Autocrine regulation of glucose metabolism in pancreatic islets

We evaluated the possible autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from normal hamsters. We measured 14CO2 and 3H2O production from d-[U-14C]glucose and d-[5-3H]glucose, respectively, in islets incubated with 0.6, 3.3, 8.3, and 16.7 mM glucose alone or with 5 or 15 mU/ml insulin, anti-insulin guinea pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured (radioimmunoassay) in islets incubated with 3.3 or 16.7 mM glucose with or without 75, 150, and 300 nM wortmannin. Insulin significantly enhanced 14CO2 and 3H2O production with 3.3 mM glucose but not with 0.6, 8.3, or 16.7 mM glucose. Addition of anti-insulin serum to the medium with 8.3 and 16.7 mM glucose decreased 14CO2 and 3H2O production significantly. A similar decrease was obtained in islets incubated with 8.3 and 16.7 mM glucose and wortmannin or nifedipine. This latter effect was reversed by adding 15 mU/ml insulin to the medium. Glucose metabolism was almost abolished when islets were incubated in a Ca2+-deprived medium, but this effect was not reversed by insulin. No changes were found in 14CO2 and 3H2O production by islets incubated with 3.3 mM glucose and anti-insulin serum, wortmannin, or nifedipine in the media. Addition of wortmannin significantly decreased insulin release induced by 16.7 mM glucose in a dose-dependent manner. Our results suggest that insulin exerts a physiological autocrine stimulatory effect on glucose metabolism in intact islets as well as on glucose-induced insulin release. Such an effect, however, depends on the glucose concentration in the incubation medium.     

A novel insulin-releasing substance, phanoside, from the plant Gynostemma pentaphyllum

Extracts from Gynostemma pentaphyllum Makino (Cucurbitaceae), a Southeast Asian herb, has been reported to affect numerous activities resulting in antitumor, cholesterol-lowering, immunopotentiating, antioxidant, and hypoglycemic effects. We have isolated one active compound by ethanol extraction, distribution in n-butyl alcohol/water, solid phase extraction/separation, and several rounds of reverse phase high pressure liquid chromatography. We have shown by NMR and mass spectrometry that this active compound is a novel saponin, a gypenoside, which we have named phanoside (21-,23-epoxy-,3beta-,20-,21-trihydroxydammar-24-ene-3-O-([alpha-d-rhamnopyranosyl(1-->2)]-[beta-d-glycopyranosyl(1-->3)]-beta-d-lyxopyranoside)), with a molecular mass of 914.5 Da. Phanoside is a dammarane-type saponin, and four stereoisomers differing in configurations at positions 21 and 23 were identified, each of which were found to stimulate insulin release from isolated rat pancreatic islets. We have also found that the stereoisomers are interconvertible. Dose-dependent insulin-releasing activities at 3.3 and 16.7 mM glucose levels were determined for the racemic mixture containing all four stereoisomers. Phanoside at 500 microM stimulates insulin release in vitro 10-fold at 3.3 mM glucose and potentiates the release almost 4-fold at 16.7 mM glucose. At these glucose levels, 2 microm glibenclamide stimulates insulin release only 2-fold. Interestingly, beta-cell sensitivity to phanoside is higher at 16.7 mM than at 3.3 mM glucose, although insulin responses were significantly increased by phanoside below 125 microM only at high glucose levels. Also when given orally to rats, phanoside (40 and 80 mg/ml) improved glucose tolerance and enhanced plasma insulin levels at hyperglycemia.     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

Decreased islet sensitivity to insulin in hamsters with dietary-induced insulin resistance

