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We investigated the effect of Cyclosporin A (100 ng/ml - 12000 ng/ml) on functional parameters of the endocrine pancreas in vitro. The insulin secretion of isolated islets in response to a variety of secretory stimulators is characterized by minimal alteration occasionally observed in the presence of very high Cyclosporin A concentrations. Cyclosporin A did not alter the glucose-induced biphasic insulin secretion, nor the arginine-stimulated glucagon secretion of the perfused rat pancreas. Exposure of isolated islets to 2000 ng Cyclosporin A/ml for 3 weeks resulted in a minimal decrease of insulin content and a marked reduction of glucagon content, which were not accompanied by drastic alterations of insulin secretion. Scanning electron micrographs of these Cyclosporin A-treated islets did not differ from cultured control islets. The results let us assume that Cyclosporin A exerts no toxic effects on the endocrine pancreas in vitro, when exposed to concentrations greater than or equal to 2000 ng/ml.

Two weeks after intraportal transplantation of 2,000 neonatal pancreatic islets, recipient rats completely recovered from streptozotocin-induced diabetes. The reversal of diabetes could be documented by the normalization of blood glucose levels, by a restored weight gain, by normal glucagon and insulin levels in blood, and by a disappearance of polyuria and polydipsia. The reversal remained stable for at least 9 months. This study determined whether intraportally transplanted pancreatic islets were reinnervated after transplantation and whether the secretion of insulin and glucagon from pancreatic islets might be modulated by the vegetative innervation of recipient livers. Predominantly catecholaminergic but also cholinergic nerve fibers were detected not only within the portal tracts around hepatic arteries, portal veins, and bile ducts, but also at the borderline of hepatocytes and β-cells and in islet cell complexes between β-cells. Corresponding electron micrographs showed β-cells in close contact with axons of nonmyelinated nerve fibers. Isolated livers were single pass perfused via both the hepatic artery and the portal vein. An increase in glucose level from 5 to 14 mmoW enhanced hepatic glucose uptake and increased insulin secretion from transplanted islets with a biphasic secretion profile but had no effect on glucagon output. Stimulation of the nerve plexus around the hepatic artery and the portal vein (7.5 Hz, 2 min), which activates primarily the sympathetic system, not only reduced glucose uptake and perfusion flow but also completely reversed the glucose-stimulated increase in insulin secretion. Nerve stimulation did not influence glucagon secretion. The α1-aradrenergic antagonist prazosin and the β-blocker propranolol completely blocked the nerve stimulation-dependent alterations in glucose balance and in perfusion flow but did not affect the reversal of the glucose-stimulated increase in insulin release caused by nerve stimulation. This indicates indirectly that the inhibition by sympathetic liver nerves of insulin secretion was mediated via α2-receptors as in normal pancreatic islets. Experiments with the α2-adrenergic antagonist yohimbine were in line with this conclusion.

Isolated pancreatic islets of normoglycemic sand rats do not respond to 2.5 mM glucose with an enhanced glucagon secretion, which could be observed in normal Wistar rats. Arginine stimulates glucagon release in the presence of 2.5 mM glucose in Wistar rats as well as in sand rats. The secretion pattern is not caused by insulin deficiency since sand rat islets are characterized by an increased insulin secretion rate in vitro. This paradoxical glucagon secretion is not caused by a changed glucagon content but might be related to this species which is able to develop a diabetic syndrome spontaneously.

Isolated pancreatic rat islets incubated in vitro were used as a bioassay system for investigating the influence of human insulin antibodies on insulin secretion. Serum samples with different titres of insulin antibodies were obtained from juvenile diabetics after various periods of insulin therapy. The insulin secretion of isolated islets is enhanced by insulin antibodies and positively correlated to the measured antibody titres in serum.

