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• W2086250538 abstract "The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 μmol/kg bolus; 30 μmol/kg·min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versussaline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg·min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation;p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 ± 1.8versus 30 min = 42.8 ± 1.4 mg/kg·min,p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4,5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling. The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 μmol/kg bolus; 30 μmol/kg·min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versussaline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg·min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation;p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 ± 1.8versus 30 min = 42.8 ± 1.4 mg/kg·min,p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4,5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling. phosphatidylinositol 3-kinase glucose infusion rate glucose uptake IRS, insulin receptor substrate high performance liquid chromatography inositol 1,3,4,5-tetraphosphate The hexosamine biosynthetic pathway in skeletal muscle serves a vital role in the production of the amino sugars that are utilized in multiple glycosylation pathways. Increased biosynthetic activity within the hexosamine pathway is associated with the development of insulin resistance (1Rossetti L. Hawkins M. Chen W. Gindi J. Barzilai N. J. Clin. Invest. 1995; 96: 132-140Crossref PubMed Scopus (242) Google Scholar, 2Hawkins M. Barzilai N. Liu R. Hu M. Chen W. Rossetti L. J. Clin. Invest. 1997; 99: 2173-2182Crossref PubMed Scopus (268) Google Scholar, 3Baron A. Zhu J.-S. Zhu J.-H. Weldon H. Maianu L. Garvey W.T. J. Clin. Invest. 1995; 96: 2792-2801Crossref PubMed Scopus (238) Google Scholar, 4Robinson K.A. Sens D.A. Buse M.G. Diabetes. 1993; 42: 1333-1346Crossref PubMed Scopus (155) Google Scholar, 5Hawkins M. Angelov I. Liu R. Barzilai N. Rossetti L. J. Biol. Chem. 1996; 272: 4889-4895Abstract Full Text Full Text PDF Scopus (109) Google Scholar). In fact, increasing the amount of flux into the GlcN pathway by various means has been shown to induce defects in insulin-stimulated glucose uptake (1Rossetti L. Hawkins M. Chen W. Gindi J. Barzilai N. J. Clin. Invest. 1995; 96: 132-140Crossref PubMed Scopus (242) Google Scholar, 2Hawkins M. Barzilai N. Liu R. Hu M. Chen W. Rossetti L. J. Clin. Invest. 1997; 99: 2173-2182Crossref PubMed Scopus (268) Google Scholar, 4Robinson K.A. Sens D.A. Buse M.G. Diabetes. 1993; 42: 1333-1346Crossref PubMed Scopus (155) Google Scholar, 5Hawkins M. Angelov I. Liu R. Barzilai N. Rossetti L. J. Biol. Chem. 1996; 272: 4889-4895Abstract Full Text Full Text PDF Scopus (109) Google Scholar, 6Robinson K.A. Weinstein M.L. Lindenmeyer G.E. Buse M.G. Diabetes. 1995; 44: 1438-1446Crossref PubMed Scopus (133) Google Scholar), GLUT4 translocation (3Baron A. Zhu J.-S. Zhu J.-H. Weldon H. Maianu L. Garvey W.T. J. Clin. Invest. 1995; 96: 2792-2801Crossref PubMed Scopus (238) Google Scholar), and glycogen synthase activation (5Hawkins M. Angelov I. Liu R. Barzilai N. Rossetti L. J. Biol. Chem. 1996; 272: 4889-4895Abstract Full Text Full Text PDF Scopus (109) Google Scholar, 7Crook E.D. McClain D.A. Diabetes. 1996; 45: 322-327Crossref PubMed Scopus (38) Google Scholar, 8Crook E.D. Zhou J. Daniels M. Neidigh J.L. McClain D.A. Diabetes. 1995; 44: 314-320Crossref PubMed Scopus (59) Google Scholar). Entry into the hexosamine pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate via the rate-limiting enzyme glutamine fructose-6-P-amidotransferase (9Traxinger R.R. Marshall S.V. J. Biol. Chem. 1991; 266: 10148-10154Abstract Full Text PDF PubMed Google Scholar). The principal end product of the pathway is UDP-GlcNAc (10Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar), which modifies intracellular proteins by glycosylation. Thus, even modest perturbations of the amount of flux through the hexosamine pathway could have diverse effects on protein functions. The amount of flux into the pathway, estimated by the accumulation of UDP-GlcNAc in muscle, is strongly correlated with the degree of impairment in peripheral insulin action (5Hawkins M. Angelov I. Liu R. Barzilai N. Rossetti L. J. Biol. Chem. 1996; 272: 4889-4895Abstract Full Text Full Text PDF Scopus (109) Google Scholar). The mechanism(s) of GlcN-induced defects in insulin action and the early sequence of events resulting in peripheral insulin resistance are still uncertain (4Robinson K.A. Sens D.A. Buse M.G. Diabetes. 