Effect of increased pancreatic islet norepinephrine,dopamine and serotonin concentration on insulin secretion in the Golden hamster

Summary The purpose of this study was to determine if increased concentrations of pancreatic islet norepinephrine, dopamine, or serotonin alter insulin secretion. Golden hamsters received intraperitoneal injections of the norepinephrine precursor DL-threo-dihydroxyphenylserine, the dopamine precursor L-3,4-dihydroxyphenylalanine, or the serotonin precursor 5-hydroxytryptophan with and without pretreatment of the hamsters with the monoamine oxidase inhibitor tranylcypromine. Administration of the monoamine precursors to animals pretreated with tranylcypromine resulted in a mean increase in plasma glucose of 192% and a mean decrease in plasma insulin of 58%. Using a collagenase isolation technique, islets from control and treated animals were evaluated for monoamine content and insulin secretory capacity. The monoamine concentrations in control islets, in mol/kg wet weight, were: norepinephrine 42±8; dopamine 8±2; and serotonin 26±9. Administration of the appropriate precursor to control hamsters resulted in a 1.9-fold (norepinephrine), 6-fold (dopamine), and 22-fold (serotonin) increase in monoamines. There was no alteration in the glucose (16.3 mmol/l)-stimulated in vitro insulin secretion from islets obtained from these hamsters. Administration of the precursors to hamsters pretreated with tranylcypromine resulted in a 3.5-fold (norepinephrine), 22-fold (dopamine), and 59-fold (serotonin) increase in monoamines. Glucose-stimulated in vitro insulin secretion from islets obtained from these hamsters was completely blocked. This study suggests that high concentrations of norepinephrine, dopamine, and serotonin in the pancreatic islets can decrease glucose-stimulated insulin secretion.     

Monoamine oxidase and catechol-o-methyltransferase activity in hamster and rat insulinomas

Hamster and rat insulinomas were assayed for norepinephrine, dopamine and serotonin concentration and for monoamine oxidase and catechol-o-ethyltransferase (COMT) activity. The concentration of norepinephrine (mean 0.55 mumol/kg, range less than 0.20 to 2.64 mumol/kg) and serotonin (mean 5.22 mucol/kg, rang less than 0.6 to 26.5 mumol/kg) in hamster insulinomas were comparable to previously reported concentrations. Dopamine conentration (mean 0.34 mumol/kg, range less than 0.20 to 0.95 mumol/kg) was only 2 to 2.5% of that reported previously. Monoamine oxidase activity of the hamster and rat insulinomas were comparable to those of normal hamster islets. In contrast, the COMT activity of both insulinomas was much greater than the COMT activity of normal pancreatic islets of both species and was greater than in several other tissues and tumours. The tumour COMT, which was predominantly in the cytosol, was Mg2+ dependent and had a comparable sensitivity to inhibition by tropolone as purified beef-liver COMT. Hamster insulinoma monoamine oxidase was more sensitive than rat insulinoma monoamine oxidase to inhibition by tranylcypromine and deprenyl, while rat insulinoma monoamine oxidase was more sensitive to inhibition by clorgyline and was more heat labile.     

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

Summary Insulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 U/ng islet DNA in GK rats and 28.8±2.5 U/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 · ng islet DNA^–1· h^–1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of ³H₂O from [5-³ H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA^–1 · h^–1) than in control islets (1.3±0.1 pmol · ng islet DNA^–1 · h^–1; p<0.05). In contrast, glucose oxidation, estimated as the production of ¹⁴CO₂ from [U-¹⁴C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with ³H₂O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.     

Effect of diabetes mellitus and chemical sympathectomy on tissue monoamine oxidase activity and norepinephrine concentration in the golden hamster

The pancreatic islets of the golden hamster have an extremely high monoamine oxidase (MAO) activity and norepinephrine (NE) concentration. To determine the cellular component responsible for these high values, we assayed MAO activity and NE concentration in collagenase-isolated islets from normal, streptozotocin-induced diabetic, and 6-hydroxydopamine (6-OHDA) induced sympathectomized golden hamsters. There was a 53% reduction in islet MAO activity in the diabetic hamsters, with no alteration in islet MAO activity in the sympathectomized ones, suggesting that the pancreatic B cells contain a large proportion of the MAO activity. There was a 98% reduction in islet NE concentration in the sympathectomized hamsters, with no alteration in islet NE concentration in the diabetic hamsters, suggesting that the adrenergic nerve endings in the islets contain the majority of the NE. There was no alteration in the MAO activity or NE concentration in the cerebral cortex, pituitary gland, liver, kidney, acinar pancreas, or heart of diabetic hamsters. Although the MAO activity of the hypothalamus was not altered, the diabetic hamsters had a 166% increase in NE and a 199% increase in dopamine in their hypothalamus, with no change in these amines in their cerebral cortex.     

