PSVII-11 Effect of exogenous fatty acids on bovine fibro-adipogenic progenitor cell proliferation and adipogenic differentiation.

Abstract Fibro-adipogenic progenitor cells (FAPs) are muscle resident stem cells that contribute to marbling- a key determinant of beef quality grade. FAPs retain their stemness into the pre-weaning period in cattle, creating opportunities for early-life interventions to promote marbling. Lipids are a critical dietary component for calves, yet there is limited information on how different dietary fatty acids influence FAPs. The objective of this experiment was to evaluate the effect of fatty acids on FAP proliferation and adipogenic differentiation in vitro. An immortalized FAP cell line was used for the initial fatty acid screen and primary bovine FAPs (N=2 cattle) for subsequent experiments. Fatty acids were supplemented as BSA conjugates in culture media between 25-200µM. Control treatments included a vehicle control (BSA) in both experiments, and a no treatment control (growth media) and metabolic control (elaidic acid) in primary cell experiments. To assess proliferation, 10µM EdU was added into the media after cell pre-treatment with fatty acids and incubated for 24 hours (immortalized cells) or 6 hours (primary cells). To assess adipogenesis, FAPs were cultured with treatments in differentiation media for 3 days then maturation media for 7 days. Lipid droplets were assessed via Oil Red O in brightfield and fluorescent microscopy; EdU, nuclei, and PPARγ intensity were assessed via immunofluorescence. Image analysis was conducted in ImageJ, and data analysis was conducted in RStudio. An immortalized FAP line was used for an initial screen of oleic, palmitic, stearic, lauric, and linoleic acid at 25, 62.5, 100, 150, and 200µM. We found significant interaction effects between fatty acid and concentration for proliferation rates (p=0.0021) and the number of lipid droplets per cell (p=0.0016), while the median lipid droplet size was only impacted by fatty acid treatment (p< 0.0001). Notably, increasing stearic acid decreased proliferation, oleic and linoleic increased lipid droplet size, and oleic, palmitic, and stearic increased lipid droplets per cell. From these results, 150µM linoleic and oleic acid were selected for further testing using primary bovine FAPs. Treatments had no significant impact on bovine FAP proliferation or PPARγ expression. In adipogenic differentiation, the addition of any fatty acid supported over 190% more lipid droplets per cell (p< 0.05) and a 225% larger median lipid droplet size (p< 0.01) compared to the vehicle control. Oleic acid increased median lipid droplet size compared to linoleic acid (p=0.0429). Overall, this preliminary work presents evidence that FAP lipid accumulation and proliferation can be differentially impacted by supplemental fatty acids. For calf nutrition, this work highlights the potential importance of dietary fat to target marbling development. For this application, further work should assess the cellular metabolism and mechanisms related to fatty acid supplementation on adipogenic differentiation of FAPs, and assess additional fatty acids.