Acetyl-CoA carboxylase 2-/- mutant mice are protected against fatty liver under high-fat, high-carbohydrate dietary and de novo lipogenic conditions

Abstract

Hepatic fat accumulation resulting from increased de novo fatty acid synthesis leads to hepatic steatosis and hepatic insulin resistance. We have shown previously that acetyl-CoA carboxylase 2 (Acc2(-/-)) mutant mice, when fed a high-fat (HF) or high-fat, high-carbohydrate (HFHC) diet, are protected against diet-induced obesity and maintained whole body and hepatic insulin sensitivity. To determine the effect of an ACC2 deletion on hepatic fat metabolism, we studied the regulation of the enzymes involved in the lipogenic pathway under Western HFHC dietary and de novo lipogenic conditions. After completing the HFHC regimen, Acc2(-/-) mutant mice were found to have lower body weight, smaller epididymal fat pads, lower blood levels of nonesterified fatty acids and triglycerides, and higher hepatic cholesterol than wild-type mice. Significant up-regulation of lipogenic enzymes and an elevation in hepatic peroxisome proliferator-activated receptor-γ (PPAR-γ) protein were found in Acc2(-/-) mutant mice under de novo lipogenic conditions. The increase in lipogenic enzyme levels was accompanied by up-regulation of the transcription factors, sterol regulatory element-binding proteins 1 and 2, and carbohydrate response element-binding protein. In contrast, hepatic levels of the PPAR-γ and PPAR-α proteins were significantly lower in the Acc2(-/-) mutant mice fed an HFHC diet. When compared with wild-type mice fed the same diet, Acc2(-/-) mutant mice exhibited a similar level of AKT but with a significant increase in pAKT. Hence, deleting ACC2 ameliorates the metabolic syndrome and protects against fatty liver despite increased de novo lipogenesis and dietary conditions known to induce obesity and diabetes.

FIGURE 1.

Body and tissue weights of Acc2^−/− mutant and control mice under different feeding conditions. The weights of 4–5-month-old male mice (n = 5) in each different feeding group, as indicated at the right of each panel, were measured. ND, mice that were maintained on a normal diet for 4–5 mo; FRFD, mice that fasted for 48 h and then fed a fat-free, high-carbohydrate diet for an additional 48 h; HFHC, 3–4-month-old male mice that were fed a high-fat, high-carbohydrate diet for an additional 2 months; FAST, 4–5-month-old mice that fasted for 48 h. Before the mice were sacrificed, blood was collected from their tails after a 4–5-h fasting period to determine the blood constituents. At the end of each experiment, the mice were weighed and sacrificed, and the extracted tissues were weighed and kept at −80 °C for further analyses.

FIGURE 2.

Representative Western blots of lipogenic enzymes, and TG and cholesterol levels in liver extracts from control and Acc2^−/− mutant mice fed a normal diet. In Western blot analyses, crude liver extracts (“Experimental Procedures”) were separated by 4–12% NuPAGE MES gels. A, 30-μg protein samples of liver extracts from control and Acc2^−/− mutant mice were separated by gel electrophoresis. Antibodies against ACL, ACC1, and FAS were used to detect their respective levels with ECL. The bottom panel shows a representative Western blot after staining with Ponceau S to indicate equal loading. B, the respective bands in the blots in A were scanned and quantified. C and D, activities of ACC and FAS in liver crude extracts, respectively. E and F, triglyceride and cholesterol levels in liver extracts from the two groups of mice were determined as described under “Experimental Procedures.”

FIGURE 3.

Body weights, representative Western blots, enzyme activities of ACC and FAS, and levels of TGs and cholesterol in samples of liver extracts from control and Acc2^−/− mutant mice fed a high-fat, high-carbohydrate diet. A, body weights of 5-week-old male mice fed a high-fat, high-carbohydrate diet for 16 weeks (n = 20). B–H, represent data from 3–4-month-old male mice fed a high-fat, high-carbohydrate diet for 2 months. B and C, representative Western blots of liver extracts and the quantification of the respective bands as described for Fig. 2. For detection of ACC, we used ACC1 antibodies, as we described previously (3). D and E, enzyme activities of ACC and FAS, respectively. F and G, levels of TGs and cholesterol in liver extracts, as determined by enzymatic methods. H, Oil Red O staining of frozen liver tissues showing the accumulation of oil droplets in orange; the bar indicates 20 μm.

FIGURE 4.

Representative Western blots of AKT and pAKT levels in samples from liver extracts of wild-type and Acc2^−/− mutant mice fed a high-fat, high-carbohydrate diet. A, 30-μg protein samples from liver extracts of wild-type and Acc2^−/− mutant mice were subjected to electrophoresis gel separation using 4–12% NuPAGE MES gels. Blots were probed with antibodies against AKT or pAKT and detected with ECL. The bottom panel shows a representative blot stained with Ponceau S as a control for equal loading. B, average ratio of the scanned values of pAKT and AKT.

