Dual-action hypoglycemic and hypocholesterolemic agents that inhibit glycogen phosphorylase and lanosterol demethylase
2004; Elsevier BV; Volume: 46; Issue: 3 Linguagem: Inglês
10.1194/jlr.m400436-jlr200
ISSN1539-7262
AutoresH. James Harwood, Stephen F. Petras, Dennis J. Hoover, Dayna C. Mankowski, Victor F. Soliman, Eliot Sugarman, Bernard Hulin, Younggil Kwon, E. Michael Gibbs, James Mayne, Judith L. Treadway,
Tópico(s)Diabetes Treatment and Management
ResumoDiabetic dyslipidemia requires simultaneous treatment with hypoglycemic agents and lipid-modulating drugs. We recently described glycogen phosphorylase inhibitors that reduce glycogenolysis in cells and lower plasma glucose in ob/ob mice (J. Med. Chem., 41: 2934, 1998). In evaluating the series prototype, CP-320626, in dogs, up to 90% reduction in plasma cholesterol was noted after 2 week treatment. Cholesterol reductions were also noted in ob/ob mice and in rats. In HepG2 cells, CP-320626 acutely and dose-dependently inhibited cholesterolgenesis without affecting fatty acid synthesis. Inhibition occurred together with a dose-dependent increase in the cholesterol precursor, lanosterol, suggesting that cholesterolgenesis inhibition was due to lanosterol 14α-demethylase (CYP51) inhibition. In ob/ob mice, acute treatment with CP-320626 resulted in a decrease in hepatic cholesterolgenesis with concomitant lanosterol accumulation, further implicating CYP51 inhibition as the mechanism of cholesterol lowering in these animals. CP-320626 and analogs directly inhibited rhCYP51, and this inhibition was highly correlated with HepG2 cell cholesterolgenesis inhibition (R2 = 0.77).These observations indicate that CP-320626 inhibits cholesterolgenesis via direct inhibition of CYP51, and that this is the mechanism whereby CP-320626 lowers plasma cholesterol in experimental animals. Dual-action glycogenolysis and cholesterolgenesis inhibitors therefore have the potential to favorably affect both the hyperglycemia and the dyslipidemia of type 2 diabetes. Diabetic dyslipidemia requires simultaneous treatment with hypoglycemic agents and lipid-modulating drugs. We recently described glycogen phosphorylase inhibitors that reduce glycogenolysis in cells and lower plasma glucose in ob/ob mice (J. Med. Chem., 41: 2934, 1998). In evaluating the series prototype, CP-320626, in dogs, up to 90% reduction in plasma cholesterol was noted after 2 week treatment. Cholesterol reductions were also noted in ob/ob mice and in rats. In HepG2 cells, CP-320626 acutely and dose-dependently inhibited cholesterolgenesis without affecting fatty acid synthesis. Inhibition occurred together with a dose-dependent increase in the cholesterol precursor, lanosterol, suggesting that cholesterolgenesis inhibition was due to lanosterol 14α-demethylase (CYP51) inhibition. In ob/ob mice, acute treatment with CP-320626 resulted in a decrease in hepatic cholesterolgenesis with concomitant lanosterol accumulation, further implicating CYP51 inhibition as the mechanism of cholesterol lowering in these animals. CP-320626 and analogs directly inhibited rhCYP51, and this inhibition was highly correlated with HepG2 cell cholesterolgenesis inhibition (R2 = 0.77). These observations indicate that CP-320626 inhibits cholesterolgenesis via direct inhibition of CYP51, and that this is the mechanism whereby CP-320626 lowers plasma cholesterol in experimental animals. Dual-action glycogenolysis and cholesterolgenesis inhibitors therefore have the potential to favorably affect both the hyperglycemia and the dyslipidemia of type 2 diabetes. Type 2 diabetes is a severe and prevalent disease in the Western world and affects roughly 16 million persons in the US, and another 14 million people have impaired glucose tolerance (1Harris M.I. Flegal K.M. Cowie C.C. Eberhardt M.S. Goldstein D.E. Little R.R. Wiedmeyer H.M. Byrd-Holt D.D. Prevalence of diabetes, impaired fasting glucose, and impaired glucose tolerance in US adults. The Third National Health and Nutrition Examination Survey, 1988–1994.Diabetes Care. 1998; 21: 518-526Crossref PubMed Scopus (2455) Google Scholar). Projections indicate that the incidence of type 2 diabetes will increase to over 25 million by 2010 in the US, and to over 300 million worldwide by 2025 (1Harris M.I. Flegal K.M. Cowie C.C. Eberhardt M.S. Goldstein D.E. Little R.R. Wiedmeyer H.M. Byrd-Holt D.D. Prevalence of diabetes, impaired fasting glucose, and impaired glucose tolerance in US adults. The Third National Health and Nutrition Examination Survey, 1988–1994.Diabetes Care. 1998; 21: 518-526Crossref PubMed Scopus (2455) Google Scholar, 2Treadway J.L. Mendys P. Hoover D.J. Glycogen phosphorylase inhibitors for treatment of type2 diabetes mellitus.Expert Opin. Investig Drugs. 2001; 10: 439-454Crossref PubMed Scopus (259) Google Scholar, 3King H. Aubert R.E. Herman W.H. Global burden of diabetes 1995–2005: prevalence, numerical estimates and projections.Diabetes Care. 1998; 21: 1414-1431Crossref PubMed Scopus (5082) Google Scholar). The annual direct medical costs associated with type 2 diabetes, which in the United States was in excess of 44 billion dollars in 1997 (4American Diabetes AssociationEconomic consequences of diabetes mellitus in the US in 1997.Diabetes Care. 1998; 21: 296-309Crossref PubMed Scopus (715) Google Scholar), result primarily from secondary hyperglycemia-related complications, such as retinopathy, nephropathy, peripheral neuropathy, and cardiovascular, peripheral vascular, and cerebrovascular disease. A high correlation exists between tighter glycemic control and reduction of these long-term complications in type 2 diabetes (5American Diabetes AssociationImplications of the United Kingdom prospective diabetes study.Diabetes Care. 2001; 24: 28-32Google Scholar). Type 2 diabetics are currently treated with interventions to improve glycemia through a progressive regimen of diet, exercise, oral antidiabetic drugs (as monotherapy or in combination), and insulin (6American Diabetes AssociationStandards of medical care for patients with diabetes mellitus.Diabetes Care. 2003; 26: 33-50PubMed Google Scholar). However, there is an ongoing need for additional oral antidiabetic agents that will achieve better glycemic control as monotherapy and/or work more safely or effectively in combination. In addition to their hyperglycemia, patients with type 2 diabetes often present with a concomitant atherogenic dyslipidemia (elevated triglycerides, low HDL cholesterol, and small, dense LDL) that increases their risk of cardiovascular disease (7American Diabetes AssociationManagement of dyslipidemia in adults with diabetes.Diabetes Care. 2003; 26: 83-86PubMed Google Scholar, 8National Cholesterol Education Program Expert PanelExecutive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III).J. Am. Med. Assoc. 2001; 285: 2486-2497Crossref PubMed Scopus (24453) Google Scholar). Because there is a high incidence of mortality for type 2 diabetics with their first myocardial infarction (7American Diabetes AssociationManagement of dyslipidemia in adults with diabetes.Diabetes Care. 2003; 26: 83-86PubMed Google Scholar), aggressive therapy for treating diabetic dyslipidemia is recommended (7American Diabetes AssociationManagement of dyslipidemia in adults with diabetes.Diabetes Care. 2003; 26: 83-86PubMed Google Scholar). It is suggested that initial lipid-modulating therapy be directed toward reducing LDL cholesterol levels to below 100 mg/dl through administration of a cholesterol synthesis inhibitor, such