# Transcriptomic Analysis of High and Low Lipid Droplet Deposition Subpopulations of Chicken Preadipocytes Based on SSC Sorting

## TL;DR

This study uses flow cytometry to sort chicken preadipocytes based on lipid droplet content and finds distinct metabolic profiles between high and low lipid groups.

## Contribution

A novel label-free method using SSC sorting identifies metabolic heterogeneity in chicken preadipocytes without relying on traditional markers.

## Key findings

- High lipid droplet cells prioritize storage and antioxidant homeostasis, while low lipid cells focus on synthesis and oxidation.
- No significant differences in adipogenesis marker genes suggest both groups are at similar differentiation stages.
- Transcriptomic analysis reveals unique metabolic pathways in each subpopulation, offering insights into avian adipocyte heterogeneity.

## Abstract

Fat deposition is a crucial economic trait that affects the production performance and meat quality of broilers. Moderate fat accumulation can enhance meat flavor, whereas excessive fat deposition decreases feed conversion rate and carcass yield. In this study, a label-free sorting method based on the Side Scatter (SSC) signal of flow cytometry was established to distinguish subpopulations of chicken preadipocytes with varying levels of lipid droplet deposition. The results demonstrated a significant positive correlation between the SSC signal and lipid droplet content (R2 > 0.81, p < 0.001), confirming that SSC is a reliable indicator for assessing lipid droplet accumulation in chicken preadipocytes. Further transcriptomic analysis revealed no significant difference in the expression of adipogenesis marker genes (PPARG, LPL, CD36, PLIN1, and PLIN2) between the high lipid droplet group (H group) and the low lipid droplet group (L group), suggesting that both are at similar differentiation levels. However, significant metabolic differences were observed between the two groups: the L group was primarily characterized by active lipid synthesis, fatty acid oxidation, and membrane lipid remodeling, whereas the H group was characterized by lipid droplet storage, lipid transport, and antioxidant homeostasis. This study suggests that these two types of cells are not at different differentiation stages but rather exhibit distinct metabolic orientations. This finding provides new insights into the molecular mechanisms underlying the metabolic heterogeneity of avian adipocytes.

Fat deposition plays a crucial role in regulating the production performance and meat quality of broilers. Although the heterogeneity of mammalian adipocytes has been extensively studied, research on the molecular mechanisms underlying differences in lipid droplet accumulation in avian adipocytes remains limited. This study confirmed a significant positive correlation (R2 > 0.81, p < 0.001) between the SSC signal and lipid droplet content via fluorescence staining of lipid droplets, Oil Red O staining, and triglyceride (TG) quantification. Based on this, a label-free sorting strategy using SSC signals was established to sort differentiated chicken preadipocytes, obtaining high lipid droplet (H) and low lipid droplet (L) subpopulations, which were subsequently subjected to transcriptome sequencing and differential gene expression (DEG) analysis, followed by GO and KEGG enrichment analysis. The results indicated no significant differences in the expression of adipogenesis marker genes (PPARG, LPL, CD36, PLIN1, PLIN2) between the high lipid droplet (H) and low lipid droplet (L) groups, suggesting that both groups are at similar stages of differentiation. KEGG analysis revealed that both the H vs. NC and L vs. NC comparisons were enriched in common pathways, including the PPAR signaling pathway, ECM–receptor interaction, focal adhesion, cytokine–receptor interaction, and calcium–Apelin signaling pathway, suggesting that both groups of cells had activated the adipogenesis program. GO analysis showed that, in both H vs. NC and L vs. NC comparisons, differentially expressed genes (DEGs) were enriched in biological processes (BPs) related to cell adhesion, nucleosome assembly, chromatin remodeling, and receptor activity, as well as cellular components (CCs) such as the extracellular matrix, cytoskeleton, and nucleosome organization, indicating extensive gene reprogramming and activation of signaling transduction during differentiation. In the H vs. L comparison, enriched pathways included ABC transporters, ECM–receptor interaction, focal adhesion, gap junctions, microtubule-related processes, and neuroactive ligand–receptor interactions, involving lipid transmembrane transport, cytoskeleton stabilization, and signal transduction regulation, suggesting that high lipid droplet cells are more mature in lipid droplet transport, storage, and homeostasis maintenance. GO enrichment results further supported this conclusion, as H vs. L specifically enriched processes related to microtubule-related processes, cell cycle, and redox reactions (BPs), as well as chromosome organization, cytoskeleton, and motor activity (CC/MF), indicating that high lipid droplet cells maintain lipid droplet fusion and metabolic homeostasis via enhanced microtubule transport and antioxidant regulation. Differential gene analysis revealed that the L group upregulated genes associated with fatty acid synthesis and elongation (ACACA, FASN, SCD, FADS2, ELOVL1), cholesterol and isoprenoid biosynthesis (HMGCR, SQLE, MSMO1, DHCR7, DHCR24, FDPS, LSS), and fatty acid oxidation (PPARA, PPARD, ACAD11, SIRT5), reflecting a metabolic characteristic of concurrent lipid synthesis and mobilization; the H group, conversely, upregulated genes associated with lipid droplet formation and storage (G0S2, MOGAT1, GPAT4, PLIN4, AUP1), lipid transport (ABCA1, ABCA2, ABCG1, OSBPL3, VLDLR), and antioxidant defense (GPX3, GPX4, HMOX1), exhibiting a storage and homeostasis-oriented metabolic state. In the NC, L, and H groups, the expression of five genes—GEM, SPP1, ABCA1, PDLIM3, and ITGA8—showed a gradual increase, suggesting that these genes were associated with preadipocyte differentiation and lipid droplet deposition. In summary, although the high and low lipid droplet subpopulations of chicken preadipocytes exhibit similar differentiation states, they form distinct metabolic orientations. The L group is characterized by active lipid synthesis, fatty acid oxidation, and membrane lipid remodeling, while the H group predominantly features lipid droplet storage, lipid transport, and antioxidant homeostasis. This study highlights the molecular mechanisms underlying the metabolic heterogeneity of avian adipocytes and provides a theoretical basis for poultry fat deposition regulation and genetic improvement.