Atomic force microscopy, biochemical analysis of 3T3-L1 cells differentiated in the absence and presence of insulin

BackgroundThere are ample evidences to demonstrate that differentiation of preadipocytes is associated with deposition of fat in cells. Still, it is unclear whether the differentiation process also alters membrane topology as well as cholesterol levels and whether insulin contributes to it.MethodsMembrane scanning of differentiated cells, along with freshly plated and 11 day preadipocytes, was performed using Atomic Force Microscopy (AFM) to gain qualitative information about cell surface properties as well as roughness. Moreover, glucose uptake, lipid analysis, expression profiling of transcription factors and signaling molecules involved in the process of differentiation was also performed.ResultsWe report (i) differentiation in the presence of 500 μM isobutylmethylxanthine (IBMX), 0.25 μM dexamethasone (DEX) with or without 0.1 μM (0.57 μg/ml) insulin directly alters membrane topology. (ii) At nano-levels, addition of insulin maintains plasma membrane roughness during differentiation in comparison with IBMX and DEX only. (iii) At macro levels, decreased fat accumulation in preadipocytes exposed to insulin during the initial stages of differentiation is a result of reduced expression and nuclear localization of sterol regulatory element binding protein (SREBP)-1.General significanceThis study reports a significant reduction of membrane cholesterol and total cholesterol (p < 0.01) in cells differentiated in the presence of insulin.