Impaired Gene and Protein Expression of Exocytotic Soluble N-Ethylmaleimide Attachment Protein Receptor Complex Proteins in Pancreatic Islets of Type 2 Diabetic Patients

Abstract

Exocytosis of insulin is dependent on the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins in the B-cells. We assessed insulin release as well as gene and protein expression of SNARE complex protein in isolated pancreatic islets of type 2 diabetic patients (n ‫؍‬ 4) and nondiabetic control subjects (n ‫؍‬ 4). In islets from the diabetic patients, insulin responses to 8.3 and 16.7 mmol/l glucose were markedly reduced compared with control islets (4.7 ؎ 0.3 and 8.4 ؎ 1.8 vs. 17.5 ؎ 0.1 and 24.3 ؎ 1.2 U ⅐ islet ؊1 ⅐ h ؊1 , respectively; P < 0.001). Western blot analysis revealed decreased amounts of islet SNARE complex and SNARE-modulating proteins in diabetes: syntaxin-1A (21 ؎ 5% of control levels), SNAP-25 (12 ؎ 4%), VAMP-2 (7 ؎ 4%), nSec1 (Munc 18; 34 ؎ 13%), Munc 13-1 (27 ؎ 4%), and synaptophysin (64 ؎ 7%). Microarray gene chip analysis, confirmed by quantitative PCR, showed that gene expression was decreased in diabetes islets: syntaxin-1A (27 ؎ 2% of control levels), SNAP-25 (31 ؎ 7%), VAMP-2 (18 ؎ 3%), nSec1 (27 ؎ 5%), synaptotagmin V (24 ؎ 2%), and synaptophysin (12 ؎ 2%). In conclusion, these data support the view that decreased islet RNA and protein expression of SNARE and SNAREmodulating proteins plays a role in impaired insulin secretion in type 2 diabetic patients. It remains unclear, however, to which extent this defect is primary or secondary to, e.g., glucotoxicity. Diabetes 55: [435][436][437][438][439][440] 2006 I n addition to insulin resistance, impaired insulin response to glucose appears to be a prerequisite for type 2 diabetes to develop (1,2). For practical and ethical reasons, most studies of molecular mechanisms behind this functional B-cell defect have been performed in animal models of the disease. One such rodent is the Goto-Kakizaki (GK) rat, which is nonobese with moderate hyperglycemia on a background of greatly impaired insulin secretion and mildly to moderately decreased insulin sensitivity (3-5). Several metabolic abnormalities with potential impact on B-cell secretory function have been demonstrated in the pancreatic islets of GK rats (4,6 -9). Exocytosis of insulin is critically dependent on the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins in the B-cells (10 -12). We have shown that expression of several of the SNARE complex proteins, such as VAMP-2, syntaxin-1A, SNAP-25, and nSec1 (Munc18), were decreased by ϳ40% in GK versus control rat islets (13,14), and similar impairments were also found in islets of the fa/fa Zucker rat (15). Moreover, in GK rat islets, dysregulation of SNARE complex protein expression was evident, because their compensatory increase by high-glucose exposure was abrogated (13).Here, we have investigated the role of the exocytotic SNARE complex proteins and several SNARE-modulating proteins in pancreatic islets obtained from patients with type 2 diabetes compared with nondiabetic control subjects. For that purpose, we studied the gene expression by Affymetrix microarray chip...

The endoplasmic reticulum in pancreatic beta cells of type 2 diabetes patients

“…Since a number of articles have recently reported that ER stress may be implicated in beta cell dysfunction and death in diabetes [10][11][12][13][14][15][16][19][20][21][22][23][24][25][26], in the present study we evaluated features of ER function/dysfunction in human beta cells from type 2 diabetic organ donors. First, we confirmed that diabetic beta cells have functional and survival defects, as previously shown by us and others [27,28,[35][36][37][38]. Specifically, decreased glucosestimulated insulin release and increased apoptosis were observed ( Figs.…”

“…1c). These findings corroborate previous observations [27,28,[35][36][37][38] and confirm that pancreatic beta cells in human type 2 diabetes have impaired function and increased demise.…”

Functional significance of repressor element 1 silencing transcription factor (REST) target genes in pancreatic beta cells

“…The integrity of the exocytotic machinery is critical for glucose homeostasis, as observed in the Goto-Kakisaki rat model of type 2 diabetes and in type 2 diabetic patients, in both of which the expression of SNARE proteins is decreased [38,39]. In view of the present knowledge on granule dynamics and distinct pools, it has been speculated that the proteins of the exocytotic machinery that are implicated in the rate-limiting priming of secretory granules are strong candidates for the triggering of beta cell dysfunctions associated with type 2 diabetes [40].…”

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

“…At resting or basal conditions, there were no differences between control and SYN-3 knockdown cells in total number of SGs, the number of SGs that were morphologically docked (<50 nm from PM [23]), or number of SGs close to the PM (within 120 nm). However, after stimulation with 11.1 mmol/l glucose plus 10 nmol/l GLP-1 to greatly potentiate the recruitment of SGs to the PM [8], the number of docked SGs (Fig.…”