Genetic Liver-Specific AMPK Activation Protects against Diet-Induced Obesity and NAFLD

Abstract

The AMP-activated protein kinase (AMPK) is a highly conserved master regulator of metabolism, whose activation has been proposed to be therapeutically beneficial for the treatment of several metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). NAFLD, characterized by excessive accumulation of hepatic lipids, is the most common chronic liver disease and a major risk factor for development of nonalcoholic steatohepatitis, type 2 diabetes, and other metabolic conditions. To assess the therapeutic potential of AMPK activation, we have generated a genetically engineered mouse model, termed iAMPK^CA, where AMPK can be inducibly activated in vivo in mice in a spatially and temporally restricted manner. Using this model, we show that liver-specific AMPK activation reprograms lipid metabolism, reduces liver steatosis, decreases expression of inflammation and fibrosis genes, and leads to significant therapeutic benefits in the context of diet-induced obesity. These findings further support AMPK as a target for the prevention and treatment of NAFLD.

Keywords: AMPK; GEMM; NAFLD; lipid metabolism; liver steatosis; obesity.

Figure 1.. Generation and Validation of iAMPK^CA Mice

(A) Diagram detailing the genetically engineered components of iAMPK^CA mice at the Col1A1 and Rosa26 loci. CAG, CMV early enhancer chicken β-actin promoter; FRT, Flp recombination target; IRES, internal ribosome entry site; LSL, loxP-Stop (neomycin-resistance gene)-loxP cassette; mkate2, next-generation red-fluorescent protein; pA, polyA signal; rtTA3, reverse tetracycline-controlled transactivator 3; TRE, tet-response element. (B) Table depicting the components required for liver-specific activation of AMPK using the iAMPK^CA mouse model. Only doxycycline-treated L-iAMPK^CA mice activate AMPK in liver. The other three groups serve as control mice. (C) Western blot analysis of the expression of constitutively active AMPK and subsequent AMPK signaling activation in livers from iAMPK^CA and L-iAMPK^CA mice. Each lane is a separate mouse. d, day; Rev, 2 weeks doxycycline and then 1 week without doxycycline (reversed); w, weeks. (D) Western blot analysis comparing the magnitude of AMPK activation in livers of iAMPK^CA and L-iAMPK^CA mice fed for 3 days with chow, plus or minus doxycycline, to AMPK activation by metformin in liver (1 hr, at indicated doses; or 3 hr where indicated). Each lane is a separate mouse. met, metformin; mpk, mg per kg. (E) Livers of iAMPK^CA and L-iAMPK^CA mice, plus or minus 4 weeks doxycycline, were subjected to cellular fractionation. The subcellular localization and phosphorylation of the indicated proteins were determined by western blot. Tubulin, TOM20, and DNMT3a were used to demonstrate fraction purity. cyto, cytoplasmic fraction; iA, iAMPK^CA mice; L-iA, L-AMPK^CA mice; mito, mitochondrial fraction; nuc, nuclear fraction.

Figure 2.. AMPK Activation Reduces Hepatic Lipid Levels

(A) Schematic of experimental design to determine the effects of liver-specific AMPK activation in chow-fed iAMPK^CA and L-iAMPK^CA mice. (B) Body weight of iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 8–10 mice per condition. (C) Fasting (6 hr) blood glucose of iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 10 mice per condition. (D) Schematic of experimental design to determine the effects of AMPK activation on de novo lipogenesis in vivo. D₂O, deuterium-labeled water. (E) Levels of newly synthesized (D₂O-labeled) lipids in livers from iAMPK^CA and L-iAMPK^CA mice treated as in (D). (F) Fatty acid oxidation rates traced from uniformly labeled [U-¹³C₁₆]-Palmitate and its oxidation into citrate in hepatocytes harvested from iAMPK^CA and L-iAMPK^CA mice, fed chow with or without doxycycline for 4 weeks. (G) Oxygen consumption rate from palmitate substrate in hepatocytes harvested from iAMPK^CA and L-iAMPK^CA mice fed chow with or without doxycycline for 4 weeks. Results from two independent experiments are plotted. (H) Total levels of palmitate (C16:0), stearate (C18:0), oleate (C18:1) and linoleate (C18:2) in livers from iAMPK^CA and L-iAMPK^CA mice fed chow with or without doxycycline for 4 weeks. n = 4 mice per condition. (I) Volcano plots showing lipidomics analysis of fold changes in triglycerides and diglycerides species in livers from iAMPK^CA and L-iAMPK^CA mice fed chow with or without doxycycline for 4 weeks. n = 4 mice per condition. All values are expressed as means, and error bars reflect SEM. Significance was determined by ANOVA (*p < 0.05; **p < 0.01; **p < 0.001; ****p < 0.0001; ns, not significant).

Figure 3.. AMPK Activation in Liver Protects against Diet-Induced Obesity

(A) Protection study: schematic of experimental design to determine the effects of liver-specific AMPK activation on iAMPK^CA and L-iAMPK^CA mice fed a high-fat diet (HFD). (B) Body weights of iAMPK^CA and L-iAMPK^CA mice during the 8-week HFD trial. n = 14 mice per condition. (C) Percentage weight gain in iAMPK^CA and L-iAMPK^CA mice at the end of the HFD trial compared to initial weight. n = 14 mice per condition. (D) Body composition and (E) percent fat mass of iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 13 mice per condition. (F) Weight of one epididymal fat depot in iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 6 mice per condition. (G) Representative images of livers from iAMPK^CA and L-iAMPK^CA mice treated as in (A) stained with H&E or Oil Red O, as indicated. Scale bar, 50 μm. (H) Quantification of the area stained by Oil Red O in livers from iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 6 mice per condition. (I) Total levels of palmitate (C16:0), stearate (C18:0), oleate (C18:1), and linoleate (C18:2) in livers from iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 6 mice per condition. (J) Western blot analysis of the indicated proteins in iAMPK^CA and L-iAMPK^CA mice treated as in (A). Each lane is a separate mouse. All values are expressed as means, and error bars reflect SEM. Significance was determined by ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant).

