SAT0062 Metabolomics and metabolic function analysis of the secretome of articular cartilage and chondrocytes in response to pro-inflammatory cytokines

Fellows, C., Quasnichka, H., Chowdhury, N.R., Budd, E., Skene, D.J. and Mobasheri, A. 2018. SAT0062 Metabolomics and metabolic function analysis of the secretome of articular cartilage and chondrocytes in response to pro-inflammatory cytokines. Presented at: Annual European Congress of Rheumatology 2018 (EULAR), Amsterdam, The Netherlands, 13-16 June 2018. Annals of the Rheumatic Diseases. , vol.77 (Supple) BMJ Publishing Group, p. 895. 10.1136/annrheumdis-2018-eular.6426

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Abstract

Background Chondrocytes rely primarily on glycolysis to meet their energy requirements, but can support cell survival and matrix synthesis during periods of nutrient stress by enhancing glycolysis with mitochondrial respiration. Accessing this ‘spare respiratory capacity’ requires optimal mitochondrial function. Impaired mitochondrial function is implicated in osteoarthritis (OA). Metabolic adaptation is evident in early-stage OA, however cartilage from late-stage disease does not seem to have this flexibility. A deeper understanding of these complex metabolic pathways may identify new markers of disease stage, and support therapeutic strategies for treating OA. Objectives Metabolomics has the potential to reveal pathological pathways and identify novel biomarkers. The aim was to identify metabolic processes involved in early stage disease by analysis of metabolites and metabolic function in pro-inflammatory models of cartilage degradation. Methods Macroscopically normal articular cartilage was obtained from equine and bovine metacarpophalangeal joints. Equine cartilage explants (n=6), and primary chondrocytes seeded at 105,000/cm2 (n=4), were cultured for 7 days in serum-free DMEM (Gibco) with or without 10 ng/ml equine interleukin-1β (IL-1β) and 10 ng/ml tumour necrosis factor-α (TNF-α). Secretome metabolite levels were measured using AbsoluteIDQ p180 targeted metabolomics kit (Biocrates), with Waters Xevo TQ-S mass spectrometer coupled to an Acquity UPLC system. PCA and OPLS-DA were performed using SIMCA-P v12.0 software. Metabolic function of primary equine (n=9) and bovine chondrocytes (n=3) was determined using Seahorse XFp and XFe24 analyzers. Cells were treated with species-specific 10 ng/ml IL-1β and/or 10 ng/ml TNF-α for 18 hour, and metabolically challenged with the Mito Stress Test. Metabolite levels, and oxygen consumption rates, were normalised to total cell protein, and values analysed by ANOVA with Tukey’s multiple comparison post-tests. Results Cytokine treatment decreased proline, ornithine and alpha-aminoadipic acid (p<0.0001) in explant secretome. Citrulline increased with cytokine treatment (p<0.0001) and glutamate, present in DMEM, was also elevated (p<0.0001). Metabolomic analysis of chondrocyte secretome showed that glutamine decreased (p<0.02) with cytokine treatment whereas citrulline was elevated (p<0.003). Metabolic analysis showed that cytokine treatment reduced basal respiration and negated spare respiratory capacity in chondrocytes (p<0.01), and the effect was due to IL-1β alone. Conclusions Explant metabolites which decreased with cytokine treatment are all downstream of glutamate. With elevated glutamate, this suggests that cytokines inhibit glutamate uptake and metabolism. Elevated citrulline in cell and explant models may be attributed to disruption of the urea cycle via induction of nitric oxide synthase. IL-1β alone negated spare respiratory capacity, and chondrocytes remained glycolytic. In conclusion, cytokines disrupt glutamate and citrulline metabolism, normally tightly regulated mitochondrial pathways, and IL-1β alone is responsible for the metabolic switch. These metabolic pathways could provide markers of early-stage inflammatory disease.

Item Type:	Conference or Workshop Item (Paper)
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	BMJ Publishing Group
ISSN:	0003-4967
Date of Acceptance:	30 April 2018
Last Modified:	11 Mar 2023 02:07
URI:	https://orca.cardiff.ac.uk/id/eprint/135026

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