Monoamines in pancreatic islets of guinea pig, hamster, rat, and mouse determined by high performance liquid chromatography

Previous studies on the occurrence of catecholamines and serotonin in pancreatic islets using various histochemical and chemical methods have given widely different results. We therefore performed a comparative analysis of these amines in whole pancreas and islet tissue from hamster, guinea pig, rat, and mouse by the use of high performance liquid chromatography. Whole pancreas of guinea pig, hamster, and rat had a norepinephrine concentration of approximately 1.1 mumol/kg of pancreatic wet weight. The mouse pancreas had less than one-half of that concentration. Epinephrine and dopamine concentrations were on the order of 0.02 mumol/kg of pancreatic wet weight in all four species. The serotonin concentration was 2.1 mumol/kg of pancreatic wet weight in the guinea pig pancreas and approximately 0.2 mumol/kg in the other three species studied. The catecholamine concentrations were much higher in the pancreatic islets than in the exocrine pancreas. Thus, the norepinephrine concentration was approximately 35 mumol/kg of islet wet weight in hamster islets and 5-10 mumol/kg in rat, guinea pig, and mouse islets. The epinephrine concentration in islet tissue ranged between 1 and 7 mumol/kg of islet wet weight and the dopamine concentration between 0.5 and 4 mumol/kg except for guinea pig islets (12 mumol/kg). The islet tissue in the mouse, rat, and guinea pig contained disproportionately more epinephrine and dopamine relative to norepinephrine than did the exocrine pancreas. Chemical sympathectomy (6-hydroxydopamine treatment) in the mouse reduced the norepinephrine and epinephrine concentrations in islet tissue to nondetectable levels, whereas the dopamine concentration was essentially unchanged, thus suggesting an extra-neuronal source of this amine in addition to its occurrence in adrenergic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Therapy of malignant hamster insulinomas with monoamine precursors

Summary The hyperinsulinaemia and hypoglycaemia produced by malignant insulinomas pose a difficult therapeutic problem. The non-polar monoamine precursors 5-hydroxytryptophan, L-3,4-dihydroxyphenylalanine and DL-threo-dihydroxyphenylserine are readily taken up by normal hamster pancreatic islets and are converted intracellularly to serotonin, dopamine and norepinephrine. These intracellular monoamines then inhibit glucose-stimulated insulin secretion. In the present study we have determined if the monoamine oxidase inhibitor pargyline, and monoamine precursors, could modify the severe hypoglycaemia of hamsters bearing a rapidly growing transplantable insulinoma. One hour after receiving the respective precursor there was an 11, 18, and 54-fold increase in the concentration of serotonin, dopamine or norepinephrine in the insulinomas. Twenty four hours after the administration of the respective precursors there were the following increases in plasma glucose in the respective groups: 5-hydroxytryptophan, 1.9 ± 0.5 to 4.9 ± 0.3 mmol/l (mean ± SEM); L-3,4-dihydroxyphenylalanine, 1.5 ± 0.3 to 3.4 ± 0.6 mmol/l; and DL-threo-dihydroxyphenylserine, 1.5 ± 0.2 to 6.4 ± 0.7 mmol/l. The change in plasma insulin concentration was statistically significant only in the group receiving DL-threo-dihydroxyphenylserine (229 ± 59 to 81 ± 42 mU/l). To determine if this therapeutic approach could modify the rapidly fatal outcome of the tumour-bearing hamsters, they received 0.154 mol/l saline, DL-threo-dihydroxyphenylserine or pargyline plus DL-threo-dihydroxyphenylserine every two days. The survival of the groups (mean ± SEM) was: saline 8.4 ± 1.1 d; DL-threo-dihydroxyphenylserine 17.9 ± 3.5 d; and pargyline plus DL-threo-dihydroxyphenylserine 24.4 ± 5.5 d. The present study suggests that increasing the monoamine content of malignant insulinomas may favourably modify the course of these devastating tumours.     

