Phase Of Glucose-stimulated Insulin Release Research Articles

Chronic effects of neuroendocrine regulatory peptide (NERP-1 and -2) on insulin secretion and gene expression in pancreatic β-cells

Neuroendocrine regulatory peptides (NERP-1 and -2) are novel amidated peptides derived from VGF, a polypeptide secreted from neurons and endocrine cells through a regulated pathway. Dr. Nakazato Masamitsu reported that NERP-1 and -2 may have a local modulator function on the human endocrine system, and clearly showed expression of NERP-1 and -2 in human pancreas islets. Based on these data, we investigated the alteration of insulin secretion, insulin granule-related protein, and pancreas-specific transcription factors in response to NERPs expression. We confirmed the expression of NERP-1 and -2 in the pancreas of a human diabetes patient, in addition to diabetic animal models. When INS1 cells and primary rat islets were incubated with 10nM NERPs for 3days, glucose-stimulated insulin secretion levels were blunted by NERP-1 and -2. The number of insulin granules released from the readily releasable pool, which is associated with the first phase of glucose-stimulated insulin release, was decreased by NERP-1 and -2. Insulin granule-related proteins and mRNAs were down-regulated by NERP-2 treatment. NERP-2 decreased the expression of BETA2/NeuroD and insulin and controlled the nucleo-cytoplasmic translocation of FOXO1 and Pdx-1. We observed that NERP-2 levels were dramatically increased in diabetic pancreas. In conclusion, NERP-2 may play an important role in insulin secretion through the regulation of insulin secretory granules and β-cell transcription factors. In addition, NERP-2 expression is increased in diabetic conditions. Therefore, we suggest that NERPs may be potent endogenous suppressors of glucose-dependent insulin secretion.

Metabolome Response to Glucose in the β-Cell Line INS-1 832/13

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mM glucose and in response to increasing concentrations of glucose in the INS-1 832/13 β-cell line. U-[(13)C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.

Islets Transplanted Intraportally into the Liver are Stimulated to Insulin and Glucagon Release Exclusively through the Hepatic Artery

Not much is known about the physiology of intraportally transplanted islets. One reason for this is that it is difficult to study such islets, since they are scattered throughout the liver. We employed a perfusion technique to characterize the functional properties of syngeneic intrahepatic 1-month-old islet grafts, and compared them to islets transplanted beneath the kidney capsule, as well as native islets. The cellular composition of the islet grafts was also examined. Glucose and arginine administered through the hepatic artery, but not through the portal vein, induced insulin release from the intraportally implanted islets. Moreover, arginine, only when administered through the hepatic artery, induced glucagon release from the same islets. The first phase of glucose-stimulated insulin release from both islets transplanted to the liver and kidney was delayed, and less prominent when compared to the pancreas. Intraportally transplanted islets contained fewer glucagon-positive cells than islets transplanted to the kidney and native islets. Our findings demonstrate that intraportally transplanted islets respond with insulin and glucagon to secretagogues, but only when stimulated through the hepatic artery. Whether intrahepatic islets may sense other substances than glucose or arginine occurring in high concentrations in the portal vein following intestinal uptake remains to be studied.

Angiotensin II type 1 receptor inhibition markedly improves the blood perfusion, oxygen tension and first phase of glucose-stimulated insulin secretion in revascularised syngeneic mouse islet grafts

We recently found evidence of an angiotensin-generating system in pancreatic islets. The present study investigated the effect of endogenously produced angiotensin II on microcirculation and function in transplanted islets. Losartan, an angiotensin II type 1 receptor inhibitor, was administered either acute intravenously to mice with 4-week-old islet renal subcapsular transplants, or added to the drinking water for the final 14 days or throughout the 4-week post-transplantation period. The graft-bearing kidney was, in some cases, dissected out and perfused in vitro to evaluate the effect of angiotensin II and losartan on glucose-stimulated insulin release from the grafts. Losartan treatment throughout the 4-week post-transplantation period had negative effects on islet revascularisation as well as on islet graft insulin release. However, administration of losartan, either intravenously or orally, after the formation of a new vascular network, improved islet graft blood perfusion. PO2 in the islet transplants was also effectively improved by the losartan treatment. Graft perfusion experiments showed a markedly better first phase of glucose-stimulated insulin release in transplanted islets when exposed to losartan. In contrast, acute administration of angiotensin II decreased islet graft blood flow, PO2 and glucose-stimulated insulin release. This study shows that inhibition of the islet reninangiotensin system may be a feasible strategy to increase the blood perfusion, PO2 and function within islet grafts. Such treatment should not be initiated, however, before the islet vascular system has been formed.