Bei 73 insulinbedurftigen Diabetikern mit klinisch differentem Stoffwechselverhalten und 7 Gesunden wurden eine 30-minutige Nicotinsaureinfusion durchgefuhrt und die Blutparameter von Glucose, Pyruvat, Lactat, FFS, Glycerin, Katecholaminen und 11-OH-CS uber einen Zeitraum von 180 min analysiert. Auserdem wurde die Insulinsekretion in vivo bei einigen diabetischen und gesunden Probanden mittels der ILA, in vitro an isolierten Langerhans'schen Inseln aus Mausepankreata mittels dem IRI unter Nikotinsaureeinwirkung untersucht. Es konnte demonstriert werden, das Nikotinsaure in vitro zu einer significanten Steigerung der Insulin-ausschuttung fuhrt. In vivo ist eine Stimulation der ILA nur bei Gesunden und stoffwechselstabilen Diabetikern nachweisbar. Das Ausmas des sekundaren Nicotinsaure-induzierten FFS-Rebound steht in negativer Relation zur Stimulierbarkeit der ILA. Es gestattet unter Einbeziehung des Blutglucoseverhaltens und des exogenen, zur Stoffwechselkompensation erforderlichen taglichen Insulinbedarfs eine metabolische Differenzierung des stoffwechsellabilen und stoffwechselstabilen Diabetestyps.

Polyelectrolyte complex capsules from cellulose sulphate can be formed by precipitation in a polycation bath. The application of this new method for encapsulation of pancreatic islets requires investigations whether and to what extent cellulose sulphate injures viability and functionality of the pancreatic islets. Islets cultures in the presence of 2% cellulose sulphate for up to 3 weeks are characterized by unchanged insulin content, secretion and biosynthesis when compared to appropriate controls.

A possible role for cyclic adenosine-3'-5'-monophosphate (cAMP) in islet cell replication was examined in collagenase-isolated pancreatic islets from Wistar rats of different age and different metabolic state (non-pregnant, pregnant, days 15.5-17.5). Islets obtained from pregnant rats released significantly more insulin in response to 10 mmol/l glucose (culture for 24 h) and their DNA synthesis (incorporation of [3H]thymidine into islet DNA) was doubled compared to islets from non-pregnant controls. Islets obtained from 4-6 days old rats showed a maximal stimulation of DNA synthesis after exposure to 0.1 mmol/l IBMX (3-isobutyl-1-methylxanthine) whereas the cAMP accumulation and the insulin biosynthesis measured in a subsequent short-term incubation were dose-dependent stimulated up to 1.0 mmol/l IBMX. In islets of 12 days old rats or 3 months old rats, however, IBMX did not stimulate DNA synthesis or insulin release measured during culture, although the cAMP content per islet was significantly enhanced after culture in the presence of IBMX.

Colony-bred sand rats were fed with rat pellet chow in restricted quantities or ad libitum for 8--10 or 28--31 weeks after weaning. The changes of glucose metabolism were characterized by an intraperitoneal glucose tolerance test. The daily food intake and the average weight gain differed only in the first 5--7 weeks of pellet nutrition. In the impaired glucose tolerance tests of all sand rats the high basal plasma IRI levels were not significantly increased by the grossly enhanced blood glucose concentrations. The insulin secretion of either acutely incubated or for 8 days cultivated isolated pancreatic islets, however, was stimulated already by low (1.7 and 5 mM) glucose concentrations in all diet groups. Otherwise the glucagon secretion of isolated islets was not suppressed by high glucose concentrations. No changes of insulin or glucagon contents of islets were found in the different diet groups. The adipocytes of all animals revealed a complete ineffectiveness of insulin on the glucose utilization to CO2 and triglycerides. The basal glucose conversion to CO2 and glycogen in skeletal muscle and the stimulatory potency of insulin was low and not distinctly different in all groups. In liver glycogen and triglyceride contents as well as gluconeogenic enzyme activities were not influenced by feeding of different quantities of pellet diet at the investigated time points. The time course of the metabolic and clinical alterations demonstrates that the peripheral organs become insensitive to insulin in the first weeks after weaning.

Release of immunoreactive insulin activity (IRI) and biological insulin like activity (ILA) from collagenase isolated pancreatic islets of sand rats (Psammomys obesus) maintained on a vegetable diet were examined at 60 min and 24-48 h intervals under culture conditions at 1.0 and 15.6 mM glucose. The glucose-insulin dose response curves for sand rats after 60 min incubation were compared with those after 24 or 48 h of incubation. The pancreatic islets responded to 5mM glucose with a high insulin release especially under culture conditions. A drastic depletion of stored insulin in the islets cultivated for 2 days at 5 or 15 mM glucose is accompanied by a continuous diminution of the glucose-induced insulin release with the prolongation of cultivation up to one week.