1993; 42: 1333-1346Crossref PubMed Scopus (155) Google Scholar, 11Hresko R. Heimberg H. Chi M.M.-Y. Mueckler M. J. Biol. Chem. 1998; 273: 20658-20668Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The variety of observed effects of GlcN may be compatible either with a proximal defect in the insulin signaling pathway or defects at more than one downstream site of insulin action. The p85/p110 phosphatidylinositol 3-kinase, (PI3K),1 is an important proximal effector in the insulin signaling cascade (12Ruderman N. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (395) Google Scholar, 13Endemann G. Yonezawa K. Roth R.A. J. Biol. Chem. 1990; 265: 396-400Abstract Full Text PDF PubMed Google Scholar). The metabolic actions of insulin mediated by PI3K include glucose uptake (14Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar), GLUT4 translocation (15Katagiri H. Asano T. Ishihara H. Inukai K. Shibasaki Y. Kikuchi M. Yazaki Y. Oka Y. J. Biol. Chem. 1996; 271: 16987-16990Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), and glycogen synthase activation (16Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (232) Google Scholar). Additionally, PI3K binds to all four insulin receptor substrates (IRS-1 to 4) as yet identified (17Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), highlighting its vital role in insulin signaling. Decreased PI3K activity in skeletal muscle has been observed in in vivo models of insulin resistance (18Heydrick S. Gautier N. Olichon-Berthe C. Van Obberghen E. Le Marchand-Brustel Y. Am. J. Physiol. 1995; 268: E604-E612Crossref PubMed Google Scholar, 19Tanti J.-F. Gremeaux T. Grillo S. Calleja V. Klippel A. Williams L. Van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1996; 271: 25227-25232Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Folli F. Saad M. Backer J.M. Kahn C.R. J. Clin. Invest. 1993; 92: 1787-1794Crossref PubMed Scopus (218) Google Scholar). Acute stimulation of PI3K by bolus insulin is known to achieve maximal levels within the first few minutes of a large bolus dose (11Hresko R. Heimberg H. Chi M.M.-Y. Mueckler M. J. Biol. Chem. 1998; 273: 20658-20668Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 18Heydrick S. Gautier N. Olichon-Berthe C. Van Obberghen E. Le Marchand-Brustel Y. Am. J. Physiol. 1995; 268: E604-E612Crossref PubMed Google Scholar, 21Goodyear L. Giorgino F. Sherman L. Carey J. Smith R. Dohm G. J. Clin. Invest. 1995; 95: 2195-2204Crossref PubMed Scopus (476) Google Scholar). However, prolonged stimulation of PI3K by insulin for 100 min has recently been demonstrated in human skeletal muscle (22Wojtaszewski J. Hansen B. Kiens B. Richter E. Diabetes. 1997; 46: 1775-1781Crossref PubMed Google Scholar). Consequently, we examined the effect of increased GlcN availability on both acute and sustained activation of skeletal muscle PI3K. Additionally, we examined the effect of GlcN on insulin-stimulated production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), probably the most metabolically important end product of PI3K (23Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1225) Google Scholar). We thereby defined the pattern and time sequence of GlcN-induced defects in proximal insulin signaling in skeletal muscle, particularly in the activation of the intracellular PI3K pool and on the peripheral metabolic actions of insulin. Eighty nine normal male Harlan Sprague-Dawley rats (Charles River Breeding Laboratories, Inc., Wilmington, MA) were housed in individual cages and subjected to a standard light (6 a.m. to 6 p.m.)