The effect of glucose stimulation on 45calcium uptake of rat pancreatic islets and their total calcium content as measured by a fluorometric micro-method

Summary Glucose-stimulated ⁴⁵calcium uptake and total calcium content of rat pancreatic islets has been studied, using a new fluorometric micro-method to estimate total calcium. Extracellular calcium was separated from incubated tissue by a rapid micro-filtration procedure. Islets incubated up to 60 min with calcium chloride 2.5 mmol/l and glucose 2.5 mmol/l maintained the same calcium content (670±7.5 pmol/g DNA). When the glucose concentration was raised to 15 mmol/l no change in the total calcium content could be detected. On incubation with glucose 2.5 mmol/l in the absence of calcium, the calcium content decreased to 488±27 pmol/g DNA. On incubation with ⁴⁵calcium chloride 2.5 mmol/l for 5 or 30 min at 2.5 mmol/l glucose, islets exchanged 21 ±2 and 28±1% of their total calcium content and, at 15 mmol/l glucose, 30±3 and 45±2%, respectively. Thus, islet calcium has a high turn-over rate. Glucose stimulation results in an increase of the calcium uptake without enhancing the total calcium content and hence must increase the calcium-exchangeable pool.     

Human pancreatic islet function at the onset of Type 1 (insulin-dependent) diabetes mellitus

Summary Viable human pancreatic islets isolated from a recent-onset Type 1 (insulin-dependent) diabetic patient were used to perform in vitro studies. Pre-proinsulin mRNA and insulin content, as well as insulin response were analysed. Insulin response to glucose and forskolin was completely absent in diabetic islets, as compared to control islets. Insulin content was reduced to only one-third of control values (395.0±3.5 vs 989.0±46.3 U/islet) and 20.7±3.9% of islets from the diabetic pancreas contained insulin-positive cells in immunofluorescence studies. Northern blot analysis revealed a severe reduction in the content of pre-proinsulin mRNA in diabetic pancreatic tissue. Our results indicate that although markedly decreased, beta cells in human pancreatic islets at the onset of Type 1 diabetes are still present. Never-theless, pancreatic islet function is disproportionately impaired with a complete absence of an insulin response.     

Glucose tolerance and plasma insulin response to intravenous glucose infusion and test meal in rats with microencapsulated islet allografts

Summary Albino Oxford rats made diabetic with 75 mg/kg streptozotocin were intraperitoneally transplanted with 2500–2900 alginate-polylysine microencapsulated Lewis islets (n=9, total islet tissue volume 8.0–11.0 l), or a similar volume of non-encapsulated Lewis islets (n=5). All rats with microencapsulated islets became normoglycaemic, and remained normoglycaemic for 5–16 weeks. In rats with non-encapsulated islet grafts, only a temporary decrease in blood glucose was observed, and all were again severely hyperglycaemic at 1 week after implantation. At 5–6 weeks after transplantation, glucose tolerance in rats with microencapsulated islets was tested by intravenous glucose infusion (10 mg/min over 20 min) and test meal administration (n=4). During glucose infusion, maximum glucose levels were 13.0±0.4 mmol/l in rats with microcapsules and 8.9±0.4 mmol/l in healthy control rats (p<0.01). Concomitant maximum plasma insulin levels were 215±17 pmol/l in rats with microcapsules and 715±85 pmol/l in controls (p<0.001). After the test meal, maximum blood glucose was 10.6±0.9 mmol/l in rats with microcapsules and 6.2±0.1 mmol/l in controls (p<0.001), with concomitant maximum plasma insulin levels of 247±11 pmol/l and 586±59 pmol/l, respectively (p<0.001). In conclusion, although the glucose tolerance is impaired and plasma insulin responses to intravenous glucose-load and test-meal are reduced, the alginate-polylysine membrane does provide adequate immunoisolation for the prolongation of allograft survival, resulting in prolonged normoglycaemia in streptozotocin diabetic rats.     

