Development and function of B cells in monolayer islet cells of 3-week-old rat

Monolayer cultures of pancreatic B cells of 3-week-old rats were kept for 7 days in medium with 5.5 mM glucose plus 1 mM 2-deoxy-2-fluoroglucose or for 4 days in medium with 5.5 mM glucose alone, following exposure for 3 days to a medium with 5.5 mM glucose plus 5 microM iodoacetic acid. Addition of the deoxyglucose or iodoacetic acid caused a selective deletion of fibroblasts, yielding large clusters that consisted mostly of islet cells. At the early stage of culture in medium with 16.7 mM glucose (day 4), the response of B cells to 16.7 mM glucose included only a small rise in insulin secreted during the first and second phases, and that to 10 mM of leucine and 2-ketoisocaproate was monophasic. After culturing for 7 days, these three secretagogues markedly stimulated insulin secretion by B cells cultured in both media, with a significant rise in secondary phase secretion. However, quantitative relationships differed. Thus, the response (total insulin secreted during a 30-min stimulation) of B cells in 2-deoxy-2-fluoroglucose to glucose was 155%, to leucine 185% and 2-ketoisocaproate 126% of that of cells exposed to iodoacetic acid. In conclusion, the present results suggest that B cells of 3-week-old rat may be immature, and that medium containing 2-deoxy-2-fluoroglucose is beneficial to continued maturation of the response in vitro.     

The effect of 2-deoxy-D-glucose on insulin release by neonatal rat B cells in culture

The culture for 7 days in medium with 5.5 mM glucose and 1 mM 2-deoxy-D-glucose enhanced the glucose sensitivity of neonatal rat B cells, and even stimulated their growth in vitro. Also, 2-deoxy-D-glucose supplementation maintained insulin release evoked by leucine and 2-ketoisocaproate from B cells at day 7 at levels several times higher than at day 1. The effect of leucine was greatly augmented by glutamine, whereas that of the 2-keto acid remained almost unchanged irrespective of the presence of glutamine. These results suggest an increase in oxidative catabolism of medium nutrients in B cells grown in medium with 2-deoxy-D-glucose for 7 days, and such metabolic changes may promote the growth of B cells in vitro.     

Maintenance of pancreatic endocrine cells of the neonatal rat: VII. The effect of 3-amino-3-deoxyglucose on insulin secretion from perifused B cells

Monolayer cultures of the pancreas of the neonatal rat were maintained in TCM 199 medium, supplemented with 5.5 mM glucose, with or without 5 mM 3-amino-3-deoxyglucose, and perifused to examine the changes which occurred in the insulin secretory response during culture. On day 0, B cells showed a monophasic insulin secretion in response to 16.7 mM glucose, whereas in the presence of 200 nM 12-o-tetradecanoyl phorbol-13-acetate, 40 microM lysophosphatidylcholine, 10 microM forskolin or 1 mM 3-isobutyl-1-methylxanthine, the same dose of glucose stimulated insulin secretion in a biphasic fashion. Under culture conditions without 3-amino-3-deoxyglucose, the response to glucose totally disappeared after 7 days, and that to 10 mM of either leucine or 2-ketoisocaproate was as low as that of day 0. In contrast, B cells that had been cultured for 7 days in medium with 3-amino-3-deoxyglucose showed an adult-like biphasic pattern in response to glucose. When stimulated by glucose at a linear gradient concentration running from 0 to 20 mM, the B cells responded to increasing concentrations of glucose in a dose-dependent fashion. Further, the response of cAMP to glucose was increased by adding forskolin or 3-isobutyl-1-methylxanthine, which also enhanced the secretion of insulin under either a step-wise or slow-rise stimulation with glucose. The effect of 12-o-tetradecanoyl phorbol-13-acetate was also outstanding. Likewise, the addition of either leucine or 2-keptoisocaproate induced a striking increase in the secondary phase secretion as well as promoting the rates of glutamine oxidation within the cells. In conclusion, it is suggested that the high response to a wider variety of stimuli may represent the reaction of neonatal B cells to the cultural milieu rather than a process of physiological development, and these effects exhibited by 3-amino-3-deoxyglucose would be related to a change in the constituents of glycoproteins in the cells.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

Evidence for secretion of 7B2 by A- and B-cells of hamster pancreatic islets.

