Increase In Glucose-stimulated Insulin Secretion Research Articles

Beta cell adaptation in pregnancy: a major difference between humans and rodents?

Increasing evidence demonstrates that the insulin-producing beta cells can dynamically adapt their size, number and function in response to a variety of experimental and pathological conditions. Whether this dynamic adaptation also takes place under physiological conditions is less clear, with the notable exception of pregnancy [1, 2]. Under this condition, it has long been known that rodents undergo a rapid and substantial expansion of beta cell mass [3–5] due to increased beta cell size (hypertrophy) and replication (hyperplasia) [4], without detectable evidence of islet neogenesis [5]. These changes are induced by prolactin, placental lactogen and growth hormone, whose receptors are upregulated in beta cells after two-thirds of the gestational duration [1, 6]. They are also rapidly reversed at about the time of delivery, due to progesterone-controlled activation of beta cell apoptosis [7]. Recent studies have implicated selected genes controlling beta cell replication, survival and apoptosis in these changes [8–10]. In rodents, the changes in beta cell mass (about threeto fivefold) cannot alone account for those in insulin output (about eightfold), implicating a further functional improvement [1]. Thus, serum insulin increases by ∼75% due to increased glucose-stimulated insulin secretion, and this happens despite a 25% reduction in plasma glucose [5, 11, 12]. The increase in glucose-stimulated insulin secretion is mostly due to a significant decrease in the threshold for glucose stimulation [1, 12, 13], possibly as a result of higher levels of GLUT2, glucokinase and beta cell to beta cell coupling [13, 14]. It has also long been known that the progressive development of insulin resistance during human pregnancy increases the metabolic demand on beta cells, as evidenced by increased insulin secretion [11]. Due to the difficulty of obtaining pancreas from pregnant women, the adaptive mechanism underlying this change remains poorly investigated. However, islet enlargement, attributed to increased beta cell proliferation and decreased beta cell apoptosis, has been reported in a limited number of human samples [15], suggesting that similar mechanisms are responsible for beta cell adaptation in rodent and human pregnancy. This tentative conclusion needs to be critically revisited in view of recent data. First, the profile of genes that control beta cell proliferation differs in rodents and humans [2, 16]. Second, several studies have now documented that, in contrast with rodents, human beta cells do not proliferate under most conditions, probably due to an age-dependent epigenetic regulation of specific genes [17]. Moreover, a new pathology study has now raised the possibility that the adaptation of human beta cells to pregnancy may be significantly less extensive than in rodents and also due to a quite different mechanism [18]. Thus, in this issue of Diabetologia, Butler et al. [18] document that the volume density of beta cells was increased by only ∼1.4-fold in human pregnancy due to increased numbers of small islets, and not to beta cell size. These changes were associated with increased numbers of scattered and duct-associated beta cells, in the absence of detectable changes in beta cell replication and apoptosis. Thus, two studies of human samples [15, 18] concur in showing that beta cells increase as a function of the increasing gestational demand, but they M. Genevay Division of Pathology, Geneva University Hospital, Geneva, Switzerland

Small-Molecule Suppressors of Cytokine-Induced β-Cell Apoptosis

Pancreatic beta-cell apoptosis is a critical event during the development of type-1 diabetes. The identification of small molecules capable of preventing cytokine-induced apoptosis could lead to avenues for therapeutic intervention. We developed a set of phenotypic cell-based assays designed to identify such small-molecule suppressors. Rat INS-1E cells were simultaneously treated with a cocktail of inflammatory cytokines and a collection of 2,240 diverse small molecules and screened using an assay for cellular ATP levels. Forty-nine top-scoring compounds included glucocorticoids, several pyrazole derivatives, and known inhibitors of glycogen synthase kinase-3beta. Two compounds were able to increase cellular ATP levels, reduce caspase-3 activity and nitrite production, and increase glucose-stimulated insulin secretion in the presence of cytokines. These results indicate that small molecules identified by this screening approach may protect beta cells from autoimmune attack and may be good candidates for therapeutic intervention in early stages of type-1 diabetes.

