S. J. H. Ashcroft

Glucoreceptor mechanisms and the control of insulin release and biosynthesis

SummaryThe models proposed for the means whereby the B-cell recognises glucose and related compounds as signals for insulin release and biosynthesis are discussed. The observed correlations between rates of metabolism and insulin release and biosynthesis are consistent with the substrate-site hypothesis. For glucose itself, the enzymes catalysing the phosphorylation of the sugar provide an explanation for the major characteristics of the islet responses, but for N-acetylglucosamine evidence is presented that the sugar transport system fulfils this discriminatory role. Possible mechanisms whereby sugar metabolism may be linked to changes in Ca2+-handling are considered and evidence is given supporting a role for the cytosolic NADPH/NADP+ ratio and the islet content of phosphoenolpyruvate. The nature of the targets for cyclic AMP and Ca2+ is discussed and some properties of islet cAMP-dependent protein kinase are summarised. Evidence is presented for the presence of calmodulin in islets and the possible involvement of calmodulin in stimulussecretion coupling. On the basis of these considerations a speculative hypothesis for the mechanisms involved in the B-cell responses to glucose is out lined.

Nesidioblastosis of the pancreas: definition of the syndrome and the management of the severe neonatal hyperinsulinaemic hypoglycaemia.

Three newborn infants are reported who developed severe non-ketotic hypoglycaemia (blood glucose less than 1.1 mmol/l; 19.8 mg/100 ml) within 6 hours of birth. All had inappropriately raised plasma insulin concentrations for the level of glycaemia, and required high rates of glucose infusion (less than 15 mg glucose/kg per minute) to prevent symptoms of hypoglycaemia. Medical treatment (hydrocortisone, diazoxide, chlorothiazide, phenytoin, propranolol, and depot glucagon) was ineffective in preventing hypoglycaemia and all 3 infants were subjected to partial and then total pancreatectomy. The pathological features of nesidioblastosis are reported from quantitative immunohistochemical studies on the pancreata. These results together with those from metabolic and endocrine studies performed on the 3 infants during the investigation of the cause of the hypoglycaemia and during the preoperative and postoperative period are presented in detail in order to define a practical approach to the management of this difficult clinical problem in the neonate.

Presence and possible role of calcium-dependent regulator (calmodulin) in rat islets of langerhans

A rise in the intracellular concentration of free Ca2+ has been implicated as a key event in the initiation of insulin secretion [ 1,2] but the mechanism by which Ca2+ triggers exocytosis is not known. In other Ca”-regulated processes, Ca2+ exerts a controlling influence by binding to specific proteins, e.g., troponin C in the initiation of muscle contraction, and phosphorylase kinase in the activation of glycogenolysis [3,4]. On the basis of amino acid sequence and X-ray crystallographic data a family of 5 homologous Ca”-modulated proteins: calcium dependent regulator (calmodulin); troponin C; myosin light chains; muscle pervalbumin; and intestinal calcium binding protein, has been described [S]. Binding of Ca2+ is suggested to depend on the presence of homologous a-helical domains referred to as EF-hands [S]. Calmodulin may be of special significance as a general intracellular Ca’+-receptor protein in view both of its ubiquitous tissue distribution in eukaryotes and of the variety of proteins to which it confers sensitivity to Ca’+. Originally discovered [6] as a heat-stable protein activator of brain cyclic nucleotide phosphodiesterase (PDE), calmodulin has been shown inter alia to activate a detergent-solubilized preparation of brain adenylyl cyclase [7] and a Ca2’-ATPase in erythrocyte membranes [8] and to be a component

Insulin secretory responses of a clonal cell line of simian virus 40-transformed B cells

