Preincubation Of Islets Research Articles

The effects of vitamin d on insulin release from isolated islets of rats.

Background:Vitamin D (vit D) affects glucose metabolism. Receptors of vitamin D have been identified in β cells and studies show that vitamin D deficiency reduces glucose-stimulated insulin secretion (GSIS).Objectives:The aim of this study was to examine the effect of vitamin D on insulin release from isolated islets of rats.Materials and Methods:Islets were isolated from male Wistar rats, weighing 200-250 grams, using the collagenase digestion method. Insulin release was assessed following 24 and 48 hours coincubation of islets with vitamin D (0.1, 1 and 10 nM) and glucose (5.6, 11.1 and 16.7 mM). In addition, islets were preincubated with vitamin D for 24 and 48 hours and GSIS was assessed for one hour in the presence of 5.6 and 16.7 mM glucose.Results:Coincubation of islets with vitamin D (10 nM) and 11.1 mM glucose increased islet insulin release (37.27 ± 3.75 vs. 24.64 ± 2.83 ng/islet/24 hours; P < 0.05), while vitamin D (1 and 10 nM) decreased insulin release in the presence of 16.7 mM glucose (21.14 ± 3.58 and 18.65 ± 3.84 vs. 37.71 ± 4.63 ng/ islet/24 hours; P < 0.05). Islets preincubation with vitamin D (1 and 10 nM) increased GSIS in the presence of 16.7 mM glucose (4.39 ± 0.73 and 4.39 ± 0.63 vs. 2.07 ± 0.43 ng/islet/1 hour; P < 0.05).Conclusions:Preincubation of islets with vitamin D increased GSIS but decreased insulin release in coincubation with high levels of glucose. Insulin secretion from β cells in the presence of glucose seems to be related to the dosage of vitamin D and duration of preincubation.

Time-dependent effect of GABA on glucose-stimulated insulin secretion from isolated islets in rat

Objective. Gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter found widely in the central nervous system and some peripheral tissues such as the islets of pancreas. The aim of this study was to determine the time dependency of the effect of GABA on glucose-stimulated insulin secretion in isolated islets of rats. Materials and Methods. The collagenase digestion technique was used to isolate the islets from pancreata of male Wistar rats (200–250 g). Insulin secretion was assessed in islets exposed to different concentrations of glucose (8.3 and 16.7 mM), in the presence or absence of GABA (50 µM), for different incubation times and also pre-incubation of islets with GABA, 30 and 45 min before 8.3 mM glucose stimulation. Furthermore insulin secretion with different concentrations of glucose (8.3 and 16.7 mM), and in the presence or absence of different concentrations of baclofen, a GABAB agonist and of saclofen, a GABAB antagonist during 1 h of incubation was assessed. Insulin release was reported as mean ± SE μU/islet/min and p values of <0.05 were considered significant. Results. GABA with concentration of 50 µM inhibited the glucose stimulated insulin secretion during a 60 min incubation period. Baclofen had no significant effect on glucose-induced insulin secretion, while saclofen (100 µM) significantly increased 16.7 mM glucose-induced insulin secretion in 60 min of incubation (p < 0.05). Pre-incubation of islets with GABA for 45 min, before glucose stimulation, increased glucose-induced insulin secretion. Conclusion. GABA could have both stimulatory and inhibitory effects on glucose-stimulated insulin secretion, depending on the time of exposure and glucose concentration.

