Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC218
Involvement of STIM and ORAI in the Differentiation of Human Preadipocytes
O. J. Ayoola1, S. Atkins1, S. Xu1
1. Diabetes and Endocrinology Group, Biomedical Research Laboratory, Hull York Medical School, Hull, United Kingdom.
Modulation of cellular calcium concentration by hormones or neurotransmitters through Ca2+ pumps, channels and transporters is central to the physiological and pathophysiological control of cellular processes, such as vesicular trafficking, gene expression, apoptosis and necrosis1. In human preadipocytes, store-operated calcium (SOC) channels have been demonstrated as a major calcium influx component2, 3. Here our aim is to detect the existence of Stim and Orai mRNAs and proteins in preadipocytes and also determine their roles in preadipocytes differentiation. Subcutaneous adipose tissues were collected from patients undergoing bariatric surgery after ethic approval and informed consent. Preadipocytes were isolated by collagenase digestion and cultured. Cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) containing 5% fetal bovine serum. Cell differentiation was induced by 3-isobutyl-1-methylxanthine, hydrocortisone, insulin and indomethacin. Immunostaining, real-time PCR, oil-red staining and Ca2+ imaging of the preadipocytes samples were performed over a 14 days’ time. The RT-PCR and immunostaining showed the presence Stim1, Stim2, Orai 1, Orai2 and Orai3 in the preadipocytes. The mRNA expression of Stim1, Stim2, Orai1 and Orai3 was significantly increased after the induction of cell differentiation for 14 days, but a small decrease for Orai2. Thapsigargin induced store-operated Ca2+ influx in both differentiated and non-differentiated preadipocytes, and the Ca2+ influx in the store-depleted preadipocytes was higher than that in cells without store-depletion. Our results showed the presence of Stim and Orai genes and proteins in human preadipocytes. These genes are up-regulated after cell differentiation, which suggests the involvement of store-operated channel molecules in preadipocyte differentiation and obesity development.
Where applicable, experiments conform with Society ethical requirements