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Obesity and development of the metabolic syndrome is related to an increased parasympathetic tone and hyperinsulinemia. We have now studied the effects of age and metabolic status on glucose-induced insulin release stimulated by the neuropeptides vasoactive intestinal polypeptide (VIP; 10 nM) and pituitary adenylate cyclase activating polypeptide (PACAP; 10 nM), that are constituents of the parasympathetic nerves in the islets, and the cholinergic agonists acetylcholine (ACh; 10 microM) and carbachol (10 microM), in isolated islets from female obese ob/ob mice and lean mice. Both VIP and PACAP enhanced insulin secretion in islets from 4-week-old hyperglycemic ob/ob mice. VIP did not increase 11.1 mM glucose-induced insulin release in islets from 4-week-old lean normoglycemic mice and neither did PACAP in the absence of bicarbonate. The neuropeptides increased insulin release in islets from 9 to 10-month-old mice but VIP and PACAP had no effect in islets from very old mice. ACh had no effect in islets from 9 to 10-months and older ob/ob mice in the absence of bicarbonate. The combination of VIP and cholinergic agonists had an additive effect in islets from ob/ob mice, and PACAP combined with carbachol potentiated insulin release in islets from 4-week-old lean mice. VIP increased early phase insulin release in perifused islets from young mice. A higher concentration of theophylline was needed to potentiate glucose-induced insulin release in islets from young lean mice than in islets from old lean mice and ob/ob mice. The present results demonstrate age-related dynamics in the effects of neuropeptides affecting cAMP in pancreatic islets. We suggest that VIP and PACAP contribute to the developing metabolic syndrome in ob/ob mice by aggravating hyperinsulinemia.

The underlying mechanistic causes of immune cell infiltration in the islets of Langerhans and beta cell failure in the non-obese diabetic (NOD) mouse is still to be completely revealed. Substance P (SP) is a substance known to have pro-inflammatory, endocrine, neuromodulatory and trophic effects, and its preferred receptor, the neurokinin receptor 1 (NK-1 R), is reported to be involved in extravasation of granulocytes and in inflammation and tissue derangement. Therefore, we have investigated the expression of NK-1 R during development of insulitis in the NOD mouse. We show that the magnitude of immunoreactivity scoring NK-1 R expression in the islets was increased in the 12-week-old NOD mouse. Expression of NK-1 R co-localized with expression of glucagon. In line with this expression pattern, we did not detect any effect of SP on glucose-induced insulin release. NK-1 R expression was particularly observed in islet cells in association with the clusters of immune cells. Expression of NK-1 R was also demonstrated in a fraction of the infiltrating B and T lymphocytes, as well as on infiltrating macrophages and dendritic cells. The observations show that the level of NK-1 R expression is increased in 12-week-old NOD mice, being correlated with the occurrence of islet mononuclear infiltration. Our data suggest that SP may act as a chemoattractant, contributing to the pathogenic mononuclear infiltration process in the NOD mouse. On the whole, the observations suggest that SP and the NK-1 R to certain extents are involved in the changes that occur during the development of insulitis in the NOD mouse.

The innervation of the islets of Langerhans may constitute a first target for the autoimmunity that develops in type 1 diabetes. Here, we report the occurrence of a decrease in general innervation within the islets in the nonobese diabetic (NOD) mouse, and the establishment of strands of Schwann cells, as detected via p75 and S-100 immunoreactivity (IR), and varicose nerve fibers expressing tyrosine kinase A (TrkA) in association with the immune cells. The findings suggest that there are marked attempts for neurotrophins to promote nerve ingrowth and survival for islet tissue and that remodeling of innervation occurs in the continuation of the insulitis process preceding the onset of type 1 diabetes.