Different effects of glucose and glyburide on insulin secretion in rat pancreatic islets pre-exposed to interleukin-1β. Possible involvement of K+ and Ca2+ channels

Summary In vitro islet exposure to interleukin 1 inhibits the beta-cell response to glucose. We have studied whether a similar inhibition also occurs in response to the sulphonylurea glyburide. Rat pancreatic islets were cultured for 24 h in the presence or absence of 50 U/ml interleukin 1 and then stimulated with either glucose or glyburide for 1 h at 37 °C. In control islets basal insulin secretion was 117±32 pg · islet^–1 · h^–1 (mean ± SEM, n=7) and greatly increased in response to 16.7 mmol/l glucose (2140±293) or 10 mol/l glyburide (1464±234). When islets were pre-exposed to interleukin 1, insulin release was significantly reduced in response to glucose (323±80, p<0.001) but not in response to glyburide (1316±185). Since both glucose and glyburide influence beta-cell K⁺ and Ca²⁺ efflux, to further investigate this different response in islets exposed to interleukin 1 we measured both Rb⁺ efflux (as index of the ATP-sensitive K⁺ channel activity) and Ca²⁺ uptake. In control islets, the increased insulin secretion in response to 16.7 mmol/l glucose or 10 mol/l glyburide was associated with a reduction of ⁸⁶Rb efflux (decrement of –50±1.2 % and –49±2.3 %, respectively, mean ± SEM, n=5). In contrast, in interleukin 1pre-exposed islets both glucose and glyburide stimulation only slightly modified ⁸⁶Rb efflux (decrement of –19±1.9% and –5.3±3.1 %, respectively, n=5, p<0.001). ⁴⁵Ca²⁺ uptake in control islets was 2.6±0.4 pmol · islet^–1 · 20 min^–1 under basal conditions (at 2.8 mmol/l glucose), and increased to 16.8±3.2 and 10.7±2.1 pmol · islet^–1 · 20 min^–1 in islets stimulated with 16.7 mmol/l glucose or 10 mol/l glyburide, respectively (mean ± SEM, n=6). ⁴⁵Ca²⁺ uptake in interleukin 1 treated islets was higher than in control islets under basal conditions (4.6±0.6 pmol · islet^–1 · 20 min^–1 at 2.8 mmol/l glucose, p<0.05), but was significantly reduced in response to glucose 16.7 mmol/l (7.1±1.1, p<0.01 with respect to control islets). In contrast to glucose, 10 mol/l glyburide was able to stimulate calcium uptake in interleukin 1 treated islets in a similar way to control islets (12.8±2.5). The present data demonstrate that rat pancreatic islets treated with interleukin 1 for 24 h lose their responsivity to glucose, but not to glyburide. The difference between the two secretagogues is associated with the persistent ability of glyburide to influence Ca²⁺ uptake even in islets with impaired K⁺-channel function.     

Better vascular engraftment and function in pancreatic islets transplanted without prior culture

Aims/hypothesis Recent studies suggest that donor endothelial cells may contribute to islet graft revascularisation. Since islet endothelial cells disappear during culture, we hypothesised that transplantation of islets without prior culture is beneficial for their engraftment.Methods Cultured (4–7 days) or freshly isolated islets (<4 h after donor pancreas extirpation) were syngeneically transplanted into Wistar–Furth rats and C57Bl/6 mice beneath the renal capsule. Islet graft revascularisation was evaluated by measuring vascular density, blood flow and tissue oxygen tension. Islet graft function was investigated by a minimal islet mass model in inbred mice (C57Bl/6).Results Four days after implantation, the partial pressure of oxygen (pO₂) in the transplanted cultured islets was less than 10 mmHg (1.33 kPa), but tended to be higher in grafts composed of freshly isolated islets. The pO₂ in the grafts of freshly isolated islets had more than doubled 4 weeks later, whereas the pO₂ in the grafts of cultured islets remained at values similar to those recorded 4 days after transplantation. Transplanted freshly isolated islets also had a higher vascular density than transplanted cultured islets (40 vs 25% of that in endogenous islets) when investigated 1 month post-implantation. When applying a minimal islet mass model in inbred mice, 200 freshly isolated islets cured alloxan-diabetic mice in all cases, whereas only 33% of the group receiving similar numbers of cultured islets were cured.Conclusions/interpretation Transplantation of pancreatic islets without prior culture is beneficial for their vascular engraftment and function.     

