Investigations on isolated islets of langerhans in vitro: 16. Modification of the glucose-dependent inhibition of glucagon secretion

Investigation of glucagon secretion in isolated Wistar rat islets was carried out to elucidate further the regulatory function of glucose and arginine on pancreatic A-cells. The suppressive effect of D-glucose could also be demonstrated with L-glucose, D-mannose, D-fructose, D-galactose, D-glyceraldehyde and DL-dihyroxyacetone, but not in the presence of 3-O-methylglucose or mannitol. Sugars other than D-glucose inhibited glucagon secretion only at much higher concentrations than those at which D-glucose was effective. Furthermore, although 7.5 mM D-glucose caused up to 80% inhibition, the effects of other sugars appeared to level off at only 50–60% inhibition. The inhibitory action of D-glucose or D-glyceraldehyde on glucagon secretion could not be overcome by L-arginine, but 3-O-methylglucose, mannoheptulose, 2-deoxy-D-glucose, iodoacetamide, theophylline, epinephrine and acetylcholine were effective. The insulin secretion in response to glucose was inhibited by the metabolic inhibitors used, whereas the B-cell response in the presence of glyceraldehyde was diminished by iodoacetamide only. Like D-glucose, a variety of other sugars markedly reduced the stimulatory effect of L-arginine in glucagon release.The results show that the suppression of glucagon secretion is not specific for D-glucose and not strongly connected on a stimulated insulin secretion.     

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets.

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. D-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only D-glucose and D-mannose were able to stimulate insulin release and cyclic [3H]AMP accumulation in the absence of other substrate. D-fructose had a stimulatory effect in the presence of 3.3 mM D-glucose only at a high concentration (33.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. D-Galactose was effective only together with 8.3 mM D-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [3H]AMP accumulation, was glucose-mannose-fructose-galactose. L-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM D-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [3H]AMP response. D-mannoheptulose and D-glucosamine inhibited the insulin and cyclic [3H]AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the beta-cell.     

Secretin uncouples glucose inhibition of glucagon-producing cells resulting in a simultaneous stimulation of both glucagon and insulin release

The effect of secretin on glucagon and insulin release and its interaction with glucose has been studied in cultured mouse pancreatic islets by column perifusion. Glucose alone showed the well-known stimulation of insulin release and inhibition of glucagon release. Addition of 10 mM secretin increased glucagon secretion at 3 mM D-glucose by 300% while no change in insulin release could be seen at this low glucose concentration. At maximal stimulation of insulin release by 20 mM D-glucose addition of 10 nM secretin increased insulin release by 30%. Despite this insulin concentration and the high glucose concentration an increase in glucagon secretion of 1800% was found. These effects of secretin were dose-dependent at 10 mM D-glucose with 1 nM secretin being the lowest effective dose.     

Stimulus-secretion coupling of arginine-induced insulin release. Functional response of islets to L-arginine and L-ornithine

L-Arginine and L-ornithine stimulate insulin release from pancreatic islets exposed to D-glucose. This coincides with an increased outflow of 86Rb and 45Ca from prelabelled islets and an increased net uptake of 45Ca by the islets. In the presence of D-glucose, L-lysine stimulates insulin secretion to the same extent as L-arginine or L-ornithine, but the hormonal release is not further enhanced by combinations of these cationic amino acids. L-Arginine or L-ornithine failed to enhance insulin release evoked by either L-leucine or 2-ketoisocaproate. The inhibitor of ornithine decarboxylase D,L-alpha-difluoromethyl ornithine failed to affect the metabolism and insulinotropic action of D-glucose in pancreatic islets, and only caused a partial inhibition of the secretory response to either L-arginine or L-ornithine. The latter amino acids inhibited modestly but significantly D-glucose utilization and oxidation by pancreatic islets. These and complementary findings suggest that the secretory response to L-arginine and L-ornithine is not attributable to any major change in the overall oxidative catabolism of nutrients, but involves mainly a biophysical component, such as the depolarization of the plasma membrane by these cationic amino acids.     

The Nitric Oxide Synthase Inhibitor NG-nitro-L-Arginine Methyl Ester Potentiates Insulin Secretion Stimulated by Glucose and L-Arginine Independently of its Action on ATP-Sensitive K+ Channels

The nature of the action of the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) on hormone release from isolated islets was investigated. We found that glucose-induced insulin release was potentiated by L-NAME in the absence or presence of diazoxide, a potent channel opener, as well as in the presence of diazoxide plus a depolarizing concentration of K⁺. At a low, physiological glucose concentration L-NAME did not influence insulin secretion induced by K⁺ but inhibited glucagon secretion. L-arginine-induced insulin release was potentiated by L-NAME. This potentiation was observed also in the presence of K⁺ plus diazoxide. Further, glucagon release induced by L-arginine as well as by L-arginine plus K⁺ and diazoxide was suppressed by L-NAME. The results strongly suggest that the L-NAME-induced potentiation of insulin secretion in response to glucose or L-arginine as well as the inhibitory effects on glucagon secretion are largely mediated by L-NAME directly suppressing islet NOS activity. Hence NO apparently affects insulin and glucagon secretion independently of membrane depolarization events.     

Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. A new mechanism of insulin resistance

We have previously shown in primary cultured rat adipocytes that insulin acts at receptor and multiple postreceptor sites to decrease insulin's subsequent ability to stimulate glucose transport. To examine whether D-glucose can regulate glucose transport activity and whether it has a role in insulin-induced insulin resistance, we cultured cells for 24 h in the absence and presence of various glucose and insulin concentrations. After washing cells and allowing the glucose transport system to deactivate, we measured basal and maximally insulin-stimulated 2-deoxyglucose uptake rates (37 degrees C) and cell surface insulin binding (16 degrees C). Alone, incubation with D-glucose had no effect on basal or maximal glucose transport activity, and incubation with insulin, in the absence of glucose, decreased maximal (but not basal) glucose transport rates only 18% at the highest preincubation concentration (50 ng/ml). However, in combination, D-glucose (1-20 mM) markedly enhanced the long-term ability of insulin (1-50 ng/ml) to decrease glucose transport rates in a dose-responsive manner. For example, at 50 ng/ml preincubation insulin concentration, the maximal glucose transport rate fell from 18 to 63%, and the basal uptake rate fell by 89%, as the preincubation D-glucose level was increased from 0 to 20 mM. Moreover, D-glucose more effectively promoted decreases in basal glucose uptake (Ki = 2.2 +/- 0.4 mM) compared with maximal transport rates (Ki = 4.1 +/- 0.4 mM) at all preincubation insulin concentrations (1-50 ng/ml). Similar results were obtained when initial rates of 3-O-methylglucose uptake were used to measure glucose transport. D-glucose, in contrast, did not influence insulin-induced receptor loss. In other studies, D-mannose and D-glucosamine could substitute for D-glucose to promote the insulin-induced changes in glucose transport, but other substrates such as L-glucose, L-arabinase, D-fructose, pyruvate, and maltose were without effect. Also, non-metabolized substrates which competitively inhibit D-glucose uptake (3-O-methylglucose, cytochalasin B) blocked the D-glucose plus insulin effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of glicentine on insulin secretion

The glucagon-like immunoreactivity of the gastrointestinal tract is heterogeneous, probably including several different peptides. One of these peptides, glicentine, has recently been extracted and highly purified. Furthermore, by immunocytochemistry a glicentine-like peptide has been reported to occur in the glucagon cell of the pancreatic islets. In the present study we investigated the effects of pure glicentine on insulin release in vivo in mice. The effects were compared with effects of two other peptides, glucagon and GIP. It was found that glicentine had no influence on basal insulin secretion. This was in contrast to equimolar doses of glucagon and GIP, which both stimulated the secretion of insulin. Glucose-induced insulin release was partially inhibited by glicentine. D-glucose, in a dose selected to give a response of 25% of its maximal, raised the plasma insulin concentrations by 44.0 +/- 5.9 microU/ml. The corresponding rise for glicentine plus D-glucose was 22.3 +/- 3.7 microU/ml, i.e. glicentine inhibited glucose-induced insulin released by about 50% (p < 0.01). GIP, on the other hand, enhanced glucose-induced insulin release. This enhancement was diminished by glicentine, a reflection of the inhibition by glicentine of the glucose-induced insulin release. Neither glicentine nor GIP in the doses tested had any effect on insulin secretion induced by cholinergic stimulation. In conclusion, glicentine seems to have no effect on basal insulin release in the mouse, but it partially inhibits glucose-induced insulin secretion. Thus, if the recently demonstrated glicentine-like peptide in the glucagon cell is authentic glicentine, the glucagon cell of the pancreatic islets may contain peptides with stimulatory (glucagon) as well as inhibitory (glicentine) effects on insulin secretion induced by glucose.     

