L-arginine stimulates insulin secretion from the pancreas of normal and diabetic rats

Summary. Several reports have shown that nitric oxide (NO) stimulates glucose-induced insulin secretion in the pancreas of normal rat but the effect of L-arginine (a NO donor) on insulin secretion from the pancreas of diabetic pancreas is unknown. Fragments of pancreatic tissue from normal and diabetic rats were incubated for 45 min in Krebs solution containing 100 mM L-arginine. The supernatant was subsequently analyzed for the insulin content using radioimmunoassay technique. L-arginine evoked large increases in insulin secretion from the pancreas of diabetic rat. The insulin secreted from the pancreas of diabetic rat was numerically but not significantly lower compared to that of normal rat pancreas. In conclusion, L-arginine, a nitric oxide donor stimulates insulin secretion from the pancreas of diabetic rats. Received October 3, 2000 Accepted November 10, 2000     

The role of calcium in insulin release from the human fetal pancreas

Previous experiments have established that the human fetal pancreas is relatively unresponsive to glucose as regards insulin release, but will secrete this hormone when exposed to agents which increase levels of cAMP or which activate protein kinase C. The current experiments were designed to establish which role another major stimulus, calcium, had in the release of insulin from this organ. For this purpose, cultured explants of human fetal pancreas were exposed to stimuli either in static or dynamic stimulation. The data show that insulin release is enhanced in the presence of 10 mM Ca2+, as well as the calcium ionophores A23187 and ionomycin, the latter agent being effective only if extracellular Ca2+ was present. A biphasic response was seen for Ca2+ but only a second phase response for A23187. Voltage-dependent calcium channels were shown to be present by the ability of the calcium channel blocker, verapamil, to inhibit insulin release caused by an agent that depolarizes membranes, potassium. The essential role of extracellular calcium in the insulinogenic effect of agents which increase cAMP levels--theophylline--and which activate protein kinase C--12-O-tetradecanoylphorbol-13-acetate--was demonstrated by showing (a) partial inhibition of insulin secretion by calcium channel blockers, (b) no enhancement of insulin release in the absence of extracellular calcium and (c) greater enhancement of insulin release in the presence of the calcium channel activator BAY-K-8644, which caused no stimulation by itself. These data put into better perspective our understanding of the mechanisms involved in insulin release from the human fetal pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyproheptadine and beta cell function in the rat: insulin secretion from pancreas segments in vitro.

Pancreatic islet cell vacuolization, hyperglycemia, and glucose intolerance develop in rats after oral administration of cyproheptadine (CPH). In order to determine whether these effects were associated with abnormal insulin secretion, pancreas segments from CPH-treated and control rats were compared for their ability to secrete insulin in response to several stimuli. Oral administration of CPH (45 mg/kg/day) to rats for 1 or 8 days inhibited glucose-mediated insulin secretion from pancreas segments obtained 3 and 24 hr after the last dose of the drug. Insulin secretion had returned to normal by 48 hr after drug administration. Intraperitoneal administration of the drug was less effective than oral administration in inhibiting in vitro insulin secretion. Other stimuli for insulin secretion (tolbutamide, glucagon, L-leucine, and dibutyryl 3',5'cyclic AMP), like glucose, were incapable of releasing normal amounts of insulin from pancreas segments of CPH-treated rats. CPH and a metabolite, desmethyl-CPH, inhibited glucose-stimulated insulin secretion when added in vitro to pancreas segments from control rats. This suggests that the inhibition of insulin secretion in pancreas segments taken from animals treated with CPH could be due, at least in part, to the presence of drug and its metabolite in the tissue. A previously observed reduction in the pancreatic content of insulin in CPH-treated rats may also contribute to the abnormal insulin release in animals given the drug.     

Distribution of vasoactive intestinal polypeptide, neuropeptide-Y and substance P and their effects on insulin secretion from the in vitro pancreas of normal and diabetic rats

This study examined the pattern of distribution of vasoactive intestinal polypeptide (VIP), neuropeptide-Y (NPY) and substance P (SP) in the pancreas of diabetic rat to determine whether there are changes in the number and pattern of distribution of these neuropeptides after the onset of diabetes. Moreover, the effect of VIP, NPY and SP on insulin secretion from the pancreas of normal and diabetic rats was also examined. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (i.p.) (60 mg kg body weight(-1)). Four weeks after the induction of DM, diabetic (n = 6) and normal (n = 6) rats were anesthetized with chloral hydrate and their pancreases removed and processed for immunohistochemistry and insulin secretion. The number of insulin-positive cells in the islets of Langerhans was reduced while that of VIP and NPY increased significantly after the onset of diabetes. The pattern of distribution of VIP, NPY and SP in the nerves innervating the pancreas was similar in both normal and diabetic rats. VIP-evoked large and significant (P < 0.02) increases in insulin secretion from the pancreas of normal and diabetic rats. NPY also induced a marked (P < 0.005) increase in insulin release from pancreatic tissue fragments of normal rat. Stimulation of pancreatic tissue fragments of diabetic rat with NPY resulted in a slight but not significant increase in insulin release. SP induced a large and significant (P < 0.005) increase in insulin secretion from the pancreas of normal rat but inhibited insulin secretion significantly (P < 0.03) from isolated pancreas of diabetic rat. In summary, VIP and NPY can stimulate insulin secretion from the pancreas after the onset of diabetes. The stimulatory effect of SP on insulin secretion is reversed to inhibitory in diabetic rats.     