Normoglycaemia after transplantation of freshly isolated and cryopreserved pancreatic islets in Type 1 (insulin-dependent) diabetes mellitus

Summary Purified islets of Langerhans and a kidney were transplanted into a 36-year-old patient who suffeded from renal failure secondary to a 25 year history of Type 1 (insulin-dependent) diabetes mellitus. The islet graft contained 243 000 fresh islets (mean islet diameter 150 m) that were syngeneic with the kidney fraft and 368 000 cryopreserved islets that had been collected from four other donors. The total of 10 000 islets/kg body weight was infused into the liver via the umbilical vein. Immunosupperession was induced with antilymphocyte globulin and maintained with prednisone, cyclosporine and azathioprine. Serum C-peptide levels (ng/ml) during fasting and after standard mixed metal feeding (Sustacal) were <0.12 preoperatively. Postoperatively, insulin secretion was restored: fasting C-peptide rose during the first 4 weeks to levels of 4 to 5 and Sustacal elicited a further rise to 6 to 7. Transplant renal function was stable. Dialy fasting glucose (mmol/l, mean±SD) was 5.6±1 and 5.3±0.6 during the first and second months respectively and post-Sustacal glucose was 5.7+-0.8. Exogenous insulin therapy was progressively withdrawn and stopped duting the ninth week. Thereafter, fasting glucose was 4.7+-0.5, 24 h mean glucose was 6.6+-0.5, and normoglycaemia was maintained after Sustacal. These data show that this mass of freshly isolated and cryopreserved islets from multiple donors provided sustained function (3 months) that reversed insulin-dependence in an immunosuppressed Type 1 diabetic patient treated with simultaneous islet-kedney transplantation.     

The location of VIP in the pancreas of man and rat

Summary VIP has powerful stimulatory effects on both endocrine and exocrine pancreas but its localisation within the gland has not been established. In this study, human pancreas was obtained fresh at surgery (eleven) or within four hours of death (seven). The pancreas was also removed from rats (twenty-two). Immunocytochemical staining showed VIP to be present in fine nerve fibres in all areas of the pancreas. Many fibres were seen in the exocrine pancreas, running between the acini, and around ducts and blood vessels. In addition, dense networks of fibres were observed forming meshes around islets and occasional ganglia were found containing immunoreactive cell bodies. In general, there were fewer VIP fibres in the rat pancreas than in the human, but overall distribution was identical. The mean VIP content of whole human pancreatic tissue was 42±10 pmol/g wet weight (38±9 pmol/g in head, 49±6 pmol/g in body and 42±11 pmol/g in tail). Whole rat pancreatic tissue contained 28±7 pmol/g wet weight while preparations of isolated islets were found to contain 374±30 pmol/g. It is possible that the heavy VIP innervation of the islets described here indicates a role in the regulation of islet hormone release.     

Evidence for metabolic regulation of pancreatic glucagon secretion by L-glutamine

To investigate effects by L-glutamine on pancreatic A-cell secretion and intermediary metabolism, isolated pancreatic islets from normal and streptozotocin treated guinea pigs (A-cell rich islets) were incubated in the presence of glucose (5.5 mM) +/- L-glutamine (10 mM). Glutamine significantly enhanced glucagon release from 297 +/- 54 to 528 +/- 53 pg/micrograms DNA/h in normal islets and from 553 +/- 31 to 806 +/- 50 pg/micrograms DNA/h in A-cell rich islets. All results were expressed on the basis of islet DNA concentration, being 66 +/- 4 ng DNA per normal islet and 32 +/- 2 ng DNA per A-cell rich islet. Simultaneously, glutamine suppressed glucose oxidation to 64 per cent in normal islets and to 47 per cent of basal oxidation in A-cell rich islets. Islet content of ATP was also reduced by glutamine to about 60 per cent in A-cell rich islets, but not significantly changed in normal islets. Glutamine oxidation, at 5.5 mM-glucose, was considerably higher in A-cell rich islets (911 +/- 65 pmol/micrograms DNA/h) than in normal islets (313 +/- 52 pmol/micrograms DNA/h). Addition of porcine insulin (25 mU/ml) counteracted these effects by glutamine, i.e. suppressed glucagon release but increased glucose oxidation and ATP content of the A-cell rich islets. The present findings demonstrate that glutamine stimulates glucagon release and is readily metabolized by the A-cells. Furthermore, the regulation of glucagon secretion by glutamine appears to be reciprocally related to factors affecting glucose metabolism and ATP-levels in the A-cell.     

