Background: The glycosaminoglycan HS is attached to
the core proteins of HS proteoglycans (HSPGs). HS is strongly expressed in
islets in situ and plays a critical role in beta cell survival.
Objective: This study examined the potential mechanism for the depletion of intra-islet HS during islet isolation.
Methods: HSPGs (collagen type XVIII, syndecan-1) and HS in B6 and B6.Hpse-KO (lacking the HS-degrading enzyme heparanase (Hpse)) islets pre- and post-isolation were examined by immunohistochemistry and Alcian blue histochemistry, respectively. Donor mice were treated with hydroethidine (80 nmol/kg i.v.) to identify oxidation products in extracts of isolated islets by liquid chromatography/ mass spectrometry. Isolated beta cells ± culture with 50 μg/ml heparin were examined for intracellular oxidants e.g., reactive oxygen species, using dihydrodichlorofluorescein diacetate (H2DCFDA) and flow cytometry. Additional donor mice were treated with the antioxidant butylated hydroxyanisole (BHA, 120 mg/kg i.p.) and the antioxidant dimethylthiourea (DMTU, 50 mM) was included in the isolation process.
Results: Islets pre- and post-isolation showed intense expression of collagen type XVIII and syndecan-1 core proteins. In contrast, B6 and B6.Hpse-KO isolated islets showed 50%-60% loss of intra-islet HS. Superoxide and non-specific oxidation products were identified in islet extracts. DCF-derived fluorescence (identifying oxidants) was detected in control isolated beta cells but not following HS replacement with heparin. Antioxidant treatment significantly improved the HS content of isolated islets.
Conclusions: These findings suggest that loss of islet HS during islet isolation is due to oxidant-mediated degradation and not to Hpse or loss of HSPG core proteins. HS replacement in beta cells prevented detection of DCF-derived fluorescence, suggesting that in situ beta cell HS may function as an antioxidant. Our findings indicate that during islet isolation, excessive levels of oxidants are generated, damaging beta cell HS. We propose that HS deficiency leads to poor islet viability and islet loss after transplantation.