AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism
2012; Elsevier BV; Volume: 54; Issue: 1 Linguagem: Inglês
10.1194/jlr.m030528
ISSN1539-7262
AutoresStephen L. Pinkosky, С. И. Филиппов, Rahul Srivastava, Jeffrey C. Hanselman, Cheryl D. Bradshaw, Timothy R. Hurley, Clay T. Cramer, Mark A. Spahr, Ashley F. Brant, Jacob L. Houghton, Chris Baker, Mark Naples, Khosrow Adeli, Roger S. Newton,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca2+/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca2+/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. acetyl-CoA carboxylase adenylate energy charge ATP-citrate lyase AMP-activated protein kinase Ca2+/calmodulin-dependent kinase β cholesteryl ester cardiovascular disease diet-induced obesity 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid free cholesterol glucose-6-phosphatase forkhead box protein O1 high-fat diet high-fat, high-cholesterol HMG-CoA reductase hepatocyte nuclear factor long-chain fatty acid LDL-cholesterol liver kinase β metabolic syndrome phosphoenolpyruvate carboxykinase type 2 diabetes triglyceride VLDL-cholesterol Cardiovascular disease (CVD) remains a leading cause of morbidity and mortality in the Western world (1Rosamond W. Flegal K. Friday G. Furie K. Go A. Greenlund K. Haase N. Ho M. Howard V. 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Statins are effective at decreasing LDL-C and CVD; however, frequent muscle-related side effects limit dosage and impede maximal risk reduction in dyslipidemic patients (10Thompson P.D. Clarkson P. Karas R.H. Statin-associated myopathy.JAMA. 2003; 289: 1681-1690Crossref PubMed Scopus (1163) Google Scholar). Furthermore, recent evidence suggests that high-dose statins may increase the risk of developing type 2 diabetes (T2D) (11Culver A.L. Ockene I.S. Balasubramanian R. Olendzki B.C. Sepavich D.M. Wactawski-Wende J. Manson J.E. Qiao Y. Liu S. Merriam P.A. et al.Statin use and risk of diabetes mellitus in postmenopausal women in the Women's Health Initiative.Arch. Intern. Med. 2012; 172: 144-152Crossref PubMed Scopus (331) Google Scholar), further justifying the need for alternative therapeutic interventions that have statin-like effects for lowering LDL-C and are designed to beneficially affect other common cardiometabolic risk factors associated with atherosclerosis and MetS. ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid), also known as ESP55016, is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. ETC-1002 favorably changes lipid profiles in preclinical models of dyslipidemia (12Cramer C.T. Goetz B. Hopson K.L. Fici G.J. Ackermann R.M. Brown S.C. Bisgaier C.L. Rajeswaran W.G. Oniciu D.C. Pape M.E. Effects of a novel dual lipid synthesis inhibitor and its potential utility in treating dyslipidemia and metabolic syndrome.J. Lipid Res. 2004; 45: 1289-1301Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), benefits glucose homeostasis in mouse models of impaired glycemic control (12Cramer C.T. Goetz B. Hopson K.L. Fici G.J. Ackermann R.M. Brown S.C. Bisgaier C.L. Rajeswaran W.G. Oniciu D.C. Pape M.E. Effects of a novel dual lipid synthesis inhibitor and its potential utility in treating dyslipidemia and metabolic syndrome.J. Lipid Res. 2004; 45: 1289-1301Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Hanselman, J. C., Bradshaw, C. D., Brant, A. F., Cramer, C. T., Hurley, T. R., Pinkosky, S. L., Spahr, M. A., Washburn, J. G., Newton, R. S., Srivastava, R. A. K., . 2011. ETC-1002 reduces circulating and hepatic triglyceride content and improves glycemic control in KKAy mice. (Abstract 554 in ATVB 2011 Scientific Sessions, Chicago, IL, April 28–30, 2011).Google Scholar), and decreases atherosclerosis in LDL-receptor-deficient mice (14Cramer, C. T., Srivastava, R. A., Lutostanski, J., Bisgaier, C. L., Newton, R. S., . 