An optimised protocol for the investigation of insulin signalling in a human cell culture model of adipogenesis.

While there is no standardized protocol for the differentiation of human adipocytes in culture, common themes exist in the use of supra-physiological glucose and hormone concentrations, and an absence of exogenous fatty acids. These factors can have detrimental effects on some aspects of adipogenesis and adipocyte function. Here, we present methods for modifying the adipogenic differentiation protocol to overcome impaired glucose uptake and insulin signalling in human adipose-derived stem cell lines derived from the stromal vascular fraction of abdominal and gluteal subcutaneous adipose tissue. By reducing the length of exposure to adipogenic hormones, in combination with a physiological glucose concentration (5 mM), and the provision of exogenous fatty acids (reflecting typical dietary fatty acids), we were able to restore early insulin signalling events and glucose uptake, which were impaired by extended use of hormones and a high glucose concentration, respectively. Furthermore, the addition of exogenous fatty acids greatly increased the storage of triglycerides and removed the artificial demand to synthesize all fatty acids by