Metabolic Nuclear Receptors PPARα And FXR Coordinate Energy Metabolism To Regulate Sox9~+ Hepatocyte Fate

The liver is a complex organ that performs many important physiological functions,and it has strong regenerative capacity.The ability of the liver repair is largely due to the ability of mature hepatocytes or hepatic stem cells to proliferate and differentiate after injury.In the case of acute liver injury or long-term chronic liver injury,the liver may lose its regenerative capacity or regenerative capacity is insufficient.Therefore,elucidating the mechanism of liver injury repair based on the regenerative ability of mature hepatocytes or hepatic stem cells can provide a theoretical basis for the treatment of liver aplastic disorders.Peroxisome proliferator-activated receptor alpha（PPARα）and farnesoid X receptor（FXR）play an important role in regulating metabolism.Metabolic shift has an important impact on the proliferation,differentiation and self-renewal of stem cells.Sox9⁺hepatocytes are able to propagate and differentiate in response to tissue injury.Studies have reported that PPARαand FXR are involved in liver injury and repair,but whether there is coordinating role is unclear.This study mainly explored the regulatory mechanism of the metabolic nuclear receptors PPARαand FXR on Sox9,and revealed that the metabolic nuclear receptors PPARαand FXR affect the proliferation,differentiation and self-renewal of Sox9⁺hepatocytes by regulating energy metabolism.The results of the study are as follows:1.PPARαand FXR regulate Sox9 transcriptionTo determine the regulation of Sox9 expression by PPARαor FXR,HepG2 cells or primary mouse hepatocytes were treated with PPARαagonist GW7647.PPARαactivation increased the expression of Sox9 and enhancing OXPHOS and ATP levels.In addition,HepG2 cells or primary mouse hepatocytes were treated with FXR agonist GW4064.FXR activation suppressed the expression of Sox9,followed by the increase in glycolysis and the decrease ATP levels.The results are the same in vivo experiments.These data show that PPARαand FXR regulate Sox9 transcription and effect energy metabolism.2.Sox9 is a target of PPARαand FXRTo further elaborate the regulatory mechanism of PPARαand FXR on Sox9,the double luciferase reporter assays,Chromatin immunoprecipitation assay and electrophoretic mobility shift assay further confirmed that predicted binding site（IR9）in Sox9 promoter region is the common binding site of FXR and PPARα.3.PPARαinduces Sox9 expression and FAO;and FXR suppresses Sox9expression and increases the glycolysis after CCl₄-induced chronic mouse liver injuryWT,PPARα^-/-or FXR^-/-mice received intraperitoneal CCl₄ injections for four weeks,and were orally gavaged with GW7647,or GW4064.We found that PPARαactivation increases Sox9 expression and FAO,and increase ATP levels,while FXR activation suppresses Sox9 expression and increases the glycolysis,and decrease ATP levels.4.PPARαpromotes the proliferation of Sox9⁺hepatocytes,and FXR prevents the proliferation of Sox9⁺hepatocytes after CCl₄-induced chronic liver injuryWT,PPARα^-/-or FXR^-/-Sox9-Cre^ERT2;Rosa26-mTmG mice received intraperitoneal CCl₄ injections for four weeks,and were orally gavaged with GW7647,or GW4064.We found that PPARαpromotes the proliferation and differentiation of Sox9⁺hepatocytes,and FXR prevents the proliferation and differentiation of Sox9⁺hepatocytes.5.PPARαinduces proliferation and differentiation of Sox9⁺hepatocytes by enhancing FAO and OXPHOS,and FXR promote self-renewal of Sox9⁺hepatocytes by increasing glycolysis and inhibiting OXPHOSSox9-Cre^ERT2;Rosa26-mTmG mice received intraperitoneal CCl₄ injections for four weeks,and were orally gavaged with GW7647,or GW4064.We found that PPARαpromoted proliferation and differentiation of Sox9⁺hepatocytes by increasing FAO,and that FXR promoted self-renewal of Sox9⁺hepatocytes by increasing glycolysis.GFP⁺primary mouse hepatocytes from Sox9-Cre^ERT2;Rosa26-mTmG mice were treated with GW7647,or GW4064.We found that GW7647 treatment promoted proliferation of GFP⁺hepatocytes,and the morphology of mitochondria became elongated,and that GW4064 treatment induced the asymmetric division of GFP⁺hepatocytes,and the number of globule-shaped mitochondria increased.These results indicated that PPARαpromoted proliferation and differentiation of Sox9⁺hepatocytes by increasing FAO and OXPHOS,and that FXR promoted self-renewal of Sox9⁺hepatocytes by increasing glycolysis and inhibiting OXPHOS.Conclusions:PPARαand FXR oppositely control Sox9⁺hepatocyte fate in tissue homeostasis and liver injury repair via direct transcriptional regulation and energy change.The specific mechanism is as follows:1)PPARαand FXR bound to the shared site in Sox9 promoter with opposite transcriptional outputs.PPARαpromote the expression of Sox9,and FXR inhibit the expression of Sox9;2)PPARαactivation enhanced the fatty acidβ-oxidation（FAO）,oxidative phosphorylation（OXPHOS）,and adenosine triphosphate（ATP）production,thus promoting proliferation and differentiation of Sox9⁺hepatocytes along periportal（PP）—perivenous（PV）axis.However,FXR activation increased glycolysis,but decreased OXPHOS and ATP production,therefore preventing proliferation of Sox9⁺hepatocytes along PP—PV axis by promoting Sox9⁺hepatocyte self-renewal.