Cold-Induced DHRS4 Promotes Thermogenesis via Enhanced Fatty Acid β-Oxidation in Porcine Subcutaneous Adipocytes

Abstract

Adipose tissue exhibits remarkable plasticity in adapting to thermal stress, yet the epigenetic mechanisms coordinating metabolic reprogramming in large mammals-particularly in livestock species lacking classical brown adipose tissue (BAT) such as swine-remain elusive. Using a porcine cold exposure model, we investigated adipose adaptation mechanisms through integrated single-cell RNA sequencing and bulk transcriptomic analyses of subcutaneous adipose tissue (subWAT). We identified a cold-induced thermogenic adipocyte subpopulation, characterized by upregulated DHRS4 expression. Mechanistically, cold exposure induced hypomethylation at the DHRS4 promoter locus, enhancing its expression to potentiate fatty acid β-oxidation, accompanied by thermogenic capacity upregulation. Our findings establish DHRS4 as an epigenetic-metabolic switch governing cold adaptation and a potential target for improving cold resistance in swine production systems.

Keywords: cold adaptation; dehydrogenase/reductase (SDR family) member 4; fatty acid β-oxidation.

Figure 1

(A) UMAP of adipocytes subpopulations. (B) Bubble chart showing scaled average expression of adipocytes subpopulations-enriched marker genes. (C) The trajectory inference and state information of adipocytes subpopulations. Each dot represents a single cell. (D) The fraction of each adipocyte’s subpopulation in dorsal subWAT from the RT group and the Cold group. (E) The top 20 enrichment pathways revealed by KEGG enrichment analysis of upregulated differentially expressed genes in Adi0, Adi5 subpopulations. The red font color indicates metabolic pathways related to thermogenesis.

Figure 2

(A) I. Venn diagram showing the overlapping genes that DEGs of Adi0 and Adi5 subpopulations significantly upregulated and RNA-seq of dorsal subWAT. (B,C) qPCR and Western Blotanalysis of DHRS4 expression in dorsal subWAT at the RT group and the Cold group. (D) Expression patterns of DHRS4 during the differentiation of SVF Cells and mouse Brown Adipocytes. ** p  ≤  0.01; *** p  ≤  0.001.

Figure 3

(A) Detection of DHRS4 overexpression efficiency in ISP4# cells. (B) qPCR analysis of adipogenic differentiation marker genes following DHRS4 overexpression in ISP4# cells at differentiation day 8. (C) Oil Red O staining of ISP4# cells following DHRS4 overexpression at differentiation day 8. (D,E) qPCR analysis of genes related to thermogenesis, lipolysis, and fatty acid β-oxidation following DHRS4 overexpression in ISP4# cells at differentiation day 8. (F) Western blot analysis of HSL, CPT1A, PPARG, UCP3, and DHRS4 expression levels in ISP4# cells overexpressing DHRS4 at differentiation day 8. (G) Free fatty acid (FFA) content in culture medium of ISP4# cells overexpressing DHRS4 at differentiation day 8. * p  ≤  0.05; ** p  ≤  0.01; *** p  ≤  0.001.

Figure 4

(A) Detection of DHRS4 overexpression efficiency and qPCR analysis of adipogenic differentiation marker genes following DHRS4 overexpression in SVF cells at differentiation day 8. (B) Oil Red O Staining of SVF cells following DHRS4 overexpression at differentiation day 8. (C,D) qPCR analysis of genes related to thermogenesis, lipolysis, and fatty acid β-oxidation following DHRS4 overexpression in SVF cells at differentiation day 8. (E) Western blot analysis of HSL, CPT1A, PPARG, UCP3, and DHRS4 expression levels in SVF cells overexpressing DHRS4 at differentiation day 8. * p  ≤  0.05; ** p  ≤  0.01; *** p  ≤  0.001.

Figure 5

(A) Prediction of CpG Islands in the DHRS4 promoter using MethPrimer software (https://www.methprimer.com/). (B) Amplification of DHRS4 CpG sites using methylation-specific primers in dorsal subWAT from RT and Cold groups. (C) Methylation patterns of CpG islands in the DHRS4 promoter region in dorsal subWAT from RT and Cold groups. Black circles represent methylated sites, while white circles denote unmethylated sites.