OBJECTIVES: Decreased beta-cell function is often observed in older individuals and may predispose to the development of type 2 diabetes. We have studied the age-related effects of M-receptor agonism on insulin release in islets isolated from female ob/ ob and lean mice. METHODS: Islets were challenged with 11.1 or 16.7 mmol/L glucose in media with HCO3/CO2 (KRBH) or without (KRH). RESULTS: Acetylcholine (ACh) (10 micromol/L) increased glucose-induced insulin release in islets from 4- to 5-week-old ob/ob mice both in KRBH and KRH. In islets from 9- to 13-month-old ob/ob mice, 10 micromol/L ACh and 10 micromol/L carbachol enhanced insulin release in KRBH but not in KRH. ACh increased insulin release in islets from 4- to 5-week-old and 16-month-old lean mice incubated in KRH but not in islets from 24-month-old lean mice. The Na/H exchange inhibitor dimethylamiloride (100 micromol/L) did not affect insulin release stimulated by M-receptor agonists. Carbachol did not enhance glucose-induced insulin secretion in islets from 9- to 10-month-old ob/ob mice in the presence of low extracellular Na concentration. ACh stimulated cytoplasmic Ca mobilization in islets from 9- to 10-month-old mice also when bicarbonate was omitted. The results suggest that cholinergic signal transduction involving extracellular bicarbonate and Na is reduced with age in mouse pancreatic islets. CONCLUSION: Chronic hyperglycemia may add to the age-related decrease in M-receptor-mediated insulin release by affecting the buffering capacity of the islets through mechanisms other than amiloride-sensitive proton exchange.

Obesity is often accompanied by hyperleptinemia, hyperinsulinemia, and an increased parasympathetic tone. Obese-hyperglycemic mice (Umeå ob/ob) have functional leptin receptors and a raised parasympathetic tone. We studied insulin release in islets isolated from 9-month-old severely obese ob/ob mice. Leptin (0.5-18 nM) did not affect insulin release together with 2.8-20 mM glucose. Leptin (18 microM) had no effect in the presence of low glucose (2.8-5.5 mM), but increased insulin secretion in islets challenged with 11.1 or 16.7 mM glucose. Leptin at 18 microM increased insulin secretion stimulated by the parasympathetic neurotransmitters acetylcholine (ACh; 10 microM) or vasoactive intestinal peptide (VIP; 10 nM), and by 5 mM theophylline or 2.5 microM forskolin. Overnight culture increased the effect of 18 microM leptin, but no effects were observed with 18 nM leptin. Pretreatment of islets with phorbol 12-myristate 13-acetate (PMA) did not suggest any involvement of protein kinase C. In summary, a high concentration of leptin stimulates insulin release in the presence of stimulatory concentrations of glucose alone and with parasympathetic neurotransmitters. Hyperleptinemia and increased parasympathetic stimulation may in part cause the hyperinsulinemia observed in obesity. This may aggravate insulin resistance and the abnormal metabolism in diabetes mellitus.

Information on the biocompatibility of glass ionomers and resin composites is sparse. To extend the scale of biological testing we evaluated the influence of those materials on insulin secretion at whole organ level in vitro. The effects on insulin secretion of three glass ionomers and two resin composites, aged for 1 week, were studied in isolated mouse islets of Langerhans at basal (5.5mM) and at stimulatory (11.1mM) D-glucose concentrations. In addition, viability of single mouse beta-cells was evaluated. The effect of glass ionomer specimens aged for 1 and 4 months on insulin secretion at 11.1mM D-glucose was also studied. None of the materials affected the viability of the beta-cells. At 5.5mM D-glucose none of the materials affected the insulin secretion. At 11.1mM D-glucose, the glass ionomers only decreased the secretion and glass ionomers aged for 1 month still decreased insulin release whereas after 4 months ageing only one of the glass ionomers affected the release. The result shows a dynamic effect on insulin release of the elements and/or compounds released from the specimens.

Objectives: To investigate the possibility of using a whole organ model for evaluating the biological effects of dental restoration materials in vitro.

Methods: The effect on insulin release of isolated Langerhans islets of a series of dental materials was examined. The islets were incubated for 1 h with extracts obtained from various dental materials and insulin was assayed radioimmunologically with crystalline mouse insulin. The results were analysed statistically using analysis of variance (ANOVA) and Scheffe's test at a significance level of p<0.05.