2011. ETC-1002: a novel small molecule inhibitor of lipogenesis improves plasma lipid profile and inhibits atherosclerosis progression in LDLr−/− Mouse by a lipid-dependent and independent mechanism. (Abstract 404 in ATVB 2011 Scientific Sessions, Chicago, IL, April 28–30, 2011).Google Scholar). Importantly, clinical studies have shown that ETC-1002 reduces LDL-C levels in subjects with mild dyslipidemia and has beneficial effects on other relevant cardio-metabolic risk factors, including insulin levels, hsCRP, and blood pressure (15Ballantyne C.M. Davidson M. MacDougall D. Margulies J. DiCarlo L. ETC-1002 lowers LDL-C and beneficially modulates other cardio-metabolic risk factors in hypercholesterolemic subjects with either normal or elevated triglycerides.J. Am. Coll. Cardiol. 2012; 59: E1625Crossref Google Scholar). In the current study, we demonstrate that these beneficial effects of ETC-1002 on lipid and carbohydrate metabolism are tightly linked to activation of hepatic AMP-activated protein kinase (AMPK), a master kinase controlling whole-body energy homeostasis. Additionally, the CoA thioester of ETC-1002 revealed potent inhibitory activity against hepatic ATP-citrate lyase (ACL), another central enzyme coordinating extra-mitochondrial carbon flux into the synthesis of lipids. The combination of these two distinct molecular mechanisms not only may regulate LDL-C but also may exhibit additional beneficial attributes for the treatment of CVD and provide clinically meaningful efficacy for other risk factors associated with MetS. DMEM, nonessential amino acids, HEPES, PBS, sodium pyruvate, and penicillin/streptomycin were obtained from Invitrogen (Logon, UT). Fetal bovine serum (FBS) was obtained from Hyclone (Grand Island, NY). Bovine albumin, fraction V, insulin, hydrocortisone, Triacsin C, Compound C, 5-amino-4-imidazolecarboxamide riboside (AICAR), glucagon, palmitate, acetyl-CoA, citrate, CoASH, HMG-CoA, malonyl-CoA, adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate were acquired from Sigma Chemical Co. (St. Louis, MO). [14C]acetic acid was obtained from American Radiolabeled Chemicals (St. Louis, MO). HepG2 cells (HB-8065) were purchased from American Type Culture Collection (Manassas, VA). Biocoat type I collagen-coated plates were purchased from Becton Dickinson Labware (Bedford, MA). Antibodies to AMPKα (threonine 172), AMPKα (total), acetyl-CoA carboxylase (ACC; Serine 79), ACC (total), forkhead box protein O1 (FOXO1), hepatocyte nuclear factor HNF-4α, and β-actin were obtained from Cell Signaling Technologies (Beverly, MA). Antibodies to HMGR (serine 872) were obtained from Millipore (Billerica, MA). Antibodies to citrate synthase and PGC-1α were obtained from Abcam (Cambridge, MA). HPLC grade reagents, solvents, and Ultima Gold scintillation cocktail were obtained from Sigma-Aldrich (St. Louis, MO). Microscint O was purchased from PerkinElmer (Waltham, MA). High-carbohydrate diet (D01121101B) was formulated by Research Diets (New Brunswick, NJ). For in vitro assays, ETC-1002 was formulated using aseptic technique at 30 and 100 mM in sterile dimethylsulfoxide (DMSO) and stored in sterile microcentrifuge tubes at 4°C for up to four weeks (stability was assessed). Working solutions of ETC-1002 were prepared in serum-free DMEM containing 12 mM HEPES, 10,000 U/ml penicillin, and 100 μg/ml streptomycin. ETC-1002-CoA was synthesized using rat liver microsomes essentially as described by Cramer et al. (12Cramer C.T. Goetz B. Hopson K.L. Fici G.J. Ackermann R.M. Brown S.C. Bisgaier C.L. Rajeswaran W.G. Oniciu D.C. Pape M.E. Effects of a novel dual lipid synthesis inhibitor and its potential utility in treating dyslipidemia and metabolic syndrome.J. Lipid Res. 2004; 45: 1289-1301Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). For in vivo experiments, ETC-1002 dosing solutions were formulated by preparing a disodium salt aqueous solution using 2:1 molar ratio of NaOH to ETC-1002 in water. Carboxymethyl cellulose (CMC) and Tween-20 were added to make a final solution containing 0.5% CMC and 0.025% Tween, with a final pH 7–8. Compound concentrations in dosing solutions were based upon a 10 ml/kg body weight dosing volume. Nutritionally staged male Sprague-Dawley [Crl:CD (SD)] rats were anesthetized with isoflurane, and livers were perfused for