Aging in the Liver: Alcohol and the Retinoic Acid Pathway - People who are 65 and older are 13% of the United States population and this will increase to 19% by 2030. Excessive alcohol use across individuals of all ages poses a challenge especially in the elderly because of the risk to a more severe alcohol-associated liver disease (ALD) and the risks of an overall healthspan decline. Lack of aging-related biomarkers and therapies for ALD that can predict the susceptibility to worse liver disease in the elderly highlights the limited number of mechanism-based studies leading to the design of therapeutics. Our published and preliminary data indicate that both mice and cultured hepatocytes with loss of a vitamin A effector, retinoic acid receptor β (RARβ), show increased oxidative stress upon EtOH treatment and failure to produce NAD+, making this model ideal to define how ethanol accelerates aging. These data led us to the central hypothesis that RARβ is necessary to protect the liver from EtOH- induced oxidative stress and aging. To test our hypothesis, we propose the following aims: Specific Aim (1): Examine if mice with a liver-specific knockout of RARβ age more rapidly and if ethanol increases their rate of aging. We will compare liver-specific RARβ knockout (AlbCre) mice (RARβ-LKO) with age matched wild type (Wt) mice treated with and without EtOH. Using spatial transcriptomics, one of the most recently developed cutting-edge technologies that will allow us to generate a detailed transcriptional mapping of the liver zones during the aging process under the effects of EtOH. To measure the rate of aging in the different experimental groups, we will determine the telomere length, intracellular β galactosidase accumulation, the senescence-associated secretory phenotype (SASP), and the NAD+/NADH ratio. Specific Aim (2): Define the mechanisms whereby RARβ protects the liver against oxidative stress. Primary hepatocytes isolated from young and old Wt and RARβ-LKO mice, and human primary hepatocytes obtained through the NIH-supported Human Hepatocyte Isolation Distribution (University of Pittsburgh) program in which we will knockdown RARβ by siRNA. We will investigate and quantify if RARβ is necessary to protect hepatocytes against EtOH-induced senescence-associated pathways (SASP) and the NAD+/NADH ratio decreases (1); and against EtOH-induced mitochondrial stress investigated at morphological and functional level (2).
