Beta-cell function abnormalities in islets from an adult subject with nesidioblastosis and autoantibodies against the islet cells.

Human islets from an adult subject with nesidioblastosis were isolated and used to perform in vitro studies. Isolated nesidioblastotic islets showed an increased basal rate of insulin secretion (nesidioblastotic islets, 81.3 +/- 6.4 vs. control islets, from cadaveric organ normal donors, 10.2 +/- 0.9 microU/islet/90 min) without any further release with increasing glucose concentration. In addition, islets isolated from the pancreas with nesidioblastosis contained more insulin than control islets, 1,547.0 +/- 128.7 vs. 935.0 +/- 51.7 microU/islet, and the levels of insulin mRNA were also higher than those measured in controls. Interestingly, the serum of the subject with nesidioblastosis contained autoantibodies that stained brightly and selectively a population of islet cells whose distribution coincided with that expected of alpha cells. In summary, pancreatic islets in nesidioblastosis display beta-cell functional abnormalities. Moreover, the finding of antibodies against islet cells is a common feature in the serum of nesidioblastotic subjects; nevertheless, their pathological significance warrants further investigation.

Functional properties of isolated human pancreatic islets beneficial effects of culture and exposure to high glucose concentrations.

In order to assess the functional properties of human pancreatic islets we have evaluated insulin secretion, glucose metabolism and insulin mRNA synthesis. Particular attention was given to evaluate how culture and exposure to high glucose concentrations "in-vitro" influence the subsequent function of these cells. Islets were cultured for 1 and 7 days at 5.5 or 16.7 mM glucose concentrations. Insulin response to glucose, glucose+forskolin and leucine+glutamine was evaluated in freshly isolated and cultured islets. Catabolism of D-glucose was studied measuring D-(6-14C) glucose oxidation and D-(5-3H) glucose utilization. Northern blot analysis was performed to measure insulin mRNA levels. In freshly isolated islets there a low insulin response to all stimulus, but especially to nutrient secretagogues. Total glucose utilization was also lower than expected, with preferential disturbances in mitochondrial oxidative events. Independently of glucose concentration, culture improved insulin release, glucose catabolism and especially glucose oxidation, and insulin mRNA synthesis. After 7-days culture, high glucose concentration in the medium exerted beneficial effects on the overall functional activities of human pancreatic islets. Our study, 1) indicates that functional properties, initially impaired in freshly isolated human islets, improve after culture and become comparable to those previously proposed for rodent islets; and 2) argues against high glucose concentrations producing a deleterious effect on human beta-cell function. Rather they point to the concept that high glucose levels are beneficial for human islets function after long-term exposure in-vitro.

Deleterious effect of dithizone-DMSO staining on insulin secretion in rat and human pancreatic islets.

Dithizone (DTZ) is a selective stain for pancreatic islets which facilitates their identification, being of special interest in human islet isolation assessment. Nevertheless, there are few studies concerning its potential toxic effects on islet function. In our study, we have evaluated the effects of DTZ (dissolved in dimethyl sulfoxide [DMSO] 1% w/v) at three different concentrations (2, 10, and 100 micrograms/ml) on insulin response to glucose in human and rat islets. Likewise, we studied the effect of incubation time, in the presence of DTZ at the above-mentioned concentrations, on insulin release. Only when DTZ was employed at low concentrations and for a short period of incubation (10 min) was there no impairment of pancreatic islet function. Moreover, even at this low concentration, DTZ became deleterious for islet function when the incubation period with the dye was prolonged for 30 min. Culture (24 h) of previously stained islets produced a partial recovery of insulin response. In conclusion, our findings indicate (a) DTZ should not be employed to collect islets for functional studies because of its deleterious effect on beta-cell function, (b) DTZ's deleterious effects on beta-cell function should be considered if this dye is used to purify islets by fluorescence-activated cell sorting for transplantation.

Regulation of islet amyloid polypeptide in human pancreatic islets.