Effects of salicylate on insulin and glucagon secretion by the isolated and perfused rat pancreas

The effects of sodium salicylate, a prostaglandin synthesis inhibitor, on glucose-induced secretion of insulin and glucagon by the isolated perfused rat pancreas have been studied. Sodium salicylate inhibited both basal (2.8 mM glucose) and stimulated (16.7 mM glucose) insulin release in a dose dependent manner (1, 5 and 10 mM). This inhibition is not interpretable in terms of a simple inhibition of cyclooxygenase by sodium salicylate. Basal glucagon release was not changed by 1 mM sodium salicylate but the latter partially blocked its inhibition by 16.7 mM glucose. Higher doses of sodium salicylate (5 and 10 mM) inhibited basal glucagon secretion without affecting its response to 16.7 mM glucose. These findings suggest a predominant stimulatory action of endogenous prostaglandins on glucagon release.     

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.     

Exchange of 3-O-methylglucose in isolated fat cells. Concentration dependence and effect of insulin.

3-O-[14C]Methylglucose was used to study the insulin action on the sugar transport in white fat cells. The experiments comprised determinations of the 3-O-methylglucose space at stationary distribution, of the rate constants for 3-O-methylglucose equilibrium exchange under various conditions, and of the 3-O-methylglucose inhibition of the lipogenesis from glucose. The following was found. The intracellular distribution space for 3-O-methylglucose at equilibrium was unaffected by insulin and was identical with the intracellular 3H2O space. The half-time for the equilibrium exchange of 3-O-methylglucose at a concentration of 25 mM was about 240 s in the absence of insulin and about 15 s with insulin (0.7 muM) present. Addition of phloridzin (5 mM) decreased the rate of the exchange process about 25-fold in both cases. The self-exchange of 3-O-methylglucose (1 mM) was at least 50 times faster than the self-exchange of L-glucose (1 mM). The concentration dependence of the 3-O-methylglucose exchange rate was approximately hyperbolic both in the absence and the presence of insulin, although the saturation of the transport mechanism at high concentrations of sugar was not as complete as predicted. In the absence of insulin the estimate of the half-saturation constant (Kt) was about 5 mM; that of the maximal exchange rate (Vmax) varied from 0.07 mmol s-1/liter of intracellular water to 0.2 mmol s-1 liter-1. In the presence of insulin Kt remained about 5 mM, whereas Vmax was increased to about 1.7 mmol s-1 liter-1. The latter estimate was reproducible within about 20%. The incorporation of trace amounts of [U-14C]glucose into intracellular lipids was inhibited by unlabeled 3-O-methylglucose pre-equilibrated over the membrane. The inhibition constant estimated from such experiments was about 5 mM both in the absence and the presence of insulin, and the insulin-induced increase in the rate of glucose incorporation was similar to the increase in the rate of the 3-O-methylglucose exchange process. It is concluded that exchange of 3-O-methylglucose proceeds via a mechanism which shows stereospecificity and saturability and that insulin acts by increasing the maximal transport capacity without changing the half-saturation constant.     

Islet amyloid polypeptide inhibits glucagon release and exerts a dual action on insulin release from isolated islets

We have studied the influence of a wide concentration range of islet amyloid polypeptide (IAPP) on both glucagon and insulin release stimulated by various types of secretagogues. In an islet incubation medium devoid of glucose, the rate of glucagon release being high, we observed a marked suppressive action by low concentrations of IAPP, 10(-10) and 10(-8) M, on glucagon release. Similarly, glucagon release stimulated by L-arginine, the cholinergic agonist carbachol, or the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX), an activator of the cyclic AMP system, was inhibited by IAPP in the 10(-10) and 10(-8) M concentration range. Moreover, basal glucagon release at 7 and 10 mM glucose was suppressed by IAPP. In contrast, IAPP exerted a dual action on insulin release. Hence, low concentrations of IAPP brought about a modest increase of basal insulin secretion at 7 mM glucose and also of insulin release stimulated by carbachol. High concentrations of IAPP, however, inhibited insulin release stimulated by glucose (10 and 16.7 mM), IBMX, carbachol and L-arginine. In conclusion, our data suggest that IAPP has complex effects on islet hormone secretion serving as an inhibitor of glucagon release and having a dual action on insulin secretion exerting mainly a negative feedback on stimulated and a positive feedback on basal insulin release.     

Effect of glucocorticoids on the glucose transport system of isolated fat cells.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.     

The dual effect of alloxan modulated by 3-O-methylglucose or somatostatin on insulin secretion in the isolated perfused rat pancreas

While alloxan treatment stimulated insulin secretion, alloxan pretreatment reduced arginine and glucose-induced insulin secretion in the isolated perfused rat pancreas. The transient insulin secretion by alloxan was inhibited by 3-O-methylglucose and somatostatin. Diminished insulin response to arginine and glucose induced by pretreatment with alloxan was restored by the addition of 3-O-methylglucose, whereas the addition of somatostatin did not improve the impaired insulin secretion. These results indicate that alloxan induced insulin secretion is not due to an uncontrolled leakage, but that the stimulatory and inhibitory action of alloxan on insulin secretion might be initiated by the binding of alloxan to the hexose transport site.     