-dark (6 p.m. to 6 a.m.) cycle. The rats were anesthetized with intraperitoneal injection of pentobarbital (50 mg/kg body weight), and indwelling catheters were inserted into the right internal jugular vein and the left carotid artery, as described previously (1Rossetti L. Hawkins M. Chen W. Gindi J. Barzilai N. J. Clin. Invest. 1995; 96: 132-140Crossref PubMed Scopus (242) Google Scholar,24Rossetti L. Laughlin M.R. J. Clin. Invest. 1989; 84: 892-899Crossref PubMed Scopus (108) Google Scholar, 25Rossetti L. Giaccari A. J. Clin. Invest. 1990; 85: 1785-1792Crossref PubMed Scopus (237) Google Scholar, 26Rossetti L. Hu M. J. Clin. Invest. 1993; 92: 2963-2974Crossref PubMed Scopus (63) Google Scholar). The venous catheter was extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. All in vivo studies were performed 5–7 days following catheter placement in awake, fasted, and unstressed rats. At the end of all the in vivo studies, rats were anesthetized (pentobarbital 60 mg/kg body weight, intravenously); the abdomen was quickly opened, and the rectus abdominal muscle was freeze-clampedin situ with aluminum tongs precooled in liquid nitrogen (1Rossetti L. Hawkins M. Chen W. Gindi J. Barzilai N. J. Clin. Invest. 1995; 96: 132-140Crossref PubMed Scopus (242) Google Scholar,24Rossetti L. Laughlin M.R. J. Clin. Invest. 1989; 84: 892-899Crossref PubMed Scopus (108) Google Scholar). The time from injection of the anesthetic to freeze clamping of the muscle was approximately 30 s. All tissue samples were stored at −80 °C for subsequent analysis. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committees of the Albert Einstein College of Medicine. Initial pilot studies (n = 12) using bolus doses of insulin between 0.5 and 1.0 unit/kg administered 1, 2, or 5 min prior to sacrifice indicated that maximal stimulation of PI3K by insulin was observed 2 min following a 1 unit/kg bolus of insulin (data not shown). Thus, in the current protocol, fasting animals received intra-arterial boluses of insulin (1 unit/kg) followed 1.5 min later by the intravenous administration of pentobarbital. Freeze-clamped rectus muscle was then obtained 2 min following the insulin bolus. The effect of primed continuous infusions of GlcN (750 μmol/kg bolus, 30 μmol/kg·min) on the ability of insulin to maximally stimulate PI3K activity was assessed by infusion of GlcN (n = 6) versussaline (n = 8) for 2 h, prior to the administration of insulin. Plasma samples were obtained via the venous catheter both before the insulin bolus and at the time of sacrifice for measurement of plasma glucose, insulin, and GlcN concentrations. Euglycemic, hyperinsulinemic (18 milliunits/kg·min) clamp studies were performed for 3 h in combination with [3-3H]glucose infusion as described previously (1Rossetti L. Hawkins M. Chen W. Gindi J. Barzilai N. J. Clin. Invest. 1995; 96: 132-140Crossref PubMed Scopus (242) Google Scholar, 24Rossetti L. Laughlin M.R. J. Clin. Invest. 1989; 84: 892-899Crossref PubMed Scopus (108) Google Scholar, 26Rossetti L. Hu M. J. Clin. Invest. 1993; 92: 2963-2974Crossref PubMed Scopus (63) Google Scholar). GlcN (750 μmol/kg bolus, 30 μmol/kg·min) was administered throughout the 3-h insulin clamp studies (n = 5), while time control euglycemic, hyperinsulinemic clamp studies were performed with infusion of saline for 3 h in additional age- and weight-matched control rats (n = 6). A primed continuous infusion of HPLC-purified [3H-3]glucose (NEN Life Science Products; 8 μCi bolus, 0.4 μCi/min) was infused throughout the studies in order to measure the rates of peripheral glucose uptake, glycolysis, and glycogen synthesis. A variable infusion of 25% glucose solution was started at time 0 and adjusted every 10 min, maintaining basal plasma glucose concentrations (∼7 mm) throughout. Plasma samples were obtained for determination of [3H]glucose-specific activity at 10-min intervals throughout the insulin infusions. Samples for measurement of plasma insulin and GlcN concentrations were obtained at times 0, 60, 120, and 180 min. The total volume of blood sampled was ∼3.0 ml/study; to prevent volume depletion and anemia, a solution (1:1 