Although adipose tissue plays important physiological roles, the increase in adiposity with aging is a common feature in several pathologies, including some forms of osteoporosis. Ion channels and ghrelin signalling have been shown to be involved in regulating many cell processes such as proliferation, differentiation and apoptosis, particularly in bone. This thesis investigated the roles of ghrelin and ion channels in the adipogenic differentiation of 7F2 preosteoblastic cells. The amount of Oil Red O stain increased by 135%, and the mRNA expression of two adipocytic markers, PPARγ (5.46 ± 0.22; p = 0.024) and C/EBPα (25.3 ± 0.21; p = 0.0048), was higher in 7F2 cells cultured with adipogenic medium compared with basal medium, indicating that 7F2 cells differentiated into adipocytes. 7F2 cells also expressed previously unreported variant isoforms of ghrelin at the mRNA level; these variants isoforms were much more abundant than native ghrelin. 7F2 cells also express the other key genes from ghrelin signalling, i.e. Ghsr and Mboat4, suggesting that ghrelin or its variant isoforms may have autocrine/paracrine effects in these cells. Ghrelin mRNA expression was higher in 7F2 cells treated with basal medium + 200 nM ghrelin (144.84 ± 0.58) and adipogenic medium + 200 nM ghrelin (123.07 ± 0.33) compared with cells cultured with basal medium alone (p = 0.01), and adipogenic medium alone (0.79 ± 0.40; p = 0.0089), respectively. Treatment with 20 nM and 200 nM ghrelin had no significant effect on lipid content and on osteoblastic and adipocytic markers mRNA levels (p > 0.05). On the contrary, cell numbers were lower in 7F2 cells cultured with the voltage-gated K+ channel blocker tetraethylammonium, and lipid droplets inside 7F2 cell-derived adipocytes were smaller compared with cells cultured with adipogenic medium alone, suggesting that voltage-gated K+ channels are involved in regulating cell proliferation and adipogenic differentiation. 7F2 cells and 7F2 cell-derived adipocytes expressed the KATP channel subunits Kcnj8 and Sur2B, and the BK channel subunits Kcnma1 and Kcnmb2. 12 types of functional ion channels were detected in electrophysiology experiments but could not be identified. In conclusion, ghrelin treatment did not inhibit adipogenic differentiation in 7F2 cells, but 7F2 cells expressed previously unreported variant isoforms of ghrelin and ion channels which may play important roles in regulating osteoblast functions and adipogenic differentiation. Future work is required to confirm the presence of ghrelin variants at the protein level, and to study the roles of these variants and ionic mechanisms in bone metabolism. A better understanding of the mechanisms underlying pathological adipogenesis in bone may provide interesting therapeutic targets for disorders such as osteoporosis.
QP Physiology, RM Therapeutics. Pharmacology
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