hepatocytes isolation according to the method of Ulrich et al. (16Ulrich R.G. Aspar D.G. Cramer C.T. Kletzien R.F. Ginsberg L.C. Isolation and culture of hepatocytes from the cynomolgus monkey (Macaca fascicularis).In Vitro Cell. Dev. Biol. 1990; 26: 815-823Crossref PubMed Scopus (15) Google Scholar). Hepatocytes were plated in high-glucose DMEM containing 20% FBS, 14 mM HEPES, 0.2% bovine albumin, 2 mM L-glutamine, 1× MEM nonessential amino acids, 100 nM insulin, 100 μg/ml dexamethasone, and 20 μg/ml gentamicin at a density of 1.5 × 105 cells/cm2 on collagen-coated 6-well plastic dishes. After the attachment period, (3–4 h), cells were washed once and cultured overnight in DMEM containing 10% FBS. HepG2 cells were grown and treated in DMEM containing 1 g/L D-glucose supplemented with 10% FBS. Reverse transfections were performed in 6-well culture plates at 2.5 × 105 cells/well using Lipofectamine 2000. Cells were incubated for 48 h with 10 nM silencer siRNA for liver kinase β (LKB)1 or negative control prior to compound treatment. Cells were placed on ice, deproteinized with ice-cold 6% perchloric acid, scraped, neutralized with 10 M NaOH, and buffered with 1 M K2HPO4 to precipitate potassium perchlorate. Solutions were transferred to microcentrifuge tubes and centrifuged for 5 min. Supernatant (20 μl) was diluted in cold HPLC-grade water and maintained at approximately 4°C until injection. Diluted sample (20 µl) was injected into the LC-MS/MS system and three m/z transitions were monitored (m/z 348.2 → 136.5 for AMP, m/z 428.1 → 136.5 for ADP, and m/z 508.2 → 136.5 for ATP) on an API-4000 triple-quadrupole mass spectrometer (AB Sciex, Framingham, MA). The relative amounts of AMP, ADP, and ATP were determined for each sample by normalizing triplicate measures of test conditions to vehicle treatment from the same plate. To determine the adenine nucleotide levels in freeze-clamped liver, approximately 500 mg of frozen liver was homogenized in ice-cold methanol and diluted in cold HPLC water before injecting 30 μl into the LC-MS/MS system. AMP, ADP, and ATP concentrations in liver were determined by comparing the sample peak area to the peak area of known calibration standard samples prepared in methanol. Rates of lipid synthesis were assessed in cultured primary rat hepatocytes using [14C]acetate or [14C]citrate. Experiments were performed in DMEM with 4.5 g/l glucose. Cells were treated with compound or vehicle (0.1% DMSO) for up to 4 h followed by lipid isolation. After metabolic labeling, saponified and nonsaponified lipids were extracted from cells essentially as described by Slayback et al. (17Slayback J.R. Cheung L.W. Geyer R.P. Quantitative extraction of microgram amounts of lipid from cultured human cells.Anal. Biochem. 1977; 83: 372-384Crossref PubMed Scopus (57) Google Scholar). Glucose production was measured in primary rat hepatocyte cultures. Cells were cultured in glucose- and phenol red-free DMEM, containing 10 mM lactate, 1 mM pyruvate, and nonessential amino acids (glucose production buffer, GPB). To assess the effects of ETC-1002 on glucagon-stimulated glucose production, cells were incubated with and without 0.3 μM glucagon (Sigma, St. Louis, MO) with various concentrations of ETC-1002 (0.1 to 100 μM). Media was sampled over time. Following specified treatments, cells were washed twice in GPB. Cells were then incubated for an additional hour to assess glucose production by adding GPB containing equivalent glucagon concentrations without ETC-1002. Cells were incubated for 1 h, and the concentration of glucose in the media was determined using a glucose oxidase assay kit (catalog #GAGO20-1KT; Sigma Chemicals). The activity of recombinant human ACL was carried out essentially as described in (18Ma Z. Chu C.H. Cheng D. A novel direct homogeneous assay for ATP citrate lyase.J. Lipid Res. 2009; 50: 2131-2135Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Briefly, 7.5× compounds were added to a 96-well PolyPlate containing 60 μl of Buffer (87 mM Tris, pH 8.0, 20 μM MgCl2, 10 mM KCl, 10 mM dithiothreitol) per