Poster Presentation Australian Diabetes Society and the Australian Diabetes Educators Association Annual Scientific Meeting 2016

Heparan sulfate (HS) loss in Type 2 diabetes (T2D)-prone db/db beta cells increases susceptibility to oxidative stress (#210)

Sarita Dhounchak 1 , Sarah Popp 1 , Harpreet Vohra 2 , Trevor Biden 3 , Ross Laybutt 3 , Christopher Parish 4 , Charmaine Simeonovic 1
  1. Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
  2. Imaging and Cytometry Facility, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
  3. Diabetes and Obesity Research Program, The Garvan Institute of Medical Research, Sydney, NSW, Australia
  4. Cancer biology and Therapeutics, The John Curtin school of Medical Research, The Australian National University, canberra, ACT, Australia

Introduction: HS is a polysaccharide which is synthesised directly onto core proteins to form heparan sulfate proteoglycans (HSPGs). HS is localised at unusually high intracellular levels in beta cells and is critical for beta cell survival; HS but not HSPG core proteins is lost during islet isolation. We have previously demonstrated progressive loss of both intra-islet HS and HSPG core proteins in db/db mice during T2D development, suggesting a role for endoplasmic reticulum (ER) stress in reducing HS synthesis. This study examined whether loss of HS in db/db beta cells contributes to oxidative stress in T2D.

Methods:  The non-fasting blood glucose levels of female wildtype (wt) and db/db mice at 3-20 weeks of age were measured using a glucometer and the mice were sacrificed (n=3/time-point). Islet HS in donor pancreata was assessed by Alcian blue histochemistry and morphometry. Using additional 5-7 weeks old mice, islets were isolated and dispersed into single cells. The beta cells were cultured for 2 days with medium alone (controls) or with heparin (50 μg/ml) to restore intracellular HS. Cell viability on day 2 was measured with/without acute exposure to 30% hydrogen peroxide (H2O2) by Sytox Green uptake (detecting dying/dead cells) and flow cytometry.

Results: Female db/db mice at 6 weeks were hyperglycaemic (18.3±3.2 mmol/L) and intra-islet HS was significantly reduced to 55.6% of wt islets. H2O2 -induced death/damage of heparin-treated wt beta cells was reduced to 14% of untreated controls (n=9 expts). Heparin-treated beta cells from normoglycaemic and hyperglycaemic db/db donors and exposed to H2O2 showed a significant decline in cell death/damage to 39.7%, and 48.7% of controls, respectively (n= 3-4 expts).

Conclusions: HS replacement protects T2D beta cells from acute oxidative damage in vitro. Our findings suggest that ER stress-induced loss of islet HS contributes to oxidative stress during T2D development in db/db mice.