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MARC1 knockdown protects liver from diet-induced NASH in mouse

ABSTRACT: Human genetic studies have identified several MARC1 variants as protective against non-alcoholic fatty liver diseases (NAFLD). The MARC1 variants are associated with reduced lipid profiles, liver enzymes, and liver-related mortality. However, the role of mitochondrial amidoxime reducing component 1 (mARC1), encoded by MARC1, in NAFLD is still unknown and the therapeutic potential of this target has never been developed. Given that mARC1 is mainly expressed in hepatocytes, we developed an N-acetylgalactosamine conjugated mouse mARC1 siRNA to address this. In ob/ob mice, knockdown of mARC1 in mouse hepatocytes resulted in decreased liver weight, serum lipid enzymes, low-density lipoprotein cholesterol, and liver triglycerides. Loss of mARC1 also improved the lipid profiles and attenuated liver pathological changes in two diet-induced nonalcoholic steatohepatitis (NASH) mouse models. A comprehensive analysis of mARC1-deficient liver in NASH by metabolomics, proteomics, and lipidomics showed that mARC1 knockdown partially restored metabolites and lipids altered by diets. Taken together, loss of mARC1 protects mouse liver from NASH, suggesting a potential therapeutic approach of NASH by downregulation of mARC1 in hepatocytes.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Matthew Rardin

PROVIDER: MSV000091820 | MassIVE | Fri Apr 28 15:37:00 BST 2023

SECONDARY ACCESSION(S): PXD041883

REPOSITORIES: MassIVE

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Liver-specific mitochondrial amidoxime-reducing component 1 (Mtarc1) knockdown protects the liver from diet-induced MASH in multiple mouse models.

Guo Yuanjun Y Gao Zhengyu Z LaGory Edward L EL Kristin Lewis Wilson LW Gupte Jamila J Gong Yan Y Rardin Matthew J MJ Liu Tongyu T Nguyen Thong T TT Long Jason J Hsu Yi-Hsiang YH Murray Justin K JK Lade Julie J Jackson Simon S Zhang Jun J

Hepatology communications 20240502 5

<h4>Background</h4>Human genetic studies have identified several mitochondrial amidoxime-reducing component 1 (MTARC1) variants as protective against metabolic dysfunction-associated steatotic liver disease. The MTARC1 variants are associated with decreased plasma lipids and liver enzymes and reduced liver-related mortality. However, the role of mARC1 in fatty liver disease is still unclear.<h4>Methods</h4>Given that mARC1 is mainly expressed in hepatocytes, we developed an N-acetylgalactosamine ...[more]

PMID: 38696369

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Project description:Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ~25% worldwide, with significant public health consequences, yet few effective treatments. Human genetics can help elucidate novel biology and identify targets of new therapeutics. Genetic variants in mitochondrial amidoxime reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality, however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods: Analyses including multi-trait colocalization and mendelian randomization were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra GAN-diet NASH mouse model treated with hepatocyte specific GalNAc-siRNA to understand the in vivo impacts of MTARC1. Results: We show that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids and body composition and these associations are due to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian Randomization, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion and in vivo GalNAc-siRNA mediated knockdown of mARC1 lowered hepatic, but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions: Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD.

Project description:Background and Aims Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ~25% worldwide, with significant public health consequences yet has few effective treatments. Human genetics can help elucidate novel biology and identify targets of new therapeutics. Genetic variants in mitochondrial amidoxime reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality, however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods Analyses including multi-trait colocalization and Mendelian randomization were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra GAN diet NASH mouse model treated with hepatocyte specific GalNAc-siRNA to understand the in vivo impacts of MTARC1. Results We show that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids and body composition and these associations are due to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian Randomization, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion and in vivo GalNAc-siRNA mediated knockdown of mARC1 lowered hepatic, but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD.