as a statin (HMG-CoA reductase inhibitor), and that this treatment be combined with a fibric acid derivative, such as fenofibrate, for patients with HDL cholesterol levels below 40 mg/dl and for patients with triglycerides that remain elevated (>150 mg/dl) after both improvement of glycemic control and initiation of statin therapy (7American Diabetes AssociationManagement of dyslipidemia in adults with diabetes.Diabetes Care. 2003; 26: 83-86PubMed Google Scholar). In pursuing new treatments for type 2 diabetes, we have targeted inhibition of glycogen phosphorylase (E.C. 2.4.1.1), the enzyme that catalyzes the hydrolytic release of glucose-1-phosphate from glycogen, as an approach to reducing hepatic glycogenolysis and thereby controlling plasma glucose levels (2Treadway J.L. Mendys P. Hoover D.J. Glycogen phosphorylase inhibitors for treatment of type2 diabetes mellitus.Expert Opin. Investig Drugs. 2001; 10: 439-454Crossref PubMed Scopus (259) Google Scholar). Through these efforts, we have identified a series of indole-2-carboxamide glycogen phosphorylase inhibitors (9Martin W.H. Hoover D.J. Armento S.J. Stock I.A. McPherson R.K. Danley D.E. Stevenson R.W. Barrett E.J. Treadway J.L. Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo.Proc. Natl. Acad. Sci. USA. 1998; 95: 1776-1781Crossref PubMed Scopus (217) Google Scholar, 10Hoover D.H. Lefkowitz-Snow S. Burgess-Henry J.A. Martin W.H. Armento S.J. Stock I.A. McPherson R.K. Genereux P.E. Gibbs E.M. Treadway J.L. Indole-2-carboxamide inhibitors of human liver glycogen phosphorylase.J. Med. Chem. 1998; 41: 2934-2938Crossref PubMed Scopus (104) Google Scholar) that inhibit the human liver isoform of glycogen phosphorylase by binding at a unique allosteric regulatory site on the enzyme (11Rath V.L. Ammirati M. Danley D.E. Ekstrom J.L. Gibbs E.M. Hynes T.R. Mathiowetz A.M. McPherson R.K. Olson T.V. Treadway J.L. Hoover D.J. Human liver glycogen phosphorylase inhibitors bind at a new allosteric site.Chem. Biol. 2000; 7: 677-682Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), reduce forskolin-induced glycogenolysis in SK-HEP-1 cells (10Hoover D.H. Lefkowitz-Snow S. Burgess-Henry J.A. Martin W.H. Armento S.J. Stock I.A. McPherson R.K. Genereux P.E. Gibbs E.M. Treadway J.L. Indole-2-carboxamide inhibitors of human liver glycogen phosphorylase.J. Med. Chem. 1998; 41: 2934-2938Crossref PubMed Scopus (104) Google Scholar, 12Treadway J.L. McPherson R.K. Genereux P.E. Zavadoski W.J. Vestergaard P. Kwon Y. Hoover D.J. Gibbs E.M. The human liver glycogen phosphorylase inhibitor CP-320626 shows sustained glucose lowering on multiple dosing in diabetic ob/ob mice (Abstract).Diabetes. 1998; 47: 287PubMed Google Scholar), and exhibit glucose-lowering activity when given orally to diabetic ob/ob mice (10Hoover D.H. Lefkowitz-Snow S. Burgess-Henry J.A. Martin W.H. Armento S.J. Stock I.A. McPherson R.K. Genereux P.E. Gibbs E.M. Treadway J.L. Indole-2-carboxamide inhibitors of human liver glycogen phosphorylase.J. Med. Chem. 1998; 41: 2934-2938Crossref PubMed Scopus (104) Google Scholar, 12Treadway J.L. McPherson R.K. Genereux P.E. Zavadoski W.J. Vestergaard P. Kwon Y. Hoover D.J. Gibbs E.M. The human liver glycogen phosphorylase inhibitor CP-320626 shows sustained glucose lowering on multiple dosing in diabetic ob/ob mice (Abstract).Diabetes. 