The protective effect of berberine on β-cell lipoapoptosis

To evaluate the protective effect of berberine on β-cell lipoapoptosis induced by palmitate and to explore the possible underlying mechanisms. HIT-T15 pancreatic β-cells were divided into the following treatment groups: untreated controls; 100 μM berberine; 0.5 mM palmitate; 0.5 mM palmitate + 0.1 μM berberine; 0.5 mM palmitate + 1 μM berberine; 0.5 mM palmitate + 10 μM berberine; and 0.5 mM palmitate + 100 μM berberine. After 48 h, cell apoptosis was assessed by flow cytometry and the Hoechst 33258 fluorescent assay. Basal and glucose-stimulated insulin levels in culture medium were measured by radioimmunoassay. Peroxisome proliferator- activated receptor-γ (PPAR-γ) mRNA and protein levels were determined by real-time PCR and immunocytochemistry, respectively. Apoptosis was significantly increased upon treatment with palmitate as compared to the untreated controls (p<0.0001). In addition, glucose-stimulated insulin secretion (GSIS), PPAR-γ mRNA and protein expression were significantly reduced in response to palmitate (p<0.0001); however, palmitate-induced apoptosis and reduction in PPAR-γ expression were reversed in response to berberine in a dose-dependent manner (p<0.05). Furthermore, there was a non-significant increase in GSIS with increasing berberine dose. Palmitate exerted lipotoxic effects on HIT-T15 cells, inducing apoptosis and reducing GSIS. Berberine reduced palmitate-induced lipoapoptosis and tended to increase GSIS in HIT-T15 cells, possibly through increased PPAR-γ expression.

Apelin is a novel islet peptide

Apelin, a recently discovered peptide with wide tissue distribution, regulates feeding behavior, improves glucose utilization, and inhibits insulin secretion. We examined whether apelin is expressed in human islets, as well as in normal and type 2 diabetic (T2D) animal islets. Further, we studied islet apelin regulation and the effect of apelin on insulin secretion. Apelin expression and regulation was examined in human and animal specimens using immunocytochemistry, in situ hybridization, and real-time PCR. Insulin secretion was studied in INS-1 (832/13) clonal beta cells. APJ-receptor expression was studied using real-time PCR. In human and murine islets apelin was predominantly expressed in beta cells and alpha cells; a subpopulation of the PP cells in human islets also harbored apelin. In porcine and feline islets apelin was mainly expressed in beta cells. APJ-receptor expression was detected in INS-1 (832/13) cells, and in human and mouse islets. A high dose (1 µM) of apelin-36 caused a moderate increase in glucose-stimulated insulin secretion (30%; p < 0.001), while lower concentrations (10–100 nM) of apelin robustly reduced insulin secretion by 50% ( p < 0.001). Apelin was upregulated in beta cells of T2D db/db mice (47% vs. controls; p < 0.02) and GK-rats (74% vs. controls; p < 0.002), but human islet apelin expression was unaffected by glucose. On the other hand, human islet apelin expression was diminished after culture in glucocorticoids (16% vs. controls; p < 0.01). We conclude that apelin is a novel insulin-regulating islet peptide in humans and several laboratory animals. Islet apelin expression is negatively regulated by glucocorticoids, and upregulated in T2D animals. The presence of apelin receptors in islets suggests a role for apelin as a paracrine or autocrine messenger within the islets.

The CB1 Antagonist Rimonabant Decreases Insulin Hypersecretion in Rat Pancreatic Islets

Type 2 diabetes and obesity are characterized by elevated nocturnal circulating free fatty acids, elevated basal insulin secretion, and blunted glucose-stimulated insulin secretion (GSIS). The CB1 receptor antagonist, Rimonabant, has been shown to improve glucose tolerance and insulin sensitivity in vivo but its direct effect on islets has been unclear. Islets from lean littermates and obese Zucker (ZF) and Zucker Diabetic Fatty (ZDF) rats were incubated for 24 h in vitro and exposed to 11 mmol/l glucose and 0.3 mmol/l palmitate (GL) with or without Rimonabant. Insulin secretion was determined at basal (3 mmol/l) or stimulatory (15 mmol/l) glucose concentrations. As expected, basal secretion was significantly elevated in islets from obese or GL-treated lean rats whereas the fold increase in GSIS was diminished. Rimonabant decreased basal hypersecretion in islets from obese rats and GL-treated lean rats without decreasing the fold increase in GSIS. However, it decreased GSIS in islets from lean rats without affecting basal secretion. These findings indicate that Rimonabant has direct effects on islets to reduce insulin secretion when secretion is elevated above normal levels by diet or in obesity. In contrast, it appears to decrease stimulated secretion in islets from lean animals but not in obese or GL-exposed islets.

