Cardiac lipotoxicity plays an important role in the pathogenesis of obesity-related cardiovascular disease. The flavonoid quercetin (QUE), a nutraceutical compound that is abundant in the “Mediterranean diet”, has been shown to be a potential therapeutic agent in cardiac and metabolic diseases. Here, we investigated the beneficial role of QUE and its derivative Q2, which demonstrates improved bioavailability and chemical stability, in cardiac lipotoxicity. To this end, H9c2 cardiomyocytes were pre-treated with QUE or Q2 and then exposed to palmitate (PA) to recapitulate the cardiac lipotoxicity occurring in obesity. Our results showed that both QUE and Q2 significantly attenuated PA-dependent cell death, although QUE was effective at a lower concentration (50 nM) when compared with Q2 (250 nM). QUE decreased the release of lactate dehydrogenase (LDH), an important indicator of cytotoxicity, and the accumulation of intracellular lipid droplets triggered by PA. On the other hand, QUE protected cardiomyocytes from PA-induced oxidative stress by counteracting the formation of malondialdehyde (MDA) and protein carbonyl groups (which are indicators of lipid peroxidation and protein oxidation, respectively) and intracellular ROS generation, and by improving the enzymatic activities of catalase and superoxide dismutase (SOD). Pre-treatment with QUE also significantly attenuated the inflammatory response induced by PA by reducing the release of key proinflammatory cytokines (IL-1β and TNF-α). Similar to QUE, Q2 (250 nM) also significantly counteracted the PA-provoked increase in intracellular lipid droplets, LDH, and MDA, improving SOD activity and decreasing the release of IL-1β and TNF-α. These results suggest that QUE and Q2 could be considered potential therapeutics for the treatment of the cardiac lipotoxicity that occurs in obesity and metabolic diseases.
Background. Premenstrual syndrome (PMS) is a set of physical, psychological, and emotional symptoms that occur during the luteal phase of the menstrual cycle. The etiopathogenesis of this condition is not fully understood, and several studies suggest a possible role of environmental factors, such as diet. The aim of this work was to investigate the relationship between dietary habits and the occurrence and severity of PMS. Methods and Results. Forty-seven women were enrolled in the study. Participants were asked to complete the Daily Record of Severity of Problems (DRSP) to diagnose PMS and to complete a three-day food record during the perimenstrual phase. Thirty women completed the study (16 with PMS and 14 controls). An analysis of the food diaries revealed no differences between the women with PMS and the control subjects in terms of total energy intake (1649 vs. 1570 kcal/day), diet composition, and the consumption of macro- or micronutrients, except for copper, whose consumption was higher in women with PMS than in the control subjects (1.27 ± 0.51 vs. 0.94 ± 0.49 mg/d, p < 0.05). Conclusions. The data presented here are very preliminary, and only a significant difference in copper intake was found when comparing women with PMS and controls. Larger studies are needed to better define how diet may contribute to the exacerbation of the psychological and somatic symptoms associated with PMS and whether PMS itself may influence macro- or micronutrient intake by changing dietary habits.
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
Background:NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain–thyroid–lung syndrome (BTLS). Methods: The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. Results: The mutation is a deletion of a cytosine at position 834 (ref. sequence NM_003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. Conclusions: We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype–phenotype correlations in the NKX2-1 deficiency syndrome.
Epigenetic mechanisms include DNA methylation, posttranslational modifications of histones, chromatin remodeling factors, and post transcriptional gene regulation by noncoding RNAs. All together, these processes regulate gene expression by changing chromatin organization and DNA accessibility. Targeting enzymatic regulators responsible for DNA and chromatin modifications hold promise for modulating the transcriptional regulation of genes that are involved in cancer, as well as in chronic noncommunicable metabolic diseases like obesity, diabetes, and cardiovascular diseases. Increasingly studies are emerging, leading to the identification of specific and effective molecules targeting epigenetic pathways involved in disease onset. In this regard, RNA interference, which uses small RNAs to reduce gene expression and nucleic acid aptamers are arising as very promising candidates in therapeutic approach. Common to all these strategies is the imperative challenge of specificity. In this regard, nucleic acid aptamers have emerged as an attractive class of carrier molecules due to their ability to bind with high affinity to specific ligands, their high chemical flexibility as well as tissue penetration capability. In this review, we will focus on the recent progress in the field of aptamers used as targeting moieties able to recognize and revert epigenetics marks involved in diseases onset.
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