1998; 47: 287PubMed Google Scholar). Because of the potential pharmacological utility of this series of glycogen phosphorylase inhibitors, we have evaluated a representative analog, CP-320626 (Fig. 1);[IC50 vs. human liver glycogen phosphorylase, 205 nM (10Hoover D.H. Lefkowitz-Snow S. Burgess-Henry J.A. Martin W.H. Armento S.J. Stock I.A. McPherson R.K. Genereux P.E. Gibbs E.M. Treadway J.L. Indole-2-carboxamide inhibitors of human liver glycogen phosphorylase.J. Med. Chem. 1998; 41: 2934-2938Crossref PubMed Scopus (104) Google Scholar)], for its subchronic effects in diabetic ob/ob mice (10Hoover D.H. Lefkowitz-Snow S. Burgess-Henry J.A. Martin W.H. Armento S.J. Stock I.A. McPherson R.K. Genereux P.E. Gibbs E.M. Treadway J.L. Indole-2-carboxamide inhibitors of human liver glycogen phosphorylase.J. Med. Chem. 1998; 41: 2934-2938Crossref PubMed Scopus (104) Google Scholar, 12Treadway J.L. McPherson R.K. Genereux P.E. Zavadoski W.J. Vestergaard P. Kwon Y. Hoover D.J. Gibbs E.M. The human liver glycogen phosphorylase inhibitor CP-320626 shows sustained glucose lowering on multiple dosing in diabetic ob/ob mice (Abstract).Diabetes. 1998; 47: 287PubMed Google Scholar), in rats, and in dogs. During the course of these studies, we discovered that CP-320626 reduced plasma cholesterol levels in a variety of normoglycemic, nondiabetic animals in a manner and magnitude inconsistent with its expected action as a glycogen phosphorylase inhibitor. Herein, we report identification of the mechanism responsible for the cholesterol-lowering action of CP-320626 as inhibition of the cholesterolgenic enzyme lanosterol 14α-demethylase (CYP51), and characterize structure–activity relationships for this activity within the series. Lanosterol, NADP+, glucose-1-phosphate, glycogen, isocitrate, isocitrate dehydrogenase, dioleoyl l-α-phosphatidylcholine, ergosterol, tyloxapol, ketoconazole, quinidine, sulfaphenazole, cytochrome-P450 (CYP) reductase, PEG400, and diagnostic kits for measuring plasma lactate and β-hydroxybutyrate were from Sigma Chemical Co. (St. Louis, MO). TMSI + Pyridine, 1:4 (Sylon TP) in 1 ml aliquots were from Supelco (Bellefonte, PA). Sodium [2-14C] acetate (56 mCi/mmol), R,S-[2-14C]mevalonolactone (58 mCi/mmol), and Aquasol-2 were from New England Nuclear (Boston, MA). Ready-Safe was from Beckman Instruments (Fullerton, CA). [2,6,11,12,15,23-3H]lanosterol was from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), l-glutamine, and gentamicin were from GIBCO Laboratories (Grand Island, NY). Heat-inactivated fetal bovine serum was from HyClone Laboratories (Logan, UT). Silica gel 60C TLC plates were from Eastman Kodak (Rochester, NY). BCA protein assay reagent was from Pierce (Rockford, IL). A-Gent™ Glucose-UV, A-Gent™ Triglyceride and A-Gent™ Cholesterol Test reagent systems were from Abbott Laboratories (Irving, TX). Pluronic P105 Block Copolymer Surfactant was from BASF (Parsippany, NY). Sprague Dawley rats and Beagle dogs were from Charles Rivers (Boston, MA). C57BL/6J-ob/ob mice were from Jackson Laboratory (Bar Harbor, ME). RMH 3200 laboratory meal and Agway Respond 2000 laboratory dog chow were from Agway, Inc. (Syracuse, NY). HepG2 cells were from the American Type Culture Collection (Rockville, MD). All other chemicals and reagents were from previously listed sources (13Harwood Jr., H.J. Barbacci-Tobin E.G. Petras S.F. Lindsey S. Pellarin L.D. 3-(4-chlorophenyl)-2-(4-diethylaminoethoxyphenyl)-A-pentenonitrile monohydrogen citrate and related analogs. Reversible, competitive, first half-reaction squalene synthetase inhibitors.Biochem. Pharmacol. 1997; 53: 839-864Crossref PubMed Scopus (23) Google Scholar, 14Petras S.F. Lindsey S. Harwood Jr, H.J. HMG-CoA reductase regulation: use of structurally diverse first half-reaction squalene synthetase inhibitors to characterize the site of mevalonate-derived nonsterol regulator production in cultured IM-9 cells.J. Lipid Res. 1999; 40: 24-38Abstract Full Text Full Text PDF PubMed Google Scholar, 15Harwood Jr., H.J. Petras S.F. Shelly L.D. Zaccaro L.M. Perry D.A. Makowski M.R. Hargrove D.M. Martin K.A. Tracey W.R. Chapman J.G. Magee W.P. Dalvie D.K. Soliman V.F. Martin W.H. Mularski