SummaryWe have evaluated the potential of the clonal insulin-secretory cell line HIT-T15 as a model system for investigating stimulus-secretion coupling in pancreatic B cells. In contrast to other cell lines, HIT cell insulin secretion was consistently stimulated 2- to 3-fold by D-glucose. The maximally effective concentration of glucose was 10 mmol/1; between 2 and 10 mmol/l glucose the increase in insulin release was paralleled by an increased rate of glucose oxidation. The main characteristics of glucose-stimulated insulin release by HIT cells were essentially similar to those of normal islets. Thus, the response was (1) specific for metabolizable sugars (D-mannose and D-glyceraldehyde stimulated insulin release but L-glucose and D-galactose were ineffective); (2) markedly dependent on extracellular Ca2+ concentration; (3) potentiated by forskolin, glucagon, acetylcholine and 12-0-tetradecanoyl phorbol 13-acetate; (4) inhibited by adrenaline or somatostatin; (5) showed a biphasic pattern of release in perifusion experiments, with both phases being potentiated by forskolin. The secretory response of the HIT cells to amino acids was also similar to that of normal islets. Thus, L-leucine and its deamination product 2-ketoisocaproate were effective stimuli, whereas L-isoleucine and L-glutamine were ineffective. Insulin release from HIT cells could also be evoked by the sulphonylureas glibenclamide and tolbutamide and by an increase in concentration of extracellular K+ to 40 mmol/1. The content of cyclic AMP in HIT cells was increased modestly by glucose but not by an increase in extracellular K+. Forskolin elicited a 4-fold increase in cyclic AMP content. We conclude that HIT cells retain the essential features of the insulin secretory response of normal B cells and represent an important tool for further biochemical characterisation of the secretory system.

Effect of adrenal steroids on insulin release from cultured rat islets of Langerhans

SummaryThe effect of additions to the culture medium of some natural or synthetic corticosteroid hormones was studied in cultured rat islets of Langerhans. The steroids decreased glucose-induced insulin release. The extent of inhibition by dexamethasone was 18–55%, prednisolone 23%, hydrocortisone 21% and aldosterone 18%. None of them affected the basal secretion of insulin or had any effect on diameter or insulin content of the islet. The inhibitory action of dexamethasone on insulin release was observed in the range 63 nmol/l to 6.3 μmol/l. At 6.3 μmol/l during two h, dexamethasone (a) inhibited insulin response to glucose concentrations above 5 mmol/l (b) caused a delay in the first phase and markedly reduced the second phase of insulin release of perifused islets, and (c) decreased the incorporation of [H3]-leucine into total islet proteins without affecting [H3]-leucine-incorporation into insulin plus proinsulin. It is suggested that steroids, by directly acting on the islets of Langerhans, may modulate the insulin-release response to secretagogues.

Insulin release from human pancreatic islets in vitro

SummaryIslets of Langerhans were isolated by collagenase digestion from the pancreas of a 39 year-old female renal transplant donor. The islets were subjected to three consecutive periods of tissue culture, after each of which they were incubated in vitro with various agents whose effects on insulin release from islets of laboratory animals have previously been established. After the first culture period, the basal insulin secretion rate of 5.2 μU/islet/h seen with 2 mmol/l glucose was increased approx. 5-fold on raising the glucose concentration to 20 mmol/l. The islets retained the insulin-secretory response to 20 mmol/l glucose throughout the period of study. Insulin secretion was also stimulated by mannose, leucine, α-ketoisocaproate, dihydroxyacetone and 3-hydroxybutyrate, but not by fructose or N-acetyl-glucosamine. Fructose however increased insulin release in the presence of 4 mmol/l glucose. Caffeine elicited insulin release in the absence of glucose and enhanced insulin release in response to 10 mmol/l glucose. Glucose-stimulated insulin release was inhibited by trifluoperazine (25 μmol/l).

Regulation and specificity of glucose‐stimulated insulin gene expression in human islets of Langerhans

The insulin response of cultured human islets of Langerhans was measured at both mRNA and polypeptide levels in response to natural and pharmacological stimuli. We report a dosage dependent stimulation of both mRNA levels and insulin secretion by extracellular glucose, and present evidence that islet responsiveness can be divided into two temporal phases: an early response, apparently under post‐transcriptional control, and a late phase in which insulin messenger accumulates. Although glucose effects in man are similar to rodents, there are important differences, especially with respect to modulation of glucose stimulation by activators of β‐cell protein kinases.

Calcitonin gene-related peptide and somatostatin inhibit insulin release from individual rat B cells

Hormone secretion from single, rat pancreatic B cells was visualised by a reverse haemolytic plaque assay for C-peptide. Quantitative analysis of the size and number of haemolytic plaques indicated that exposure to 3, 5, 10 and 20 mM glucose resulted in a dose-dependent increase in both the magnitude of C-peptide, and thus, insulin release by individual B cells and the recruitment of activity secreting B cells. Somatostatin and calcitonin gene-related peptide, fragment 28-37 (CGRP28-37) were shown to inhibit glucose-stimulated insulin release as assessed by the size of individual plaques and the number of recruited B cells, and hence to reduce the total area of plaques formed. In the presence of 15 mM glucose, a dose-dependent effect of CGRP28-37 on the secretion of insulin was observed, with the size of plaques formed by individual B cells reduced at concentrations of CGRP28-37 between 10(-5) and 10(-11) M. Thus, both somatostatin and CGRP28-37 can act directly on individual B cells to inhibit their secretory response to increasing levels of glucose. We suggest that these peptides which can be immunolocalised in islet cells may have a role in the regulation of insulin secretion.

The hypoglycaemic and insulinotropic activity of Tinospora crispa: studies with human and rat islets and HIT-T15 B cells

SummaryIn Malaysia, Tinospora crispa extract is taken orally by Type 2 (non-insulin-dependent) diabetic patients to treat hyperglycaemia. We have evaluated the claimed hypoglycaemic property by adding aqueous extract to the drinking water of normal and alloxan-diabetic rats. After one week, fasting blood glucose levels were significantly (p<0.01) lower and serum insulin levels were significantly (p<0.01) higher in treated diabetic animals (10.4±1.0 mmol/l and 12.8±1.1 μU/ml respectively) compared to untreated diabetic controls (17.4±1.7 mmol/l and 8.0±0.7 μU/ml respectively). The insulinotropic action of T. crispa was further investigated in vitro using isolated human or rat islets of Langerhans and HIT-T15 cells. In static incubations with rat islets and HIT-T15 B cells, the extract induced a dosage dependent stimulation and potentiation of basal and glucose-stimulated insulin secretion respectively. This insulinotropic effect was also evident in perifused human and rat islets and HIT-T5 B-cells. The observations that (i) in all three models insulin secretory rates rapidly returned to basal levels on removal of the extract and (ii) in rat islets, a second challenge with T. crispa induced an additional, stimulated response, are all consistent with physiological release of insulin by B cells. Moreover, the rate of HIT-T15 glucose utilisation was not affected by incubation with T. crispa, suggesting that the cells were viable throughout. These are the first studies to provide biochemical evidence which substantiates the traditional claims for an oral hypoglycaemic effect of Tinospora crispa, and which also show that the hypoglycaemic effect is associated with increased insulin secretion.

Effects of growth hormone on insulin release in the rat

SummaryGrowth hormone injected intravenously in the rat elicited a 6-fold spike change in immunoreactive insulin with little variation in glucose. Subcutaneous administration of growth hormone for 4 days augmented by 56% the insulin-secretory response to glucose of isolated islets from hypophysectomised rats but not the response of control rat islets. When islets were cultured in the presence of growth hormone, the glucose-induced insulin release was increased by 35% in batch incubations of islets from both normal and hypophysectomised rats and by 70–110% in perifused islets. Thus the capacity for stimulated release of insulin is limited by hypophysectomy, and growth hormone is capable of directly influencing the secretory function of the β- cell.