P2Y2R Deficiency Ameliorates Hepatic Steatosis by Reducing Lipogenesis and Enhancing Fatty Acid β-Oxidation through AMPK and PGC-1α Induction in High-Fat Diet-Fed Mice

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease associated with obesity and insulin resistance. Activation of the purinergic receptor P2Y2R has been reported to promote adipogenesis, inflammation and dyslipidemia in adipose tissues in obese mice. However, the role of P2Y2R and its mechanisms in NAFLD remain unknown. We hypothesized that P2Y2R deficiency may play a protective role in NAFLD by modulating lipid metabolism in the liver. In this study, we fed wild type and P2Y2R knockout mice with a high-fat diet (HFD) for 12 weeks and analyzed metabolic phenotypes. First, P2Y2R deficiency effectively improved insulin resistance with a reduction in body weight and plasma insulin. Second, P2Y2R deficiency attenuated hepatic lipid accumulation and injury with reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Third, P2Y2R deficiency decreased the expression of fatty acid synthesis mediators (cluster of differentiation (CD36), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1)); and increased the expression of adipose triglyceride lipase (ATGL), a lipolytic enzyme. Mechanistically, P2Y2R deficiency increased the AMP-activated protein kinase (AMPK) activity to improve mitochondrial fatty acid β-oxidation (FAO) by regulating acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase 1A (CPT1A)-mediated FAO pathway. In addition, P2Y2R deficiency increased peroxisome proliferator-activated gamma co-activator-1α (PGC-1α)-mediated mitochondrial biogenesis. Conclusively, P2Y2R deficiency ameliorated HFD-induced hepatic steatosis by enhancing FAO through AMPK signaling and PGC-1α pathway, suggesting P2Y2R as a promising therapeutic target for NAFLD.

Keywords: AMPK; NAFLD; P2Y2R; fatty acid β-oxidation; hepatic steatosis.

Figure 1

P2Y2R deficiency reduces insulin resistance in HFD-fed mice. (A) Experimental scheme for generating HFD-induced hepatic steatosis, where WT and P2Y2R KO mice were fed with a normal chow diet (NCD) or HFD for 12 weeks. (B) Weekly changes in the body weight in WT and KO mice for 12 weeks of feeding (n = 9) (C) Blood glucose levels were determined in WT or KO mice after overnight (15 h) fasting (n = 9). (D) Plasma insulin levels were measured after sacrifice in mice fed for 12 weeks (n = 8). (E) Insulin tolerance test (ITT) were performed in WT or KO mice fed for 12 weeks, and the corresponding areas under the curves (AUC) were calculated (n = 9). Data are presented as the mean ± SEM. One-way ANOVA was used for statistical analysis followed by Bonferroni’s multiple comparison test. * p < 0.05, *** p < 0.001 vs. WT control mice; and ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. WT HFD mice; and ^$ p < 0.05, ^$$$ p < 0.001 vs. KO control mice.

Figure 2

P2Y2R deficiency attenuates hepatic steatosis and cellular injury in HFD-fed mice. (A) Plasma ALT and AST levels were measured to assess hepatic injury (n = 8). (B,C) Plasma total cholesterol and triglyceride (n = 8), and hepatic triglyceride levels were determined to assess lipid accumulation (n = 5). (D) Representative images of H&E staining from liver sections were presented for evaluating hepatic steatosis and pathological changes (n = 5). (E) Hepatic steatosis, ballooning, and lobular inflammation were semi-quantitatively evaluated from H&E-stained sections to determine NAFLD activity score (NAS) as detailed in materials and methods. Data are presented as the mean ± SEM. One-way ANOVA was used for statistical analysis followed by Bonferroni’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT control mice; and ^# p < 0.05, ^## p < 0.01, ^### p vs. WT HFD mice; and ^$ p < 0.05, ^$$ p < 0.01, ^$$$ p < 0.001 vs. KO control mice. Scale bar, 100 µm.

Figure 3

P2Y2R deficiency decreases de novo lipogenesis in HFD-fed mice. (A) Liver tissues were lysed to perform western blot analysis and the levels of proteins regulating lipid metabolism (CD36, FAS, SCD1 and ATGL) and β-actin (a loading control), were examined. Quantitative analysis of each protein was shown (n = 5). (B) The expression of genes involved in lipogenesis and lipolysis was determined by real-time PCR analysis. Relative mRNA levels were normalized to those of GAPDH (n = 3–5). Data are presented as the mean ± SEM. One-way ANOVA was used for statistical analysis followed by Bonferroni’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT control mice; and ^## p < 0.01, ^### p < 0.001 vs. WT HFD mice.

Figure 4

P2Y2R deficiency enhances mitochondrial fatty acid β-oxidation in HFD-fed mice. (A,B) Liver tissues were lysed to perform western blot analysis, and the protein levels of p-AMPK, p-LKB1, p-ACC, PGC-1α, CPT1A, and β-actin (a loading control), were examined. Quantitative analysis of each protein was shown (n = 5). (C) The mRNA expression of genes involved in fatty acid β-oxidation was determined by real-time PCR analysis. Relative mRNA levels were normalized to those of GAPDH (n = 3–5). Data are presented as the mean ± SEM. One-way ANOVA was used for statistical analysis followed by Bonferroni’s multiple comparison test. * p < 0.05, *** p < 0.001 vs. WT control mice; and ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. WT HFD mice; and ^$$ p < 0.01, ^$$$ p < 0.001 vs. KO control mice.

Figure 5

P2Y2R deficiency attenuates hepatic mitochondrial dysfunction in HFD-fed mice. (A) Liver tissues were lysed to perform western blot analysis, and the protein levels of DRP1, MFN2, OPA1 and β-actin (a loading control) were examined. Quantitative analysis of each protein was shown (n = 5). (B,C) The mRNA expression of genes involved in mitochondrial dynamics and respiratory and antioxidant functions was determined by real-time PCR analysis. Relative mRNA levels were normalized to those of GAPDH (n = 3–5). Data are presented as the mean ± SEM. One-way ANOVA was used for statistical analysis followed by Bonferroni’s multiple comparison test. ** p < 0.01, *** p < 0.001 vs. WT control mice; and ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. WT HFD mice; and ^$$ p < 0.01 vs. KO control mice.

Figure 6

A schematic diagram illustrating the role of P2Y2R in HFD-induced hepatic steatosis. P2Y2R increases triglyceride accumulation and lipogenic gene expression, while inhibiting fatty acid β-oxidation and mitochondrial homeostasis through AMPK inhibition; thus, P2Y2R aggravates hepatic steatosis in HFD-induced obesity. In P2Y2R deficiency, AMPK activation enhances ATGL-mediated lipolysis and inhibits ACC activity, promoting CPT1A-mediated FAO and PGC-1α-mediated mitochondrial homeostasis, thereby ameliorating hepatic steatosis.