The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets☆
Section snippets
Isolation and culture of pancreatic islets
Islets were isolated from the pancreas of 7- to 8-week-old male DBA/2 mice (Walter and Eliza Hall Institute Animal Research Facility; Kew, Victoria, Australia) by collagenase digestion using a modification of the methods of Lacy and Kostianovsky [10] and Gotoh et al. [11]. Briefly, pancreata were digested by intraductal injection of 3 mL Collagenase P (0.5 mg/mL) in RPMI 1640 (with l-glutamine) containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 11.1 mM glucose. Following incubation at 37 °C
Results
Incubation of mouse islets with azaserine for 24 h resulted in a marked reduction in GSIS (Fig. 1A). Insulin secretion in response to a cocktail of secretagogues was also decreased following islet treatment with azaserine (Fig. 1B). The coincubation of azaserine-treated islets with GlcNAc, however, resulted in a reversal of the inhibition of secretion seen with azaserine alone, while incubation with GlcNAc alone, did not affect insulin secretion (Fig. 2A). Taken together, these data suggest that
Discussion
The HBP has been described as a cellular sensor of energy availability [16], [17], [18], [19], capable of modifying many proteins via O-linked glycosylation [5], [20], [21], [22]. While this phenomenon has been extensively investigated in a number of systems, including the pancreatic β-cell, there have been no studies to suggest a link between protein glycosylation and insulin secretion. In this study, we provide evidence that the HBP is associated with the secretion of insulin via
Acknowledgements
We thank Dr. David Thorburn for his kind assistance in measuring islet ATP levels and Paul Brazzoduro for his technical assistance.
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The work was supported by a grant from the National Health and Medical Research Council of Australia (Grant 114163 to S.A., J.P.) and a Grant in Aid from the Diabetes Australia Research Trust Fund.