Mechanisms of action of the dipeptidyl peptidase-4 inhibitor vildagliptin in humans.

Inhibition of dipeptidyl peptidase-4 (DPP-4) by vildagliptin prevents degradation of glucagon-like peptide-1 (GLP-1) and reduces glycaemia in patients with type 2 diabetes mellitus, with low risk for hypoglycaemia and no weight gain. Vildagliptin binds covalently to the catalytic site of DPP-4, eliciting prolonged enzyme inhibition. This raises intact GLP-1 levels, both after meal ingestion and in the fasting state. Vildagliptin has been shown to stimulate insulin secretion and inhibit glucagon secretion in a glucose-dependent manner. At hypoglycaemic levels, the counterregulatory glucagon response is enhanced relative to baseline by vildagliptin. Vildagliptin also inhibits hepatic glucose production, mainly through changes in islet hormone secretion, and improves insulin sensitivity, as determined with a variety of methods. These effects underlie the improved glycaemia with low risk for hypoglycaemia. Vildagliptin also suppresses postprandial triglyceride (TG)-rich lipoprotein levels after ingestion of a fat-rich meal and reduces fasting lipolysis, suggesting inhibition of fat absorption and reduced TG stores in non-fat tissues. The large body of knowledge on vildagliptin regarding enzyme binding, incretin and islet hormone secretion and glucose and lipid metabolism is summarized, with discussion of the integrated mechanisms and comparison with other DPP-4 inhibitors and GLP-1 receptor activators, where appropriate.

Evidence that vildagliptin attenuates deterioration of glycaemic control during 2-year treatment of patients with type 2 diabetes and mild hyperglycaemia.

AIM: To assess the 2-year efficacy and tolerability of vildagliptin (50 mg once daily) in patients with type 2 diabetes (T2DM) and mild hyperglycaemia. METHODS: This was a multicentre, randomized, double-blind, placebo-controlled trial comprising a 52-week core study with a 4-week, active treatment-free washout followed by a 52-week extension study with another washout period conducted in 131 drug-naïve patients with T2DM and mild hyperglycaemia [glycosylated haemoglobin (HbA(1c)) 6.2-7.2%]. All patients received lifestyle counselling at each study visit. Efficacy and tolerability were assessed during visits at weeks 0 (core study baseline), 4, 8, 12, 16, 24, 32, 40 and 52 of active treatment; at week 56 (i.e. after the first washout period); at weeks 68, 80, 96 and 108 and at week 112 (i.e. after the second washout period). Standard meal tests were also performed at weeks 0, 24, 52, 56, 80, 108 and 112 to assess postprandial glycaemia and beta-cell function, which was quantified by glucose area under the curve (AUC(0-2) (h))/insulin secretory rate (ISR) AUC(0-2) (h) (ISR/G). Changes from baseline and between-treatment differences (placebo-adjusted changes from baseline during vildagliptin treatment) were analysed by ancova. RESULTS: The placebo-adjusted change from week 0 in HbA(1c) was -0.3 +/- 0.1% after 1 year of vildagliptin treatment (p < 0.001) and -0.5 +/- 0.2% after 2 years (p = 0.008). The placebo-adjusted change from core study baseline in fasting plasma glucose, in glucose AUC(0-2) (h) and in the beta-cell function parameter, ISR/G, tended to be greater after 2 years than after 1 year of treatment with vildagliptin. Even after a 4-week washout, the placebo-adjusted change from week 0 to week 112 in ISR/G was 3.2 +/- 1.6 pmol/min/m(2)/mM (p = 0.058) and the placebo-adjusted difference in the change from week 0 to week 112 in HbA(1c) was -0.3 +/- 0.1% (p = 0.051). The incidences of adverse events (AEs), serious AEs and discontinuations because of AEs were similar in the two treatment groups, and hypoglycaemic episodes were reported by no patient receiving vildagliptin and by two patients receiving placebo. CONCLUSIONS: In drug-naïve patients with mild hyperglycaemia, 2-year treatment with vildagliptin 50 mg once daily attenuated the progressive loss of glycaemic control seen in patients receiving only lifestyle counselling (and placebo). This appears to be because of a corresponding attenuation of the deterioration of beta-cell function as assessed by ISR/G.

