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Proteomic and phosphoproteomic characterization of steatotic liver from diet-induced obese mice in response to dietary fat

ABSTRACT: One of the metabolic consequences of obesity is abnormal lipid accumulation in the liver, or hepatosteatosis, which can develop intomore serious diseases in the non-alcoholic liver disease (NAFLD) spectrum including non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma. Therefore, it is necessary to understand the metabolic changes that occur in hepatosteatosisin order toprevent disease progression.The liver plays a major role in regulating macronutrient and energy homeostasis in both the fed and fasted states. However, whether hepatosteatosis influences thepostprandial molecularresponse, specifically to dietary fat,has not been investigated.Therefore, the goal of this study was to comparethe proteome and phosphoproteome of steatotic livers from diet-induced obese (DIO) mice and control livers from lean mice in the postprandial response to dietary fat. Using untargeted LC-MS/MS analysis, we identified significant alterations in the levels of proteins involved in macronutrient and energy metabolism in livers of DIO compared to lean mice. In addition, uniquely phosphorylated proteins in livers of DIO andlean mice reflect regulatory mechanisms controlling cellular processes contributingto hepatosteatosis. Theresultsof this studyexpandourknowledge ofthe metabolic consequences that occur duringhepatosteatosisandtheinfluence of dietary fat on NAFLD progression.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

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

SUBMITTER: Uma K Aryal

PROVIDER: MSV000085612 | MassIVE | Fri Jun 19 12:08:00 BST 2020

REPOSITORIES: MassIVE

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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.

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Proteomic and phosphoproteomic characterization of steatotic liver from diet-induced obese mice in response to dietary fat

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