Acetyl-CoA Carboxylase Inhibition Reverses NAFLD and Hepatic Insulin Resistance but Promotes Hypertriglyceridemia in Rodents

Abstract

Pharmacologic inhibition of acetyl-CoA carboxylase (ACC) enzymes, ACC1 and ACC2, offers an attractive therapeutic strategy for nonalcoholic fatty liver disease (NAFLD) through simultaneous inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. However, the effects of ACC inhibition on hepatic mitochondrial oxidation, anaplerosis, and ketogenesis in vivo are unknown. Here, we evaluated the effect of a liver-directed allosteric inhibitor of ACC1 and ACC2 (Compound 1) on these parameters, as well as glucose and lipid metabolism, in control and diet-induced rodent models of NAFLD. Oral administration of Compound 1 preferentially inhibited ACC enzymatic activity in the liver, reduced hepatic malonyl-CoA levels, and enhanced hepatic ketogenesis by 50%. Furthermore, administration for 6 days to high-fructose-fed rats resulted in a 20% reduction in hepatic de novo lipogenesis. Importantly, long-term treatment (21 days) significantly reduced high-fat sucrose diet-induced hepatic steatosis, protein kinase C epsilon activation, and hepatic insulin resistance. ACCi treatment was associated with a significant increase in plasma triglycerides (approximately 30% to 130%, depending on the length of fasting). ACCi-mediated hypertriglyceridemia could be attributed to approximately a 15% increase in hepatic very low-density lipoprotein production and approximately a 20% reduction in triglyceride clearance by lipoprotein lipase (P ≤ 0.05). At the molecular level, these changes were associated with increases in liver X receptor/sterol response element-binding protein-1 and decreases in peroxisome proliferator-activated receptor-α target activation and could be reversed with fenofibrate co-treatment in a high-fat diet mouse model. Conclusion: Collectively, these studies warrant further investigation into the therapeutic utility of liver-directed ACC inhibition for the treatment of NAFLD and hepatic insulin resistance.

Conflict of interest statement

Conflicts of interest: JB, TW, MS, and ASR are employed by and may own stock in Gilead.

© 2018 by the American Association for the Study of Liver Diseases.

Figures

Figure 1. Liver-directed inhibition of ACC by Compound 1

(A) Chemical structure of Compound 1. (B) Diagram depicting the use of AMPK phosphorylation sites as a biomarker of ACC engagement by Compound 1. (C-D) Representative Western blots depicting pACC (S79) and total ACC (C) and malonyl-CoA (D) in the liver, gastrocnemius muscle (muscle), heart, epididymal white adipose (eWAT) and brain of chow-fed male Sprague-Dawley (SD) rats treated with an intragastric bolus of 10 mg/kg Compound 1 (ACCi) or vehicle control for 1.5 h. HSP90 was used as a loading control. Quantification of blots shown to the right. Data are presented as mean ± SEM. n = 6-9 per treatment group. *P ≤ 0.05 by unpaired student's t-test compared to vehicle control.

Figure 2. Inhibition of ACC enhances hepatic ketogenesis and reduces hepatic DNL in chow and high-fructose fed rats, respectively

(A-B). Plasma βOHB (A) and whole-body βOHB turnover (B) in chow-fed male Sprague-Dawley (SD) rats fasted for 6 h, infused with 0.1 mg/(kg-min) [13C4]βOHB (basal) and treated with an intragastric bolus of 10 mg/kg Compound 1 (ACCi) or vehicle control for 1.5 h (treatment). n = 6-9 per treatment group. (C-E) Endogenous glucose production (C), hepatic pyruvate carboxylase flux (VPC) (D) and hepatic mitochondrial citrate flux (VCS) (E) in male SD rats fasted overnight, infused with 0.1 mg/(kg-min) [1,2,3,4,5,6,6-2H7]glucose and 40 uM/(kg-min) [3-13C]lactate and treated as in (A). n = 7 per treatment group. (F) Body weight, % de novo lipogenesis (DNL) and hepatic triglyceride content in male SD rats fed a high fructose diet (60%) for 6 days and treated with ACCi [10 mg/(kg BW per day)] or vehicle control. Rates of DNL were assessed in the hepatic fatty acid pool after 3 d treatment with deuterated water. Rats were sacrificed 12 h (n = 12-14 per treatment group) or 2 h (n = 6 per treatment group) after dosing. Data are presented as mean ± SEM. *P ≤ 0.05 by paired student's t-test compared to basal in each treatment group (A-B). *P ≤ 0.05 by unpaired student's t-test compared to vehicle control (C-F).

Figure 3. Compound 1 reduces hepatic steatosis in high-fat diet fed rats

(A) Male Sprague-Dawley (SD) rats were fed a high-fat diet (60% Safflower oil) supplemented with 1% sucrose drinking water (HFSD) for 3 days and treated with 10 mg/kg/day Compound 1 (ACCi) or vehicle control for 21 days. (B) Plasma, liver and muscle pharmacokinetics following single oral administration of 10 mg/kg Compound 1 to male SD-rats fed a chow diet (Chow) or rats treated as in (A) and treated with ACCi 2 or 6 h before sacrifice (HFSD). n = 3-6 per treatment group. (C) Hepatic malonyl-CoA levels in overnight fasted rats treated as in (A). (D) Representative livers (left) and liver sections stained with H&E (middle) and Oil Red O (right) of overnight rats treated as in (A). Macroscopic scale bar = 6.4 mm; microscopic scale bar = 50 μm. (E) Hepatic triglyceride (TAG) content in overnight fasted rats treated as in (A). Data are presented as mean ± SEM. *P ≤ 0.05 by unpaired student's t-test compared to vehicle control. n = 5 per treatment group.

