Glucose regulation of arginine-induced pancreatic somatostatin release from the isolated perfused rat pancreas

The effects of glucose alone, combinations of glucose with arginine or tolbutamide and either arginine or tolbutamide alone, on somatostatin, insulin, and glucagon secretion were investigated using the isolated perfused rat pancreas. When glucose alone was raised in graded increments at 15-min intervals from an initial concentration of 0 mM to a maximum of 16.7 mM, somatostatin as well as insulin in the perfusate increased with the glucose, while glucagon decreased. The similarity of the glucose stimulated somatostatin and insulin release was especially evident when the perfusate glucose was increased from an initial dose of 4.4 mM rather than 0 mM to 8.8 mM or 16.7 mM. In addition, glucose at concentrations varying from 4.4 mM to 11 mM dose-dependently enhanced arginine-induced somatostatin and insulin release and suppressed glucagon release dose-dependently as before. Arginine in the absence of glucose was not capable of stimulating somatostatin secretion whereas tolbutamide, in contrast, was capable of stimulating somatostatin secretion even in the absence of glucose.     

Effects of exogenous somatostatin and insulin on islet amyloid polypeptide (amylin) release from perfused rat pancreas.

This study examined the effects of exogenous somatostatin and insulin on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas. Somatostatin inhibited the release of both amylin and insulin from the perfused pancreas to the same extent. The infusion of 10 nM somatostatin resulted in 40% inhibition of the secretion of both amylin and insulin induced by 11.1 mM glucose and 10 mM arginine, and this inhibition was significantly increased to 70% by the infusion of 100 nM somatostatin (p less than 0.05). The amylin/insulin molar ratios remained constant at 0.8% and were not changed by the infusion of somatostatin. On the other hand exogenous insulin at a concentration of 1.8 nM did not affect the release of amylin induced by 11.1 mM glucose and 10 mM arginine, whereas 180 nM insulin slightly, although not significantly, inhibited the release of amylin by 15%. These findings suggest that the release of amylin may be negatively regulated by somatostatin and that circulating insulin may have no direct effect on the release of amylin at least at a physiological concentration.     

Regulation of immunoreactive-insulin release from a rat cell line (RINm5F).

1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.     

Interaction of somatostatin and calcium in regulating insulin release from isolated pancreatic islets of rats.

The effect of synthetic somatostatin on insulin release was studied in vitro by using isolated islets of rats. Somatostatin, with concentrations from 10 ng/ml to 10μg/ml, inhibited insulin release induced by 16.7 mM glucose. Insulin release elicited by 10 μg/ml glucagon or 2 mM dibutyryl cyclic AMP was likewise inhibited by 100ng/ml somatostatin. By raising the calcium concentration of the incubation medium to 6 mM, glucose-induced insulin release was fully restored even in the presence of somatostatin.However, the same maneuver only partially counteracted the somatostatin inhibition of dibutyryl cyclic AMP-induced insulin release. These results suggest the involvement of calcium mobilization process in the inhibitory action of somatostatin.     

Exendin-4 dose-dependently stimulates somatostatin and insulin secretion in perfused rat pancreas.

We have investigated the effect of exendin-4, a GLP-1 analogue, on somatostatin and insulin secretion in perfused rat pancreas. At constant glucose concentration within the type 2 diabetic range (9 mM), exendin-4 stimulated somatostatin and insulin secretion in a dose-dependent manner. Dose-response curves were sigmoidal (R (2) = 0.9954 and R (2) = 0.9973, respectively; p < 0.01) and the EC (50) was 4.3 nM for somatostatin secretion and 1.4 nM for insulin secretion. Exendin-4 stimulated somatostatin output at low (3.2 mM), normal (5.5 mM) and high (9 mM) glucose concentrations, while the insulinotropic effect of exendin-4 was not found at low glucose levels. On the other hand, exendin-4 potentiated somatostatin and insulin responses to an increase in perfusate glucose levels, and to arginine and carbachol. Finally, the insulinotropic effect of exendin-4 was maintained in the absence of a somatostatin response as induced by cysteamine pretreatment, indicating a direct effect of exendin-4 on the B-cell. In summary, exendin-4 behaves as a general stimulatory agent of both insulin and somatostatin release in the perfused rat pancreas. Given that exendin-4 has also been shown to increase gastric somatostatin secretion, it is tempting to speculate that exendin-4 might behave as a general stimulator of D-cell function in other tissues, a point worthy of further investigation.     

