Co-ordinated Ca(2+)-signalling within pancreatic islets: does beta-cell entrainment require a secreted messenger.

Isolated beta-cells are heterogeneous in sensory, biosynthetic and secretory capabilities, however, to enable efficient and appropriate secretion, cellular activity within the intact islet is synchronised. Historically, the entrainment of activity to a common pattern has been attributed to gap-junction mediated cell-to-cell communication. Although clearly influential, the possibility remains for other local synchronising mechanisms. In this study, we have used small clusters of insulin-secreting MIN6 cells to assess how contact-dependent, homotypic interactions between cells influences nutrient- and non-nutrient- evoked Ca(2+)-handling and insulin secretion, and to determine whether a secreted product plays a role in the synchronisation of oscillatory activity. Tolbutamide evoked a concentration-dependent recruitment of active cells within cell clusters, both in terms of numbers of cells and amplitude of the evoked Ca(2+)-response. The change in [Ca(2+)](i) was characteristically oscillatory above a mean elevated plateau, and was in phase between member cells of an individual cluster. Even at maximal concentrations (100 microM) some cells within a cluster responded before their immediate neighbours. Subsequent oscillatory behaviour then became entrained between member cells within that cluster. Inhibiting exocytosis using the microtubule inhibitors vincristine and nocodazole, or the adrenergic agent noradrenaline, did not prevent tolbutamide-evoked oscillatory changes in [Ca(2+)](i) but did reduce the probability of obtaining synchronous activity within an individual cluster. Above a threshold glucose concentration, the number of cells secreting insulin increased, without a commensurate change in secretory efficiency. This recruitment of cells secreting insulin mirrored Ca(2+) data that showed a glucose-dependent increase in cell number, without a change in the mean basal-to-peak change in [Ca(2+)](i). Together these data suggest that synchronised behaviour in MIN6 cells is dependent, in part, on a secreted factor that acts in a local paracrine fashion to recruit heterogeneous individual cellular activity into an organised group response.

Apoptosis and disease progression in the spontaneously diabetic BB/S rat.

AIMS/HYPOTHESIS: Type I (insulin-dependent) diabetes mellitus is an autoimmune disease culminating in pancreatic beta-cell destruction. A role for apoptosis in this destruction has been suggested, although controversy exists over the identity of the apoptotic cells and the time of onset of apoptosis. This study investigates the extent and timing of islet cell apoptosis in vivo in the spontaneously diabetic BB/S rat. METHODS: Pancreatic biopsies were taken from 30 diabetes-prone and 6 diabetes-resistant BB/S rats matched for age. Animals were serially biopsied before, during and after development of diabetes and apoptotic cells analysed in serial sections. The diabetes-prone group included animals (n = 6) that had insulitis but did not develop diabetes. RESULTS: Apoptosis was not detected in any pancreatic sections from diabetes resistant animals at any age investigated or from any animal before 50 days of age. By 68 days, apoptosis was, however, detectable in both the diabetes-prone group and in the group that had insulitus but did not develop diabetes and this correlated with a decrease in pancreatic insulin staining and a development of insulitis. There was a further increase in apoptosis in the diabetes-prone group at 85 days, which coincided with the time of onset of diabetes (84 days). In addition, there was a sixfold increase in intra-islet apoptosis between 68 and 85 days in the diabetes-prone group and at 85 days intra-islet apoptosis was threefold higher in the diabetes-prone group than in the group that had insulitus but did not develop diabetes. At 107 days, apoptosis (total and intra-islet) was higher in the group that had insulitus but did not develop diabetes (OND-DP) than in either the diabetes resistant (DR) or diabetes-prone (DP) groups. CONCLUSION/INTERPRETATION: We have shown significant islet cell apoptosis in the pancreas of diabetes-prone BB/S rats, which coincides with the appearance of insulitis and the onset of diabetes. We have also detected differences in the levels of apoptosis between diabetic and non-diabetic animals and suggest that such differences could be an important determinant of disease progression in this animal model of Type I diabetes.

Glycation of insulin in the islets of Langerhans of normal and diabetic animals.

The glycation of immunoreactive insulin (IRI) was assessed in extracts of pancreas and islets from control and hyperglycemic animal models. Glycated and nonglycated IRI were separated by affinity chromatography and quantified by radioimmunoassay. Hydrocortisone-treated Wistar rats (80 mg x kg-1 x day-1 and obese hyperglycemic (ob/ob) mice showed significant increases in plasma glucose (P < 0.001), percentage glycated hemoglobin (P < 0.001), plasma IRI (P < 0.01), and total pancreatic IRI content (P < 0.01), compared with their respective controls. These diabetic groups also demonstrated significant increases (P < 0.05) in the percentage of glycated pancreatic IRI above the controls. Streptozotocin-treated (200 mg/kg) Swiss TO mice exhibited significant increases in plasma glucose (P < 0.001), glycated hemoglobin (P < 0.001), and percentage glycated pancreatic IRI (P < 0.05), compared with untreated controls, despite a marked decrease in both plasma IRI (P < 0.001) and total pancreatic IRI content (P < 0.001). Significant elevations in the percentage of glycated IRI were also observed in islets isolated from obese hyperglycemic (ob/ob) mice (P < 0.001), compared with islets from lean controls, and when lean mouse islets were cultured in hyperglycemic media for 24 h (33.3 vs. 5.6 mmol/l D-glucose; P < 0.001). The contribution of glycated plus nonglycated insulin and proinsulin to the total IRI was estimated in lean and obese mouse pancreatic extracts following high-performance liquid chromatography separation. The contribution of proinsulin to the total IRI was approximately 10%. Proinsulin represented 27-28% of the total glycated IRI. These data indicate that the glycation of insulin and proinsulin occurs within the pancreatic islets and is elevated in both insulin-deficient and insulin-resistant diabetic animal models.

Effects of okadaic acid on insulin secretion from rat islets of Langerhans.

We have studied the effects of the phosphatase inhibitor, okadaic acid, on insulin secretion and protein phosphorylation in intact and electrically permeabilized pancreatic islets. Okadaic acid inhibited glucose-induced insulin secretion from intact islets, although this effect was probably non-specific since similar effects were obtained using 1-nor-okadaone, a virtually inactive analogue of okadaic acid. In permeabilized islets, okadaic acid enhanced basal and cyclic-AMP-induced insulin secretion and protein phosphorylation. These results indicate that protein phosphatases may play a role in the regulation of insulin release.

Noradrenaline uptake inhibition increases melatonin secretion, a measure of noradrenergic neurotransmission, in depressed patients.

Eight patients with endogenous depression who had received no antidepressant treatment for the previous year were treated with the noradrenaline (NA) uptake inhibitor, desipramine (DMI). Pre-treatment plasma melatonin concentrations were normal. After one day of DMI treatment plasma melatonin concentrations were increased but the response was impaired compared to normal subjects. The acute effect of DMI on plasma melatonin persisted after six weeks of treatment. These findings question the hypothesis that beta adrenoceptors are supersensitive in depression and that antidepressant drugs act by down-regulating these receptors.