Implanted islets in the anterior chamber of the eye are prone to autoimmune attack in a mouse model of diabetes

Aims/hypothesis

Type 1 diabetes is an autoimmune disease resulting from the destruction of insulin-producing beta cells. Along with advances in generating replacement beta cells for treating diabetes, there is also increasing demand for non-invasive tools to evaluate the recurrence of autoimmune attack on transplanted tissue. Here, we examined the anterior chamber of the eye as a potential islet transplant site, and also evaluated whether in vivo imaging of the islets transplanted in the eye could enable real-time visualisation of autoimmune processes underway in the pancreas.

Methods

Syngeneic islet equivalents were transplanted into the eye or kidney capsule of streptozotocin-induced diabetic C57BL/6 mice to compare islet dose (25–125 islet equivalents) and function across transplant sites. Autoimmune attack of syngeneic islets was evaluated in the pancreas and eye tissues of NOD and NOD-severe combined immunodeficient (SCID) mice given diabetogenic splenocytes.

Results

Islet transplantation in the eye decreased fasting plasma glucose levels and increased weight gain and survival in an islet-dose-dependent manner. Even 50 islets in the eye reduced blood glucose levels, whereas ≥200 islets were required in the kidney for a similar effect. Autoimmune destruction of pancreatic islets in the eye mirrored that in the pancreas and could be visualised in real time by non-invasive imaging.

Conclusions/interpretation

We found that far fewer islets were required to restore normoglycaemia when transplanted into the anterior chamber of the eye vs the kidney capsule. However, our results suggest that islets are not protected against autoimmune attack in the eye, making this a suitable site for visualising autoimmune processes against transplanted tissue.     

Antibodies directed against the pancreatic islet cell plasma membrane detection and specificity

Summary Rabbits were immunised with suspensions of viable, insulin-producing islet cells prepared from collagenase-isolated rat or ob/ob mouse pancreatic islets. Antibodies reactive with the surface of dispersed rat islet cells were present in both the rabbit anti-rat and the rabbit anti -ob/ob mouse islet sera as revealed by indirect immunofluorescence or by a radioligandassay using ¹²⁵I-Protein A as a measure of cell bound IgG. In a competition assay the binding of ¹²⁵I-Protein A was displaced in a concentration dependent manner by non-radioactive Protein A. Maximal displacement was found at concentrations of Protein A higher than 0.1 g. added to 10⁵ islet cells. Although not always detected by immunofluorescence there was a several-fold increase above normal rabbit serum of ¹²⁵I-Protein A-binding to rat hepatocytes and spleen lymphocytes incubated with the islet cell antisera. Conversely, rabbit antisera against rat spleen lymphocytes or against a rat liver plasma membrane preparation reacted with rat islet cells. The rabbit anti-rat islet cell antiserum was absorbed to both spleen lymphocytes and hepatocytes until there was no binding of ¹²⁵I-Protein A to either cell type. Islet specific antibodies were still present since this doubly absorbed antiserum induced cell surface immunofluorescence as well as ¹²⁵I-Protein A-binding to rat islet cells. It is concluded that apart from common antigenic determinants immunisation with viable islet cells induces formation of antibodies directed against specific islet cell surface components.     

Characterization of pancreatic islet monoamine oxidase

Monoamine oxidase (MAO) is present in isolated islets of Langerhans of rabbits, golden hamsters, and rats. Tryptamine, tyramine, serotonin, and dopamine can serve as substrates for this enzyme. We compared the properties of islet and liver MAO in the rabbit. The Michaelis constant (K_m) for tryptamine of islet MAO (6.5 × 10^?5M) is greater than the K_m of liver MAO (3 × 10^?5M). The K_m for tyramine of islet MAO (1.5 × 10^?4M) is similar to the K_m of liver MAO (1.8 × 10^?4M). Islet MAO appeared to be more susceptible to heat inactivation (50°C) than did liver MAO. This may be an artifact produced by the collagenase technique used in the preparation of the islets, as collagenase treatment of liver increased the thermal lability of the MAO in this tissue. Liver and islet MAO have a comparable sensitivity to MAO inhibitors such as clorgyline, deprenyl, tranylcypromine, pargyline, and harmine. The present report, along with previous reports that MAO inhibitors alter insulin secretion, suggests that islet MAO may modify insulin secretion.     

