Pancreastatin-dependent inflammatory signaling mediates obesity-induced insulin resistance.

Chromogranin A knockout (Chga-KO) mice exhibit enhanced insulin sensitivity despite obesity. Here, we probed the role of the chromogranin A-derived peptide pancreastatin (PST: CHGA(273-301)) by investigating the effect of diet-induced obesity (DIO) on insulin sensitivity of these mice. We found that on a high-fat diet (HFD), Chga-KO mice (KO-DIO) remain more insulin sensitive than wild-type DIO (WT-DIO) mice. Concomitant with this phenotype is enhanced Akt and AMPK signaling in muscle and white adipose tissue (WAT) as well as increased FoxO1 phosphorylation and expression of mature Srebp-1c in liver and downregulation of the hepatic gluconeogenic genes, Pepck and G6pase. KO-DIO mice also exhibited downregulation of cytokines and proinflammatory genes and upregulation of anti-inflammatory genes in WAT, and peritoneal macrophages from KO mice displayed similarly reduced proinflammatory gene expression. The insulin-sensitive, anti-inflammatory phenotype of KO-DIO mice is masked by supplementing PST. Conversely, a PST variant peptide PSTv1 (PST-NΔ3: CHGA(276-301)), lacking PST activity, simulated the KO phenotype by sensitizing WT-DIO mice to insulin. In summary, the reduced inflammation due to PST deficiency prevented the development of insulin resistance in KO-DIO mice. Thus, obesity manifests insulin resistance only in the presence of PST, and in its absence obesity is dissociated from insulin resistance.

Naturally occurring variants of the dysglycemic peptide pancreastatin: differential potencies for multiple cellular functions and structure-function correlation.

Pancreastatin (PST), a chromogranin A-derived peptide, is a potent physiological inhibitor of glucose-induced insulin secretion. PST also triggers glycogenolysis in liver and reduces glucose uptake in adipocytes and hepatocytes. Here, we probed for genetic variations in PST sequence and identified two variants within its functionally important carboxyl terminus domain: E287K and G297S. To understand functional implications of these amino acid substitutions, we tested the effects of wild-type (PST-WT), PST-287K, and PST-297S peptides on various cellular processes/events. The rank order of efficacy to inhibit insulin-stimulated glucose uptake was: PST-297S > PST-287K > PST-WT. The PST peptides also displayed the same order of efficacy for enhancing intracellular nitric oxide and Ca(2+) levels in various cell types. In addition, PST peptides activated gluconeogenic genes in the following order: PST-297S ≈ PST-287K > PST-WT. Consistent with these in vitro results, the common PST variant allele Ser-297 was associated with significantly higher (by ∼17 mg/dl, as compared with the wild-type Gly-297 allele) plasma glucose level in our study population (n = 410). Molecular modeling and molecular dynamics simulations predicted the following rank order of α-helical content: PST-297S > PST-287K > PST-WT. Corroboratively, circular dichroism analysis of PST peptides revealed significant differences in global structures (e.g. the order of propensity to form α-helix was: PST-297S ≈ PST-287K > PST-WT). This study provides a molecular basis for enhanced potencies/efficacies of human PST variants (likely to occur in ∼300 million people worldwide) and has quantitative implications for inter-individual variations in glucose/insulin homeostasis.

Pancreastatin is an endogenous peptide that regulates glucose homeostasis.

Pancreastatin (PST) is a regulatory peptide containing 49 amino acids, first isolated from porcine pancreas. Intracellular and extracellular processing of the prohormone Chromogranin A (Chga) results various bioactive peptides of which PST has dysglycemic activity. PST regulates glucose, lipid, and protein metabolism in liver and adipose tissues. It also regulates the secretion of leptin and expression of leptin and uncoupling protein 2 in adipose tissue. In Chga knockout mice, PST induces gluconeogenesis in the liver. PST reduces glucose uptake in mice hepatocytes and adipocytes. In rat hepatocytes, PST induces glycogenolysis and glycolysis and inhibits glycogen synthesis. In rat adipocytes, PST inhibits lactate production and lipogenesis. These metabolic effects are confirmed in humans. In the dual signaling mechanism of PST receptor, mostly PST activates Gαq/11 protein leads to the activation of phospholipase C ß3-isoform, therefore increasing cytoplasmic free calcium and stimulating protein kinase C. PST inhibits the cell growth in rat HTC hepatoma cells, mediated by nitric oxide and cyclic GMP production. Elevated levels of PST correlating with catecholamines have been found in gestational diabetes and essential hypertension. Rise in the blood PST level in Type 2 diabetes suggests that PST is a negative regulator of insulin sensitivity and glucose homeostasis.

