Decreased expression of t-SNARE, syntaxin 1, and SNAP-25 in pancreatic beta-cells is involved in impaired insulin secretion from diabetic GK rat islets: restoration of decreased t-SNARE proteins improves impaired insulin secretion.

Abstract

The physiological role of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in insulin exocytosis has been reported in pancreatic beta-cells. To determine whether the beta-cells of GK rats, a nonobese rodent model of type 2 diabetes, exhibit abnormalities in their SNARE proteins, we studied the expression and function of target (t)-SNAREs, syntaxin 1A, and synaptosomal-associated protein of 25 kDa (SNAP-25) in GK rat islets. Although insulin release and insulin content of islets isolated from 12-week-old GK rats were reduced, the proinsulin biosynthetic rate was about twofold higher than that in control rat islets, and no change in the preproinsulin mRNA level was observed. Pulse-chase experiments suggested the increased degradation of insulin in GK rat islets. Immunoblot analysis revealed that protein levels of syntaxin 1A and SNAP-25 in GK rat islets decreased to approximately 60% of the levels in control rat islets. We then examined whether the restoration of the decreased expression of t-SNAREs to the normal level in GK rat islets affected insulin secretion. Restoration was achieved by the overexpression of syntaxin 1A and SNAP-25 via the recombinant adenovirus-mediated gene transduction system, which recovered levels of these proteins to almost control levels. Glucose-stimulated insulin release from AdexlCA syntaxin 1A and Adex1CA SNAP-25-infected GK rat islets increased up to approximately 135 and 200%, respectively, of those from uninfected GK rat islets, although no difference in basal (2.2 mmol/l glucose) insulin release was evident between them. We conclude that decreased expression of t-SNAREs in GK rat islets is in part the defect responsible for impaired insulin secretion.

Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analysed by total internal reflection fluorescence microscopy

“…It is of note that the recovery of the decreased number of SNAP-25 clusters in GK beta cells to normal levels by adenovirus treatment or insulin treatment could restore the number of docked insulin granules, which caused an increased number of fusion events from previously docked granules. We previously reported that restoration of decreased t-SNARE levels improved the impaired insulin secretion in GK islets [10]. At that time, however, we could not know how t-SNARE restoration affects the insulin exocytosis.…”

“…For the normalisation of hyperglycaemia, human insulin (Humalin N; Lilly, Indianapolis, Ala., USA) was injected subcutaneously at 08.00 (2 U/rat) and 20.00 (4 U/rat) hours daily for 2 weeks. Pancreatic islets of Langerhans were isolated by collagenase digestion [10], with some modification. Isolated islets were dissociated into single cells by incubation in Ca 2+ -free KRB containing 1 mmol/l EGTA, and cultured on fibronectin-coated (KOKEN, Tokyo, Japan) high-refractive-index glass (Olympus, Tokyo, Japan) in RPMI 1640 medium supplemented with 10% FBS (Invitrogen Gibco BRL, Carlsbad, Calif., USA), 200 U/ml penicillin, and 200 µg/ml streptomycin at 37°C in an atmosphere of 5% CO 2 .…”

“…The construction of expression vectors and recombinant adenovirus encoding insulin-GFP and SNAP-25 has been previously described [10,21]. To label Fig.…”

“…Islets prepared from insulin-treated and/or insulin-untreated GK or normal Wistar rats were disrupted by sonication, boiled in SDS sample buffer with 10 mmol/l dithiothreitol, subjected to SDS-PAGE, and then transferred onto nitrocellulose filters. Immunoblotting procedures were performed as described previously [10] using anti-HPC-l-antibodies, anti-SNAP-25 antibodies, and anti-actin monoclonal antibody (Chemicon International, Temecula, Calif., USA). The protein bands were scanned and analysed by NIH Image.…”

“…These components, including N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment protein (SNAP), and membrane-associated SNAP receptors (SNAREs) such as syntaxin-1 and SNAP-25, are also expressed in pancreatic beta cells [3,4,5], which play an important role in insulin exocytosis [6,7,8,9]. We [10] and others [11,12] have demonstrated that the expression of SNARE proteins is decreased in diabetic animal models. It is conceivable that the expression of SNARE proteins is closely related to the docking/ fusion of insulin exocytosis; however, there has been no apparent evidence to show the direct interaction between target membrane (t-)SNAREs and docking/ fusion of insulin granules.…”