Pancreatic noradrenergic nerves are activated by neuroglucopenia but not by hypotension or hypoxia in the dog. Evidence for stress-specific and regionally selective activation of the sympathetic nervous system.

To determine if acute stress activates pancreatic noradrenergic nerves, pancreatic norepinephrine (NE) output (spillover) was measured in halothane-anesthetized dogs. Central neuroglucopenia, induced by intravenous 2-deoxy-D-glucose [( 2-DG] 600 mg/kg + 13.5 mg/kg-1 per min-1) increased pancreatic NE output from a baseline of 380 +/- 100 to 1,490 +/- 340 pg/min (delta = +1,110 +/- 290 pg/min, P less than 0.01). Surgical denervation of the pancreas reduced this response by 90% (delta = +120 +/- 50 pg/min, P less than 0.01 vs. intact innervation), suggesting that 2-DG activated pancreatic nerves by increasing the central sympathetic outflow to the pancreas rather than by acting directly on nerves within the pancreas itself. These experiments provide the first direct evidence of stress-induced activation of pancreatic noradrenergic nerves in vivo. In contrast, neither hemorrhagic hypotension (50 mmHg) nor hypoxia (6-8% O2) increased pancreatic NE output (delta = +80 +/- 110 and -20 +/- 60 pg/min, respectively, P less than 0.01 vs. neuroglucopenia) despite both producing increases of arterial plasma NE and epinephrine similar to glucopenia. The activation of pancreatic noradrenergic nerves is thus stress specific. Furthermore, because both glucopenia and hypotension increased arterial NE, yet only glucopenia activated pancreatic nerves, it is suggested that a regionally selective pattern of sympathetic activation can be elicited by acute stress, a condition in which sympathetic activation has traditionally been thought to be generalized and nondiscrete.

Vildagliptin, a dipeptidyl peptidase-IV inhibitor, improves model-assessed beta-cell function in patients with type 2 diabetes.

AIMS/HYPOTHESIS: The dipeptidyl peptidase IV inhibitor, vildagliptin, increases levels of intact glucagon-like peptide-1 (GLP-1) and improves glycemic control in patients with type 2 diabetes. Although GLP-1 is known to stimulate insulin secretion, vildagliptin does not affect plasma insulin levels in diabetic patients, suggesting that more sophisticated measures are necessary to ascertain the influence of vildagliptin on beta-cell function. METHODS: This study examined the effects of 28-d treatment with vildagliptin (100 mg, twice daily; n = 9) vs. placebo (n = 11) on beta-cell function in diabetic patients using a mathematical model that describes the insulin secretory rate as a function of glucose levels (beta-cell dose response), the change in glucose with time (derivative component), and a potentiation factor, which is a function of time and may reflect the actions of nonglucose secretagogues and other factors. RESULTS: Vildagliptin significantly increased the insulin secretory rate at 7 mmol/liter glucose (secretory tone), calculated from the dose response; the difference in least squares mean (deltaLSM) was 101 +/- 51 pmol.min(-1).m(-2) (P = 0.002). The slope of the beta-cell dose response, the derivative component, and the potentiation factor were not affected. Vildagliptin also significantly decreased mean prandial glucose (deltaLSM, -1.2 +/- 0.4 mmol/liter; P = 0.01) and glucagon (deltaLSM, -10.7 +/- 4.8 ng/liter; P = 0.03) levels and increased plasma levels of intact GLP-1 (deltaLSM, +10.8 +/- 1.6 pmol/liter; P < 0.0001) and gastric inhibitory polypeptide (deltaLSM, +43.4 +/- 9.4 pmol/liter; P < 0.0001) relative to placebo. CONCLUSION: Vildagliptin is an incretin degradation inhibitor that improves beta-cell function in diabetic patients by increasing the insulin secretory tone.

Galanin--sympathetic neurotransmitter in endocrine pancreas?

