Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-α (PKCα) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms α and δ, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKδ, but not of DGKα expression, was sufficient to prevent the effect of high glucose on PKCα activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCα activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism. Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-α (PKCα) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms α and δ, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKδ, but not of DGKα expression, was sufficient to prevent the effect of high glucose on PKCα activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCα activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism. Prolonged hyperglycemia is a key contributor in development and progression of diabetic complications (1Stratton I.M. Kim A.I. Neil H.A. Matthews D.R. Manley S.E. Cull C.A. Hadden D. Turner R.C. Holman R.R. Br. Med. J. 2000; 321: 405-412Crossref PubMed Scopus (6958) Google Scholar). It has also been established that chronically elevated glucose levels may worsen insulin sensitivity and impair insulin control of glucose metabolism (2Tomas E. Kim Y.S. Dagher Z. Saha A. Luo Z. Ido Y. Ruderman N.B. Ann. N. Y. Acad. Sci. 2002; 967: 43-51Crossref PubMed Scopus (111) Google Scholar). This is due, at least in part, to persistent activation of conventional isoforms of the protein kinase C (PKC), 3The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; DGK, diacylglycerol kinase; PA, phosphatidic acid; 2-DG, 2-deoxyglucose; IR, insulin receptor; IRS, IR substrate. which in turn, down-regulate insulin signaling either by direct phosphorylation of insulin receptor (IR) and insulin receptor substrates (IRSs) or by indirect mechanisms (3Shmueli E. Kim K.G. Record C.O. J. Intern. Med. 1993; 234: 397-400Crossref PubMed Scopus (58) Google Scholar, 4Way K.J. Kim N. King G.L. Diabet. Med. 2001; 12: 945-959Crossref Scopus (276) Google Scholar, 5Miele C. Kim A. Maitan M.A. Oriente F. Romano C. Formisano P. Giudicelli J. Beguinot F. Van Obberghen E. J. Biol. Chem. 2003; 278: 47376-47387Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The latter include regulation of gene expression and stress-related responses (6Green K. Kim M.D. Murphy M.P. Diabetes. 2004; 53: S110-S118Crossref PubMed Google Scholar, 7Stenina O.I. Curr. Pharm. Res. 2005; 11: 2367-2381Crossref PubMed Scopus (38) Google Scholar). Increased activity of the conventional PKC and elevated levels of diacylglycerol (DAG), the main endogenous activator, have also been documented in several tissues from animal models and diabetic individuals (4Way K.J. Kim N. King G.L. Diabet. Med. 2001; 12: 945-959Crossref Scopus (276) Google Scholar, 8Considine R.V. Kim M.R. Allen L.E. Morales L.M. Triester S. Serrano J. Colberg J. Lanza-Jacoby S. Caro J.F. J. Clin. Invest. 1995; 95: 2938-2944Crossref PubMed Scopus (136) Google Scholar, 9Ceolotto G. Kim A. Miola M. Sartori M. Trevisan R. Del Prato S. Semplicioni A. Avogaro A. Diabetes. 1999; 48: 1316-1322Crossref PubMed Scopus (89) Google Scholar, 10Wolf B.A. Kim J.R. Easom R.A. Chang K. Sherman W.R. Turk J. J. Clin. Invest. 1991; 87: 31-38Crossref PubMed Scopus (172) Google Scholar). On the other hand, evidence exists indicating that glucose acutely activates its own utilization, both in vitro and in vivo (11Capaldo B. Kim D. Riccardi G. Pernotti N. Sacca L. J. Clin. Invest. 1986; 77: 1285-1290Crossref PubMed Scopus (53) Google Scholar, 12Galante P. Kim L. Kellerer M. Berti L. Tippmer S. Bossenmaier B. Fujiwara T. Okuno A. Horikoshi H. Haring H.U. Diabetes. 