Role of Intra-Islet Endothelial Cells in Islet Allo-Immunity

Intra-islet endothelial cells (IECs) express high levels of major histocompatibility complex (MHC) and are pivotal for posttransplant islet revascularization. We postulated that donor-specific sensitization would result in hyperacute rejection of IECs and prevent islet engraftment. Furthermore, ligation of endothelial cells with subsaturating concentrations of anti-MHC class I antibody (Ab) results in "accommodation" conferring protection against Ab/complement-mediated lysis. Therefore, we investigated whether accommodation of IECs would prevent hyperacute rejection of islets in sensitized recipients. Islets were transplanted beneath the kidney capsule and allograft survival monitored using daily blood glucose (diabetes >300 mg/dL, normoglycemia <150 mg/dL). Recipients were presensitized with donor islets, splenocytes, or skin. Accommodation was induced by incubating human or murine islets with varying concentrations of anti-MHC class I Ab ex vivo. Isografts remained functional for >100 days, whereas allografts were rejected by day 14. Islet allo-transplantation induced donor-specific but not third-party anti-MHC Abs. Donor-specific, but not third-party, sensitization induced hyperacute rejection of subsequent islet allografts (median survival 1 day) associated with complement deposition. Preincubation of islets with subsaturating concentrations of anti-MHC-I Abs (1-100 ng/mL) up-regulated Bcl-2, Bcl-xl, and HO-1 within CD31+ IEC. These accommodated islets were resistant against hyperacute rejection when transplanted into donor-(splenocyte) sensitized recipients without any immunosuppression (median survival 6 days). Pretransplant sensitization against donor antigens results in hyperacute rejection of murine islets. IECs may play a crucial role in development of donor-specific immunity after islet transplantation. Significantly, accommodation of IEC may confer resistance to hyperacute rejection in sensitized recipients.

Leptin Constrains Phospholipase C-Protein Kinase C-Induced Insulin Secretion via a Phosphatidylinositol 3-Kinase-Dependent Pathway

Leptin-deficient Lep(ob)/Lep(ob)mice hypersecrete insulin in response to acetylcholine stimulation of the phospholipase C-protein kinase C (PLC-PKC) pathway, and leptin constrains this hypersecretion. Leptin has been reported to activate phosphatidylinositol 3-kinase (PI 3-K) and subsequently phosphodiesterase (PDE) to impair protein kinase A (PKA)-induced insulin secretion from cultured islets of neonatal rats. We determined if PKA-induced insulin secretion was also hyperresponsive in islets from Lep(ob)/Lep(ob)mice, and if leptin impaired this pathway in islets from these mice. Additionally, the possible role for PI 3-K and PDE in leptin-induced control of acetylcholine-induced insulin secretion was examined. Stimulation of insulin secretion with GLP-1, forskolin (an activator of adenylyl cyclase), or IBMX (an inhibitor of PDE) did not cause hypersecretion of insulin from islets of young Lep(ob)/Lep(ob)mice, and leptin did not inhibit GLP-1-induced insulin secretion from islets of these mice. Inhibition of PDE with IBMX also did not block leptin-induced inhibition of acetylcholine-mediated insulin secretion from islets of Lep(ob)/Lep(ob)mice. But, preincubation of islets with wortmannin, an inhibitor of PI 3-K activity, blocked the ability of leptin to constrain acetylcholine-induced insulin secretion from islets of Lep(ob)/Lep(ob)mice. We conclude that the capacity of the PKA pathway to stimulate insulin secretion is not increased in islets from young Lep(ob)/Lep(ob)mice, and that leptin does not regulate this pathway in islets from mice. Leptin may stimulate PI 3-K to constrain PLC-PKC-induced insulin secretion from islets of Lep(ob)/Lep(ob)mice.

Chronic chloroquine treatment enhances insulin release in rats

The effects of chronic chloroquine treatment on intravenous glucose tolerance, and the plasma insulin response to intravenous glucose were studied in rats. Plasma glucose disappearance constants ( K g) were significantly greater (6.2 ± 0.5 %min −1) than in corresponding controls (3.7 ± 0.4 %min −1, P < 0.001). This improved glucose tolerance was associated with significantly higher plasma immunoreactive insulin levels in response to glucose injection. Islets isolated from rats treated with chloroquine showed significantly enhanced ( P < 0.05) insulin release when incubated with 16.7 mM glucose but not with lower glucose concentrations (3 and 8 mM). Pre-incubation of islets with streptozotocin (0.05–1.5 mg/ml) produced a dose-dependent reduction in glucose-stimulated insulin secretion which was not modified in islets from chloroquine-treated rats. The concentration of chloroquine in the pancreas increased rapidly during administration and reached a value of 20.2 ± 0.7 μg/g (fresh weight) after 20 weeks of treatment. It is concluded that chronic chloroquine treatment results in an improved glucose tolerance associated with an enhanced glucose-induced insulin secretion. Although earlier work showed chloroquine to reduce the severity of diabetes induced subsequently with streptozotocin, the present study shows that such an amelioration was not due to a protective effect against the β-cell cytotoxic action of streptozotocin.

