Abstracts: British Society for Matrix Biology The Grey Area – Age and the Extracellular Matrix

摘要

SIRT1, an enzyme vital to cartilage homeostasis, is regulated by osmolarity and glucose levels >H. K. Heywood*, P. W Caton^† and D. A Lee* *The Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary university of London, Mile End Road, London, E1 4NS, UK, ^†Diabetes & Nutritional Sciences Division King's College London, Fourth Floor Franklin‐Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK Introduction Sirtuins (SIRT1‐7) are a family of NAD⁺‐dependent lysine de‐acetylases which play a protective role in numerous age‐associated pathologies. Age is a major risk factor for osteoarthritis, a degenerative disease of articular cartilage which has recently been linked to SIRT1 deficiency. The physiological mechanisms regulating cartilage sirtuin expression remain to be elucidated, but would enhance our understanding of risk factors for osteoarthritis and may reveal novel targets for therapeutic intervention. Chondrocytes experience scarce glucose and elevated osmolality relative to most tissues. Glucose becomes depleted to levels as low as 1 mM in the deep zone, whilst osmolarity increases from 350–480 mOsm, according to the matrix fixed charge density distribution. Therefore, we ask whether these biochemical parameters regulate chondrocyte sirtuin expression. Materials and Methods Freshly isolated bovine chondrocytes were cultured as a suspension culture with different media compositions: (A) Osmotically‐balanced media with 1 mM or 10 mM glucose or (B) 10 mM glucose media with increasing osmolarity, achieved by adding mannitol. After specified times, samples were processed for qPCR and Western blot to ascertain SIRT1 expression. As sirtuin activity is coupled to bioenergetic status, NAD/NADH ratio was monitored fluorometrically (Ex 355, Em 460 nm), and mitochondria activated by 0.5 mM dichloroacetate as control. Results Chondrocyte SIRT1 mRNA expression was upregulated 2‐fold by physiological glucose level of 1 mM compared to 10 mM glucose. SIRT1 mRNA increased exponentially with osmolarity, reaching a 3–5‐fold increase at 500 mOsm in 4 cell donors. Western blot confirmed this correlated with significantly increased protein expression, peaking after 10 h exposure (P < 0.05). Elevated osmolarity and dichloroacetate treatment was associated with increased NADH fluorescence, a known SIRT1 modulator. Discussion SIRT1 expression is highly responsive to osmolarity, increasing over the physiological range from 350–500 mOsm. Thus conditions which reduce tissue osmolarity, such as matrix degradation, could negatively influence SIRT1 expression. By contrast, high glucose environments, such as those found in diabetes and typical culture media, reduced SIRT1 expression. By providing context to the in vivo niche, these findings may enhance current understanding of risk factors for osteoarthritis. Additionally, optimising glucose and osmolarity may drive up sirtuin expression for cell‐based cartilage repair therapies.