Acetyl CoA Carboxylase Inhibition Reduces Hepatic Steatosis but Elevates Plasma Triglycerides in Mice and Humans: A Bedside to Bench Investigation

Abstract

Inhibiting lipogenesis prevents hepatic steatosis in rodents with insulin resistance. To determine if reducing lipogenesis functions similarly in humans, we developed MK-4074, a liver-specific inhibitor of acetyl-CoA carboxylase (ACC1) and (ACC2), enzymes that produce malonyl-CoA for fatty acid synthesis. MK-4074 administered to subjects with hepatic steatosis for 1 month lowered lipogenesis, increased ketones, and reduced liver triglycerides by 36%. Unexpectedly, MK-4074 increased plasma triglycerides by 200%. To further investigate, mice that lack ACC1 and ACC2 in hepatocytes (ACC dLKO) were generated. Deletion of ACCs decreased polyunsaturated fatty acid (PUFA) concentrations in liver due to reduced malonyl-CoA, which is required for elongation of essential fatty acids. PUFA deficiency induced SREBP-1c, which increased GPAT1 expression and VLDL secretion. PUFA supplementation or siRNA-mediated knockdown of GPAT1 normalized plasma triglycerides. Thus, inhibiting lipogenesis in humans reduced hepatic steatosis, but inhibiting ACC resulted in hypertriglyceridemia due to activation of SREBP-1c and increased VLDL secretion.

Keywords: SREBPs; acetyl-CoA carboxylase; hepatic steatosis; hypertriglyceridemia; inhibitors; lipogenesis; malonyl-CoA.

Figure 1. Liver Targeting ACC1 and ACC2 Inhibitor MK-4074

(A) Chemical structure of MK-4074. (B) Male KKAy mice (n=10–11/group) were administered a single oral dose of MK-4074 (0.3 to 3 mg/kg) and DNL rates was measured from the liver slices as described in Experimental Procedures. (C) Male KKAy mice (n=5/group) were administered a single oral dose of MK-4074 (3 to 30 mg/kg) and liver DNL rates were measured. (D) Male KKAy mice (n=8/group) were administered a single oral dose of MK-4074 (10 to 100 mg/kg) and plasma ketone bodies were measured at the indicated times. (E) Male C57BL/6J mice (n=5, veh; n=10, MK-4074) were fed chow or a high fat/high sucrose (HF/HS) diet for 7 weeks and vehicle or MK-4074 was administered orally (10 or 30 mg/kg/day) for 4 weeks. At the end of 4 weeks, mice were euthanized and liver weights obtained. (F) Liver TG concentrations from mice described in (E). Statistical analysis was performed with Williams test except for Figure 1E, which used a two-tailed Student’s t test (* denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001).

Figure 2. Phase 1 Clinical Pharmacology

(A) Healthy subjects (n=11) were administered a single dose (140 mg), or divided doses (70 mg b.i.d.) of MK-4074 for 7 days and fructose-stimulated DNL assay was measured with stable isotope (¹³C-acetate) as described in Experimental Procedures. (B) A single dose of MK-4074 (200 mg) was administered to healthy subjects (n=12) after an overnight (8-hour) fast. Pre-dose ketone bodies were obtained, followed immediately by MK-4074 dosing. Ketone bodies were then measured under fasted state (Fasted) at 2, 3, 4, 5, 6, 7, and 8 hours after MK-4074 dosing. To determine ketone bodies under fed state (Fed), all subjects were given a breakfast at 1.5 hours after MK-4074 dosing and ketone bodies measured at 3, 4, 5, 6, 7, and 8 hours after MK-4074 dosing. The least square geometric mean change from pre-dose in acetoacetate and β-hydroxybutyrate plasma concentrations were plotted.

Figure 3. MK-4074 Decreased Hepatic TGs but Increased Plasma TGs in Humans

(A) Thirty male or female patients between the ages of 18 and 60 (Table S2) were randomized to: 1) twice daily 200 mg dose of MK-4074; 2) once daily pioglitazone (30 mg); or 3) placebo for 4 weeks. Hepatic TG content was assessed using magnetic resonance imaging (MRI) prior to first administration and following 4 weeks of treatment. ^† Statistically lower than placebo, ‡ statistically lower than pioglitazone. Statistic methods are described in Experimental Procedures and Table S3. (B) Plasma TG concentrations from subjects described in (A). (C) Plasma was obtained from subjects described in (A), lipoproteins were size-fractionated by FPLC, and TG content measured as described in Experimental Procedures.

