ABSTRACT: Abstract Background: Intramuscular fat (IMF) and subcutaneous fat (SF) are two major adipose depots in cattle, each playing distinct roles in meat quality and systemic metabolism. The development of adipose tissue requires proliferation and differentiation of preadipocytes into adipocytes. However, the identity and characteristics of preadipocytes forming IMF and SF, especially the former, remain poorly defined. Results: In this study, we derived four single-preadipocyte clones from stromal vascular fraction (SVF) of IMF and SF tissues of four adult steers and characterized their gene expression signatures. All single-cell clones of IMF and SF preadipocytes were capable of forming adipocytes, as evidenced by lipid droplets accumulation and upregulation of adipocyte marker genes such as PPARG, CEBPA, FABP4, and ADIPOQ upon adipogenic induction. Transcriptomic analysis revealed that although both IMF and SF preadipocyte clones expressed classical preadipocyte markers such as PDGFRA, DLK1, PPARG, CD34, and ZNF423, they had significantly different gene expression profiles. Over 2,000 genes were differentially expressed between IMF and SF preadipocyte clones. Surprisingly, many muscle-specific genes such as MYOG, CKM, MYH2, and MYH3 were expressed at high levels in IMF preadipocyte clones while barely detectable in SF preadipocyte clones. These muscle-specific genes remained elevated in adipocytes differentiated from IMF preadipocyte clones. Functional enrichment analysis revealed that genes upregulated in IMF preadipocyte clones compared to SF preadipocyte clones were significantly enriched in pathways such as PI3K-Akt signaling, MAPK signaling, calcium signaling, cholesterol biosynthesis, muscle structure development, and neuromuscular junction formation, further suggesting that IMF preadipocytes bear characteristics of muscle cells. Differentiation assays demonstrated that IMF SVF cells had strong adipogenic potential, as they readily formed adipocytes upon adipogenic induction, but showed little myogenic potential, as they did not form myotubes upon myogenic induction. In contrast, bovine satellite cells, the widely considered myogenic progenitor cells in skeletal muscle, exhibited both myogenic and adipogenic potential, forming both myotubes and adipocytes under respective induction conditions. Conclusions: These results demonstrate that IMF and SF preadipocytes differ significantly in gene expression profiles with the former expressing many muscle-specific genes. Our findings suggest that IMF preadipocytes may originate from muscle satellite cells or share a common progenitor, whereas SF preadipocytes arise from non-myogenic adipose progenitor cells.