well with substrates CoA (200 μM), ATP (400 μM), and [14C]citrate (specific activity: 2 μCi/μmol) (150 μM). Reaction was started with 4 μl (300 ng/well) ACL, and the plate was incubated at 37°C for 3 h. The reaction was terminated by the addition of 3.5 μl 500 mM EDTA. MicroScint-O (200 μl) was then added to the reaction mixture and incubated at room temperature overnight with gentle shaking. The [14C]acetyl- CoA signal was detected (5 min/well) in a TopCount NXT liquid scintillation counter (Perkin-Elmer, Waltham, MA). Hepatocyte cell lysates were prepared using approximately 150–400 μl 1× lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 1 mM PMSF, and 1× phosphatase inhibitor cocktail (Sigma). Total lysate protein concentrations were determined using the BCA Protein Assay (Bio-Rad Laboratories, Hercules, CA). Protein concentrations were adjusted and diluted in 4× LDS (lithium dodecyl sulfate gel sample buffer) containing 50 mM dithiothreitol. Proteins were separated using SDS-PAGE (4–12%) Bis/Tris, MOPS running buffer (Invitrogen, Logon, UT). Separated proteins were electrophoretically transferred to PVDF membranes. Nonspecific binding was blocked, and membranes were probed with antibodies against β-actin, total and phosphorylated ACC, AMPK α, hepatocyte nuclear factor (HNF)-4α, FOXO1, and LKB1 (Cell Signaling Technologies, Danvers, MA), PGC-1α (BioVision, Mountain View, CA), citrate synthase, phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) (Abcam, Cambridge, MA), and HMGR-ser872 (Millipore, Billerica, MA). For in vitro studies, cell culture samples were prepared as previously described for nucleotide measurements. For in vivo studies, freeze-clamped liver was homogenized in ice-cold methanol, and then samples were prepared by liquid-liquid extraction using chloroform and 0.1% formic acid in water. A portion of the aqueous phase was transferred and injected into the LC-MS/MS system monitoring five m/z transitions (808.0 → 461.2 for acetyl-CoA, 191.0 → 111.3 for citrate, 766.0 → 408.4 for CoASH, 910.0 → 408.2 for HMG-CoA, and 852.0 → 808.4 for malonyl CoA) on an API-4000 triple-quadrupole mass spectrometer (AB Sciex, Framingham, MA). Acetyl-CoA, citrate, CoASH, HMG-CoA, and malonyl-CoA concentrations were determined by comparing the sample peak area to the peak area of known calibration standard samples prepared in methanol. Male Wistar Han [Crl:WI] rats (Charles River Laboratories) weighing 225–250 g were acclimated to the laboratory environment for seven days, housed 2–3 per cage in a temperature controlled room, and maintained on a 12 h light and dark cycle with ad libitum access to food and water. Prior to single-dose ETC-1002 administration, rats were fasted for 48 h and refed a high-carbohydrate diet for an additional 48 h. For two-week assessment, rats were maintained on standard chow diet (Purina 5001) and dosed by oral gavage with ETC-1002 at 30 mg/kg/day for two weeks in the morning. Following nutritional staging and/or dosing, food was withdrawn 2 h prior to last the oral dose of vehicle control or ETC-1002. Blood and liver were collected from isofluorane-anesthetized animals 2 or 8 h after the last dose, blood was collected from the subclavian vein, and liver tissue was harvested by freeze clamp. The freeze-clamped liver samples were held frozen in liquid nitrogen immediately following excision and stored at −70°C. Plasma triglycerides, β-hydroxybutyrate (β-HBA), and total cholesterol levels were measured with commercially available kits (Wako Diagnostics, Richmond, VA) adapted to a 96-well format. All animal procedures were conducted in accordance with protocols approved by an Institutional Animal Care and Use Committee at Michigan Life Science and Innovation Center. Male golden Syrian hamsters were obtained from Charles River (Montreal, QC) at 8–10 weeks of age and weighed 100–120 g. Animals were maintained on Prolab RMH 1000 standard rodent chow diet (PMI Nutrition International, St. Louis, MO) during a seven-day quarantine period. Following randomization into treatment groups (n = 6), hyperlipidemia was induced by feeding high-fat, high-cholesterol (HFHC) Prolab RMH 1000 diet containing: 11.5% coconut oil, 11.5% corn oil, 5% fructose, and 0.5% cholesterol. During the study, animals were individually housed in an environmentally controlled room with a 12 h light and dark cycle. Following two weeks on HFHC diet, hamsters were dosed by oral gavage once daily with vehicle (0.5% carboxymethyl cellulose and 0.025% Tween-20, pH 7–8) or vehicle plus ETC-1002 (30 mg/kg) for three weeks. Body weights were recorded every two days at the beginning of dosing, and food consumption was measured every four days. Blood samples were collected by administering isoflurane anesthesia and bleeding from the orbital venous plexus in lithium heparinized tubes during the study and by cardiac puncture under anesthesia at the end of the study. Plasma samples were analyzed for triglycerides, total cholesterol, nonesterified fatty acids, and β-hydroxybutyrate on an automated chemistry analyzer. Liver and epididymal fat were collected, weighed, frozen in liquid nitrogen, and stored at −80°C until processing. All hamster procedures were conducted in accordance with the current guidelines for animal welfare at the Hospital for Sick Children and were in compliance with National Institutes of Health Publication 86-23, 1985; Animal Welfare act, 1966, as amended in 1970, 1976, and 1985, 9 CFR Parts 1, 2, and 3. Male C57BL/6N mice were obtained from Taconic (Germantown, NY) at 8 weeks of age and singly housed on α-dri paper bedding on a normal 12 h light and dark cycle (6 AM to 6 PM). Upon arrival mice, were fed a high-fat diet (HFD) containing 60% kcal fat (D12492; Research Diets, New Brunswick, NJ) for 12 weeks. Mice were randomized into two treatment arms at 20 weeks of age based on 4 h fasted blood glucose and body weight and received oral dosing of either CMC/Tween vehicle or 30 mg/kg/day ETC-1002 q.d in the morning for an additional two weeks. Body weight and food consumption were monitored throughout the study. Following the two-week dosing period, food was removed at 8 AM, and bedding was changed 2 h prior to oral administration of ETC-1002. Two hours post dose, fasting samples were collected. Fasting blood glucose levels were measured immediately prior to anesthesia using a hand-held Alphatrak glucometer (Abbott, Chicago, IL), with blood collected by unrestrained tail snip. For insulin determinations, blood was collected under isoflurane anesthesia via retro-orbital sinus into EDTA-coated tubes, and plasma was isolated by centrifugation. Plasma insulin levels were measured with a commercially available ELISA (Crystal Chem Associates, Downers Grove, IL). Plasma samples were transferred to autosampler vials and maintained at 4°C until injection onto the FPLC system (Waters Alliance 2695 Separations Module) utilizing size-exclusion chromatography with a Superose 6 10/300GL column (GE Healthcare Biosciences, Uppsala, Sweden) and 0.9% sodium chloride/0.02% sodium azide in water. Postcolumn effluent and CHOL CHOD-PAP cholesterol reagent (Roche Diagnostics, Indianapolis, IN) were mixed in-line and reacted in a 37°C heated knitted coil prior to monitor at 490 nm (Waters 2996) PDA. Very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) ratios were determined by calculation of the peak area for each protein as a percentage of the total peak area of all proteins detected in the sample. Approximately 100 mg of frozen liver was homogenized in a glass screw-top vial in 0.50 ml 150 mM sodium chloride (NaCl)/5 mM 3-(N-morpholino)propanesulfonic acid (MOPS)/1 mM EDTA/0.01% phenylmethylsulfonyl fluoride (PMSF) and extracted twice with 1.0 ml each of 2:1 dichloromethane:methanol (CH2Cl2:MeOH). The aqueous layer (top, approximately 0.8 ml) was retained for ETC-1002-CoA determinations. The combined organic phase (bottom, approximately 1.8 ml) was concentrated to dryness at 37°C under a stream of nitrogen and reconstituted into 1.0 ml 95:5:5 trimethylpentane:CH2Cl2:MeOH (TDM). A 10 μl aliquot of the prepared sample was injected into the