In the present study, we evaluated the autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from hamsters with insulin resistance (IR) induced by administration of a sucrose-rich diet (SRD) during 5 weeks. We used an approach of two metabolic pathways (glucose oxidation and utilization) based on the measurement of 14CO2 and 3H2O production from D-[U-14C]-glucose and D-[5-(3)H]-glucose, respectively, in isolated islets incubated with 3.3 and 16.7 mM glucose alone, or with 5 or 15 mU/ml insulin, anti-insulin guinea-pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured by radioimmunoassay in islets incubated with 3.3 or 16.7 mM glucose, with or without 75, 150, and 300 nM wortmannin. Results showed that the stimulatory effect of insulin upon 14CO2 and 3H2O production in control islets was not observed in SRD islets. Addition of anti-insulin serum, nifedipine or wortmannin to the medium with 16.7 mM glucose decreased 14CO2 and 3H2O production in control but not in SRD islets. Whereas wortmannin did not decrease insulin release induced by 16.7 mM glucose in SRD hamsters, it did in controls. We can conclude that the autocrine stimulatory effect of insulin upon glucose metabolism observed in normal islets is attenuated or even absent in islets from IR animals. Such decreased islet sensitivity to insulin did not prevent the compensatory secretion of insulin from maintaining glucose homeostasis, suggesting that, at least in this model, the islets can put forward alternative mechanisms to overcome such defect.     

Mastoparan, a wasp venom, stimulates insulin release by pancreatic islets through pertussis toxin sensitive GTP-binding protein

A wasp venom, mastoparan, rapidly stimulated insulin release by rat pancreatic islets in a dose-related manner. The amount of insulin released in response to 58 microM mastoparan far exceeded that induced by 27.8 mM glucose. Mastoparan stimulated insulin release to similar degrees at ambient glucose concentrations of 1.7 mM and 5.6 mM. The islets obtained from pertussis toxin-treated rats showed unequivocally less response to mastoparan. Pretreatment of islets with bromophenacyl bromide, a phospholipase A2 inhibitor, abolished their responsiveness to mastoparan. Pretreatment of islets with nifedipine, a Ca2+ channel blocker, was without effect. Mastoparan is a unique stimulator of insulin release by the pancreatic islets, which acts through GTP-binding protein(s) and phospholipase A2.     

Augmented Insulinotropic Action of Arachidonic Acid through the Lipoxygenase Pathway in the Obese Zucker Rat

Objective: The metabolism of arachidonic acid (AA) has been shown to be altered in severe insulin resistance that is present in obese (fa/fa) Zucker rats. We examined the effects and mechanism of action of AA on basal and glucose‐stimulated insulin secretion in pancreatic islets isolated from obese (fa/fa) Zucker rats and their homozygous lean (Fa/Fa) littermates. Research Methods and Procedures: Islets were isolated from 10‐ to 12‐week‐old rats and incubated for 45 minutes in glucose concentrations ranging from 3.3 to 16.7 mM with or without inhibitors of the cyclooxygenase or lipoxygenase pathways. Medium insulin concentrations were measured by radioimmunoassay, and islet production of the 12‐lipoxygenase metabolite, 12‐hydroxyeicosatetraenoic acid (12‐HETE), was measured by enzyme immunoassay. Results: In islets from lean animals, AA stimulated insulin secretion at submaximally stimulatory glucose levels (< 11.1 mM) but not at 16.7 mM glucose. In contrast, in islets derived from obese rats, AA potentiated insulin secretion at all glucose concentrations. AA‐induced insulin secretion was augmented in islets from obese compared with lean rats at high concentrations of AA in the presence of 3.3 mM glucose. Furthermore, the inhibitor of 12‐lipoxygenase, esculetin (0.5 μM), inhibited AA‐stimulated insulin secretion in islets from obese but not lean rats. Finally, the islet production of the 12‐HETE was markedly enhanced in islets from obese rats, both in response to 16.7 mM glucose and to AA. Discussion: The insulin secretory response to AA is augmented in islets from obese Zucker rats by a mechanism related to enhanced activity of the 12‐lipoxygenase pathway. Therefore, augmented action of AA may be a mechanism underlying the adaptation of insulin secretion to the increased demand caused by insulin resistance in these animals.     

Evidence for glucose-responsive and -unresponsive pools of phospholipid in pancreatic islets

