Secretory, enzymatic, and morphological characterization of rat pancreatic endocrine tumours induced by streptozotocin and nicotinamide

Rat pancreatic endocrine tumours were induced by administration of streptozotocin plus nicotinamide. Fifteen to eighteen months later tumours with wet weights of 0.1 to 224 mg were isolated. These tumours were compared with normal rat pancreatic islets. Insulin release from perifused tumours was stimulated by D-glucose, L-leucine, 2-ketoisocaproate, and D-glyceraldehyde, potentiated by theophylline and inhibited by norepinephrine. Compared with isolated rat pancreatic islets, however, insulin secretory responsiveness to glucose stimulation and insulin content were reduced in tumour tissue. Hypoglycaemia in tumour bearing rats and impaired diffusion of insulin out of the tumours may explain this difference. The pattern of enzyme activities observed in tumour tissue was typical for pancreatic endocrine tissue. The activities of succinate dehydrogenase, the two types of the monoamine oxidase, and alpha-glucosidase were in the normal range in tumour tissue. Only the activities of 5'nucleotidase and glutamate dehydrogenase were decreased. Immunocytochemical analysis of the tumours revealed that they contained an average of 91% B-cells. In addition 8% of D-cells were encountered. Proportions of A-cells and PP-cells ranged below 1%. Thus this endocrine tumour of the pancreas with a high proportion of functionally intact B-cells is an interesting model for studying regulation of secretion and endocrine tumour development.     

Experimental hypoglycemizing tumor of B-cells of Langerhans islets produced by the combined action of streptozotocin annd nicotinamide in the rat.

Young male and female Wistar-Velaz rats were treated with streptozotocin-nicotinamide combination according to the method of Rakieten and examined periodically for 23 months for fasting and postprandial glycemia, by intravenous, intraperitoneal or intragastric glucose tolerance and tolbutamide tests with the aim to detect in vivo the experimentally produced nesidiomas. One male rat with severe hypoglycemia, apparent first on tolbutamide test after twelve months, later also on glucose tolerance test and fasting hypoglycemia associated with paraplegia with macroscopically and microscopically documented nesidioma is described. The exstirpation of this nesidioma was followed first by normalization and later by development of latent diabetes.     

Fine structure of rat islet cell tumors induced by streptozotocin and nicotinamide

Summary Young male Holtzman rats were injected intravenously with 50 mg/kg of Streptozotocin, preceded and followed by a single intraperitoneal injection of 350 mg/kg of nicotinamide, according to the method of Rakieten et al. [16]. After 245 to 323 days, 27 pancreatic islet cell tumors measuring up to 0.6 cm were demonstrable in 20 of 41 rats so treated; they were solitary in 15 and multiple (two or three neoplasms each) in five animals. It was not possible to distinguish between tumor-bearing and tumor-free rats on the basis of periodic blood sugar determinations and serum insulin assays. Mean insulin concentration in grossly tumor-free pancreatic specimens was 0.661 units of insulin/g of wet tissue, but amounted to 5.385 units/g in specimens containing tumor. The islet cell tumors were rounded and well delineated. They were located in all parts of the pancreas. In general, their cells stained deeply with aldehyde-fuchsin. Ultra-structurally, most tumors consisted of well granulated B cells. A or D cells were not encountered while occasional EC cells were identified. Nucleoli were frequently prominent. Some necrotic B cells and others with few or unusually small secretory granules were present. Extravasated erythrocytes as well as hemosiderin deposits were seen in many tumors, and tumor cell particles were occasionally noted within the lumina of capillaries. Distant metastases were not demonstrable in this group of animals.Supported by a grant from the National Institutes of health, No. A.2203Presented at the Eighth Congress of the International Diabetes Federation, Brussels, Belgium, June, 1973     

Human beta cells are exceedingly resistant to streptozotocin in vivo

Streptozotocin (STZ) causes beta cell death in rodents via the mechanism of DNA damage precipitating poly(ADP-ribose) synthetase activation followed by lethal nicotinamide adenine dinucleotide depletion. It is unclear whether humans are susceptible to this mechanism. Islets were isolated from STZ-sensitive (CD1 mice and Lewis rats) and resistant [fish (tilapia)] species and from man and then were transplanted into diabetic nude mice under the kidney capsule. Normoglycemic recipients with normal glucose tolerance tests on d 30 were injected with increasing iv doses of STZ and their plasma glucose levels followed for 5 d; glucose tolerance tests were repeated on nondiabetic mice. Mice were then killed; grafts and native pancreata were examined. Based upon three criteria (i.e. nonfasting plasma glucose levels, glucose tolerance tests, and islet histology), the following observations were made: 1) Recipients of rat islets were resistant to 25 mg/kg but were uniformly diabetic at doses of 50 or 75 mg/kg. 2) Recipients of mouse islets were resistant to 75 mg/kg but were uniformly diabetic at 150 or 200 mg/kg. 3) Recipients of the fish islets were resistant to 300, 400, and 450 mg/kg. 4) Recipients of human islets were resistant to 100, 200, 300, 400, and 450 mg/kg. The results in recipient mice bearing long-term rat, mouse, or fish islet grafts were the same as previously published dose-response data for each donor species. We extrapolate from our results based on human islet grafts in mice that human beta cells are exceedingly resistant to STZ.     

