Characterization of the 2-deoxyglucose effect on the adrenocortical axis.

In the present study we examined the mechanisms involved in the activation of the hypothalamo-pituitary-adrenal (HPA) axis after administration of the glucose analog 2-deoxyglucose (2-DG), which inhibits intracellular glucose use. Adult male rats were injected with either 2-DG (400 mg/kg body wt ip) or vehicle and were killed 60 min later. 2-DG increased serum ACTH and corticosterone (CS) by 3- and 7-fold, respectively, as compared to vehicle-treated rats. Bilateral lesions of the lateral hypothalamic area completely inhibited the 2-DG-induced HPA axis activation. Administration of 2-DG caused a significant depletion in the CRF-41 content of the median eminence (ME). Pretreatment with dexamethasone (80 micrograms/kg body wt ip) inhibited the 2-DG-induced depletion of ME CRF-41 and the increase in serum ACTH and CS. To investigate the role of type I and type II corticosteroid receptors in mediating the feedback effect of endogenous glucocorticoids on the responses to 2-DG, specific type I (RU-28318) or type II (RU-38486) receptor antagonists were injected intracerebroventricularly (icv) (1 microgram/kg body wt). In rats pretreated with these antagonists, the recovery of serum ACTH and CS to basal levels after 2-DG was markedly inhibited. Injection of the serotonin (5-HT) neurotoxin, 5,7-dihydroxy-tryptamine, either into the raphe nuclei or icv, which caused a 50 and 70% depletion of the hypothalamic 5-HT content, respectively, did not affect the HPA axis responses to 2-DG. In contrast, icv injection of ketanserin, a 5-HT2 receptor antagonist, completely inhibited the 2-DG-induced activation of the HPA axis. The results suggest that: 1) the lateral hypothalamic area is involved in the mediation of the HPA axis responses to 2-DG; 2) CRF-41 released from the ME plays a dynamic role in mediating the 2-DG-induced adrenocortical response; 3) the effect of 2-DG is sensitive to inhibition by dexamethasone, and the feedback effect exerted by endogenous glucocorticoids is mediated by both type I and type II corticosteroid receptors; and 4) 5-HT is involved in the activation of the HPA axis after 2-DG via its interactions with 5-HT2 receptors.

Monolayer culture of adult rat pancreatic islets on extracellular matrix: modulation of B-cell function by chronic exposure to high glucose.

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.

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.

Long QT syndrome and complete situs inversus. Preliminary report of a family.

The familial long QT syndrome is a rare condition that may occur with or without deafness. Dextrocardia with complete situs inversus is a familial syndrome generally found in normal subjects with a high incidence of consanguinity among the parents. In this report we describe a Jerbian family with both disorders and with several cases of sudden death. Of 27 members of this family (3 generations) in whom ECG was performed, 15 had QT prolongation (QTc greater than 0.45). Four members (2 generations) had complete situs inversus, 3 of them also had ECG evidence of QT prolongation. The combined occurrence of these rare diseases within the same family has not been previously reported. It may be due to the high incidence of consanguinity and may raise the possibility that the loci responsible for the 2 conditions are closely related and located on the same chromosome. A more extensive study of the family is being carried out.

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.

ACTH and corticosterone secretion following 2-deoxyglucose administration in intact and in hypothalamic deafferentated male rats.

Adult male rats were given a single i.p. injection of 2-deoxyglucose (2-DG, 100-400 mg/kg b. wt). The animals were decapitated 1-4 h later and trunk blood was collected for ACTH, corticosterone (CS) and glucose determinations. Serum ACTH and CS were markedly elevated when compared with saline-treated animals; these elevations were correlated with given doses of 2-DG so that with the higher dose, an approximately 6-fold increase in serum levels of both hormones was observed. Injection of 2-DG up to 200 mg/kg did not change serum glucose levels; injection of 400 mg/kg of 2-DG increased serum glucose by approximately 2-fold. A time course study showed that levels of serum ACTH, CS and glucose were maximal 1 h after 2-DG administration and returned to basal values 3 h later. Injection of 2-DG to animals with complete hypothalamic deafferentation failed to induce any change in serum ACTH, CS or glucose. This study demonstrates that: (1) 2-DG can stimulate the hypothalamo-hypophyseal-adrenal (HHA) axis as measured by ACTH and CS; this effect is not related to blood glucose levels; (2) the HHA response to 2-DG is mediated by sites outside the mediobasal hypothalamus.

Correction by bromocriptine of hypothalamic dysfunction and post-prandial hypoglycaemic symptoms in a 31-year-old woman.

A 31-year old female presented with recurrent episodes of post-prandial hypoglycaemic symptoms. Basal serum levels of ACTH, cortisol, GH, insulin and glucagon were normal. An adrenaline test demonstrated a normal peripheral response. An exercise test failed to produce ACTH, cortisol or FFA responses. Insulin (0.1 u/kg)-induced-hypoglycaemia failed to elevate serum ACTH, cortisol or GH. Metyrapone and ACTH tests were normal, demonstrating adequate hypophyseal and adrenal function. These findings suggested that the patient suffered from hypothalamic dysfunction. Bromocriptine (Parlodel, 7.5 mg/d for 5 weeks) resulted in an improved general condition, accompanied by a decrease in sugar consumption. Following treatment, FFA, ACTH and cortisol responses to exercise test were normal, as were ACTH, cortisol and GH responses to insulin-induced hypoglycaemia. It is concluded that bromocriptine may be useful in the treatment of post-prandial hypoglycaemic symptoms associated with hypothalamic dysfunction.

Effect of 6-hydroxydopamine on in vitro hippocampal corticosterone binding capacity in the male rat.

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.