Fat depot differences in lipids and genes of Shanxia black pigs

Pork fat has long been treated as a single, undifferentiated commodity in both agricultural science and the kitchen, but a new multi-omics study of a Chinese indigenous pig breed demonstrates that fat taken from different parts of the same animal is molecularly distinct—and that these differences are deep enough to serve as chemical fingerprints for each anatomical site. By combining high-resolution lipidomics with whole-transcriptome sequencing, a research team led by Li Zhang, Longyun Li and Yizhong Huang has mapped the lipid landscapes of four distinct adipose depots in the Shanxia long black pig, revealing a fundamental divide between subcutaneous fat, which is built for energy storage, and visceral fat, which appears wired for signaling, immune activity and membrane remodeling.

The study, published in the Journal of Agriculture and Food Research, examined six Shanxia long black pigs with an average live weight of 105 kilograms, reared under identical conditions at a breeding company in Jiangxi province. From each animal, the researchers harvested four depots: abdominal backfat and thoracic backfat, both from the subcutaneous layer; visceral fat from the greater omentum, the apron-like tissue draped over the intestines; and leaf fat, the perirenal depot surrounding the kidneys. Each depot yielded six biological replicates, frozen within thirty minutes of slaughter to preserve the molecular state of the tissue.

To profile the lipids, the team ground each frozen sample under liquid nitrogen and extracted lipids with a methyl tert-butyl ether and methanol mixture spiked with internal standards. The extracts were then separated on a C30 ultra-performance liquid chromatography column and analyzed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring mode, a targeted technique that allows precise quantification of hundreds of individual lipid molecules by tracking their characteristic fragmentation transitions. In total, the researchers identified 668 lipid species spanning five major categories and thirty-four subcategories, including 317 triglycerides, more than a hundred ceramides, and dozens of phosphatidylcholines, phosphatidylethanolamines and related membrane lipids.

The most striking finding concerns what dominates each depot. Triglycerides, the classic storage lipids, made up between roughly 81 and 91 percent of the lipid content in all four tissues, but free fatty acids were significantly more abundant in the two visceral depots—the greater omentum fat and the leaf fat—than in the two subcutaneous depots. When the researchers applied principal component analysis and orthogonal partial least-squares discriminant analysis to the full lipid dataset, all four tissues separated clearly, with the strongest divergence between thoracic backfat and visceral fat and the mildest between thoracic backfat and leaf fat. The statistical models showed high explanatory power and predictive ability, with permutation testing over 200 iterations confirming that the separations were not artifacts of overfitting.

Perhaps most practically, the team identified signature lipids that can distinguish the depots from one another. Ceramides of the alpha-hydroxy fatty acid-sphingosine class, combined with phosphatidylcholine, reliably separate thoracic backfat, omental fat and leaf fat, while triglycerides distinguish abdominal backfat from leaf fat. In the comparison between thoracic backfat and leaf fat, nine of the ten lipid markers with the greatest statistical weight were ceramides, all far more abundant in the backfat. Ceramides are not passive structural molecules; they are bioactive signaling lipids implicated in obesity, insulin resistance, type 2 diabetes and even thermogenesis regulation, which means their differential distribution across pig depots may matter both for lard quality and for understanding metabolic disease.

The transcriptomic arm of the study reinforced and extended the lipid picture. The researchers sequenced strand-specific libraries from the same tissues, generating more than 182 gigabase pairs of clean data aligned to the pig reference genome, with an average mapping rate of nearly 96.5 percent. Quantitative PCR validation of eight selected genes confirmed the reliability of the sequencing. The numbers of differentially expressed genes between depots ranged from 555 to 2,433, and a consistent hierarchy emerged: omental visceral fat showed the most transcriptionally active profile, with up to 79 percent of differentially expressed genes upregulated in comparisons against other depots, while leaf fat was transcriptionally the quietest. Enrichment analysis showed that visceral fat was dominated by immune and signaling pathways—cytokine-cytokine receptor interactions, leukocyte-mediated immunity, chemokine signaling—whereas comparisons between the two subcutaneous depots highlighted metabolic pathways, fatty acid metabolism and the PPAR signaling pathway.

The researchers then took the analysis a step further by integrating the two data types, a strategy they describe as transcriptomic-lipidomic association analysis. Because only certain pathways were enriched in both datasets, they focused on two comparisons: thoracic backfat versus leaf fat, and omental fat versus leaf fat. In the first, Pearson correlation analysis within the shared metabolic pathways revealed 103 lipids and 22 genes with strong, statistically significant correlations, defined as a correlation coefficient exceeding 0.8 in absolute value. Six genes stood out as hubs connected to more than ten lipid species each: ENPP6, PLD4, CA13, HDC, CBR2 and SCD.

The biology behind these correlations is suggestive. ENPP6 encodes a choline-specific phosphodiesterase that breaks down lysophosphatidylcholine, and its concurrent elevation with ceramide abundance raises the possibility that phospholipid catabolism feeds intermediates into ceramide biosynthesis or reshapes membrane microdomains that govern ceramide signaling. CA13, a carbonic anhydrase, may support de novo fat synthesis by supplying bicarbonate for pyruvate carboxylase. HDC, the rate-limiting enzyme of histamine production, is known from mouse studies to influence energy balance—mice lacking the gene develop visceral obesity and impaired glucose tolerance. Most intriguingly, SCD, the stearoyl-CoA desaturase that converts saturated fatty acids into monounsaturated ones, showed negative correlations with most ceramide species, hinting that subcutaneous fat actively keeps its ceramide pool unsaturated to preserve membrane fluidity, while visceral fat, with lower SCD expression, may accumulate more saturated ceramides and thus greater metabolic risk. In the visceral comparison, the growth factor gene FGF7 correlated most strongly with phosphatidylcholine and lysophosphatidylcholine species, suggesting a possible role in driving the conversion of lysophosphatidylcholine into phosphatidylcholine through the lipid remodeling pathway known as the Lands cycle.

The authors are careful to note the limits of their study. With six animals per depot, the statistical power is adequate for profiling but limited for detecting subtle effects, and no gene knockdown or overexpression experiments were performed, so the gene-lipid associations remain correlative rather than causal. They propose future functional work—such as CRISPR-mediated suppression of ENPP6 or SCD in pig adipocytes followed by targeted lipidomics—to test whether these genes genuinely control depot lipid composition. They also suggest that the lipid markers identified here could eventually serve in market supervision and authenticity testing, distinguishing the anatomical origin of fat products, though larger sample sets would be needed to validate such applications.

The broader implications run in two directions. For the swine industry, the results offer a molecular basis for the differentiated use of pig fat, a resource of considerable economic and culinary importance in China, where lard remains a traditional cooking oil. Knowing that subcutaneous depots specialize in triglyceride storage while visceral depots concentrate signaling lipids could guide breeding programs seeking to reduce unwanted fat deposition without sacrificing the intramuscular fat that underpins tenderness, juiciness and flavor. For human health, the parallels are direct: human adipose biology shows the same subcutaneous-versus-visceral dichotomy, with visceral fat strongly linked to insulin resistance and cardiometabolic disease. A molecular atlas of how these depots diverge in a large animal model—one far closer to human physiology than rodents—may help researchers pinpoint which lipid species and regulatory genes drive those differences, and how they might be therapeutically targeted. In showing that a pig’s fat is really four chemically distinct organs wearing the same name, the study turns a humble by-product into a rich system for exploring one of metabolism’s oldest questions: why fat stored in different places behaves so differently.