Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.

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.

Deletion of hepatocyte cysteine dioxygenase type 1, a bile acid repressed gene, enhances glutathione synthesis and ameliorates acetaminophen hepatotoxicity.

Liver is a major organ that metabolizes sulfur amino acids cysteine, which is the substrate for the synthesis of many essential cellular molecules including GSH, taurine, and coenzyme A. Bile acid-activated farnesoid x receptor (FXR) inhibits cysteine dioxygenase type 1 (CDO1), which mediates hepatic cysteine catabolism and taurine synthesis. To define the impact of bile acid inhibition of CDO1 on hepatic sulfur amino acid metabolism and antioxidant capacity, we developed hepatocyte-specific CDO1 knockout mice (Hep-CDO1 KO) and hepatocyte specific CDO1 transgenic mice (Hep-CDO1 Tg). Liver metabolomics revealed that genetic deletion of hepatic CDO1 reduced de novo taurine synthesis but had no impact on hepatic taurine abundance or bile acid conjugation. Consistent with reduced cysteine catabolism, Hep-CDO1 KO mice showed increased hepatic cysteine abundance but unaltered methionine cycle intermediates and coenzyme A synthesis. Upon acetaminophen overdose, Hep-CDO1 KO mice showed increased GSH synthesis capacity and alleviated liver injury. In contrast, hepatic CDO1 overexpression in Hep-CDO1 Tg mice stimulated hepatic cysteine to taurine conversion, resulting in reduced hepatic cysteine abundance. However, Hep-CDO1 Tg mice and WT showed similar susceptibility to acetaminophen-induced liver injury. Hep-CDO1 Tg mice showed similar hepatic taurine and coenzyme A compared to WT mice. In summary, these findings suggest that bile acid and FXR signaling inhibition of CDO1-mediated hepatic cysteine catabolism preferentially modulates hepatic GSH synthesis capacity and antioxidant defense, but has minimal effect on hepatic taurine and coenzyme A abundance. Repression of hepatic CDO1 may contribute to the hepatoprotective effects of FXR activation under certain pathologic conditions.

Combining ASBT inhibitor and FGF15 treatments enhances therapeutic efficacy against cholangiopathy in female but not male Cyp2c70 KO mice.

Therapeutic reduction of hydrophobic bile acids exposure is considered beneficial in cholestasis. The Cyp2c70 KO mice lack hydrophilic muricholic acids and have a human-like hydrophobic bile acid pool resulting in hepatobiliary injury. This study investigates if combining an apical sodium-dependent bile acid transporter inhibitor GSK2330672 (GSK) and fibroblast growth factor-15 (FGF15) overexpression, via simultaneous inhibition of bile acid synthesis and gut bile acid uptake, achieves enhanced therapeutic efficacy in alleviating hepatobiliary injury in Cyp2c70 KO mice. The effects of GSK, adeno-associated virus (AAV)-FGF15, and the combined treatment on bile acid metabolism and cholangiopathy were compared in Cyp2c70 KO mice. In female Cyp2c70 KO mice with more severe cholangiopathy than male Cyp2c70 KO mice, the combined treatment was more effective in reversing portal inflammation, ductular reaction, and fibrosis than AAV-FGF15, while GSK was largely ineffective. The combined treatment reduced bile acid pool by ∼80% compared to ∼50% reduction by GSK or AAV-FGF15, and enriched tauro-conjugated ursodeoxycholic acid in the bile. Interestingly, the male Cyp2c70 KO mice treated with AAV-FGF15 or GSK showed attenuated cholangiopathy and portal fibrosis but the combined treatment was ineffective despite reducing bile acid pool. Both male and female Cyp2c70 KO mice showed impaired gut barrier integrity. AAV-FGF15 and the combined treatment, but not GSK, reduced gut exposure to lithocholic acid and improved gut barrier function. In conclusion, the combined treatment improved therapeutic efficacy against cholangiopathy than either single treatment in the female but not male Cyp2c70 KO mice by reducing bile acid pool size and hydrophobicity.

TFEB regulates sulfur amino acid and coenzyme A metabolism to support hepatic metabolic adaptation and redox homeostasis.

Fatty liver is a highly heterogenous condition driven by various pathogenic factors in addition to the severity of steatosis. Protein insufficiency has been causally linked to fatty liver with incompletely defined mechanisms. Here we report that fatty liver is a sulfur amino acid insufficient state that promotes metabolic inflexibility via limiting coenzyme A availability. We demonstrate that the nutrient-sensing transcriptional factor EB synergistically stimulates lysosome proteolysis and methionine adenosyltransferase to increase cysteine pool that drives the production of coenzyme A and glutathione, which support metabolic adaptation and antioxidant defense during increased lipid influx. Intriguingly, mice consuming an isocaloric protein-deficient Western diet exhibit selective hepatic cysteine, coenzyme A and glutathione deficiency and acylcarnitine accumulation, which are reversed by cystine supplementation without normalizing dietary protein intake. These findings support a pathogenic link of dysregulated sulfur amino acid metabolism to metabolic inflexibility that underlies both overnutrition and protein malnutrition-associated fatty liver development.