HPLC-ELSD system utilizing a Spherisorb S5W Silica gel 5 μm, 100 × 4.6 mm ID HPLC column (Waters) running a 99:1 isooctane:tetrahydrofuran (mobile phase A), 2:1 acetone:CH2Cl2 (mobile phase B), 85:15 isopropanol:7.5 mM acetic acid/7.5 mM ethanolamine (mobile phase C) gradient before being detected using evaporative light-scattering detection (ELSD) on an SEDEX 75 detector (Sedere, Lawrenceville, NJ). Cholesteryl ester, cholesterol, and triglyceride concentrations were determined by comparing the sample peak area to the peak area of known calibration standard samples prepared in TDM. A 15 μl aliquot of the aqueous layer (top, approximately 0.8 ml) from the extraction procedure described above was injected into the HPLC system utilizing an Alltima C8 5μ, 250 × 4.6 mm ID HPLC column (Alltech Associates, Deerfield, IL) running a 15-40% acetonitrile in 25 mM potassium hydrogen phosphate (pH 7.0) gradient before being UV detection at 254 nm on a G1314A detector (Agilent Technologies, Santa Clara, CA). ETC-1002-CoA concentrations were determined by comparing the sample peak area to the peak area of a ETC-1002-CoA calibration standard. ETC-1002 has been previously shown to inhibit de novo sterol and fatty acid synthesis in primary rat hepatocytes in vitro and in vivo, with equal potency (12Cramer C.T. Goetz B. Hopson K.L. Fici G.J. Ackermann R.M. Brown S.C. Bisgaier C.L. Rajeswaran W.G. Oniciu D.C. Pape M.E. Effects of a novel dual lipid synthesis inhibitor and its potential utility in treating dyslipidemia and metabolic syndrome.J. Lipid Res. 2004; 45: 1289-1301Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In these studies, ETC-1002-CoA thioester was identified as the primary active form of ETC-1002 and was shown to inhibit partially purified ACC (IC50= 29 μM) without activating the AMPK pathway in vitro (12Cramer C.T. Goetz B. Hopson K.L. Fici G.J. Ackermann R.M. Brown S.C. Bisgaier C.L. Rajeswaran W.G. Oniciu D.C. Pape M.E. Effects of a novel dual lipid synthesis inhibitor and its potential utility in treating dyslipidemia and metabolic syndrome.J. Lipid Res. 2004; 45: 1289-1301Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In a follow-up study reported here, we unexpectedly found a marked and sustained increase in AMPK (T172) (358.3% ± 48.14; P = 0.0007) and ACC (S79) phosphorylation (164.7% ± 12.39; P = 0.001) (Fig. 1) in rat livers following two weeks of treatment with ETC-1002. Interestingly, in liver extracts, the ETC-1002 free acid concentration was approximately 110:1 molar ratio compared with the CoA thioester indicating that previously uncharacterized free acid may be involved in regulating ETC-1002-mediated metabolic activities. Furthermore, while ACC inhibition has been attributed to ETC-1002-CoA, this only explains the inhibition of fatty acid synthesis, leaving the mechanism for the equipotent inhibition of sterol synthesis unidentified. To obtain better insight into the molecular targets for ETC-1002 free acid and the CoA thioester, we first characterized the temporal nature of ETC-1002 uptake and CoA thioesterification in primary rat hepatocytes. Treatment with ETC-1002 resulted in rapid uptake and CoA thioesterification (Fig. 2A), which was associated with immediate inhibition (≤ 5 min) of de novo lipid synthesis (Fig. 2B) and transient increases in phosphorylation of AMPK (T172), ACC (S79) and HMGR (S182) (Fig. 2C). These data revealed that ETC-1002 uptake in primary rat hepatocytes is closely linked to CoA thioesterification and, unlike in vivo, results in an approximately 1:1 to 2:1 molar ratio (ETC-1002 free acid:ETC-1002-CoA) and only transient AMPK activation (Fig. 2C). The identification of relatively low intracellular ETC-1002 free acid concentrations and lack of sustained AMPK activation in primary rat hepatocytes further suggested that, in vivo, the ETC-1002 free acid may indeed be linked to the AMPK activation, while ETC-1002-CoA may mediate its effects through a mechanism distinct from AMPK. To characterize the link between ET
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