C.J. Eisenbeis S.A. Isozyme-nonselective N-substituted bipiperidylcarboxamide acetyl-CoA carboxylase inhibitors reduce tissue malonyl-CoA concentrations, inhibit fatty acid synthesis, and increase fatty acid oxidation in cultured cells and in experimental animals.J. Biol. Chem. 2003; 278: 37099-37111Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 16Chandler C.E. Wilder D.E. Pettini J.L. Savoy Y.E. Petras S.F. Chang G. Vincent J. Harwood Jr, H.J. CP-346086: an MTP inhibitor that lowers plasma cholesterol and triglycerides in experimental animals and in humans.J. Lipid Res. 2003; 44: 1886-1901Abstract Full Text Full Text PDF Scopus (175) Google Scholar). All procedures using experimental animals were approved by the Institutional Animal Care and Use Procedures Review Board. Sprague Dawley rats, C57BL/6J-ob/ob mice, and Beagle dogs were given food and water ad libitum and treated orally at a volume of 1.0 ml/200 g body weight (rats), 0.25 ml/25 g body weight (mice), or 1.0 ml/kg body weight (dogs) with either an aqueous solution of 0.1% pluronic P-105 in 10% DMSO (vehicle) or an aqueous solution of 0.1% pluronic P-105 in 10% DMSO plus CP-320626. Serum glucose triglyceride and total cholesterol concentrations were determined by the Abbott VP™ and VP Super System® Autoanalyzer using the A-Gent™ Glucose-UV, A-Gent™ Triglyceride and A-Gent™ Cholesterol Test reagent systems. Serum insulin and glucagon concentrations were determined by radioimmunoassay (RIA) using kits from Binax (Portland, ME) and Amersham Corp. (Arlington Heights, IL), respectively. β-Hydroxybutyrate concentration was determined spectrophotometrically using kits from Sigma. Free fatty acid concentration was determined using a kit from Wako (Richmond, VA). The serum glucose, insulin, glucagon, triglyceride, total cholesterol, β-hydroxybutyrate, and free fatty acid–lowering activity of test compounds were determined by statistical analysis (unpaired t-test) with the vehicle-treated control group. To 100 μl aliquots of plasma were added 50 μl of an internal standard (2 μg/ml CP-89816 in methanol), 5 ml methyl tert-butyl ether, and 1 ml of 0.5 M sodium carbonate (pH 9). After vigorous mixing and centrifugation, the ether layers were removed and evaporated to dryness, and the resulting solid was reconstituted with 75 μl mobile phase [45% acetonitrile, 55% 50 mM sodium phosphate monobasic, and 30 mM triethylamine (pH 3)]. Aliquots (30 μl) of reconstituted samples were injected onto a 4 μm Waters Nova-Pak C-18 (Waters Corp, Bedford, MA) reverse phase column (3.9 × 150 mm), with a mobile phase flow rate of 1 ml/min. CP-320626 and internal standard were detected by fluorescence (excitation at 290 nm and emission at 348 nm). The linear dynamic range was between 0.1 μg/ml (lower limit of quantification) and 1 μg/ml (upper limit of quantification). Cmax was the concentration in the blood sample in which the highest plasma concentration was measured. The area under the plasma concentration time curve (AUC) from 0 to tlast (AUC0–tlast) was calculated using a linear trapezoidal approximation, where tlast is the time point of the last quantifiable plasma concentration. Nonsaponifiable lipids were isolated from liver and plasma and quantitated by gas chromatography-mass spectrometry (GC/MS). Samples of plasma (0.75 ml) and liver (0.75 g) were saponified at 70°C for 120 min in 2.5 ml of 2.5 M NaOH, then 5 ml of absolute EtOH was added to each sample and the solutions were mixed. Ten milliliters of petroleum ether was then added to each sample, and the mixtures were shaken vigorously for 2 min then centrifuged at 2,000 g in a bench-top Sorvall for 10 min. After a second petroleum ether extraction, the resultant petroleum ether layers were removed, dried