Cullin neddylation inhibitor attenuates hyperglycemia by enhancing hepatic insulin signaling through insulin receptor substrate stabilization.

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.

Effects of apical sodium-bile acid transporter inhibitor and obeticholic acid co-treatment in experimental non-alcoholic steatohepatitis.

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.

Combined ASBT Inhibitor and FGF15 Treatment Improves Therapeutic Efficacy in Experimental Nonalcoholic Steatohepatitis.

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.

Gut-restricted apical sodium-dependent bile acid transporter inhibitor attenuates alcohol-induced liver steatosis and injury in mice.

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.

Liver-specific microRNA-185 knockout promotes cholesterol dysregulation in mice.

BACKGROUND: The liver plays a key role in regulating whole body cholesterol homeostasis. Hepatic cholesterol accumulation causes liver injury in fatty liver disease and hypercholesterolemia increases the risk of cardiovascular disease. MicroRNAs (miRNAs, miRs) have been shown to regulate various pathways in cholesterol metabolism. Recently, miR-185 has been shown to regulate sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR) to modulate cholesterol synthesis and uptake. MATERIALS AND METHODS: The role of miR-185 in regulating diet-induced metabolic disorders were studied in liver-specific miRNA-185 knockout (L-miR-185 KO) mice. RESULTS: L-miR-185 KO mice developed worsened hepatic steatosis upon high fat high cholesterol Western diet feeding with accumulation of triglyceride and cholesterol in the liver. In addition, L-miR-185 KO mice developed hypercholesterolemia upon Western diet feeding. Gene expression analysis showed that L-miR-185 KO mice did not show increased hepatic mRNA expression of SREBP2 or its targets LDLR and HMG-CoA reductase (HMGCR). Although expression of miR-185 mimic inhibited the mRNA of SREBP2, HMGCR and LDLR in HepG2 cells, miR-185 inhibitor did not increase the mRNA of SREBP2, HMGCR or LDLR in HepG2 cells. CONCLUSIONS: In conclusion, we reported that L-miR-185 KO mice were more sensitive to Western diet induced hepatic steatosis and hypercholesterolemia. The molecular mechanisms underlying these metabolic changes remain to be investigated in future studies.

An FGF15/19-TFEB regulatory loop controls hepatic cholesterol and bile acid homeostasis.

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.

Hepatocyte sortilin 1 knockout and treatment with a sortilin 1 inhibitor reduced plasma cholesterol in Western diet-fed mice.

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.

HNF4α Regulates CSAD to Couple Hepatic Taurine Production to Bile Acid Synthesis in Mice.

Cysteine dioxygenase 1 (CDO1) converts cysteine to cysteine sulfinic acid, which can be further converted by cysteine sulfinic acid decarboxylase (CSAD) to hypotaurine for taurine production. This cysteine catabolic pathway plays a major role in regulating hepatic cysteine homeostasis. Furthermore, taurine is used for bile acid conjugation, which enhances bile acid solubility and physiological function in the gut. Recent studies show that this cysteine catabolic pathway is repressed by bile acid signaling, but the molecular mechanisms have not been fully elucidated. The mechanisms of bile acid and farnesoid X receptor (FXR) regulation of hepatic CSAD expression were studied in mice and hepatocytes. We showed that hepatocyte nuclear factor 4α (HNF4α) bound the mouse CSAD proximal promoter and induced CSAD transcription. FXR-induced small heterodimer partner (SHP) repressed mouse CSAD gene transcription via interacting with HNF4α as a repressor. Consistent with this model, cholic acid feeding, obeticholic acid administration, and liver HNF4α knockdown reduced hepatic CSAD expression, while liver SHP knockout and apical sodium-dependent bile acid transporter (ASBT) inhibitor treatment induced hepatic CSAD expression in mice. Furthermore, TNF-α also inhibited CSAD expression, which may be partially mediated by reduced HNF4α in mouse hepatocytes. In contrast, bile acids and GW4064 did not inhibit CSAD expression in human hepatocytes. This study identified mouse CSAD as a novel transcriptional target of HNF4α. Bile acids and cytokines repress hepatic CSAD, which closely couples taurine production to bile acid synthesis in mice. The species-specific regulation of CSAD reflects the differential preference of bile acid conjugation to glycine and taurine in humans and mice, respectively.

Bile acids regulate cysteine catabolism and glutathione regeneration to modulate hepatic sensitivity to oxidative injury.

