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Dynamic MTORC1-TFEB feedback signaling regulates hepatic autophagy, steatosis and liver injury in long-term nutrient oversupply.

ABSTRACT: Normal metabolism requires a controlled balance between anabolism and catabolism. It is not completely known how this balance can be retained when the level of nutrient supply changes in the long term. We found that in murine liver anabolism, as represented by the phosphorylation of RPS6KB (ribosomal protein S6 kinase), was soon elevated while catabolism, as represented by TFEB (transcription factor EB)-directed gene transcription and lysosomal activities, was downregulated after the administration of a high-fat diet (HFD). Surprisingly, neither the alteration in RPS6KB phosphorylation nor that in TFEB functions was static over the long course of HFD feeding. Instead, the 2 signals exhibited dynamic alterations in opposite directions, which could be explained by the dependence of MTORC1 (MTOR complex 1) activation on TFEB-supported lysosome function and the feedback suppression of TFEB by MTORC1. Disruption of the dynamics by enforced expression of TFEB in HFD-fed mice at the peaks of MTORC1 activation restored lysosome function. Consistently, interference of MTORC1 activation with rapamycin or with a constitutively activated RRAGA mutant at the peak or nadir of MTORC1 oscillation enhanced or reduced the lysosome function, respectively. These treatments also improved or exacerbated hepatic steatosis and liver injury, respectively. Finally, there was a significant inverse correlation between TFEB activation and steatosis severity in the livers of patients with non-alcohol fatty liver diseases, supporting the clinical relevance of TFEB-regulated events. Thus, maintaining catabolic function through feedback mechanisms during enhanced anabolism, which is caused by nutrient oversupply, is important for reducing liver pathology.

SUBMITTER: Zhang H

PROVIDER: S-EPMC6135624 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Project description:Liraglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA), has been demonstrated to alleviate non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanism has not been fully elucidated. Increasing evidence suggests that autophagy is involved in the pathogenesis of hepatic steatosis. In this study, we examined whether liraglutide could alleviate hepatic steatosis through autophagy-dependent lipid degradation and investigated the underlying mechanisms. Herein, the effects of liraglutide on NAFLD were evaluated in a high-fat diet (HFD)-induced mouse model of NAFLD as well as in mouse primary and HepG2 hepatocytes exposed to palmitic acid (PA). The expression of the GLP-1 receptor (GLP-1R) was measured in vivo and in vitro. Oil red O staining was performed to detect lipid accumulation in hepatocytes. Electron microscopy was used to observe the morphology of autophagic vesicles and autolysosomes. Autophagic flux activity was measured by infecting HepG2 cells with mRFP-GFP-LC3 adenovirus. The roles of GLP-1R and transcription factor EB (TFEB) in autophagy-lysosomal activation were explored using small interfering RNA. Liraglutide treatment alleviated hepatic steatosis in vivo and in vitro. In models of hepatic steatosis, microtubule-associated protein 1B light chain-3-II (LC3-II) and SQSTM1/P62 levels were elevated in parallel to blockade of autophagic flux. Liraglutide treatment restored autophagic activity by improving lysosomal function. Furthermore, treatment with autophagy inhibitor chloroquine weakened liraglutide-induced autophagy activation and lipid degradation. TFEB has been identified as a key regulator of lysosome biogenesis and autophagy. The protein levels of nuclear TFEB and its downstream targets CTSB and LAMP1 were decreased in hepatocytes treated with PA, and these decreases were reversed by liraglutide treatment. Knockdown of TFEB expression compromised the effects of liraglutide on lysosome biogenesis and hepatic lipid accumulation. Mechanistically, GLP-1R expression was decreased in HFD mouse livers as well as PA-stimulated hepatocytes, and liraglutide treatment reversed the downregulation of GLP-1R expression in vivo and in vitro. Moreover, GLP-1R inhibition could mimic the effect of the TFEB downregulation-mediated decrease in lysosome biogenesis. Thus, our findings suggest that liraglutide attenuated hepatic steatosis via restoring autophagic flux, specifically the GLP-1R-TFEB-mediated autophagy-lysosomal pathway.