Direct vasoactive effects of the chromogranin A (CHGA) peptide catestatin in humans in vivo.

Catestatin is a bioactive peptide of chromogranin A (CHGA) that is co-released with catecholamines from secretory vesicles. Catestatin may function as a vasodilator and is diminished in hypertension. To evaluate this potential vasodilator in vivo without systemic counterregulation, we infused catestatin to target concentrations of approximately 50, approximately 500, approximately 5000 nM into dorsal hand veins of 18 normotensive men and women, after pharmacologic venoconstriction with phenylephrine. Pancreastatin, another CHGA peptide, was infused as a negative control. After preconstriction to approximately 69%, increasing concentrations of catestatin resulted in dose-dependent vasodilation (P = 0.019), in female subjects (to approximately 44%) predominantly. The EC(50) (approximately 30 nM) for vasodilation induced by catestatin was the same order of magnitude to circulating endogenous catestatin (4.4 nM). No vasodilation occurred during the control infusion with pancreastatin. Plasma CHGA, catestatin, and CHGA-to-catestatin processing were then determined in 622 healthy subjects without hypertension. Female subjects had higher plasma catestatin levels than males (P = 0.001), yet lower CHGA precursor concentrations (P = 0.006), reflecting increased processing of CHGA-to-catestatin (P < 0.001). Our results demonstrate that catestatin dilates human blood vessels in vivo, especially in females. Catestatin may contribute to sex differences in endogenous vascular tone, thereby influencing the complex predisposition to hypertension.

Secretion of ghrelin from rat stomach ghrelin cells in response to local microinfusion of candidate messenger compounds: a microdialysis study.

Ghrelin is produced by A-like cells (ghrelin cells) in the mucosa of the acid-producing part of the stomach. The mobilization of ghrelin is stimulated by nutritional deficiency and suppressed by nutritional abundance. In an attempt to identify neurotransmitters and regulatory peptides that may contribute to the physiological, nutrient-related regulation of ghrelin secretion, we challenged the ghrelin cells in situ with a wide variety of candidate messengers, including known neurotransmitters (e.g. acetylcholine, catecholamines), candidate neurotransmitters (e.g. neuropeptides), local tissue hormones (e.g. serotonin, histamine, bradykinin, endothelin), circulating gut hormones (e.g. gastrin, CCK, GIP, neurotensin, PYY, secretin) and other circulating hormones/regulatory peptides (e.g. calcitonin, glucagon, insulin, PTH). Microdialysis probes were placed in the submucosa of the acid-producing part of the rat stomach. Three days later, the putative messenger compounds were administered via the microdialysis probe (reverse microdialysis) at a screening dose of 0.1 mmol l(-1) for regulatory peptides and 0.1 and 1 mmol l(-1) for amines and amino acids. The rats were awake during the experiments. The resulting microdialysate ghrelin concentration was monitored continuously for 3 h (radioimmunoassay), thereby revealing stimulators or inhibitors of ghrelin secretion. Dose-response curves were constructed for each candidate messenger that significantly (p<0.05) affected ghrelin mobilization at the screening dose. Peptides that showed a (non-significant) tendency to affect ghrelin release at the screening dose were also given at a dose of 0.3 or 1 mmol l(-1). Adrenaline, noradrenaline, endothelin and secretin stimulated ghrelin release, while somatostatin and GRP inhibited. Whether these agents act directly or indirectly on the ghrelin cells remains to be investigated. All other candidate messengers were without measurable effects, including acetylcholine, serotonin, histamine, GABA, aspartic acid, glutamic acid, glycine, VIP, PACAP, CGRP, substance P, NPY, PYY, PP, gastrin, CCK, GIP, insulin, glucagon, GLP and glucose.

Antral gastric motility impairement and autonomic nervous system dysfunction in patients with pancreatic cancer (preliminary results).

The aim of the study was to evaluate the influence of pancreatic carcinoma on gastric motility and autonomic system activity in patients before surgery. Patients with histologically confirmed pancreatic cancer were studied. Our results show that gastric dysrrhythmias are continuously present in patients with pancreatic cancer both with and without gastric emptying delay. Antral distribution of meal suggests the presence of impaired proximal gastric response to meal due to diminished autonomic nervous system function and impaired myoelectric gastric activity in patients with pancreatic cancer.