Age-Related Impairment in the Short Term Regulation of Insulin Biosynthesis by Glucose in Rat Pancreatic Islets*

The pancreatic beta-cell is differentiated to express the insulin biosynthetic secretory pathway and to regulate this pathway principally by sensing the extracellular glucose concentration. With aging, glucose stimulation of both insulin synthesis and secretion is significantly impaired. The mechanism of the 40-50% decrease in the short term stimulation of insulin synthesis by glucose in rat pancreatic islets was studied in an effort to elucidate the specific stage at which the impairment occurs. In isolated islets from both young (4- to 5-month-old) and old (18- to 28-month-old) rats at normal (5.5 mM) and elevated (11 mM) glucose concentrations, insulin I was the predominant mature insulin species that accumulated during a 4-h labeling period. After preincubation of islets for 16 h in Minimum Essential Medium (with 5.5 mM glucose), total preproinsulin mRNA levels were similar in old and young islets; after preincubation in an enriched medium, RPMI-1640 (with 11 mM glucose), old and young levels were still similar, although preproinsulin mRNA levels were 6-fold higher. Proinsulin synthesis during a 30-min pulse label period was stimulated 10-fold in young islets and 7-fold in old islets at 16.7 mM glucose compared with synthesis at 2.8 mM glucose and was not affected by 5 micrograms/ml actinomycin-D. Furthermore, stimulation of the two major known intracellular second messenger systems in beta-cells, the cAMP system and the protein kinase-C system, with 8-bromo-cAMP, 3-isobutyl-1-methylxanthine, or a phorbol ester did not further increase proinsulin synthesis over a 30-min pulse label period in islets already stimulated by 16.7 mM glucose. Thus, the impairments in the short term effects of glucose on proinsulin synthesis in old islets are posttranscription of preproinsulin mRNA and likely to lie at the level of enhancement of translation of preexisting preproinsulin mRNA by factors activated or inhibited by the ambient glucose concentration. These age-impaired factors are not modulated through the cAMP or protein kinase-C system.

Enzymes of phospholipid metabolism in rat pancreatic islets: subcellular distribution and the effect of glucose and calcium.

The effect of glucose and calcium on the activities of the phosphatidylinositol cycle enzymes, CDP-diglyceride inositol transferase, diacylglycerokinase, and lysophosphatidylcholine 2-acyltransferase in rat pancreatic islets was studied. Calcium inhibited the activity of CDP-diglyceride inositol transferase but had no effect on lysophosphatidylcholine 2-acyltransferase and diacylglycerokinase activities. Upon preincubation of islets in a concentration of glucose known to stimulate insulin release, the activity of lysophosphatidylcholine 2-acyltransferase, but not that of diacylglycerokinase or the CDP-diglyceride inositol transferase, was stimulated. Subcellular fractionation of pancreatic islets showed that secretory granule membranes were enriched in CDP-diglyceride inositol transferase, whereas lysophosphatidylcholine 2-acyltransferase activity was highest in the microsomal membranes. The activation of 2-acyltransferase by incubating islets in insulinotropic glucose, and the calcium sensitivity of CDP-diglyceride inositol transferase, suggest that these enzymes may have roles in regulation of insulin secretion.

Evidence for a direct action of exogenous insulin on the pancreatic islets of diabetic mice: islet response to insulin pre-incubation.