Role of insulin and glucagon in oxytocin effects on glucose metabolism.

Oxytocin (OT) infusion in normal dogs increases plasma insulin and glucagon levels and increases rates of glucose production and uptake. The purpose of this study was to determine whether the effects of OT on glucose metabolism were direct or indirect. The studies were carried out in normal, unanesthetized dogs in which OT infusion was superimposed on infusion of either somatostatin, which suppresses insulin and glucagon secretion, or clonidine, which suppresses insulin secretion only. Infusion of 0.2 microgram/kg/min of somatostatin suppressed basal levels of plasma insulin and glucagon and inhibited the OT-induced rise of these hormones by about 60-80% of that seen with OT alone. The rates of glucose production and uptake by tissues, measured with [6-3H] glucose, were significantly lower than those seen with OT alone, and the rise in glucose clearance was completely inhibited. Clonidine (30 micrograms/kg, sc), given along with an insulin infusion to replace basal levels of insulin, completely prevented the OT-induced rise in plasma insulin and markedly reduced the glucose uptake seen with OT alone, but did not reduce the usual increase in plasma glucose and glucagon levels or glucose production. To determine whether the OT-induced rise in plasma insulin was in response to the concomitant increase in plasma glucose, similar plasma glucose levels were established in normal dogs by a continuous infusion of glucose and an OT infusion was superimposed. OT did not raise plasma glucose levels further, but plasma insulin levels were increased, indicating that OT can stimulate insulin secretion independently of the plasma glucose changes. Studies by others have shown that the addition of OT to pancreatic islets or intact pancreas can stimulate insulin and glucagon secretion, indicating a direct effect. Our studies agree with that and suggest that in vivo, OT raises plasma insulin levels, at least in part, through a direct action on the pancreas. These studies also show that OT increases glucose production by increasing glucagon secretion and, in addition, a direct effect of OT on glucose production is likely. The OT-induced increase in glucose uptake is mediated largely by increased insulin secretion.     

Alternate models for shared carriers or a single maturing carrier in hexose uptake into rabbit jejunum in vitro

The uptake (tissue accumulation) of three hexoses into rabbit jejunum was measured in a flux chamber in conditions of effective stirring. Glucose uptake was inhibited by galactose or 3-O-methylglucose: 1-40 mM galactose caused a progressive decline in glucose uptake; 1-5 mM 3-O-methylglucose inhibited glucose uptake but higher concentrations of 3-O-methylglucose had no further effect. When 1-40 mM 3-O-methylglucose was added to glucose plus galactose there was a further decrease in the uptake of glucose; adding 1-40 mM galactose to glucose plus 3-O-methylglucose also produced a decrease in glucose uptake. Both glucose and 3-O-methylglucose inhibited uptake of galactose but the pattern of inhibition varied between the two sugars. The uptake of 3-O-methylglucose was also inhibited by glucose and by galactose, but the uptake of 3-O-methylglucose in the presence of either galactose or glucose was no further reduced by adding the third hexose. Graphical analysis and analysis by non-linear regression both showed that neither the single Michaelis-Menten function, nor the single Michaelis-Menten-plus-competitive-inhibition function was appropriate for any of these data. The results are consistent with the hypothesis that either there are multiple (at least three) intestinal carriers for hexoses; alternatively that there is a single carrier whose transport properties for the three hexoses change differentially during cell maturation and migration up the villus.     

Selective amylin inhibition of the glucagon response to arginine is extrinsic to the pancreas

Amylin, a peptide hormone from pancreatic beta-cells, is reported to inhibit insulin secretion in vitro and in vivo and to inhibit nutrient-stimulated glucagon secretion in vivo. However, it has been reported not to affect arginine-stimulated glucagon secretion in vitro. To resolve if the latter resulted from inactive peptide (a problem in the early literature), those experiments were repeated here with well-characterized peptide and found to be valid. In isolated perfused rat pancreas preparations, coperfusion with 1 nM amylin had no effect on arginine-, carbachol-, or vasoactive intestinal peptide-stimulated glucagon secretion. Amylin also had no effect on glucagon output stimulated by decreasing glucose concentration from 11 to 3.2 mM or on glucagon suppression caused by increasing glucose from 3.2 to 7 mM. Amylin at 100 nM had no effect in isolated islets in which glucagon secretion was stimulated by exposure to 10 mM arginine, even though glucagon secretion in the same preparation was inhibited by somatostatin. In anesthetized rats, amylin coinfusion had no effect on glucagon secretion stimulated by insulin-induced hypoglycemia. To reconcile reports of glucagon inhibition with the absence of effect in the experiments just described, anesthetized rats coinfused with rat amylin or with saline were exposed sequentially to intravenous L-arginine (during a euglycemic clamp) and then to hypoglycemia. Amylin inhibited arginine-induced, but not hypoglycemia-induced, glucagon secretion in the same animal. In conclusion, we newly identify a selective glucagonostatic effect of amylin that appears to be extrinsic to the isolated pancreas and may be centrally mediated.     