Pancreastatin: multiple actions on human intermediary metabolism in vivo, variation in disease, and naturally occurring functional genetic polymorphism.

RATIONALE: The chromogranin A (CHGA) fragment pancreastatin (human CHGA250-301) impairs glucose metabolism, but the role of human pancreastatin in vivo remains unexplored. METHODS: We studied brachial arterial infusion of pancreastatin (CHGA273-301-amide at approximately 200 nm) on forearm metabolism of glucose, free fatty acids, and amino acids. Plasma pancreastatin was measured in obesity or type 2 diabetes. Systematic discovery of amino acid variation was performed, and the potency of one variant in the active carboxyl terminus (Gly297Ser) was tested. RESULTS: Pancreastatin decreased glucose uptake by approximately 48-50%; the lack of change in forearm plasma flow indicated a metabolic, rather than hemodynamic, mechanism. A control CHGA peptide (catestatin, CHGA352-372) did not affect glucose. Insulin increased glucose uptake, but pancreastatin did not antagonize this action. Pancreastatin increased spillover of free fatty acids by about 4.5- to 6.4-fold, but not spillover of amino acids. Insulin diminished spillover of both free fatty acids and amino acids, but these actions were not reversed by pancreastatin. Plasma pancreastatin was elevated approximately 3.7-fold in diabetes, but was unchanged during weight loss. Proteolytic cleavage sites for pancreastatin in vivo were documented by matrix-assisted laser desorption ionization/time of flight mass spectrometry. Three pancreastatin variants were discovered: Arg253Trp, Ala256Gly, and Gly297Ser. The Gly297Ser variant had unexpectedly increased potency to inhibit glucose uptake. CONCLUSIONS: The dysglycemic peptide pancreastatin is specifically and potently active in humans on multiple facets of intermediary metabolism, although it did not antagonize insulin. Pancreastatin is elevated in diabetes, and the variant Gly297Ser had increased potency to inhibit glucose uptake. The importance of human pancreastatin in vivo as well as its natural variants is established.

GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets 1.

BACKGROUND & AIMS: Our aim was to determine whether the intestinotrophic effects of GLP-2 are mediated by acute up-regulation of intestinal substrate utilization in TPN-fed piglets. METHODS: Twenty-four 12-day-old pigs, fitted with a portal flow probe and carotid, jugular and portal catheters, were fed by TPN for 7 days. On day 8, a group of pigs (n = 8) was infused intravenously with saline (control) for 4 hours and then with GLP-2 (500 pmol x kg(-1) x hour(-1), GLP-2) for 4 hours. (2)H-glucose and (13)C-phenylalanine were infused to estimate their kinetics and protein turnover. Another group (n = 8) received consecutive intravenous infusions of saline, GLP-2, and GLP-2 plus N(G)-Nitro-L-arginine methyl ester (L-NAME, 50 micromol x kg(-1) x hour(-1)) for 4 hours each. RESULTS: GLP-2 acutely increased portal-drained visceral (PDV) blood flow rate (+25%) and intestinal blood volume (+51%) in TPN-fed piglets. GLP-2 also increased intestinal constitutive nitric oxide synthase (NOS) activity and endothelial NOS protein abundance. GLP-2 acutely increased PDV glucose uptake (+90%) and net lactate production (+79%). Co-infusion of GLP-2 plus L-NAME did not increase either PDV blood flow rate or glucose uptake. GLP-2 increased PDV indispensable amino acid uptake by 220% and protein synthesis by 125%, but did not decrease protein breakdown or phenylalanine oxidation. CONCLUSIONS: We conclude that in TPN-fed neonatal pigs, GLP-2 acutely stimulates intestinal blood flow and glucose utilization, and this response is nitric oxide-dependent. These findings suggest that GLP-2 may play an important physiological role in the regulation of intestinal blood flow and that nitric oxide is involved in GLP-2 receptor function.

Pancreastatin, a chromogranin-A-derived peptide, inhibits insulin-stimulated glycogen synthesis by activating GSK-3 in rat adipocytes.