Project description:The mechanistic target of rapamycin kinase complex 1 (MTORC1) is a central cellular kinase that integrates major signaling pathways, allowing for regulation of anabolic and catabolic processes including macroautophagy/autophagy and lysosomal biogenesis. Essential to these processes is the regulatory activity of TFEB (transcription factor EB). In a regulatory feedback loop modulating transcriptional levels of RRAG/Rag GTPases, TFEB controls MTORC1 tethering to membranes and induction of anabolic processes upon nutrient replenishment. We now show that TFEB promotes expression of endocytic genes and increases rates of cellular endocytosis during homeostatic baseline and starvation conditions. TFEB-mediated endocytosis drives assembly of the MTORC1-containing nutrient sensing complex through the formation of endosomes that carry the associated proteins RRAGD, the amino acid transporter SLC38A9, and activate AKT/protein kinase B (AKT p-T308). TFEB-induced signaling endosomes en route to lysosomes are induced by amino acid starvation and are required to dissociate TSC2, re-tether and activate MTORC1 on endolysosomal membranes. This study characterizes TFEB-mediated endocytosis as a critical process leading to activation of MTORC1 and autophagic function, thus identifying the importance of the dynamic endolysosomal system in cellular clearance. Abbreviations: CAD: central adrenergic tyrosine hydroxylase-expressing-a-differentiated; ChIP-seq: chromosome immunoprecipitation sequencing; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; EEA1: early endosomal antigen 1; EGF: epidermal growth factor; FBS: fetal bovine serum; GFP: green fluorescent protein; GTPase: guanosine triphosphatase; HEK293T: human embryonic kidney 293 cells expressing a temperature-sensitive mutant of the SV40 large T antigen; LAMP: lysosomal-associated membrane protein; LYNUS: lysosomal nutrient-sensing complex; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha/beta; MTOR: mechanistic target of rapamycin kinase; MTORC: mechanistic target of rapamycin kinase complex; OE: overexpression; PH: pleckstrin homology; PtdIns(3,4,5)P3: phosphatidylinositol 3,4,5-trisphosphate; RRAGD: Ras related GTPase binding D; RHEB: Ras homolog enriched in brain; SLC38A9: solute carrier family 38 member 9; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TSC2: tuberous sclerosis 2; TMR: tetramethylrhodamine; ULK1: unc-51 like kinase 1; WT: wild type.

Project description:TRIM37 gene mutations cause mulibrey (muscle-liver-brain-eye) nanism, a severe growth disorder with prenatal onset. Although TRIM37 depletion normally induces apoptosis, patients with TRIM37 mutations have a high risk of developing tumors, suggesting that there may be an alternative pro-survival mechanism for TRIM37-deficient tumor cells. We find that TRIM37 interacts with MTOR and RRAGB proteins, enhances the MTOR-RRAGB interaction and promotes lysosomal localization of MTOR, thereby activating amino acid-stimulated MTORC1 signaling. In response to loss of TRIM37 functions, phosphorylation of TFEB is significantly reduced, resulting in its translocation into the nucleus enabling its transcriptional activation of genes involved in lysosome biogenesis and macroautophagy/autophagy. The enhanced autophagy depends on the inhibition of MTORC1 signaling and may serve as an alternative mechanism to survive the loss of TRIM37 functions. Our study unveils a positive role of TRIM37 in regulating the MTORC1-TFEB axis and provides mechanistic insights into the pathogenesis of mulibrey nanism, as well as potential therapeutic treatment. ABBREVIATIONS:ACTB: actin beta; ATG: autophagy related; CASP3: caspase3; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; CTS: cathepsin proteases; CTSL: cathepsin L; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LMNB1: lamin B1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mulibrey: muscle-liver-brain-eye; NAC: N-acetyl-L-cysteine; PARP1: poly(ADP-ribose) polymerase 1; RAP2A: member of RAS oncogene family; RHEB: Ras homolog enriched in brain; ROS: reactive oxygen species; RPS6KB1: ribosomal protein S6 kinase B1; RRAGB: Ras related GTP binding B; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TRIM37: tripartite motif containing 37.

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Dynamic MTORC1-TFEB feedback signaling regulates hepatic autophagy, steatosis and liver injury in long-term nutrient oversupply.

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