Higher confluency enhances lipid droplet size, triglyceride content and endocrine function on white-like adipocytes

The murine fibroblast cell line 3T3-L1, due to their capability to differentiate into adipocytes, is extensively employed in studies of adipogenesis, metabolism and inflammation. However, the molecular phenotype and endocrine function is unclear with respect to initial confluency at the onset of the differentiation into adipocytes. This study aims to evaluate preadipocytes confluency implication during adipogenesis with morphological, biochemical, and molecular phenotype, and endocrine activity. Cells were stimulated by differentiation medium varying the initial confluency of either 75-85 % (low confluency group) or >95 % (high confluency group) and were analyzed 10 and 14 days after the onset of the differentiation protocol, assessing lipid droplets, triglycerides, molecular phenotype, and endocrine function. The high confluency group showed higher frequencies of lipid droplets in the ranges 3.0-5.0 μm², 35-50 and > 50 μm², with increased triglycerides synthesis at days 10 and 14, respectively, compared to either preadipocytes (2.51 ± 0.27 and 5.10 ± 2.09 -fold increase) or the low confluency group (2.51 ± 0.27 and 5.10 ± 2.09 -fold). Pref-1 expression decreased proportional to the differentiation time, and PGC-1α expression decreased on day 14 only for the high confluency group. PPARγ and adiponectin expression were directly proportional to confluency and time-differentiation. Adiponectin release increased independently of confluency and time, on the contrary, leptin release increased proportional to confluency and time-differentiation. In conclusion, a high confluency, compared to regular confluency, for white-like adipocyte differentiation increases lipid droplets biogenesis and triglycerides storage without altering molecular phenotype, enhancing endocrine function as observed by increased adiponectin and leptin release maintained up to 14 days. Our findings allow us to clarify the importance of confluency in the molecular phenotype and the endocrine function of white-like adipocytes.