HIV-1 Protease Inhibitor Induced Oxidative Stress Suppresses Glucose Stimulated Insulin Release: Protection with Thymoquinone

The highly active anti-retroviral therapy (HAART) regimen has considerably reduced the mortality rate in HIV-1 positive patients. However, long-term exposure to HAART is associated with a metabolic syndrome manifesting cardiovascular dysfunction, lipodystrophy, and insulin resistance syndrome (IRS). The inclusion of HIV-1 protease inhibitors (PIs) in HAART has been linked to the induction of IRS. Although several molecular mechanisms of PI-induced effects on insulin action have been postulated, the deleterious effects of PIs on insulin production by pancreatic beta-cells have not been fully investigated and therapeutic strategies to ameliorate insulin dysregulation at this level have not been targeted. The present study showed that exposure to several different PIs, nelfinavir (5-10 microM), saquinavir (5-10 microM) and atazanavir (8-20 microM), decreases glucose stimulated insulin secretion from rat pancreatic beta-cells (INS-1). Nelfinavir significantly increased reactive oxygen species (ROS) generation and suppressed cytosolic, but not mitochondrial superoxide dismutase (SOD) levels. Nelfinvair also decreased both glutathione and ATP and increased UCP2 levels in these cells. Simultaneous treatment with thymoquinone (TQ) (2.5 microM), an active ingredient of black seed oil, significantly inhibited the effect of nelfinavir on augmented ROS production and suppressed SOD levels. Both TQ and black seed oil exposure increased glucose stimulated insulin secretion and ameliorated the suppressive effect of nelfinavir. The present findings imply a direct role of ROS in PI induced deleterious effects on pancreatic beta-cells. Our findings also suggest that TQ may be used as a potential therapeutic agent to normalize the dysregulated insulin production observed in HAART treated patients.

Role for malic enzyme, pyruvate carboxylation, and mitochondrial malate import in glucose-stimulated insulin secretion

Pyruvate cycling has been implicated in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. The operation of some pyruvate cycling pathways is proposed to necessitate malate export from the mitochondria and NADP(+)-dependent decarboxylation of malate to pyruvate by cytosolic malic enzyme (ME1). Evidence in favor of and against a role of ME1 in GSIS has been presented by others using small interfering RNA-mediated suppression of ME1. ME1 was also proposed to account for methyl succinate-stimulated insulin secretion (MSSIS), which has been hypothesized to occur via succinate entry into the mitochondria in exchange for malate and subsequent malate conversion to pyruvate. In contrast to rat, mouse beta-cells lack ME1 activity, which was suggested to explain their lack of MSSIS. However, this hypothesis was not tested. In this report, we demonstrate that although adenoviral-mediated overexpression of ME1 greatly augments GSIS in rat insulinoma INS-1 832/13 cells, it does not restore MSSIS, nor does it significantly affect GSIS in mouse islets. The increase in GSIS following ME1 overexpression in INS-1 832/13 cells did not alter the ATP-to-ADP ratio but was accompanied by increases in malate and citrate levels. Increased malate and citrate levels were also observed after INS-1 832/13 cells were treated with the malate-permeable analog dimethyl malate. These data suggest that although ME1 overexpression augments anaplerosis and GSIS in INS-1 832/13 cells, it is not likely involved in MSSIS and GSIS in pancreatic islets.

Endogenous and synthetic agonists of GPR119 differ in signalling pathways and their effects on insulin secretion in MIN6c4 insulinoma cells