The effect of glucose on the metabolism of phospholipids in pancreatic islets was studied with three radioactive phospholipid precursors, [32P]orthophosphate, [3H]myoinositol, and [3H]arachidonic acid, to determine the conditions necessary for studying the breakdown of prelabeled phospholipids. Islets were incubated in the presence of a radioactive precursor for 60 or 90 min and in the presence of either 3.3 or 16.7 mM glucose to prelabel phospholipids. To study the breakdown of prelabeled phospholipid, the unincorporated precursor was removed and the islets were reincubated for 15 or 20 min under conditions that either did or did not stimulate insulin release. Prelabeling in the presence of a noninsulinotropic concentration of glucose (3.3 mM) supported the incorporation of precursors into almost all islet phospholipids studied. Prelabeling in an insulinotropic concentration of glucose (16.7 mM) increased the incorporation of precursors into a number of phospholipids even more; and reincubation in 16.7 mM glucose caused a rapid loss of radioactivity from specific phospholipids (phosphatidylinositol and/or phosphatidylcholine, depending on the precursor). This breakdown was observed only when islets had been prelabeled in 16.7 mM glucose. The amount of radioactivity lost from phospholipid corresponded roughly to the additional amount incorporated during the prelabeling in the high concentration of glucose. Radioactivity in phospholipids in islets prelabeled in 3.3 mM glucose or in nonsecretagogue metabolic fuels, such as malate plus pyruvate, did not decrease when the islets were subsequently exposed to 16.7 mM glucose, nor did it decrease in 3.3 mM glucose when these islets had been prelabeled in 16.7 mM glucose. Glyceraldehyde, an insulin secretagogue, but not galactose or L-glucose which are not insulin secretagogues, stimulated phospholipid breakdown in islets that had been prelabeled in 16.7 mM glucose. Depriving islets of extracellular calcium, a condition that inhibits insulin release, inhibited phospholipid breakdown. The results suggest that pancreatic islets contain a glucose-responsive and a glucose-unresponsive phospholipid pool. The glucose-responsive pool becomes labeled and undergoes rapid turnover only under stimulatory conditions and may play a role in the stimulus-secretion coupling of insulin release.     

Homogeneous magnetic fields influence pancreatic islet function in vitro

Pancreatic islets were isolated from newborn rats and exposed to homogeneous magnetic fields for 48 hours. Under these conditions insulin release, measured at low (5.6 mM) and high (16.7 mM) glucose concentrations, was significant and dose-dependent only at low glucose concentrations. High glucose and aminophylline (10 mM) inhibited insulin release. Thus, in the absence of stimulatory glucose concentrations, low-intensity magnetic fields (1 to 10 Gauss) significantly influence insulin discharge from rat islets in vitro.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

A new method for the simultaneous estimation of phosphate efflux and influx during glucose stimulation of isolated pancreatic islets

Isolated rat pancreatic islets were prelabeled with [33Pi] and then incubated with basal (2.8 mM) or stimulatory (16.7 mM) glucose in the presence of [32Pi]. Subsequent changes in islet [33P] and [32P] were utilized as respective indices of net efflux and influx. During the initial eight min, (the period usually spanning the first phase of stimulated insulin secretion) efflux was significantly greater with 16.7 than 2.8 mM glucose whereas the lesser amount of phosphate influx did not differ in the two systems. During the subsequent seven min (a time usually associated with the onset of the second phase of stimulated insulin secretion), efflux was dampened in the presence of 16.7 mM glucose and Pi influx significantly exceeded the 2.8 mM glucose values. Thus, acute stimulation with glucose effects an initial phosphate depletion in pancreatic islets as efflux exceeds influx and repletion occurs thereafter as efflux is attenuated and influx is enhanced. These oscillations in islet phosphate may contribute to the biphasic pattern of glucose-stimulated insulin release.     

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.     

Chronic exposure to high leucine impairs glucose-induced insulin release by lowering the ATP-to-ADP ratio

Exposure of rat pancreatic islets to 20 mM leucine for 24 h reduced insulin release in response to glucose (16.7 and 22.2 mM). Insulin release was normal when the same islets were stimulated with leucine (40 mM) or glyburide (1 microM). To investigate the mechanisms responsible for the different effect of these secretagogues, we studied several steps of glucose-induced insulin secretion. Glucose utilization and oxidation rates in leucine-precultured islets were similar to those of control islets. Also, the ATP-sensitive K(+) channel-independent pathway of glucose-stimulated insulin release, studied in the presence of 30 mM K(+) and 250 microM diazoxide, was normal. In contrast, the ATP-to-ADP ratio after stimulation with 22.2 mM glucose was reduced in leucine-exposed islets with respect to control islets. The decrease of the ATP-to-ADP ratio was due to an increase of ADP levels. In conclusion, prolonged exposure of pancreatic islets to high leucine levels selectively impairs glucose-induced insulin release. This secretory abnormality is associated with (and might be due to) a reduced ATP-to-ADP ratio. The abnormal plasma amino acid levels often present in obesity and diabetes may, therefore, affect pancreatic islet insulin secretion in these patients.     

