Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas.

The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK-8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP-1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods, 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 microM) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system.     

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).     

DPP IV resistance and insulin releasing activity of a novel di-substituted analogue of glucose-dependent insulinotropic polypeptide, (Ser2-Asp13)GIP

Structure-function studies suggest that preservation of the N-terminus and secondary structure of glucose-dependent insulinotropic polypeptide (GIP) is important for biological activity. Therefore, a novel di-substituted analogue of GIP, (Ser(2)-Asp(13))GIP, containing a negatively charged Asp residue in place of an Ala in position 13, was synthesised and evaluated for in vitro biological activity. Incubation with dipeptidyl peptidase IV (DPP IV) showed the half-lives of GIP and (Ser(2)-Asp(13))GIP to be 2.3 and >4h, respectively. Insulin releasing studies in clonal pancreatic BRIN-BD11 cells demonstrated that (Ser(2)-Asp(13))GIP (10(-12)to 10(-7)mol/l) was significantly less potent (60-90%; P<0.05 to P<0.001) than native GIP. The peptide failed to display antagonistic properties as it did not significantly alter insulin secretion when incubated in the presence of GIP (10(-7)mol/l). These results demonstrate that despite increased resistance to DPP IV, substituting Ala in position 13 with a negatively charged Asp, thus producing the di-substituted analogue (Ser(2)-Asp(13))GIP, significantly reduces biological activity, most likely due to modifications within the secondary structure.     

Functional Enhancement of Electrofusion-derived BRIN-BD11 Insulin-secreting Cells After Implantation into Diabetic Mice

Electrofusion-derived BRIN-BD11 cells are glucosesensitive insulin-secreting cells which provide an archetypal bioengineered surrogate β-cell for insulin replacement therapy in diabetes mellitus, 5x10⁶ BRIN-BD11 cells were implanted intraperitoneally into severely hyperglycaemic (>24mmol/l) streptozotocin-induced insulin-treated diabetic athymic nude (nu/nu) mice. The implants reduced hyperglycaemia such that insulin injections were discontinued by 5–16 days (<17mmol/l) and normoglycaemia (<9mmol/l) was achieved by 7–20 days. Implanted cells were removed after 28 days and re-established in culture. After re-culture for 20 days, glucose-stimulated (16.7mmol/l) insulin release was enhanced by 121% (p<0.001) compared to non-implanted cells. Insulin responses to glucagon-like peptide-1 (10⁻⁹mol/l), cholecystokinin-8 (10⁻⁸ mol/l) and L-alanine (10 mmol/l) were increased by 32%, 31% and 68% respectively (p<0.05–0.01). Insulin content of the cells was 148% greater at 20 days after re-culture than before implantation (p<0.001), but basal insulin release (at 5.6 mmol/l glucose) was not changed. After re-culture for 40 days, insulin content declined to 68% of the content before implantation (p<0.01), although basal insulin release was unchanged. However, the insulin secretory responses to glucose, glucagonlike peptide-1, cholecystokinin-8 and L-alanine were decreased after 40 days of re-culture to 65%, 72%, 73% and 42% respectively of the values before implantation (p<0.05–0.01). The functional enhancement of electrofusion-derived surrogate β-cells that were re-cultured for 20 days after implantation and restoration of normoglycaemia indicates that the in vivo environment could greatly assist β-cell engineering approaches to therapy for diabetes.     

Hexose-specific inhibition in vitro of human red cell Ca(2+)-ATPase activity.

In a concentration-dependent manner (5.5-27.5 mmol/l), D-glucose incubated in vitro with human erythrocyte membranes at 37 degrees C for 1 h inhibited membrane Ca(2+)-ATPase activity by up to 75%. The IC50 was 11 mmol/l. L-Glucose was ineffective, as were 3-O-methylglucose, 2-deoxyglucose, sorbitol and myo-inositol. In contrast, D-fructose decreased Ca(2+)-ATPase activity nearly as effectively as D-glucose and mannose and galactose at 11 mmol/l were less than 50% as effective as D-glucose. Tunicamycin (12 pmol/l), but not 10 mmol/l aminoguanidine, progressively antagonized in vitro the D-glucose effect on the enzyme. Erythrocyte membrane Ca(2+)-ATPase activity may be regulated by glycosylation, rather than nonenzymatic glycation.     