Phospholipase D1 Regulates Secretagogue-stimulated Insulin Release in Pancreatic β-Cells

Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.

Acute pancreatitis, expression of inducible nitric oxide synthase and defective insulin secretion.

A high level of nitric oxide (NO) produced by inducible NO synthase (iNOS) is involved in pancreatic beta-cell dysfunction and apoptosis. In the present study, we examined whether iNOS is also expressed in beta cells after induction of acute pancreatitis (AP) in the rat. Pancreatic islets taken from AP animals and incubated for 60 min in the presence of 20.0 mmol/l glucose showed a decreased insulin secretory response to glucose. The basal insulin release at 1.0 mmol/l glucose was also moderately reduced. Experiments on the dynamics of insulin secretion from perfused pancreas revealed an impairment of both first and second phase of glucose-stimulated insulin release after the induction of AP. Confocal microscopy demonstrated that most of the beta cells in pancreas of rat with AP expressed strong immunoreactivity for iNOS. This was further confirmed by biochemical and Western blot analysis that showed a marked increase in iNOS protein expression and enzyme activity concomitant with a modest reduction in the cNOS protein and activity. Although the mechanisms underlying the defective insulin secretory response of beta cells seen during the early stage of AP are complex, the present finding suggests that the expression of iNOS and a marked iNOS-derived NO production in the beta cells may play at least a contributory role in the impairment of beta-cell function.

Glucose-stimulated signaling pathways in biphasic insulin secretion.

Glucose-stimulated biphasic insulin secretion involves at least two signaling pathways, the KATP channel-dependent and KATP channel-independent pathways, respectively. In the former, enhanced glucose metabolism increases the cellular adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, closes KATP channels and depolarizes the cell. Activation of voltage-dependent Ca(2+) channels increases Ca(2+) entry and [Ca(2+)]i and stimulates insulin release. The KATP channel-independent pathways augment the response to increased [Ca(2+)]i by mechanisms that are currently unknown. However, they affect different pools of insulin-containing granules in a highly coordinated manner. The beta-cell granule pools can be minimally described as reserve, morphologically docked, readily and immediately releasable. Activation of the KATP channel-dependent pathway results in exocytosis of an immediately releasable pool that is responsible for the first phase of glucose-stimulated insulin release. Following glucose metabolism, the rate-limiting step for the first phase lies in the rate of signal transduction between sensing the rise in [Ca(2+)]i and exocytosis of the immediately releasable granules. The immediately releasable pool of granules can be enlarged by previous exposure to glucose (by time-dependent potentiation, TDP), and by second messengers such as cyclic adenosine monophosphate (cyclic AMP) and diacylglycerol (DAG). The second phase of glucose-stimulated insulin secretion is due mainly to the KATP channel-independent pathways acting in synergy with the KATP channel-dependent pathway. The rate-limiting step here is the conversion of readily releasable granules to the state of immediate releasability, following which, in an activated cell they will undergo exocytosis. In the rat and human beta-cell the KATP channel-independent pathways induce a time-dependent increase in the rate of this step that results in the typical rising second-phase response. In the mouse beta-cell the rate appears not to be changed much by glucose. Potential intermediates involved in controlling the rate-limiting step include increases in cytosolic long-chain acyl-CoA levels, adenosine triphosphate (ATP) and guanosine triphosphate (GTP), DAG binding proteins, including some isoforms of protein kinase (PKC), and protein acyl transferases. Agonists that can change the rate-limiting steps for both phases of insulin release include those like glucagon-like peptide 1 (GLP-1) that raise cyclic AMP levels and those like acetylcholine that act via DAG.

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release.

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.

Hyposmotic shock stimulates insulin secretion by two distinct mechanisms. Studies with the betaHC9 cell.