A subpopulation (n = 27) of normoglycaemic Sand rats was characterized as carbohydrate-intolerant by intraperitoneal glucose loading. Five of these animals did not show any rise in peripheral insulin concentrations when injected with glucose. However, when isolated by collagenase digestion their islets still exhibited a significant enhancement of insulin secretion in response to glucose, glyceraldehyde, mannose and theophylline. The in vitro secretory responses were comparable to those of islets from carbohydrate-tolerant Sand rats. The results underline the importance of the natural environment for the B-cell response in vivo.

Goldthioglucose enhanced independently of glucose but related to dose the insulin secretion of pancreatic rat islets in vitro. The stimulatory action is additive to that of glucose, is not inhibited by mannoheptulose and not connected with an altered glucose-utilization. The gold thioglucose-induced insulin secretion is diminished in the presence of epinephrine and Mg++, respectively, and characterized by a uniphasic response peaking at 15 min. In the absence of glucose goldthioglucose did not modify the pancreatic glucagon secretion.

Starvation of Wistar rats induced a shift of glucose threshold for insulin secretion of isolated islets above 5 mM, which can be restored by pretreatment of the tissue with glucose, mannose, glyceraldehyde, an theophylline, but not with acetylcholine or lactate. The improved insulin secretion is not connected with an enhanced glucose utilization.

Irradiation of pancreatic rat islets up to a dose of 2.5 Gy did neither alter glucose-nor IBMX-induced insulin secretion studied in vitro. The insulin as well as glucagon content of irradiated islets were similar as in the control tissue. This was also true in islets irradiated with 25 Gy which were characterized by a decreased insulin secretion in the presence of glucose and IBMX, respectively. Since we did not find indications of an enhanced hormone output in the radiation medium, we want to suggest that higher irradiation doses affect insulin release of pancreatic islets in vitro. This observation has to be taken into account for application of radioimmunosuppression for transplantation.

In normoglycemic sand rats the insulin secretion of isolated islets was investigated dependent on the time of artificial nutrition in captivity. Whereas the islets of short-term adapted animals did not respond within 15 min with an enhanced insulin secretion after stimulation with 16.5 mM glucose or 10 mM arginine, prolongation of incubation time resulted in a significantly enhanced hormone release. An intraveneous glucose injection (1 g/kg body weight) 45 min prior to the excision of the pancreas caused an immediate insulin secretion of the isolated islets in response to glucose or arginine. Islets of sand rats adapted for more than 10 weeks to artificial nutrition responded immediately with an enhanced insulin release in contrast to the islets of short-term adapted animals. These differences could not be correlated to the insulin content, the A/B cell ratio, and ultrastructural alterations of the islets.

The secretion of insulin and glucagon was investigated in pancreatic islets from diabetic and nondiabetic sand rats of similar age and weight. The metabolic characterization was based on an intraperitoneal glucose tolerance test. Compared to nondiabetic animals diabetic sand rats had a diminished insulin content in their islets and a decreased insulin secretory response to glucose, glyceraldehyde and theophylline. Diazoxide inhibited insulin release in diabetic as well as in nondiabetic sand rats whereas mannoheptulose was effective only in the nondiabetic rats. There was no significant difference in glucagon content between the two groups. The glucagon secretion by pancreatic islets of diabetic animals was not suppressed by glucose, as in nondiabetic sand rats islets, but by glyceraldehyde. This indicates that the sensitivity to glucose rather than the suppressibility of glucagon release was altered.

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-dihydroxyacetone, 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 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-glyceraldedyde 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.

Tissue culture of human foetal pancreas slices, obtained at a gestational age between 10 and 19 weeks, as a method of preservation and to pool the material before transplantation was investigated. Before and after 2 weeks of culture the pancreatic insulin content, the insulin secretion in response to glucose and isobutylmethylxanthin (IBMX) as well as the protein biosynthesis were measured. In addition, the distribution of the insulin immunoreactive cells was examined, as well as their relative volume density. After 2 weeks' culture an increase of the basal insulin secretion was observed; this was probably due to the glucose content (10 mmol/l) in the culture medium. Neither the stimulated insulin secretion, nor the protein biosynthesis, the insulin content, and the B-cell volume were altered by the used culture conditions. It was concluded that tissue culture is a suitable method to preserve human foetal pancreas slices before transplantation.