v/v) of ∼3.0 ml of fresh blood (obtained by heart puncture from a littermate of the test animal) and heparinized saline (10 units/ml) was infused throughout. Primed continuous infusions of GlcN were administered for variable time intervals during the 2-h euglycemic, hyperinsulinemic (18 milliunits/kg·min) clamp studies to establish a time course for the effects of GlcN on insulin-mediated glucose uptake and on insulin-stimulated PI3K activity. Thus, GlcN (750 μmol/kg bolus, 30 μmol/kg·min) was administered throughout the 2-h insulin clamp studies (designated 120 min, n = 5) or during the final 30 (30 min, n = 5), 60 (60 min,n = 5), or 90 min (90 min, n = 5) of the protocols. Time control euglycemic hyperinsulinemic (18 milliunits/kg·min) clamp studies were performed with infusion of saline for 2 h in an additional n = 12 age- and weight-matched control rats. Primed continuous infusions of HPLC-purified [3-3H]glucose (8 μCi bolus, 0.4 μCi/min) were infused throughout the studies, and plasma samples were obtained for determination of [3H]glucose-specific activity, insulin, and GlcN concentrations as described above. Assessment of basal PI3K activity was performed in additional fasting animals in the presence of 2-h infusions of GlcN (750 μmol/kg bolus, then 30 μmol/kg·min;n = 8) or saline (n = 12). The rates of glycolysis were estimated as described previously (24Rossetti L. Laughlin M.R. J. Clin. Invest. 1989; 84: 892-899Crossref PubMed Scopus (108) Google Scholar, 25Rossetti L. Giaccari A. J. Clin. Invest. 1990; 85: 1785-1792Crossref PubMed Scopus (237) Google Scholar, 26Rossetti L. Hu M. J. Clin. Invest. 1993; 92: 2963-2974Crossref PubMed Scopus (63) Google Scholar). Briefly, plasma-tritiated water-specific activity was determined by liquid scintillation counting of the protein-free supernatant (Somogyi filtrate) before and after evaporation to dryness. Since tritium on the C-3 position of glucose is lost to water during glycolysis, it can be assumed that plasma tritium is present either in the form of tritiated water or [3-3H]glucose (24Rossetti L. Laughlin M.R. J. Clin. Invest. 1989; 84: 892-899Crossref PubMed Scopus (108) Google Scholar). The rates of peripheral glycogen synthesis during the insulin clamp studies were estimated as the difference between the rates of glucose uptake and glycolysis. Muscle glycogen concentration was determined following digestion with amyloglucosidase as described previously (24Rossetti L. Laughlin M.R. J. Clin. Invest. 1989; 84: 892-899Crossref PubMed Scopus (108) Google Scholar). Muscle glycogen synthase activity was measured by a modification (24Rossetti L. Laughlin M.R. J. Clin. Invest. 1989; 84: 892-899Crossref PubMed Scopus (108) Google Scholar, 26Rossetti L. Hu M. J. Clin. Invest. 1993; 92: 2963-2974Crossref PubMed Scopus (63) Google Scholar) of the method of Thomas et al. (27Thomas J.A. Schlender K.K. Larner J. Anal. Biochem. 1968; 25: 486-499Crossref PubMed Scopus (949) Google Scholar) and is based on the measurement of the incorporation of radioactivity into glycogen from UDP-[U-14C]glucose. Frozen rectus muscle samples were pulverized in liquid nitrogen and placed in ice-cold lysis buffer (NaCl 140 mm, Tris·HCl 10 mm, CaCl2 1 mm, MgCl2 1 mm, aprotinin 10 μg/ml, leupeptin 50 μm, sodium vanadate 2 mm, phenylmethylsulfonyl fluoride 1 mm, glycerol 10%; total dilution 1:3). Samples were immediately homogenized on ice with a Tissumizer at moderate speed, using 3 cycles of 20 s each. Nonidet P-40 1% by volume was added to each tube, and the mixture was rotated for 1 h at 4 °C. Post-13,000 × g supernatants were immunoprecipitated with αp85 antibody (28Backer J.M. Myers Jr., M.G. Sun X.-J. Chin D.J. Shoelson S. Miralpeix M. White M.F. J. Biol. Chem. 1993; 268: 8204-8212Abstract Full Text PDF PubMed Google Scholar) overnight and then assayed for PI3K activity by the method of Ruderman (12Ruderman N. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (395) Google Scholar). Each set of assayed samples included tissues from at least 2 fasted and insulin-stimulated animals, respectively, and PI3K activity in each sample was expressed as a multiple of the average fasting activity in that sample set. PtdIns(3,4,5)P3 concentration in skeletal muscle samples was determined by a highly specific radioligand displacement assay, adapted from the method of van der Kaay et al. (29van der Kaay J. Batty I.H. Cross D.A.E. Watt P.W. Downes C.P. J. Biol. Chem. 1997; 272: 5477-5481Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Ins(1,3,4,5)P4-binding protein was generated from BL21 cells transfected with GST-ΔC2 GAP1IP4BP (kind gift of Drs. Derek Brazil and Morris White of Boston) following induction with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside. The cell lysate was applied twice to a ProBond Resin column containing 50% glutathione-Sepharose. The glutathione-Sepharose beads were washed twice with phosphate-buffered saline (with EGTA 1 mm, EDTA 1 mm, and β-mercaptoethanol 1 mm) and twice with 75 mm Tris, pH 8.0, and 300 mm NaCl and then suspended in 75 mm Tris and 300 mm NaCl with 50% glycerol and stored at −20 °C. Preparation of skeletal muscle samples involved alkaline hydrolysis of tissue phospholipid extract to generate Ins(1,3,4,5)P4 from PtdIns(3,4,5)P3. Briefly, 200 mg of frozen muscle was homogenized in 2 ml of 10% trichloroacetic acid and then centrifuged at 3,000 rpm for 10 min. 3 ml of EDTA 10 mm were added to the pellet, which was then centrifuged at 3,000 rpm for 10 min. The pellet was washed twice with chloroform/MeOH and then extracted with chloroform/MeOH/HCl (40:80:1). Following centrifugation at 3,000 rpm for 20 min, the chloroform was taken to dryness under N2. 200 μl of 1 m KOH was added to the pellet, which was put in a boiling water bath for 30 min. The pH was then raised to 5.0 with 1 m acetic acid. Two extractions were performed with 2 ml of butanol/petroleum ether/ethyl acetate (20:4:1) to remove fatty acids, and then the samples were dried and stored at −20 °C. On the day of the assay the samples were resuspended in 200 μl of 40 mm acetic acid. The assay was performed by measurement of displaced labeled [3H]Ins(1,3,4,5)P4 (NEN Life Science Products) by the tissue extract. To 10 μl of a suspension of protein-bound glutathione beads, 40 μl of the tissue extract and 1.5 × 104 dpm of [3H]Ins(1,3,4,5)P4 were added, to a total volume of 200 μl in assay buffer (0.1 m NaAc, 0.1m KH2PO4, pH 5.0, 4 mmEDTA). The samples were incubated in the cold for 30 min and the beads separated by centrifugation and washed once. Radioactivity was determined by scintillation counting of the washed beads. All samples were assayed in duplicate. Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Inc., Palo Alto, CA) and plasma insulin by radioimmunoassay using rat and porcine insulin standards. Plasma [3H]glucose radioactivity was measured in duplicate on the supernatants of Ba(OH)2 and ZnSO4 precipitates of plasma samples after evaporation to dryness to eliminate tritiated water. Regression analysis of the slopes of 3H2O rate of appearance (used in the calculation of the rates of glycolysis) was performed at 60-min intervals throughout the insulin clamp studies. Muscle UDP-Glc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc concentrations were obtained through 2 sequential chromatographic separations and UV detection (30Rossetti L. Lee Y. Ruiz J. Aldridge S. Shamoon H. Boden G. Am. J. Physiol. 1993; 265: E761-E769PubMed Google Scholar, 31Giaccari A. Rossetti L. J. Clin. Invest. 1992; 89: 36-45Crossref PubMed Scopus (75) Google Scholar). UDP-GlcNAc and UDP-GalNAc co-elute with UDP-Glc and UDP-Gal during the solid phase extraction. The retention times for UDP-Glc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc were 28.5, 30.7, 33.9, and 35.4 min, respectively. Plasma GlcN concentrations were determined by high performance liquid chromatography (HPLC) following quantitative derivatization with phenylisothiocyanate as described by Anumula and Taylor (32Anumula K.R. Taylor P.B. Anal. Biochem. 