We have previously found that pancreastatin (PST) inhibits glucose uptake in rat adipocytes by preventing GLUT4 translocation to the plasma membrane. We have also described that this effect is mediated by the cross-talk with insulin signaling, inhibiting Tyr-phosphorylation and PI3-kinase (PI3K) activity, via protein kinase C (PKC) activation. In the present work, we have further investigated the effects of PST on glucose metabolism and the signaling pathways involved in its regulation. As expected, we found that PST inhibited insulin-stimulated PKB activity, since it depends on PI3-kinase activity. Next, we studied the activity of the target enzyme of PKB, glycogen synthase kinase-3 (GSK-3). PST not only prevented the insulin effect decreasing GSK-3 activity, but PST itself was able to activate GSK-3 activity in rat adipocytes. As previously described, phosphorylation level of GSK-3 was negatively correlated with the activity. Thus, insulin stimulated GSK-3 serine phosphorylation, whereas PST inhibited this effect, and even decreased basal phosphorylation. The PST stimulation of GSK-3 activity seems to be mediated by PKC since it can be prevented by a specific PKC inhibitor (bisindolylmaleimide). Finally, the PST effect on GSK-3 activity resulted in an inhibition on both basal and insulin stimulated glycogen synthesis in rat adipocytes. This effect of PST can also be prevented by using a PKC inhibitor. In conclusion, the chromogranin-A-derived peptide PST inhibits glycogen synthesis in rat adipocytes by activating GSK-3 activity through the activation of PKC.

Pancreastatin, a chromogranin A-derived peptide, inhibits DNA and protein synthesis by producing nitric oxide in HTC rat hepatoma cells.

BACKGROUND/AIMS: Pancreastatin, a chromogranin A-derived peptide, has a counter-regulatory effect on insulin action. We have previously characterized pancreastatin receptor and signalling in rat liver and HTC hepatoma cells. A G alpha(q/11)-PLC-beta pathway leads to an increase in [Ca2+]i, PKC and mitogen activated protein kinase (MAPK) activation. These data suggested that pancreastatin might have a role in growth and proliferation, similar to other calcium-mobilizing hormones. METHODS: DNA and protein synthesis were measured as [3H]-thymidine and [3H]-leucine incorporation. Nitric oxide (NO) was determined by the Griess method and cGMP production was quantified by enzyme-linked immunoassay. RESULTS: Contrary to the expected results, we have found that pancreastatin inhibits protein and DNA synthesis in HTC hepatoma cells. On the other hand, when the activity of NO synthase was inhibited by N-monomethyl-L-arginine (NMLA), the inhibitory effect of pancreastatin on DNA and protein synthesis was not only reverted, but a dose-dependent stimulatory effect was observed, probably due to MAPK activation, since it was prevented by PD98059. These data strongly suggested the role of NO in the inhibitory effect of pancreastatin on protein and DNA synthesis, which is overcoming the effect on MAPK activation. Moreover, pancreastatin dose-dependently increased NO production in parallel to cyclic guanosine monophosphate (cGMP). Both effects were prevented by NMLA. Finally, an indirect effect of pancreastatin through the induction of apoptosis was ruled out. CONCLUSIONS: Therefore, the NO and the cGMP produced by the NO-activated guanylate cyclase may mediate the dose-dependent inhibitory effect of pancreastatin on growth and proliferation in HTC hepatoma cells.

Effects of arginine on pancreatic hormones and hepatic metabolism in rainbow trout.

Arginine (Arg), injected intraperitoneally into rainbow trout (Oncorhynchus mykiss), increases plasma concentrations of glucagon, glucagon-like peptide-1 (GLP-1), and insulin by three- to 10-fold. Resulting ratios of glucagon and GLP-1 over insulin are unchanged in 20-d food-deprived fish (saline, 1.28 vs. Arg, 0.93; not significant) while slightly increased in feeding trout (saline, 0.70 vs. Arg, 0.92; P<0.05). In food-deprived juveniles, Arg injection leads to significant decreases in plasma fatty acids (saline, 1.65 mM L(-1) vs. Arg, 1.09 mM L(-1); P<0.05) and increases in glycogen phosphorylase total activity (saline, 3.7 units g(-1) vs. Arg, 4.6 units g(-1); P<0.05) and degree of phosphorylation (saline, 1.7 units g(-1) vs. Arg, 2.33 units g(-1); P<0.05). Plasma and liver glucose and liver enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, phosphoenolpyruvate carboxykinase, lactate dehydrogenase, and malic enzyme) are unaffected. Otherwise, fish show the changes in plasma metabolites expected with food deprivation. Arg injection into feeding fish results in decreases in plasma fatty acids, liver glycogen, and glucose, while liver glucose 6-phosphate concentrations increase. Hepatocytes isolated from feeding fish injected with Arg 2 h previously show significantly lower rates of lactate oxidation than controls (85% of control), while rates of gluconeogenesis and hormonal responses to mammalian glucagon and GLP-1 remain unchanged. Rates of lactate oxidation and gluconeogenesis are significantly decreased by 5%-10% on treatment with porcine insulin. Complete immunoneutralization of insulin with rabbit antisalmon insulin serum decreases hepatic glucose 6-phosphate concentrations and abolishes the Arg-dependent effects on glycogen phosphorylase. It appears that short-term increases in pancreatic hormones cause only minor metabolic readjustments in the relatively short time frame covered in these experiments. Surprisingly, complete removal of insulin does not have immediate altering or detrimental effects on key metabolites and metabolic pathways, even if glucagon and GLP-1 concentrations are concurrently several-fold higher than usual. Our data clearly show the dual role of Arg in fish metabolism.