Fat-induced changes in mouse pancreatic islet insulin secretion,insulin biosynthesis and glucose metabolism

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with -ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.     

Islet amyloid polypeptide in pancreatic islets from type 1 diabetic subjects

Aims/hypothesis:

Islet amyloid polypeptide is originally isolated as the chief constituent of amyloid deposits in type 2 diabetic islets. Islet amyloid polypeptide hyposecretion was known in type 1 diabetics and this study aimed to detect possibly reduced islet amyloid polypeptide-positive cells in type 1 diabetic islets.

Results:

Non-diabetic control islets showed about 60% of islet cells were insulin cells, and 60% of insulin cells were positive for IAPP. In type 1 diabetic islets, islets were generally smaller than control islets, consisting of weaker positive cells for insulin and islet amyloid polypeptide. Medium-sized islets still retained some insulin positive cells, whereas islet amyloid polypeptide positive cells were much less or even absent, but some insulin-negative cells were weakly islet amyloid polypeptide positive. An occasional extra-large islet, representing regenerating islets, consisting of more than 100 islet cells revealed less than 35% insulin and 20% islet amyloid polypeptide positive cells with relatively increased glucagon and somatostatin cells. Both normal and type 1 diabetic islets revealed scattered, densely insulin and islet amyloid polypeptide positive sickle-shaped cytoplasm without granular appearance, consistent with degenerating insulin cells.

Methods:

Using commercially available rabbit anti-islet amyloid polypeptide antibody, immunostaning was performed on ten cases of type 1 diabetic pancreata and eight non-diabetic controls. Both control and type 1 diabetic pancreata were systematically immunostained for insulin, glucagon, somatostatin and islet amyloid polypeptide.

Conclusion/Interpretation:

Control islets consisted of about 60% insulin cells, and about 34% of islet cells were amyloid polypeptide positive with scattered and densely positive for insulin and islet amyloid polypeptide without granular appearance, consistent with degenerating β-cells. All islets, including occasional extra-large islets from type 1 diabetics, showed less insulin cells and less islet amyloid polypeptide positive cells with twice increased glucagon and somatostatin cells of the control islets, but some insulin-negative cells were positive for islet amyloid polypeptide, suggesting the presence of islet amyloid polypeptide in degenerating and extra large regenerating islets. Thus, this immunocytochemical staining revealed generally less islet amyloid positive cells in type 1 diabetic islets, corresponding to severe hyposecretion of islet amyloid polypeptide in type 1 diabetics.Key words: immunocytochemistry, islet amyloid polypeptide, pancreatic islets, type 1 diabetes     

Monoamines in the pancreatic islets of the mouse

Summary By application of autoradiographic technique the cellular and subcellular distribution of radio-activity in mouse pancreatic islets was investigated following intravenous administration of³H-5-hydroxytryptophan. Autoradiographic silver grains, most of which probably represent 5-hydroxytryptamine formed from the labelled precursor, appeared over A₂ and B cells, whereas very few grains were recorded over A₁ cells at any time investigated (20 min–16 hours) and also when monoamine oxidase was inhibited. Quantitative analysis of autoradiographic sections revealed that the concentration of silver grains over the specific granules of A₂ and B cells was 5–10 times higher than over the remaining parts of these cells. In A₂ cells the highest grain count was recorded at 20 minutes, in B cells at 1 hour after the injection of label. After 8 hours very few, and after 16 hours no silver grains appeared over islet cells. Inhibition of monoamine oxidase caused an increased retention of label over islet cells, most pronounced over A₂ cells. Pretreatment with reserpine abolished the autoradiographic reaction.This study was supported by Grant K71-12X-3352-01 from the Swedish Medical Research Council.