PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis

Abstract

Phosphatidylinositol 3-kinase (PI3K) signaling plays an important role in the regulation of cellular lipid metabolism and non-alcoholic fatty liver disease (NAFLD). However, little is known about the role of the regulatory subunits of PI3K in lipid metabolism and NAFLD. In this study, we characterized the functional role of PIK3R3 in fasting-induced hepatic lipid metabolism. In this study, we showed that the overexpression of PIK3R3 promoted hepatic fatty acid oxidation via PIK3R3-induced expression of PPARα, thus improving the fatty liver phenotype in high-fat diet (HFD)-induced mice. By contrast, hepatic PIK3R3 knockout in normal mice led to increased hepatic TG levels. Our study also showed that PIK3R3-induced expression of PPARα was dependent on HNF4α. The novel PIK3R3-HNF4α-PPARα signaling axis plays a significant role in hepatic lipid metabolism. As the activation of PIK3R3 decreased hepatosteatosis, PIK3R3 can be considered a promising novel target for developing NAFLD and metabolic syndrome therapies.

Figure 1

PIK3R3 expression is regulated by nutritional status and is upregulated in HFD-induced fatty liver mice. (a) Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained liver sections from C57BL/6J mice fed a normal chow or high-fat diet (HFD) for 8 weeks (n=6/group). (b) Serum ketone body (top panel) and hepatic TG (bottom panel) levels of mice fed a normal chow diet or HFD. (c) Quantitative PCR (top panel) analysis and western blot (bottom panel) analysis showing the expression level changes of Pik3r3 in mice livers during HFD feeding. (d) Quantitative PCR (left) analysis and western blot (right) analysis showing expression levels of hepatic Pik3r3 in C57BL/6J mice subjected to ad libitum feeding, 24 h fasting and 24 h fasted/24 h re-fed conditions (n=2/group). Data are expressed as the means±s.e.m. *P<0.05; **P<0.01.

Figure 2

Overexpression of PIK3R3 regulated lipid metabolism in vitro and alleviated the fatty liver phenotype in vivo. (a) The indicated protein expression levels were detected by western blot after hepatic HepG2 and LO2 cells were transfected with the PIK3R3 overexpression vector (PIK3R3) or control vector (Vector). (b) Quantitative PCR analysis and chemical colorimetric diagnostic analysis showing the levels of genes involved in fatty acid oxidation in mice livers. (c) Mice were fed HFD for 8 weeks (n=6/group) and injected with Ad-control or Ad-Pik3r3 as described in the Materials and methods. Mice were killed 5 days after the last injection. Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained sections of mouse livers. (d, e) Serum ketone body levels (d) and hepatic TG levels (e). Data are expressed as the means±s.e.m. *P<0.05.

Figure 3

Down-regulation of PIK3R3 impairs lipid homeostasis in vitro and in vivo. (a) PIK3R3 was knocked down in hepatic HepG2 and LO2 cells by siRNA transfection; the indicated protein expression levels were detected by western blot. (b) Quantitative PCR analysis and chemical colorimetric diagnostic analysis showing the levels of genes involved in fatty acid oxidation in mice livers. (c) Mice were fed HFD for 8 weeks (n=6/group) and injected with si-control or si-Pik3r3 as described in the Materials and methods. Mice were killed 5 days after the last injection. Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained sections of mouse livers. (d, e) Serum ketone body levels (d) and hepatic TG levels (e). Data are expressed as the means±s.e.m. *P<0.05.

Figure 4

PIK3R3 regulates the expression of PPARα. (a) Quantitative PCR (upper panel) analysis and western blot (bottom panel) analysis showing the expression level changes of Pparα in mice livers during HFD feeding. Data are expressed as the means±s.e.m. *P<0.05; **P<0.01. (b) Immunohistochemistry analysis showing the expression level changes of hepatic Pparα in the livers of the mice in Figure 2c. (c) Western blot analysis showing the expression level changes of hepatic Pparα in the livers of the mice in Figure 2c. (d) Immunohistochemistry analysis showing the expression level changes of hepatic Pparα in the livers of the mice in Figure 3c. (e) Western blot analysis showing the expression level changes of hepatic Pparα in the livers of the mice in Figure 3c. (f) PIK3R3 was knocked down in HepG2 and LO2 cells by siRNAs transfection. Western blot analysis showing the expression levels of PPARA and PPARG. (g) PIK3R3 was overexpressed in hepatic HepG2 and LO2 cells by plasmid transfection. Western blot analysis showing the expression levels of PPARA and PPARG.

Figure 5

PPARα mediates the effects of PIK3R3 on cellular and hepatic lipid metabolism. (a) PIK3R3 was overexpressed in LO2 cells with or without the knockdown of PPARA; western blot analysis showing the protein levels of PIK3R3, PPARA, ACADM and CPT1A. (b) PIK3R3 was knocked down in HepG2 cells with or without the overexpression of PPARA; western blot analysis showing the protein levels of PIK3R3, PPARA, ACADM and CPT1A. (c) Mice were fed HFD for 8 weeks (n=6/group) and injected with Ad-control, Ad-Pik3r3 or Ad-Pik3r3+si-Pparα as described in the Materials and methods. Mice were killed for further analysis 5 days after the last injection. Representative H&E, Oil Red O, Pparα, Acadm and Cpt1a stained sections of mouse livers. (d) Pik3r3 and Pparα expression levels were detected by western blot. (e) Chemical colorimetric diagnostic analysis showing the changes in hepatic TG. (f) Chemical colorimetric diagnostic analysis showing changes in serum ketone bodies. Data are expressed as the means±s.e.m. *P<0.05.

Figure 6

PIK3R3 regulation of PPARα expression was dependent on HNF4α. (a) PIK3R3 was overexpressed in LO2 cells and knocked down in HepG2 cells; western blots were used to detect the protein levels of PIK3R3 and HNF4α. (b) PIK3R3 was overexpressed in LO2 cells with or without HNF4α knockdown; western blot analysis showing the expression levels of PIK3R3, HNF4α and PPARα. (c) PIK3R3 was knocked down in LO2 cells with or without HNF4α overexpression; western blot analysis showing the expression levels of PIK3R3, HNF4α and PPARα. (d) ChIP assay and quantitative PCR analysis showing the binding activity of HNF4α to the PPARα promoter after the overexpression of PIK3R3. (e) ChIP assay and quantitative PCR analysis showing the binding activity of HNF4α to the PPARα promoter after the downregulation of PIK3R3. Data are expressed as the means±s.e.m. *P<0.05.