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

Abstract

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 Ca(2+)/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.

Figures

Fig. 1.

ETC-1002 activates liver AMPK in chow fed rats. Phospho-AMPKα (T172) (P-AMPK), total AMPKα (T-AMPK), phosphor-ACC (S79) (P-ACC), total ACC (T-ACC), and β-actin were determined by Western blotting of freeze-clamped liver homogenates from chow-fed rats dosed with vehicle or vehicle containing 30 mg/kg/day ETC-1002 for 14 days. Phospho/total protein ratios were calculated and are expressed as mean % vehicle ± SEM; n = 5. Comparisons were made using an unpaired Student t-test; *P < 0.05.

Fig. 2.

Primary rat hepatocytes rapidly convert ETC-1002 to a CoA thioester, which inhibits de novo lipid synthesis and transiently activates the AMPK pathway. (A) Total ETC-1002 cellular uptake, and ETC-1002-CoA levels were determined using 30 μM [14C]ETC-1002 (∼40 μci/μmol) and HPLC, respectively, following the indicated time. (B) Hepatocytes were pretreated for 5–30 min with 10 and 100 μM ETC-1002 or 500 μM of the AMP analog and AMPK activator, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), prior to a 15 min [14C]acetate pulse. Counts incorporated into the nonsaponifiable (sterols), and saponifiable (fatty acids) lipid fractions were determined as described in Materials and Methods and shown as dpm/well. (C) AMPK (T172), ACC (S79), and HMGR (S872) phosphorylation was measured in primary rat hepatocytes treated with 30 μM ETC-1002 for the indicated times. Phospho/total protein ratio was calculated and expressed as percentage of time zero. Data are representative of multiple experiments and expressed as mean ± SEM of 2–3 replicate wells. Comparisons to vehicle treatment were made using two-way ANOVA; *P < 0.05.

Fig. 3.

ETC-1002-CoA thioesterification and inhibition of total lipid synthesis. (A) Primary rat hepatocytes were treated for 2 h with the indicated concentration of ETC-1002, and intracellular CoASH, malonyl-CoA, HMG-CoA, acetyl-CoA, and citrate were measured by LC-MS/MS. (B) Hepatocytes and HepG2 cells were treated with the indicated concentration of ETC-1002 for 30 min, and the intracellular concentration of ETC-1002-CoA was determined by HPLC. (C) [14C]acetate incorporation into total lipids was measured in hepatocytes treated for 4 h with 10 μM ETC-1002 and 1, 3, or 10 μM Triacsin C. Data (A, C) were normalized to vehicle controls and expressed a mean percentage of vehicle ± SEM of triplicate wells. Data from (B) are expressed as pmol/well and shown as mean ± SEM of triplicate wells. Data are representative of multiple experiments. Comparisons were made with (C) using one-way ANOVA; *P < 0.05.

Fig. 4.

ETC-1002-CoA inhibits recombinant human ACL in a cell-free system and reduces lipid synthesis intermediates downstream of ACL in vivo. (A) Inhibition of ACL by ETC-1002-CoA is concentration-dependent and competitive for CoASH. The controls, vehicle, hydroxycitric acid (HCA), and ETC-1002-FA (free acid) are shown. Data are representative of multiple experiments and shown as mean percentage of vehicle ± SEM of triplicate wells. (B) Normal fasted/refed Wistar rats were treated with a single 30 mg/kg ETC-1002 dose. Acetyl-CoA, malonyl-CoA, HMG-CoA, and citrate were measured in freeze-clamped liver samples 2 h post dose and expressed as ng/g liver (wet weight). Data are mean ± SEM, n = 5. Comparisons were made using unpaired Student t-test; *P < 0.05.

Fig. 5.

ETC-1002 activates the AMPK pathway independent of energy depletion and calcium signaling in HepG2 cells. (A) HepG2 cells were treated for 4 h with vehicle (Veh) or the indicated concentration of ETC-1002 or metformin, and the phosphorylation of AMPKα (T172) and acetyl-CoA carboxylase (ACC) (S79) were assessed from cell lysates by Western blotting. Data are expressed as a ratio of phospho/total and presented as mean percentage of vehicle ± SEM of two wells. (B) HepG2 cells were pretreated with vehicle, 40 μM Compound C (Cmpd C), or 2.5 μM STO-609 prior to exposure to 100 μM ETC-1002 or 1,000 μM metformin for 4 h. Cell lysates were blotted for phospho-AMPKα (T172) (P-AMPK), total AMPKα, phosphor-ACC (S79) (P-ACC), total ACC, and β-actin. (C) HepG2 cells were treated with 100 μM ETC-1002 or 10 μM rotenone for 4 h. ATP, ADP, and AMP levels were determined using LC-MS/MS. ADP/ATP, AMP/ATP, and adenylate energy charge (AEC) were calculated. (D) HepG2 cells were grown in glucose or galactose media and exposed to vehicle, 3, 10, 30, 100, 300 μM ETC-1002, 10 μM CCCP, or 10 μM rotenone for 24 h. Relative intracellular ATP concentrations were determined using the ATPlite Luminescence ATP detection assay system (PerkinElmer) according to manufacturer's directions. Representative data from multiple experiments are shown. Data (A, D) were normalized to vehicle treatment and expressed as mean percentage of vehicle ± SEM of duplicate wells. Data (C) are expressed as mean ± SEM triplicate wells, and ADP/ATP, AMP/ATP, and AEC were calculated. Comparisons were made by one-way ANOVA; *P < 0.05 compared with vehicle treatment in same medium and ΨP < 0.05 between media within same compound treatment.

