Pork is a globally significant protein source, and its quality directly impacts human health. Intramuscular fat (IMF) deposition, or marbling, is crucial for pork quality, enhancing flavor, tenderness, and juiciness. However, the cellular and transcriptional mechanisms driving IMF deposition remain unclear. While high IMF content is desirable, understanding its regulation is vital for sustainable pork production, especially in China, the world's largest pork market. Previous studies have identified various cell types contributing to IMF formation using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST). However, these methods are limited by the size of myofibers. Single-nucleus RNA sequencing (snRNA-seq) overcomes this limitation by allowing analysis of both mono- and multinucleated cells. This study uses Laiwu pigs, a Chinese breed with naturally varying IMF content, as a model to investigate the cellular and transcriptional mechanisms of IMF deposition through a multi-omics approach integrating lipidomics, snRNA-seq, and RNA-seq.

Existing literature highlights the importance of marbling in enhancing pork quality traits. Studies have explored the association between fat infiltration in skeletal muscle and age-related diseases in humans. However, the specific cell sources and regulatory mechanisms of IMF deposition are poorly understood. High-throughput sequencing techniques like scRNA-seq and spatial transcriptomics have been employed to identify cell types involved in IMF formation, including satellite cells, fibro/adipogenic progenitors (FAPs), and adipocytes. The use of snRNA-seq in livestock studies, particularly for investigating IMF deposition, has been limited.

The study used Laiwu pigs categorized into high (HLW) and low (LLW) IMF content groups based on intramuscular fat content. Longissimus dorsi muscle (LDM) samples were collected for lipidomic analysis, snRNA-seq, and RNA-seq. Lipidomics utilized mass spectrometry to profile lipid classes and species. For snRNA-seq, nuclei were isolated from LDM, processed using the 10x Genomics Chromium platform, and sequenced. Data analysis involved quality control, clustering, and identification of cell types using Seurat. Subcluster analysis focused on adipocytes and FAPs. RNA-seq was performed on LDM samples from both groups, analyzing differentially expressed genes (DEGs). In addition to the pig studies, the researchers used a glycerol (GLY)-injured model in Pdgfracre-ER/ROSAMTmG mice to study the lineage tracing of FAPs and their differentiation into adipocytes in vivo. Primary FAP isolation, magnetic-activated cell sorting, and cell culture techniques were used to confirm the differentiation potential of FAPs in vitro. Quantitative real-time PCR (qRT-PCR) was used for gene expression analysis. Pathway enrichment analysis was conducted using GO and KEGG databases to determine the significant pathways affected by changes in gene expression. Statistical analysis employed unpaired two-tailed Student’s t-tests.

The HLW group exhibited significantly higher IMF content but lower drip loss compared to the LLW group. Lipidomics revealed increased levels of glycerolipids (triglycerides, diglycerides, monoglycerides) and sphingolipids (ceramides, monohexose ceramide) in the HLW group. snRNA-seq identified nine distinct cell clusters, with HLW pigs showing a higher percentage of adipocytes (1.40% vs. 0.17%). Three adipocyte subpopulations were identified: PDE4D+/PDE7B+, DGAT2+/SCD+, and FABP5+/SIAH1+. The DGAT2+/SCD+ and FABP5+/SIAH1+ subpopulations were significantly enriched in the HLW group. Subcluster analysis of FAPs/fibroblasts revealed three subpopulations: FAPs, fibroblasts, and PDE4D+/PDE7B+. Trajectory analysis showed that FAPs can differentiate into both fibroblasts and PDE4D+/PDE7B+ cells. In vivo lineage tracing using a mouse model confirmed that FAPs contributed to 43.35% of adipocytes in the tibialis anterior muscle. RNA-seq identified 1034 differentially expressed genes (DEGs), with significant changes in genes related to adipogenesis, lipid metabolism (including increased FABP4, FABP5, LIPE, ELOVL1, CPT1A, and decreased LPL), and fatty acid elongation. KEGG pathway analysis revealed enrichment of pathways such as glycerophospholipid metabolism, cGMP-PKG signaling, calcium signaling, and Notch signaling.

The study's findings highlight the complex interplay of cell types and transcriptional programs in marbling formation. The increased proportion of adipocytes in HLW pigs, particularly the DGAT2+/SCD+ and FABP5+/SIAH1+ subpopulations, emphasizes the role of specific adipocyte subtypes in IMF deposition. The identification of FAPs as a significant source of IMF adipocytes, but not the sole source, suggests other cell types like ECs, SPs and pericytes might also contribute. The altered expression of genes involved in lipid metabolism, particularly the downregulation of LPL, and the activation of fatty acid elongation provide mechanistic insights into the increased TG and Cer accumulation in HLW pigs. The enrichment of signaling pathways such as calcium signaling and Notch signaling suggests these pathways may regulate the process of marbling formation.

This study provides a comprehensive understanding of the cellular and molecular mechanisms underlying IMF deposition in high-marbled pork. The identification of key adipocyte subpopulations, the contribution of FAPs, and the altered expression of genes and signaling pathways offer novel insights into marbling formation. Future research should investigate the specific roles of different cell types and signaling pathways in regulating IMF deposition, potentially leading to strategies for enhancing marbling and pork quality.

The study primarily focused on Laiwu pigs, limiting the generalizability of the findings to other pig breeds. The in vivo lineage tracing experiment in mice may not perfectly reflect the dynamics in pigs. While the study identified several key genes and pathways, further research is necessary to elucidate the detailed functional relationships between these components and the mechanisms of IMF deposition.