In addition, PTP 1B activity, as well as total PTPase activity, was
Track 4. Basic Research
inversely correlated to serum glucose concentrations (r = -0.434, p < 0.02) and insulin levels (r = -0.408, p < 0.05). These findings suggest that PTP 1B is not involved in the genetic susceptibility to insulin resistance in these animals but is secondarily impaired by some factors in the diabetic milieu. The overexpression of PTP 1B in skeletal muscle may represent a compensatory mechanism against the impairment of the enzyme.
P626 Redistribution of Signaling Target for Insulin-Mimetic CijNTER MiiLLER, Stefan Deutschlond GmbH Disease Main, Germany
Proteins from Caveolae as Potential Compounds Welte, Susanne Wied. Aventis Pharma Group Metabolic Diseases, Frankfurt am
Phosphoinositolglycans (PIG) representing the polar glycan head group of the glycosylphosphatidylinositol (GPI) membrane anchor of GPI-modified plasma membrane proteins (GPI proteins) have been demonstrated to exert potent insulin-mimetic metabolic and signaling activity in isolated adipose and muscle cells. In rat adipocytes this activity depends on the presence of a 115-kDa polypeptide residing in caveolae of the plasma membrane where it may interact with GPI roteins as the natural ligands. Purpose of the Study: We investigated whether caveolar localization of certain inactive signaling molecules could provide a compartmental basis for their regulated activation by PIG and explain cross-talk with insulin signaling. Methods: It has been shown that caveolin, a principle component of caveolae membranes, functionally supresses the activity of dual acylated Src-family tyrosine kinases (e.g. p~59~Y”) through the so-called caveolin-scaffolding domain. Results: PIG (0.5 PM) as well as caveolin scaffolding domain peptide (25 PM, introduced into cells by electroporation) induced tyrosine phosphorylation of the insulin receptor substrates 1, 2 and 3 (IRS) independent of the insulin receptor kinase thereby eliciting potent stimulation of glucose transport and metabolism in isolated rat adipocytes accompanied by up to 7.5- and 3.7-fold, respectively, activation of ~~59~““. In correlation to p~59~Y” activation by PIG, several GPI proteins (e.g. Gcel) were significantly deprived from tbe caveolae relative to the resident proteins caveolin and the 115-kDa receptor. Conclusion: The dissociation of GPl proteins from the 115-kDa receptor protein by PIG structures may abolish the inhibitory effect of caveolin on p~59~fl via causing their dissociation and subsequent movement of pp59LY” into non-caveolar areas of the plasma membrane, where tyrosine phosphorylationof IRS and thereby initiation of metabolic insulin signaling occurs. Thus, redistribution of GPI proteins from caveolae may represent a novel target for insulin signal transduction therapy.
P627 The Acute and Chronic Stimulatory Effects of Endothelin-1 on Glucose lkansport Are Mediated by Distinct Pathways in 3T3-Ll AdipoCytes KENICHI ISHIBASHI, Takeshi Imamura, Prem M. Sharma, Satoshi Ugi, Jerrold M. Olefsky. Endocrinology and Metabolism, University of California LA Jolla, CA, United States of America We have recently shown that pretreatment with Endothelin-1 (ET-l) for 20 min stimulates GLUT4 translocation in a PI3-kinase dependent manner in 3T3-Ll adipocytes (JBC. 274.33691-5). This study presents another pathway by which ET-l potentiates glucose transport in 3T3-Ll adipocytes. ET-l treatment (10 nM) exhibits a biphasic effect on 2-deoxyglucose (2-DOG) uptake, with a rapid increase up to -2.5 fold within 20 min, followed by a transient decline at 1 h, reaching a maximal effect (-4 fold) at approximately 6 h and recovering almost to basal levels after 24 h. Insulin stimulation (3 ng/ml) leads to a monophasic effect on 2-DOG uptake, with a maximum increase at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-l induced glucose uptake both at 20 min and 6 h, while the ETB receptor
s157
antagonist, BQ 788, was without effect. Interestingly, ET-l stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-l involve distinct mechanisms. GLUT4 translocation assay showed that 20 min. but not 6 h, exposure to ET-l led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min. exposure to ET-l increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-l induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the Gai inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-l induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-Ll adipocytes.
P628 Relationship of Insulin Activation of Protein Kinase B in Human Muscle Cells with Myogeuic Diffenmtiation but Not Glucose Uptake or Glycogen Synthesis JAN KRUETZPBLDT, Christiana Kausch, Hans U. Haering, Michael Stumvoll. Medizinische Klinik n! Universitaet Tuebingen, Tuebingen, Germany Aim: Activation of protein kinase B (PKB) has been implicated in myogenic differentiation and insulin‘s metabolic effects in animal muscle cell lines. To evaluate analogous functions of PKB-activation in human muscle cells we compared cultured myotubes (MT) with high and low insulin-stimulated phosphorylation of serine 473 in PKB (SeP73). Methods: Myotubes derived from muscle biopsies from 20 healthy volunteers were screened for insulin-stimulated Se#73-phosphorylation by immunoblotting. Cell lines with high (H, 2.73+0.22relU, n=3) and low (L, 0.86f0.02relU. n=3; ~~0.05) insulin-induced Ser473-phosphorylation were selected and compared for muscle-specific markers, insulin signaling and action. Results: H exhibited thicker and longer multinucleated MT compared to L. Creatine kinase activity was higher in H (2.90f0.23 vs 0.62fO.O2relU, ~~0.05) as was expression of a-sarcomeric actin (2.35f0.06 vs 1.08f0.34relU, pcO.05). Insulin activated insulin receptor kinase (2.07f0.27 vs 1.78fO.l3relU, p=O.38) and IRS-l-associatedphosphatidylinositol 3-kinase (PI3K. 2.02f0.22 vs 1.76&0.27relU, p&51) were not different between H and L. In addition, insulin-stimulated glucose uptake (1.38f0.11 vs 1.47f0.08-fold basal, p=O.53) and glycogen synthesis (2.1 lf0.21 vs 2.05f0.18-fold basal, p&86) were not different. Reduced insulin-stimulated Ser“73-phosphorylation in L was not compensated for by protein kinase C-dependent mechanisms, since also in tbe presence of 20wM PKC inhibitor RO 31-8220 (RO) insulin-stimulated glucose uptake was not different between H and L (1.2540.07 vs 1.34f0.07-fold basal, p=O.40), while RO significantly reduced basal glucose uptake by 45% and 52%. respectively. Conclusions: These results suggest a potential regulatory role of PKB in myogenic differentiation of human muscle cells. Such a regulation seems not to involve PI3K. Maximal insulin activation of PKB is not a prerequisite for maximal insulin-stimulated glucose uptake or glycogen synthesis.
P629 GLUT4 Gene Expression in Insulin-Resistant Obese Rats Involves Post-Ihmscriptional Poly-A ‘Igil mRNA Modulation PATRICIA M. SERAPHIM, Thiago G.R. Assis, Claudia B. Volpato, Maria T. Nunes, Ubiratan E Machado. Physiology and Biophysics, Institute of Biomedical Sciences. University of &io Paulo, Srio Paula, Srio Paula, Brazil Recent reports have demonstrated that spontaneously obese12 month-old Wistar rats have insulin resistance which is accompanied by tissue specific