Characterization of pancreastatin receptor and signaling in rat HTC hepatoma cells.

Pancreastatin, a chromogranin A-derived peptide widely distributed throughout the neuroendocrine system, has a general inhibitory effect on endocrine secretion and a counterregulatory effect on insulin action. We have recently described the cross-talk of pancreastatin with insulin signaling in rat hepatoma cells (HTC), where it inhibits insulin action and signaling through the serine phosphorylation of the insulin receptor, thereby impairing tyrosine kinase activity. Here, we have characterized pancreastatin receptors and signaling in HTC cells. The pancreastatin effector systems were studied by determining phospholipase C activity in HTC membranes and mitogen-activated protein kinase (MAPK) phosphorylation activity in HTC cells. Binding studies with radiolabeled pancreastatin showed a population of high affinity binding sites, with a B(max) of 8 fmol/mg protein and a K(d) of 0.6 nM. Moreover, we assessed the coupling of the receptor with a G protein system by inhibiting the binding with guanine nucleotide and by measuring the GTP binding to HTC membranes. We found that pancreastatin receptor was coupled with a G alpha(q/11) protein which activates phospholipase C-beta(1) and phospholipase C-beta(3), in addition to MAPK via both beta gamma and alpha(q/11).

Peptidergic regulation of maturation of the stimulus-secretion coupling in fetal islet beta cells.

The stimulus-secretion coupling of the insulin-producing pancreatic islet beta cell is subject to functional maturation during fetal life. We studied the maturation of a glucose-responsive insulin release from fetal rat islets and specifically investigated the impact of peptidergic regulation. To this end, islets were isolated from 21-day-old fetal rats and maintained for 7 days in tissue culture at 3.3 or 11.1 mM glucose and various supplements. In islets cultured in low glucose, acutely raising the ambient glucose concentration to 16.7 mM evoked a modest stimulation of short-term insulin release that was more pronounced in islets maintained in high glucose. Moreover, the insulin content was much higher in islets cultured in high than in low glucose. Culture with growth hormone (GH) markedly amplified both basal and stimulated short-term insulin secretion from islets maintained in either low or high glucose. Additionally, GH significantly elevated the insulin content in islets maintained in low glucose. Transforming growth factor alpha (TGF-alpha) increased basal, but not glucose-stimulated, insulin release and insulin content in islets cultured in low glucose. Gastrin, expressed in islets during fetal life, did not affect basal or glucose-stimulated insulin release, or insulin content, in islets maintained in either low or high glucose. The addition of gastrin to TGF-alpha did not affect the results obtained with the latter peptide. Gastrin-releasing peptide failed to influence basal or glucose-responsive insulin secretory rates, and insulin content, at either glucose concentration during culture. The somatostatin analog Sandostatin (octreotide acetate) neither influenced basal nor stimulated short-term insulin release at any glucose concentration present during culture, whereas the hormone significantly decreased the insulin content of islets cultured in high glucose. Pancreastatin, produced by porcine islet beta and delta cells, failed to influence basal or glucose-responsive insulin secretory rates, and islet insulin content, at either glucose concentration during culture. Culture with gastric inhibitory peptide (GIP) or glucagon-like peptide I (GLP-1), two proposed incretins, did not affect short-term insulin secretion in response to 3.3 or 16.7 mM glucose irrespective of the ambient glucose concentration during culture. To the contrary, GLP-1, but not GIP, increased the content of insulin in islets cultured in low glucose. We conclude that islet beta-cell differentiation and functional maturation of the stimulus-secretion coupling can be modulated in vitro in fetal rat pancreatic tissue by peptidergic regulation and glycemic stimulation. We suggest that GH and TGF-alpha stimulate, while somatostatin, through paracrine interaction, may inhibit, these processes. These effectors may be of regulatory significance in the in vivo development of glucose-sensitive beta cells, and defects in these mechanisms may result in glucose intolerance in adult subjects.