The effects of sympathetic neural activation on basal pancreatic hormone secretion cannot be explained solely by the actions of the classic sympathetic neurotransmitter norepinephrine. The nonadrenergic component may be mediated by the 29-amino acid peptide galanin in that this neuropeptide meets several of the criteria necessary to be considered a sympathetic neurotransmitter in the endocrine pancreas. 1) Galanin administration inhibits basal insulin and somatostatin secretion and stimulates basal glucagon secretion from the pancreas, qualitatively reproducing the effects of sympathetic nerve stimulation. These sympathomimetic effects appear to be mediated by direct actions of galanin on the islet. 2) Galanin-like immunoreactivity exists in fibers that innervate pancreatic islets. 3) Galanin is released during electrical stimulation of pancreatic nerves. The quantity released is sufficient to reproduce sympathetic nerve stimulation-induced effects on insulin secretion and to contribute to the neural effects on somatostatin and glucagon release. 4) Whether interference with galanin action or release reduces the islet response to sympathetic nerve stimulation remains to be determined. We hypothesize that galanin and norepinephrine act together to mediate the islet response to sympathetic neural activation. If galanin is a sympathetic neurotransmitter in the endocrine pancreas, it may contribute to the inhibition of insulin secretion that occurs during stress and thereby to the hyperglycemic response. Moreover, the local presence of this potent beta-cell inhibitor in the islet leads to speculation on galanin's contribution to the impairment of insulin secretion that occurs in non-insulin-dependent diabetes mellitus and therefore on the potential utility of a galanin antagonist in the treatment of this disease.

Glucosamine inhibits glucokinase in vitro and produces a glucose-specific impairment of in vivo insulin secretion in rats.

A characteristic feature of non-insulin-dependent diabetes mellitus (NIDDM) is the lack of an acute insulin response to intravenous glucose with maintenance of the response to other secretagogues. It has been hypothesized that impaired glucose sensing stems from defective beta-cell glucokinase. It remains unclear whether decreased pancreatic glucokinase activity will produce defects of insulin secretion similar to those observed in NIDDM. In this study, the effects of glucosamine on glucokinase activity and on islet function were assessed in vitro and in vivo. Glucosamine (5 mmol/l) reduced glucokinase activity in islet homogenate and diminished the insulin response to glucose (200 mg/dl) by isolated islets, whereas the response to arginine (20 mmol/l at 100 mg/dl glucose) was unaffected. In conscious normal rats, glucosamine lowered plasma insulin, followed by an increase in blood glucose. Administration of glucosamine 10 min before an infusion of glucose (10 mg.min-1. 15 min) reduced the insulin response. The primary effect was an attenuation of the first-phase insulin response relative to the decreased basal insulin levels. Arginine (10 mg.min-1.15 min) induced biphasic insulin release in both groups. Although glucosamine slightly reduced the absolute insulin response, it was normal relative to preinfusion levels. In all experiments, glucagon secretion was unaffected by glucosamine. The results indicate that glucosamine inhibits beta-cell glucokinase activity in vitro. In addition, glucosamine impairs glucose- but not arginine-induced insulin secretion. We conclude that glucosamine, probably via a reduction of glucokinase activity, impairs insulin secretion in a manner comparable to that seen in NIDDM.

Role for autonomic nervous system to increase pancreatic glucagon secretion during marked insulin-induced hypoglycemia in dogs.