Fig. 6.

ETC-1002 inhibits glucagon-dependent glucose production and reduces PEPCK, G6Pase, and FOXO1 levels in primary rat hepatocytes. (A) Primary rat hepatocytes were untreated, or stimulated with 0.3 μM glucagon alone or with 100 nM insulin, or stimulated with 10 μM ETC-1002. Glucose in the media was measured 1.5, 3.5, 5.5 and 20 h poststimulation. (B) ETC-1002 glucagon-dependent glucose production concentration response (0.1 to 100 μM) following overnight exposure to ETC-1002 and glucagon. (C, D) Effects of 30 μM ETC-1002 on glucagon-dependent PEPCK and G6Pase expression and basal (no glucagon) FOXO1 protein expression. Data are presented as mean ± SEM of triplicate wells (A, B), or mean percentage of vehicle ± SEM of duplicate wells (D) Comparisons between groups were made by one-way ANOVA; *P < 0.05.

Fig. 7.

ETC-1002 activates the AMPK pathway in a LKB1-dependent manner in HepG2 cells. HepG2 cells were treated for 24 h with vehicle, 100 μM ETC-1002, or 1,000 μM metformin following a 48 h transfection with siRNA (LKB1 or mock control). (A) Western blotting was used for endogenous LKB1. The effect of LKB1 knockdown on ETC-1002 and metformin-dependent (B) ACC (S79) phosphorylation, (C) HNF4α protein level, (D) AEC, (E) total cholesterol, and (F) triglycerides are shown. Data are expressed as mean percentage of vehicle ± SEM for Western blot and mean ± SEM for AEC, total cholesterol, and triglycerides of duplicate wells. Comparisons (within siRNA treatment) to vehicle treatment were made using unpaired Student t-test; *P < 0.05.

Fig. 8.

Energy independent activation of hepatic AMPK by ETC-1002 is associated with reduced liver triglyceride, FOXO1 and HNF-4α protein levels, and markers of oxidative metabolism. (A) Plasma AST and ALT from male and female Wistar rats treated for four weeks with ETC-1002 (30 mg/kg/day). (B) Liver from the same animals as in Fig. 1 were analyzed for AEC, TG, CE, and FC content, and expressed as mg/g liver wet weight. (C) Protein levels of PGC-1α, citrate synthase, FOXO1, and HNF-4α were measured by Western blot, normalized to β-actin, and expressed as mean percentage of vehicle control ± SEM, n = 5. Comparisons were made using unpaired Student t-test; *P < 0.05.

Fig. 9.

ETC-1002 enhances β-oxidation, and reduces hepatic steatosis and plasma proatherogenic lipoproteins in hyperlipidemic male golden Syrian hamsters. (A) Hamsters fed a high-fat, high-cholesterol diet for two weeks followed by daily oral dosing with 30 mg/kg ETC-1002. Following three weeks of treatment, plasma, epididymal fat, and liver were collected. (A) Epididymal fat mass, plasma NEFA, and β-HBA levels. (B) Hepatic TG, CE, and FC levels. (C) Plasma TG, total plasma cholesterol, VLDL-C, LDL-C levels. Data are expressed mean ± SEM; n = 6. Comparisons were made using unpaired Student t-test; *P < 0.05 versus vehicle.

Fig. 10.

ETC-1002 treatment reduces body weight gain and improves glycemic control in a mouse model of diet-induced obesity. HFD-fed mice were orally administered 30 mg/kg/day ETC-1002 for two weeks. Body weight and food consumption were measured throughout the study. No effect on food consumption was observed. Following a 4 h fast, plasma glucose and insulin were measured. Data are expressed as mean ± SEM; n = 10. Comparisons were made using unpaired Student t-test; *P < 0.05 versus vehicle.

Fig. 11.

ETC-1002 activates AMPK and inhibits ACL to regulate lipid and carbohydrate metabolism. In liver, ETC-1002 free acid activates the AMPK pathway, which reduces the activities of key rate-limiting enzymes of lipid (HMGR and ACC) and carbohydrate (PEPCK and G6Pase) synthesis. ETC-1002 is also converted to ETC-1002-CoA by ACS and directly inhibits ACL. Inhibition of ACL results in a reduction in cytosolic acetyl-CoA, a common substrate supporting fatty acid and sterol synthesis, as well as reduces HMG-CoA and malonyl-CoA levels. AMPK-dependent inhibitory ACC phosphorylation and reduced enzyme substrate decrease malonyl-CoA levels, deinhibit CPT-1, and increase fatty acid mitochondrial transport for β-oxidation. The combination of AMPK activation and ACL inhibition reduces lipid and glucose synthesis from the substrate and signals transduction levels. ACC, acetyl-CoA carboxylase; ACS, acyl-CoA synthetase; β-Ox, β-oxidation; CPT-1, carnitine palmitoyltransferase-1; G6Pase, glucose 6-phosphatase; HMGR, HMG-CoA reductase; PEPCK, phosphoenolpyruvate carboxykinase; TCA, tricarboxylic acid.