Modulation of somatostatin release by endogenous glucagon and insulin: physiological relationship between A, B and D cells in rat pancreatic islets

In order to understand the physiological role of endogenous insulin or glucagon in somatostatin release, isolated rat pancreatic islets were treated with antiinsulin or antiglucagon antiserum in the presence of physiological amounts of glucose. The release of somatostatin was unchanged by treatment with antiinsulin antiserum which neutralized insulin released by 3.3, 8.3 and 16.7 mM of glucose. However, somatostatin release after treatment with antiglucagon antiserum was much reduced at all concentrations of glucose when compared with the release from control serum. Exogenous rat insulin (0.11, 1.11 micrograms/ml) had no effect, but exogenous glucagon (1, 5 micrograms/ml) resulted in a significant increase. Somatostatin release was stimulated by glucose, but the effect was insignificant. These results clearly indicate the physiological role of endogenous glucagon in the modulation of somatostatin release from the islets of Langerhans. Furthermore, the physiological relationship between A, B and D cells may be mediated through the paracrine mechanism.     

Insulin release from collagenase-isolated islets of rat pancreas in the presence of cyclic AMP and somatostatin.

In order to study the role of cyclic AMP in the inhibition by somatostatin of glucose-induced insulin release, the effect of somatostatin on the potentiation by dibutyryl-cyclic AMP (db-cAMP) of insulin release from isolated pancreatic islets of rats was examined. Isolated islets were obtained from the rat pancreas by the collagenase method. Ten islets were incubated for periods of 30 min in Krebs-Ringer bicarbonate buffer containg albumin and glucose 2.0 mg/ml in the presence or absence of somatostatin (1 microgram/ml or 100 ng/ml) and/or db-cAMP 1 mM. Glucose-induced insulin release was reduced by somatostatin in concentrations of 1 microgram/ml. Somatostatin in a concentration of 100 ng/ml significantly abolished the potentiation by db-cAMP of insulin release (p less than 0;01), in spite of exerting no inhibition of glucose-induced insulin release. However, in the presence of theophylline 5 mM, somatostatin 100 ng/ml did not show that inhibitory effect on the potentiated insulin release.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets.

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumulation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5--1 microgram/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 microgram/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 microgram/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 microgram/ml). Somatostatin (1 microgram/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated. The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Inhibition of the glucose induced insulin release by somatostatin in the isolated perfused rat pancreas. Action of cyclic AMP, glucagon and glibenclamide.

Insulin release in the perfused isolated rat pancreas was measured after stimulation with 16.5 mM glucose with and without somatostatin (cycle form, 100 ng/ml) in the medium. A complete blockage of the typical biphasic pattern of insulin release ocurred with somatostatin in the medium. Such blockage was abolished when cAMP (2.5 mM) and a 0.5 ml solution of glucagon (1 mg/ml) were continuously perfused for 20-minute periods and for 30-second periods correspondently. It did not take place when glibenclamide (HB-419) was perfused for a 20-minute period at a rate of 10 mug/ml. The results suggest that the adenylcyclase dependent mechanisms of glucose-induced insulin release are involved in the inhibition of the glucose-induced insulin secretion by somatostatin.     

