Article Text

PDF
SAT0039 Insulin-Induced Cartilage Degradation in Osteoarthritis is Associated to Defective Autophagy
  1. B. Carames1,
  2. M. Ribeiro1,2,
  3. P. Lopez de Figueroa1,
  4. A.F. Mendes2,3,
  5. F.J. Blanco1
  1. 1Rheumatology Division. Cartilage Biology Group, Biomedical Research Institute of A Coruña (INIBIC), A coruña, Spain
  2. 2Centre for Neuroscience and Cell Biology
  3. 3Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

Abstract

Background Autophagy, a key cellular quality control mechanism, is defective in Osteoarthritis (OA) and Type 2 Diabetes (T2D) (1,2). T2D has been proposed as a risk factor for OA. Although epidemiological studies suggest a strong association between these diseases (3), how T2D may have an effect on the deterioration of articular cartilage is still unknown.

Objectives The objective of this study is to understand the role of autophagy in the articular cartilage function under diabetic conditions.

Methods Human chondrocyte cell line (TC28a2) and primary human chondrocytes (HC) were cultivated in DMEM high glucose (25 mM) and treated with Insulin (10, 100, 500 nM) for 2, 6 and 24 hours. Activity of LC3-II, Akt and rpS6 was evaluated by Western blotting (WB). To investigate whether autophagy activation protects from diabetic conditions, autophagy was induce by Rapamycin (10 μM). Human cartilage explants were cultivated in DMEM 25mM glucose and insulin (100, 500, 1000nM) for 24 hours to evaluate histopathological changes. MMP-13 and IL-1β expression was determined by immunohistochemistry and WB, respectively. Expression of LC3 and p-rpS6 was determined by WB in human chondrocytes from Non Diabetic-OA and Diabetic-OA patients.

Results In the presence of high glucose and increased doses of insulin autophagy was decreased in a dose dependent-manner in human chondrocytes, as indicated by LC3II expression, the main marker of autophagy activation (TC28-a2; p<0.05 at 6 hours post-treatment; HC; p<0.01 at 24 hours post-treatment). To investigate the mechanism by which autophagy is reduced by insulin, Akt and rpS6 phosphorylation was analyzed. We observed a significant increase in p-AKT and p-rpS6 activity, suggesting that insulin effect is mediated by AKT/mTOR pathway (TC28-a2 p<0.05 at 6 hours; HC; p<0.01 at 2 hours). Autophagy activation by Rapamycin reversed insulin effects on LC3 and p-rbS6 expression (Tc28a2 and HC:p<0.05), indicating that autophagy induction prevents insulin-mediated autophagy signaling downregulation. To evaluate the impact of insulin-mediated autophagy regulation in the context of articular cartilage biology, cartilage explants were treated with insulin (100, 500 and 1000 nM) for 24 hours. Histological analysis indicated a loss of proteoglycans and increased MMP-13 and IL-1β expression (p<0.01) after insulin treatment. Remarkably, chondrocytes from OA-diabetic patients showed decreased LC3 and increased p-rpS6 expression compared to Non-Diabetic OA patients.

Conclusions Our findings demonstrate that diabetic conditions decrease autophagy by an AKT/mTOR dependent mechanism. Pharmacological activation of autophagy might protect against T2D in human chondrocytes. Our data also indicate that chondrocytes from OA-diabetic patients exhibit a deficient autophagy. Taking together, these results suggest that impaired autophagy might be one of the mechanisms by which T2D diabetes accelerates cartilage degradation.

References

  1. Caramés, B., et al., Arthritis Rheum, 2010. 62(3): p. 791-801

  2. Murrow, L., et al. Annu Rev Pathol, 2013. 8: p. 105-37.

  3. Berenbaum, F., Ann Rheum Dis, 2011. 70(8): p. 1354-6.

Acknowledgements This study was supported by Instituto de Salud Carlos III- Ministerio de Economía y Competitividad, Spain-CP11/00095. M.R is supported by FCT (SFRH/BD/78188/2011).

Disclosure of Interest None declared

Statistics from Altmetric.com

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.