Regulation of insulin release in persistent hyperinsulinaemic hypoglycaemia of infancy studied in long-term culture of pancreatic tissue.

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.

Monolayer culture of adult rat pancreatic islets on extracellular matrix: long term maintenance of differentiated B-cell function.

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.

Binding, degradation, and biological activity of insulin in vascular smooth muscle cells.

The interaction of insulin with the vascular smooth muscle was studied using cultures derived from the bovine aortic arch. The cultured cells exhibited specific binding of 125I-insulin that was reversible and dependent on pH. Both insulin and insulinlike growth factor (IGF) I competed for 125I-insulin binding; IGF I, however, was less effective than insulin by at least an order of magnitude. Insulin binding was accompanied by internalization and degradation of the hormone in a temperature- and time-dependent manner. Chloroquine and other lysosomotropic agents elevated the internalized insulin and reduced its degradation. Pre-exposure of cell cultures to insulin resulted in downregulation of cell surface receptors. Insulin stimulated alpha-aminoisobutyric acid transport in confluent smooth muscle cells. The maximal response was observed at 100 ng/ml insulin with a half-maximal effect at 10 ng/ml. Sparse, serum-starved smooth muscle cells responded to insulin with a dose-dependent increase in [3H]-thymidine incorporation into DNA. Although the effect was already apparent at 1 ng/ml insulin, it reached near maximal level only at 10,000 ng/ml. IGF I also stimulated DNA synthesis in smooth muscle cells; however, at low concentrations insulin was more efficient in this respect. Human growth hormone was inactive. The data indicate the presence of specific receptors for insulin in bovine aortic smooth muscle cells. These receptors appear to mediate the metabolic activity as well as part of the mitogenic effect of insulin in these cells.

Insulin binding and degradation in vascular endothelial cells: modulation by cell growth and culture organization.

The interaction of insulin with the vascular endothelium and its modulation by cell growth and culture organization was studied using bovine aortic endothelial cells in monolayer cultures. Three types of cultures were investigated: 1) confluent nondividing cultures, organized and differentiated as the in vivo tissue; 2) subconfluent, not yet organized cell cultures, representing proliferating endothelium; and 3) endothelial cell cultures modified to lose their property of contact inhibition, growing in multiple layers. All three types of cultures exhibited specific binding of 125I-insulin to high and low affinity cell surface receptor sites, and were capable of degrading 125I-insulin. Preexposure of the cultures to insulin resulted in a time dependent reduction in the availability of cell surface receptors (down-regulation). Insulin binding per cell was 2.4-fold and 10-fold higher in the subconfluent and modified cultures, respectively, as compared to the contact-inhibited confluent cultures. Similarly, the rate of insulin degradation was higher in the subconfluent and modified cultures (2.3-fold and 20-fold, respectively). Subconfluent cultures were more sensitive than confluent cultures to the down-regulatory effect of insulin. They exhibited a 60% decrease in insulin binding as compared to a 40% decrease in confluent cultures after preexposure to 50 ng/ml insulin. The increase in insulin binding and degradation in growing endothelial cells suggests a role for the hormone in the regulation of endothelial cell growth, e.g. in response to injury. This was further supported by the observation of a dose-dependent stimulation of [3H]thymidine incorporation into sparse, serum-starved endothelial cells by physiological concentrations of the hormone.

Binding, internalization, and degradation of insulin in vascular endothelial cells.

The interaction of insulin with the vascular endothelium was studied using bovine aortic endothelial cells in monolayer cultures. Confluent cell cultures exhibited specific binding of 125I-insulin to high- and low-affinity cell surface receptor sites. Binding was reversible, saturable, and accompanied by internalization and degradation of the bound hormone in a temperature- and time-dependent manner. Pre-exposure of the cultures to insulin resulted in a time-dependent reduction in the availability of cell surface receptors (downregulation). It is concluded that the occurrence of reversible insulin binding and of insulin degradation in endothelial cells supports the concept that the vascular endothelium compartment may regulate the level of insulin in the circulation.