Key result
Liver-directed CARF overexpression reduces hepatic triglyceride burden and restores CPT2 levels in obese mice.
Why the study?
Impaired mitochondrial fatty acid β-oxidation contributes to MASLD in obesity, prompting investigation into whether CARF mitigates hepatic steatosis by reprogramming these pathways.
CARF overexpression activates mitochondrial fatty acid oxidation and reduces hepatic steatosis in obese mice, suggesting a potential therapeutic target for MASLD.
Supports CARF as a MASLD target in obese models; leaves open translation to human therapy.
Introduction and Objective: Metabolic dysfunction-associated steatotic liver disease (MASLD) is highly prevalent in obesity, and impaired mitochondrial fatty acid β-oxidation (FAO) is a key contributor to its pathogenesis. Building on our prior report that CARF loss exacerbates steatosis and CARF overexpression improves metabolic phenotypes, we tested whether CARF mitigates obesity-associated hepatic steatosis by reprogramming mitochondrial FAO pathways. Methods: CARF was knocked down in hepatocytes, and changes in FAO pathway gene expression were assessed by transcriptomics. Palmitate-driven FAO was measured by Seahorse oxygen consumption rate (OCR) after CARF overexpression. Diet-induced obese (DIO) mice were fed a high-fat diet (HFD) for 16 weeks (n=8/group) and received liver-directed CARF overexpression (AAV-CARF; HFD-COE) or vector control. Hepatic triglyceride (TG), liver histology (H & E staining), RNA sequencing, RT-CCR, and western blotting for CARF/FAO pathway genes, and metabolomics profiling were assessed. Results: CARF ablation significantly suppressed the FAO/PPAR pathway genes, including CPT2 and ACADM. RT-PCR and immunoblotting validated that CARF regulates CPT2 at the mRNA and protein levels. In agreement, CARF overexpression increased palmitate-driven OCR, indicating enhanced FAO. In DIO mice, CARF overexpression reduced hepatic TG burden and restored CPT2 protein levels compared with the vector controls. In addition, CARF-CPT2 restoration was accompanied by metabolite remodeling, with clear separation of NCD, HFD, and HFD-COE groups by PCA and heatmap. To provide mechanistic support, we observed that pharmacologic AMPK activation (AICAR) increased CARF/CPT2 levels and reduced TG, whereas AMPK inhibition (Compound C) decreased CARF/CPT2 levels and increased TG. Conclusion: CARF activates a mitochondrial fatty acid oxidation program in obesity, restoring FAO flux and hepatic metabolite profiles while reducing steatosis—supporting CARF as a mechanistic, potentially druggable node in MASLD. Disclosure J. Salinas: None. D.M. Clayborn: None. T.C. Friedman: Advisory Panel; Ended; Acella Pharmaceuticals. K.M. Hasan: None. Funding NIGMS-5R16GM153632NIGMS-5SC2GM125551
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Salinas et al. (2026) studied Metabolic dysfunction-associated steatotic liver disease (MASLD). Liver-directed CARF overexpression (AAV-CARF) vs. Vector control was evaluated on Hepatic triglyceride (TG) burden and CPT2 protein levels. Liver-directed CARF overexpression reduced hepatic triglyceride burden and restored CPT2 protein levels in diet-induced obese mice compared with vector controls.
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