The dynamics of insulin release from spiny mouse perifused pancreatic islets was investigated. In agreement with earlier studies we found no initial (0-10 min) insulin secretion in response to glucose in islets prepared from mice up to 35 weeks of age. The islets of animals older than 40 weeks, however, were characterized by greater insulin content, and a restored typical biphasic insulin secretion profile. The results suggest that immediate glucose response is modified by environmental or other factors related to age.

Pancreatic islets of wistar rats, isolated after 15 min of digestion with collagenase, secreted insulin in response to 15.0 mM glucose within 2 min and showed the typical sigmoidal glucose response during an incubation time of 15 and 60 min, respectively. Islets, isolated after 35 min of digestion with collagenase, responded with delay after stimulation with glucose (after 15 min of incubation), and are characterized by an increased "release" in the presence of 2.5 mM glucose.

Islets were isolated from the pancreas of female rats by using the collagenase technique. After culturing for 4 days, the islets were taken for measurement of insulin release biosynthesis as well as glucose utilization in subsequent short-time incubations. A low glucose concentration was insufficient to maintain a glucose-stimulated insulin release in vitro. A high glucose concentration had a protecting and restoring effect on the insulin release: ultrastructurally, such islets showed signs of an active biosynthesis in the electron micrograph. The enhancement of Mg++ in the culture medium resulted in an improvement of insulin storage in the islets, accompanied by a well-preserved action of glucose in a subsequent incubation.

Three different methods of legal abortion were applied to 23 patients, who had asked for termination of pregnancy, between the eleventh and 14th weeks of pregnancy. In vitro studies into human foetal pancreas were taken into consideration, when the choice was made. Carbohydrate tolerance was normal in all cases (50 g oGTT). Timing of intra-uterine foetal death, in the context of the three above methods (B-scan ultrasonic diagnosis and HPL level measurement) as well as studies into in vitro stimulation of foetopancreatic insulin secretion have shown purely mechanical, surgical abortion, sometimes following prostaglandin cervical gel priming, to be indispensable a condition for successful in vitro elucidation of the aetiopathogenesis of foetal diabetes. Extra-amniotic prostaglandin induction of abortion has failed to yield any relevant information, in that context.3 different methods of legal abortion were administered to 23 patients who had requested termination of pregnancy between weeks 11-14 of pregnancy. In vitro studies into human fetal pancreas were taken into consideration when the choice was made. Carbohydrate tolerance was normal in all cases (50 g oGTT). Timing of intrauterine fetal death, in the context of the 3 above methods (B-scan ultrasonic diagnosis and HPL level measurement), as well as studies into in vitro stimulation of fetopancreatic insulin secretion, have shown purely mechanical, surgical abortion, sometimes following prostaglandin (PG) cervical gel priming, to be indispensable as a condition for successful in vitro elucidation of the etiopathogenesis of fetal diabetes. Extraamniotic PG induction of abortion has failed to yield any relevant information in that context. (author's)

The effect of various inhibitors of insulin secretion such as mannoheptulose (20 mM), atropine (1 mM), diphenylhydantoin (20 microng/ml), high concentration of Mg++ (5.3 mM) in the presence of 20 mM glucose (control) on insulin content and secretion from collagenase-isolated rat pancreatic islets was studied in vitro by cultivation of islets up to 5 or 9 days in glass Petri dishes without attachment. In a following short-term incubation for 60 min the glucose-induced insulin release without and with theophylline (5 mM) was investigated. Islets cultivated at 5 mM glucose and at 20 mM glucose with the inhibitors mannoheptulose or atropine lost the responsiveness to glucose and theophylline whereas such islets cultivated at 20 mM glucose alone or with diphenylhydantoin (DPH) or 5.3 mg Mg++ showed a stimulation of insulin secretion by glucose and theophylline. Compared, however, with freshly isolated islets all cultivated islets were restricted in their maximal glucose response and this defect was not evoked alone by quantitative changes in islet insulin content. Nevertheless, culture conditions which facilitate a net increase of insulin (content and release) during cultivation influenced also positively the glucose-induced insulin release without and with 5 mM theophylline in the following short-term experiments.