Background and purpose:GPR119 is a G protein-coupled receptor that is preferentially expressed in islet cells and mediates insulin secretion. Oleoyl-lysophosphatidylcholine and oleoylethanolamide (OEA) act as endogenous ligands for this receptor, whereas PSN375963 and PSN632408 are two recently reported synthetic agonists. In this study, we explored mechanisms underlying GPR119-induced insulin secretion. In addition, we assessed the potential utility of the synthetic agonists as tools for exploring GPR119 biology.Experimental approach:We examined natural and synthetic GPR119 agonist activity at GPR119 in MIN6c4 and RINm5f insulinoma cells. We evaluated insulin secretion, intracellular calcium [Ca2+]i, ion channel involvement and levels of cAMP.Key results:We report that increases in insulin secretion induced by OEA were associated with increased cAMP and a potentiation of glucose-stimulated increases in [Ca2+]i. We also demonstrate that ATP-sensitive K+ and voltage-dependent calcium channels were required for GPR119-mediated increases in glucose-stimulated insulin secretion. In contrast to OEA, the synthetic GPR119 agonist PSN375963 and PSN632408 have divergent effects on insulin secretion, cAMP and intracellular calcium in MIN6c4 cells.Conclusions and implications:The endogenous ligand OEA signals through GPR119 in a manner similar to glucagon-like peptide-1 (GLP-1) and its receptor with respect to insulin secretion, [Ca2+]i and cAMP. In addition, PSN375963 and PSN632408 substantially differ from OEA and from one another. These studies suggest that the commercially available synthetic agonists, although they do activate GPR119, may also activate GPR119-independent pathways and are thus unsuitable as GPR119-specific pharmacological tools.

Glucose Metabolism In Vivo in Four Commonly Used Inbred Mouse Strains

OBJECTIVE—To characterize differences in whole-body glucose metabolism between commonly used inbred mouse strains.RESEARCH DESIGN AND METHODS—Hyperinsulinemic-euglycemic (∼8.5 mmol/l) and -hypoglycemic (∼3.0 mmol/l) clamps were done in catheterized, 5-h-fasted mice to assess insulin action and hypoglycemic counter-regulatory responsiveness. Hyperglycemic clamps (∼15 mmol/l) were done to assess insulin secretion and compared with results in perifused islets.RESULTS—Insulin action and hypoglycemic counter-regulatory and insulin secretory phenotypes varied considerably in four inbred mouse strains. In vivo insulin secretion was greatest in 129X1/Sv mice, but the counter-regulatory response to hypoglycemia was blunted. FVB/N mice in vivo showed no increase in glucose-stimulated insulin secretion, relative hepatic insulin resistance, and the highest counter-regulatory response to hypoglycemia. In DBA/2 mice, insulin action was lowest among the strains, and islets isolated had the greatest glucose-stimulated insulin secretion in vitro. In C57BL/6 mice, in vivo physiological responses to hyperinsulinemia at euglycemia and hypoglycemia were intermediate relative to other strains. Insulin secretion by C57BL/6 mice was similar to that in other strains in contrast to the blunted glucose-stimulated insulin secretion from isolated islets.CONCLUSIONS—Strain-dependent differences exist in four inbred mouse strains frequently used for genetic manipulation and study of glucose metabolism. These results are important for selecting inbred mice to study glucose metabolism and for interpreting and designing experiments.

Effects of Oral Fat Load on Insulin Output and Glucose Tolerance in Healthy Control Subjects and Obese Patients Without Diabetes

Elevated plasma nonesterified fatty acid (NEFA) concentrations cause peripheral and hepatic insulin resistance and may play an important role in regulating glucose-induced insulin secretion. The aim of our study was to investigate the influence of physiologically elevated NEFA levels on glucose-stimulated insulin secretion in order to find evidence that NEFAs are a potential factor predisposing for type 2 diabetes and related metabolic disorders, which are known risk factors for cardiovascular disease. We combined an orally administered fat emulsion with an intravenous glucose tolerance test and measured the time course of NEFA, insulin, and glucose. In order to find pathological conditions we applied the experiment to healthy and obese subjects. The main findings are a significant increase in glucose-stimulated insulin secretion after oral fat load in both groups compared with the condition without preceding fat ingestion and a prolonged insulin secretion after fat load in obese patients compared with control subjects. The results provide evidence that fat ingestion modulates beta-cell function and that NEFA is a plausible mediator that acts as a link between fat and glucose metabolism by modulating glucose-stimulated insulin secretion. Under the condition of elevated plasma levels of NEFA, this mechanism may be responsible for hyperinsulinemia in obese patients and a potential target of type 2 diabetes prevention strategies.