Effect of glucose on adenosine 3', 5'-monophosphate levels in rat pancreatic islets.

Time course of the changes in insulin release and cyclic AMP levels in isolated rat islets incubated in media containing 5 or 16.7 mM of glucose were followed. The higher glucose concentration caused a slight but significant increase of cyclic AMP levels after 10 min incubation, but not 5 min incubation, whereas the stimulation of insulin release by 16.7 mM of glucose was apparent in both incubation times. Theophylline increased cyclic AMP levels markedly but did not stimulate insulin release when the glucose concentration was 5 mM. A slight augmentation by theophylline of insulin release was observed in the incubation medium containing 16.7 mM glucose. All these findings suggest that the elevation of cyclic AMP in islets may not play a role for the initiation of the insulin release induced by glucose, though it may act to modulate the glucose effect.     

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.     

Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose.

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.     

Progesterone inhibits insulin secretion by a membrane delimited,non-genomic action

In rat islets, progesterone caused a prompt concentration-dependent inhibition of glucose-stimulated insulin release with an IC50 of 10 microM at 8.4mM glucose. The inhibition was specific since both testosterone and 17beta-estradiol had no such effect. The degree of inhibition was similar in islets from male and female rats. The inhibition was not blocked in PTX-treated islets thus ruling out the Gi/Go proteins as mediators of the inhibition. Progesterone inhibited both glucose- and BayK-8644-stimulated insulin secretion in HIT-T15 cells and the IC50 vs. 10 mM glucose was also 10 microM. There was no effect on intracellular cyclic AMP concentration in the presence 0.2 and 10 mM glucose. Progesterone decreased [Ca2+]i under all conditions tested. The decrease in [Ca2+]i was due to blockade of the L-type voltage-dependent Ca2+ channels. Under Ca(2+)-free conditions, progesterone did not inhibit the stimulation of insulin release due to the combination of glucose, phorbol ester and forskolin. Thus blockade of Ca2+ entry appears to be the sole mechanism by which progesterone inhibits insulin release. As progesterone covalently linked to albumin had a similar inhibitory effect as progesterone itself, it is concluded that the steroid acts at the outer surface of the beta-cell plasma membrane. These effects would be classified as either AI or AIIb in the Mannheim classification of nongenomically initiated steroid actions.     

Bombesin inhibits insulin release from isolated pancreatic islets of rats in vitro.

The effect of bombesin on insulin release from isolated pancreatic islets of rats was examined in vitro. Bombesin, at the doses ranging from 10 ng/ml to 1 microgram/ml, significantly inhibited 16.7 mM glucose-induced insulin release, while bombesin had no inhibitory effect on insulin release at 8.3 mM and 3.3 mM glucose. Moreover, bombesin also suppressed insulin release elicited by 10 mM arginine at the doses of 100 ng/ml and 1 microgram/ml. These results indicate that bombesin has a direct inhibitory action on insulin release.     

Expression of glucagon-like peptide-1 (GLP-1) receptor and the effect of GLP-1-(7-36) amide on insulin release by pancreatic islets during rat ontogenic development.

The expression of glucagon-like peptide-1 (GLP-1) receptor and the effects of GLP-1-(7-36) amide (t-GLP-1) on glucose metabolism and insulin release by pancreatic islets during rat development were studied. GLP-1 receptor mRNA was found in significant amounts in pancreatic islets from all age groups studied, GLP-1 receptor expression being maximal when pancreatic islets were incubated at physiological glucose concentration (5.5 mM), but decreasing significantly when incubated with either 1.67 or 16.7 mM glucose. Glucose utilization and oxidation by pancreatic islets from fetal and adult rats rose as a function of glucose concentration, always being higher in fetal than in adult islets. The addition of t-GLP-1 to the incubation medium did not modify glucose metabolism but gastric inhibitory polypeptide and glucagon significantly increased glucose utilization by fetal and adult pancreatic islets at 16.7 mM glucose. At this concentration, glucose produced a significant increase in insulin release by the pancreatic islets from 10-day-old and 20-day-old suckling rats and adult rats, whereas those from fetuses showed only a significant increase when glucose was raised from 1.67 to 5.5 mM. t-GLP-1 elicited an increase in insulin release by pancreatic islets from all the experimental groups when the higher glucose concentrations were used. Our findings indicate that GLP-1 receptors and the effect of t-GLP-1 on insulin release are already present in the fetus, and they therefore exclude the possibility that alterations in the action of t-GLP-1 are responsible for the unresponsiveness of pancreatic beta cells to glucose in the fetus, but stimulation of t-GLP-1 release by food ingestion in newborns may partially confer glucose competence on beta cells.     

