Elevated SNAP-25 is associated with fatty acid-induced impairment of mouse islet function

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Abstract

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in insulin secretion following chronic exposure to non-esterified fatty acids (NEFAs) has not been extensively investigated. Here, we show that synaptosome-associated protein of 25 kDa (SNAP-25) levels were predominantly elevated in the soluble fraction of mouse islets exposed to palmitate. This coincided with an impairment of insulin secretion to glucose and non-glucose secretagogues, consistent with a defect at a distal regulatory step in exocytosis. Removal of palmitate from the media restored both SNAP-25 protein levels and insulin secretion to control levels. We conclude that increased expression of SNAP-25 is associated with NEFA-induced impairment of insulin secretion in mouse islets.

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Materials and methods

Isolation and culture of pancreatic islets. Islets were isolated from the pancreata of 7–8-week-old male C57BL/6 mice (Walter and Eliza Hall Institute Animal Research Facility; Kew, Victoria, Australia) by collagenase digestion using methods previously described [16] and approved by the Royal Melbourne Hospital Animal Research Ethics Committee. The concentration of glucose in the culture media was 11.1 mmol/L.

Evaluation of insulin secretion and content. Following overnight culture, batches of

Results

Incubation of mouse islets with NEFA (1 mmol/L palmitate) for 48 h resulted in a marked reduction in glucose-stimulated insulin secretion (GSIS) (Fig. 1A). Insulin secretion was also reduced in response to a cocktail of secretagogues following chronic incubation with NEFA (Fig. 1B). The cocktail, containing IBMX, arginine, and carbachol, stimulates Ca2+ entry, raises cAMP levels, and activates protein kinase C, thereby stimulating insulin exocytosis through other pathways in addition to glucose

Discussion

In this study, we demonstrate that incubation of β-cells with NEFA for 48 h causes an increase in the t-SNARE SNAP-25, and that this increase is associated with an impairment in insulin secretion. The impairment in insulin secretion was present to all tested insulin secretagogues including a cocktail containing IBMX, arginine, and carbachol and to glutamate and KCl, consistent with a defect occurring in the final common pathway, namely the movement and fusion of insulin-containing granules to

Acknowledgements

This 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. We thank Dr. David Thorburn for his kind assistance in measuring islet ATP levels and Paul Brazzoduro and Joanne McKie for technical assistance.

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    Abbreviations: GSIS, glucose-stimulated insulin secretion; NEFA, non-esterified fatty acids; SNAP-25, synaptosome-associated protein of 25kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.

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