Vasoactive intestinal polypeptide enhances hormone content and insulin release in cultured fetal rat islets

The effect of porcine vasoactive intestinal polypeptide (VIP) on development of the biphasic insulin release response in cultured fetal rat islets was investigated. Fetal islets, 21.5 days gestational age, were cultured for 7 days in RPMI 1640 culture medium containing either 2.8 or 11.1 mM glucose adn subsequently challenged with 16.7 mM glucose in a perfusion system. Islets were exposed to VIP at a final concentration of 13.2 nM by adding the peptide to the perifusion buffer (acute exposure) or by adding it to the culture medium throughout the culture period (chronic exposure). Islet hormone and DNA contents were also quantitated at the end of the culture period. Acute exposure to VIP resulted in no alterations of the insulin release pattern after culture in the presence of either glucose concentration. However, chronic treatment of islets with 13.2 nM VIP in the presence of 2.8 mM glucose resulted in significant increases in the maximum rate of insulin release during the first phase and the total amount of insulin release during both phases. Similarly, islets cultured in the presence of 11.1 mM glucose and 13.2 nM VIP demonstrated enhanced biphasic insulin release patterns with increased maximum rate and total amount of release during both phases. The presence of VIP and 2.8 mM glucose increased islet glucagon and somatostatin contents, but islet DNA and insulin contents remained unchanged. These findings indicate that VIP plays a significant role in the in vitro development of the biphasic insulin release pattern and may be a factor controlling the maturation of the fetal islet in vivo.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5–1 μg/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 μg/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 μg/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 μg/ml). Somatostatin (1 μg/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated.The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Isolated mouse islets respond to glucose with an initial peak of glucagon release followed by pulses of insulin and somatostatin in antisynchrony with glucagon

Recent studies of isolated human islets have shown that glucose induces hormone release with repetitive pulses of insulin and somatostatin in antisynchrony with those of glucagon. Since the mouse is the most important animal model we studied the temporal relation between hormones released from mouse islets. Batches of 5-10 islets were perifused and the hormones measured with radioimmunoassay in 30s fractions. At 3mM glucose, hormone secretion was stable with no detectable pulses of glucagon, insulin or somatostatin. Increase of glucose to 20mM resulted in an early secretory phase with a glucagon peak followed by peaks of insulin and somatostatin. Subsequent hormone secretion was pulsatile with a periodicity of 5min. Cross-correlation analyses showed that the glucagon pulses were antisynchronous to those of insulin and somatostatin. In contrast to the marked stimulation of insulin and somatostatin secretion, the pulsatility resulted in inhibition of overall glucagon release. The cytoarchitecture of mouse islets differs from that of human islets, which may affect the interactions between the hormone-producing cells. Although indicating that paracrine regulation is important for the characteristic patterns of pulsatile hormone secretion, the mouse data mimic those of human islets with more than 20-fold variations of the insulin/glucagon ratio. The data indicate that the mouse serves as an appropriate animal model for studying the temporal relation between the islet hormones controlling glucose production in the liver.     

Somatostatin inhibition of insulin release from freshly isolated and organ cultured rat islets of langerhans in vitro

1×10^?6M somatostatin causes a 37–44% inhibition of glucose induced insulin release from freshly isolated rat islets of Langerhans. A 81 to 95% inhibition is observed when the isolated islets are maintained in organ culture for 2 days prior to the somatostatin treatment. The dose curve of somatostatin on cultured islets shows an apparent K_I of 1.4×10^?9. The tetradecapeptide also causes a reversible inhibition of the stimulation of insulin release by 5 mM theophylline and 23 mM K⁺.     

Suppression of insulin release by galanin and somatostatin is mediated by a G-protein. An effect involving repolarization and reduction in cytoplasmic free Ca2+ concentration

The effects of galanin and somatostatin on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration [( Ca2+]i) were investigated using beta-cells isolated from obese hyperglycemic mice. Whereas insulin release was measured in a column perifusion system, membrane potential and [Ca2+]i were measured with the fluorescent indicators bisoxonol (bis-(1,3-diethylthiobarbiturate)trimethineoxonol) and quin 2, in cell suspensions in a cuvette. Galanin (16 nM) and somatostatin (400 nM) suppressed glucose-stimulated insulin release in parallel to promoting repolarization and a reduction in [Ca2+]i. The reduction in [Ca2+]i comprised an initial nadir followed by a slow rise and the establishment of a new steady state level. The slow rise in [Ca2+]i was abolished by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. Both peptides suppressed insulin release even when [Ca2+]i was raised by 25 mM K+. Under these conditions the inhibition of insulin release was partly reversed by an increase in the glucose concentration. Addition of 5 mM Ca2+ to a cell suspension, incubated in the presence of 20 mM glucose and either galanin, somatostatin, or the alpha 2-adrenergic agonist clonidine (10 nM), induced oscillations in [Ca2+]i, this effect disappearing subsequent to the addition of D-600. The effects of galanin, somatostatin, and clonidine on [Ca2+]i were abolished in beta-cells treated with pertussis toxin. In accordance with measurements of [Ca2+]i, treatment with pertussis toxin reversed the inhibitory effect of galanin on insulin release. The inhibitory action of galanin and somatostatin on insulin release is probably accounted for by not only a repolarization-induced reduction in [Ca2+]i and a decreased sensitivity of the secretory machinery to Ca2+, but also by a direct interaction with the exocytotic process. It is proposed that these effects are mediated by a pertussis toxin-sensitive GTP-binding protein.     

Somatostatin-induced inhibition of insulin secretion from isolated islets of rat pancreas in presence of glucagon.

In order to study the oeffect of somatostatin on the endocrine pancreas directly, islets isolated from rat pancreas by collagenase were incubated for 2 hrs 1) at 50 and 200 mg/100 ml glucose in the absence and presence of somatostatin (1, 10 and 100 mg/ml) and2) at 200 mg/100 ml glucose together with glucagon (5 mug/ml), with or without somatostatin (100 ng/ml). Immunologically measurable insulin was determined in the incubation media at 0, 1 and 2 hrs. Insulin release was not statistically affected by any concentration stomatostatin. On the other hand, somatostatin exerted a significant inhibitory action on glucagon-potentiated insulin secretion (mean +/- SEM, mu1/2 hrs/10 islets: glucose and glucagon: 1253 +/- 92; glucose, glucagon and somatostatin: 786 +/- 76). The insulin output in th epresence of glucose, glucagon and somatostatin was also significantly smaller than in thepresence of glucose alone (1104 +/- 126) or of glucose and somatostatin (1061 +/- 122). The failure of somatostatin to affect glucose-stimulated release of insulin from isolated islets contrasts its inhibitory action on insulin secretion as observed in the isolated perfused pancreas and in vivo. This discrepancy might be ascribed to the isolation procedure using collagenase. However, somatostatin inhibited glucagon-potentiated insulin secretion in isolated islets which resulted in even lower insulin levels than obtained in the parallel experiments without glucagon. It is concluded that the hormone of the alpha cells, or the cyclic AMP system, might play a part in the machanism of somatostatin-induced inhibition of insulin release from the beta-cell.     

The effect of ventromedial hypothalamic nuclei destruction on somatostatin and insulin release from the isolated perfused rat pancreas

The effect of electrolytic lesions in the ventromedial hypothalamic nuclei (VMH) on somatostatin and insulin release was studied using the isolated perfused rat pancreas. Obesity gradually developed in the rats after placement of the VMH lesions, and fasting insulin levels determined immediately before the isolation of the pancreas were significantly higher than those in sham-operated controls. In the presence of 4.4 mM glucose, both perfusate somatostatin and insulin responses to arginine were significantly greater than in the controls, suggesting that VMH lesions cause not only hypersecretion of insulin but hypersecretion of somatostatin as well.     

Effects of porcine diazepam-binding inhibitor on insulin and glucagon secretion in vitro from the rat endocrine pancreas.