Exposure of betaHC9 cells to a Krebs-Ringer bicarbonate-HEPES buffer (KRBH) made hypotonic by a reduction of 25 mM NaCl resulted in a prompt stimulation of insulin release. The stimulation was transient, and release rates returned to basal levels after 10 min. The response resembles that of the first phase of glucose-stimulated insulin release. The response did not occur if the reduction in NaCl was compensated for by the addition of an equivalent osmolar amount of sorbitol, so the stimulation of release was due to the osmolarity change and not the reduction in NaCl. The hyposmotic shock released insulin in KRBH with or without Ca(2+). The L-type Ca(2+) channel blocker nitrendipine inhibited the response in normal KRBH but had no effect in KRBH without Ca(2+) despite the latter response being larger than in the presence of extracellular Ca(2+). Similar data were obtained with calciseptine, which also blocks L-type channels. The T-type Ca(2+) channel blocker flunarizine was without effect, as was the chloride channel blocker DIDS. In parallel studies, the readily releasable pool of insulin-containing granules was monitored. Immunoprecipitation of the target-SNARE protein syntaxin and co-immunoprecipitation of the vesicle-SNARE VAMP-2 was used as an indicator of the readily releasable granule pool. After hypotonic shock in the presence of extracellular Ca(2+), the amount of VAMP-2 coimmunoprecipitated by antibodies against syntaxin was much reduced compared with controls. Therefore, under these conditions, hypotonic shock stimulates exocytosis of the readily releasable pool of insulin-containing granules. No such reduction was seen in the absence of extracellular Ca(2+). In conclusion, after reexamination of the effect of hyposmotic shock on insulin secretion in the presence and absence of Ca(2+) (with EGTA in the medium), it is clear that two different mechanisms are operative under these conditions. Moreover, these two mechanisms may be associated with the release of two distinct pools of insulin-containing granules.

Identification of the docked granule pool responsible for the first phase of glucose-stimulated insulin secretion.

The mechanisms underlying the first phase of glucose-stimulated insulin release, the deterioration of which marks the early stages of both type 1 and type 2 diabetes, are essentially unknown. Among many hypotheses, one holds that the first phase is due to a readily releasable pool of insulin-containing granules. We used current knowledge of the mechanisms of exocytosis and the proteins involved in docking granules at the plasma membrane to test this hypothesis. A docked pool of readily releasable granules was identified by immunoprecipitation of the plasma membrane protein syntaxin with a specific antibody and by co-immunoprecipitation of soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25) and the granule proteins synaptobrevin and synaptotagmin. The four SNARE proteins co-immunoprecipitated each other, thus identifying the core complex associated with docked granules. Using co-immunoprecipitation as a marker for docked granules, we found that the docked pool was rapidly discharged during the first phase of glucose-stimulated insulin release and refilled during the second phase. Other secretagogues also released the pool, whereas the physiological inhibitor norepinephrine blocked its release. Further studies on the nature of this pool of granules should shed light on the causes of its deterioration in the early stages of diabetes and the reasons for deficient insulin release.

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: enzymatic, metabolic and cationic determinants.

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.

Augmentation of insulin release by glucose in the absence of extracellular Ca2+: new insights into stimulus-secretion coupling.

Glucose stimulates insulin secretion in the pancreatic beta-cell by means of a synergistic interaction between at least two signaling pathways. One, the K(ATP) channel-dependent pathway, increases the entry of Ca2+ through voltage-gated channels by closure of the K(ATP) channels and depolarization of the beta-cell membrane. The resulting increase in [Ca2+]i stimulates insulin exocytosis. The other, a K(ATP) channel-independent pathway, requires that [Ca2+]i be elevated and augments the Ca2+-stimulated release. These mechanisms are in accord with the belief that glucose-stimulated insulin secretion has an essential requirement for extracellular Ca2+ and increased [Ca2+]i. However, when protein kinases A and C are activated simultaneously, a large effect of glucose to augment insulin release can be seen in the absence of extracellular Ca2+, under conditions in which [Ca2+]i is not increased, and even when [Ca2+]i is decreased to low levels by intracellular chelation with BAPTA. In the presence or absence of Ca2+, there are similarities in the characteristics of augmentation of insulin release that suggest that only one augmentation mechanism may be involved. These similarities include time course, glucose dose-responses, augmentation by nutrients other than glucose such as alpha-ketoisocaproate (alpha-KIC), and augmentation by the fatty acids palmitate and myristate. However, augmentation in the presence and absence of Ca2+ is distinctly different in GTP dependency. Therefore, exocytosis under these two conditions appears to be triggered differently-one by Ca2+ and the other by GTP or a GTP-dependent mechanism. The augmentation pathways are likely responsible for time-dependent potentiation of secretion and for the second phase of glucose-stimulated insulin release.

