The Role of Mitochondria-Linked Fatty-Acid Uptake-Driven Adipogenesis in Graves Orbitopathy

Context: Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty acid (FA)-uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs). Objective:This work sought a role for mitochondria in OAT adipogenesis in GO. Methods: Confluent PFs from healthy OAT (OAT-H), OAT from GO (OAT-GO) and white adipose tissue in culture medium compared with culture medium containing a mixed hormonal cocktail as adipogenic medium (ADM), or culture-medium containing FA-supplementation, oleate:palmitate:linoleate (45:30:25%) with/without different concentration of mitochondrial biosubstrate adenosine 5'-diphosphate/guanosine 5'-diphosphate (ADP/GDP), AICAR (adenosine analogue), or inhibitor oligomycin-A for 17 days. Main outcome measures included oil-red-O staining and foci count of differentiated adipocytes for in vitro adipogenesis, flow cytometry, relative quantitative polymerase chain reaction, MTS-assay/10(6) cells, total cellular-ATP detection kit, and Seahorse-XFe96-Analyzer for mitochondria and oxidative-phosphorylation (OXPHOS)/glycolysis-ATP production analysis. Results: During early adipogenesis before adipocyte formation (days 0, 4, and7), we observed OAT-specific cellular ATP production via mitochondrial OXPHOS in PFs both from OAT-H and OAT-GO, and substantially disrupted OXPHOS-ATP/glycolysis-ATP production in PFs from OAT-GO, for example, a 40% reduction in OXPHOS-ATP and trend-increased glycolysis-ATP production on days 4 and 7 compared with day 0, which contrasted with the stable levels in OAT-H. FA supplementation in culture-medium triggered adipogenesis in PFs both from OAT-H and OAT-GO, which was substantially enhanced by 1-mM GDP reaching 7% to 18% of ADM adipogenesis. The FA-uptake-driven adipogenesis was diminished by oligomycin-A but unaffected by treatment with ADP or AICAR. Furthermore, we observed a significant positive correlation between FA-uptake-driven adipogenesis by GDP and the ratios of OXPHOS-ATP/glycolysis-ATP through adipogenesis of PFs from OAT-GO. Conclusion: Our study confirmed that FA uptake can drive OAT adipogenesis and revealed a fundamental role for mitochondria-OXPHOS in GO development, which provides potential for therapeutic interventions.