1995; 44: 646-651Crossref PubMed Google Scholar, 13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In vivo, these regulatory actions have been attributed to the mass action effect of glucose (14Saccà L. Kim D. Cicala M. Trimarco B. Ungano B. Metabolism. 1981; 30: 457-461Abstract Full Text PDF PubMed Scopus (9) Google Scholar). Subsequently, it has been shown that acute hyperglycemia per se may increase muscle membrane content of GLUT4 (12Galante P. Kim L. Kellerer M. Berti L. Tippmer S. Bossenmaier B. Fujiwara T. Okuno A. Horikoshi H. Haring H.U. Diabetes. 1995; 44: 646-651Crossref PubMed Google Scholar, 15Nolte L.A. Kim J. Wahlstrom E.O. Craig B.W. Zierath J.R. Wallberg-Henriksson H. Diabetes. 1995; 44: 1345-1348Crossref PubMed Scopus (52) Google Scholar). Although several studies have investigated the chronic effect of high glucose concentrations on PKC activity (2Tomas E. Kim Y.S. Dagher Z. Saha A. Luo Z. Ido Y. Ruderman N.B. Ann. N. Y. Acad. Sci. 2002; 967: 43-51Crossref PubMed Scopus (111) Google Scholar, 3Shmueli E. Kim K.G. Record C.O. J. Intern. Med. 1993; 234: 397-400Crossref PubMed Scopus (58) Google Scholar, 4Way K.J. Kim N. King G.L. Diabet. Med. 2001; 12: 945-959Crossref Scopus (276) Google Scholar), the molecular mechanisms of the short term autoregulatory effect have been only partially elucidated. In a previous work we have shown that, in a skeletal muscle cell model, acute exposure to increasing glucose concentrations determined a parallel increase in glucose uptake and its intracellular metabolism (13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Glucose autoregulation involves PKCα retrotranslocation from the membrane to the cytoplasm and dissociation from the IR. This is followed by the transient trans-activation of the IR tyrosine kinase and a consequent induction of glucose uptake (13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, how glucose acutely modulates PKCα remains to be investigated. The regulation of DAG intracellular levels by acute hyperglycemia represents an attractive hypothesis. DAG is a lipid second messenger with important signaling functions (3Shmueli E. Kim K.G. Record C.O. J. Intern. Med. 1993; 234: 397-400Crossref PubMed Scopus (58) Google Scholar, 15Nolte L.A. Kim J. Wahlstrom E.O. Craig B.W. Zierath J.R. Wallberg-Henriksson H. Diabetes. 1995; 44: 1345-1348Crossref PubMed Scopus (52) Google Scholar). Generation and removal of diacylglycerol are indeed critical for different intracellular signaling pathways (16Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5760) Google Scholar, 17Hou W. Kim Y. Morisset J. Cell.. Signal. 1996; 8: 487-496Crossref PubMed Scopus (9) Google Scholar, 18Bagnato C. Kim R.A. J. Biol. Chem. 2003; 278: 52203-52211Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In response to many extracellular stimuli DAG is generated through the action of phospholipases (mainly PLC and PLD) (17Hou W. Kim Y. Morisset J. Cell.. Signal. 1996; 8: 487-496Crossref PubMed Scopus (9) Google Scholar). The removal of DAG is largely operated by specific enzymes of the diacylglycerol kinase (DGK) family. DGK phosphorylates DAG to produce phosphatidic acid (PA) and plays an important role in signal transduction by modulating the balance between these two lipids (17Hou W. Kim Y. Morisset J. Cell.. Signal. 1996; 8: 487-496Crossref PubMed Scopus (9) Google Scholar). By controlling the cellular levels of DAG, DGK can serve as a negative regulator of PKC (16Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5760) Google Scholar). To date, ten DGK isozymes have been identified in mammals and divided into five classes based on their primary structure (19Kanoh H. Kim K. Sakane F. J. Biochem. 2002; 131: 629-633Crossref PubMed Scopus (116) Google Scholar, 20Imai S. Kim M. Yasuda S. Kanoh H. Sakane F. J. Biol. Chem. 2005; 280: 39870-39881Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). DGKs feature a conserved catalytic domain at the C terminus and, within the regulatory domain at the N terminus, possess two or three cysteine-rich regions homologous to the C1A and C1B motifs of PKC (21Van Blitterswijk W.J. Kim B. Cell Signal. 2000; 12: 595-605Crossref PubMed Scopus (229) Google Scholar). Moreover, these enzymes share other conserved motifs that are likely to play a role in lipid-protein and protein-protein interactions in various signaling pathways dependent on DAG and/or PA production (22Topham M.K. J. Cell. Biochem. 2006; 97: 474-484Crossref PubMed Scopus (119) Google Scholar). The differences in the regulatory domains of the various sub-types, together with the differential tissue expression pattern of the different isoforms suggest that the regulation of DGKs varies among cell types and/or in response to different stimuli and that the DGK isoforms serve distinct although related functions (23Kai M. Kim F. Imai S. Wada I. Kanoh H. J. Biol. Chem. 1994; 269: 18492-18498Abstract Full Text PDF PubMed Google Scholar, 24Ding L. Kim M. Topham M.K. McIntyre Zimmerman T.M. Prescott S.M. G.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5519-5524Crossref PubMed Scopus (56) Google Scholar). DGK may serve as an off switch controlling PKC activation, thereby mediating the acute glucose regulation of its own metabolism. In the present work we have investigated the mechanism involved in acute regulation of PKCα activity by glucose. We have found that the acute exposure to high glucose concentration leads to a decrease in DAG levels and concomitantly impairs the enzymatic activity and translocation of PKCα. This effect is mediated by glucose action on DGK activity and subcellular localization. Materials—Media and sera for tissue culture and the transfection reagent, N-[1-(2,3-diomeoyloxy)propyl]-N,N,N-trimethylammonium chloride/dioleoylphosphatidylethanolamine, were purchased from Invitrogen. Electrophoresis reagents were from Bio-Rad. Protein A-Sepharose beads was from Pierce. Radiochemicals, Western blot, ECL reagents, and the DAG quantification test kit and the PKC enzyme assay system were purchased from Amersham Biosciences. Polyclonal GLUT4 antibody was from Biogenesis (Sandown, NH). Polyclonal antibody toward PKC was from Invitrogen. Polyclonal antibodies toward DGKα and DGKδ have been previously generated and characterized (25Kanoh H. Kim T. Ono T. Suzuki T. J. Biol. Chem. 1986; 261: 5597-5602Abstract Full Text PDF PubMed Google Scholar, 26Sakane F. Kim S. Kai M. Wada I. Kanoh H. J. Biol. Chem. 1996; 271: 8394-8401Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). DGKζ antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents, including the DGK inhibitor R59949, were from Sigma. Cell Culture—The L6 cell clones expressing the wild-type human insulin receptors have been previously characterized and described (13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium containing 25 mm glucose and supplemented with 2% fetal bovine serum as described previously (13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and used at the myotube stage of differentiation. Determination of DAG Cellular Content—DAG content was quantified radioenzymatically by incubating aliquots of the lipid extract with DAG kinase and [32P]ATP, as described by Preiss et al. (27Preiss J. Kim C.R. Bishop R.W. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar). The manufacturer's instructions for the commercially available DAG test kit were followed. The 32P-labeled PA was purified using chloroform/methanol/acetic acid (65: 15:5, v/v) as a solvent system and quantified with a Storm 860 PhosphorImager (Amersham Biosciences). Western Blot Analysis—Western blot analysis was performed as previously reported (12Galante P. Kim L. Kellerer M. Berti L. Tippmer S. Bossenmaier B. Fujiwara T. Okuno A. Horikoshi H. Haring H.U. Diabetes. 1995; 44: 646-651Crossref PubMed Google Scholar, 28Fiory F. Kim A.T. Miele C. Oriente F. Esposito I. Corbo V. Ruvo M. Tizzano B. Rasmussen T.E. Grammeltoft S. Formisano P. Beguinot F. Mol. Cell. Biol. 2005; 25: 10803-10814Crossref PubMed Scopus (13) Google Scholar). Briefly, cells were rinsed and incubated in glucose-free buffer (20 mm Hepes, pH 7.8, 120 mm NaCl, 5 mm KCl, 2.5 mm MgSO4, 10 mm NaHCO3, 1,3 mm CaCl2, 1.2 mm KH2PO4, 0.25% bovine serum albumin) for 3 h. The cells were subsequently incubated for 5 and 30 min in the same buffer supplemented with the indicated concentrations of glucose or R59949, as indicated. Then the cells were solubilized in lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 10 mm EDTA, 10 mm Na2P2O7, 2 mm Na3VO4, 100 mm NaF, 10% glycerol, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin) for 2 h at 4°C. Cell lysates were clarified by centrifugation at 5000 × g for 20 min, separated by SDS-PAGE, and transferred into 0.45-μm Immobilon-P membranes (Millipore, Bedford, MA). Upon incubation with primary and secondary antibodies, immunoreactive bands were detected by ECL according to the manufacturer's instructions. Purified Plasma Membrane Preparations—Purified plasma membrane preparations were obtained as in Caruso et al. (13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) with slight modifications. Briefly, the cells, after exposure to glucose or R59949 for the indicated times, were further incubated for 10 min in glucose-free buffer, washed in ice-cold phosphate-buffered saline, and homogenized in 500 μl of ice-cold fractionation buffer (20 mm HEPES-NaOH, pH 7.4, 250 mm sucrose, 25 mm sodium fluoride, 1 mm sodium pyrophosphate, 0.1 mm sodium orthovanadate, 2 μm microcystin LR, 1 mm benzamidine) by passing them 10 times through a 22-gauge needle. The cell lysates were centrifuged at 800 × g for 5 min at 4 °C. Supernatants were further centrifuged at 100,000 × g for 20 min at 4 °C. The final supernatants were collected and used as the cytosolic fraction. The membrane pellet was solubilized in Buffer A containing 1% Triton X-100 by bath sonication and centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was used as the membrane fraction. Cytosolic and membrane fractions were then analyzed by Western blot. Purity (>90%) of the subcellular fractions was assessed by immunoblot with antibody against the β-subunit of the insulin-like growth factor-1 receptor (as control of membrane fraction) and against β-actin (as control of cytosolic fraction). Determinations of 2-Deoxy-d-glucose Uptake—For 2-deoxyglucose (2-DG) uptake studies, cells were rinsed and incubated in glucose-free buffer (20 mm Hepes, pH 7.8, 120 mm NaCl, 5 mm KCl, 2.5 mm MgSO4, 10 mm NaHCO3, 1,3 mm CaCl2, 1.2 mm KH2PO4, 0.25% bovine serum albumin) for 3 h. The cells were subsequently incubated for 3 min in the same buffer supplemented with the indicated concentrations of glucose or/and R59949, washed again, and incubated for further 10 min in glucose-free buffer containing 2-DG (final concentration, 0.15 mm) and 0.5 μCi/assay [14C]2-DG (13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The cells were finally lysed, and 2-DG uptake was determined by liquid scintillation