To determine the role of the autonomic nervous system (ANS) in mediating the glucagon response to marked insulin-induced hypoglycemia in dogs, we measured arterial and pancreatic venous glucagon responses to insulin-induced hypoglycemia during acute, terminal experiments in halothane-anesthetized dogs in which the ANS was intact (control; n = 9), pharmacologically blocked by the nicotinic ganglionic antagonist hexamethonium (n = 6), or surgically ablated by cervical vagotomy and cervical spinal cord section (n = 6). In control dogs, insulin injection caused plasma glucose to fall by 4.4 +/- 0.2 mM to a nadir of 1.7 +/- 0.2 mM. Arterial epinephrine (EPI) levels increased by 13,980 +/- 1860 pM (P less than 0.005), confirming marked activation of the ANS. Pancreatic output of glucagon increased from 0.53 +/- 0.12 to 2.04 +/- 0.38 ng/min during hypoglycemia (change [delta] +1.51 +/- 0.33 ng/min, P less than 0.005). This increased arterial plasma glucagon from 27 +/- 3 to 80 +/- 15 ng/L (delta +52 +/- 14 ng/L, P less than 0.025). Hexamethonium markedly reduced the ANS response to insulin injection (delta EPI +2130 +/- 600 pM, P less than 0.025 vs. control) despite a similar fall of plasma glucose (delta -4.1 +/- 0.2 mM) and a lower nadir (0.6 +/- 0.1 mM). Both the pancreatic glucagon response (delta glucagon output +0.45 +/- 0.2 ng/min) and the arterial immunoreactive glucagon response (delta +5 +/- 4 ng/L) were substantially reduced by hexamethonium (P less than 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of early insulin secretion: comparison of nateglinide and glyburide in previously diet-treated patients with type 2 diabetes.

OBJECTIVE: This study compared the effects of nateglinide, glyburide, and placebo on postmeal glucose excursions and insulin secretion in previously diet-treated patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: This randomized, double-blind, placebo-controlled multicenter study was conducted in 152 patients who received either nateglinide (120 mg before three meals daily, n = 51), glyburide (5 mg q.d. titrated to 10 mg q.d. after 2 weeks, n = 50), or placebo (n = 51) for 8 weeks. Glucose, insulin, and C-peptide profiles during liquid meal challenges were measured at weeks 0 and 8. At weeks -1 and 7, 19-point daytime glucose and insulin profiles, comprising three solid meals, were measured. RESULTS: During the liquid-meal challenge, nateglinide reduced the incremental glucose area under the curve (AUC) more effectively than glyburide ( = -4.94 vs. -2.71 mmol. h/l, P < 0.05), whereas glyburide reduced fasting plasma glucose more effectively than nateglinide ( = -2.9 vs. -1.0 mmol/l, respectively, P < 0.001). In contrast, C-peptide induced by glyburide was greater than that induced by nateglinide ( = +1.83 vs. +0.95 nmol. h/l, P < 0.01), and only glyburide increased fasting insulin levels. During the solid meal challenges, nateglinide and glyburide elicited similar overall glucose control ( 12-h incremental AUC = -13.2 vs. -15.3 mmol. h/l), but the insulin AUC induced by nateglinide was significantly less than that induced by glyburide ( 12-h AUC = +866 vs. +1,702 pmol. h/l, P = 0.01). CONCLUSIONS: This study demonstrated that nateglinide selectively enhanced early insulin release and provided better mealtime glucose control with less total insulin exposure than glyburide.

Calcitonin gene-related peptide: a potent and selective stimulator of gastrointestinal somatostatin secretion.

Calcitonin gene-related peptide (CGRP) exists in nerves throughout the gastrointestinal tract and pancreas, and exogenous CGRP has been reported to inhibit many endocrine and exocrine secretions of the gut and pancreas. Because somatostatin also has widespread inhibitory actions and because both gut and pancreatic somatostatin secretion may be under peptidergic control, we examined the influence of CGRP on circulating levels of somatostatin-like immunoreactivity (SLI) and on hormone output from the duodenal lobe of the dog pancreas in situ. Intravenous infusion of human CGRP in anesthetized dogs increased arterial SLI in a dose-dependent manner. During iv infusion of CGRP at 500 pmol/min, the increment of circulating SLI (change at 20 min, +175 +/- 24 fmol/ml) was composed of nearly equimolar amounts of SLI-14 and SLI-28, suggesting an effect of CGRP on both gastric and intestinal somatostatin secretion. The effect of iv CGRP (500 pmol/min) on arterial SLI exceeded those of iv CCK-8 (440 pmol/min), iv isoproterenol (10 nmol/min), and intragastric administration of acidified liver extract. In contrast, salmon calcitonin (500 pmol/min, iv) was without effect. CGRP did not stimulate pancreatic SLI output when infused iv (500 pmol/min) or when infused directly into a pancreatic artery (5 pmol/min). The pancreatic infusion of CGRP decreased insulin output slightly (change at 20 min, -21 +/- 8%), but did not affect glucagon output. We conclude that CGRP is a most effective yet selective stimulator of gastrointestinal somatostatin release, with little influence on islet function. We suggest that exogenous and possibly endogenous neuronal CGRP could exert inhibitory effects on gastrointestinal function via the release of somatostatin.