Project description:Objective: Nonalcoholic fatty liver disease (NAFLD) is linked to obesity and diabetes, suggesting an important role of adipose tissue in the pathogenesis of NAFLD. Here we aim to investigate the interaction between adipose tissue and liver in NAFLD, and identify potential early plasma markers that predict NASH. Research Design and Methods: C57Bl/6 mice were chronically fed a high fat diet to induce NAFLD and compared with mice fed low fat diet. Extensive histological and phenotypical analyses coupled with a time-course study of plasma proteins using multiplex assay was performed. Results: Mice exhibited pronounced heterogeneity in liver histological scoring, leading to classification into 4 subgroups: LF-low (LFL) responders displaying normal liver morphology, LF-high (LFH) responders showing benign hepatic steatosis, HF-low (HFL) responders displaying pre-NASH with macrovesicular lipid droplets, and HF-high (HFH) responders exhibiting overt NASH characterized by ballooning of hepatocytes, presence of Mallory bodies, and activated inflammatory cells. Compared to HFL responders, HFH mice gained weight more rapidly and exhibited adipose tissue dysfunction characterized by decreased final fat mass, enhanced macrophage infiltration and inflammation, and adipose tissue remodelling. Plasma haptoglobin, IL-1β, TIMP-1, adiponectin and leptin were significantly changed in HFH mice. Multivariate analysis indicated that in addition to leptin, plasma CRP, haptoglobin, eotaxin and MIP-1α early in the intervention were positively associated with liver triglycerides. Intermediate prognostic markers of liver triglycerides included IL-18, IL-1β, MIP-1γ and MIP-2, whereas insulin, TIMP-1, GCP-2 and MPO emerged as late markers. Conclusions: Our data support the existence of a tight relationship between adipose tissue dysfunction and NASH pathogenesis and point to several novel potential predictive biomarkers for NASH. Keywords: Expression profiling by array Male wildtype C57Bl/6 mice were fed LFD or HFD for 21 weeks. Mice were divided into 4 groups based on liver histology.

Project description:Non-alcoholic fatty liver disease (NAFLD), defined as fatty infiltration in >5% of hepatocytes (steatosis) in the absence of excessive alcohol consumption, is emerging as a leading cause of liver disease worldwide (1). Subjects with NAFLD have an increased risk of progressing into non-alcoholic steatohepatitis (NASH) as well as developing type 2 diabetes, cardiovascular disease, and liver cancer (2-4). Thus, understanding the pathobiology of NAFLD is of high medical relevance for the efficient prevention and treatment of a range of complex metabolic diseases. In NAFLD, excess lipids accumulate within intrahepatocellular lipid droplets (LDs), which are composed of a neutral lipid core of mainly triacylglycerol (TAG) and cholesterol esters surrounded by a phospholipid monolayer that harbors specific proteins. Once thought to be only inert energy storage depots, hepatic LDs are increasingly recognized as organelles that orchestrate liver lipid partitioning but also play a critical role in protein quality control and storage, cell signaling, and viral replication (5). Notably, LDs are shown to dynamically interact with a variety of cellular organelles including the endoplasmic reticulum (ER), mitochondria, peroxisomes, and endosomes (6). Liver LD surface-associated proteins regulate the functional properties of LDs, and dietary fat content as well as liver metabolic status have been shown to dynamically influence the composition of hepatic LD proteins (7, 8). Consequently, exploring how LD-associated proteins affect hepatic metabolism is important for understanding the molecular pathophysiology of NAFLD. In the search for novel targets that regulate ectopic lipid accumulation in the context of nutritional stress and obesity, we identified serine/threonine protein kinase (STK)25, a member of the Ste20 kinase superfamily (9), as a hepatic LD-associated protein, which critically controls the development and progression of NAFLD (10-15). We found that STK25 knockdown attenuates lipid deposition in human hepatocytes and mouse liver by repressing lipid synthesis and enhancing β-oxidation and very-low-density lipoprotein (VLDL)-TAG secretion, with the reciprocal phenotype seen when STK25 protein is overexpressed (10-15). Notably, administration of hepatocyte-targeting GalNAc-conjugated Stk25 antisense oligonucleotide (ASO) in obese mice effectively ameliorates diet-induced hepatic steatosis as well as NASH features (i.e., liver inflammation, hepatocellular ballooning, and fibrosis), providing in vivo nonclinical proof-of-principle for the metabolic benefit of pharmacological STK25 inhibitors (15). Furthermore, we observed that STK25 levels in human liver biopsies correlate with the severity of NASH, and several common non-linked single nucleotide polymorphisms (SNPs) in the human STK25 gene are associated with altered liver fat content (12-14). Although these studies suggest a key role for STK25 in the control of liver LD dynamics, the mechanism-of-action of STK25 in regulation of hepatic lipid partitioning has remained elusive. To provide novel insights into biological function of STK25 in the liver under steatotic conditions, we here characterized the hepatic LD-associated phosphoproteome in Stk25 knockout mice and their wild-type littermates following high-fat diet feeding using non-biased approach by isobaric tagging for relative quantification. Of the 4,515 proteins and 982 phosphoproteins identified with a 1% false discovery rate, we report a total of 131 proteins and 69 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, antioxidant defense, and ubiquitination-mediated proteolysis were coordinately regulated in the livers from high-fat-fed Stk25-/- mice compared with wild-type controls.