7B2 is a neuroendocrine protein, and in the pancreatic islets the presence of 7B2 in A- and B-cells was immunohistochemically demonstrated. In order to examine 7B2 secretion by A- and B-cells of pancreatic islets, we prepared isolated hamster pancreatic islet cells as well as an A-cell-rich culture, and studied 7B2 secretion under certain stimulations. 7B2 was secreted by isolated hamster pancreatic islet cells. This secretion was stimulated by theophylline and arginine, but glucose had a weak effect on the 7B2 secretion. Such a response of 7B2 to the stimulations was different from that of insulin or glucagon. 7B2 secretion was also noted in the A-cell-rich culture. These results suggest that 7B2 is secreted by both A- and B-cells of the hamster pancreatic islets and its secretion is regulated under certain conditions.     

Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones

Cells derived from rat islet tumor and grown in culture (parent cells-RIN-m) and two clones obtained from them were used to study the effect of various secretagogues on insulin, glucagon, and somatostatin secretion. Parent cells secreted all three hormones in various quantities, while clone 5F secreted predominantly insulin and clone 14B secreted predominantly somatostatin. The secretory behavior of these cells were compared to each other and to that of normal islets. In general, as in the case of normal islets, insulin secretion was stimulated by calcium, potassium, tolbutamide, theophylline, and glucagon. It was inhibited by somatostatin. Glucagon secretion was stimulated by calcium, arginine, and theophylline. Somatostatin secretion was stimulated in clone 14B by arginine, tolbutamide, theophylline, and insulin. These cells differ from normal islets, in that they do not respond to glucose or arginine with increased insulin secretion. Also somatostatin failed to inhibit glucagon secretion. The similarity in insulin secretory responses of parent cells and clone 5F suggests that local or paracrine islet hormone secretion plays only a negligible role in the control of other hormone secretion in these cells.     

Glucagon and insulin secretion by dispersed islet cells: possible paracrine relationships

The ability of dispersed islet cells in a perifusion system to secret glucagon and insulin in response to physiologic stimuli was investigated. Normal hamster islets were isolated by collagenase digestion and the cells dispersed by sequential digestion with collagenase and trypsin. Following a 50-min period of equilibrium in buffer with high glucose concentrations (5.0 mg/ml), glucagon secretion was stimulated by glucopenia and subsequently, inhibited by increasing the concentration of glucose. The responsiveness to glucose inhibition was significantly less in dispersed islet cells than in intact islets. However, the dispersed islet cells showed significantly greater response to arginine. Glucagon secretion by dispersed islet cells was stimulated to tolbutamide and epinephrine but somatostatin had no effect. Dispersed islet cell preparations did not augment insulin secretion in response to glucose but did secrete more insulin in response to arginine. Intact islets secreted insulin in response to glucose but not arginine. We conclude that A cells in cell suspension do not need direct contact or an intact intra-islet environment in order to respond to glucose, arginine, epinephrine, or tolbutamide but the extent of response may be influenced by paracrine effects. However, paracrine relationships may be important in determining the response of B cells to secretagogues.     

Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system

Excessive secretion of glucagon is a major contributor to the development of diabetic hyperglycemia. Secretion of glucagon is regulated by various nutrients, with glucose being a primary determinant of the rate of alpha cell glucagon secretion. The intra-islet action of insulin is essential to exert the effect of glucose on the alpha cells since, in the absence of insulin, glucose is not able to suppress glucagon release in vivo. However, the precise mechanism by which insulin suppresses glucagon secretion from alpha cells is unknown. In this study, we show that insulin induces activation of GABAA receptors in the alpha cells by receptor translocation via an Akt kinase-dependent pathway. This leads to membrane hyperpolarization in the alpha cells and, ultimately, suppression of glucagon secretion. We propose that defects in this pathway(s) contribute to diabetic hyperglycemia.     

