Oral Presentation Australian Diabetes Society and the Australian Diabetes Educators Association Annual Scientific Meeting 2014

Depletion of beta cell heparan sulfate (HS) in T2D-prone db/db mice results from diminished heparan sulfate proteoglycan (HSPG) core protein levels (#103)

Sarita Dhounchak 1 , Sarah Popp 1 , Harpreet Vohra 2 , Trevor Biden 3 , Ross Laybutt 3 , Christopher Parish 1 , Charmaine Simeonovic 1
  1. Department of Immunology, The John Curtin School of Medical Research, Canberra, ACT, Australia
  2. Microscopy and Cytometry Resource Facility, The John Curtin School of Medical Research, Canberra, ACT, Australia
  3. Diabetes and Obesity Research Program, The Garvan Institute of Medical Research, Sydney, NSW, Australia

Introduction: HS is a complex polysaccharide which is synthesised directly onto HSPG core proteins. High intracellular levels of HS are essential for islet beta cell survival (1). T2D-prone db/db mice are characterised by obesity, insulin resistance, endoplasmic reticulum (ER) stress, defective protein maturation and ultimately beta cell failure.
Objective: This study investigated whether ER stress constitutes the molecular basis for HS loss in islet beta cells of T2D-prone db/db mice.
Methods: The intra-islet expression of HSPG core proteins (collagen type XVIII (Col18), syndecan-1 (sdc1) and CD44) and HS was examined by immunohistochemistry and Alcian blue histochemistry, respectively, in pancreases of db/db female mice and control db/+ mice at 4, 5, 6, 9 and 12 weeks of age (n=3/time-point). The %HS+ve and %HSPG+ve islet tissue was determined by morphometry. ER stress was induced in MIN6 beta cells using thapsigargin (50nM) and confirmed by real-time RT-PCR analysis of Atf3, CHOP and BiP gene expression. Intracellular staining of HSPG core proteins and HS in MIN6 cells and apoptosis by 7AAD uptake was examined by flow cytometry.
Results: Intra-islet HS in db/db islets declined to 56% and 44% of control db/+ islets at 4 and 12 (p<0.001) weeks, respectively. Intra-islet levels of col18, CD44 and sdc1 declined to 67%, 29%(p<0.001) and 41%(p<0.05) of db/+ controls at 4 weeks, respectively, and were significantly reduced to 12%, 25% and 9% of controls by 12 weeks (p<0.001) at which time 67% of db/db mice were hyperglycaemic and 100% were obese. ER-stressed MIN6 cells showed decreased intracellular sdc1 (p<0.01) and CD44 (p<0.05) core proteins to 24%-42% of controls and a significant 1.4-fold increase in apoptosis (p<0.05).
Conclusions: Unlike T1D where beta cell HS is degraded by heparanase, our findings suggest that ER stress in T2D down-regulates HSPG core protein levels, leading to diminished HS synthesis and beta cell failure.

  1. Ziolkowski AF, Popp SK, Freeman C, Parish CR, Simeonovic CJ. J Clin Invest (2012) 122:132-141