RESUMO
Fructose over-consumption contributes to the development of liver steatosis in part by stimulating ChREBPα-driven de novo lipogenesis. However, the mechanisms by which fructose activates ChREBP pathway remain largely undefined. Here we performed affinity purification of ChREBPα followed by mass spectrometry and identified DDB1 as a novel interaction protein of ChREBPα in the presence of fructose. Depletion and overexpression of Ddb1 showed opposite effects on the ChREBPα stability in hepatocytes. We next tested the impact of hepatic Ddb1 deficiency on the fructose-induced ChREBP pathway. After 3-week high-fructose diet feeding, both Ddb1 liver-specific knockout and AAV-TBG-Cre-injected Ddb1flox/flox mice showed significantly reduced ChREBPα, lipogenic enzymes, as well as triglycerides in the liver. Mechanistically, DDB1 stabilizes ChREBPα through CRY1, a known ubiquitination target of DDB1 E3 ligase. Finally, overexpression of a degradation-resistant CRY1 mutant (CRY1-585KA) reduces ChREBPα and its target genes in the mouse liver following high-fructose diet feeding. Our data revealed DDB1 as an intracellular sensor of fructose intake to promote hepatic de novo lipogenesis and liver steatosis by stabilizing ChREBPα in a CRY1-dependent manner.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Criptocromos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Carboidratos da Dieta/farmacologia , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Frutose/farmacologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Membrana/metabolismo , Animais , Hepatócitos/metabolismo , Proteínas Imediatamente Precoces/genética , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Cultura Primária de Células , UbiquitinaçãoRESUMO
Targeted protein degradation through ubiquitination is an important step in the regulation of glucose metabolism. Here, we present evidence that the DDB1-CUL4A ubiquitin E3 ligase functions as a novel metabolic regulator that promotes FOXO1-driven hepatic gluconeogenesis. In vivo, hepatocyte-specific Ddb1 deletion leads to impaired hepatic gluconeogenesis in the mouse liver but protects mice from high-fat diet-induced hyperglycemia. Lack of Ddb1 downregulates FOXO1 protein expression and impairs FOXO1-driven gluconeogenic response. Mechanistically, we discovered that DDB1 enhances FOXO1 protein stability via degrading the circadian protein cryptochrome 1 (CRY1), a known target of DDB1 E3 ligase. In the Cry1 depletion condition, insulin fails to reduce the nuclear FOXO1 abundance and suppress gluconeogenic gene expression. Chronic depletion of Cry1 in the mouse liver not only increases FOXO1 protein but also enhances hepatic gluconeogenesis. Thus, we have identified the DDB1-mediated CRY1 degradation as an important target of insulin action on glucose homeostasis.
Assuntos
Criptocromos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Forkhead Box O1/metabolismo , Gluconeogênese/fisiologia , Hiperglicemia/metabolismo , Fígado/metabolismo , Animais , Western Blotting , Linhagem Celular , Criptocromos/genética , Proteínas de Ligação a DNA/genética , Dieta Hiperlipídica/efeitos adversos , Proteína Forkhead Box O1/genética , Gluconeogênese/genética , Glucose/metabolismo , Humanos , Hiperglicemia/etiologia , Hiperglicemia/genética , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
Epidemiologic and animal studies implicate overconsumption of fructose in the development of nonalcoholic fatty liver disease, but the molecular mechanisms underlying fructose-induced chronic liver diseases remain largely unknown. Here, we have presented evidence supporting the essential function of the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP) in mediating adaptive responses to fructose and protecting against fructose-induced hepatotoxicity. In WT mice, a high-fructose diet (HFrD) activated hepatic lipogenesis in a ChREBP-dependent manner; however, in Chrebp-KO mice, a HFrD induced steatohepatitis. In Chrebp-KO mouse livers, a HFrD reduced levels of molecular chaperones and activated the C/EBP homologous protein-dependent (CHOP-dependent) unfolded protein response, whereas administration of a chemical chaperone or Chop shRNA rescued liver injury. Elevated expression levels of cholesterol biosynthesis genes in HFrD-fed Chrebp-KO livers were paralleled by an increased nuclear abundance of sterol regulatory element-binding protein 2 (SREBP2). Atorvastatin-mediated inhibition of hepatic cholesterol biosynthesis or depletion of hepatic Srebp2 reversed fructose-induced liver injury in Chrebp-KO mice. Mechanistically, we determined that ChREBP binds to nuclear SREBP2 to promote its ubiquitination and destabilization in cultured cells. Therefore, our findings demonstrate that ChREBP provides hepatoprotection against a HFrD by preventing overactivation of cholesterol biosynthesis and the subsequent CHOP-mediated, proapoptotic unfolded protein response. Our findings also identified a role for ChREBP in regulating SREBP2-dependent cholesterol metabolism.