Effects of oxytocin on in vivo release of insulin and glucagon studied by microdialysis in the rat pancreas and autoradiographic evidence for [3H]oxytocin binding sites within the islets of Langerhans

In the present investigation it was studied whether oxytocin administered directly in the pancreas of the rat stimulates the release of insulin and glucagon. In order to study such effects in vivo, a new experimental model applying the microdialysis technique was developed. To test the validity of the method, glucose or arginine were infused i.v. and it was shown that perfusate concentrations of insulin and glucagon increased significantly to 344 and 292% of basal overflow, respectively. Administration of oxytocin via the dialysis probe into the splenic portion of the pancreas resulted in significant elevations of insulin and glucagon concentrations to 210 (P less than 0.05) and 528% (P less than 0.01), respectively. The present study also includes a combined autoradiographic and immunohistochemical investigation of binding sites for oxytocin in the rat pancreas. A high density of [3H]oxytocin binding was present in the periphery of the islets of Langerhans, corresponding to the localization of the glucagon-producing alpha-cells. Both oxytocin and arginine(A)-vasopressin displaced [3H]oxytocin. The IC50 values were 10 and 180 nM, respectively. In conclusion, the oxytocin-induced release of insulin and glucagon as previously demonstrated in a number of species, may be due to a stimulation exerted by the peptide directly within the pancreas.     

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.     

A closed-loop artificial pancreas based on model predictive control: Human-friendly identification and automatic meal disturbance rejection

Type 1 diabetes is characterized by a lack of insulin production by the pancreas, causing high blood glucose concentrations and requiring external insulin infusion to regulate blood glucose. Continuous glucose sensors can be coupled with continuous insulin infusion pumps to create a closed-loop artificial pancreas. A novel procedure of “human-friendly” identification testing using multisine inputs is developed to estimate suitable models for use in an artificial pancreas. A constrained model predictive control (MPC) strategy is developed to reduce risks of hypo- and hyperglycemia (low and high blood glucose concentration). Meal detection and meal size estimation algorithms are developed to improve meal glucose disturbance rejection when incoming meals are not announced. Closed-loop performance is evaluated through simulation studies of a type 1 diabetic individual, illustrating the ability of the MPC-based artificial pancreas control strategy to handle announced and unannounced meal disturbances.     

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional L-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of L-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (less than 30 s) paired-tracer dilution technique. In the non-diabetic pancreas L-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 microU/ml). Transport of L-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of L-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.     

First advances in molecular endocrinology obtained using the Cre/LoxP system]

The insulin-like growth factor I (IGF-I) is produced in the liver and is considered mediating the effect of the growth hormone (GH). However, a knock-out only in liver IGF-I slightly disturbs the growth and development of mice. Such mice develop insulin resistance of various organs, including muscles. A knock-out in the liver insulin gene also results in insulin resistance. Selective inactivation of the gene for glucokinase (a target of insulin) in pancreatic islets or in the liver suppresses insulin secretion in the pancreas.     

First Advances Made with the cre/LoxPTechnique in Molecular Endocrinology

The insulin-like growth factor I (IGF-I) is produced in the liver and is believed to mediate the effect of the growth hormone. However, a knockout only in liver IGF-I but slightly disturbs the growth and development of mice. Such mice develop insulin resistance of various organs, including muscle. A knockout in the liver insulin receptor gene also results in insulin resistance. Selective inactivation of the gene for glucokinase (a target of insulin) in pancreatic islets or in the liver suppresses insulin secretion in the pancreas.     

Potassium ions and the secretion of insulin by islets of Langerhans incubated in vitro

1. A method was devised for the isolation of islets of Langerhans from rabbit pancreas by collagenase digestion in order to study the influx and efflux of K(+) in islets during insulin secretion. 2. Glucose-induced insulin release was accompanied by an increased rate of uptake of (42)K(+) by the islets of Langerhans, though this was not the case for secretion in response to tolbutamide. Ouabain significantly inhibited the uptake of (42)K(+) by islet tissue. 3. No significant increase in the rate of efflux of (42)K(+) was demonstrated during active insulin secretion. 4. Slices of rabbit pancreas were incubated in media of different K(+) content, and rates of insulin release were determined. Alteration of the K(+) concentration of the medium between 3 and 8mm had no effect on the rate of insulin release by pancreas slices. However, decrease of the K(+) concentration to 1mm resulted in inhibition of secretion in response to both glucose and to tolbutamide. Conversely, an increase in K(+) concentration increased rates of insulin release in response to both these stimuli. 5. It is concluded that, though unphysiological concentrations of K(+) may influence the secretion of insulin, fluxes of K(+) in the islets do not appear to be important in the initiation of insulin secretion.     