Analysis of the Pancreatic β Cell in the Mouse with Targeted Disruption of the Pancreatic β Cell-Specific Glucokinase Gene

This is the first systematic study on the pancreatic β cell function in the heterozygous mouse with targeted disruption of the β cell glucokinase gene. The hetetrozygotes’ β cell displayed the following characteristics: (1) impaired glucose sensitivity with normal glucose responsiveness, (2) poor discrimination of α and β glucose anomers, and (3) normal response to glucose in the presence of 25 mM K+and 150 μM diazoxide. Both the first and the second phases of glucose-stimulated insulin release were depressed. Although the heterozygotes were mildly hyperglycemic, insulin treatment further suppressed β cell function, implying the β cell glucose toxicity is not the cause of impaired glucose sensitivity. The data are compatible with the glucokinase glucose sensor concept inasmuch as glucose sensitivity is reduced in the heterozygotes’ β cell. The anomeric malaise and preservation of the ATP-sensitive K+channel-independent glucose action were considered due to chronic hyperglycemia.

Dynamics of glucose-induced insulin release from mouse islets transplanted under the kidney capsule.

Mouse pancreatic islet grafts under the kidney capsule of syngeneic hosts were removed and perifused in vitro 1-40 weeks after the transplantation. In comparison with fresh islets, 12- to 40-week-old grafts exhibited an attenuated first phase of glucose-stimulated insulin release. In grafts 1, 12, 28, or 40 weeks old, but not in fresh islets, the mean secretory rate during the initial 10 min of stimulation was significantly lower than that during the subsequent 15 min. When expressed in relation to insulin content, the insulin output in response to 11 mmol/L glucose was no less from grafts than from fresh islets; in grafts 12 or 40 weeks old at 16.7 mmol/L glucose, the fractional output above baseline was significantly diminished during the initial 10 min, but not subsequently. Immediately on switching from basal to stimulatory glucose concentration, there was a transient drop in insulin secretion from the grafts, especially after more than 12 weeks of transplantation and in response to 16.7, as compared with 11, mmol/L glucose. When glucose was switched back from stimulatory to basal concentration, grafts also frequently exhibited a transient increase in the insulin secretory rate. Neither initial drops nor "off responses" were seen in untransplanted islets. The modifications of the secretory dynamics in islet grafts suggest that transplantation influences the balance between the stimulatory and inhibitory influences of glucose on the beta-cell's secretory machinery.

The role of islet secretory function in the development of diabetes in the GK Wistar rat

Insulin secretion and glucose metabolism were compared in islets isolated from GK Wistar rats (a non-obese, spontaneous model of non-insulin-dependent diabetes mellitus) and control Wistars aged 8 and 14 weeks. By 8 weeks of age, GK Wistar rats were clearly diabetic as indicated by non-fasting plasma glucose concentrations and impaired glucose tolerance. Islet insulin content was not significantly different to controls at either age. In islets from 14-week-old GK Wistar rats glucose-stimulated insulin release (6-16 mmol/l glucose) was significantly reduced to 25-50% of controls in static incubations (p < 0.001). In perifusion, glucose-stimulated insulin release was reduced by 90% for first phase (p < 0.01) and by 75% for second phase (p < 0.05). The responses to arginine and 2 alpha Ketoisocaproate in islets were similar to those in controls. In contrast, islets isolated from 8-week-old GK Wistar rats exhibited no significant reduction in glucose-stimulated insulin secretion in static incubations. In perifusion, although both first and second phases of glucose-stimulated insulin release were slightly reduced, these were not significantly different to controls. Islets from 8-week-old GK Wistar rats failed however to respond to stimulation by glyceraldehyde. Raising the medium glucose concentration to 16 mmol/l significantly increased rates of glucose utilisation ([3H] H2O production from 5-[3H] glucose) and oxidation ([14C] CO2 production from U-[14C] glucose) in islets isolated from 8-week-old control and GK Wistar rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of hexose transport, glycerol phosphate shuttle and Krebs cycle in islets of adult rats injected with streptozotocin during the neonatal period

