Background: Functional studies on uncoupling protein 1 (UCP1) are important to identify potential pharmacological targets which interfere with energy metabolism. However, current cell models of human origin are scarce due to limited tissue availability. In this study, we generated human preadipocytes and adipocytes with an overexpression of UCP1 and studied the metabolic function of these cells.
Methods: Human Simpson-Golabi-Behmel syndrome (SGBS) cells were used as a model system. Cells were transduced in the preadipocyte state with lentivirus encoding human UCP1. Stable cultures were obtained by antibiotic selection. SGBS-UCP1 cells were characterized by qPCR, Western blot, and immunofluorescence. Mitochondrial content was determined by quantification of mitochondrial DNA and citrate synthase activity. Metabolic processes were assessed using an XFe96 Extracellular Flux Analyzer.
Results: UCP1 was not detectable in parental SGBS preadipocytes, but a weak expression on both mRNA and protein level was observed in late adipogenesis. Using lentiviral transduction, we achieved a robust overexpression of UCP1 in preadipocytes. UCP1-overexpressing cells displayed an adipogenic differentiation capacity comparable to control cells. In differentiated adipocytes, we achieved a UCP1 overexpression by ~12-fold on protein level, without affecting the mitochondrial content and the expression of other brown adipose tissue (BAT)-associated genes. Both preadipocytes and adipocytes overexpressing UCP1 showed a significantly increased basal respiration rate (~2-fold and ~1.5-fold, respectively) and a reduced coupling efficiency compared to control cells (30% vs 100% and 12% vs 70%, respectively). Acute stimulation with dibutyryl-cAMP markedly increased respiration in UCP1-overexpressing adipocytes, which was dependent on hormone sensitive lipase (HSL) activity. UCP1 activity could also be induced by treating SGBS-UCP1 cells with free fatty acids.
Conclusion: Our findings demonstrate that UCP1 overexpressed in a homologous cell system is fully functional and displays the expected uncoupling of the respiratory chain. We introduce these cells as a novel human model system to study UCP1 function and activation.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology