DEP domain-containing mTOR-interacting protein suppresses lipogenesis and ameliorates hepatic steatosis and acute-on-chronic liver injury in alcoholic liver disease

Abstract

Alcoholic liver disease (ALD) is characterized by lipid accumulation and liver injury. However, how chronic alcohol consumption causes hepatic lipid accumulation remains elusive. The present study demonstrates that activation of the mechanistic target of rapamycin complex 1 (mTORC1) plays a causal role in alcoholic steatosis, inflammation, and liver injury. Chronic-plus-binge ethanol feeding led to hyperactivation of mTORC1, as evidenced by increased phosphorylation of mTOR and its downstream kinase S6 kinase 1 (S6K1) in hepatocytes. Aberrant activation of mTORC1 was likely attributed to the defects of the DEP domain-containing mTOR-interacting protein (DEPTOR) and the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) in the liver of chronic-plus-binge ethanol-fed mice and in the liver of patients with ALD. Conversely, adenoviral overexpression of hepatic DEPTOR suppressed mTORC1 signaling and ameliorated alcoholic hepatosteatosis, inflammation, and acute-on-chronic liver injury. Mechanistically, the lipid-lowering effect of hepatic DEPTOR was attributable to decreased proteolytic processing, nuclear translocation, and transcriptional activity of the lipogenic transcription factor sterol regulatory element-binding protein-1 (SREBP-1). DEPTOR-dependent inhibition of mTORC1 also attenuated alcohol-induced cytoplasmic accumulation of the lipogenic regulator lipin 1 and prevented alcohol-mediated inhibition of fatty acid oxidation. Pharmacological intervention with rapamycin alleviated the ability of alcohol to up-regulate lipogenesis, to down-regulate fatty acid oxidation, and to induce steatogenic phenotypes. Chronic-plus-binge ethanol feeding led to activation of SREBP-1 and lipin 1 through S6K1-dependent and independent mechanisms. Furthermore, hepatocyte-specific deletion of SIRT1 disrupted DEPTOR function, enhanced mTORC1 activity, and exacerbated alcoholic fatty liver, inflammation, and liver injury in mice.

Conclusion: The dysregulation of SIRT1-DEPTOR-mTORC1 signaling is a critical determinant of ALD pathology; targeting SIRT1 and DEPTOR and selectively inhibiting mTORC1-S6K1 signaling may have therapeutic potential for treating ALD in humans. (Hepatology 2018).

Fig.1. Chronic-plus-binge ethanol feeding leads to inhibition of DEPTOR and activation of mTORC1 signaling and promotes the development of fatty liver in mice

A. Effect of chronic-binge ethanol feeding on metabolic parameters and plasma alanine aminotransferase (ALT) levels. Representative H&E staining of hepatic steatosis in a mouse model of chronic-plus-binge ethanol feeding. Original magnification: 10X or 20X. B. Representative immunoblots for phosphorylation of mTOR (pmTOR), S6K1 (pS6K1), S6 (pS6), and 4E-BP1 (p4E-BP1) in livers from four mice each group. C. Positive immunostaining for phosphorylated S6 (a brown color) was predominantly located in lipid-rich hepatocytes (yellow arrows) in ethanol-fed mice. D. Effect of chronic-binge ethanol feeding on key regulators of mTORC1 and mTORC2. E. Chronic-binge ethanol feeding leads to an impairment of hepatic DEPTOR. Notably, positive staining for DEPTOR is visualized mainly in the cytoplasm of hepatocytes in pair-fed mice, and this effect is reduced in ethanol-fed mice. Original magnification: 20X or 40X. Linear regression between hepatic DEPTOR levels and mTOR phosphorylation in normal and ethanol-fed mice. The data are presented as mean ± SEM, n = 6–8 each group. *P < 0.05 vs. pair-fed mice. F. The effect of ethanol on DEPTOR and mTORC1 signaling in AML12 mouse hepatocytes.

