Role of hepatocyte PEMT in metabolic dysfunction-associated steatohepatitis (MASH) - ABSTRACT Metabolic dysfunction-associated steatotic liver disease (MASLD) is the leading cause of chronic liver disease. MASLD can progress to metabolic dysfunction-associated steatohepatitis (MASH) increasing the risk of cirrhosis, liver cancer, and death. MASLD is characterized by liver steatosis and at least one cardiometabolic risk factor. MASH is characterized by inflammation, fibrosis, and hepatocellular ballooning. Currently, MASLD/MASH treatments are limited to lifestyle modifications, such as diet and exercise, with only two FDA-approved therapies for advanced MASH with fibrosis. Therefore, identifying molecular mechanisms driving MASLD progression is crucial for developing targeted therapies. Hepatic steatosis results from dysregulated fatty acid (FA) synthesis, storage, and utilization. FAs are also incorporated into phospholipids, primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which are essential for maintaining cellular function. PC and PE are synthesized in the Kennedy pathway. In hepatocytes, PE can also be methylated by phosphatidylethanolamine methyltransferase (PEMT) to generate PC. Of note, increased steatosis and altered PC and PE levels are associated with MASLD progression. Interestingly, single-nucleotide polymorphisms of the human PEMT gene, which lead to reduced PEMT activity, are associated with MASLD. Moreover, hepatic PEMT expression is reduced in patients with MASH. In mice, the knockout of PEMT exacerbates steatosis and accelerates MASH. However, the precise mechanisms by which the loss or reduced activity of hepatocyte PEMT contribute to MASLD progression remain unclear. In this project, we sought to elucidate the role of hepatocyte PEMT in maintaining liver health and mitochondrial function, and how the loss of hepatocyte-specific PEMT expression in mice may lead to the development of MASH. To achieve this goal, in Aim 1, we will characterize how hepatocyte- specific PEMT knockout alters lipid metabolism and promotes diet-induced MASH. We will feed mice a high-fat and cholesterol diet supplemented with fructose in the drinking water for 4 weeks. We will assess metabolic parameters, hepatic pathology, and liver lipidomic profiles with liquid chromatography coupled with mass spectrometry to evaluate lipid homeostasis. In Aim 2 of this project, we will investigate the role of hepatocyte PEMT in maintaining mitochondrial function to prevent the progression of MASLD. Mouse primary hepatocytes will be isolated from mice with different expressions of hepatocyte PEMT and treated with PEMT activators and/or inhibitors, cultured under in vitro MASH conditions. Then, we will assess mitochondrial respiration, function, and dynamics using Seahorse and fluorescence-based microscopy. Overall, these studies will provide mechanistic insights into hepatocyte PEMT’s role in maintaining liver health and mitochondrial function, and how reduced hepatocyte PEMT contributes to the progression of MASLD.