under nitrogen, and dissolved in 500 μl dry pyridine solution. A 500 μl aliquot of TMSI + Pyridine, 1:4 (Sylon TP) was then added to each sample, and derivatization was allowed to continue at room temperature (RT) for 1 h. Derivatized samples were analyzed using an HP-6890 Series gas chromatograph equipped with a 6890 Series GC Injector and interfaced with an HP-5973N mass selective detector. Separation was achieved using a Supelco SAC-5 (15 m × 0.25 mm × 0.25 μm) GC column. The oven temperature was held at 250°C for 0 min, then heated to 300°C at a rate of 2°C/min. The ions monitored using full scan electron ionization (70 eV) corresponded to the molecular ions of the trimethylsilyl derivatives. Retention times of detectable precursor sterols relative to that of cholesterol were cholestanol (dihydrocholesterol), 1.03; 8-dehydrocholesterol, 1.06; desmosterol, 1.07; 7-dehydrocholesterol, 1.07; lathosterol, 1.10; 4-methylsterol, 1.20; 4,4-dimethylsterol, 1.20; lanosterol, 1.42; and dihydrolanosterol, 1.42, similar to those previously reported for C-3 hydroxyl-derivatized sterols (17Patterson G.W. Relation between structure and retention time of sterols in gas chromatography.Anal. Chem. 1971; 43: 1165-1170Crossref Scopus (201) Google Scholar, 18Nes W.R. A comparison of methods for the identification of sterols.Methods Enzymol. 1985; 111: 3-51Crossref PubMed Google Scholar). Based on GC/MS analysis, the sterol composition of control rat liver was ∼98% cholesterol, with 0.04% cholestanol and 8-dehydrocholesterol, 0.17% desmosterol and 7-dehydrocholesterol, 0.13% lathosterol, 1.11% monomethyl and dimethyl sterols, and 0.49% trimethylsterols (lanosterol and dihydrolanosterol). The sterol composition of control rat plasma was >99% cholesterol, with 0.57% monomethyl and dimethyl sterols and 0.38% trimethylsterols (lanosterol and dihydrolanosterol). Levels of cholestanol, 8-dehydrocholesterol, desmosterol, 7-dehydrocholesterol, and lathosterol in control rat plasma were all below the limits of detection. The activity of recombinant human CYP51, expressed in TOPP3 cells and partially purified by the method of Stromstedt, Rozman, and Waterman (19Stromstedt M. Rozman D. Waterman M.R. The ubiquitously expressed human CYP51 encodes lanosterol 14α-demethylase, a cytochrome P450 whose expression is regulated by oxysterols.Arch. Biochem. Biophys. 1996; 329: 73-81Crossref PubMed Scopus (154) Google Scholar), was determined by measuring the conversion of lanosterol to 4,4-dimethylcholesta-8,14,24-trien-3β-ol as previously described (19Stromstedt M. Rozman D. Waterman M.R. The ubiquitously expressed human CYP51 encodes lanosterol 14α-demethylase, a cytochrome P450 whose expression is regulated by oxysterols.Arch. Biochem. Biophys. 1996; 329: 73-81Crossref PubMed Scopus (154) Google Scholar), with the following modifications: Briefly, 25 μl of a 1 mM suspension of lanosterol in a mixture of tyloxapol-acetone (1:1; v/v), dioleylphosphatidylcholine micelles (5 mg/ml), methanol, and 100 mM potassium phosphate buffer (pH 7.4) was added to 5 ml glass tubes to provide a final lanosterol concentration of 50 μM (approximate Km). Inhibitors, dissolved in methanol for final concentrations ranging between 0.01 and 200 μM, were then added, and the lanosterol/inhibitor mixtures were allowed to dry under nitrogen for 10 min. To the residues were added 20 pmol partially purified recombinant human CYP51, 125 pmol human CYP reductase, and 50 μl rat lipid. After incubation at RT for 10–15 min for enzyme reconstitution, 540 μl of 100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.1 mM DTT, 0.1 mM EDTA, and 0.5 mM KCN was added to each tube. Reaction mixtures were preincubated for 2 min at 37°C, then reactions were initiated by the addition