Fatty acids (FAs) are essential for energy storage and metabolic homeostasis, but disruptions to the regulation of FA transport contribute to metabolic diseases such as obesity and type 2 diabetes. Circulating FAs must first pass through endothelial cells (ECs) lining the blood vessels before reaching metabolic tissue, such as adipocytes (fat cells). Previous research has shown that insulin can stimulate adipocytes to release lactate, signaling neighboring ECs to increase FA uptake and transport. However, these findings relied on isolated murine adipocytes, which are costly and difficult to maintain. My research aims to establish a more accessible model by using differentiated 3T3-L1 adipocytes. In this protocol, I induced 3T3L1 fibroblasts to differentiate into adipocytes over 14 days using a specialized chemical cocktail. I then treated the differentiated adipocytes with varying concentrations of rosiglitazone and insulin and applied the conditioned media (containing secreted molecules) to ECs. Finally, I quantified the endothelial FA uptake using BODIPY C-12, a fluorescent FA analog.
After establishing that I can reliably differentiate 3T3L1 cells, I carried out a time-course experiment, in which I tested conditioned media collected at different stages of the differentiation protocol and their potency for inducing endothelial fat uptake. Strikingly, I found that day 11 media stimulated endothelial FA uptake the most, suggesting that the adipocyte metabolic signaling peaks during the second half of the protocol, and begins to decline as differentiation concludes. With further experiments, I have also found that conditioned media from adipocytes exposed to higher concentrations of insulin dose-dependently increased endothelial FA uptake. Finally, my most recent results revealed that rosiglitazone treatment is not only necessary to generate active conditioned media but also that sustained exposure of differentiating adipocytes to rosiglitazone leads to greater FA uptake than a single exposure.
Future work will further investigate the nature of the active factor within the conditioned media responsible for this signaling by interrogating its physical and chemical features. Understanding how adipocytes regulate endothelial FA uptake is crucial for identifying the mechanisms that cause metabolic diseases. Developing a reliable and accessible model for studying these interactions will support future research on diabetes and other related diseases.