Somatostatin-28 does not regulate islet function in the dog.

Somatostatin-28 (S-28) is a naturally occurring N-terminally extended form of the tetradecapeptide somatostatin (S-14). The concept has arisen that S-28 is a gut hormone that regulates insulin secretion. This concept is based on 1) reports that S-28 is a more potent inhibitor of insulin secretion than S-14; 2) the finding that S-28 is present in D-cells of the intestine and is released after a meal; and 3) the demonstration of selective binding of S-28 to B-cells of the rat islet. To critically test this hypothesis we have 1) measured the circulating levels of somatostatin-like immunoreactivity (SLI) during infusions of S-28 and S-14 to accurately compare their potencies to inhibit insulin and glucagon secretion from the in vivo dog pancreas, and 2) measured the circulating levels of endogenous SLI released after a meal and compared these to the circulating levels of infused S-28 needed to inhibit insulin secretion. Infusion of S-28 at rates of 170 and 500 pmol/min raised arterial SLI levels by 282 +/- 26 and 885 +/- 98 fmol/ml, respectively. Immunoreactive insulin (IRI) output was inhibited by 20 +/- 11% (P less than 0.05) and 52 +/- 7% (P less than 0.0005), respectively. Immunoreactive glucagon (IRG) output was not significantly altered by either dose. Pancreatic SLI output was inhibited by 32 +/- 5% (P less than 0.0005) by the 500 pmol/min infusion. Infusion of S-28 at 50 pmol/min increased arterial SLI by 108 +/- 17 fmol/ml, but did not alter IRI output (+4 +/- 20%). In comparison, infusion of S-14 (100 pmol/min) raised arterial SLI levels by a similar amount (110 +/- 21 fmol/ml), but, unlike S-28, inhibited both IRI (-50 +/- 6%, P less than 0.0005) and IRG output (-17 +/- 8%; P less than 0.05). Thus, comparable increments in arterial S-28 failed to reproduce the inhibition of IRI secretion seen during the S-14 infusion, while similar inhibition was seen with an 8-fold increment. This suggests that S-28 is significantly less potent than S-14 in the dog. After a mixed meal, endogenous SLI levels increased by 35 +/- 3 fmol/ml in arterial plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

Human galanin: primary structure and identification of two molecular forms.

From acid/ethanol extracts of surgical specimens of human large intestine we isolated two peptides, in approximately equal amounts, that reacted with an antiserum against porcine galanin. By amino acid analysis, sequence analysis and mass spectrometry, the larger of the two peptides was found to consist of 30 amino acid residues, the sequence of which was identical to that of porcine galanin except for the following substitutions: Val16, Asn17, Asn26, Thr29 and Ser30. Unlike porcine galanin, the carboxy-terminus was not amidated. The smaller peptide corresponded to the first 19 amino acid residues counted from the N-terminus of the 30 residue peptide (again without amidation). The structural analysis was repeated on another batch of tissue with identical results. By HPLC analysis of extracts of specimens from a further 4 patients, the same peptides were identified. Thus, human galanin includes two peptides of 19 and 30 amino acids that share the sequence of the N-terminal 15 residues with other mammalian galanins, but exhibit characteristic differences in the remaining part of the molecules.

Rapid acting insulinotropic agents: restoration of early insulin secretion as a physiologic approach to improve glucose control.