N-terminally modified glucagon-like peptide-1(7-36) amide exhibits resistance to enzymatic degradation while maintaining its antihyperglycaemic activity in vivo

Glucagon-like peptide-1(7-36)amide (tGLP-1) is inactivated by dipeptidyl peptidase (DPP) IV by removal of the NH(2)-terminal dipeptide His(7)-Ala(8). We examined the degradation of NH(2)-terminally modified His(7)99% of His(7)-glucitol tGLP-1 remained intact at 12 h. His(7)-glucitol tGLP-1 was similarly resistant to plasma degradation in vitro. His(7)-glucitol tGLP-1 showed greater resistance to degradation in vivo (92% intact) compared to tGLP-1 (27% intact) 10 min after i.p. administration to Wistar rats. Glucose homeostasis was examined following i.p. injection of both peptides (12 nmol/kg) together with glucose (18 mmol/kg). Plasma glucose concentrations were significantly reduced and insulin concentrations elevated following peptides administration compared with glucose alone. The area under the curve (AUC) for glucose for controls (AUC 691+/-35 mM/min) was significantly lower after administration of tGLP-1 and His(7)-glucitol tGLP-1 (36 and 49% less; AUC 440+/-40 and 353+/-31 mM/min, respectively; P<0.01). This was associated with a significantly higher AUC for insulin (98-99% greater; AUC 834+/-46 and 838+/-39 ng/ml/min, respectively; P<0.01) after tGLP-1 and His(7)-glucitol tGLP-1 administration compared to controls (421+/-30 ng/ml/min). In conclusion, His(7)-glucitol tGLP-1 resists plasma DPP IV degradation while retaining potent antihyperglycaemic and insulin-releasing activities in vivo.     

Degradation and glycemic effects of His(7)-glucitol glucagon-like peptide-1(7-36)amide in obese diabetic ob/ob mice

Glucagon-like peptide-1(7-36)amide (tGLP-1) has attracted considerable potential as a possible therapeutic agent for type 2 diabetes. However, tGLP-1 is rapidly inactivated in vivo by the exopeptidase dipeptidyl peptidase IV (DPP IV), thereby terminating its insulin releasing activity. The present study has examined the ability of a novel analogue, His(7)-glucitol tGLP-1 to resist plasma degradation and enhance the insulin-releasing and antihyperglycemic activity of the peptide in 20-25-week-old obese diabetic ob/ob mice. Degradation of native tGLP-1 by incubation at 37 degrees C with obese mouse plasma was clearly evident after 3 h (35% intact). After 6 h, more than 87% of tGLP-1 was converted to GLP-1(9-36)amide and two further N-terminal fragments, GLP-1(7-28) and GLP-1(9-28). In contrast, His(7)-glucitol tGLP-1 was completely resistant to N-terminal degradation. The formation of GLP-1(9-36)amide from native tGLP-1 was almost totally abolished by addition of diprotin A, a specific inhibitor of DPP IV. Effects of tGLP-1 and His(7)-glucitol tGLP-1 were examined in overnight fasted obese mice following i.p. injection of either peptide (30 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Plasma glucose was significantly lower and insulin response greater following administration of His(7)-glucitol tGLP-1 as compared to glucose alone. Native tGLP-1 lacked antidiabetic effects under the conditions employed, and neither peptide influenced the glucose-lowering action of exogenous insulin (50 units/kg). Twice daily s.c. injection of ob/ob mice with His(7)-glucitol tGLP-1 (10 nmol/kg) for 7 days reduced fasting hyperglycemia and greatly augmented the plasma insulin response to the peptides given in association with feeding. These data demonstrate that His(7)-glucitol tGLP-1 displays resistance to plasma DPP IV degradation and exhibits antihyperglycemic activity and substantially enhanced insulin-releasing action in a commonly used animal model of type 2 diabetes.     

Oral administration of a fusion protein containing eight GLP-1 analogues produced in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3) in streptozotocin-induced diabetic rats

Glucagon-like peptide-1 (7-36) amide (GLP-1), a gut hormone released into the blood stream after feeding, can stimulate insulin secretion by potentiating the insulinotropic action of glucose. An expression vector pET-22bG8, encoding a fusion protein containing eight tandem repeat GLP-1 ([Ser(8), Gln(26), Asp(34)]-GLP-1) analogues, was constructed and transformed into the Escherichia coli BL21(DE3) strain over-expressing the His-tagged fusion protein under the IPTG promoter. SDS-PAGE and Western blot analysis demonstrated that the His-tagged GLP-1 fusion protein migrated as a single protein with a molecular weight of 32 kDa. Following chronic (10 days) oral administration (20 mg kg(-1) day(-1)) of the fusion protein to diabetic rats, serum glucose levels were significantly lowered from 26 +/- 2.5 to 7.9 +/- 1.4 mmol/l. Further studies are needed to evaluate the potential use for GLP-1 analogue short peptide in the treatment of diabetes mellitus.     

Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are both incretin hormones regulating postprandial insulin secretion. Their relative importance in this respect under normal physiological conditions is unclear, however, and the aim of the present investigation was to evaluate this. Eight healthy male volunteers (mean age: 23 (range 20-25) years; mean body mass index: 22.2 (range 19.3-25.4) kg/m2) participated in studies involving stepwise glucose clamping at fasting plasma glucose levels and at 6 and 7 mmol/l. Physiological amounts of either GIP (1.5 pmol/kg/min), GLP-1(7-36)amide (0.33 pmol/kg/min) or saline were infused for three periods of 30 min at each glucose level, with 1 h "washout" between the infusions. On a separate day, a standard meal test (566 kcal) was performed. During the meal test, peak insulin concentrations were observed after 30 min and amounted to 223+/-27 pmol/l. Glucose+saline infusions induced only minor increases in insulin concentrations. GLP-1 and GIP infusions induced significant and similar increases at fasting glucose levels and at 6 mmol/l. At 7 mmol/l, further increases were seen, with GLP-1 effects exceeding those of GIP. Insulin concentrations at the end of the three infusion periods (60, 150 and 240 min) during the GIP clamp amounted to 53+/-5, 79+/-8 and 113+/-15 pmol/l, respectively. Corresponding results were 47+/-7, 95+/-10 and 171+/-21 pmol/l, respectively, during the GLP-1 clamp. C-peptide responses were similar. Total and intact incretin hormone concentrations during the clamp studies were higher compared to the meal test, but within physiological limits. Glucose infusion alone significantly inhibited glucagon secretion, which was further inhibited by GLP-1 but not by GIP infusion. We conclude that during normal physiological plasma glucose levels, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide contribute nearly equally to the incretin effect in humans, because their differences in concentration and potency outweigh each other.     

Mechanism of actions of cholecystokinin octapeptide on food intake and insulin and pancreatic polypeptide release in the dog

We investigated the mechanism by which CCK-8 injected into the third cerebral ventricle (ITV administration) inhibits food intake and stimulates insulin and pancreatic polypeptide (PP) secretion in the dog. ITV administration of CCK-8 (4.08 micrograms/5 min) resulted in a significant elevation of plasma insulin and PP concentrations. This effect was abolished by truncal vagotomy and promptly inhibited by ITV administration of atropine (20 micrograms) and proglumide (10 mg). CCK-8 was less effective in increasing insulin and PP concentrations than in reducing feeding. Thus, 1.36 micrograms of ITV CCK-8 markedly reduced food intake to 14, 15, 29 and 31% of control values at 10, 30, 60 and 120 min, respectively. Atropine and naloxone (50 micrograms) had no blocking effect on CCK-8-induced satiety, whereas proglumide antagonized it. These results indicate that ITV CCK-8 effects the endocrine pancreas and food intake through atropine-sensitive and atropine-insensitive mechanisms, respectively, both of which are likely to be mediated by CNS CCK receptors. Intravenous CCK-8 also stimulated PP and insulin release, through mechanisms that were atropine-sensitive and atropine-insensitive, respectively. However, its mode of action, especially on insulin secretion, was quite different from that of ITV CCK-8. Therefore, exogenous CCK appears to act in the brain and the periphery in concert with and independently from cholinergic systems.     

