Project description:Abstract Background: Intramuscular fat refers to the white adipose tissue deposited between muscle fibers, and its quantity and distribution directly impact the quality and value of beef. Compared to subcutaneous fat, intramuscular fat develops later and accumulates more slowly in cattle. The reasons for the delayed development and slower growth of intramuscular fat in cattle remain unclear. Results: Histological analysis showed that adipocytes in intramuscular fat were smaller than those in subcutaneous fat from the same mature cattle, indicating a delayed development or slower growth of intramuscular fat compared to subcutaneous fat. Intramuscular fat had a lower capacity for retaining or incorporating long-chain fatty acids into triglycerides than subcutaneous fat. Comparing the transcriptomes of intramuscular and subcutaneous fat by RNA sequencing identified more than 1,000 genes differentially expressed (DEGs) between the two adipose depots. Genes upregulated in intramuscular fat included FOXO6, SLC27A1, HDAC9, WWTR1, and PIK3C2A, which are known to inhibit adipose tissue development and growth. Genes downregulated in intramuscular fat included FABP4, AGPAT2, ADIG, ADIRF, and PLIN2, which are known to promote adipose tissue development and growth. Functional enrichment analyses of these DEGs suggested that intramuscular fat may have a lower capacity for fatty acid binding and adipogenesis compared to subcutaneous fat. Furthermore, genes downregulated in intramuscular fat were enriched in signaling pathways such as the PPAR signaling pathway, whereas genes upregulated in intramuscular fat were enriched in pathways including the Wnt signaling pathway. Stromal vascular fraction (SVF) cells from intramuscular fat exhibited a lower adipogenic potential than those from subcutaneous fat. Conclusion: Multiple factors may contribute to the delayed and slower deposition of intramuscular fat compared to subcutaneous fat in cattle, including reduced fatty acid binding capacity, lower triglyceride synthesis, and decreased adipogenesis in intramuscular fat. These differences are possibly driven by lower expressions of genes such as AGPAT2, FABP4, and ADIG, higher expression of genes such as FOXO6, HDAC9, and SLC27A1, reduced activation of the PPAR signaling pathway, and increased activation of the Wnt signaling pathway in intramuscular fat.

Project description:*Background: Adipocytes mainly function as energy storage and endocrine cells. The amount and distribution of fat are important factor that influence the meat quality in the beef industry. Fat depot can be found around internal organ (ometal), beneath the skin (subcutaneous), and between muscles (intramuscular). Different adipose depot showed the biological and genetic difference depending on their location. This inter-depot variation might be influenced by the inherent genetic programing for development of adipose depots. In this study, we used RNA-seq data to investigate the difference in transcriptome of various adipose depots in Hanwoo. *Results: Using RNA-seq, we identified 5797, 2156, and 5455 DEGs in the comparison between OI, OS, and IS respectively (FDR<0.01) and found 853, 48, and 979 DEGs specific to subcutaneous, intramuscular and omental fat respectively. DEGs in intramuscular fat were highly enriched the metabolism related pathways compared to other fat depots. DEGs specific to the omental fat is significantly enriched in PPAR signaling pathway and cell-junction related pathway. In subcutaneous fat, cytokine-cytokine receptor interaction with chemokines (CXC and CC subfamily) was the most significantly enriched the pathways. Interestingly, melanogenesis pathway was associated with the subcutaneous depot. Even though the adipose tissues shared the same pathways for adipocyte differentiation, the regulation of genes were different based on the depot. *Conclusions: We comparatively analyzed the transcripome profile from different adipose tissues using NGS and identified DEGs between adipose depot and specific to depot in Hanwoo animals. The functional annotation analysis of DEGs found that transcriptome profile difference in various adipose tissue of intramuscular, subcutaneous, and ometal fat. whole mRNA sequencing profiles of nine Korean native cattle (nine profiles of omental fat tissue, nine profiles of intramuscular fat tissue, nine profiles of subcutaneous fat tissue and eight profiles of muscle tissue)

Project description:Intramuscular fat (IM; marbling) deposition is the deciding factor of beef quality grade in the U.S. Defining molecular mechanisms regulating adipogenesis in distinct anatomical areas in beef cattle is key to the development of strategies for marbling enhancement while limiting accumulation of excess subcutaneous adipose tissue (SAT; backfat). Our objective was to define the IM and SAT transcriptional heterogeneity at the whole tissue and single-cell levels in beef cattle. Longissimus dorsi muscle (9-11th rib) samples were collected from two harvested finished beef steers to dissect matched IM and adjacent SAT. Nuclei were isolated by dounce homogenization, then sequenced via bulk RNA sequencing (RNAseq) and single-nuclei RNA sequencing (snRNAseq) with 10x Genomics in an Illumina NovaSeq 6000. Analysis was conducted via Cell Ranger pipeline and Seurat in R Studio. By the expression of signature marker genes, 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 with 19% of the population, EC with 28%, IMC with 7% and SMC with 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 cattle, 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.

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