Characterization of β-cell function impairment in first-degree relatives of type 2 diabetic subjects: modeling analysis of 24-h triple-meal tests

To investigate early secretory defects in prediabetes, we evaluated beta-Cell function and insulin sensitivity (M value, by euglycemic clamp) in 26 normotolerant first-degree relatives of type 2 diabetic patients (FDR) and 17 age- and weight-matched control subjects. beta-Cell function was assessed by modeling analysis of glucose and C-peptide concentrations measured during 24 h of standardized living conditions. Fasting and total insulin secretion (ISR) were increased in FDR, as was ISR at a reference 5 mM glucose level (ISR5, 107 +/- 6 vs. 87 +/- 6 pmol x min(-1) x m(-2), P < 0.05). ISR5 was inversely related to M in controls (ISR5 = k/M1.23, rho = -0.74, P < 0.005) but not in FDR; when M was accounted for (by calculating a compensation index ISR5 x M1.23), compensation for insulin resistance was impaired in FDR (10.8 +/- 1.0 vs. 13.4 +/- 0.6 units, P < 0.05). Potentiation of ISR, expressing relative transient increases in glucose-stimulated ISR during meals, was impaired in FDR (1.29 +/- 0.08 vs. 1.62 +/- 0.08 during 1st meal, P < 0.02). Moreover, the potentiation time course was related to glucose-dependent insulin-releasing polypeptide (GIP) concentrations in both groups, and the sensitivity of potentiation to GIP derived from this relationship tended to be impaired in FDR. Compensation index, potentiation, and sensitivity to GIP were interrelated parameters (P < 0.05 or less). beta-Cell function parameters were also related to mean 24-h glucose levels (r2 = 0.63, P < 0.0001, multivariate model). In conclusion, although in absolute terms ISR is increased in insulin-resistant FDR, beta-cell function shows a cluster of interrelated abnormalities involving compensation for insulin resistance, potentiation, and sensitivity to GIP, suggesting a beta-cell defect in the amplifying pathway of insulin secretion.

The Effect of Prematurity and Intrauterine Growth Restriction on Glucose Metabolism in the Newborn

After completing this article, readers should be able to: 1. List the three components required for the transition to independent glucose homeostasis in the newborn. 2. Explain why preterm infants are at increased risk for neonatal hypoglycemia. 3. Compare the capacity for gluconeogenesis in the term and preterm infant and in the infant who has intrauterine growth restriction. 4. Describe the effects of enteral feedings on glucose homeostasis in the newborn. At birth, the transplacental supply of nutrients, especially glucose, to the newborn is interrupted abruptly. This sets into motion a complex cascade of metabolic and endocrine events that allow a healthy term newborn to adjust to his or her new environment. This cascade is triggered by a surge in the concentrations of glucagon and catecholamines and cessation of insulin secretion. Glucagon and catecholamines increase glycogenolysis, gluconeogenesis, lipolysis, and ketogenesis. The complex enzyme machinery responsible for these processes is mature in term healthy newborns. In contrast, the metabolic and endocrine processes allowing the transition to the extrauterine environment are not developed fully in preterm infants and those who have suffered intrauterine growth restriction (IUGR), placing these newborns at significant risk of hypoglycemia. This review focuses on the regulation of glucose metabolism during the fetal period and the metabolic and endocrine changes that occur at birth and in the postnatal period, with special reference to preterm infants and infants who have experienced IUGR. Carbohydrate is transported to the fetus as glucose, which is taken up from the maternal plasma by glucose transporters. GLUT1 and GLUT3 are the primary glucose transporters involved in the transplacental movement of glucose and are present on both the microvillus and basal membranes of the syncytial barrier. Glucose is transported to the fetus by facilitative diffusion according to concentration-dependent kinetics, with placental glucose transport capacity increasing with gestational age. The …

Glucagon-like peptide-1 causes pancreatic duodenal homeobox-1 protein translocation from the cytoplasm to the nucleus of pancreatic beta-cells by a cyclic adenosine monophosphate/protein kinase A-dependent mechanism.