At 3–4°C, the transport of 3-O- methyl- d - glucose (30 mM) was severely impaired in islets prepared from adult rats injected with streptozotocin during the neonatal period. However, at 37°C, the first and second phase of glucose-stimulated insulin release were decreased to the same relative extent in perifused islets of diabetic, as compared to control, animals. Moreover, the time-related increase in the oxidative response of the islets to 16.7 mM d-glucose was less pronounced in diabetic than control rats. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase in islet homogenates of diabetic rats only represented one-fifth of that found in control rats, whereas the activity of the cytosolic NAD-glycerophosphate dehydrogenase was comparable in both types of rats. This coincided with the fact that a rise in d-glucose concentration from 2.8 to 16.7 mM failed to increase significantly l-[2- 3H]glycerol conversion to 3HOH in islets from diabetic rats, in contrast to the situation found in control animals. The activity of 2-ketoglutarate dehydrogenase in islet homogenates when expressed per μg protein was not different in control and diabetic rats. Likewise, the ratio between d-[6− 14C]glucose oxidation and d-[3,4- 14C]glucose oxidation and the capacity of either a non-metabolized analog of l-leucine or 3-phenylpyruvate to preferentially stimulated d-[6− 14C]glucose oxidation relative to d-[5- 3H]glucose utilization were both unaffected in islets from diabetic rats. These findings argue against the existence of a primary defect in the Krebs cycle of diabetic rats. It is proposed that, despite an obvious alteration of the hexose transport system in the islet cells of diabetic rats, the preferential impairment of the B-cell secretory response to d-glucose, as distinct from other secretagogues, in this model of non-insulin-dependent diabetes is mainly attributable to the low activity of FAD-linked glycerophosphate dehydrogenase, resulting in a decreased metabolic flow through the glycerol phosphate shuttle and a reduced rate of aerobic glycolysis.

Inhibition of Glucose-Stimulated Insulin Release in the Perfused Rat Pancreas by Parathyroid Secretory Protein-I (Chromogranin-A)*

The effect of graded doses (10(-10)-10(8) M) of highly purified bovine parathyroid secretory protein-I (SP-I; chromogranin-A) or synthetic porcine pancreastatin on glucose-stimulated insulin release in the perfused rat pancreas was examined. SP-I (10(-9) M) inhibited the first phase of glucose-stimulated insulin release, and 10(-8) M SP-I inhibited both the first and second phases of glucose-stimulated insulin release; 10(-10) M SP-I was inactive. In comparison, pancreastatin at 10(-10) M inhibited the first phase of insulin release, and at 10(-9) and 10(-8) M, pancreastatin inhibited both phases of insulin release. The inhibition by SP-I was achieved at concentrations that normally exist in the general circulation of man. These and other data suggest that circulating SP-I plays a physiological role in the regulation of insulin secretion.

Inhibition of insulin release after passive transfer of immunoglobulin from insulin-dependent diabetic children to mice.

We used the mouse passive transfer model to test whether islet cell antibodies affect beta-cell function. The immunoglobulin (Ig) fraction of plasma from 5 islet cell surface antibody-positive, newly diagnosed insulin-dependent diabetic children or of a pool of plasma from 12 normal subjects was injected daily (7-16 mg IgG/day) for 14 days into normal immunosuppressed BALB/c mice. Insulin secretory responses in the Ig-injected mice were then examined by perfusing the rodent pancreata in vitro. Insulin release induced by 20 mmol/liter D-glucose during 30 min of stimulation decreased from 900 ng insulin (median; range, 814-1138) from pancreata of mice injected with control Ig to 511 ng (range, 130-786) from pancreata of mice injected with diabetic Ig (P less than 0.003). Both the initial peak and the sustained second phase of glucose-stimulated insulin release were depressed in 4 of the 5 pancreata from mice injected with diabetic Ig. These results indicate that circulating antibodies in diabetic children may alter beta-cell function and possibly contribute to the pathogenesis of insulin-dependent diabetes.

Chloride modulation of insulin release, 86Rb+ efflux, and 45Ca2+ fluxes in rat islets stimulated by various secretagogues.