Fig. 2. Adenoviral overexpression of hepatic DEPTOR inhibits mTORC1 and improves hepatic steatosis in chronic-binge ethanol-fed mice

A. Schematic representation of the interaction regions between mTOR and DEPTOR. DEPTOR inhibits mTOR activity by binding the PDZ domain of DEPTOR to the FAT domain of the mTOR kinase. Immunoblotting analyses confirmed adenovirus-mediated overexpression of DEPTOR in human HepG2 cells. B. Hepatic overexpression of either DEPTOR protein (∼46 kDa) or GFP protein (∼27 kDa) in mice is confirmed. C. Overexpression of DEPTOR improves hepatic steatosis and lowers triglyceride accumulation in ethanol-fed mice. D. Overexpression of DEPTOR represses the induction of mTORC1 toward the downstream signaling in ethanol-fed mice. E. Positive staining for phosphorylated S6 in hepatocytes (yellow arrows) in ethanol-fed mice is reduced by overexpressing DEPTOR. F. Increased DEPTOR levels correlate with decreased S6 phosphorylation and lowered hepatic triglyceride content in mice. The data are presented as mean ± SEM, n = 6–8. *P < 0.05 vs. ethanol-fed mice with Ad-GFP injection.

Fig. 3. Hepatic overexpression of DEPTOR ameliorates alcohol-mediated dysregulation of lipid metabolism in chronic-binge ethanol-fed mice

A. The active, nuclear form of SREBP-1 is increased in ethanol-fed mice and decreased by DEPTOR overexpression. P and N denote the precursor (~125 kDa) and cleaved nuclear (~68 kDa) forms of SREBP-1. B-C. The nuclear translocation of SREBP-1 as well as expression of SREBP-1c and its targets including ACC1, FAS, and SCD1 are reduced by DEPTOR overexpression. Notable, positive staining for SREBP-1 is primarily located in the nuclei of the hepatocytes of ethanol-fed mice (red arrows). D–E. Overexpression of DEPTOR represses the expression and cytoplasmic translocation of lipin-1 in chronic-binge ethanol-fed mice. Immunofluorescent staining showed that strong staining for lipin 1 (green) was predominantly located in the nuclear (blue) of hepatocytes (red arrows) of pair-fed mice, but it was elevated and mainly presented in the cytoplasm in hepatocytes (yellow arrows) surrounding central or portal veins of ethanol-fed mice. Notably, strong staining for lipin 1 is visualized mainly in the cytoplasm of hepatocytes (yellow arrows) of control mice on the ethanol diet, and this induction is inhibited by overexpression of DEPTOR. F. Expression of key genes involving fatty acid oxidation such as PPARα, CPT-1α and PGC-1α is analyzed. The data are presented as mean ± SEM, n = 6–8. *P < 0.05 vs. pair-fed mice. ^#P < 0.05 vs. ethanol-fed mice with Ad-GFP injection.

Fig. 4. Pharmacologic intervention via mTORC1 inhibition reduces hepatic lipogenesis, stimulates fatty acid oxidation, and ameliorates hepatic steatosis in chronic-binge ethanol-fed mice

A. Schematic structure and rapamycin binding site of the mTOR kinase. The FRB domain is the docking site of the FK506-binding protein 12 (FKBP12)–rapamycin complex. B. Phosphorylation of mTOR and its downstream effectors was sensitive to treatment with rapamycin in ethanol-fed mice. C. Positive staining for phosphorylated S6 was primarily localized in the cytoplasm of lipid-laden hepatocytes (yellow arrows) in vehicle control mice, and the staining intensity is reduced in rapamycin-treated mice. D. The cleavage and nuclear translocation of SREBP-1 and induction of FAS are diminished by rapamycin treatment. Representative photomicrographs of immunofluorescence for SREBP-1 (red) and DAPI (blue) in liver sections of mice. E-F. mTORC1 is required for alcohol to induce hepatic lipogenesis and to inhibit PPARα-mediated fatty acid oxidation in mice. The data are presented as the mean ± SEM, n = 6–8. *P < 0.05, vs. ethanol-fed mice with vehicle administration.

Fig. 5. Hepatic S6K1 activity is required for the proteolytic activation of SREBP-1 and its lipogenic process in mice after chronic-binge ethanol feeding

A. Schematic structure and phosphorylation sites of p70S6K1. The dominant negative form of S6K1 bears a mutation of lysine 100 to arginine (K100R) in the ATP binding site of the kinase domain. Immunoblots confirmed adenovirus-mediated overexpression of the dominant negative form of S6K1 (DN-S6K1) in HepG2 hepatocytes. B–C. Overexpression of DN-S6K1 inhibits S6K1 activity and attenuates hepatic steatosis in ethanol-fed mice. Notably, strong positive staining for phosphorylated S6 in the cytoplasm of hepatocytes (yellow arrows) in ethanol-fed mice is markedly decreased by overexpression of DN-S6K1. D–E. Overexpression of DN-S6K1 suppresses the accumulation of nuclear SREBP-1 and induction of lipogenic genes in response to ethanol feeding. Notably, positive staining for the nuclear translocation of SREBP-1 was presented in hepatocytes (red arrows) around central and portal veins in GFP-expressed mice. F. Alcohol-mediated induction of lipin 1 is unaffected by overexpression of DN-S6K1. The data are presented as mean ± SEM, n = 6–8. *P < 0.05 vs. ethanol-fed mice with Ad-GFP injection.

