Background/Purpose: Many signaling pathways activated under inflammatory and hypoxic conditions have profound effects on intracellular metabolism to support cell growth and survival. Cancer cells consume glucose and glutamine at a high rate compared to normal cells. Recent studies have identified cancer-specific metabolic changes that provide new therapeutic targets. The microenvironment in inflamed joints in RA is also characterized by hypoxia and low concentration of nutrients, and fibroblast-like synoviocytes from RA patients (RA-FLS) is known to have several tumor-like characteristics. However, little is known about how glucose or glutamine metabolism are involved in the aberrant proliferation of RA-FLS. We aimed to evaluate the role of these metabolisms in RA-FLS, and discover a metabolic characteristics of RA-FLS.

Methods: The expression of glycolysis- or glutaminolysis-related enzymes was evaluated by real-time PCR and Western blotting. The intracellular metabolites in FLS were analyzed by gas chromatography mass spectrometry (GC/MS) and capillary electrophoresis (CE)-MS. We used glucose- or glutamine-free medium for investigating the effects of glucose or glutamine on cell growth of RA-FLS. siRNA or compound 968 was used to inhibit glutaminase 1 (GLS1). Arthritis was induced in SKG mice by zymosan A injection. SKG mice were treated with compound 968 three days per week. Ki-67-positive cells were analyzed by immunohistochemistry.

Results: GLS1 expression was increased in RA-FLS and metabolome analyses suggested that glutamine metabolism was upregulated in RA-FLS. Cell proliferation of RA-FLS was significantly decreased under the glutamine-derived condition, but not under glucose-derived condition. Cell proliferation of RA-FLS was significantly suppressed by GLS1 inhibition. The levels of GLS1 mRNA was increased by treatment with IL-17 or PDGF in RA-FLS. Administration of GLS1 inhibitor, compound 968, ameliorated autoimmune arthritis in SKG mice. Histologic score of arthritis in compound 968-treated mice were significantly lower than those in control mice. Furthermore, immunohistochemical analysis revealed that compound 968 treatment significantly downregulated the Ki-67-positive synovial cells. The number of Th17 cells and Treg cells in spleens from compound 968-treated and control mice were not changed by compound 968 treatment.

Conclusion: Glutamine metabolism was involved in the pathogenesis of RA. Glutamine deprivation or inhibition of GLS1 inhibited cell proliferation of RA-FLS. Administration of GLS1 inhibitor ameliorated the inflammatory arthritis in SKG mice by suppressing the proliferation of RA-FLS. GLS1 inhibition directly affected the cell cycle progression of RA-FLS and suppresses the aberrant cell proliferation. GLS1 may play an important role in regulating RA-FLS proliferation, and could be a novel therapeutic target in RA.