Figure 4. Liver-directed inhibition of ACC improves hepatic insulin sensitivity

(A) Plasma glucose and glucose infusion rate (GIR) during a hyperinsulinemia-euglycemic clamp (4 mU/[kg-min]) insulin) in rats fed a high fat diet supplemented with 1% sucrose drinking water (HFSD) for 3 days and treated with 10 mg/kg/day Compound 1 (ACCi) or vehicle control for 21 days (n = 10 per group). (B) Insulin-stimulated glucose disposal (Rd). (C) Endogenous glucose production (EGP) during the basal and steady-state period of the clamp. (D) Hepatic acetyl-CoA levels in basal (overnight fasted) and clamped rats treated as in (A). (E) Insulin-mediated suppression of EGP during the clamp. (F) Insulin-mediated suppression of plasma non-esterified fatty acids (NEFAs) during the clamp. In all panels, n = 10 per treatment group. Data are presented as mean ± SEM. *P ≤ 0.05 by unpaired student's t-test compared to vehicle control.

Figure 5. Inhibition of hepatic ACC reduces membrane DAG content and PKCε translocation and increases hepatic insulin action in rats fed a HFSD

(A-C) Membrane-associated diacylglycerol (DAG) (A), total ceramides (B), and representative Western blot of protein kinase C epsilon (PKCε) membrane to cytosol translocation (C) in the livers of 6 h fasted rats that were fed a high-fat diet (60% Safflower oil) supplemented with 1% sucrose drinking water (HFSD) for 3 days and treated with 10 mg/kg/day Compound 1 (ACCi) or vehicle control for 21 days. GAPDH and NA-K ATPase were used as cytosolic and membrane loading controls, respectively. Quantification of blot shown in the right panel. (D) Representative Western blot of pAKTS473, AKT2, pIRKY1162, and INSRβ in clamped livers of rats treated as in (A). Quantification of blot shown to the right. n = 8-9 (A and C) or 12-13 (B) per treatment group. Data are presented as mean ± SEM. *P ≤ 0.05 by unpaired student's t-test compared to vehicle control.

Figure 6. Long-term inhibition of ACC increases hepatic TG secretion and reduces plasma lipid clearance in HFSD-fed rats

(A) Plasma triglyceride content of FPLC-fractionated lipoproteins from pooled plasma (n = 4 per group) of overnight fasted rats that were fed a high-fat diet (60% Safflower oil) supplemented with 1% sucrose drinking water (HFSD) for 3 days and treated with 10 mg/kg/day Compound 1 (ACCi) or vehicle control for 21 days. (B) Fasting plasma triglycerides in rats treated as in (A). n = 12 per group. (C-D) Hepatic triglyceride production in overnight fasted rats treated as in (A) and injected with Poloxamer 407 to inhibit lipolysis of triglyceride rich lipoproteins (TRL). n = 10-14 per treatment group. (E) Lipid clearance test in overnight fasted rats treated as in (A) and given an intravenous bolus of 20% Intralipid conjugated with 3H-labeled triolein. n = 6 per treatment group. (F) Post-heparin plasma LPL activity in rats treated as in (A). n = 7-9 per treatment group. (G–I) mRNA (G) and protein (H) expression of indicated genes in the livers of overnight fasted rats treated as in (A). n = 7–10 per treatment group. In panel (H), HSP90 was used as a loading control. Quantification of blot shown in panel (I). (J) Plasma APOC3 concentration in rats treated as in (A). n = 7 per treatment group. (K) Plasma ANGPTL3 concentration in rats treated as in (A). n = 12 per treatment group. Data are presented as mean ± SEM. In panels (B-G and J-K), *P ≤ 0.05 by unpaired student's t-test compared to vehicle control.

Figure 7. Fenofibrate reverses ACCi-mediated hypertriglyceridemia in FFD-fed mice

(A) Male B6 mice were fed a fast food diet (FFD) for 5 months and treated with 1 mg/kg (BID), 5 mg/kg (BID), 10 mg/kg (BID) Compound 1 (ACCi) or vehicle control for 28 days. (B–C) Liver (B) and plasma (C) triglycerides in mice treated as in (A). n = 8–13 per treatment group. (C) Male B6 mice were fed a fast food diet (FFD) for 5 months and treated with vehicle, 10 mg/kg (BID) Compound 1 (ACCi), or 10 mg/kg ACCi (BID) and fenofibrate (0.1% in chow, feno) for 14 days. (D) Plasma triglycerides (pooled samples collected at 2, 6, and 24 h post dose) in mice treated as in (C). Values are expressed as percent of vehicle-treated animals collected at the same time of day after treatment with vehicle, ACCi or ACCi + fenofibrate. (D) mRNA expression of SREBP1c and PLIN2 (a canonical PPARα target) in the livers of mice treated as in (C). Data are presented as mean ± SEM. In panels (B-C), *P ≤ 0.05 compared to vehicle control by one-way ANOVA with Dunnett correction for multiple comparisons. In panels (D-E), *P ≤ 0.05 by unpaired student's t-test. BID: twice daily; QD: once daily.