Abstract
Disclosure: J. Salinas: None. J.S. Burgos: None. J.R. Camano: None. A.P. Sinha-Hikim: None. T.C. Friedman: None. K.M. Hasan: None. Metabolic dysfunction-associated steatotic liver disease (MASLD) is highly prevalent in the U.S. population, affecting approximately 30-35% of individuals. It is considered a critical risk factor for type 2 diabetes (T2D), cardiovascular diseases (CVD), and hepatic cancer. However, no effective therapeutics are currently available to cure this chronic metabolic condition, making it imperative to understand its molecular mechanisms. CARF (CDKN2AIP) is a novel multifunctional gene that has been shown to regulate cellular fate in response to various stresses, including DNA damage, oxidative stress, and replication stress. Until recently, its response to metabolic or over-nutrition stress was unknown. Our recent study demonstrated that CARF expression is dramatically reduced in response to free fatty acid (FFA) deposition in the liver, suggesting that CARF may play a protective role against MASLD. Supporting this hypothesis, overexpression of CARF was found to suppress MASLD in a high-fat diet (HFD)-induced mouse model of the disease. However, the underlying mechanisms by which CARF suppresses MASLD remain largely unknown. To address this, transcriptome analysis by RNA sequencing upon silencing of CARF revealed that the PPARα signaling pathway, which regulates cellular fatty acid oxidation (FAO), was altered in hepatocytes. We hypothesize that silencing CARF attenuates hepatic FAO, leading to fat deposition in hepatocytes. Using RT-PCR and western blotting, we confirmed that the expression of multiple PPARα signaling pathway genes, including CPT2, MCAD, and ACAD9, was reduced in CARF-depleted HepG2 cells. Furthermore, using palmitate as a substrate in Seahorse metabolic analysis, we demonstrated that CARF overexpression enhances the oxygen consumption rate (OCR) in HepG2 cells, suggesting that CARF regulates mitochondrial β-oxidation. Consistent with this, CPT2 expression, along with CARF, was significantly reduced in HFD-fed livers, indicating that the CARF-CPT2 axis could play a vital role in hepatic FAO. Intriguingly, hepatic overexpression of CARF restored CPT2 expression in HFD-fed livers, which could be accounted for CARF's protective role against MASLD in mice. In conclusion, these results establish CARF as a novel regulator of cellular fatty acid metabolism. Its dysregulation could contribute to the development of MASLD, warranting further research to evaluate its clinical significance. Presentation: Saturday, July 12, 2025