Glucagon-like peptide-1 (GLP-1) enhances insulin secretion and synthesis. It also regulates the insulin, glucokinase, and GLUT2 genes. It mediates increases in glucose-stimulated insulin secretion by activating adenylyl cyclase and elevating free cytosolic calcium levels in the beta-cell. In addition, GLP-1 has been shown, both in vitro and in vivo, to be involved in regulation of the transcription factor, pancreatic duodenal homeobox-1 protein (PDX-1), by increasing its total protein levels, its translocation to the nucleus and its binding and resultant increase in activity of the insulin gene promoter in beta-cells of the pancreas. Here we have investigated the role of protein kinase A (PKA) in these processes in RIN 1046-38 cells. Three separate inhibitors of PKA, and a cAMP antagonist, inhibited the effects of GLP-1 on PDX-1. Furthermore, forskolin, (which stimulates adenylyl cyclase and insulin secretion), and 8-Bromo-cAMP, (an analog of cAMP which also stimulates insulin secretion), mimicked the effects of GLP-1 on PDX-1. These effects were also prevented by PKA inhibitors. Glucose-mediated increases in nuclear translocation of PDX-1 were not prevented by PKA inhibitors. Our results suggest that regulation of PDX-1 by GLP-1 occurs through activation of adenylyl cyclase and the resultant increase in intracellular cAMP, in turn, activates PKA, which ultimately leads to increases in PDX-1 protein levels and translocation of the protein to the nuclei of beta-cells.

Prolonged elevation of plasma free fatty acids impairs pancreatic beta-cell function in obese nondiabetic humans but not in individuals with type 2 diabetes.

Our recent in vivo observations in healthy nonobese humans have demonstrated that prolonged elevation of plasma free fatty acids (FFAs) results in diminished glucose-stimulated insulin secretion (GSIS) when the FFA-mediated decrease in insulin sensitivity is taken into account. In the present study, we investigated whether obese individuals and patients with type 2 diabetes are more sensitive than healthy control subjects to the inhibitory effect of prolonged elevation of plasma FFAs on GSIS. In seven patients with type 2 diabetes and seven healthy nondiabetic obese individuals, we assessed GSIS with a programmed graded intravenous glucose infusion on two occasions, 6-8 weeks apart, with and without a prior 48-h infusion of heparin and Intralipid, which was designed to raise plasma FFA concentration approximately twofold over basal. The nondiabetic obese subjects had a significant 21% decrease in GSIS (P = 0.0008) with the heparin and Intralipid infusion, associated with a decrease in whole body insulin clearance. The impairment in GSIS was evident at low (<11 mmol/l) but not at higher plasma glucose concentrations. In contrast, the patients with type 2 diabetes had a slight increase in GSIS (P = 0.027) and no change in insulin clearance, although there was marked interindividual variability in response. Plasma proinsulin concentrations measured in a subset of subjects were not altered in either group by the infusion of heparin and Intralipid. In summary, 1) obese nondiabetic individuals are susceptible to a desensitization of GSIS with heparin and Intralipid infusion, and 2) patients with type 2 diabetes do not demonstrate such susceptibility when FFAs are elevated approximately twofold above basal with heparin and Intralipid. Our results suggest that FFAs could play an important role in the development of beta-cell failure in obese individuals who are at risk for developing type 2 diabetes. They do not, however, seem to further deteriorate the beta-cell function of patients who already have established type 2 diabetes and may even result in a slight increase in GSIS in this latter group.

Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy.

Islets undergo a number of upregulatory changes to meet the increased demand for insulin during pregnancy, including an increase in glucose-stimulated insulin secretion with a reduction in the stimulation threshold. Treatment with the lactogenic hormone prolactin (PRL) in vitro has been shown to induce changes in islets similar to those observed during pregnancy. We examined cAMP production in islets treated with PRL to determine if changes in cAMP are involved in the upregulation of insulin secretion. Insulin secretion and cAMP concentrations were measured from islets in response to a suprathreshold (6.8 mmol/l) or high (16.8 mmol/l) glucose concentration in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Insulin secretion increased by 2.1-, 5.0-, and 5.9-fold at the suprathreshold glucose concentration and by 1.6-, 2.3-, and 2.9-fold at the higher glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. After a similar pattern, cAMP metabolism increased by 1.2-, 1.6-, and 2.1-fold at the suprathreshold glucose concentration and by 1.2-, 1.7-, and 2.2-fold at the high glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. The similar increases in insulin secretion and cAMP concentration suggest that changes in cAMP metabolism are involved in lactogen-induced upregulation of insulin secretion. To gain additional insight into the role of cAMP in the upregulation of islet function after lactogen treatment, we examined the relationship between changes in cAMP concentration and insulin secretion. Under all conditions (differing glucose concentrations and time periods), the increase in insulin release was directly proportional to the increase in cAMP. Thus increased glucose-stimulated insulin secretion from lactogen-treated islets could be accounted for by increased generation of cAMP and did not appear to require any further specific changes in intracellular processes mediated by cAMP. Because the PRL receptor is not directly involved in cAMP metabolism, the lactogen-induced increase in cAMP was most likely due to the increase in glucose metabolism that we have previously demonstrated in PRL-treated islets and in islets during pregnancy.

