Abstract: 【Objective】 This study aimed to investigate the gene expression differences and associated signaling pathways in white adipocytes and beige adipocytes of Tibetan pigs before and after thermogenic activation,providing a theoretical basis for understanding adipocyte thermogenesis mechanisms and improving cold resistance in piglets.【Method】 Stromal vascular fraction (SVF) cells were isolated from the adipose tissue of Tibetan pigs and induced to differentiate into white adipocytes and beige adipocytes.Thermogenesis was activated by treating with 5 μmol/L forskolin (FSK) for 24 hours.The experiment comprised five groups:Untreated SVF cells group (C), white adipocytes group (W),FSK-treated white adipocytes group (WF), beige adipocytes group (B),and FSK-treated beige adipocytes group (BF).RNA-Seq was performed using the Illumina HiSeq platform.Differentially expressed genes were identified using DESeq2.Functional and metabolic pathway analyses of differentially expressed genes were conducted using GO and KEGG,and the expression of key genes was validated by Real-time quantitative PCR.Additionally,data from cold-stimulated Tibetan pigs were integrated with the in vitro beige adipocyte thermogenesis model (BF vs B) to identify co-expressed thermogenesis-related genes and transcription factors.【Result】 The transcriptome sequencing results demonstrated that FSK treatment significantly altered the gene expression profiles of both white adipocytes and beige adipocytes.Principal component analysis (PCA) and heatmaps revealed a marked separation in gene expression patterns before and after FSK treatment.Among them,beige adipocytes exhibited a stronger response to FSK treatment compared to white adipocytes.After treatment with FSK on beige adipocytes,a total of 1 514 genes were up-regulated and 1 544 genes were down-regulated.After treatment with FSK,a total of 815 genes were up-regulated and 1 096 genes were down-regulated in white adipocytes.GO and KEGG analyses indicated that the upregulated genes in beige adipocytes were significantly enriched in lipid metabolism pathways (fatty acid oxidation),cAMP signaling pathways,and immune-related pathways,and the enrichment score was higher than that of white adipocytes.Compared with the results before treatment after FSK treatment,there were a total of 485 up-regulated and 525 down-regulated differentially expressed genes in the two types of adipocytes.The up-regulated genes were enriched in thermogenic pathways such as cAMP,AMPK and PPAR,while the down-regulated genes were enriched in adiposynthetic pathways (such as unsaturated fatty acid biosynthesis).The results of Real-time quantitative PCR showed that the expression levels of KLF11 and PDK4 genes in white adipocytes after FSK treatment were extremely significantly upregulated (P＜0.01).By integrating the transcriptome sequencing data of cold-stimulated adipose tissue in Tibetan pigs and beige adipocytes,81 up-regulated co-expressed differentially expressed gene both in vivo and in vitro . There were 7 transcription factors such as PPARGC1A and NCOA3，and these transcription factors were related to processes such as mitochondrial biosynthesis and lipid metabolism reprogramming,suggesting their potential role in thermogenesis regulation.【Conclusion】 In this experiment,the transcriptome data of Tibetan pigs adipocytes were processed by FSK,revealing the key signaling pathways and genes in the thermogenesis process of adipocytes.Genes such as KLF11 and PDK4 that potentially regulated thermogenesis were screened out from the in vitro transcriptome data.Combined with the in vivo transcriptome data,7 transcription factors that potentially regulated thermogenesis in adipose tissue were screened out.The results provided potential targets for improving the cold resistance and fat deposition characteristics of pigs.

Key words: Tibetan pigs; beige adipocytes; white adipocytes; thermogenesis; RNA-Seq