The loss of early insulin secretion appears to be a critical event in the deterioration in glucose tolerance during the development of type 2 diabetes. There is therefore a strong rationale for developing new antidiabetic agents aimed at restoring or replacing early prandial insulin secretion and thereby curbing mealtime glucose excursions in patients with type 2 diabetes. Four such new agents are either now available (repaglinide and nateglinide) or in clinical development (KAD-1229 and BTS 67 582). Preclinical studies suggest that each of these new insulinotropic agents share a common receptor/effector mechanism with the sulfonylureas (SUs) but that each may have distinct characteristics that differentiate them from the SUs and from each other. Nateglinide and KAD-1229 clearly stimulate biphasic insulin secretion in vitro and in vivo and their effects are rapidly reversible, whereas the effects of repaglinide and BTS 67 582 are prolonged well beyond their removal from perfusion media in vitro or their clearance in vivo. Available data from human studies indicate that the pharmacokinetics of repaglinide and nateglinide are similar, i.e., they are both rapidly absorbed and eliminated, but consistent with findings from animal studies, the insulinotropic and glucose-lowering effects of repaglinide are slower in onset and more prolonged than those of nateglinide. Repaglinide and nateglinide have been shown to be safe and well-tolerated in patients with type 2 diabetes and to produce clinically-meaningful reductions of HbA1c, both alone and in combination with agents with complementary modes of action (e.g., metformin and thiazolidinediones). Because these new agents can potentially bring patients to near normoglycemia without an undue risk of hypoglycemia, they are important additions to the therapeutic armamentarium.

Extraction of epinephrine and norepinephrine by the dog pancreas in vivo.

This study determined the fractional extraction of epinephrine and norepinephrine by the in situ dog pancreas. Plasma samples for epinephrine measurements were taken simultaneously from the femoral artery and the superior pancreaticoduodenal vein. Pancreatic extraction of epinephrine was 73 +/- 5% when basal arterial epinephrine levels were 380 +/- 93 pg/mL, 76 +/- 4% when arterial levels were 896 +/- 123 pg/mL (epinephrine infused intravenously at 20 ng/kg/min), and 84 +/- 1% when arterial levels were 2,956 +/- 414 pg/mL (epinephrine infused intravenously at 80 ng/kg/min) suggesting that the process of epinephrine extraction by the pancreas is not saturable over this range. During a similar sampling protocol, norepinephrine was infused intravenously at 4 micrograms/kg/min; pancreatic extraction of norepinephrine was then 65 +/- 7% when arterial norepinephrine levels were 107,000 +/- 28,000 pg/mL. In separate experiments, lower rates of norepinephrine (12 to 1,200 ng/min) were infused directly into the pancreatic artery and pancreatic norepinephrine extraction was calculated; it ranged between 66% and 75%. Because the pancreas produces as well as extracts norepinephrine, a third technique was required to determine pancreatic norepinephrine extraction at the lower endogenous levels of norepinephrine; 3H-norepinephrine was infused intravenously and the arteriovenous difference of 3H-norepinephrine was measured. Fractional extraction of 3H-norepinephrine was 74 +/- 4% both in the basal state (arterial norepinephrine level = 202 +/- 44 pg/mL) and during systemic, glucopenic, stress induced by 2-deoxy-glucose (arterial norepinephrine level = 636 +/- 70 pg/mL). These data suggest that also the norepinephrine extraction process by the pancreas is not saturable.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of food on the oral bioavailability and the pharmacodynamic actions of the insulinotropic agent nateglinide in healthy subjects.