Characterization of pancreastatin receptors and signaling in adipocyte membranes.

Pancreastatin (PST), a chromogranin A derived peptide with an array of effects in different tissues, has a role as a counterregulatory hormone of insulin action in hepatocytes and adipocytes, regulating glucose, lipid and protein metabolism. We have previously characterized PST receptors and signaling in rat hepatocytes, in which PST functions as a calcium-mobilizing hormone. In the present work we have studied PST receptors as well as the signal transduction pathways generated upon PST binding in adipocyte membranes. First, we have characterized PST receptors using radiolabeled PST as a ligand. Analysis of binding data indicated the existence of one class of binding sites, with a B(max) of 5 fmol/mg of protein and a K(d) of 1 nM. In addition, we have studied the G protein system that couples the PST receptor by gamma-(35)S-GTP binding studies. We have found that two G protein systems are involved, pertussis toxin-sensitive and -insensitive respectively. Specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Galpha(q/11) and to a lesser extent Galpha(i1,2) are activated by PST in rat adipocyte membranes. On the other hand, adenylate cyclase activity was not affected by PST. Finally, we have studied the specific phospholipase C isoform that is activated in response to PST. We have found that PST receptor is coupled to PLC-beta(3) via Galpha(q/11) activation in adipocyte membranes.

Effect of pancreastatin on cerulein-stimulated pancreatic blood flow and exocrine secretion in anaesthetized rats.

BACKGROUND: Pancreastatin (PST) is an inhibitor of pancreatic exocrine secretion in vivo but not in vitro, which suggests that the inhibitory effect of PST is indirect, that is, not mediated by a specific receptor on pancreatic acinar cells. In this study, we investigated the effects of PST on pancreatic exocrine secretion and local pancreatic blood flow in anaesthetized rats to elucidate the participation of PST in indirect regulation of pancreatic exocrine function through blood supply. METHODS: Pancreastatin (100, 200 or 500 pmol/kg per h) was administered intravenously under background infusion of cerulein (0.5 microg/kg per h), a cholecystokinin analogue. Pancreatic exocrine secretion was monitored by volume and protein output of the pancreatic juice and local pancreatic blood flow was measured by the hydrogen gas clearance method. RESULTS: Pancreastatin significantly reduced cerulein-induced local pancreatic blood flow in a dose-dependent manner. Pancreatic exocrine secretion was also reduced significantly by PST dose-dependently. Pancreastatin did not change systemic blood pressure. These results suggested that the reduction of pancreatic blood flow is associated with the reduction of pancreatic exocrine secretion. CONCLUSIONS: We conclude that the mechanism of PST-induced inhibition of pancreatic exocrine secretion is, at least, partly mediated by the reduction of local pancreatic blood flow through blockade, caused by the action of cerulein on pancreatic blood flow.

Interaction of islet hormones with cholecystokinin octapeptide-evoked secretory responses in the isolated pancreas of normal and diabetic rats.

