Wednesday, May 1, 2024
5/1/2024
PROJECT SUMMARY Non-Alcoholic Fatty Liver Disease (NAFLD) and the more severe Non-Alcoholic Steatohepatitis (NASH) are driving increased rates of cirrhosis and hepatocellular carcinoma in the USA. Though obesity is a known risk factor, the overlap between patients with obesity and NAFLD is incomplete, and a full understanding of the mechanisms that govern onset and progression of NAFLD is critically lacking. In this proposal, I will probe the role of the mitophagy receptor BNIP3 in the onset and progression of lipid accumulation in the liver to inform future disease treatment. BNIP3 is a known mediator of selective mitochondrial autophagy (mitophagy), and we showed previously that hepatic BNIP3 expression is highly induced by fasting and has a marked effect on metabolic homeostasis by decreasing mitochondrial mass. We have also shown that a liver-specific deletion of BNIP3 causes spontaneous lipid accumulation in adult mouse liver. BNIP3 is more highly expressed around the central vein of the liver, which is also the region where most lipid accumulates in Bnip3 null liver and in human and mouse NAFLD. Our findings indicate that BNIP3 is a critical regulator of hepatic lipid homeostasis, and the goal of this proposal is to define the mechanism of this regulation. My central hypothesis is that BNIP3 decreases lipid droplet (LD) content by promoting both mitophagy and LD autophagy (lipophagy) in pericentral regions of the liver. I propose that it does so indirectly by facilitating the engulfment of small LDs that are tethered to mitochondria targeted by BNIP3 for degradation at the lysosome. I have shown for the first time that isolated primary hepatocytes retain their patterns of zonal gene expression for over 24 hours following culture outside their hepatic nutrient microenvironment. This allows me to define for the first time the cellular processes modulating lipid metabolism in distinct zonal populations of hepatocytes ex vivo. In Aim 1, I will use this finding to determine whether BNIP3 drives lipophagy to clear LDs predominantly in pericentral hepatocytes. I will exploit machine-learning image analysis tools for unbiased quantification of rates of mitophagic and lipophagic flux in discrete populations of primary hepatocytes, sorted by their zone of origin, to test whether BNIP3 drives lipophagy more highly in pericentral vs. periportal hepatocytes. I will test this finding in vivo by assessing whether exogenous re-expression of BNIP3 is sufficient to clear lipid from pericentral regions of the liver in vivo. In Aim 2, I will test the hypothesis that BNIP3 promotes small LD turnover by targeting small LDs “hitchhiking” on mitochondria to the autolysosome. I propose that knockdown of well characterized LD-mitochondrial tethering proteins, such as PLIN5, will ablate the LD clearance capabilities of BNIP3 by disrupting LD-mitochondrial contacts. My work elucidating the mechanisms that lead to lipid accumulation in the liver may suggest novel ways to prevent NAFLD and NASH in the future.
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