Nateglinide (Starlix, SDZ DJN 608 or A-4166), a new insulinotropic agent, is intended to be administered prior to a meal in order to improve early insulin release in non-insulin-dependent diabetes mellitus patients. The effects of a meal on the oral bioavailability and pharmacodynamic actions of nateglinide were investigated. Twelve healthy male subjects completed this randomized, single-dose, four-way crossover study in which each subject received a 60 mg dose of nateglinide 10 minutes before the start of and immediately after a high-fat breakfast meal. In addition, each subject received a single 30 and 60 mg dose of nateglinide underfasting conditions. Plasma and urine concentrations of nateglinide were determined by an HPLC method while plasma glucose and insulin concentrations were measured by standard immunoassay methods. Compared to the fasted state, administration of nateglinide 10 minutes before the meal was associated with an increase in the rate of absorption (12% increase in Cmax and 52% decrease in tmax), while there was no significant effect on the extent of absorption (AUC). Alternatively, when nateglinide was given after the meal, a food effect was observed that was characterized by a decrease in the rate of absorption: 34% decrease in Cmax and a 22% increase in tmax but no significant effect on AUC. Nateglinide was rapidly eliminated with plasma t 1/2 = 1.4 hours. Its plasma renal clearance, 20.7 ml/min, appears to be due mostly to active tubular secretion. However, only 13% to 14% of the dose is recovered as nateglinide in the urine. The 30 and 60 mg tablets were dose proportional in terms of both AUC and Cmax; both tmax and t 1/2 were dose independent. Regardless of timing, the combination of a meal and nateglinide produced a larger increase in insulin levels than did nateglinide alone. Meal-related glucose excursions were eliminated when nateglinide was taken prior to the meal. Thus, the rapid onset/short duration stimulation of insulin release by nateglinide should allow good control of prandial hyperglycemia while limiting exposure to hyperinsulinemia.

Modulation of insulin and glucagon secretion from the perfused rat pancreas by the neurohypophysial hormones and by desamino-D-arginine vasopressin (DDAVP).

Marked stimulation of glucagon release and modest stimulation of insulin release were observed during in situ perfusion of the rat pancreas with AVP or OT. Glucagon release in response to AVP or OT (200 pg/ml) gradually increased over a 45 min perfusion period reaching maxima of 500% and 300% of the pre-stimulatory levels, respectively. Insulin release transiently increased by 100%. In each case release rates returned to control values immediately after withdrawal of the peptides. Total glucagon release was concentration dependent and linear from 20 pg to 20 ng AVP or OT/ml (r greater than .97). Pancreatic response to DDAVP perfused at 20 ng/ml was virtually indistinguishable from that induced by AVP at 200 pg/ml. This demonstration of a glucagonotrophic action of the neurohypophysial hormones in the in situ perfused rat pancreas confirms earlier studies using isolated islets and bolus IV injection.

The presence and actions of NPY in the canine endocrine pancreas.

Immunofluorescent staining for neuropeptide Y (NPY) in canine pancreatic tissue was performed together with an evaluation of the effects of synthetic NPY on the release of insulin (IRI), glucagon (IRG) and somatostatin (SLI) from the duodenal lobe of the canine pancreas in situ. NPY-like immunoreactivity was localized in perivascular nerve fibers throughout the acinar tissue. NPY-immunoreactive fibers were also demonstrated in the islets, usually surrounding blood vessels but also occasionally in fibers associated with endocrine cells, primarily at the periphery of islets. In addition, the ganglia dispersed in the pancreatic parenchyma were densely innervated by NPY-immunoreactive fibers, and these ganglia regularly contained cell bodies staining for NPY. Direct infusion of NPY into the pancreatic artery (p.a.) produced a dose-dependent decrease of pancreatic SLI output and of pancreatic venous blood flow. Low-dose p.a. infusion of NPY (50 pmol/min) had no effect on basal IRI or IRG output or on the islet response to glucose (5-g bolus, i.v.). High-dose p.a. infusion of NPY (500 pmol/min) transiently stimulated IRI output and modestly increased IRG output. However, the comparatively sparse innervation of canine islets with NPY-like immunoreactive fibers and the relatively minor effects of large doses of synthetic NPY on pancreatic hormone release lead us to conclude that this peptide is not an important neuromodulator of islet function in the dog.

Evidence that vasoactive intestinal polypeptide is a parasympathetic neurotransmitter in the endocrine pancreas in dogs.

