RESUMO
Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.
Assuntos
Fígado Gorduroso , Hiperglicemia , Resistência à Insulina , Animais , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , NAD/metabolismo , Proteínas Culina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Camundongos Knockout , LipídeosRESUMO
Hepatic insulin resistance is a hallmark feature of nonalcoholic fatty liver disease and type-2 diabetes and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress-sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin-RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover, but the pathophysiological roles and therapeutic implications of this cellular signaling regulation is largely unknown. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a cullin scaffold. Here, we report that pharmacological inhibition of cullin neddylation by MLN4924 (Pevonedistat) rapidly decreases hepatic glucose production and attenuates hyperglycemia in mice. Mechanistically, neddylation inhibition delays CRL-mediated IRS protein turnover to prolong insulin action in hepatocytes. In vitro knockdown of either cullin 1 or cullin 3, but not other cullin members, attenuates insulin-induced IRS protein degradation and enhances cellular insulin signaling activation. In contrast, in vivo knockdown of liver cullin 3, but not cullin 1, stabilizes hepatic IRS and decreases blood glucose, which recapitulates the effect of MLN4924 treatment. In summary, these findings suggest that pharmacological inhibition of cullin neddylation represents a therapeutic approach for improving hepatic insulin signaling and lowering blood glucose.
Assuntos
Proteínas Culina/metabolismo , Ciclopentanos/farmacologia , Hiperglicemia/tratamento farmacológico , Insulina/metabolismo , Fígado/efeitos dos fármacos , Proteína NEDD8/metabolismo , Pirimidinas/farmacologia , Receptor de Insulina/metabolismo , Animais , Linhagem Celular , Hiperglicemia/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinas/metabolismoRESUMO
Background and aims: Several bile acids-based monotherapies have been developed for non-alcoholic steatohepatitis (NASH) treatment but clinical trial findings suggest that they do not satisfactorily improve NASH and liver fibrosis in many patients. Recently, we have shown that combining a gut-restricted apical sodium-bile acid transporter (ASBT) inhibitor GSK2330672 (GSK) with adeno-associated virus (AAV)-mediated liver fibroblast growth factor 15 (FGF15) overexpression provides significantly improved efficacy than either single treatment against NASH and liver fibrosis in a high fat, cholesterol, and fructose (HFCFr) diet-induced NASH mouse model. The beneficial effects of the combined treatment can be attributed to the markedly reduced bile acid pool that reduces liver bile acid burden and intestinal lipid absorption. The aim of this study is to further investigate if combining GSK treatment with the orally bioavailable obeticholic acid (OCA), which induces endogenous FGF15 and inhibits hepatic bile acid synthesis, can achieve similar anti-NASH effect as the GSK+AAV-FGF15 co-treatment in HFCFr-diet-fed mice. Materials and methods: Male C57BL/6J mice were fed HFCFr diet to induce NASH and liver fibrosis. The effect of GSK, OCA, and GSK+OCA treatments on NASH development was compared and contrasted among all groups. Results: Findings from this study showed that the GSK+OCA co-treatment did not cause persistent reduction of obesity over a 12-week treatment period. Neither single treatment nor the GSK+OCA co-treatment reduce hepatic steatosis, but all three treatments reduced hepatic inflammatory cytokines and fibrosis by a similar magnitude. The GSK+OCA co-treatment caused a higher degree of total bile acid pool reduction (~55%) than either GSK or OCA treatment alone. However, such bile acid pool reduction was insufficient to cause increased fecal lipid loss. The GSK+OCA co-treatment prevented GSK-mediated induction of hepatic cholesterol 7alpha-hydroxylase but failed to induce ileal FGF15 expression. GSK did not reduce gallbladder OCA amount in the GSK+OCA group compared to the OCA group, suggesting that ASBT inhibition does not reduce hepatic OCA distribution. Conclusions: Unlike the GSK+AAV-FGF15 co-treatment, the GSK+OCA co-treatment does not provide improved efficacy against NASH and liver fibrosis than either single treatment in mice. The lack of synergistic effect may be partly attributed to the moderate reduction of total bile acid pool and the lack of high level of FGF15 exposure as seen in the GSK+AAV-FGF15 co-treatment.
