Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.

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 de novo lipogenesis. Thus, modifying the adipogenic cocktail can enhance functional aspects of human adipocytes in vitro and is an important variable to consider prior to in vitro investigations into adipocyte biology.