The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets

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Abstract

The hexosamine biosynthesis pathway plays a role in the modification of cellular proteins via the provision of substrate for addition of O-linked N-acetylglucosamine (GlcNAc). The relative importance of the GlcNAc modification of proteins to insulin secretion from pancreatic β-cells has not been investigated and so remains unclear. In the present study, we show that inhibition of the hexosamine biosynthesis pathway decreases insulin secretion from mouse islets in response to a number of secretagogues, including glucose. This impairment in β-cell function could not be attributed to reduced islet insulin content, altered ATP levels, or cell death and was restored with the addition of N-acetylglucosamine, a substrate that enters the pathway below the point of inhibition. Western blot analysis revealed that decreased islet protein glycosylation paralleled the decrease in insulin secretion following inhibition of the pathway. In conclusion, the data suggest a role for the hexosamine biosynthesis pathway in regulating the secretion of insulin by altering protein glycosylation. This finding may have implications for the development of type 2 diabetes, as chronic increase in flux through the hexosamine biosynthesis pathway may lead to the deterioration of β-cell function via abnormal protein glycosylation.

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.

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