Involvement of nitric oxide in endothelium-dependent arterial relaxation by leptin

Leptin is a polypeptide, mainly produced in white adipose tissue, and increases sympathetic nerve activity. A few studies investigated leptin's effect on peripheral vessels. We examined the vasorelaxant effects of human leptin on rat arteries. Arterial rings were precontracted with 1 x 10(-6) mol/l of phenylephrine, and leptin was superfused. Leptin relaxed phenylephrine-precontracted arterial rings in a dose-dependent manner. ED50 was calculated to 8.4 microg/ml. Removal of endothelium abolished the effects of leptin. Indomethacin (1 x 10(-5) mol/l) did not affect the vasorelaxation by leptin, whereas 1 x 10(-4) mol/l of N(omega)-nitro-L-arginine methyl ester (L-NAME) completely suppressed it. The inhibition was antagonized by 1 x 10(-4) mol/l of L-arginine. Leptin normally relaxed arterial rings during superfusion of K channel blockers, including 3 x 10(-5) mol/l of glibenclamide, 1 x 10(-6) of mol/l apamin, and 5 x 10(-7) mol/l of charybdotoxin. Low Cl(-) solution (8. 3 mmol/l) inhibited leptin-induced relaxation, but endothelium-independent vasodilatation by nitroprusside was not impaired at low Cl(-) solution. These results suggest that arterial relaxation by leptin is mediated by nitric oxide released from endothelium, and Cl(-) plays an important role in leptin-induced nitric oxide release.     

Lipolysis induced by alloxan in rat adipocytes is not inhibited by insulin.

Isolated rat adipocytes were incubated with adrenaline, adrenaline plus insulin, alloxan or alloxan plus insulin. Glycerol release was taken as a measure of lipolysis. It was observed that alloxan in the concentration of 3, 10 and 20 mmol/l intensifies lipolysis in adipocytes in the absence of adrenaline. Insulin (10(-6) mol/l) treatment of cells did not inhibit lipolysis caused by this compound, but significantly restricted lipolysis induced by adrenaline (10(-6) mol/l). It was also shown that alloxan in the concentration of 3 and 10 mmol/l intensified lipolysis stimulated by adrenaline (10(-6) mol/l). Addition of 20 mmol/l of alloxan strongly inhibited glycerol release in the presence of adrenaline. The results presented here clearly indicate that the action of alloxan concerns cells of the white adipose tissue.     

Guanosine 3':5'cyclic monophosphate and activators of guanylate cyclase inhibit secretagogue-induced corticotropin release by rat anterior pituitary cells

The secretion of corticotropin by perfused rat anterior pituitary cell columns was studied. Forty-one residue corticotropin releasing factor, vasopressin and high extracellular KC1 all stimulated the secretion of corticotropin. The hormonal response to corticotropin-releasing factor (10(-10) mol/l), vasopressin (10(-9) mol/l) as well as KC1 (48 mmol/l) was reduced by membrane permeant analogs of cGMP, such as 8-BrcGMP and dibutyryl-cGMP. The 8-BrcGMP analog (10(-5) mol/l) inhibited corticotropin release in response to corticotropin-releasing factor by 30%, that to vasopressin by 70%, and that to KCl by 50%. Atriopeptin1-28 (10(-8) and 10(-7) mol/l), a peptide known to activate membrane-bound guanylate cyclase in the anterior pituitary gland, decreased the release of corticotropin induced by vasopressin to about 30% of control. Similarly, activators of soluble guanylate cyclase, such as glyceryltrinitrate and sodium nitroprusside (10(-5) mol/l) inhibited vasopressin-stimulated corticotropin release by 60%. In conclusion, the data show that purported activators of particulate and soluble guanylate cyclase, as well as derivatives of cGMP itself are strong inhibitors of secretagogue-induced corticotropin release by corticotroph cells of the anterior pituitary gland.     

Synthesis and biological activity of some new flavonyl-2,4-thiazolidinediones

A new series of flavonyl-2,4-thiazolidinediones (Va-c, VIa-c) was prepared by Knoevenagel reaction. The synthesized compounds were tested for their ability to inhibit rat kidney aldose reductase (AR) and for their insulinotropic activities in INS-1 cells. Compound Vb was able to increase insulin release in the presence of 5.6mmol/l glucose. Compounds VIa-c displayed moderate to high AR inhibitory activity levels. Particularly, compound VIa showed the highest AR inhibitory activity (86.57%).     