This study investigated the effect of glucose on islet amyloid polypeptide secretion, content, and mRNA synthesis of human pancreatic islets. The release of islet amyloid polypeptide from fresh isolated islets in response to glucose was parallel to that of insulin. The islet amyloid polypeptide-to-insulin molar ratios in response to 5.5 and 16.7 mM glucose were 1:16 and 1:15 respectively. Islets were cultured for 1 and 7 days at two different glucose concentrations (5.5 and 16.7 mM). The islet amyloid polypeptide response to the 1-day culture was similar to that of the fresh islets; however, after the 7-day culture the islet amyloid polypeptide and insulin secretory responses to glucose were dissociated. The insulin response of islets to a high-glucose stimulus was significantly (P < 0.001) increased, whereas the islet amyloid polypeptide response of islets to the same stimulus was blunted. The IAPP content was greater than insulin content in a molar ratio (1:50 to 1:30) after long exposure of islets to concentrations of high glucose even though the increase was significant for both peptides (P < 0.005). Northern blot analysis of each cultured condition showed an increase of both mRNA IAPP and insulin signals after exposure of islets at 16.7 mM glucose, the maximum mRNA expression being after long exposure to high-glucose concentrations. Quantification of both signals by densitometry showed a greater increase for islet amyloid polypeptide than for insulin. These findings suggest that IAPP can be accumulated in beta-cells after long exposure of human islets to high-glucose concentrations, because glucose increases IAPP synthesis but not secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of glutamic acid decarboxylase (GAD) in the alpha, beta and delta cells of normal and diabetic pancreas: implications for the pathogenesis of type I diabetes.

One of the paradoxes of insulin-dependent diabetes mellitus is that the destruction of the pancreatic islets' endocrine cells is restricted to the insulin-producing beta cells, whereas the main autoantibodies, islet cell antibodies (ICA), are directed against all endocrine islet cells. GAD has recently been proposed as the main target of the humoral and cellular autoimmune attack to the islets, and since in rat pancreas this enzyme was expressed only in the beta cells, this provided an explanation for the cell specificity of the destructive process. The finding of GAD-positive cells in the islets of two diabetic patients, one of whom had completely lost the beta cells, led us to study in detail the distribution of GAD in normal human islet cells using a panel of GAD antisera and the double indirect immunofluorescence technique on cryostat sections, monolayer cultures and cytosmears. The results showed that GAD is present not only in the cytoplasm of beta cells but also in 69% of the alpha and 27% of the delta cells. GAD was not present, however, on the surface of the islet cells. These results suggest that the cellular distribution of GAD can not by itself explain the selectivity of beta cell destruction in insulin-dependent diabetes mellitus.

Human pancreatic islet function at the onset of type 1 (insulin-dependent) diabetes mellitus.

Viable human pancreatic islets isolated from a recent-onset Type 1 (insulin-dependent) diabetic patient were used to perform in vitro studies. Pre-proinsulin mRNA and insulin content, as well as insulin response were analysed. Insulin response to glucose and forskolin was completely absent in diabetic islets, as compared to control islets. Insulin content was reduced to only one-third of control values (395.0 +/- 3.5 vs 989.0 +/- 46.3 microU/islet) and 20.7 +/- 3.9% of islets from the diabetic pancreas contained insulin-positive cells in immunofluorescence studies. Northern blot analysis revealed a severe reduction in the content of pre-proinsulin mRNA in diabetic pancreatic tissue. Our results indicate that although markedly decreased, beta cells in human pancreatic islets at the onset of Type 1 diabetes are still present. Nevertheless, pancreatic islet function is disproportionately impaired with a complete absence of an insulin response.

Effects of single dose irradiation on pancreatic beta-cell function.

We have irradiated abdominal cavity of 23 rats with 10 Gy irradiation-induced hypoglycemia on the fourth day after intervention. Islets collected at this time showed an impaired insulin secretion without affecting insulin content. This impairment persisted after one month follow-up with reduced number of beta-cells in morphological examination.