counting. Protein Kinase C Assays—Determination of PKC activity was achieved with a commercially available kit (Invitrogen, catalogue number 13161-013). This assay kit is based on measurement of phosphorylation of the synthetic peptide from myelin basic protein Ac-MBP (4Way K.J. Kim N. King G.L. Diabet. Med. 2001; 12: 945-959Crossref Scopus (276) Google Scholar, 5Miele C. Kim A. Maitan M.A. Oriente F. Romano C. Formisano P. Giudicelli J. Beguinot F. Van Obberghen E. J. Biol. Chem. 2003; 278: 47376-47387Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 6Green K. Kim M.D. Murphy M.P. Diabetes. 2004; 53: S110-S118Crossref PubMed Google Scholar, 7Stenina O.I. Curr. Pharm. Res. 2005; 11: 2367-2381Crossref PubMed Scopus (38) Google Scholar, 8Considine R.V. Kim M.R. Allen L.E. Morales L.M. Triester S. Serrano J. Colberg J. Lanza-Jacoby S. Caro J.F. J. Clin. Invest. 1995; 95: 2938-2944Crossref PubMed Scopus (136) Google Scholar, 9Ceolotto G. Kim A. Miola M. Sartori M. Trevisan R. Del Prato S. Semplicioni A. Avogaro A. Diabetes. 1999; 48: 1316-1322Crossref PubMed Scopus (89) Google Scholar, 10Wolf B.A. Kim J.R. Easom R.A. Chang K. Sherman W.R. Turk J. J. Clin. Invest. 1991; 87: 31-38Crossref PubMed Scopus (172) Google Scholar, 11Capaldo B. Kim D. Riccardi G. Pernotti N. Sacca L. J. Clin. Invest. 1986; 77: 1285-1290Crossref PubMed Scopus (53) Google Scholar, 12Galante P. Kim L. Kellerer M. Berti L. Tippmer S. Bossenmaier B. Fujiwara T. Okuno A. Horikoshi H. Haring H.U. Diabetes. 1995; 44: 646-651Crossref PubMed Google Scholar, 13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 14Saccà L. Kim D. Cicala M. Trimarco B. Ungano B. Metabolism. 1981; 30: 457-461Abstract Full Text PDF PubMed Scopus (9) Google Scholar) by PKC (in the presence of activators) as described by Yasuda et al. (29Yasuda I. Kim A. Tanaka S. Tominaga M. Sakurai A. Nishizuka Y. Biochem. Biophys. Res. Commun. 1990; 166: 1220-1227Crossref PubMed Scopus (309) Google Scholar). PKC specificity is confirmed by using the PKC pseudosubstrate inhibitor peptide. For analyzing PKC activity, L6 cells were deprived from serum and glucose as described above and then exposed to 25 mm glucose as indicated. PKC activity was then quantitated in total cell lysates or cell fractions or in immunoprecipitates as previously reported (30Formisano P. Kim F. Miele C. Caruso M. Auricchio R. Vigliotta G. Condorelli G. Beguinot F. J. Biol. Chem. 1998; 273: 13197-13202Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and according to the manufacturer's instructions. Briefly, cells were solubilized in the extraction buffer (20 mm Tris, pH 7.5, 0.5 mm EDTA, 0.5 mm EGTA, 0.5% Triton X-100, 25 mg/ml aprotinin, and 25 mg/ml leupeptin) and then clarified by centrifugation at 10,000 × g for 15 min at 4 °C. Upon protein quantitation, equal aliquots of the extract were added to the lipid activators (10 μm phorbol 12-myristate,13-acetate, 0.28 mg/ml phosphatidylserine, and 4 mg/ml dioleine, final concentrations) and the 32P-substrate solution (50 mm Ac-MBP (4Way K.J. Kim N. King G.L. Diabet. Med. 2001; 12: 945-959Crossref Scopus (276) Google Scholar, 5Miele C. Kim A. Maitan M.A. Oriente F. Romano C. Formisano P. Giudicelli J. Beguinot F. Van Obberghen E. J. Biol. Chem. 2003; 278: 47376-47387Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 6Green K. Kim M.D. Murphy M.P. Diabetes. 2004; 53: S110-S118Crossref PubMed Google Scholar, 7Stenina O.I. Curr. Pharm. Res. 2005; 11: 2367-2381Crossref PubMed Scopus (38) Google Scholar, 8Considine R.V. Kim M.R. Allen L.E. Morales L.M. Triester S. Serrano J. Colberg J. Lanza-Jacoby S. Caro J.F. J. Clin. Invest. 