The influence of a single i.v. injection of 30 mg/kg streptozotocin (SZ) into female Wistar rats on plasma glucose, pancreatic insulin content, volume and function of the remaining beta-cells has been investigated. 14 days after SZ treatment most animals remained normoglycemic; only about 15% showed permanent hyperglycemia. The pancreatic insulin content in normoglycemic rats was reduced to 30%, whereas in hyperglycemic rats only 6% of control values could be detected. The remaining beta-cell volume was comparable in the two groups, indicating no correlation between pancreatic insulin content and residual beta-cell volume. The glucose- and IBMX-stimulated insulin release of islets isolated from normoglycemic cats 14 days after SZ application was not significantly altered whereas the islets insulin content of SZ treated animals was significantly lowered in comparison to controls.

For investigating insulin secretion in vitro human fetal pancreatic slices prepared from women with a normal carbohydrate tolerance at a mean gestational age of 12 +/- 1 weeks were incubated in the presence of various secretagogues. Glucose concentration up to 20 mmol/l failed to enhance insulin secretion, whereas an increase of glucose up to 40 mmol/l, the addition of the phosphodiesterase inhibitor IBMX or a priming period of 30 min in the presence of 20 mmol/l glucose resulted in an enhancement of hormone release, calculated per microgram dry weight. For the first time the results demonstrates an intact secretory machinery of the B-cells under controlled conditions in an early stage of gestational development.

The effect of somatostatin on insulin secretion in response to a variety of stimuli was investigated in micro-dissected as well as collagenase isolated pancreatic rat islets. Somatostatin inhibited insulin secretion in both islet preparations in the presence of different initiators, but failed to affect the hormone output induced by theophylline, 1-methyl-3-isobutylxanthin (IBMX) or p-chloromercuriphenylsulfonic acid (CMBS). The inhibitory action was associated with decreased glucose metabolism by islets (measured as conversion of U-14C-glucose to 14CO2).

Insulin secretion, biosynthesis and content of isolated pancreatic islets prepared for fetuses of nondiabetic or insulin-dependent diabetic women were investigated in vitro. Glucose failed to stimulate the hormone release during an incubation period of 60 min in islets of fetuses from non-diabetic women. Prolongation of the glucose exposure or the addition of theophylline resulted in a significantly enhanced insulin release. The islets isolated from fetuses of insulin-dependent mothers are glucose-sensitive with respect to hormone release and biosynthesis. The results demonstrated that the human fetal B-cell is glucose-sensitive after glucose exposure. The differences between the islets of fetuses of nondiabetic and diabetic women are discussed as a response to the fetal plasma glucose levels.

Insulin (IRI) secretion pattern of collagenase isolated islets from Wistar rats were investigated in a batch type incubation (60 min) and under organ culture conditions (up to 7 days) with different concentrations of glucose as stimulus. The B-cell response within 60 min of incubation was determined in Krebs-Ringer bicarbonate buffer with 1 mg/ml bovine serum albumin and 16 mM HEPES (KRB-HEPES) and compared with the hormone release in a culture medium (TC 199) containing 10% calf serum. Additionally the insulin content of islets before and after 7 days of culture was assayed radioimmunologically. In the presence of culture medium the insulin secretion was enhanced by 5.8 mM glucose whereas this glucose concentration does not stimulate the insulin secretion in KRB-HEPES. During organ culture the insulin secretion was identical in media with 1.0 and 5.8 mM glucose, respectively, but 15 mM glucose raised the hormon output more than 10-fold. The insulin content of islets, cultured for 7 days was decreased in the presence of 15 mM glucose up to 30% and in the presence of 5.8 mM glucose up to 13% of that of islets after isolation. The recovery rate of insulin calculated as the sum of secretion and content after cultivation at 15 mM glucose was higher than 100%, whereas the experiments in the presence of 1.0-5.8 mM glucose are characterized by a recovery rate of 25%. The results are discussed in connection with a altered intracellular breakdown of insulin in the B-cells.

Mouse islets of Langerhans, isolated by microdissection after treatment with collagenase, were incubated either with pure insulin antibodies (IAB), which were prepared by immune precipitation, or with exogenous insulin. Insulin release was enhanced with increased concentrations of IAB and was inhibited by exogenous insulin. The results suggest that it was not the insulin per se, but probably its biological effect on the β-cells that influenced insulin secretion.