RESUMO
BACKGROUND & AIMS: Pharmacologic agents targeting bile acid signaling show promise for treating nonalcoholic steatohepatitis (NASH). However, clinical findings suggest that new treatment strategies with enhanced therapeutic efficacy and minimized undesired effects are needed. This preclinical study investigates whether combining an apical sodium-bile acid transporter (ASBT) inhibitor GSK233072 (GSK672) and fibroblast growth factor-15 (FGF15) signaling activation improves anti-NASH efficacy. METHODS: Mice with high fat, cholesterol, and fructose (HFCFr) diet-induced NASH and stage 2 fibrosis are used as a NASH model. GSK672 or AAV8-TBG-FGF15 interventions are administered alone or in combination to HFCFr diet-fed mice. RESULTS: The combined treatment significantly enhances therapeutic efficacy against steatosis, inflammation, ballooning, and fibrosis than either single treatment. Mechanistically, the synergistic actions of GSK672 and FGF15 on inhibiting gut bile acid reuptake and hepatic bile acid synthesis achieve greater magnitude of bile acid pool reduction that not only decreases bile acid burden in NASH livers but also limits intestinal lipid absorption, which, together with FGF15 signaling activation, produces weight loss, reduction of adipose inflammation, and attenuated hepatocellular organelle stress. Furthermore, the combined treatment attenuates increased fecal bile acid excretion and repressed bile acid synthesis, which underlie diarrhea and hypercholesterolemia associated with ASBT inhibition and FGF19 analogue, respectively, in clinical settings. CONCLUSIONS: Concomitant ASBT inhibition and FGF15 signaling activation produce metabolic changes that partially mimic the bariatric surgery condition whereby lipid malabsorption and increased FGF15/19 signaling synergistically mediate weight loss and metabolic improvement. Further clinical studies may be warranted to investigate whether combining ASBT inhibitor and FGF19 analogue enhances anti-NASH efficacy and reduced treatment-associated adverse events in humans.
Assuntos
Colesterol/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Metilaminas/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/terapia , Tiazepinas/administração & dosagem , Animais , Ácidos e Sais Biliares/metabolismo , Terapia Combinada , Dependovirus/genética , Modelos Animais de Doenças , Frutose/efeitos adversos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacologia , Masculino , Metilaminas/farmacologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismo , Tiazepinas/farmacologia , Resultado do TratamentoRESUMO
BACKGROUND: Recent studies have shown that human and experimental alcohol-related liver disease (ALD) is robustly associated with dysregulation of bile acid homeostasis, which may in turn modulate disease severity. Pharmacological agents targeting bile acid metabolism and signaling may be potential therapeutics for ALD. METHODS: The potential beneficial effects of a gut-restricted apical sodium-dependent bile acid transporter (ASBT) inhibitor were studied in a chronic-plus-binge ALD mouse model. RESULTS: Blocking intestinal bile acid reabsorption by the gut-restricted ASBT inhibitor GSK2330672 attenuated hepatic steatosis and liver injury in a chronic-plus-binge ALD mouse model. Alcohol feeding is associated with intestinal bile acid accumulation but paradoxically impaired ileal farnesoid × receptor (FXR) function, and repressed hepatic cholesterol 7α-hydrolase (CYP7A1) expression despite decreased hepatic small heterodimer partner (SHP) and ileal fibroblast growth factor 15 (FGF15) expression. ASBT inhibitor treatment decreased intestinal bile acid accumulation and increased hepatic CYP7A1 expression, but further decreased ileal FXR activity. Alcohol feeding induces serum bile acid concentration that strongly correlates with a liver injury marker. However, alcohol-induced serum bile acid elevation is not due to intrahepatic bile acid accumulation but is strongly and positively associated with hepatic multidrug resistance-associated protein 3 (MRP4) and MRP4 induction but poorly associated with sodium-taurocholate cotransporting peptide (NTCP) expression. ASBT inhibitor treatment decreases serum bile acid concentration without affecting hepatocyte basolateral bile acid uptake and efflux transporters. CONCLUSION: ASBT inhibitor treatment corrects alcohol-induced bile acid dysregulation and attenuates liver injury in experimental ALD.
Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatias Alcoólicas/tratamento farmacológico , Fígado/efeitos dos fármacos , Metilaminas/uso terapêutico , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Simportadores/antagonistas & inibidores , Tiazepinas/uso terapêutico , Proteínas Angiogênicas/metabolismo , Animais , Ácidos e Sais Biliares/sangue , Avaliação Pré-Clínica de Medicamentos , Fígado/metabolismo , Masculino , Metilaminas/farmacologia , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Tiazepinas/farmacologia , Transaminases/sangueRESUMO
Bile acid synthesis plays a key role in regulating whole body cholesterol homeostasis. Transcriptional factor EB (TFEB) is a nutrient and stress-sensing transcriptional factor that promotes lysosomal biogenesis. Here we report a role of TFEB in regulating hepatic bile acid synthesis. We show that TFEB induces cholesterol 7α-hydroxylase (CYP7A1) in human hepatocytes and mouse livers and prevents hepatic cholesterol accumulation and hypercholesterolemia in Western diet-fed mice. Furthermore, we find that cholesterol-induced lysosomal stress feed-forward activates TFEB via promoting TFEB nuclear translocation, while bile acid-induced fibroblast growth factor 19 (FGF19), acting via mTOR/ERK signaling and TFEB phosphorylation, feedback inhibits TFEB nuclear translocation in hepatocytes. Consistently, blocking intestinal bile acid uptake by an apical sodium-bile acid transporter (ASBT) inhibitor decreases ileal FGF15, enhances hepatic TFEB nuclear localization and improves cholesterol homeostasis in Western diet-fed mice. This study has identified a TFEB-mediated gut-liver signaling axis that regulates hepatic cholesterol and bile acid homeostasis.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Linhagem Celular , Colesterol 7-alfa-Hidroxilase/metabolismo , Dieta Ocidental/efeitos adversos , Modelos Animais de Doenças , Células Hep G2 , Humanos , Hipercolesterolemia/etiologia , Hipercolesterolemia/metabolismo , Hipercolesterolemia/prevenção & controle , Íleo/efeitos dos fármacos , Íleo/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Simportadores/antagonistas & inibidoresRESUMO
Sortilin 1 (Sort1) is a member of the Vps10p domain intracellular trafficking receptor family. Genetic variations of the SORT1 gene are strongly associated with plasma cholesterol levels in humans. Recent studies have linked Sort1 to regulation of cholesterol metabolism in hepatocytes and pro-inflammatory response in macrophages, but the tissue-specific roles of Sort1 in lipid metabolism have not been well defined. We developed Sort1 floxed mice and investigated the development of Western diet (WD)-induced steatosis, hepatic inflammatory response, and hyperlipidemia in hepatocyte Sort1 KO mice and myeloid cell Sort1 KO mice. Our findings suggest that hepatocyte Sort1 deficiency attenuated diet-induced hepatic steatosis and hypercholesterolemia in mice. In contrast, myeloid Sort1 deficiency did not reduce hepatic cytokine expression or plasma cholesterol levels, but exacerbated hepatic triglyceride accumulation in WD-fed mice. Finally, we showed that treating WD-fed mice with an orally bioavailable Sort1 inhibitor, AF38469, decreased plasma cholesterol and hepatic cytokine expression. AF38469 treatment did not affect diet-induced obesity or insulin resistance, but was associated with reduced hepatic VLDL secretion and higher hepatic cholesterol 7α-hydrolase expression in WD-fed mice. In conclusion, findings from this study suggest that Sort1 loss-of-function in hepatocytes contributes to lower plasma cholesterol, and pharmacological inhibition of Sort1 attenuates diet-induced hypercholesterolemia in mice.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Colesterol/sangue , Dieta Ocidental/efeitos adversos , Técnicas de Inativação de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Glicemia/metabolismo , Citocinas/metabolismo , Jejum/sangue , Fígado Gorduroso/sangue , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Sortilin 1(Sort1) is a vesicle trafficking receptor that mediates protein sorting in the endocytic and exocytic pathways. Sort1 is a component of the GLUT4 storage vesicles in adipocytes and is also involved in the regulation of adipogenesis. Sort1 protein is reduced in adipose of obese mice and humans, but the underlying cause is not fully understood. Here we report that insulin/PI3K/AKT signaling cascade critically regulates adipose Sort1 protein abundance. Administration of a PI3K inhibitor rapidly decreased Sort1 protein but not mRNA in adipose of chow-fed mice. In 3T3-L1 adipocytes, serum-starvation or inhibition of the PI3K/AKT signaling also decreased Sort1 protein without affecting Sort1 mRNA expression. Sort1 protein downregulation upon PI3K inhibition was blocked by pretreatment of MG132 but not Bafilomycin A1, suggesting that PI3K inhibition caused Sort1 degradation via the proteasome pathway. Using a phospho-specific Sort1 antibody, we showed that endogenous Sort1 was phosphorylated at S825 adjacent to the DXXLL sorting motif on the cytoplasmic tail. We demonstrated that mutagenesis that abolished Sort1 S825 phosphorylation decreased insulin-stimulated Sort1 localization on the plasma membrane and Sort1 protein stability in 3T3-L1 adipocytes. However, endogenous Sort1 phosphorylation at S825 was not affected by insulin stimulation or by inhibition of PI3K. In conclusion, this study revealed an important role of insulin signaling in regulating adipose Sort1 protein stability, and further suggests that impaired insulin signaling may underlie reduced adipose Sort1 in obesity. The cellular events downstream of insulin/PI3K/AKT signaling that mediates insulin regulation of Sort1 stability requires further investigation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adipócitos/metabolismo , Insulina/metabolismo , Obesidade/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células 3T3-L1 , Adipócitos/patologia , Animais , Leupeptinas/farmacologia , Macrolídeos/farmacologia , Masculino , Camundongos , Camundongos Obesos , Obesidade/patologia , Fosforilação/efeitos dos fármacosRESUMO