FIGURE 5.

Representative Western blots and activities of lipogenic enzymes and lipid levels in samples from liver extracts of wild-type and Acc2^−/− mutant mice under fasted-refed dietary conditions. A, 30-μg protein samples from liver extracts of wild-type and Acc2^−/− mutant mice were subjected to separation by gel electrophoresis using 4–12% NuPAGE MES gels and probed with the antibodies indicated, as described in the legends to Figs. 2–4, and avidin-peroxidase to detect ACC. B, the relative abundance of lipogenic enzymes in A, determined by scanning the respective bands. C and D, ACC and FAS activities in liver crude extracts, respectively. E and F, hepatic levels of TGs and cholesterol determined by enzymatic methods.

FIGURE 6.

Representative Western blots, activities of lipogenic enzymes, and TG and cholesterol levels in samples from liver extracts of control and Acc2^−/− mutant mice after 48-h fasting. A, 30-μg protein samples from liver extracts of wild-type and Acc2^−/− mutant mice were separated by gel electrophoresis using 4–12% NuPAGE MES gels and probed with the indicated antibodies, as described in the legend to Fig. 2. B, relative values of scanned bands shown in A. C and D, ACC and FAS activities, respectively, in liver extracts. E and F, levels of TGs and cholesterol, respectively.

FIGURE 7.

Relative mRNA levels of transcription factors and target genes in liver. RNA was isolated from livers of both wild-type and Acc2^−/− mutant mice (n = 5) and measured by real-time RT-PCR as described previously (17). The values were normalized to 18 S ribosomal RNA and are depicted as mean ± S.D. (*, p < 0.05).

FIGURE 8.

Levels of transcription factors PPAR-γ and PPAR-α in liver extracts of control and Acc2^−/− mutant mice under fasted-refed (FRFD) conditions. A, representative Western blots for PPAR-γ and PPAR-α as indicated by arrows. Bottom panel shows a representative blot stained with Ponceau S as a control for equal loading. B and C, scanned values of PPAR-γ and PPAR-α, respectively. D, representative Northern blot of total RNA, which was electrophoresed on a 1% agarose gel in the presence of formalin and transferred to Hybond N filters. The filters were hybridized with ³²P-labeled cDNA probes of PPAR-γ and PPAR-α. Bottom panel shows an ethidium bromide-stained agarose gel for control of equal loading; the ribosomal RNA 28 S and 18 S are indicated. E, scanned values of the PPAR-γ and PPAR-α bands. Data are expressed as mean ± S.D. (n = 5; *, p < 0.05).

FIGURE 9.

Levels of transcription factors PPAR-γ and PPAR-α in liver extracts of control and Acc2^−/− mutant mice under high-fat, high-carbohydrate feeding conditions. A, representative Western blots of PPAR-γ and PPAR-α, and Ponceau S-stained filters as a control for equal loading. B and C, relative levels of PPAR-γ and PPAR-α as determined by scanning the respective bands. D, relative mRNA levels of PPAR-γ and PPAR-α transcripts determined by real-time quantitative PCR. Data are expressed as mean ± S.D. (n = 5; *, p < 0.05).

FIGURE 10.

Schematic proposed model for major metabolic pathways affected by ACC2 deletion in the livers of wild-type mice fed FFHC and HFHC diets. Master regulators of fat metabolic enzymes (SREBP1, SREBP2, ChREBP, and PPAR-γ) are induced under FFHC feeding conditions that result in up-regulation of key enzymes in fatty acid and cholesterol metabolism (ACL, ACC1, ACC2, FAS, mevalonate pyrophosphate decarboxylase (MVD), and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR)). In livers of wild-type mice, where ACC2 is highly active, the malonyl-CoA produced blocks the activity of CPT1, thus inhibiting fatty acid oxidation and leading to excessive accumulation of excessive TGs and more fat (A). When ACC2 is deleted, there is a further increase in the transcriptional factors of fat synthesis, resulting in even more up-regulation of the lipogenic enzymes. However, the lack of CPT1 inhibition by ACC2-produced malonyl-CoA results in increased fatty acid oxidation and significant reduction in the accumulation of long-chain fatty acids and TGs and prevents fatty liver (B). Under HFHC feeding conditions, the livers of wild-type mice accumulate high levels of TGs as a result of exogenous fatty acids and de novo synthesis from the high carbohydrate in the diet (C). In livers of ACC2 mutant mice, fewer TGs accumulate due both to down-regulation of lipogenic enzymes and increased fatty acid oxidation, thus preventing excessive accumulation of TGs in these livers (D). The decrease in acylglycerides in livers of Acc2^−/− mutant mice on a HFHC diet will result in an increase in pAKT that will enhance insulin sensitivity and increase glucose uptake (D). Thick arrows indicate up-regulation.