Bile acids are signaling molecules that critically control hepatocellular function. Disrupted bile acid homeostasis may be implicated in the pathogenesis of chronic liver diseases. Glutathione is an important antioxidant that protects the liver against oxidative injury. Various forms of liver disease share the common characteristics of reduced cellular glutathione and elevated oxidative stress. This study reports a potentially novel physiological function of bile acids in regulating hepatic sulfur amino acid and glutathione metabolism. We found that bile acids strongly inhibited the cysteine dioxygenase type-1-mediated (CDO1-mediated) cysteine catabolic pathway via a farnesoid X receptor-dependent mechanism. Attenuating this bile acid repressive effect depleted the free cysteine pool and reduced the glutathione concentration in mouse liver. Upon acetaminophen challenge, cholestyramine-fed mice showed impaired hepatic glutathione regeneration capacity and markedly worsened liver injury, which was fully prevented by N-acetylcysteine administration. These effects were recapitulated in CDO1-overexpressing hepatocytes. Findings from this study support the importance of maintaining bile acid homeostasis under physiological and pathophysiological conditions, as altered hepatic bile acid signaling may negatively impact the antioxidant defense mechanism and sensitivity to oxidative injury. Furthermore, this finding provides a possible explanation for the reported mild hepatotoxicity associated with the clinical use of bile acid sequestrants in human patients.

Sortilin 1 Loss-of-Function Protects Against Cholestatic Liver Injury by Attenuating Hepatic Bile Acid Accumulation in Bile Duct Ligated Mice.

Sortilin 1 (Sort1) is an intracellular trafficking receptor that mediates protein sorting in the endocytic or secretory pathways. Recent studies revealed a role of Sort1 in the regulation of cholesterol and bile acid (BA) metabolism. This study further investigated the role of Sort1 in modulating BA detoxification and cholestatic liver injury in bile duct ligated mice. We found that Sort1 knockout (KO) mice had attenuated liver injury 24 h after bile duct ligation (BDL), which was mainly attributed to less bile infarct formation. Sham-operated Sort1 KO mice had about 20% larger BA pool size than sham-operated wildtype (WT) mice, but 24 h after BDL Sort1 KO mice had significantly attenuated hepatic BA accumulation and smaller BA pool size. After 14 days BDL, Sort1 KO mice showed significantly lower hepatic BA concentration and reduced expression of inflammatory and fibrotic marker genes, but similar degree of liver fibrosis compared with WT mice. Unbiased quantitative proteomics revealed that Sort1 KO mice had increased hepatic BA sulfotransferase 2A1, but unaltered phase-I BA metabolizing cytochrome P450s or phase-III BA efflux transporters. Consistently, Sort1 KO mice showed elevated plasma sulfated taurocholate after BDL. Finally, we found that liver Sort1 was repressed after BDL, which may be due to BA activation of farnesoid x receptor. In conclusion, we report a role of Sort1 in the regulation of hepatic BA detoxification and cholestatic liver injury in mice. The mechanisms underlying increased hepatic BA elimination in Sort1 KO mice after BDL require further investigation.

Inhibition of insulin/PI3K/AKT signaling decreases adipose Sortilin 1 in mice and 3T3-L1 adipocytes.

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.

Targeting the Enterohepatic Bile Acid Signaling Induces Hepatic Autophagy via a CYP7A1-AKT-mTOR Axis in Mice.

BACKGROUND & AIMS: Hepatic cholesterol accumulation and autophagy defects contribute to hepatocyte injury in fatty liver disease. Bile acid synthesis is a major pathway for cholesterol catabolism in the liver. This study aims to understand the molecular link between cholesterol and bile acid metabolism and hepatic autophagy activity. METHODS: The effects of cholesterol and cholesterol 7α-hydroxylase (CYP7A1) expression on autophagy and lysosome function were studied in cell models. The effects and mechanism of disrupting enterohepatic bile acid circulation on hepatic autophagy were studied in mice. RESULTS: The results first showed differential regulation of hepatic autophagy by free cholesterol and cholesterol ester, whereby a modest increase of cellular free cholesterol, but not cholesterol ester, impaired lysosome function and caused marked autolysosome accumulation. We found that CYP7A1 induction, either by cholestyramine feeding in mice or adenovirus-mediated CYP7A1 expression in hepatocytes, caused strong autophagy induction. Mechanistically, we showed that CYP7A1 expression markedly attenuated growth factor/AKT signaling activation of mechanistic target of rapamycin (mTOR), but not amino acid signaling to mTOR in vitro and in vivo. Metabolomics analysis further found that CYP7A1 induction not only decreased hepatic cholesterol but also altered phospholipid and sphingolipid compositions. Collectively, these results suggest that CYP7A1 induction interferes with growth factor activation of AKT/mTOR signaling possibly by altering membrane lipid composition. Finally, we showed that cholestyramine feeding restored impaired hepatic autophagy and improved metabolic homeostasis in Western diet-fed mice. CONCLUSIONS: This study identified a novel CYP7A1-AKT-mTOR signaling axis that selectively induces hepatic autophagy, which helps improve hepatocellular integrity and metabolic homeostasis.

Sortilin 1 knockout alters basal adipose glucose metabolism but not diet-induced obesity in mice.

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.

Sortilin 1 Modulates Hepatic Cholesterol Lipotoxicity in Mice via Functional Interaction with Liver Carboxylesterase 1.

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.

Insulin resistance induces posttranslational hepatic sortilin 1 degradation in mice.

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.