Effect of exercise training on insulin and glucagon release from perfused rat pancreas

The effect of physical training on insulin and glucagon release in perfused rat pancreas was examined in the spontaneously exercised group running in a wheel cage an average of 1.4 km/day for 3 weeks and in the sedentary control group kept in the cage whose rotatory wheel was fixed on purpose. Pancreatic immunoreactive insulin (IRI) responses to glucose and arginine were reduced by 28% and 47.8% respectively in trained rats compared with untrained rats, while IRI content of the pancreas was similar in these two groups. The demonstrated decrease in insulin secretion of the beta-cell of the trained rats, in response to the glucose and arginine stimulations, may be functional in nature. On the other hand, neither pancreatic glucagon immunoreactivity (GI) response to glucose and arginine nor GI content of the pancreas was modified by exercise training. These results demonstrate that exercise training reduces IRI responses to glucose as well as to arginine stimulations, but does not modify any secretory response of pancreatic GI.     

Maintenance of pancreatic endocrine cells of the neonatal rat: IV. The effect of 3-amino-3-deoxyglucose

Monolayer islet cells prepared from neonatal rat pancreases were cultured in media with 5.5 mM glucose alone or further supplemented with 5 mM 3-amino-3-deoxyglucose (3A3dG) for a total of 7 days. After culture for 7 days, 3A3dG-supplementation maintained the recovery of insulin released into the medium during the last 2 days of a 7-day culture at a level 2.9 fold higher that at day 0. Similarly, the insulin content of the cells was significantly higher than the initial level at day 0 (2.8-fold) and that of the cells grown in medium with glucose alone (4.5-fold). The maximum secretory responses to glucose (2.8-16.7 mM), leucine (2.5-10 mM) and 2-ketoisocaproate (2.5-10 mM) were several times as high as the initial. Further, 3A3dG caused a selective deletion of fibroblasts mostly consisting of endocrine cells. In these monolayer cells, either the cAMP response to glucose or the cellular cAMP content were significant. In conclusion, it is suggested that the beneficial effect of 3A3dG may be associated with an increase in either the oxidative catabolism of amino acids or the activity of adenylate cyclase in the B cell.     

Secretion of glucagon and insulin in hypophysectomized rats: effect of tolbutamide.

Normal and hypophysectomized (hypox) rats, fed ad libitum, received intraperitoneal injections of tolbutamide (75 mg/kg/day) or of saline for 6 weeks. 24 h after the last injection, blood samples were taken for glucose, insulin and glucagon determinations. In normal rats, tolbutamide treatment did not alter serum glucose, insulin and glucagon, although it suppressed the secretion of insulin and glucagon by the pancreatic islets. In hypox rats, tolbutamide decreased serum glucose and insulin, elevated serum glucagon and stimulated the secretion of glucagon, but not that of insulin by the pancreatic islets. In addition, tolbutamide treatment increased the glucagon response to arginine in normal, but not in hypox rats. The serum glucose response to arginine was decreased by tolbutamide treatment and by hypophysectomy and, thus, appeared independent of the glucagon rise or preexisting glucagon level. We conclude that tolbutamide treatment decreased the secretion of glucagon and insulin in normal rats and stimulated that of glucagon in hypox rats, perhaps because of the low levels of insulin in the serum and in the pancreas of the latter. Our results are compatible with the hypothesis that the pancreatic action of tolbutamide is influenced by the pituitary.     

Insulin as a physiological modulator of glucagon secretion

Glucose homeostasis is regulated primarily by the opposing actions of insulin and glucagon, hormones that are secreted by pancreatic islets from beta-cells and alpha-cells, respectively. Insulin secretion is increased in response to elevated blood glucose to maintain normoglycemia by stimulating glucose transport in muscle and adipocytes and reducing glucose production by inhibiting gluconeogenesis in the liver. Whereas glucagon secretion is suppressed by hyperglycemia, it is stimulated during hypoglycemia, promoting hepatic glucose production and ultimately raising blood glucose levels. Diabetic hyperglycemia occurs as the result of insufficient insulin secretion from the beta-cells and/or lack of insulin action due to peripheral insulin resistance. Remarkably, excessive secretion of glucagon from the alpha-cells is also a major contributor to the development of diabetic hyperglycemia. Insulin is a physiological suppressor of glucagon secretion; however, at the cellular and molecular levels, how intraislet insulin exerts its suppressive effect on the alpha-cells is not very clear. Although the inhibitory effect of insulin on glucagon gene expression is an important means to regulate glucagon secretion, recent studies suggest that the underlying mechanisms of the intraislet insulin on suppression of glucagon secretion involve the modulation of K(ATP) channel activity and the activation of the GABA-GABA(A) receptor system. Nevertheless, regulation of glucagon secretion is multifactorial and yet to be fully understood.     

