Deposition of intramuscular fat (IM), also known as marbling, is the deciding factor of beef quality grade in the U.S. Defining molecular mechanisms underlying the differential deposition of adipose tissue in distinct anatomical areas in beef cattle is key to the development of strategies for marbling enhancement while limiting the accumulation of excessive subcutaneous adipose tissue (SAT). The objective of this exploratory study was to define the IM and SAT transcriptional heterogeneity at the whole tissue and single-nuclei levels in beef steers. Longissimus dorsi muscle samples (9–11th rib) were collected from two finished beef steers at harvest to dissect matched IM and adjacent SAT (backfat). Total RNA from IM and SAT was isolated and sequenced in an Illumina NovaSeq 6000. Nuclei from the same samples were isolated by dounce homogenization, libraries generated with 10× Genomics, and sequenced in an Illumina NovaSeq 6000, followed by analysis via Cell Ranger pipeline and Seurat in RStudio (v4.3.2) By the expression of signature marker genes, single-nuclei RNA sequencing (snRNAseq) analysis identified mature adipocytes (AD; ADIPOQ, LEP), adipose stromal and progenitor cells (ASPC; PDGFRA), endothelial cells (EC; VWF, PECAM1), smooth muscle cells (SMC; NOTCH3, MYL9) and immune cells (IMC; CD163, MRC1). We detected six cell clusters in SAT and nine in IM. Across IM and SAT, AD was the most abundant cell type, followed by ASPC, SMC, and IMC. In SAT, AD made up 50% of the cellular population, followed by ASPC (31%), EC (14%), IMC (1%), and SMC (4%). In IM depot, AD made up 23% of the cellular population, followed by ASPC at 19% of the population, EC at 28%, IMC at 7% and SMC at 12%. The abundance of ASPC and AD was lower in IM vs. SAT, while IMC was increased, suggesting a potential involvement of immune cells on IM deposition. Accordingly, both bulk RNAseq and snRNAseq analyses identified activated pathways of inflammation and metabolic function in IM. These results demonstrate distinct transcriptional cellular heterogeneity between SAT and IM depots in beef steers, which may underly the mechanisms by which fat deposits in each depot. The identification of depot-specific cell populations in IM and SAT via snRNAseq analysis has the potential to reveal target genes for the modulation of fat deposition in beef cattle.

Deposition of intramuscular adipose tissue (IMAT) is the primary determinant for beef quality grade in the U.S. Accumulation of subcutaneous (SCAT) and visceral (VIAT) adipose tissue precedes that of IMAT and often leads to excessive adiposity in beef cattle. Approaches to increase marbling while limiting subcutaneous and visceral adiposity are limited. Our objective is to define the depot-specific transcriptome profile and adipocyte function in IMAT, SCAT, and VIAT in beef steers. Transcriptomics revealed the upregulation of adipogenic and lipogenic genes in SCAT and VIAT vs. IMAT. Functional transcriptome analysis demonstrated the activation of pathways for lipid metabolic processes and biosynthesis in SCAT, accompanied by increased preadipocyte proliferation, adipocyte size, and insulin responses of SCAT in vitro. While IMAT had a greater abundance of preadipocytes, they proliferated at a lower rate and differentiated into adipocytes that were smaller and less responsive to insulin compared to SCAT. The upregulation of extracellular matrix genes in IMAT suggests that fat accumulation may be limited by the muscle microenvironment. The activation of inflammatory and immune response pathways, combined with a higher abundance of immune cells, highlighted VIAT as an immune-responsive depot. Our findings reveal transcriptional and cellular profiles underlying fat deposition in SCAT, VIAT, and IMAT in beef cattle.