1991; 197: 113-120Crossref PubMed Scopus (39) Google Scholar). All HPLC analyses were performed on a Waters HPLC system using a reverse-phase, ion pairing isocratic method, on two C18T (Supelco) reverse-phase columns (0.46 × 25 cm) in series. ATP concentrations in skeletal muscle were measured as follows: freeze-clamped skeletal muscle samples were finely pulverized in liquid nitrogen and then stirred into ice-cold 3 mHClO4 (3 ml/g) in an alcohol bath maintained at −10 °C. Following complete penetration of acid into tissue powder, tissue extracts were diluted with 1 ml of H2O, 0.3 ml of extract, rotated at 4 °C for 10 min, then centrifuged at 5,000 ×g for 10 min. Supernatants were removed and neutralized to pH 7 with 2 m KOH and 0.4 m imidazole base. ATP concentrations in neutralized supernatants were calculated from fluorometric measurements of NADPH generated in a two-step reaction, following the method of Passonneau and Lowry (33Passonneau J.V. Lowry O.H. Enzymatic Analysis: A Practical Guide. Humana Press Inc., Totawa, NJ1993: 122-123Google Scholar). All values are presented as the mean ± S.E. Comparisons between groups were made using repeated measures analysis of variance where appropriate. Where F ratios were significant, further comparisons were made using Student's t tests (paired difference test and small sample test for independent samples, as appropriate). At the time of the study, the mean body weight of the animals was similar in all groups and averaged 309 ± 16 g. The mean plasma glucose, free fatty acid, and insulin concentrations at base line (0 h) were also similar in all groups. We assessed the effect of 2-h infusions of GlcN versus saline on acute insulin stimulation of PI3K, 2 min following intra-arterial bolus of insulin (1 units/kg) in normal rats (Fig. 2). The administration of bolus insulin resulted in rapid elevations in plasma insulin levels from fasting levels of 25 ± 5 microunits/ml to >1000 microunits/ml after 2 min. Whereas in saline-infused animals, bolus insulin induced a 3.3-fold stimulation of PI3K activity, there was only a 1.9-fold stimulation by insulin in the glucosamine-treated group. Of note, there was a tendency toward small increases in basal (non-insulin-stimulated) PI3K activity following 2 h of GlcN infusion, but these did not achieve statistical significance. We next examined the effect of more prolonged (3 h) insulin administration on the activation of PI3K in skeletal muscle and whether this was also impaired in the presence of increased GlcN availability. Primed continuous infusion of GlcN for 3 h during the insulin clamp studies resulted in sustained elevations in plasma GlcN levels, achieving a plateau by 90 min of infusion and maximal circulating GlcN levels of 2.3 ± 0.5 mm following 3 h. The plasma glucose concentration was maintained at basal levels throughout all clamp studies. The plasma insulin levels were maintained constant at ∼600 microunits/ml throughout. The coefficients of variation in plasma glucose and insulin levels were less than 5 and 10%, respectively, in all studies. These elevations in plasma GlcN levels resulted in marked and progressive decreases in insulin-mediated glucose disposal (Fig. 3 A). There were comparable decreases in both glucose infusion rates (GIR, 180 min = 31.1 ± 2.0versus 30–60 min = 37.8 ± 1.4 mg/kg·min;p < 0.005) and glucose uptake (Rd, 180 min = 37.2 ± 1.8 versus 30–60 min = 44.6 ± 1.2 mg/kg·min; p < 0.005) with GlcN infusion, with a similar time of onset (p < 0.05 at 110 min). Consistent with previous observations (1Rossetti L. Hawkins M. Chen W. Gindi J. Barzilai N. J. Clin. Invest. 1995; 96: 132-140Crossref PubMed Scopus (242) Google Scholar), there were no significant reductions in either the GIR (180 min = 30.5 ± 3.6versus 30–60 min = 32.3 ± 3.0 mg/kg·min;p > 0.05) or Rd (180 min = 38.6 ± 3.1versus 30–60 min = 40.9 ± 2.7 mg/kg·min;p > 0.05) during the 3-h time control insulin clamp studies with saline infusion. Fig. 3 A depicts the GIR at 10-min intervals throughout the 3-h insulin clamp studies with GlcN infusion (GlcN 180 min), and throughout the time control studies (SalI 80 min). Following the observed decreases in Rd and GIR in the presence of increased GlcN availability, more prolonged infusion of GlcN also resulted in