Aberrant energy metabolism causes chondrocyte dysfunction in osteoarthritis

Abstract

Purpose: Metabolic pathways are a series of chemical reactions by which cells break-down nutrients to acquire the energy and processes building blocks that they need to maintain critical cellular processes. The details of chondrocyte metabolism and how it rewires in osteoarthritis (OA) disease progression is not well characterised. Central carbon metabolism (CCM) is an interconnected metabolic system that transforms carbon through glycolysis, gluconeogenesis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle pathway into energy that is essential for the physiology of chondrocytes. The aim of this research is to identify how changes in the energy metabolic state, in particular the central carbon metabolism, contributes to OA. Methods: OA and non-OA cartilage specimens were harvested from patients who required total knee arthroplasty (n=8 patient samples). In order to investigate the metabolism differences an integrated isotype assisted U-13C metabolic flux labelling approach was utilized on chondrocytes growing in limited glucose conditions. Isotopologue profiles of key metabolites were obtained by liquid chromatography/mass spectrometry (LC/MS). Expression profiles of genes regulating energy metabolism of OA cartilage of human and OA rat models were determined using RNA-Seq analysis. We further used extracellular flux assays, which allow for direct evaluation of cellular bioenergetic profiles ex vivo by measuring oxygen consumption rate (OCR, measure of oxidative phosphorylation) and extracellular acidification rate (ECAR, measure of aerobic glycolysis), to test the bio-energetic profile of OA chondrocytes. Results: Firstly, U-13C-labeling identified significant metabolic differences between OA chondrocytes vs. non-OA chondrocytes. In particular, OA chondrocytes displayed remarkably diverse rate of glucose utilization. Higher levels of glucose 6-phosphate (G6P), fructose 1,6-disphosphate (F16DP), glyceraldehyde 3-phosphate (GA3P) and lactate (LAC) were detected in OA chondrocytes. Furthermore, there was also a marked increase in 13C labelled enrichment of oxaloacetic acid (OAA) in TCA cycle of OA chondrocytes, while decrease in the rest substrates such as alpha-Ketoglutarate (aKG) and succinate (SUC). Together, these results demonstrated altered metabolism in OA chondrocytes (Figure 1). Secondly, analysis of the metabolic pathway differentially expressed genes revealed that genes involved in central carbon metabolism were altered in OA chondrocytes compared to non-OA cells. Thirdly, mitochondria and glycolysis stress tests utilizing seahorse technology revealed that basal glycolysis was increased in OA chondrocytes (Non-OA: 46.72±11.03; OA: 81.45±24.91 mpH/min, p<0.05) while basal mitochondrial oxidative phosphorylation was decreased (Non-OA: 24.26±10.35; OA: 6.94±3.06 pmol/min, p<0.05). Stress tests also showed that maximum glycolysis capacity was higher in OA chondrocytes (Non-OA: 47.86±15.01; OA: 91.67±36.31 mpH/min, p<0.05) while maximum oxidative phosphorylation capacity increased in non-OA chondrocytes (Non-OA: 121.22±54.70; OA: 40.25±9.83 pmol/min, p<0.01). These results demonstrated that OA chondrocytes switch to conditions of higher glycolysis and lower mitochondrial oxidative during OA progression. Conclusions: Altogether, this study, which is the first comprehensive comparative analysis of metabolism in chondrocytes, provides a foundation for targeting metabolism for therapeutic benefit in OA.