THU0011 ANALYSIS OF METABOLIC STATUS IN CYBRIDS REVEALED IMPAIRED METABOLIC FLEXIBILITY IN OA PROCESS.
Authors: A. Dalmao-Fernandez, J. Lund, T. Hermida Gómez, M. E. Vazquez Mosquera, I. Rego-Perez, F. J. Blanco, M. Fernandez-Moreno
Journal: Annals of the Rheumatic Diseases (2020)

Background: There are several metabolic pathways involved in cell metabolism, including glycolysis, tricarboxylic acid (TCA) cycle and fatty acid (FA) oxidation. Metabolic flexibility has previously described as the ability to respond or adapt to changes in metabolic demand; assessed by the ability to switch from fat to carbohydrate oxidation. In the last years there is a growing interest to assess the influence of metabolic flexibility, as a mechanism to explain how lipids can accumulate in the tissue. During OA, it has been established a relationship between mitochondrial dysfunction and cellular damage due to impairments in mitochondrial function and metabolic flexibility. Several studies have suggested that fatty acids may play an important role in OA development and progression. Objectives: The aim of this work was to examine the differences in glucose and fatty acid metabolism, with special focus on metabolic flexibility, in cybrids from healthy (N) or OA donors. Methods: Cybrids were developed using 143B.TK - Rho-0 cell line (nuclear donor) and platelets (mitochondrial donors) from healthy (N) and OA donors. Glucose and FA metabolism were measured using D-[ 14 C(U)]glucose and [1- 14 C]oleic acid respectively. Metabolic flexibility was evaluated by co-culturing with glucose and oleic acid acutely by using inhibitors against glucose and FA oxidation, 20µM UK5099 and 10µM etomoxir, respectively. Incorporation of FA into lipid droplet (LD) was evaluated by thin layer chromatography and LD were stained by LD540 and analyzed by confocal microscope and flow cytometry. Mitochondrial dynamics was measured by real-time PCR method. Percentage of mitochondrial Anion Superoxide (O 2 - ) production was evaluated incubating cells with MitoSox® using Flow Cytometer. Appropriate statistical analyses were performed with GraphPad Prism v6. Results: There were no changes in basal glucose metabolism between cybrids. N cybrids had higher acid-soluble metabolites, reflecting incomplete FA β-oxidation than OA cybrids. Comparing glucose and FA metabolism showed that both types of cybrids preferred to oxidize glucose. Co-culturing with glucose and Oleic acid, increased total cellular uptake and oxidation of glucose in N compared to basal condition (Figure-1) and in this condition the OA cybrids showed an increase in mitochondrial O 2 - production. Inhibition of FA oxidation by etomoxir increased complete glucose oxidation of N cybrids but not in OA cybrids that had a preference to oxidize oleic acid compared to basal condition. Gene expression of mitofusin-2 ( MFN2 ) was higher in N than OA cybrids under inhibiting conditions. Combine these data indicate that N cybrids are more metabolically flexible and have better adaptative response than OA. Cybrids presented different lipid distribution patterns. Lipid droplet (LD) formation increased in both groups incubated in presence of FA. Furthermore, N cybrids showed less LD formation than OA. Conclusion: The results indicated that cybrids from OA patients had reduced metabolic flexibility compared to N cybrids. These results enhance our understanding of the mitochondria metabolism in OA, suggesting a mitochondrial dysfunction and impairment of metabolic flexibility during the OA process. Disclosure of Interests: Andrea Dalmao-Fernandez: None declared, Jenny Lund: None declared, Tamara Hermida Gómez: None declared, Maria Eugenia Vazquez Mosquera: None declared, Ignacio Rego-Perez: None declared, Francisco J. Blanco Grant/research support from: Sanofi-Aventis, Lilly, Bristol MS, Amgen, Pfizer, Abbvie, TRB Chemedica International, Glaxo SmithKline, Archigen Biotech Limited, Novartis, Nichi-iko pharmaceutical Co, Genentech, Jannsen Research &amp; Development, UCB Biopharma, Centrexion Theurapeutics, Celgene, Roche, Regeneron Pharmaceuticals Inc, Biohope, Corbus Pharmaceutical, Tedec Meiji Pharma, Kiniksa Pharmaceuticals, Ltd, Gilead Sciences Inc, Consultant of: Lilly, Bristol MS, Pfizer, Mercedes Fernandez-Moreno: None declared