Cyproheptadine and beta cell function in the rat: insulin secretion from pancreas segments in vitro.

Pancreatic islet cell vacuolization, hyperglycemia, and glucose intolerance develop in rats after oral administration of cyproheptadine (CPH). In order to determine whether these effects were associated with abnormal insulin secretion, pancreas segments from CPH-treated and control rats were compared for their ability to secrete insulin in response to several stimuli. Oral administration of CPH (45 mg/kg/day) to rats for 1 or 8 days inhibited glucose-mediated insulin secretion from pancreas segments obtained 3 and 24 hr after the last dose of the drug. Insulin secretion had returned to normal by 48 hr after drug administration. Intraperitoneal administration of the drug was less effective than oral administration in inhibiting in vitro insulin secretion. Other stimuli for insulin secretion (tolbutamide, glucagon, L-leucine, and dibutyryl 3',5'cyclic AMP), like glucose, were incapable of releasing normal amounts of insulin from pancreas segments of CPH-treated rats. CPH and a metabolite, desmethyl-CPH, inhibited glucose-stimulated insulin secretion when added in vitro to pancreas segments from control rats. This suggests that the inhibition of insulin secretion in pancreas segments taken from animals treated with CPH could be due, at least in part, to the presence of drug and its metabolite in the tissue. A previously observed reduction in the pancreatic content of insulin in CPH-treated rats may also contribute to the abnormal insulin release in animals given the drug.     

The ontogeny of insulin secretion mechanisms

Insulin secretion from pieces of pancreas of rabbits aged 6 weeks or 1 day, or of 24-day foetuses was studied in vitro in response to glucose, glucagon and theophylline. Glucose did not stimulate insulin release from foetal pancreas but was effective postnatally. Glucagon in medium containing 3.0 mg glucose/ml stimulated insulin secretion equally at each stage of development. Theophylline in medium containing 0.6 or 3.0 mg glucose/ml stimulated insulin secretion from foetal pancreas but was effective on postnatal pancreas only in the presence of 3.0 mg glucose/ml. Glucose potentiated the action of theophylline on the foetal β cell and theophylline potentiated the action of glucose on the adult β cell.     

Arachidonic acid metabolism in isolated pancreatic islets. VI. Carbohydrate insulin secretagogues must be metabolized to induce eicosanoid release.

Pancreatic islets stimulated with D-glucose are known to liberate arachidonic acid from membrane phospholipids and release prostaglandin E2 (PGE2). A component of the eicosanoid release induced by D-glucose has been demonstrated to occur without calcium influx and must be triggered by other coupling mechanisms. In this study, we have attempted to identify mechanisms other than calcium influx which might couple D-glucose stimulation to hydrolysis of arachidonate from membrane phospholipids in islet cells. We have found that occupancy of the beta cell plasma membrane D-glucose transporter is insufficient and that D-glucose metabolism is required to induce islet PGE2 release because 3-O-methylglucose fails to induce and mannoheptulose prevents PGE2 release otherwise induced by 17 mM D-glucose. The carbohydrate insulin secretagogues mannose and D-glyceraldehyde have also been found to induce islet PGE2 release, but the non-secretagogue carbohydrates L-glucose and lactate do not. Carbohydrate secretagogues are known to be metabolized to yield ATP and induce depolarization of the beta cell plasma membrane. We have found that depolarization by 40 mM KCl induces PGE2 release only in the presence and not in the absence of extracellular calcium, but exogenous ATP induces islet PGE2 release with or without extracellular calcium. Carbachol is demonstrated here to interact synergistically with increasing concentrations of glucose to amplify PGE2 release and insulin secretion. Pertussis toxin treatment is shown here not to prevent PGE2 release induced by glucose or carbachol but to increase the basal rate of PGE2 release and the islet cyclic AMP content. Theophylline (10 mM) exerts similar effects. Eicosanoid release in pancreatic islets can thus be activated by multiple pathways including muscarinic receptor occupancy, calcium influx, increasing cAMP content, and a metabolic signal derived from nutrient secretagogues, such as ATP.     

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.