Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

AIMS: Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. METHODS: The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. RESULTS: M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. CONCLUSIONS: Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes.

Prolonged culture of human islets induces ER stress.

Type 2 diabetes (T2D) is characterized by islet dysfunction and beta-cell deficiency caused by apoptosis. One mechanism underlying induction of beta-cell apoptosis is stress in the endoplasmic reticulum (ER). Isolated human islets are a frequently used model to examine islet pathophysiology in T2D. Therefore it is important to establish how function and beta-cell turnover of human islets change in culture. Islets from four organ donors were cultured over four weeks. At 0, 1, 2, 3 and 4 weeks aliquots of islets were used for analysis of a) islet-cell turnover (replication by Ki-67 and apoptosis by TUNEL staining), b) the ER stress level (CHOP and phospho-eIF2alpha staining), c) fractional beta-cell content (insulin staining) and d) islet function (2 h static incubation). Culture duration positively correlated to replication (p=0.03) and negatively correlated to apoptosis (p=0.003). In comparison to islets in situ islet cell turnover is accelerated (>10-fold). The ER stress level was stable during the first three weeks, but showed a sharp increase (p<0.05) at four weeks. The fractional beta-cell content increased from 29+/-2% to 41+/-2% (p=0.0004). Islet function improved (p<0.0001). In conclusion, isolated human islets may be used for in vitro experiments for up to three weeks. During this time islet function and islet-cell turnover are stable. If islet culture is extended beyond three weeks ER stress may impair islet viability. Studies analyzing the pathophysiology of human T2D at the level of the endocrine pancreas need to confirm results obtained with isolated human islets by analysis of primary human pancreatic tissue.