The adenylate cyclase system and calcitonin secretion from perfused dog thyroid lobes

The aim of the study was to assess the involvement of the adenylate cyclase system in calcitonin (CT) secretion from thyroidal C-cells. The cAMP analogues Br-cAMP (10(-6) and 10(-4) mol/l) and DB-cAMP (10(-4) mol/l) and the activators of adenylate cyclase cholera toxin (0.1 microgram/ml and 5 micrograms/ml) and forskolin (10(-7) mol/l and 10(-5) mol/l) were infused for 6 min periods in perfused dog thyroid lobes. CT was measured in thyroid effluent by radioimmunoassay. Br-cAMP and cholera toxin did not alter basal CT secretion. DB-cAMP had a minimal stimulatory effect and forskolin 10(-5) mol/l a moderate stimulatory effect. This was much less than the effect of increasing perfusate Ca++ from 1.5 to 2.0 mmol/l. 10(-4) mol/l Br-cAMP increased the response to Ca++ with approximately 50 per cent. These results suggest that the activity of the adenylate cyclase system of the C-cells by itself is of little importance for CT secretion, but that it may have a role as modulator of the response to Ca++.     

Effects of cholecystokinin on appetite and pyloric motility during physiological hyperglycemia

Recent studies suggest that the interaction between small intestinal nutrient stimulation and the blood glucose concentration is important in the regulation of gastric motility and appetite. The purpose of this study was to determine whether the effects of cholecystokinin octapeptide (CCK-8) on antropyloric motility and appetite are influenced by changes in the blood glucose concentration within the normal postprandial range. Seven healthy volunteers were studied on 4 separate days. A catheter incorporating a sleeve sensor was positioned across the pylorus, and the blood glucose was stabilized at either 4 mmol/l (2 days) or 8 mmol/l (2 days). After the desired blood glucose had been maintained for 90 min, an intravenous infusion of either CCK-8 (2 ng. kg(-1). min(-1)) or saline (control) was given for 60 min. Thirty minutes after the infusion began, the catheter was removed and subjects drank 400 ml of water with guar gum before being offered a buffet meal. The amount of food consumed (kcal) was quantified. The order of the studies was randomized and single-blinded. There were fewer antral waves at a blood glucose of 8 than at 4 mmol/l during the 90-min period before the infusions (P<0.05) and during the first 30 min of CCK-8 or saline infusion (P = 0.07). CCK-8 suppressed antral waves (P<0.05), stimulated isolated pyloric pressure waves (IPPWs) (P<0.01), and increased basal pyloric pressure (P<0.005) compared with control. During administration of CCK-8, basal pyloric pressure (P<0.01), but not the number of IPPWs, was greater at a blood glucose of 8 mmol/l than at 4 mmol/l. CCK-8 suppressed the energy intake at the buffet meal (P<0.01), with no significant difference between the two blood glucose concentrations. We conclude that the acute effect of exogenous CCK-8 on basal pyloric pressure, but not appetite, is modulated by physiological changes in the blood glucose concentration.     

Characterization of the cellular and metabolic effects of a novel enzyme-resistant antagonist of glucose-dependent insulinotropic polypeptide

A novel N-terminally substituted Pro(3) analogue of glucose-dependent insulinotropic polypeptide (GIP) was synthesized and tested for plasma stability and biological activity both in vitro and in vivo. Native GIP was rapidly degraded by human plasma with only 39 +/- 6% remaining intact after 8 h, whereas (Pro(3))GIP was completely stable even after 24 h. In CHL cells expressing the human GIP receptor, (Pro(3))GIP antagonized the cyclic adenosine monophosphate (cAMP) stimulatory ability of 10(-7) M native GIP, with an IC(50) value of 2.6 microM. In the clonal pancreatic beta cell line BRIN-BD11, (Pro(3))GIP over the concentration range 10(-13) to 10(-8) M dose dependently inhibited GIP-stimulated (10(-7) M) insulin release (1.2- to 1.7-fold; P < 0.05 to P < 0.001). In obese diabetic (ob/ob) mice, intraperitoneal administration of (Pro(3))GIP (25 nmol/kg body wt) countered the ability of native GIP to stimulate plasma insulin (2.4-fold decrease; P < 0.001) and lower the glycemic excursion (1.5-fold decrease; P < 0.001) induced by a glucose load (18 mmol/kg body wt). Collectively these data demonstrate that (Pro(3))GIP is a novel and potent enzyme-resistant GIP receptor antagonist capable of blocking the ability of native GIP to increase cAMP, stimulate insulin secretion, and improve glucose homeostasis in a commonly employed animal model of type 2 diabetes.     

