Background:We recently reported that the inhibitor of hyaluronan (HA) biosynthesis, 4-methylumbelliferone (4-MU) blocked IL-1β activation of MMP13 mRNA and protein expression in human osteoarthritic (OA), bovine as well as bovine or OA cartilage explants [1]. This was a somewhat counterintuitive observation because we have also demonstrated that the overexpression of HAS2 (HAS2-OE) exerted the same chondroprotective effects on human and bovine chondrocytes. Others [2] have reported that HAS2-OE in tumor cells generates a flux in intracellular UDP-sugar pools that resulted in changes in cell metabolism; switching from a dependence on glycolysis to aerobic respiration. HAS2-OE and 4-MU likely also cause dramatic fluxes in intracellular UDP-GlcUA pools. From these results, we hypothesized that the effect of HAS2-OE and 4-MU relate to changing metabolism and the possibility of inhibition of glycolysis induce chondroprotective effect. To determine that, we used the glycolysis inhibitor, 2-Deoxyglucose (2DG) as an alternative agent to change metabolism in chondrocytes.Objectives:The objective of this study was to investigate the mechanism of chondroprotective effects of 2DGMethods:Bovine and human chondrocyte were stimulated with IL-1β (2ng/ml) in the presence or absence of 4MU (1.0 mM), 2DG (0.2-20 mM). Bovine chondrocytes were tested using Seahorse Flux Analyzer (Agilent Tech) to determine rate changes in medium accumulation of +H protons (indicative of lactic acid accumulation: ECAR) and for O2 consumption (indicative of mitochondrial respiration: OCR). Accumulation of MMP13 and phosphor AMPK (pAMPK) protein was quantified with Western blotting. Human and Bovine cartilage explants were cultured with L-1β in the presence or absence of 2DG (20 mM) and d 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) to pharmacologically induce AMPK for 7 days and stained with Safranin O.Results:Reduced mitochondrial potential and enhanced dependence on glycolysis was observed in IL-1β stimulated chondrocytes. Co-treatment with 4-MU and 2DG returned the cell metabolism to levels at or below baseline (Fig 1A, B). The Seahorse ATP Rate Assay means the contributions of glycolysis and mitochondrial respiration to chondrocyte ATP production (Fig 1C). In control chondrocytes, the use of glycolysis contributes to the majority of ATP produced (grey bars) approximately 1/5th from the TCA cycle (red bars). IL1β-activated chondrocytes display increase in glycolysis and decrease in mitochondrial contributions. These changes are reversed by co-treatment with 4MU and 2DG. As shown in Figs 2A, 2DG reversed the IL1β-induced increases accumulation of MMP13 protein in human OA chondrocytes by Western blotting analysis. Although IL-1β lost safranin O staining in human and bovine samples, co-incubation with 2DG blocked in the loss of proteoglycan (Fig 2B). pAMPK is associate with energy homeostasis in chondrocytes. IL-1β treatment decreased accumulation of phosphor AMPK. Co-treatment with 4-MU and 2DG resulted in a rescue of the pAMPK status (Figure 3A). Co treatment with AICAR, which is inducer of AMPK, also blocked in the loss of proteoglycan (Fig 3B).Conclusion:4-MU and 2DG have chondroprotective effect by changing metabolism and upregulate AMPK. We propose that 4MU and 2DG become useful when these endogenous responses are not enough to rescue cells from a pro-catabolic phenotype.References:[1]J. Biol. Chem. 291:12087, 2016; [2] J. Biol. Chem. 291:24105, 2016Disclosure of Interests:KENYA TERABE: None declared, Nobunori Takahashi Speakers bureau: AbbVie, Asahi Kasei, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eisai, Eli Lilly, Janssen, Mitsubishi Tanabe, Ono, Pfizer, Takeda, and UCB Japan, Ohashi Yoshifumi: None declared, Maeda Masataka: None declared, Warren Knudson: None declared, Cheryl Knudson: None declared, Toshihisa Kojima Grant/research support from: Chugai, Eli Lilly, Astellas, Abbvie, and Novartis, Consultant of: AbbVie, Speakers bureau: AbbVie, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eli Lilly, Janssen, Mitsubishi Tanabe, Pfizer, and Takeda, Naoki Ishiguro Grant/research support from: AbbVie, Asahi Kasei, Astellas, Chugai, Daiichi-Sankyo, Eisai, Kaken, Mitsubishi Tanabe, Otsuka, Pfizer, Takeda, and Zimmer Biomet, Consultant of: Ono, Speakers bureau: Astellas, Bristol-Myers Squibb, Daiichi-Sankyo, Eli Lilly, Pfizer, and Taisho Toyama
NF-κB controls energy homeostasis and metabolic adaptation by upregulating mitochondrial respiration