Chronic sympathetic innervation of islets in transgenic mice results in differential desensitization of alpha-adrenergic inhibition of insulin secretion.

The effects of chronic sympathetic hyperinnervation on pancreatic beta-cell insulin secretion were investigated utilizing the in vitro perfused pancreas from transgenic mice. These mice exhibit islet hyperinnervation of sympathetic neurons resulting from overexpression of nerve growth factor in their beta-cells (1). The goal was to determine whether sympathetic hyperinnervation increased classic alpha-adrenergic inhibition of beta-cell insulin secretion or, in contrast, down-regulated beta-cell sensitivity to adrenergic input resulting in enhanced insulin secretion. Both fasting and fed blood sugars and pancreatic insulin content were normal in the transgenics. Response of the transgenic perfused pancreas to low glucose (7 mM) was primarily first phase and normal whereas high glucose (22 mM) caused enhanced, rather than reduced, insulin secretion of both first and second phases. The alpha-antagonist, phentolamine, caused a six-fold increase in glucose-stimulated insulin secretion from the control pancreas, an effect that was blunted for the transgenic pancreas. A similarly blunted response to phentolamine occurred when this agent was superimposed on a combined glucose-forskolin stimulus. (The positive effect on insulin secretion by phentolamine in normal beta-cell preparations has arguably been ascribed to non-specific ionic effects.) Therefore, as a test of possible changes in the ATP regulated K+ channel or the linked Ca++ channels, glyburide was perfused during glucose stimulation. Insulin secretion in response to glyburide was increased two fold in the control pancreas. However, with the transgenic pancreas, in contrast to the enhanced response to glucose, the effect of glyburide was almost completely inhibited. It is concluded that: 1) chronic adrenergic hyperinnervation results in enhanced glucose-stimulated insulin secretion by desensitization of a major alpha-adrenergic inhibitory site(s); and 2) adrenergic hyperinnervation acts directly or indirectly on ion flux to partially inhibit insulin release, an effect which is not desensitized. Since down-regulation of a single alpha-adrenergic receptor would be expected to desensitize both phenomena the observed differential desensitization indicates that different post receptor events or more than one adrenergic receptor are involved.

Endothelin-1 stimulates insulin secretion by direct action on the islets of Langerhans in mice.

Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, is secreted in response to insulin. Elevated circulating ET-1 levels have been found in patients with diabetes mellitus and vascular dysfunction. The question arises whether ET-1 acts as a direct modulator of insulin secretion. To test this, we studied the effects of ET-1 on isolated mouse islets of Langerhans. ET-1 (1 nmol/l-1 mumol/l) dose-dependently stimulated insulin secretion from islets incubated in the presence of 16.7 mmol/l glucose (p < 0.05). The effect of ET-1 is glucose-dependent since no potentiation was found at 3.3 mmol/l glucose. Furthermore, ET-1 induced a large, transient increase in glucose-stimulated insulin secretion during islet perifusion in the presence (p < 0.001), but not in the absence, of extracellular Ca2+. The rate of 45Ca(2+)-efflux from 45Ca(2+)-prelabelled islets was transiently stimulated by ET-1 during perifusion at 16.7 mmol/l glucose in the presence of extracellular Ca2+ (p < 0.001). A short-lived increase in 45Ca(2+)-efflux was also observed in the absence of extracellular Ca2+ (p < 0.05). It is suggested that the effects of ET-1 on insulin secretion are critically dependent on influx via Ca(2+)-channels. In addition, ET-1 transiently enhanced 86Rb(+)-efflux from 86Rb(+)-prelabelled islets both in the presence (p < 0.001) and in the absence (p < 0.001) of extracellular Ca2+ suggesting that ET-1 does not elicit insulin secretion by inhibition of the potassium permeability. Our study provides evidence that ET-1 stimulates insulin secretion via a direct effect on the islets of Langerhans.

Glucokinase, hexokinase, glucose transporter 2, and glucose metabolism in islets during pregnancy and prolactin-treated islets in vitro: mechanisms for long term up-regulation of islets.