Effects of pancreastatin and chromogranin A on insulin release stimulated by various insulinotropic agents

The effects of porcine pancreastatin on insulin release stimulated by insulinotropic agents, glucagon, cholecystokinin-octapeptide (CCK-8), gastric inhibitory polypeptide (GIP) and L-arginine, were compared to those of bovine chromogranin A (CGA) using the isolated perfused rat pancreas. Pancreastatin significantly potentiated glucagon-stimulated insulin release (first phase: 12.5 +/- 0.9 ng/8 min; second phase: 34.5 +/- 1.6 ng/25 min in controls; 16.5 +/- 1.1 ng/8 min and 44.0 +/- 2.2 ng/25 min in pancreastatin group), whereas CGA was ineffective. The first phase of L-arginine-stimulated insulin release was also potentiated by pancreastatin (6.9 +/- 0.5 ng/5 min in controls, 8.4 +/- 0.6 ng/5 min in pancreastatin group), but not by CGA. Pancreastatin did not affect CCK-8 or GIP-stimulated insulin release. Similarly, CGA did not affect insulin release stimulated by CCK-8 or GIP. These findings suggest that pancreastatin stimulates insulin release in the presence of glucagon. Because pancreastatin can have multiple effects on insulin release, which are dependent upon the local concentration of insulin effectors, pancreastatin may participate in the fine tuning of insulin release from B cells.     

Effects of epidermal growth factor and calcium omission on cholecystokinin-stimulated Cl- conductance in rat pancreatic zymogen granules.

Evidence suggests that cholecystokinin-octapeptide (CCK-8)-induced activation of a Cl- conductance in the membrane of zymogen granules (ZG) is closely related to pancreatic enzyme secretion. Following stimulation of isolated pancreatic acinar cells with increasing concentrations of CCK-8, the Cl- conductance in the ZG from these acini increased, reached a maximum of 40 +/- 7% above basal Cl- conductance at 10(-12) M CCK-8, and then decreased at CCK-8 concentrations higher than 10(-9) M to a level comparable to the basal Cl- conductance. We had interpreted the inhibitory action of high CCK-8 concentrations to be due to the generation of high concentrations of diacylglycerol and/or its metabolites by an "overstimulation" of phospholipase C at supramaximal CCK-8 concentrations. We now show that EGF abolishes the downstroke of the dose response curve for CCK-8-induced ZG Cl- conductance and shifts the stimulatory response to higher CCK-8 concentrations. Similarly in a nominally "Ca(2+)-free buffer" (free [Ca2+] approximately 0.2 nM), stimulated Cl- conductance at 10(-12) M CCK-8 is nearly abolished and the decreased Cl- conductance at 10(-8) M CCK-8 is increased to the level of maximal stimulation at 10(-12) M CCK-8. We conclude that both EGF and low [Ca2+] affect CCK-8-induced ZG Cl- conductance by decreasing phospholipase C activity.     

Adenosine triphosphate increases the reactivity of the afferent arteriole to low concentrations of norepinephrine

Adenosine triphosphate (ATP) and norepinephrine (NE) interact in the control of blood flow in the kidney. A combined effect of NE and ATP has not been previously investigated at the level of the afferent arteriole (Af). We studied the effects of ATP on the contractile response of the Af to NE. Vascular reactivity to ATP, NE, and their combination was investigated in isolated perfused Af from mice. The roles of alpha-adrenoceptors and P2-ATP-receptors were investigated by use of specific agonists and antagonists. Cytosolic calcium was measured using the fluorescent calcium dye fura-2. ATP in concentrations from 10(-12) to 10(-4) mol/l induced transient contractions. NE constricted the Af in a dose-dependent manner and induced significant contractions at > 10(-7) mol/l. Treatment with ATP (10(-8) and 10(-6) mol/l) increased the NE response. Diameters were reduced by 20% already at 10(-11) mol/l NE during ATP treatment of 10(-6) mol/l. ATP increased the calcium response to NE significantly at 10(-8) and 10(-7)mol/l NE. The P2-type ATP receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10(-5) mol/l) abolished the sensitization of the NE response by ATP. The alpha(1)-blocker prazosin (10(-7) mol/l) inhibited the ATP effect, as did the alpha 2-blocker yohimbine (10(-7) mol/l). Neither the phenylephrine- nor clonidine-induced concentration response curves was affected by ATP in the bath solution. Costimulation with ATP enhances the response of the Af to NE. This effect is mediated by increased cytosolic calcium. The enhancing effect involves P2-type ATP receptors and both alpha (1)- and alpha 2-adrenoceptors.