1995; 95: 2938-2944Crossref PubMed Scopus (136) Google Scholar, 9Ceolotto G. Kim A. Miola M. Sartori M. Trevisan R. Del Prato S. Semplicioni A. Avogaro A. Diabetes. 1999; 48: 1316-1322Crossref PubMed Scopus (89) Google Scholar, 10Wolf B.A. Kim J.R. Easom R.A. Chang K. Sherman W.R. Turk J. J. Clin. Invest. 1991; 87: 31-38Crossref PubMed Scopus (172) Google Scholar, 11Capaldo B. Kim D. Riccardi G. Pernotti N. Sacca L. J. Clin. Invest. 1986; 77: 1285-1290Crossref PubMed Scopus (53) Google Scholar, 12Galante P. Kim L. Kellerer M. Berti L. Tippmer S. Bossenmaier B. Fujiwara T. Okuno A. Horikoshi H. Haring H.U. Diabetes. 1995; 44: 646-651Crossref PubMed Google Scholar, 13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 14Saccà L. Kim D. Cicala M. Trimarco B. Ungano B. Metabolism. 1981; 30: 457-461Abstract Full Text PDF PubMed Scopus (9) Google Scholar), 20 μm ATP, 1 mm CaCl2, 20 mm MgCl2, 4 mm Tris, pH 7.5, and 10 mCi/ml (3,000 Ci/mm) [γ-32P]ATP), in the presence or the absence of the substrate peptide and/or of the inhibitor. The samples were incubated for 20 min at room temperature and rapidly cooled on ice, and 20-μl aliquots were spotted on phosphocellulose disc papers (Invitrogen). Discs were washed twice with 1% H3PO4, followed by two additional washes in water, and the disc-bound radioactivity was quantitated by liquid scintillation counting. DGK Antisense silencing—For antisense studies, a phosphorothioate DGKα oligodeoxynucleotide was generated with the following sequence, 5′-TACCGGTTTCTGTTTTCACA-3′ and a phosphorothioate DGKδ oligodeoxynucleotide was generated with the following sequence, 5′-TACCTGGTAAAGAGTCCCTG-3′. For control, scrambled oligodeoxynucleotides (POs) with the sequences 5′-CTTGATATTCCCGTCGGACC-3′ and 5′-GGTCCACGTGCCACTTGGAC-3′ were also obtained. L6hIR cells were grown in 6-well plates. The cells were then rinsed with 3 ml of serum-free Dulbecco's modified minimum essential medium and 3 ml of medium containing 2 μg/ml N-[1-(2,3-diomeoyloxy)propyl]-N,N,N-trimethylammonium chloride/dioleoylphosphatidylethanolamine transfection reagent, and 4 μg/ml antisense were added for 16 h. The cells were washed with serum-free Dulbecco's modified minimum essential medium and incubated for 18 h in the same medium supplemented with 0.25% bovine serum albumin. Transfected cells were exposed to 25 mm glucose as indicated and assayed for DAG levels and PKCα activation as described above. DGK Enzymatic Assays—DGK activity was assayed in vitro as previously described (31Bregoli L. Kim J.J. Raben D.M. J. Biol. Chem. 2001; 276: 23288-23295Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Briefly, octylglucoside/DAG-mixed micelles were prepared as follows: a mixture of 0.25 mm DAG, 55 mm octylglucoside, and phosphatidylserine (either 1 mm, resulting in 1.8 mol% in micelles, or 5 mm, resulting in 8.3 mol% in micelles) was resuspended in 1 mm diethylenetriaminepentaacetic acid, pH 7.4, by vortex-mixing and sonication until the suspension appeared clear. 20 μl of mixed micelles was added to 70 μl of reaction mix (final concentration: 100 μm diethylenetriaminepentaacetic acid, pH 7.4, 50 mm imidazole-HCl, 50 mm NaCl, 12.5 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 1 mm [γ-32P]ATP). 10 μl of total homogenate was added to 90 μl of mixed-micelles reaction mix solution. The reaction was started by vortex-mixing for 3 s and sonication for 5 s. After 30-min incubation at 25 °C, the reaction was terminated by the addition of chloroform/methanol/1% perchloric acid (1:2:0.75, v/v), then vortex-mixed. 