of 50 μl of an NADPH regenerating system (final incubation concentration, 10 mM MgCl2, 0.54 mM NADPH, 6.2 mM DL-isocitric acid, 0.5 U/ml isocitrate dehydrogenase). After 60 min incubation at 37°C, reactions were terminated by addition of a 25 μl volume of ethyl acetate that also contained the internal standard, ergosterol (1 mg/ml), followed by extraction with 5 ml ethyl acetate. After vigorous mixing and centrifugation to facilitate phase separation, 3–4 ml of the ethyl acetate phase was transferred to fresh tubes and evaporated to dryness under nitrogen at 50°C. Samples were reconstituted in 150 μl mobile phase (see below), and 25–50 μl was applied to an HPLC system. Ergosterol (internal standard) and 4,4-dimethylcholesta-8,14,24-trien-3β-ol (reaction product) were separated on a Waters Novapak C18 column (4.0 μM, 150 mm × 3.9 mm), with a mobile phase consisting of methanol-acetonitrile-HPLC-grade water (45:45:10 v/v/v), at a flow rate of 1.5 ml/min. Ergosterol and 4,4-dimethylcholesta-8,14,24-trien-3β-ol were monitored by UV detection at 248 nm using a SpectroMonitor variable wavelength detector (LDC Analytical; Riviera Beach, FL), and the Multichrom™ data acquisiton system (Version. 2.11; Fisons Instruments, Beverly, MA) was used for data collection and analysis. Approximate retention times for 4,4-dimethylcholesta-8,14,24-trien-3β-ol and ergosterol were 25 and 30 min, respectively. The activity of recombinant human liver glycogen phosphorylase, expressed in baculovirus, purified to >95% homogeneity, and fully activated by phosphorylase kinase as previously described (20Coats W.S. Browner M.F. Fletterick R.J. Newgard C.B. An engineered liver glycogen phosphorylase with AMP allo-steric activation.J. Biol. Chem. 1991; 266: 16113-16119Abstract Full Text PDF PubMed Google Scholar, 21Luong C.B.H. Browner M.F. Fletterick R.J. Haymore B.L. Purification of glycogen phosphorylase isozymes by metal-affinity chromatography.J. Chromatogr. 1992; 584: 77-84Crossref PubMed Scopus (19) Google Scholar, 22Engers H.D. Shechosky S. Madsen N.B. Kinetic mechanism of phosphorylase a. I. Initial velocity studies.Can. J. Biochem. 1970; 48: 746-754Crossref PubMed Scopus (70) Google Scholar), was determined by measuring glycogen synthesis from glucose-1-phosphate by assessing the release of inorganic phosphate (reverse reaction) at 22°C in 100 μl of 50 mM HEPES buffer (pH 7.2) containing 100 mM KCl, 2.5 mM EGTA, 2.5 mM MgCl2, 0.5 mM dithiothreitol, 0.63 mM glucose-1-phosphate, 1.25 mg/ml glycogen, 9.4 mM glucose, 0.7% DMSO, and up to 2 μg of partially purified, activated human liver glycogen phosphorylase, based on the method of Engers, Shechosky, and Madsen (22Engers H.D. Shechosky S. Madsen N.B. Kinetic mechanism of phosphorylase a. I. Initial velocity studies.Can. J. Biochem. 1970; 48: 746-754Crossref PubMed Scopus (70) Google Scholar), as previously described (9Martin W.H. Hoover D.J. Armento S.J. Stock I.A. McPherson R.K. Danley D.E. Stevenson R.W. Barrett E.J. Treadway J.L. Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo.Proc. Natl. Acad. Sci. USA. 1998; 95: 1776-1781Crossref PubMed Scopus (217) Google Scholar). The inorganic phosphate released during a 60 min incubation was measured at 620 nm, 20 min after the addition of 150 μl of 1 M HCl containing 10 mg/ml ammonium molybdate and 0.38 mg/ml malachite green (23Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. An improved assay for nanomole amounts of inorganic phosphate.Anal. Biochem. 1992; 100: 95-97Crossref Scopus (1821) Google Scholar). Sterol and fatty acid synthesis were evaluated in HepG2 cells by measuring incorporation of [2-14C]acetate into cellular lipids as previously described (13Harwood Jr., H.J. Barbacci-Tobin E.G. Petras S.F. Lindsey S. Pellarin L.D. 3-(4-chlorophenyl)-2-(4-diethylaminoethoxyphenyl)-A-pentenonitrile monohydrogen citrate and related analogs. Reversible, competitive, first half-reaction squalene synthetase inhibitors.Biochem. Pharmacol. 