impairments in the rates of peripheral glycogen synthesis (180 min = 21.7 ± 1.7 versus 30 min = 26.3 ± 2.0 mg/kg·min; p < 0.01) which first became significant by 160 min of GlcN infusion (p < 0.05). We measured PI3K activity following 3-h euglycemic, hyperinsulinemic clamp studies with infusions of either GlcN or saline throughout. The infusion of GlcN for 3 h throughout insulin clamp studies resulted in a marked impairment in the ability of insulin (∼600 microunits/ml) to stimulate PI3K activity (Fig. 3 B), such that there was only a 1.2-fold stimulation of PI3K activity with GlcN infusion, relative to a 2.4-fold stimulation in the time control group. Once we established that the defect in insulin-stimulated PI3K activity was also detectable following prolonged insulin infusions, we performed time course studies to determine the sequence of various GlcN-induced defects in insulin action and the length of time required for their onset. These observations allowed us to examine the time course of the effect of GlcN on PI3K vis à vis other metabolic actions of insulin. Primed continuous infusion of GlcN for variable durations during the course of 2-h insulin clamp studies resulted in very rapid elevations in plasma GlcN levels, to 1.1 ± 0.2 mm by 60 min of primed continuous infusion (Table I), again reaching a plateau by 90 min of infusion. Fig. 4 Adepicts the GIR at 10-min intervals throughout the 2-h insulin clamp studies with GlcN infusion over variable time intervals and throughout time control studies with saline infusion. Again, significant decreases in Rd and GIR were observed following 110 min of GlcN infusion (p < 0.05 versus 30 min) and were thus only seen in the group which received GlcN infusion throughout the 2-h studies (GlcN 120 min).Table IPlasma concentrations of glucose, free fatty acids (FFA), glucosamine, an" @default.
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• W2086250538 title "Discordant Effects of Glucosamine on Insulin-stimulated Glucose Metabolism and Phosphatidylinositol 3-Kinase Activity" @default.
• W2086250538 cites W1502152603 @default.
• W2086250538 cites W1525154317 @default.
• W2086250538 cites W1541173869 @default.
• W2086250538 cites W1551443142 @default.
• W2086250538 cites W1568605582 @default.
• W2086250538 cites W1568866400 @default.
• W2086250538 cites W1569718989 @default.
• W2086250538 cites W1588589182 @default.
• W2086250538 cites W1590732591 @default.
• W2086250538 cites W1612870394 @default.
• W2086250538 cites W1896937632 @default.
• W2086250538 cites W1900171978 @default.
• W2086250538 cites W1947575656 @default.
• W2086250538 cites W1971547187 @default.
• W2086250538 cites W1974052445 @default.
• W2086250538 cites W1989294105 @default.
• W2086250538 cites W1993842351 @default.
• W2086250538 cites W1994667169 @default.
• W2086250538 cites W1999351520 @default.
• W2086250538 cites W2001866120 @default.
• W2086250538 cites W2005659898 @default.
• W2086250538 cites W2031087463 @default.
• W2086250538 cites W2031504013 @default.
• W2086250538 cites W2034397029 @default.
• W2086250538 cites W2037526260 @default.
• W2086250538 cites W2044211587 @default.
• W2086250538 cites W2045025174 @default.
• W2086250538 cites W2047965820 @default.
• W2086250538 cites W2048670619 @default.
• W2086250538 cites W2049113370 @default.
• W2086250538 cites W2056925433 @default.
• W2086250538 cites W2058138221 @default.
• W2086250538 cites W2077903723 @default.
• W2086250538 cites W2081698610 @default.
• W2086250538 cites W2092045274 @default.
• W2086250538 cites W2095352859 @default.
• W2086250538 cites W2095736731 @default.
• W2086250538 cites W2101318760 @default.
• W2086250538 cites W2103642791 @default.
• W2086250538 cites W2109300167 @default.
• W2086250538 cites W2109607186 @default.
• W2086250538 cites W2114508339 @default.
• W2086250538 cites W2133550632 @default.
• W2086250538 cites W2153074450 @default.
• W2086250538 cites W2155674538 @default.
• W2086250538 cites W2156053266 @default.
• W2086250538 cites W2163166760 @default.
• W2086250538 cites W89133559 @default.
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