1% perchloric acid and chloroform (1:1, v/v) were added, and the mixture was centrifuged for 5 min at 2,000 rpm in a tabletop Sorval centrifuge at 25 °C. The organic phase was washed twice in 1% perchloric acid, and an aliquot was dried under a stream of nitrogen and spotted onto a silica gel 60 TLC plate. PA was separated by chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, v/v). The amount of [γ-32P]PA was measured by using a liquid scintillation spectrophotometer. Glucose Effect on DAG Levels in L6 Skeletal Muscle Cells—We investigated whether the intracellular levels of DAG were changed by exposure of L6hIR myotubes to high glucose concentrations. The L6hIR (L6 Cl Wt1 and Wt2) are clones of L6 cells overexpressing human insulin receptors and have been previously generated and characterized (13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In both clones of L6hIR myotubes, incubation with 25 mm glucose for 5 min induced a decrease in DAG levels of ∼2-fold as compared with untreated cells (p < 0.001). Prolonging glucose exposure at 30 and 60 min, DAG levels were increased by 1.4- and 1.6-fold over basal levels, respectively (Fig. 1). To evaluate the specificity of glucose effect, DAG levels were measured in the same cells treated with 25 mm xylose. No variation occurred in DAG levels after xylose treatment (Fig. 1). In addition, acute glucose exposure of the cells following preincubation with 25 mm pyruvate in the glucose-free medium elicited similar effects (Fig. 1), indicating that glucose effect was not due to changes in osmolarity or to energy depletion. Glucose Effect on DGK Activity in L6 Myotubes—To investigate the mechanism by which glucose regulates DAG levels, we measured cellular DGK activity in L6hIR (L6 Cl Wt1 and Wt2) myotubes after exposure to 25 mm glucose for 5 and 30 min. After 5 min of glucose stimulation, the levels of PA were increased by 12-fold (p < 0.001) (Fig. 2A). However, PA levels returned close to basal levels by prolonging the incubation with high glucose concentrations for up to 30 min. In addition, pharmacological inhibition of DGK with a well characterized non-isoform specific inhibitor, R59949 (1 mm), prevented the acute glucose-dependent increase in PA levels, suggesting that this increase was due to DGK activation. We next analyzed the effect of R59949 on the glucose-induced decrease in DAG levels (Fig. 2B). Interestingly, in the presence of the DGK inhibitor, DAG levels were slightly increased by glucose stimulation for 5 min, compared with basal levels. Thus, glucose-induced reciprocal changes of PA and DAG were largely prevented by DGK inhibition. Effect of DGK Inhibition on Glucose-induced PKCα Activity and Translocation—To investigate the role of DGK in glucose regulation of the DAG/PKC pathway, we evaluated the effect of DGK inhibition on PKCα activity and translocation to the plasma membrane after exposure to 25 mm glucose. As previously shown (13Caruso M. Kim C. Oriente F. Maitan M.A. Bifulco G. Andreozzi F. Condorelli G. Formisano P. Beguinot F. J. Biol. Chem. 1999; 274: 28637-28644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), incubation of L6hIR myotubes with 25 mm glucose for 5 min induced a 2-fold decrease in PKCα activity (Fig. 3A). Inhibition of DGK activity with R59949 prevented the negative effect of glucose on PKCα activity, which, instead, was increased by 1.5-fold over basal levels after 5 min of high glucose stimulation. Incubation with PA did not affect PKCα activity in in vitro assays, suggesting that its accumulation was not responsible for PKC inhibition (data not shown). Treatment with R59949 also prevented glucose-induced cytosolic retrotranslocation of PKCα (Fig. 3B), indicating that DGK is involved in acute glucose regulation of PKCα activity and translocation. PKC may directly regulate DGK activity and modify intracellular DAG concentration (29Yasuda I. Kim A. Tanaka S. Tominaga M. Sakurai A. Nishizuka Y. Biochem. Biophys. Res. Commun. 1990; 166: 1220-1227Crossref PubMed Scopus (309) Google Scholar). We therefore measur