Exocrine and endocrine secretion from isolated perfused rat pancreas with islet cell tumors induced by streptozotocin and nicotinamide

Pancreatic exocrine and endocrine function in the rat with islet cell tumors induced by streptozotocin and nicotinamide was studied in thein vitro isolated perfused pancreas. The tumor-bearing pancreas secreted significant amounts of insulin even at 2.8 mM glucose stimulation. Further, insulin response to 8.3 mM glucose stimulation was greater in the tumor-bearing pancreas than in the control. Not only endocrine, but also exocrine, disorders were found in the rat pancreas bearing islet cell tumors. In contrast to the increased response of insulin, amylase output in response to 0.1 ng/ml cerulein was significantly lower in the tumor-bearing than in the control pancreas, although there was no difference in pancreatic juice flows from both groups. These results suggest that enzyme secretory function of the pancreas with islet cell tumors may be suppressed in the presence of some interrelationship between the exocrine and endocrine portion of the pancreas with islet cell tumors.     

Recovery of pancreatic beta cells in response to long-term normoglycemia after pancreas or islet transplantation in severely streptozotocin diabetic adult rats

In the well-established, high-dose streptozotocin diabetic rat model, it is unknown whether normoglycemia after pancreas or islet transplantation may induce the expression of the glucose recognition structures and stimulate the replication of the few surviving pancreatic beta cells. Therefore, the endocrine pancreatic tissue was examined immunocytochemically in streptozotocin-treated major histocompatibility complex congenic Lewis rats at 10 and 100 days after transplantation of whole pancreata or isolated islets implanted under the kidney capsule. In the diabetic state the pancreatic beta cells displayed a weak immunostaining for insulin and glucokinase together with a lack of GLUT2 glucose transporter immunoreactivity in the plasma membrane. Ten days after transplantation, the surviving beta cells had regained their normal immunostaining for insulin and for the glucose recognition structures glucokinase and the A single high dose of streptozotocin causes severe experimental insulin-dependent diabetes mellitus in adult rats due to a selective destruction of the pancreatic beta cells in the islets of Langerhans. At doses between 50 and 60 mg/kg of body weight, only very few beta cells survive in the pancreas (1-3). The diabetic state is irreversible and insulin-dependent, thus representing an experimental animal model for type I diabetes (2). Because of the prevailing hyperglycemia, even the few residual beta cells in the pancreas do not function properly and therefore cannot contribute even to a basal supply of insulin to the organism (4). Pancreatic beta cells can function properly in a diabetic organism apparently only after restitution of normoglycemia (5). GLUT2 glucose transporter. One hundred days after transplantation, both of whole pancreas or isolated islets, the number of surviving beta cells in islets of the pancreata of the recipient animals had increased by two- to threefold. The regenerated beta cells were surrounded by a rim of other endocrine cells. The increase in the number of beta cells was not accompanied by signs of neogenesis from ductal structures in the pancreata. The authors' observations support the concept that strict long-term maintenance of normoglycemia through adequate supply of insulin from endocrine grafts is the ideal prerequisite for beta-cell recovery and restitution of the glucose recognition structures, as well as replication of beta cells in pancreata with end-stage diabetic beta-cell destruction after high-dose streptozotocin treatment.     

The uptake in the pancreatic islets of nicotinamide, nicotinic acid and tryptophan and their ability to prevent streptozotocin diabetes in mice.

The uptake of the nicotinamide adenine dinucleotide (NAD)-precursors nicotinamide, nicotinic acid and tryptophan in the pancreatic islets of mice was studied by use of autoradiographic methods. The ability of these substances to prevent streptoxotocin diabetes was studied in the same species. It was found that only nicotinamide was strongly accumulated in the pancreatic islets and nicotinamide was also the only NAD-precursor which protected against the streptoxotocin diabetes. Apparently there is a relationship between the ability of the NAD-precursors to be taken up in the pancreatic islets and their ability to prevent streptoxotocin diabetes.     