Insulin release and eflux of [32P]Phosphate from islets of rat Langerhans treated with iodoacetic acid and the anomers of D-glucose.

To clarify the insulin-releasing mechanism, we studied insulin release and the efflux of [32P]phosphate by glucose at 0.1 mM/min of gradient level or at 16.7 mM, and other metabolism in islets of rat Langerhans. When treated with 1 mM iodoacetic acid (IAA) plus the anomers of D-glucose at 2.8 mM for 6 min at 37 degrees C, islets elicited insulin at half the control rate under the step-wise stimulation by glucose and at the same rate as the control under the slow-rise stimulation by glucose. Using islets treated with IAA plus the alpha anomer at 16.7 mM, the step-wise stimulation secreted insulin at half a rate of the control and the slow-rise stimulation at the rate lower than the control, which was not significantly different from the control rate. Treatment with IAA plus the beta anomer at 16.7 mM inhibited insulin release under both types of stimulations by glucose. The step-wise stimulation caused the same rapid efflux of [32P]phosphate from IAA-treated islets as from the control islets, except for islets treated with IAA plus the beta anomer at 16.7 mM. The rate of glucose utilization in islets was inhibited by all IAA-treatments to the same extent, being merely half the control rate. Treatments with IAA plus the anomers at 16.7 mM significantly reduced the formation of [3H]-cAMP and the activity of protein phosphokinase in islets, while in the presence of the anomers at 2.8 mM IAA produced no significant effect. Neither IAA-treatments altered the uptake of 45Ca and the ATP content in islets. The uptake of [14C]IAA was significantly enhanced by the presence of the beta anomer at 16.7 mM to two times the control level. On the basis of these results, we suggested that the B cell might contain both glucoreceptors and rate-sensors of glucose controlling insulin release and the former might be less sensitive to IAA as compared with the latter.     

Expression of Protein Kinase C Isoforms in Pancreatic Islets and Liver of Male Goto-Kakizaki Rats,a Model of Type 2 Diabetes

Protein kinase C (PKC) is a family of protein kinases controlling protein phosphorylation and playing important roles in the regulation of metabolism. We have investigated expression levels of PKC isoforms in pancreatic islets and liver of diabetic Goto-Kakizaki (GK) rats with and without insulin treatment to evaluate their association with glucose homeostasis. mRNA and protein expression levels of PKC isoforms were assessed in pancreatic islets and liver of Wistar rats and GK rats with or without insulin treatment. PKCα and PKCζ mRNA expressions were down-regulated in islets of GK compared with Wistar rats. PKCα and phosphorylated PKCα (p-PKCα) protein expressions were decreased in islets of GK compared with insulin-treated GK and Wistar rats. PKCζ protein expression in islets was reduced in GK and insulin-treated GK compared with Wistar rats, but p-PKCζ was decreased only in GK rats. Islet PKCε mRNA and protein expressions were lower in GK compared with insulin-treated GK and Wistar rats. In liver, PKCδ and PKCζ mRNA expressions were decreased in both GK and insulin-treated GK compared with Wistar rats. Hepatic PKCζ protein expression was diminished in both GK rats with and without insulin treatment compared with Wistar rats. Hepatic PKCε mRNA expression was down-regulated in insulin-treated GK compared with GK and Wistar rats. PKCα, PKCε, and p-PKCζ expressions were secondary to hyperglycaemia in GK rat islets. Hepatic PKCδ and PKCζ mRNA expressions were primarily linked to hyperglycaemia. Additionally, hepatic PKCε mRNA expression could be under control of insulin.