Chronic exposure to polychlorinated biphenyls (PCBs), a class of ubiquitous environmental toxicants, causes neurocognitive anomalies. The transcription factor repressor element 1-silencing transcription factor (REST) plays a critical role in neuronal phenotype elaboration in both neural progenitor cells and non-neuronal cells. Here, we investigated the possible relationship between PCBs and REST in neuroblastoma SH-SY5Y cells. In these cells, chronic exposure to the PCB mixture Aroclor 1254 (A-1254; 5-30 μg/ml) caused dose-dependent cell death via the induction of calpain but not caspase-3. Intriguingly, this effect was prevented by the calpain inhibitor calpeptin. Furthermore, A-1254 enhanced REST mRNA and protein expression levels after both 24 and 48 h. REST down-regulation by small interfering RNA prevented A-1254-induced cell death. In addition, A-1254 enhanced the binding of REST to the synapsin 1 gene promoter, and synapsin 1 knockdown potentiated A-1254-induced cell death. A-1254 (10 μg/ml) also increased the expression of the two REST cofactors, the REST corepressor and the mammalian SIN3 homolog A transcription regulator. Moreover, the PCB mixture decreased acetylation of the histone proteins H3 and H4. It is noteworthy that the histone deacetylase inhibitor trichostatin A prevented such decreases and reduced the A-1254-induced neurotoxic effect. Collectively, these results suggest that A-1254 exerts its toxic effect via REST by down-regulating synapsin 1 and decreasing H3 and H4 acetylation.
Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.
The phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (ped/pea-15) gene is overexpressed in human diabetes and causes this abnormality in mice. Transgenic mice with β-cell–specific overexpression of ped/pea-15 (β-tg) exhibited decreased glucose tolerance but were not insulin resistant. However, they showed impaired insulin response to hyperglycemia. Islets from the β-tg also exhibited little response to glucose. mRNAs encoding the Sur1 and Kir6.2 potassium channel subunits and their upstream regulator Foxa2 were specifically reduced in these islets. Overexpression of PED/PEA-15 inhibited the induction of the atypical protein kinase C (PKC)-ζ by glucose in mouse islets and in β-cells of the MIN-6 and INS-1 lines. Rescue of PKC-ζ activity elicited recovery of the expression of the Sur1, Kir6.2, and Foxa2 genes and of glucose-induced insulin secretion in PED/PEA-15–overexpressing β-cells. Islets from ped/pea-15–null mice exhibited a twofold increased activation of PKC-ζ by glucose; increased abundance of the Sur1, Kir6.2, and Foxa2 mRNAs; and enhanced glucose effect on insulin secretion. In conclusion, PED/PEA-15 is an endogenous regulator of glucose-induced insulin secretion, which restrains potassium channel expression in pancreatic β-cells. Overexpression of PED/PEA-15 dysregulates β-cell function and is sufficient to impair glucose tolerance in mice.
Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-alpha activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6(PED/PEA-15)) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (K(D) = 0.37 +/- 0.13 mum), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6(PED/PEA-15) cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by approximately 70%. Similar results are obtained by expression of D4 in L6(PED/PEA-15). All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.