Sortilin 1 (Sort1) is a trafficking receptor that has been implicated in the regulation of plasma cholesterol in humans and mice. Here, we use metabolomics and hyperinsulinemic-euglycemic clamp approaches to obtain further understanding of the in vivo effects of Sort1 deletion on diet-induced obesity as well as on adipose lipid and glucose metabolism. Results show that Sort1 knockout (KO) does not affect Western diet-induced obesity nor adipose fatty acid and ceramide concentrations. Under the basal fasting state, chow-fed Sort1 KO mice have decreased adipose glycolytic metabolites, but Sort1 deletion does not affect insulin-stimulated tissue glucose uptake during the insulin clamp. These results suggest that Sort1 loss-of-function in vivo does not affect obesity development, but differentially modulates adipose glucose metabolism under fasting and insulin-stimulated states.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Tecido Adiposo/metabolismo , Glucose/metabolismo , Obesidade/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Metabolismo Basal , Ceramidas/metabolismo , Dieta Ocidental/efeitos adversos , Jejum , Ácidos Graxos/metabolismo , Técnica Clamp de Glucose , Glicólise , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/metabolismo , Insulina/administração & dosagem , Insulina/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/genéticaRESUMO
The liver plays a key role in cholesterol metabolism. Impaired hepatic cholesterol homeostasis causes intracellular free cholesterol accumulation and hepatocyte injury. Sortilin 1 (SORT1) is a lysosomal trafficking receptor that was identified by genome-wide association studies (GWAS) as a novel regulator of cholesterol metabolism in humans. Here we report that SORT1 deficiency protected against cholesterol accumulation-induced liver injury and inflammation in mice. Using an LC-MS/MS-based proteomics approach, we identified liver carboxylesterase 1 (CES1) as a novel SORT1-interacting protein. Mechanistic studies further showed that SORT1 may regulate CES1 lysosomal targeting and degradation and that SORT1 deficiency resulted in higher liver CES1 protein abundance. Previous studies have established an important role of hepatic CES1 in promoting intracellular cholesterol mobilization, cholesterol efflux, and bile acid synthesis. Consistently, high cholesterol atherogenic diet-challenged Sort1 knock-out mice showed less hepatic free cholesterol accumulation, increased bile acid synthesis, decreased biliary cholesterol secretion, and the absence of gallstone formation. SORT1 deficiency did not alter hepatic ceramide and fatty acid metabolism in high cholesterol atherogenic diet-fed mice. Finally, knockdown of liver CES1 in mice markedly increased the susceptibility to high cholesterol diet-induced liver injury and abolished the protective effect against cholesterol lipotoxicity in Sort1 knock-out mice. In summary, this study identified a novel SORT1-CES1 axis that regulates cholesterol-induced liver injury, which provides novel insights that improve our current understanding of the molecular links between SORT1 and cholesterol metabolism. This study further suggests that therapeutic inhibition of SORT1 may be beneficial in improving hepatic cholesterol homeostasis in metabolic and inflammatory liver diseases.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Hidrolases de Éster Carboxílico/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colesterol/toxicidade , Hepatócitos/patologia , Inflamação/patologia , Animais , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/genética , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Feminino , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genéticaRESUMO
Insulin promotes hepatic apolipoprotein B100 (apoB100) degradation, whereas insulin resistance is a major cause of hepatic apoB100/triglyceride overproduction in type 2 diabetes. The cellular trafficking receptor sortilin 1 (Sort1) was recently identified to transport apoB100 to the lysosome for degradation in the liver and thus regulate plasma cholesterol and triglyceride levels. Genetic variation of SORT1 was strongly associated with cardiovascular disease risk in humans. The major goal of this study is to investigate the effect and molecular mechanism of insulin regulation of Sort1. Results showed that insulin induced Sort1 protein, but not mRNA, in AML12 cells. Treatment of PI3K or AKT inhibitors decreased Sort1 protein, whereas expression of constitutively active AKT induced Sort1 protein in AML12 cells. Consistently, hepatic Sort1 was down-regulated in diabetic mice, which was partially restored after the administration of the insulin sensitizer metformin. LC-MS/MS analysis further revealed that serine phosphorylation of Sort1 protein was required for insulin induction of Sort1 in a casein kinase 2-dependent manner and that inhibition of PI3K signaling or prevention of Sort1 phosphorylation accelerated proteasome-dependent Sort1 degradation. Administration of a PI3K inhibitor to mice decreased hepatic Sort1 protein and increased plasma cholesterol and triglyceride levels. Adenovirus-mediated overexpression of Sort1 in the liver prevented PI3K inhibitor-induced Sort1 down-regulation and decreased plasma triglyceride but had no effect on plasma cholesterol in mice. This study identified Sort1 as a novel target of insulin signaling and suggests that Sort1 may play a role in altered hepatic apoB100 metabolism in insulin-resistant conditions.