Responses of pancreatic b cells to alloxan and streptozotocin in the guinea pig

Guinea pigs injected with streptozotocin were significantly hyperglycemic on day 1 after injection but only mildly so on day 14. However, serum insulin levels were significantly depressed on day 14; at this time the animals had lost 25% of their initial body weights and were severely glycosuric. The volume fraction of immunostainable B cells in the pancreas was reduced to one third of control values by day 1 after injection and remained at this level by day 14. Animals that received alloxan were slightly hyperglycemic on day 1 but not day 14. Both serum insulin and volume fraction of B cells in the pancreas were reduced by 70% on day 1 but had returned to control levels by day 14. Body weights for this group were equivalent to controls at both time points. These data indicate that: streptozotocin treatment of guinea pigs causes a diabetes-like condition characterized by insulin deficiency, pancreatic B cell loss, glycosuria, and weight loss, which are not reversed in the first 2 weeks after injection, whereas hyperglycemia is only transitory; alloxan also produces a diabetes-like condition early after injection, but all signs of diabetes disappear within 2 weeks, by which time serum insulin levels and the volume fraction of B cells in the pancreas have returned to normal. The experimental results suggest that regeneration of islet B cells following destruction by alloxan may be the primary cause of the recovery of alloxan-injected guinea pigs from the effects of the drug, whereas the persistence of insulin deficiency is consistent with an absence of islet B cell regeneration in the streptozotocin-treated animals.     

Heterogeneous expression of glucokinase among pancreatic beta cells.

The cellular location of glucokinase (GK), a key component of the glucose-sensing mechanism of the pancreatic islet, was determined using immunocytochemical techniques. In rat islets, GK immunoreactivity was detected only in beta cells with no immunoreactivity detected in alpha, delta, or pancreatic polypeptide-containing (PP) cells. However, within various beta cells, GK immunoreactivity varied considerably. Most beta cells displayed relatively low levels of cytoplasmic immunoreactivity whereas other beta cells stained intensely for this enzyme. Colocalization studies of GK and GLUT2, the high Km glucose transporter of beta cells, confirmed that these proteins are located in different subcellular domains of beta cells. The lack of GK immunoreactivity in glucagon- and somatostatin-secreting cells in islets suggests that these cells are not directly responsive to glucose or utilize a fundamentally different mechanism for sensing glucose fluctuations. Moreover, the differential expression of GK among pancreatic beta cells suggests that glucose phosphorylation is the probable enzymatic control point for the functional diversity of these cells.     

Life and death of the pancreatic beta cells.

Pancreatic beta cells are responsible for maintaining the body's glucose levels within a very narrow range; their population is dynamic, with compensatory changes to maintain euglycemia. Throughout the lifetime of a mammal, low levels of beta-cell replication and apoptosis are balanced and result in a slowly increasing mass of beta cells. The emphasis in this review is on recent insights on the natural history of the beta cell in a normal pancreas: sources of renewal, survival and changes in differentiation.     

Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells.

Glucose is a well-known stimulus of proinsulin biosynthesis. In purified beta cells, the sugar induces a 25-fold increase in the synthesis of insulin immunoreactive material over 60-min incubation. Autoradiographic analysis of the individual cells shows that this effect is achieved via dose-dependent recruitment of pancreatic beta cells to biosynthetic activity. Recruitment of beta cells is also seen in isolated islets exposed to glucose. The sigmoidal dose-response curve for glucose-induced proinsulin biosynthesis thus reflects a heterogeneous responsiveness of pancreatic beta cells rather than a progressively increasing activity of functionally homogeneous cells. Dose-dependent recruitment of functionally diverse cells may be a ubiquitous mechanism in tissue function.     

Pancreatic islet circulation in relation to the diabetogenic action of streptozotocin in the rat

A possible relationship between pancreatic islet blood flow (IBF) and the diabetogenic action of streptozotocin (STZ) has been investigated in rats injected ip with saline, glucose, propranolol, phentolamine, yohimbine, or adrenaline. A low dose (25 mg/kg BW) of STZ was given iv 10 min later, and serum glucose concentrations were determined after 3 and 7 days. Measurements of IBF were performed in separate experiments with a microsphere technique 10 min after injection of the drugs. Administration of phentolamine, glucose, or yohimbine was followed by overt diabetes, whereas animals injected with saline, adrenaline, or propranolol failed to become diabetics. Blood flow measurements showed an increase in IBF 10 min after the injection of glucose or propranolol, while there were no significant effects of the other drugs. It is concluded that changes in IBF are of minor importance for the action of agents modulating the diabetogenic effect of STZ. However, the potentiated hyperglycemic effects by glucose, phentolamine, or yohimbine in combination with STZ seemed to coincide with increased insulin secretion, as reflected by elevated serum insulin concentrations at the time of STZ administration.     