1997; 53: 839-864Crossref PubMed Scopus (23) Google Scholar, 14Petras S.F. Lindsey S. Harwood Jr, H.J. HMG-CoA reductase regulation: use of structurally diverse first half-reaction squalene synthetase inhibitors to characterize the site of mevalonate-derived nonsterol regulator production in cultured IM-9 cells.J. Lipid Res. 1999; 40: 24-38Abstract Full Text Full Text PDF PubMed Google Scholar), with modifications (15Harwood Jr., H.J. Petras S.F. Shelly L.D. Zaccaro L.M. Perry D.A. Makowski M.R. Hargrove D.M. Martin K.A. Tracey W.R. Chapman J.G. Magee W.P. Dalvie D.K. Soliman V.F. Martin W.H. Mularski C.J. Eisenbeis S.A. Isozyme-nonselective N-substituted bipiperidylcarboxamide acetyl-CoA carboxylase inhibitors reduce tissue malonyl-CoA concentrations, inhibit fatty acid synthesis, and increase fatty acid oxidation in cultured cells and in experimental animals.J. Biol. Chem. 2003; 278: 37099-37111Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 16Chandler C.E. Wilder D.E. Pettini J.L. Savoy Y.E. Petras S.F. Chang G. Vincent J. Harwood Jr, H.J. CP-346086: an MTP inhibitor that lowers plasma cholesterol and triglycerides in experimental animals and in humans.J. Lipid Res. 2003; 44: 1886-1901Abstract Full Text Full Text PDF Scopus (175) Google Scholar) to allow simultaneous assessment of both sterol and fatty acid synthesis. HepG2 cells grown in T-75 flasks as previously described (13Harwood Jr., H.J. Barbacci-Tobin E.G. Petras S.F. Lindsey S. Pellarin L.D. 3-(4-chlorophenyl)-2-(4-diethylaminoethoxyphenyl)-A-pentenonitrile monohydrogen citrate and related analogs. Reversible, competitive, first half-reaction squalene synthetase inhibitors.Biochem. Pharmacol. 1997; 53: 839-864Crossref PubMed Scopus (23) Google Scholar, 14Petras S.F. Lindsey S. Harwood Jr, H.J. HMG-CoA reductase regulation: use of structurally diverse first half-reaction squalene synthetase inhibitors to characterize the site of mevalonate-derived nonsterol regulator production in cultured IM-9 cells.J. Lipid Res. 1999; 40: 24-38Abstract Full Text Full Text PDF PubMed Google Scholar) were seeded into 24-well plates at a density of 1.2 × 105 cells/well and maintained in 1.0 ml of supplemented DMEM (DMEM containing 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 40 μg/ml gentamicin) for 7 days in a 37°C, 5% CO2 incubator with medium changes on days 3 and 5. On day 8, the medium was removed and replaced with fresh medium containing 1% DMSO ± effector compounds. Immediately after compound addition, 25 μl of media containing 4 μCi of [2-14C]acetate (56 mCi/mmol) was added to each incubation well. Plates were then sealed with parafilm to prevent evaporation, and cells were incubated at 37°C for 6 h with gentle shaking. After incubation, the samples were saponified by addition to each well of 1 ml of 5 N KOH in MeOH, followed first by incubation for 2 h at 70°C and then by overnight incubation at RT. Mixtures were transferred to glass conical tubes and extracted three times with 4.5 ml hexane. The pooled organic fractions (containing cholesterol, post-squalene cholesterol precursors, and other nonsaponifiable lipids) were dried under nitrogen, resuspended in 25 μl chloroform, and applied to 1 × 20 cm channels of Silica Gel 60C TLC plates. Channels containing nonradioactive cholesterol, lanosterol, and squalene were included on selected TLC plates as separation markers. TLC plates were developed in hexane-diethyl ether-acetic acid (70:30:2 v/v/v), air dried, and assessed for radio
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