Vasoactive intestinal polypeptide (VIP) has been found in pancreatic nerves in several species. Studies were conducted to determine if VIP could be a parasympathetic neurotransmitter in the canine endocrine pancreas. To verify that VIP is localized in pancreatic parasympathetic nerves, sections of canine pancreas were immunostained for VIP. VIP staining was identified in the majority of neuronal cell bodies in intrapancreatic parasympathetic ganglia. In addition. VIP was localized in nerve fibers innervating pancreatic islets in the proximity of alpha cells. Next, to determine if VIP is released during electrical stimulation of parasympathetic nerves, pancreatic spillover of VIP was measured during vagal nerve stimulation (VNS) in anesthetized dogs. VIP spillover increased from a baseline of 630+/-540 pg/min to 2580+/-540 pg/min (delta = +1950+/-490 pg/min, p <0.01). Pancreatic VIP release during VNS was not affected by atropine, whereas ganglionic blockade with hexamethonium nearly abolished the VIP response to VNS (p<0.005 vs control), suggesting that VIP is a postganglionic neurotransmitter in the dog pancreas. To examine the effects of VIP on pancreatic hormone secretion, synthetic VIP was infused locally into the pancreatic artery. VIP, at a low dose (5 pmol/min), increased glucagon secretion from 1750+/-599 to 3800+/-990 pg/min (delta = +2060+/-870 pg/min, p<0.05), but did not affect insulin secretion (delta = -1030+/-760 microU/min, NS). Thus, VIP is contained in and released from pancreatic parasympathetic nerves in proximity to islet alpha cells and exogenous VIP, at a dose which approximates the increase of VIP spillover during VNS, preferentially stimulates glucagon vs insulin secretion. Therefore, VIP is likely to function as a parasympathetic neurotransmitter in the endocrine pancreas in dogs. We hypothesize that VIP could mediate the glucagon response to parasympathetic activation which has been shown to resistant to cholinergic blockade with atropine in several species.

Reduced pancreatic content of the inhibitory neurotransmitter galanin in genetically obese, hyperinsulinemic mice.

Because abnormalities of the autonomic nervous system have been described in several animal models of obesity, and because galanin has been proposed to be a sympathetic neurotransmitter in the endocrine pancreas, we hypothesized that the hyperinsulinemia observed in genetically obese (ob/ob) mice may result either from defective ability of galanin to inhibit insulin release or from a reduced degree of pancreatic galaninergic innervation. To address these possibilities, we examined the effect of exogenous galanin on immunoreactive insulin (IRI) levels in ob/ob mice and compared the pancreatic content of galaninlike immunoreactivity (GLIR) in ob/ob mice with that in lean littermates. Intravenous administration of synthetic porcine galanin significantly reduced basal IRI levels in ob/ob mice, suggesting that a defect in galanin action is unlikely to account for the hyperinsulinemia in this model. In contrast, reduced pancreatic galaninergic innervation was supported by findings that pancreatic content of GLIR in ob/ob mice was less than 10% of that in age- and sex-matched lean littermates. The reduction of pancreatic GLIR in ob/ob mice appeared organ specific; no such reduction was observed in adrenal GLIR content when comparing obese and lean mice. In addition, the relationship between pancreatic GLIR content and plasma IRI levels was examined in groups of obese and lean mice. It was found in young females, young males, and older mice of mixed sex that there was a significant negative correlation between pancreatic GLIR and plasma IRI in lean mice, whereas no such correlation was observed in obese mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Neural control of islet function by norepinephrine and sympathetic neuropeptides.

It is clear that the sympathoadrenal system has a role in the regulation of endocrine pancreatic function and that the sympathetic nerves of the pancreas can change pancreatic hormone secretion to increase the availability of metabolic fuels. It seems likely that the classical sympathetic neurotransmitter, NE, acts in concert with peptide co-transmitters, such as galanin and NPY. Each is released during the stimulation of pancreatic sympathetic nerves and each is capable of influencing either islet function or pancreatic blood flow. There is considerable indirect evidence that the sympathetic innervation of the pancreas is activated during acute stress and influences the endocrine pancreas. However, proving such a physiologic role is difficult because of redundant mechanisms that influence the secretion of the metabolically-crucial hormones, insulin and glucagon. Such definitive proof therefore awaits the development of new techniques to dissect and dissociate these mechanisms.