This study investigates the effects of the islet hormones, insulin (Ins), glucagon (Glu) and somatostatin (Som) with cholecystokinin octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca2+]i and their pattern of distribution in the isolated pancreas of normal and diabetic rats. Ins and Glu evoked small increases in amylase output from pancreatic segments compared with a much enhanced effect of CCK-8. In contrast, Som induced a biphasic response comprising an initial decrease followed by a secondary increase and this biphasic response may be dependent upon the concentration. Combining the islet hormones with CCK-8 resulted in marked potentiation in amylase output compared with either CCK-8 alone or the individual hormone. Genistein and tyrphostin A25, the tyrosine kinase inhibitors, evoked a small decrease in amylase output from pancreatic segments. They had no effect on the CCK-8-evoked secretory response but markedly inhibited the potentiation of the islet hormones with CCK-8. In pancreatic acini and acinar cells Ins, Glu and Som individually evoked small increases in amylase output compared with a much larger response with CCK-8. When the islet hormones were combined with CCK-8 there was no potentiation of amylase output. Similarly, when rats were rendered diabetic by prior treatment with streptozotocin Ins, Glu and Som failed to potentiate the secretory response of CCK-8. In fura-2-loaded pancreatic acinar cells Ins or Glu evoked small increases in [Ca2+]i compared with a much larger elevation with CCK-8. Ins, Glu and Som each enhanced the CCK-8-evoked [Ca2+]i. Genistein elicited a decrease in [Ca2+]i both in the absence and presence of the islet hormones. It also decreased the elevation in [Ca2+]i resulting from the combined presence of CCK-8 with either Ins or Glu but it had no effect on CCK-8 in combination with Som. In pancreatic acinar cells from diabetic rat Ins, Glu and Som had no detectable effect on CCK-8-evoked elevation in [Ca2+]i compared with the response obtained with CCK-8 alone. CCK-8-immunopositive cells were distributed around the walls of blood vessels, numerous Ins-positive cells in the central and peripheral parts of the islets of Langerhans, Glu-immunoreactive cells in the periphery of islets and Som-positive cells in the outer part of the islets. During diabetes, the number of CCK-immunopositive cells remained unchanged whereas the number of Ins-positive cells decreased coupled with an increase in the number of Glu-positive cells. The results indicate that both tyrosine kinase and cellular Ca2+ seem to be the intracellular mediators involved with the enhanced secretory responses obtained with a combination of the islet hormones with CCK-8. Moreover, the presence of viable pancreatic islets of Langerhans seems to be associated with the potentiation of the islet hormones with CCK-8.

Modulation of insulin receptor signalling by pancreastatin in HTC hepatoma cells.

Pancreastatin, a neuropeptide derived from chromogranin A, has a glycogenolytic and counterregulatory effect to insulin in the rat liver. This effect is mediated by calcium and protein kinase C activity. Our aim was to study the possible cross-talk between pancreastatin and the insulin signalling system, by using the well-studied insulin sensitive rat hepatoma HTC cells. First, we checked the counterregulatory effect of pancreastatin on insulin action. Pancreastatin dose-dependently inhibited insulin stimulated glycogen synthesis. This effect was not due to competition for insulin receptors. Moreover, when protein kinase C activation was blocked with staurosporine, this effect of pancreastatin was not observed. Next, we found a dose-dependent inhibition of insulin receptor autophosphorylation by pancreastatin. In addition, phosphorylation of the major substrates of insulin receptor in HTC, i. e. insulin-receptor substrate (IRS)-1/IRS-2 and p62 was also blunted and so was its association with p85 regulatory subunit of phosphatidylinositol-3-kinase. Moreover, the insulin activation of S6 kinase was also blocked by pancreastatin. Again, all these inhibitory effects of pancreastatin were prevented by staurosporine. Furthermore, pancreastatin produced Ser/Thr phosphorylation of insulin receptor by a staurosporine-sensitive mechanism. Finally, we checked the pancreastatin activation of protein kinase C in HTC cells and found that a "classical" isoform of this protein is translocated to the plasma membrane. These findings suggest that pancreastatin could exert its anti-insulin effect in the hepatocyte by interrupting the stimulation of early insulin receptor signalling as a result of phosphorylation. This interaction might have a role in the mechanisms of insulin resistance.

Pancreastatin inhibits insulin action in rat adipocytes.

Pancreastatin (PST), a regulatory peptide with a general inhibitory effect on secretion, is derived from chromogranin A, a glycoprotein present throughout the neuroendocrine system. We have previously demonstrated the counterregulatory role of PST on insulin action in rat hepatocytes. Here, we are reporting the PST effects on rat adipocytes. PST dose dependently inhibits basal and insulin-stimulated glucose transport, lactate production, and lipogenesis, impairing the main metabolic actions of insulin in adipocytes. These effects were observed in a wide range of insulin concentrations, leading to a shift to the right in the dose-response curve. Maximal effect was observed at 10 nM PST, and the IC50 value was approximately 1 nM. Moreover, PST has a lipolytic effect in rat adipocytes (ED50 0.1 nM), although it was completely inhibited by insulin. In contrast, PST dose dependently stimulated protein synthesis and enhanced insulin-stimulated protein synthesis. In summary, these data show the lipokinetic effect of PST and the inhibitory effect of PST on insulin metabolic action within a range of physiological concentrations. Therefore, these results give new pathophysiological basis for the association of PST with insulin resistance.

