Studies in vivo indicate that chronic hyperglycemia is deleterious for insulin secretion. We have used an improved islet monolayer culture system to study chronic modulations of B-cell function. Adult rat islets maintained over several weeks on extracellular matrix in the presence of 11.1 mM glucose responded to an acute stimulation with 16.7 mM glucose by a 5- to 8-fold increase in insulin secretion. When cultured in the presence of higher glucose concentrations, the response to an acute glucose stimulus diminished time and dose dependently. In islets desensitized by exposure to 33.3 mM glucose for 1 week, reduction of the glucose level to 11.1 mM reversed the desensitization within 2 weeks. This desensitization was not limited to the glucose stimulus; responses to other nutrient secretagogues, such as glyceraldehyde and alpha-ketoisocaproic acid, were also reduced. In contrast, responses of insulin secretion to nonnutrient stimulators (tolbutamide and quinine) and amplifiers (isobutylmethylxanthine and carbachol) showed no desensitization in islets exposed to 33.3 mM glucose. Desensitization similar to that caused by 33.3 mM glucose could be induced by 11.1 mM glucose together with 0.1 mM isobutylmethylxanthine. High glucose also caused a time-dependent loss in compact monolayer organization with disruption of cell contacts. Our studies suggest that 1) generation of the reduced insulin response may be related to the prolonged high insulin secretion rate; 2) expression of the functional change is specific to the nutrient stimulus-secretion coupling; and 3) modifications in intercellular contacts may be involved in B-cell desensitization.

The unpredictable behavior of uncontrolled type 1 diabetes often involves frequent swings in blood glucose levels that impact maintenance of a daily routine. An intensified insulin regimen is often unsuccessful, while other therapeutic options, such as amylin analog injections, use of continuous glucose sensors, and islet or pancreas transplantation are of limited clinical use. In efforts to provide patients with a more compliable treatment method, Oramed Pharmaceuticals tested the capacity of its oral insulin capsule (ORMD-0801, 8 mg insulin) in addressing this resistant clinical state. Eight Type I diabetes patients with uncontrolled diabetes (HbA1c: 7.5–10%) were monitored throughout the 15-day study period by means of a blind continuous glucose monitoring device. Baseline patient blood glucose behavior was monitored and recorded over a five-day pretreatment screening period. During the ensuing ten-day treatment phase, patients were asked to conduct themselves as usual and to self-administer an oral insulin capsule three times daily, just prior to meal intake. CGM data sufficient for pharmacodynamics analyses were obtained from 6 of the 8 subjects. Treatment with ORMD-0801 was associated with a significant 24.4% reduction in the frequencies of glucose readings >200 mg/dL (60.1±7.9% pretreatment vs. 45.4±4.9% during ORMD-0801 treatment; p = 0.023) and a significant mean 16.6% decrease in glucose area under the curve (AUC) (66055±5547 mg/dL/24 hours vs. 55060±3068 mg/dL/24 hours, p = 0.023), with a greater decrease during the early evening hours. In conclusion, ORMD-0801 oral insulin capsules in conjunction with subcutaneous insulin injections, well tolerated and effectively reduced glycemia throughout the day.Trial RegistrationClinicaltrials.gov NCT00867594.

Fragmented islets, obtained by mild overdigestion of the adult rat pancreas with collagenase, readily formed monolayer cultures on dishes coated with extracellular matrix derived from bovine corneal endothelial cells. Contaminating fibroblasts were removed by treatment with sodium ethylmercurithiosalicylate. The cultured islets remained functional for over 6 weeks in primary culture and up to 9 weeks in secondary culture, as indicated by their substantial insulin response to an acute glucose stimulus. Insulin secretion from islet monolayers showed biphasic kinetics. The functional competence of the monolayers was further evaluated by studying glucose-stimulated insulin release in the presence of various modulators of B-cell function. The response to physiological agents such as somatostatin, epinephrine, glucagon, and arginine was retained for at least 4 weeks in culture. The sensitivity to inhibition by somatostatin and epinephrine (ID50 = 10 ng/ml) and that to stimulation by glucagon (ED50 = 3 ng/ml) were similar to or better than those for freshly isolated islets. We have thus obtained a fibroblast-free monolayer culture of pancreatic islets from adult rats containing B-cells that retain normal function for long periods. This experimental system appears ideally suited for studying chronic modulations of islet cell function under controlled in vitro conditions, which can allow the stimulation of normal and diabetic environments.

Pancreatic tissue was obtained during therapeutic subtotal pancreatectomy from five infants with persistent hyperinsulinaemic hypoglycaemia of infancy (so-called nesidioblastosis). Collagenase digests of the specimens were cultured in RPMI 1640 medium on extracellular matrix-coated plates. Acute insulin secretion showed minimal sensitivity to changes in glucose concentration. Sensitivity to other nutrient secretagogues such as glyceraldehyde, leucine, alpha-ketoisocaproic acid and arginine was variable, showing either diminished or absent response. On the other hand, stimulators of Beta cell cAMP and modulators of the phosphoinositide-protein kinase C pathway were effective inducers of insulin release. The response to cAMP stimulators was independent of the glucose concentration. Although insulin output was high in the absence of glucose, this was not due to passive leak of hormone, since both removal of calcium and addition of somatostatin and epinephrine inhibited the secretion. Beta cells were more sensitive to somatostatin than epinephrine; however, both agents failed to completely suppress the release even at suprapharmacological concentrations. Although it cannot be excluded that the culture conditions affected Beta cell function, the present findings may suggest that cultured Beta cells in persistent hyperinsulinaemic hypoglycaemia of infancy behave like fetal Beta cells at early developmental stages.

Adult male rats were injected with 6-hydroxydopamine, either into the lateral brain ventricle or directly into the dorsal hippocampus. They were adrenalectomized 5-7 days later, and following an additional 24 hours, the specific in vitro 3H-corticosterone binding capacity of dorsal hippocampal slices was determined by estimation of uptake of radioactivity by the nuclear fraction. Specific corticosterone binding was reduced by 40-50% in both experimental groups, as compared to vehicle-treated controls. These results suggest that the maintenance of normal dorsal hippocampal corticosterone binding capacity is dependent upon the integrity of endogenous brain catecholaminergic neural systems.