Deciphering the Mechanisms by which Long Chain Fatty Acid Oxidation Influences Spatial Microvesicular Steatosis - PROJECT SUMMARY Metabolic dysfunction-associated steatotic liver disease (MASLD) is associated with a 4-year decrease in lifespan and is expected to become the leading cause of liver related morbidity and mortality within the next 15 years. A hallmark of MASLD is the accumulation of small (microvesicular steatosis) and large (macrovesicular steatosis) lipid droplets in the liver. However, mechanisms driving the size and zonal deposition (e.g. accumulation in periportal versus pericentral hepatocytes) of lipid droplets remains poorly understood. The carnitine palmitoyltransferase 1a (Cpt1a) enzyme facilitates long chain fatty acid oxidation, but impairments in this pathway have been associated with microvesicular steatosis and reductions in circulating polyunsaturated fatty acids (PUFAs) in humans. Therefore, this proposal aims to identify the mechanisms explaining these associations. Using a mouse model of impaired long chain fatty acid oxidation, preliminary data shows a sexually dimorphic response to liver-specific deletion of Cpt1a characterized by panlobular microvesicular steatosis in female mice while only slight periportal steatosis in male mice. In addition, female knockout mice develop exacerbated inflammation which coincides with reductions in ω3-PUFAs in the liver. Therefore, Aim 1 utilizes in- vitro mechanistic approaches to quantify lipid droplet dynamics and peroxisomal oxidation of PUFAs in response to Cpt1a-deletion in cultured hepatocytes. Aim 2 utilizes gain- and loss-of-function approaches to modulate Cpt1a expression specifically in periportal and pericentral hepatocytes across male and female mice. To assess the impact of zonal-specific Cpt1a deletion and overexpression on the progression from MASLD to metabolic dysfunction-associated steatohepatitis (MASH), livers will be used for dual spatial transcriptomics and lipidomics, and for flow cytometry to assess the immune cell profiles in the liver. Completion of these aims will yield mechanistic insight linking mitochondrial and peroxisomal oxidation to spatial lipid deposition in the liver. The proposed research reveals several innovative mechanisms that have yet to be explored, including how Cpt1a alters PUFA oxidation and lipid droplet formation across the periportal-pericentral axis. In addition, the proposed study uses innovative approaches including the use of zonal-specific gain- and loss-of-function mouse models in conjunction with next generation spatial lipidomic and transcriptomic technology to address the overall hypothesis. Strong collaborations among the University of Kentucky Arts and Sciences Imaging Center, Oncogenomics Shared Resource Facility, COCVD Analytical research Core, Biostatistics and Bioinformatics Shared Resource Facility, and the Center for Advanced Biomolecule Research at the University of Florida ensure successful completion of the proposed research by the candidate. This R01 will support the applicant in reaching his ultimate goal of becoming an independent yet collaborative, versatile PI leveraging his unique skill set in both basic and clinical research arenas to identify novel therapeutics to treat cardiometabolic disease.
