Role of ghrelin gene-derived peptides in the induction of browning and thermogenic functions in murine 3T3-L1 cell line and primary human white adipocytes

Obesity is characterized by excessive energy intake and reduced energy expenditure in adipose tissue (AT). Unlike white adipose tissue (WAT), which stores energy, brown adipose tissue (BAT) dissipates energy as heat, through the mitochondrial uncoupling protein 1 (UCP1). Furthermore, white adipocytes may be converted into thermogenic beige/brown cells, through a process known as browning. Hence, the development of effective anti-obesity molecules enhancing browning and energy expenditure has attracted considerable attention. Ghrelin genederived peptides, acylated ghrelin (GHRL), des-acyl ghrelin (D-AG), and obestatin (OBE) are key regulators of energy homeostasis; however, their potential role in WAT browning and BAT thermogenesis is still unknown. The results of this study, show that murine 3T3-L1 preadipocytes, as well as human white adipocytes, isolated from subcutaneous adipose tissue (hSAT) and visceral adipose tissue (hVAT), can be efficiently converted into thermogenic beige adipocytes after treatment with the ghrelin gene peptides. Specifically, in 3T3-L1 transdifferentiated adipocytes, D-AG and OBE, but not GHRL, increased the mRNA levels of brown/beige markers and batokines. Moreover, D-AG and OBE enhanced isoproterenolinduced lipolysis and increased the mRNA and protein levels of lipolytic markers, while reducing lipogenic effectors. Additionally, D-AG and OBE, but not GHRL, increased glucose uptake and the translocation of intracellular GLUT4 in beige adipocytes. Morphologically, these cells showed characteristics of brown adipocytes, including small lipid droplets and round-shaped mitochondria. Importantly, mitochondrial biogenesis, content and activity was enhanced by treatment with D-AG- and OBE, but not GHRL. Accordingly, the browning and thermogenic effects of D-AG and OBE involved activation of cAMP/PKA/CREB and AMPK/SIRT1 pathways. Finally, D-AG and OBE, but not GHRL, elevated the mRNA and protein levels of brown markers in both hVAT and hSAT, and increased mitochondrial respiration in hSAT-derived beige adipocytes. Overall, these findings indicate that while GHRL has no effect, D-AG and OBE promote browning and increase thermogenesis, suggesting the potential therapeutic importance of these peptides in obesity and metabolic dysfunctions.