During pregnancy, islets undergo a number of up-regulatory changes to meet the increased need for insulin. One of the most important changes is an increase in glucose-stimulated insulin secretion with a reduction in the glucose-stimulated threshold. Similarly, placental lactogen and PRL induce the same changes in islets as pregnancy. In this study, we examined the effects of pregnancy and PRL treatment of islets in vitro on insulin secretion; glucokinase and hexokinase activities; glucokinase, hexokinase, and glucose transporter 2 protein levels; and rates of glucose utilization and oxidation. Glucokinase activity was 4.9 +/- 0.4 pmol glucose/ng DNA.h in control islets and was significantly increased by 50% in islets on day 15 of pregnancy and by 60% on day 20 of pregnancy. Hexokinase activity was 11.7 +/- 0.9 pmol glucose/ng DNA.h in control islets and was increased by 20% in islets on day 15 of pregnancy and by 90% on day 20 of pregnancy. In the in vitro studies, glucokinase activity was 7.4 +/- 0.89 pmol glucose/ng DNA.h in control islets. PRL treatment of islets in vitro increased glucokinase activity by 60%, an effect similar to that observed in the pregnancy islets. In contrast, hexokinase activity was nearly undetectable in cultured islets, whether control or PRL treated. Quantitative Western blot analysis of glucokinase and hexokinase was performed using equivalent number of protein per lane for all experimental groups. On a protein equivalency basis, glucokinase expression levels were the same in control islets on days 15 and 20 of pregnancy. Likewise, hexokinase levels were not different between control islets and islets on days 15 and 20 of pregnancy. Similarly, Western blot analysis of cultured islets indicated that there were not effect of PRL on glucokinase or hexokinase levels. However, when enzyme levels were normalized on the basis of DNA, the levels of expression appeared to be commensurate with their activities. In cultured islets, the very low level of hexokinase activity corresponded to the low level of hexokinase detected by Western blots. Glucose transporter 2, as determined by Western blot quantification, was increased 2-fold in pregnancy islets on day 15 and increased by 45% in pregnancy islets on day 20. Similar results were observed in cultured islets where glucose transporter 2 was increased 2-fold in PRL-treated islets. Islet glucose utilization and oxidation rates on day 15 of pregnancy were significantly greater than those in control islets at all glucose concentrations examined. This enhanced glucose sensitivity resulted in a shift of the glucose utilization and oxidation response curves to the left. Comparable results were obtained from islets on day 20 of pregnancy. PRL treatment of islets in vitro resulted in the same changes in glucose utilization and oxidation rates that were observed during pregnancy. These results demonstrate changes in glucokinase, hexokinase, and glucose transporter 2 levels and glucose metabolism that occur as islets adapt to an increased need for insulin secretion during pregnancy. The results also indicate that these same changes can be induced by PRL treatment of islets in vitro. This provides further evidence that the long term adaptive changes that occur under the normoglycemic conditions of pregnancy are mediated by lactogen-regulated events.

Insulin-resistant syndromes in children.

Puberty is normally associated with a decline in tissue sensitivity to insulin. However, normal glucose homoeostasis is maintained by compensatory increases in glucose-stimulated insulin secretion. Here we describe studies performed in healthy children which have determined the site of insulin resistance (hepatic vs. peripheral) and whether this resistance extends to other substrates such as amino acid and free fatty acid metabolism. The changes in insulin action and secretion that are normally seen during puberty lead us to question the role of insulin resistance in other childhood conditions that are complicated by the later development of type I or type II diabetes, namely thalassaemia major and Turner's syndrome. These studies showed that in patients with thalassaemia and Turner's syndrome, insulin resistance and increased insulin secretion are very early metabolic defects that appear before the development of diabetes.

Long-term safety and efficacy of glipizide

We studied the effect of long-term glipizide therapy in a small group of closely monitored patients. Fasting plasma glucose, glucose tolerance, glucose-stimulated insulin secretion, and insulin sensitivity were measured on multiple occasions throughout the investigation. Our observations show that glipizide therapy is effective in certain patients with noninsulin-dependent diabetes mellitus over a prolonged time period. The major effect of the drug appears to be mediated by its alteration of insulin sensitivity, but glipizide also causes a sustained increase in glucose-stimulated insulin secretion in most patients.