Serum insulin concentration and arginine tolerance test in the cynomolgus monkey (Macaca fascicularis)

The standard value of serum insulin was determined to be less than 75 microU/ml with ninety-eight female adult cynomolgus monkeys of wild origin. Then, fifteen apparently healthy laboratory-bred female cynomolgus monkeys aged 6-8 years were studied to know the usefulness of the arginine tolerance test (ATT) by measuring blood glucose, insulin and glucagon. Prior to ATT, all animals had been diagnosed as non-diabetic by the intravenous glucose tolerance test (IVGTT). Arginine hydrochloride was infused intravenously at a dose of 0.5 g/kg. BW under anesthesia. According to the standard value of insulin, fifteen animals were divided into two groups, that is, the low (n = 7) and the high (n = 8) value groups. In the low value group, glucose and insulin value did not change significantly after arginine infusion and their responses were similar to those in the control group (saline infused, n = 4). But glucagon markedly increased from 10 to 45 minutes post infusion. In the high value group, glucagon response was similar to that in the low value group, while glucose and insulin values significantly decreased. It is concluded that the pancreatic alpha-cell function (glucagon secretion) can be judged by the ATT in the cynomolgus monkey but the beta-cell function (insulin secretion) can not be diagnosed.     

Factors influencing insulin and glucagon secretion in lean and genetically obese mice.

The control of insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.     

A comparative study of several serotonin receptor antagonists on basal and stimulus induced release of insulin and glucagon in the intact rat.

Several commonly used serotonin receptor antagonists were studied for their ability to influence basal plasma insulin and glucagon (using 30K antibody) levels as well as the response of these hormones to a glucose or arginine challenge administered systematically to overnight fasted rats. Cyproheptadine, in contrast to other antagonists employed, induced large increases of insulin, glucagon and glucose, although this hyperinsulinemia was of a smaller magnitude when compared with hormone levels observed during an equivalent hyperglycemia resulting from glucose administration. The pancreatic response to a glucose load (increased insulin and decreased glucagon release) and an arginine load (increased insulin and glucagon release) were prevented by cyproheptadine pretreatment. Basal insulin levels were bot consistently altered by methysergide or cinanserin and were slightly elevated by metergoline. Basal glucagon levels were unaffected by these drugs. These three agents potentiated the insulinotropic effect of an arginine load whereas only metergoline exerted a similar effect on the response to glucose loading. Glucagon release in response to these stimuli was not significantly altered by drug pretreatment.     

Secretory response and immunochemical heterogeneity of glucagon in plasma and tumor extracts of a patient with glucagonoma

The secretory response and immunoreactive heterogeneity of glucagon was investigated in a patient with glucagonoma syndrome. After glucose administration, abnormal insulin release accompanied by glucose intolerance were observed, whereas the high glucagon circulating levels were only partially blocked after glucose or somatostatin infusion. Chromatographic fractionation of plasma samples, before and after arginine administration showed that most of the immunoreactivity eluted as true glucagon. Furthermore, when aliquots of the tumor extracts were fractionated by column chromatography or by polyacrylamide gel electrophoresis, most of the immunoreactivity eluted in the 3,500 molecular weight peak. In contrast with previous reports, our results indicate that neoplasia A cells can also manufacture and release into the bloodstream great amounts of genuine glucagon rather than larger glucagon immunoreactive forms. In spite of such findings, in this patient neither diabetes nor hyperglycemia were present.     

Glucagon secretion by dispersed alpha cell enriched islets from streptozotocin treated hamsters in perifusion