Pancreatic islets were isolated by collagenase digest from normal (+/+) and severely diabetic (db/db) mice of the C57BL/Ks strain. Batches of islets from the diabetic mice were incubated for 1 h at 37 degrees C in Krebs-Ringer medium containing glucose (3 mmol/l), with or without mouse insulin (50 mU/l), before perifusion. When compared with untreated islets from the same digest, pre-incubation of islets from diabetic mice with insulin elicited a restoration of the biphasic insulin response to a glucose challenge which was indistinguishable from the response of the untreated, control islets from non-diabetic mice. Untreated diabetic islets showed no insulin response to glucose challenge, insulin values being very significantly lower than those of either non-diabetic or treated diabetic mouse islets (p = less than 0.005). The restoration of secretion in treated islets of diabetic mice was shown not to be an artefact. These observations provide evidence for a direct, as yet undefined, action of insulin on the beta cell of the diabetic mouse.

Phospholipase A 2 activity in pancreatic islets is calcium-dependent and stimulated by glucose

Phospholipase A 2 activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from E. coli membranes. Phospholipase A 2 activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A 2 activity which was assayable in islet homogenates. Glucose stimulated phospholipase A 2 in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A 2 activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A 2 activity measured in the presence of high calcium concentrations. Phospholipase A 2 activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A 2 activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.

Cyclic AMP metabolism and insulin release in pancreatic islets of the rat Effects of agents which alter microtubular function

In order to investigate the relationship between microtubular function, insulin release and islet cyclic AMP metabolism, the effects of 2H 2O, colchicine and vincristine were studied in rat islets prelabeled with [ 3H]adenine. Glucose-induced insulin secretion and efflux of cyclic [ 3H]AMP was markedly inhibited by 8–50% 2H 2O. At a higher concentration (75%), deuterated water still suppressed insulin release, while the inhibition of nucleotide release was abolished. Glucose-induced intra-islet cyclic [ 3H]AMP accumulation was augmented by 2H 2O progressively with time. With 75% 2H 2O, although efflux of cyclic AMP was no more inhibited, intra-islet accumulation of the nucleotide was still enhanced. The cyclic AMP efflux induced by cholera toxin, or a high concentration of 3-isobutyl-1-methylxanthine was suppressed and the intra-islet nucleotide accumulation was enhanced by 2H 2O. The latter effect tended to be less pronounced than when glucose was the stimulator. All the effects of 2H 2O on glocuse-stimulated islets were mimicked by incubating the tissue in H 2O at 28°C. Colchicine and vincristine had no significant effect on glucose-induced insulin release, and did not enhance the intra-islet cyclic [ 3H]AMP response; efflux of the nucleotide was, however, significantly inhibited. This pattern of response was shared with probenecid. Preincubation of islets with colchicine did not influence the subsequent effects of 2H 2O on insulin release and cyclic AMP metabolism. It is concluded that: (1) enhancement of intra-islet cyclic AMP accumulation by 2H 2O is not due to inhibition of the nucleotide efflux; (2) the effects on cyclic AMP metabolism described here are not exclusive for microtubular affecting agents and do not seem to be related to the microtubular system of the islet.

Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

1. The effects of azaserine and nicotinamide, agents which inhibit and stimulate hepatic NAD synthesis respectively, on the content of NAD(+) and NADH in isolated rat islets of Langerhans incubated in vitro were studied. The effects of these compounds on the rates of insulin release, from isolated islets incubated in vitro, in response to various secretagogues were also measured. 2. Preincubation of islets in the presence of azaserine (0.3mm) caused a marked depletion of the normal islet-cell content of both NAD(+) and NADH and prevented the secretion of insulin in response to stimulatory concentrations of d-glucose, xylitol, d-xylulose, l-arginine hydrochloride and l-leucine. 3. Preincubation of islets in the presence of nicotinamide (2mm) increased the islet content of NAD(+) and enhanced the rate of release of insulin in response to d-glucose. Also when nicotinamide was present the inhibitory effect of azaserine on insulin release and the azaserine-induced depletion of the islet content of NAD(+) and NADH was prevented. 4. Preincubation with azaserine was without effect on the stimulation of insulin release caused by theophylline or dibutyryl cyclic AMP. 5. It is suggested that insulin release caused by sugars and amino acids is dependent on the maintenance of NAD concentrations, though this may not be the case for release due to theophylline and dibutyryl cyclic AMP.