Figure 4.. AMPK Activation in Liver Modestly Improves Glucose Homeostasis in the Context of Diet-Induced Obesity

(A) Fasting (6 hr) blood glucose in iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline for 8 weeks. n = 13 mice per condition. (B) Fasting (6 hours) plasma insulin levels in iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline for 8 weeks. n = 6 mice per condition. (C) Glucose tolerance test (GTT) on iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline for 8 weeks. n = 6 mice per condition. (D) Quantification of the area under the curve in (C). (E) Insulin tolerance test (ITT) in iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline for 8 weeks. n = 6 mice per condition. (F) Quantification of the area under the curve in (E). (G) Metabolic cage analysis of the respiratory exchange ratio (RER) in iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline for 8 weeks. Shaded area (light gray) delineates night (6:00 p.m. to 6:00 a.m.). n = 4 mice per condition. (H) Measurement of diacylglycerol (DAG) in livers of iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline for 8 weeks. n = 5 mice per condition. (I) Western blot analysis of PKCε levels in fractionated livers from iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline for 8 weeks. Tubulin and caveolin-1 were used to demonstrate fraction purity. Cyto, cytoplasmic fraction; mem, cell membrane fraction. (J) Quantification of PKCε membrane translocation in fractionated livers from iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline for 8 weeks. The ratio of membrane-associated PKCε (normalized to caveolin-1) to cytoplasmic PKCε (normalized to tubulin) is plotted. n = 4 mice per condition. All values are expressed as means, and error bars reflect SEM. Significance was determined by ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).

Figure 5.. AMPK Activation in Liver Inhibits the Onset of Diet-Induced Obesity

(A) Prevention study: schematic of experimental design to determine whether prior liver-specific AMPK activation can inhibit the onset of diet-induced obesity in L-iAMPK^CA mice. (B) Body weights of iAMPK^CA and L-iAMPK^CA mice during the HFD trial. n = 8 mice per condition. (C) Weight of iAMPK^CA and L-iAMPK^CA mice at the end of trial described in (A). n = 8 mice per condition. (D) Body composition and (E) percent fat mass of iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 8 mice per condition. (F) Fasting (6 hr) blood glucose levels in iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 7 mice per condition. (G) GTT in iAMPK^CA and L-iAMPK^CA mice treated as in (A). n = 7 mice per condition. (H) Quantification of the area under the curve in (G). All values are expressed as means, and error bars reflect SEM. Significance was determined by ANOVA (*p < 0.05; **p < 0.01).

Figure 6.. AMPK Activation in Liver Inhibits Progression of Established Diet-Induced Obesity

(A) Intervention study. Schematic of experimental design to determine the effects of liver-specific AMPK activation on established obesity in iAMPK^CA and L-iAMPK^CA mice. (B) Weights of iAMPK^CA and L-iAMPK^CA mice during the intervention study. n = 9 mice per condition. (C) Percent weight gain in iAMPK^CA and L-iAMPK^CA mice at end of the intervention study relative to weight on week 8. n = 9 mice per condition. (D and E) Body composition and (E) percent fat mass of iAMPK^CA and L-iAMPK^CA mice at the end of intervention study. n = 9 mice per condition. (F) Weight of one epididymal fat depot in iAMPK^CA and L-iAMPK^CA mice at the end of intervention study. n = 8 mice per condition. (G) Representative images of H&E- and Oil Red O-stained livers from iAMPK^CA and L-iAMPK^CA mice at the end of intervention study. Scale bar, 50 μm. (H) Quantification of Oil Red O-stained area in livers from iAMPK^CA and L-iAMPK^CA mice at the end of the intervention study. n = 6 mice per condition. (I) Fasting (6 hr) blood glucose levels in iAMPK^CA and L-iAMPK^CA mice at the end of the intervention study. n = 8 mice per condition. (J) GTT on iAMPK^CA and L-iAMPK^CA mice at the end of intervention study. n = 7 mice per condition. (K) Quantification of the area under the curve in (J). All values are expressed as means. and error bars reflect SEM. Significance was determined by ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant).

Figure 7.. AMPK Activation Downregulates Inflammation and Fibrosis-Related Transcriptional Programs

Whole-transcriptome sequencing (RNA-seq analysis) was performed on whole-liver RNA from iAMPK^CA and L-iAMPK^CA mice fed a HFD with or without doxycycline 8 weeks. (A) Heatmap representation of 283 genes upregulated after AMPK activation in liver (adjusted p < 0.05 from pairwise comparison of doxycycline-treated L-iAMPK^CA samples to each of the 3 control groups). Prkaa1 (AMPKα1) is pointed out. (B) Heatmap representation of 360 genes downregulated after AMPK activation in liver (adjusted p < 0.05 from pairwise comparison of doxycycline-treated L-iAMPK^CA samples to each of the 3 control groups). (C) Metascape pathway analysis on the genes upregulated by AMPK activation. (D) Metascape pathway analysis on the genes downregulated by AMPK activation. (E) Heatmaps showing upregulated genes in the terms “Carbohydrate catabolic process” and “Monocarboxylic acid metabolic process” from Metascape pathway analysis in (C). (F) Heatmaps showing downregulated genes in the terms “Extracellular matrix organization” and “Regulation of cell migration” from Metascape pathway analysis in (D).