Heterogeneity in glutamic acid decarboxylase expression among single rat pancreatic beta cells

Aims/hypothesis. An isoform of glutamic acid decarboxylase, (GAD)65 has been identified as a pancreatic beta-cell autoantigen in Type I (insulin dependent) diabetes mellitus. We investigated the expression of GAD isoforms among single rat beta cells in culture, under different conditions and the correlation between GAD65 expression and insulin secretion-rate. Results. Independent of culture conditions, 100 % of fresh and cultured beta cells express GAD67. In contrast, considerable heterogeneity in GAD65 expression among single beta cells was observed. After 2 days in culture in 2.6 mmol/l glucose, only 24 % of the beta cells express GAD65. This percentage increases to 39 % in 5.6 mmol/l glucose and to 54 % and 56 % in 11.6 mmol/l and 20.6 mmol/l glucose, respectively. Moreover, reducing glucose concentration from 11.6 to 2.5 mmol/l for 2 days, reduces GAD65 expression by nearly 30 %. After 11 days in culture with 11.6 mmol/l glucose, 50 % of beta cells continue expressing GAD65, this percentage is further increased to nearly 75 % by including either nerve growth factor or dibutyryl cyclic AMP or both in the culture medium. When beta cells are challenged for 1 h with 20.6 mmol/l glucose, 67 % respond forming insulin-immunoplaques. More than two-thirds of insulin-secretors are GAD65-positive, in contrast to only 11 % of the non-secreting cells. Moreover, 87 % of beta cells that have a high insulin secretory rate express GAD65. Conclusion/interpretation. These results show that the most active beta cells, which secrete more insulin, also express GAD65 and that manipulating extracellular glucose may modify the expression of the enzyme and possibly the autoimmune attack in Type I diabetes. [Diabetologia (1999) 42: 1086–1092] Received: 28 December 1998 and in revised : 05 May 1999     

Glucose and a-ketoisocaproate induce transient inward currents in rat pancreatic beta cells

Summary The perforated patch technique was used to study changes in membrane potential and whole-cell currents in single isolated rat pancreatic beta-cells during stimulation with glucose or α-ketoisocaproate. Increasing the glucose concentration from 4 to 20 mmol/l, or addition of 15 mmol/l α-ketoisocaproate, caused depolarization and, in most cases, initiation of action potentials. Under voltage-clamp conditions close to a potassium equilibrium potential (E_K) (–60 to –70 mV) these effects were accompanied by the appearance of transient inward currents. These transient currents resembled those elicited during cell swelling in response to a 10 % hypotonic bath solution, a manoeuvre which also caused beta-cell depolarization and electrical activity. Tolbutamide (0.2 mmol/l), in the absence of glucose depolarized beta-cells but did not induce transient inward currents. Nutrient-induced electrical activity and inward currents were abolished by the anion channel inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid, compounds which also inhibited glucose-induced insulin release. It is concluded that nutrient secretagogues induce transient inward currents in isolated rat beta-cells, possibly by activating a volume-sensitive anion conductance. These inward currents could enhance the intensity of electrical, and hence secretory, activity in the beta-cell during nutrient stimulation. [Diabetologia (1997) 40: 1–6] Received: 9 May 1996 and in revised form: 16 September 1996     

Tolbutamide potentiates the volume-regulated anion channel current in rat pancreatic beta cells

Aims/hypothesis Hypoglycaemic sulphonylureas are thought to stimulate insulin release by binding to a sulphonylurea receptor, closing K_ATP channels and inducing electrical activity. However, the fact that these drugs stimulate insulin release at high glucose concentrations where K_ATP channels are closed suggests additional ionic actions. The aim of this study was to test the hypothesis that sulphonylureas influence the current of the glucose- and volume-regulated anion channel.Methods Electrical and ion-channel activity were recorded in isolated rat beta cells using the patch-clamp technique. ⁸⁶Rb⁺ efflux was measured using intact islets. Beta cell volume was measured using a video-imaging technique.Results In the absence of glucose, tolbutamide (100 µmol/l) transiently depolarised the cells. In the presence of glucose (5 mmol/l), tolbutamide evoked a sustained period of electrical activity, whilst at 10 mmol/l glucose, the drug evoked a pronounced silent depolarisation. In the absence of glucose, tolbutamide inhibited ⁸⁶Rb⁺ efflux. However, at 10 mmol/l glucose, tolbutamide induced a transient stimulation of efflux. Tolbutamide potentiated the whole-cell volume-regulated anion conductance in a glucose-dependent manner with an EC₅₀ of 85 µmol/l. In single channel recordings, tolbutamide increased the channel-open probability. Tolbutamide caused beta cell swelling in the presence of glucose, but not in its absence.Conclusions/interpretation Tolbutamide can induce beta cell electrical activity by potentiating the glucose- and volume-regulated anion channel current. This effect is probably not due to a direct effect of the drug on the channel, but could be secondary to a metabolic action in the beta cell.