Cellular distribution and amount of chromogranin A in bovine endocrine pancreas

We determined the cellular distribution and the amount of chromogranin A in endocrine cells of bovine pancreas using a polyclonal antibody against bovine adrenomedullary chromogranin A. The relative amounts of chromogranin A in the different cells of the endocrine pancreas were determined by computer-assisted analyses of the optical densities of the immunoreactivities in the stained sections. More than 80% of the immunoreactive chromogranin A was located in the pancreatic B-cells. In immunoblots of acid tissue extracts, only one chromogranin A band (MW 74 KD) was observed. Quantification of the immunoblots revealed that 3 micrograms of chromogranin A and 918 micrograms of insulin were present per gram pancreas (wet weight), equivalent to a molar ratio of 460 mumol chromogranin A per mol insulin.     

GABA in the endocrine pancreas: cellular localization and function in normal and diabetic rats

Gamma amino butyric acid (GABA) and its related enzymes have been demonstrated in pancreatic beta cells of normal rat. Antibodies against GABA-synthesizing enzymes have been implicated in the pathogenesis of Type I diabetes. In spite of the importance of GABA in the aetiology of diabetes mellitus, detailed morphological data on the pattern of distribution of GABA in the pancreas of normal and diabetic rats are lacking. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (60 mg kg body weight(-1)). Four weeks after the induction of DM, normal (n = 6) and diabetic (n = 6) rats were anesthetized with chloral hydrate and their pancreata were removed and processed for the localization and effect of GABA on insulin secretion using immunohistochemistry and radioimmunoassay techniques. The number of GABA-like immunoreactive (GABA-LIR) cells in the pancreatic islets of STZ-diabetic rats decreased significantly (P<0.0001) when compared to non-diabetic control rats. The pattern and percentage distribution of GABA in the islet of Langerhans of normal and diabetic rat was similar to that of insulin. GABA induced a significant (P<0.0007) increase in insulin secretion from the pancreas of normal rats. In diabetic pancreas, GABA evoked a higher but not significant (P<0.1) increase in insulin secretion. These findings showed that the number of GABA-LIR cells is reduced significantly in diabetes. Moreover, GABA is a strong secretagogue of insulin from the pancreas of normal rat.     

Effect of aging on the sensitivity of pancreatic beta-cells to insulin secretagogues in rats.

Glucose-stimulated insulin release from rat pancreas is known to be blunted by aging. In the present study, we examined the effect of aging on insulin release induced by various secretagogues using the isolated perfused pancreas of female rats. Insulin release from the perfused pancreas in response to 16.7 mM glucose in 8-month-old rats (older rats) was much less than that in 2-month-old rats (young rats). The first phase of insulin release after glucose stimulation was attenuated in older rats. The addition of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) potentiated glucose-induced insulin secretion in both groups of rats. However, the second phase of insulin secretion in older rats was lower than that in younger rats. The phorbol ester 12-O-tetradecanoyl phorbol ester (TPA, 200 nM) enhanced both the first and the second phases of insulin release induced by glucose in both groups of rats. The amount of first phase insulin release induced by TPA with glucose in young rats was greater than that in older rats, whereas the second phase of insulin release was similar in both groups of rats. On the other hand, tolbutamide (200 uM) similarly stimulated the first phase of insulin release in both age groups of rat. In addition, the amount of cumulative insulin secretion induced by tolbutamide during the second phase was slightly but significantly greater in older rats than in young controls. Insulin content in the pancreas was significantly greater in older rats than in young rats and increased after the stimulation with TPA and tolbutamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiating synergism between alpha, beta-methylene ADP and acetylcholine on the secretion of insulin from the isolated pancreas of the newborn dog

A possible interaction between alpha,beta-methylene ADP, a stable analogue of ADP, and acetylcholine, was studied on insulin secretion from isolated new-born dog pancreas, perfused in the presence of a non stimulating glucose concentration (4.2 mM). alpha,beta-methylene ADP alone (16.5 microM) and acetylcholine alone (0.5 microM) induced a comparable increment of insulin secretion. When the two substances were simultaneously infused, the increment of secretion was significantly higher than the sum of the increments induced by alpha,beta-methylene ADP and acetylcholine infused separately (p less than 0.005). Our results show a potentiating synergism between alpha,beta-methylene ADP and acetylcholine, on insulin secretion, alpha,beta-methylene ADP acting via a P2 purinoceptor and acetylcholine via a muscarinic receptor. Similar results were previously obtained on the rat pancreas.