Results: One dental ceramic, Vita VMK 95, significantly ( p<0.01) decreased the insulin release, whereas another dental ceramic, Empress ( p<0.01), a partly re-cast high-noble gold alloy ( p<0.001), a modified high-noble gold alloy ( p<0,05), and unalloyed copper ( p<0.001) significantly increased the release of insulin.

Significance: The results demonstrate a new instance of examining the biological effects of dental restoration materials. The method provides information about the effect of different materials on organ level in vitro that can complement other in vitro tests.

Tyrosine hydroxylase (TH) is co-expressed with islet hormones in the fetal mouse pancreas. In the adult animal, the enzyme has been considered as a marker of ageing beta-cells. By immunohistochemical staining, we analyzed the expression of TH-like immunoreactivity (TH-LI), insulin-LI (INS-LI) and somatostatin-LI (SOM-LI) in adult mouse islets, in situ and after isolation and transplantation to kidney. In pancreas in situ, most TH-LI cells expressed INS-LI while less than 5% expressed SOM-LI. The total number of TH-LI cells/mm2 was significantly increased directly after isolation and in 0-day, 12-week and 52-week old grafts, but not in 3-day grafts. The proportion of TH-LI cells expressing SOM-LI increased after transplantation, amounting to about one-third by 52 weeks. As expressed per unit islet area, the frequencies of both TH/INS and TH/SOM cells increased significantly in the transplants. The results demonstrate that TH occurs in both beta-cells and D-cells of adult islets. In both cell types the enzyme appears to be responsive to the microenvironmental changes inherent in transplantation. This cellular phenotype plasticity might contribute to the altered insulin secretory dynamics in islet grafts.

Mouse islets cultured for 1 or 4 days with or without 10 nM vasoactive intestinal polypeptide (VIP) were stained for tyrosine hydroxylase (TH) and examined for insulin secretion during culture and in a postculture perifusion system. Exposure to exogenous VIP for 4 days increased the frequency of islet cells expressing TH-like immunoreactivity. Regardless of the culturing conditions, the islets exhibited significant insulin secretory responses to 16.7 mM glucose, the effect being potentiated by 10 nM VIP in the perifusion medium. The insulin-releasing action of glucose and the potentiating effect of VIP were less pronounced in islets cultured for 1 day with VIP than in islets cultured without this neuropeptide. The following conclusions are suggested: (a) VIP stimulates the expression of TH in mouse islet cells; (b) the latency of the VIP-induced TH is a postreceptor phenomenon; (c) islet cultures exposed to VIP represent a new instance of the association between increased functional demands on beta cells and enhanced expression of TH and a new instance of VIP having trophic effects.

The neuroinsular complex type 1 is composed of pancreatic endocrine islet cells and nerve cell bodies intrinsic to the islet. The details of the relation between nerve cells and between endocrine cells and nerve cells in the complex are unknown. Pancreata from newborn and 18-day-old mice were analysed by electron microscopy to establish the ultrastructural morphology of the neuroinsular complex. Immunohistochemical staining for protein gene-product 9.5 was also performed. The study showed that nerve cell bodies were closely associated to each other in the periphery of the islets with no connective tissue separating the cells. The nerve cells were closely associated to both beta-cells and alpha-cells. Direct intercellular contacts were observed between nerve cells and endocrine cells and between Schwann cells and endocrine cells. Varicose nerve endings were frequently observed in the neuroinsular complex. In the peripheral parts the varicosities were mostly being associated to the nerve cell bodies. The varicosities contained small clear or small clear and larger dense cored vesicles, suggesting cholinergic and peptidergic contents. The varicosities made specialized synaptic connections with adjacently located nerve cells. The study shows that the neuronal part of the neuroinsular complex is closely associated to the endocrine islet cells and that it is richly innervated, indicating an important regulatory function of the nerve cell component in the neuroinsular complex.