Porcine diazepam-binding inhibitor (pDBI) is a novel peptide that has been isolated from the small bowel of the pig, and that occurs also in the islet D-cells. We have studied its effects on hormone release in vitro from the endocrine pancreas of the rat. In isolated islets, pDBI (10(-9)-10(-6)M) did not affect basal insulin release at 3.3 mM glucose, whereas stimulated release at 8.3 mM glucose was dose-dependently suppressed by 32-69% (P less than 0.01). Furthermore, insulin secretion stimulated by either 16.7 mM glucose or 1 mM IBMX (3-isobutyl-1-methylxanthine) or 1 micrograms/ml glibenclamide was suppressed by pDBI at 10(-8) M (by 28-30%, P less than 0.05) and 10(-7) M (by 43-47%, P less than 0.01). In contrast, islet insulin secretion induced by 20 mM arginine was unaffected by these concentrations of pDBI. In the perfused rat pancreas, pDBI (10(-8) M) enhanced by 30% (P less than 0.05) the first phase (0-5 min) of arginine-stimulated insulin release, whereas the second phase (5-20 min) was unchanged. Moreover, pDBI suppressed by 28% (P less than 0.05) the second phase of arginine-induced glucagon release. Arginine-induced somatostatin release was not significantly affected by the peptide. Since pDBI immunoreactivity has been localized also to islet D-cells, the present results suggest that pDBI may act as a local modulator of islet hormone release.     

Homologous pancreastatin inhibits insulin secretion without affecting glucagon and somatostatin release in the perfused rat pancreas.

The identification of pancreastatin in pancreatic extracts prompted the investigation of its effects on islet cell function. However, in most of the investigations to date, pig pancreastatin was tested in heterologous species. Since there is great interspecies variability in the amino acid sequence of pancreastatin, we have investigated the influence of rat pancreastatin on insulin, glucagon and somatostatin secretion in a homologous animal model, namely the perfused rat pancreas. During 5.5 mM glucose infusion, pancreastatin (40 nM) inhibited insulin secretion (ca. 40%, P less than 0.025) as well as the insulin responses to 10 mM arginine (ca. 50%, P less than 0.025) and to 1 nM vasoactive intestinal polypeptide (ca. 50%; P less than 0.05). Pancreastatin failed to significantly modify glucagon or somatostatin release under any of the above experimental conditions. In addition, a lower pancreastatin concentration (15.7 nM) markedly suppressed the insulin release evoked by 11 mM glucose (ca. 85%, P less than 0.05). Our present observations reinforce the concept that pancreastatin is an effective inhibitor of insulin secretion, influencing the B-cell function directly and not through an A-cell or D-cell paracrine effect.     

The effects of d- and l-glyceraldehyde on glucose oxidation, insulin secretion and insulin biosynthesis by pancreatic islets of the rat

d-glyceraldehyde stimulated insulin secretion from isolated rat pancreatic islets in static incubation and perifusion systems. At low concentrations (2–4 mM) d-glyceraldehyde was a more potent secretagogue than glucose. The insulinotropic action of 15 mM d-glyceraldehyde was not affected by d-mannoheptulose, was potentiated by cytochalasin B (5 μg/ml) and theophylline (4 mM), and was inhibited by both adrenalin (2 μM) and somatostatin (10 μg/ml). D-glyceraldehyde at a concentration of 1.5 mM produced a 10-fold increase of l-[4,5-³ H]leucine incorporation into proinsulin and insulin without a significant increase into other islet proteins. Glucose at 1.5 mM did not stimulate proinsulin biosynthesis. d-Glyceraldehyde at concentrations higher than 1.5 mM, in marked contrast to glucose, progressively inhibited incorporation of labelled leucine into proinsulin + insulin and other islet proteins. d-glyceraldehyde also inhibited the oxidation of glucose. l-Glyceraldehyde did not stimulate proinsulin biosynthesis and had less effect than the d-isomer on insulin release and glucose oxidation. The results strongly suggest that metabolites below d-glyceraldehyde-3-P are signals for insulin biosynthesisand release. Interaction of d-glyceraldehyde with a “membrane receptor” cannot, however, be excluded with certainty.     

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.