CLCN3 regulates adipogenesis and intramuscular fat deposition in pigs

Introduction Intramuscular fat (IMF) content is a key determinant of pork quality; however, its genetic regulation remains poorly understood. This study aimed to elucidate the mechanisms underlying IMF deposition using integrated multi-omics analyses. Specifically, CLCN3 was identified as a candidate gene, and its functional role and regulatory pathways in adipogenesis were investigated. Methods CLCN3 expression levels were analyzed in longissimus dorsi muscle tissues of pigs exhibiting divergent IMF content. Functional validation was performed using CLCN3 heterozygous mutant [ CLCN3 (−/+)] 3T3-L1 preadipocytes generated via the CRISPR/Cas9 system. Transcriptomic analysis revealed significant enrichment of the PI3K/AKT pathway in CLCN3 (−/+) cells compared with wild-type (WT) cells. Results and discussion CLCN3 expression was significantly higher in muscles with elevated IMF content and was predominantly expressed in adipose tissue. Heterozygous knockout of CLCN3 in 3T3-L1 preadipocytes markedly impaired adipocyte differentiation and downregulated key adipogenic transcription factors, including PPARγ and C/EBPα . Transcriptomic analysis revealed significant enrichment of the PI3K/AKT signaling pathway in CLCN3 (−/+) cells. Mechanistically, CLCN3 deficiency reduced both total and phosphorylated AKT levels, inhibited mTOR expression and phosphorylation, and attenuated PPARγ transcriptional activity. This led to decreased expression of lipogenic enzymes ( ACACA and FASN ), as well as lipid-binding protein FABP4 and glucose transporter GLUT4 . Overall, these findings indicate that CLCN3 promotes adipogenesis via the PI3K/AKT signaling pathway, thereby contributing to IMF deposition in pigs and providing new insights into the genetic regulation of pork quality.