Fig. 6. Hepatic inhibition of mTORC1 protects against alcohol-induced hepatic inflammation, apoptosis, and liver injury in mice

A. Liver mRNA levels of pro-inflammatory regulators, neutrophil markers (Ly6G, MPO and CD11b), and macrophage marker (F4/80) were analyzed by real-time PCR. B. Hepatic activity of Akt, the major hepatocyte survival regulator, is upregulated by DEPTOR in chronic-binge ethanol-fed mice. C. Liver injury is assessed by measuring serum ALT levels. D. Overexpressing DEPTOR protects against alcohol-induced hepatocyte apoptosis, as evidenced by reduced caspase-3 cleavage. E. Rapamycin administration alleviates alcohol-mediated inhibition of Akt and induction of hepatic apoptosis and injury. F. Overexpression of DN-S6K1 ameliorates alcohol-induced hepatic apoptosis and liver damage. The data are presented as the mean ± SEM, n = 4–7. *P < 0.05, vs. pair-fed mice; ^#P < 0.05, vs. corresponding ethanol-fed mice.

Fig. 7. Hepatocyte-specific deletion of SIRT1 disrupts DEPTOR signaling, stimulates mTORC1 activity, and exacerbates the development of alcoholic fatty liver and liver injury in mice

A. Protein levels of SIRT1 are measured in AML12 hepatocytes and in livers of wild-type (WT) mice and SIRT1 liver-specific knockout (SIRT1 LKO) mice under conditions of normal and ethanol exposure. B. Hepatocyte-specific deletion of SIRT1 downregulates DEPTOR and enhances mTORC1 activation in mice after chronic-binge ethanol feeding. C. Genetic ablation of SIRT1 in the liver increases the susceptibility to alcohol-induced fatty liver. D. Hepatic SIRT1 deficiency exacerbates alcohol-mediated abnormalities of lipid metabolism in mice. E–F. Hepatic SIRT1 loss aggravates alcohol-induced inflammation, apoptosis, and liver injury. The data are presented as the mean ± SEM, n = 4–8. *P < 0.05, vs. pair-fed WT mice; ^#P < 0.05, vs. ethanol-fed WT mice.

Fig. 8. Integrated downregulation of SIRT1 and DEPTOR contributes to the pathogenesis in patients with ALD

A–E. Representative immunoblots and densitometric quantification for SIRT1, mTORC1 signaling, lipid metabolic regulators, and apoptosis in normal liver tissues (n= 6) and liver tissues from patients with ALD (n =8). The data are presented as the mean ± SEM. *P < 0.05 vs. normal liver tissue. F. The proposed model for the deregulation of the SIRT1-DEPTOR-mTORC1 axis in the pathogenesis of ALD in mice and humans. Chronic alcohol consumption causes SIRT1 suppression in hepatocytes, which is coupled to the downregulation of DEPTOR and activation of mTORC1 and S6K1. Aberrant activation of mTORC1 by alcohol stimulates the proteolytic processing, nuclear translocation, and transcriptional activity of SREBP-1, promotes the cytoplasmic translocation of lipin 1, and inhibits the transcriptional activity of PPARα, which in turn increases fatty acid synthesis and downregulates fatty acid oxidation. Alcohol-induces hepatic lipogenic process acts through parallel S6K1-dependent and independent pathway. Alcohol feeding acts largely via SIRT1 inhibition and mTORC1 activation to induce excess fat accumulation and apoptosis in hepatocytes. Hepatic lipotoxicity and inflammation likely contribute to the development of acute-on-chronic alcoholic liver injury. Hepatic loss of SIRT1 impairs DEPTOR function, stimulates mTORC1 and lipogenesis, and promotes triglyceride overproduction, thereby leading to inflammation and liver injury in ALD. DEPTOR-dependent inhibition of mTORC1 provides a potential druggable target for treating ALD in humans.