Effects of islet hormones on nerve-mediated and acetylcholine-evoked secretory responses in the isolated pancreas of normal and diabetic rats.

This study employs the pancreas of normal and diabetic rats to investigate the relationship between the endocrine and exocrine pancreas in the control of exocrine secretion employing enzyme and immunohistochemical and physiological techniques. Acetylcholine esterase (ACh-E) positive nerves were distributed in the interacinar regions of the pancreas lying close to the exocrine cells. There was no difference between the cholinergic innervation of the pancreas in normal and diabetic rat. Insulin (INS) immunopositive cells were observed in the peripheral and central portions of the Islet of Langerhans in the pancreas of normal rat. In the diabetic animals the number of INS-positive cells were decreased. In contrast, glucagon (GLU) and somatostatin (SOM)-immunopositive cells were identified mainly in the peripheral parts of the Islets of Langerhans and their numbers increased markedly in the diabetic pancreas. Insulin alone had no significant effect on amylase secretion in the normal pancreas whereas GLU and SOM evoked small increases in amylase out compared to basal. In contrast, the islet hormones have no detectable secretory effect on the diabetic pancreas compared to control. Both electrical field stimulation (EFS) of intrinsic secretomotor nerves and exogenous application of acetylcholine (ACh) resulted in marked increases in amylase secretion. In pancreatic acini and acinar cells ACh evoked dose-dependent increases in amylase release. In normal pancreatic segments a combination of either INS or GLU with EFS or ACh resulted in marked potentiation of amylase output. In contrast, SOM inhibited the EFS-evoked amylase output but enhanced the secretory response to ACh. In pancreatic acini and acinar cells from normal rat and in pancreatic segments from diabetic rats, the islet hormones had no potentiating effect on the ACh-evoked secretory response. Similarly, in the diabetic rat the islet hormone had no effect on EFS-evoked amylase output. In fura-2 loaded pancreatic acinar cells ACh-induced a marked increase in intracellular free calcium concentration [Ca2+]i compared to basal. Either INS or GLU, but not SOM, elicited a small increase in [Ca2+]i. Combining either INS or GLU with ACh resulted in a potentiation of [Ca2+]i compared with ACh alone. In contrast, SOM had no significant effect on the ACh-induced [Ca2+]i compared to the response obtained with ACh alone. In pancreatic acinar cells of diabetic rat ACh-elicited similar magnitude of [Ca2+]i compared to acinar cells of normal rat. However, when the islet hormones were combined with ACh there was no enhancement of [Ca2+]i compared to ACh alone. The results indicate that the potentiation of either EFS or ACh-evoked secretory responses by the islet hormones seem to occur only in pancreatic segments which have intact viable Islets of Langerhans and not in either acini and acinar cells or from the pancreas of diabetic rat. Moreover, it is apparent that cellular Ca2+ is involved with the interaction of ACh with either INS or GLU.

Pancreastatin activates beta3 isoform of phospholipase C via G(alpha)11 protein stimulation in rat liver membranes.

Pancreastatin (PST) receptors have been recently shown to mediate activation of phospholipase C (PLC) in rat liver membranes. There is evidence that the G protein that links pancreastatin receptor with PLC-beta is pertussis toxin-insensitive and belongs to the G(alpha)q family. Here, we have employed blocking antisera to sort out the specific PLC-beta isoform as well as the specific G(alpha) subunit activated by PST receptor in rat liver membranes. The presence of different PLC-beta isoforms was checked by immunoblot analysis. Only PLC-beta4 was not detected, whereas PLC-beta1, beta2 and beta3 were abundant in rat liver membranes. However, only anti-PLC-beta3 serum was able to block the PST receptor response. We also checked the expression of G(alpha)q and Galpha11 in rat liver membranes by immunoblot. Even though both isoforms were present. only anti-Galpha11 serum was able to block the PST receptor response. In order to check the specificity of the blocking antisera, we employed them to block the effect of ADP and thrombin stimulating PLC activity in platelet membranes, a system lacking Galpha11. Anti-G(alpha)q but not anti-Galpha11 sera were able to block the agonist stimulated PLC activity. These data suggest that PST receptor response is mediated by the activation of the beta3 isoform of PLC via Galpha11 protein stimulation in rat liver membranes.