Streptozotocin (70 mg/kg) was administered intravenously to female Syrian hamsters. The hamsters received insulin (5U/animal/day). Insulin treatment was withdrawn 3 days before sacrifice in one group, while another group was maintained on insulin until sacrifice. Ten to 14 days following streptozotocin administration the animals were killed, and the pancreatic islets isolated and subsequently dispersed. Islet DNA content was decreased while the glucagon content was elevated by streptozotocin treatment. The glucagon secretory responsiveness of the dispersed alpha cells of control animals was stimulated by glucopenia and decreased by glucose. Alpha cells of streptozotocin hamsters were not only suppressed but were actually stimulated by high glucose concentrations. Treatment with insulin in vivo but not in vitro, resulted in a restoration of the alpha cells responsiveness to glucose suppression. Dispersed alpha cells from control and streptozotocin treated animals were stimulated by arginine. Basal and total glucagon secretion was greatest in dispersed alpha cells from streptozotocin treated animals. We concluded: that the paradoxical response of alpha cells to glucose noted in diabetes is not due to short term insulin deprivation or the lack of morphologic contact with beta cells; that the alpha cells require and insulin stimulated islet metabolite and extra islet materials to respond appropriately to glucose; and that the alpha cells response to arginine is mediated independently of glucose regulation.     

Diminished arginine-stimulated insulin secretion in trained men

Glucose-stimulated insulin secretion is depressed by training. To further elucidate the beta-cell adaptation to training, a nonglucose secretagogue was applied. Arginine was infused for 90 min to seven trained and seven untrained young men. Arginine and glucose concentrations increased identically in the groups. The insulin response was biphasic and waned despite increasing arginine concentrations. Both these phases as well as C-peptide responses were reduced in trained subjects, whereas proinsulin responses were similar in the groups. Identical increases were found in glucagon, growth hormone, catecholamines, and production and disappearance of glucose; identical decreases were found in free fatty acids, glycerol, and beta-hydroxybutyrate. In conclusion, in men training diminishes both arginine- and glucose-stimulated insulin secretion, indicating a profound beta-cell adaptation. Being enhanced, the effects of insulin on both production and disposal of glucose are changed in the opposite direction to beta-cell secretion by training. The responses of glucagon- and growth hormone-secreting cells to arginine do not change with training.     

Combined administration of sodium chloro-2-propionate and insulin on the secretion of glucagon by the pancreas in the diabetic rat

This work was designed to study the effects of sodium 2-chloropropionate (2CP) alone or combined with insulin, in vitro, on glucagon secretion from pancreas isolated from rats, made diabetic by streptozotocin (66 mg/kg i.p.). The pancreata were perfused with a physiological solution containing 2.8 mM glucose (0.5 g/l) and glucagon secretion was stimulated by an arginine infusion (5 mM) for 30 min. When 2CP (1 mM) and/or insulin (4 IU/l) were applied, they were infused from the start of the organ perfusion. In the presence of glucose alone, a marked decrease in glucagon output was observed in diabetic rat pancreas. The arginine perfusion induced a biphasic glucagon secretion both in normal and diabetic rat pancreas; this response was however clearly reduced in diabetic rat pancreas. In diabetic rat pancreas, the infusion of either 2CP or insulin had no effect on glucagon output in presence of glucose alone, nor did it modify the response to arginine. In contrast, the combined infusion of insulin and 2CP induced different effects depending on the conditions: whereas in presence of glucose alone it restored a glucagon output close to that recorded in normal rat pancreas, it did not modify the response to arginine.     

Lack of glucose-induced functional maturation during long-term culture of human fetal islet cells

To investigate the long-term effects of glucose on the function of human fetal islets we cultured islet-like cell clusters (ICC) obtained from 12 human fetuses with a mean age of 16.1 weeks in media containing 2.8, 11.1 or 16.7 mM glucose. On the 8th day of culture, the ICC that had been maintained in 16.7 mM glucose contained 60% less insulin than the ICC cultured in 2.8 mM glucose. However, insulin release was similar in both groups, and was not affected by a 24-h incubation in high vs. low glucose. Also (pro) insulin biosynthesis was not significantly affected. During a 24-day culture period, the total release of insulin and glucagon was similar in all glucose concentrations. The ICC released about 75% of their insulin content but only 15% of their glucagon content during the last 48 h of the 24-day culture period, again regardless of glucose concentration in media. Insulin release was insensitive to acute glucose and leucine challenges in perifusion experiments after culture for 1, 5, 8 or 16 days in 11.1 mM glucose, whereas glucagon was always a potent stimulus. In conclusion, the function of cultured young human fetal islet cells is remarkably independent of glucose, even during prolonged exposure. Moreover, the primary role of glucagon in fetal life may be that of a paracrine stimulator of beta-cell function.