Substitution of extracellular Cl- by impermeant isethionate (5 mM residual Cl-) caused a monophasic inhibition of glucose-stimulated insulin release, accompanied by an initial transient increase and a secondary lasting decrease in 86Rb+ efflux from perifused islets. Cl- reintroduction restored insulin release with an overshoot above control values and successively produced a small decrease and a large increase in efflux. Theophylline potentiated the insulinotropic effect of glucose more markedly at low Cl- than at normal Cl-, but did not restore a normal rate of 86Rb+ efflux. Lowering the concentration of Cl- did not alter the effect of glucose, tolbutamide, or arginine on 86Rb+ efflux, but simply shifted the efflux rates to lower values. The first phase of glucose-stimulated insulin release was not modified, but the second phase was inhibited. The insulinotropic effect of tolbutamide was augmented at low Cl- and that of arginine (at 7 mM glucose) was not affected. In incubated islets, the stimulation of insulin release by glyceraldehyde was barely inhibited when Cl- was substituted by isethionate and the marked decrease of the effect of glucose could be prevented by glutamine. In a glucose-free, low Cl- medium, the insulinotropic effect of leucine, arginine, and lysine was inhibited; this inhibition was reversed by glutamine, but not by theophylline. Lowering the concentration of Cl- had no effect on 45Ca2+ influx or efflux in the absence of glucose, did not alter the increase in influx and efflux during the first 5 min of glucose stimulation, but impaired both influx and efflux during the second phase. Leucine-induced 45Ca2+ uptake was inhibited at low Cl- and this inhibition was prevented by glutamine. In conclusion, islet cells possess a Cl- -activated modality of K efflux, which does not seem to play a role in the stimulus-secretion coupling. Since Cl- substitution by an impermeant anion does not inhibit the stimulation of insulin release by all agents, the role of Cl- ions does not appear to be restricted to a chemiosmotic mechanism of exocytosis. No single mechanism explains the multiple changes in B-cell function resulting from the decrease in Cl- concentration, but it is proposed that some of them could result from modifications of intracellular pH.

Effects of trifluoperazine and pimozide on stimulus-secretion coupling in pancreatic B-cells. Suggestion for a role of calmodulin?

The possible involvement of calmodulin in insulin release was evaluated by studying the effects on intact islets of trifluoperazine and pimozide, two antipsychotic agents known to bind strongly to calmodulin in cell-free systems. Trifluoperazine (10-100mum) produced a dose- and time-dependent inhibition of the two phases of glucose-stimulated insulin release. The effect was not reversible by simple washing of the drug, but could be prevented by cytochalasin B or theophylline. Trifluoperazine also inhibited the release induced by glyceraldehyde, oxoisocaproate, tolbutamide or barium, but not that stimulated by 10mm-theophylline or 1mm-3-isobutyl-1-methylxanthine. Pimozide (0.5-10mum) also produced a dose-dependent inhibition of insulin release triggered by glucose, leucine or barium, but did not affect the release induced by methylxanthines. Glucose utilization by islet cells was not modified by trifluoperazine (25mum), which slightly increased cyclic AMP concentration in islets incubated without glucose. The drug did not prevent the increase in cyclic AMP concentration observed after 10min of glucose stimulation, but suppressed it after 60min. Basal or glucose-stimulated Ca(2+) influx (5min) was unaffected by 25mum-trifluoperazine, whereas Ca(2+)net uptake (60min) was inhibited by 20%. Glucose-stimulated Ca(2+) uptake was almost unaffected by pimozide. In a Ca(2+)-free medium, trifluoperazine decreased Ca(2+) efflux from the islets and did not prevent the further decrease by glucose; in the presence of Ca(2+), the drug again decreased Ca(2+) efflux and inhibited the stimulation normally produced by glucose. In the absence of glucose, trifluoperazine lowered the rate of Rb(+) efflux from the islets, decreased Rb(+) influx (10min), but did not affect Rb(+) net uptake (60min). It did not interfere with the ability of glucose to decrease Rb(+) efflux rate further and to increase Rb(+) net uptake. The results show thus that trifluoperazine does not alter the initial key events of the stimulus-secretion coupling. Its inhibition of insulin release suggests a role of calmodulin at late stages of the secretory process.