Depot-specific regulation of adipogenesis

Two most important white fat depots of mammals are visceral fat depot and subcutaneous fat depot. In human, excessive white fat accumulation has long been viewed as bad for health. However, recent studies showed that the impact of adipose tissue on human health varies dependent on the fat depot where fat is accumulated. People with high visceral fat content are prone to diabetes and other metabolic disorders. However, the association between subcutaneous fat accumulation and obesity was found much weaker. Interestingly, large variation in the fat accumulation at individual fat depots has been observed among different person.

In addition, intramuscular fat depot, an important fat depot in beef industry, is another major white fat depot. In human, intramuscular fat accumulation is a typical symptom of muscular dystrophies, which greatly affects muscle function. In beef cattle, adipogenesis at intramuscular fat depot is responsible for marbling, which is highly desired due to its positive influence on meat quality. The grade of marbling is highly variable between different cattle breeds. Two good examples are Wagyu cattle and Brahman cattle which are known for abundant marbling and extreme low marbling respectively. However, subcutaneous fat accumulation in Wagyu cattle and Brahman cattle are overall similar. 

All of these suggest the presence of depot-specific regulation of adipogenesis. Currently, we are using both cattle and mouse as models to study the mechanism regulating adipogenesis at different fat depots.

Recently, it was found that the adipogenic cells giving rise to adipocytes in intramuscular fat depots also have fibrogenic capacity and are therefore named fibro/adipogenic progenitor (FAP) cells. We are currently studying the signaling pathways regulating the fate determination of FAP cells.

Background

Intramuscular fat (IMF) and intramuscular connective tissue (IMC) are often seen in human myopathies and are central to beef quality. The mechanisms regulating their accumulation remain poorly understood. Here, we explored the possibility of using beef cattle as a novel model for mechanistic studies of intramuscular adipogenesis and fibrogenesis.

Methods

Skeletal muscle single-cell RNAseq was performed on three cattle breeds, including Wagyu (high IMF), Brahman (abundant IMC but scarce IMF), and Wagyu/Brahman cross. Sophisticated bioinformatics analyses, including clustering analysis, gene set enrichment analyses, gene regulatory network construction, RNA velocity, pseudotime analysis, and cell-cell communication analysis, were performed to elucidate heterogeneities and differentiation processes of individual cell types and differences between cattle breeds. Experiments were conducted to validate the function and specificity of identified key regulatory and marker genes. Integrated analysis with multiple published human and non-human primate datasets was performed to identify common mechanisms.

Results

A total of 32,708 cells and 21 clusters were identified, including fibro/adipogenic progenitor (FAP) and other resident and infiltrating cell types. We identified an endomysial adipogenic FAP subpopulation enriched for COL4A1 and CFD (log2FC=3.19 and 1.92, respectively; p<0.0001) and a perimysial fibrogenic FAP subpopulation enriched for COL1A1 and POSTN (log2FC=1.83 and 0.87, respectively; p<0.0001), both of which were likely derived from an unspecified subpopulation. Further analysis revealed more progressed adipogenic programming of Wagyu FAPs and more advanced fibrogenic programming of Brahman FAPs. Mechanistically, NAB2 drives CFD expression, which in turn promotes adipogenesis. CFD expression in FAPs of young cattle before the onset of intramuscular adipogenesis was predictive of IMF contents in adulthood (R2=0.885, p<0.01). Similar adipogenic and fibrogenic FAPs were identified in humans and monkeys. In aged humans with metabolic syndrome and progressed Duchenne muscular dystrophy (DMD) patients, increased CFD expression was observed (p<0.05 and p<0.0001, respectively), which was positively correlated with adipogenic marker expression, including ADIPOQ (R2=0.303, p<0.01; and R2=0.348, p<0.01, respectively). The specificity of Postn/POSTN as a fibrogenic FAP marker was validated using a lineage-tracing mouse line. POSTN expression was elevated in Brahman FAPs (p<0.0001) and DMD patients (p<0.01) but not in aged humans. Strong interactions between vascular cells and FAPs were also identified.

Conclusions

Our study demonstrates the feasibility of beef cattle as a model for studying IMF and IMC. We illustrate the FAP programming during intramuscular adipogenesis and fibrogenesis and reveal the reliability of CFD as a predictor and biomarker of IMF accumulation in cattle and humans.

Journal of Cachexia, Sarcopenia and Muscle, Revised manuscript submitted.