Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin

Abstract

The obesity epidemic has led to an increased incidence of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. AMP-activated protein kinase (Ampk) regulates energy homeostasis and is activated by cellular stress, hormones and the widely prescribed type 2 diabetes drug metformin. Ampk phosphorylates mouse acetyl-CoA carboxylase 1 (Acc1; refs. 3,4) at Ser79 and Acc2 at Ser212, inhibiting the conversion of acetyl-CoA to malonyl-CoA. The latter metabolite is a precursor in fatty acid synthesis and an allosteric inhibitor of fatty acid transport into mitochondria for oxidation. To test the physiological impact of these phosphorylation events, we generated mice with alanine knock-in mutations in both Acc1 (at Ser79) and Acc2 (at Ser212) (Acc double knock-in, AccDKI). Compared to wild-type mice, these mice have elevated lipogenesis and lower fatty acid oxidation, which contribute to the progression of insulin resistance, glucose intolerance and NAFLD, but not obesity. Notably, AccDKI mice made obese by high-fat feeding are refractory to the lipid-lowering and insulin-sensitizing effects of metformin. These findings establish that inhibitory phosphorylation of Acc by Ampk is essential for the control of lipid metabolism and, in the setting of obesity, for metformin-induced improvements in insulin action.

Fig. 1. Acc1 Ser79 and Acc2 Ser212 are essential for inhibiting enzyme activity and regulating liver fatty acid metabolism

(a) Representative Western blot of Ampk–αThr172, zcc1 Ser79 (bottom band) and Acc2 Ser212 (top band) phosphorylation in liver of WT, Acc1KI and Acc2KI and AccDKI mice. (b) Acc1 and (c) Acc2 activity with and without citrate (10 mM) in WT and AccDKI liver (n = 5 WT and 6 DKI). (d) Liver malonyl–CoA in the fed–state (n = 8). (e) The incorporation of [³H]–acetate into TAG as a measure of de novo lipogenesis and (f) [¹⁴C]–palmitate oxidation in primary hepatocytes (n = 3, from at least 3 separate experiments). (g) Total adiposity in chow-fed WT and AccDKI (n = 10 WT and 14 DKI). (h) Liver DAG and TAG (n = 6 WT and 8 DKI). (i) Histological representation (scale bar is 100 μm) and quantification of collagen staining in liver sections (n = 6). (j) Activation of liver Pkc–ε as demonstrated by membrane–association (n = 7). Data are expressed as means ± SEM, * P < 0.05, ** P < 0.01 and *** P < 0.001 relative to WT, as determined by ANOVA and Bonferonni post hoc test or a Student’s t test. For Pkc activation, Gapdh and caveolin–1 were used for cytosolic and membrane normalization, respectively, and blots shown are from duplicate gels.

Fig. 2. AccDKI mice fed a control diet are glucose intolerant and have hepatic insulin resistance

(a) Fasting blood glucose, (b) fasting serum insulin levels, (c) glucose tolerance test (2 g/kg) and (d) insulin tolerance test (0.6 U/kg) (n = 10 WT and 14 DKI) in WT and AccDKI mice. Hyperinsulinemic–euglycemic clamp results: (e) glucose infusion rate (GINF) and glucose disposal rate (GDR), (f) hepatic glucose production (HGP), and (g) suppression of hepatic glucose production (n = 7 WT and 8 DKI). (h) Liver Akt (Ser473) phosphorylation, (i) liver FoxO1 (Ser253) phosphorylation and (j) gluconeogenic gene expression (G6p and Pck) in the liver at the completion of the clamp (n = 7 WT and 8 DKI). Data are expressed as means ± SEM, * P < 0.05 and ** P < 0.01 relative to WT, as determined by Student’s t test. Relative gene expression was normalized to Actb and duplicate gels were run for quantification of total Akt and Gapdh.

Fig. 3. Metformin improves hepatic lipid metabolism via inhibition of Acc

(a) Ampk and Acc phosphorylation and (b) liver malonyl–CoA levels from saline– or metformin– (150 mg/kg) injected WT and AccDKI mice in the fed–state (n = 6 WT and 8 DKI). (c) Incorporation of [³H]–acetate into the total lipid fraction (de novo lipogenesis) in primary hepatocytes (n = 3 from at least 3 separate experiments). (d) In vivo incorporation of [³H]–acetate into total liver lipid (de novo lipogenesis) in HFD–fed WT and AccDKI mice treated with vehicle or metformin (50 mg/kg) (n = 11 for vehicle and n = 6 for metformin). (e) Representative staining (H&E) of hepatic sections (scale bar is 100 μm) as well as determination of hepatic (f) DAG and (g) TAG (n = 7 WT and 8 DKI) from WT and AccDKI mice fed a HFD for 12 weeks, with or without concurrent metformin (50 mg/kg/day) starting after 6 weeks of HFD diet. (h) Activation of hepatic Pkc–ε, shown as the ratio of membrane/cytosolic expression and expressed relative to chow WT control (n = 7) (cytosol normalized to Gapdh and membrane normalized to caveolin–1; blots shown are from duplicate gels). Data are expressed as means ± SEM, * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to WT control and ^# P < 0.05 and ^### P < 0.01 are differences between treatment, as calculated by two–way ANOVA and Bonferonni post hoc test.

Fig. 4. Obese AccDKI mice are insensitive to metformin–induced improvements in liver insulin sensitivity

WT and AccDKI mice fed a HFD for 6 weeks were given daily metformin (50 mg/kg) for an additional 6 weeks. (a) Fasting blood glucose and (b) insulin tolerance test (1 U/kg) (n = 10 HFD–fed WT and DKI; n = 12 WT and 16 DKI HFD–metformin). The effect of metformin treatment on (c) hepatic glucose production (HGP) and (d) suppression of HGP by insulin (n = 7 WT and 9 DKI). (e) Akt (Ser473) and FoxO1 (Ser253) phosphorylation, as well as (f) G6p and Pck expression, in isolated hepatocytes treated with chronic palmitate (18 h) and stimulated with insulin, where gene expression is shown relative to the WT condition without palmitate. (g) Hepatic glucose production, following chronic (18 h) exposure to palmitate (0.5 mM) in the presence or absence of metformin (0.5 mM), then in response to Bt₂–cAMP (100 μM) and insulin (10 nM) for 4 h, in the absence of acute metformin. (n = 3, from at least 3 separate experiments). Hatched line represents control hepatocytes not stimulated with Bt₂–cAMP for glucose production. (h) Schematic representation of metformin’s therapeutic effects on hepatic action during differential nutrient and hormonal programs. Data are expressed as means ± SEM, * P < 0.05, ** P < 0.01 and *** P < 0.001 represent differences between genotype and ^# P < 0.05, ^## P < 0.01 and ^### P < 0.001 are differences between treatment, as calculated by two–way ANOVA and Bonferonni post hoc test.