Figure 4. Liver TGs are Reduced in ACC dLKO Mice, but Plasma TGs are Elevated in ACC dLKO Mice

(A) Liver TG concentrations from 6 male wild type and 6 ACC dLKO mice fed chow ad lib. (B) Liver TG concentrations from 6 male wild type and 6 ACC dLKO mice fed a western diet ad lib for 1 month. (C) Liver TG concentrations from 6 male wild type and 6 ACC dLKO mice fed a high fat diet ad lib for 4 months. (D) Liver TG concentrations from 6 male ob/ob and 6 ob/ob;ACC dLKO mice fed chow ad lib. (E) Plasma TGs from mice in (A). (F) Plasma TGs from mice in (B). (G) Plasma TGs from mice in (C). (H) Plasma TGs from mice in (D). (I) FPLC of plasma lipoproteins of wild type and ACC dLKO mice fed chow described in (A). Pooled plasma (500 µl) was size-fractionated using a superose 6 column and TG concentrations were measured in each fraction as described in Experimental Procedures. Statistical analysis was performed using the two-tailed Student’s t test (* denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001).

Figure 5. SREBP-1c Levels are Increased in ACC dLKO Mouse Livers

(A) Aliquots of livers (100 µg) from the mice described in Figure 4A were homogenized and nuclear and post-nuclear fractions were prepared as described in Experimental Procedures. Pooled nuclear and post-nuclear fractions (30 µg) were subjected to SDS-PAGE and immunoblot analysis was carried out using an anti-SREBP-1 rabbit monoclonal antibody. (B) Liver RNA was prepared from the mice described in Figure 4A and quantitative PCR was performed as described in Experimental Procedures. Statistical analysis was performed with the two-tailed Student’s t test (* denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001).

Figure 6. Dietary PUFA Supplementation Normalizes SREBP-1c in ACC dLKO Mouse Livers

(A) Wild type and ACC dLKO (n=6 per group) male mice were fed chow or chow supplemented with 1% (wt/wt) ARA and 0.4% (wt/wt) DHA. Livers were harvested and lipids were extracted and fatty acids analyzed using gas chromatography as described in Experimental Procedures. (B) Nuclear and post nuclear fractions were prepared from each liver of mice in (A) and equal aliquots pooled (30 µg), subjected to SDS-PAGE, and immunoblot analysis carried out as described in Figure 5. (C) Total RNA was prepared from each mouse liver in (A) and subjected to quantitative PCR as described in Experimental Procedures (Black represents chow fed group; red represents chow supplemented with ARA and DHA) (D) Plasma TGs from mice in (A) were measured as described in Experimental Procedures. (E) Liver TGs from mice in (A) were measured as described in Experimental Procedures. Statistical analysis was performed with the two-tailed Student’s t test.

Figure 7. Liver TG Secretion from Livers of ACC dLKO Mice is Increased and Knockdown of GPAT1 Expression Normalizes Plasma TGs

(A) Wild type and ACC dLKO male mice (6 mice per group) were fasted for 2 hours and Triton WR 1339 was injected to mouse intravenously. After injection, blood was collected at 0 minutes, 30 minutes, 1 hour, and 2 hours and TGs were measured in plasma. (B) Saline or siRNA nanoparticles directed against GPAT1 (7.5 mg/kg) was administered to wild type and ACC dLKO mice (6 male mice per group) by subcutaneous injection. Mice were sacrificed 2 weeks after injection and total RNA was extracted from each liver and GPAT1 mRNA was quantified using quantitative RT-PCR as described in Experimental Procedures. (C) Plasma TGs were measured as described in Experimental Procedures. (D) Liver TGs were measured as described in Experimental Procedures. Statistical analysis was performed with the two-tailed Student’s t test (* denotes p<0.05).