Development of monoclonal mouse antibodies that specifically recognize pancreatic polypeptide.

Pancreatic polypeptide (PP) is a 36-amino acid peptide encoded by the Ppy gene, which is produced by a small population of cells located in the periphery of the islets of Langerhans. Owing to the high amino acid sequence similarity among neuropeptide Y family members, antibodies against PP that are currently available are not convincingly specific to PP. Here we report the development of mouse monoclonal antibodies that specifically bind to PP. We generated Ppy knockout (Ppy-KO) mice in which the Ppy-coding region was replaced by Cre recombinase. The Ppy-KO mice were immunized with mouse PP peptide, and stable hybridoma cell lines producing anti-PP antibodies were isolated. Firstly, positive clones were selected in an enzyme-linked immunosorbent assay for reactivity with PP coupled to bovine serum albumin. During the screening, hybridoma clones producing antibodies that cross-react to the peptide YY (PYY) were excluded. In the second screening, hybridoma clones in which their culture media produce no signal in Ppy-KO islets but detect specific cells in the peripheral region of wild-type islets, were selected. Further studies demonstrated that the selected monoclonal antibody (23-2D3) specifically recognizes PP-producing cells, not only in mouse, but also in human and rat islets. The monoclonal antibodies with high binding specificity for PP developed in this study will be fundamental for future studies towards elucidating the expression profiles and the physiological roles of PP.

Oxidative grafting vs. monolayers self-assembling on gold surface for the preparation of electrochemical immunosensors. Application to the determination of peptide YY.

A comparison of the performance of two electrochemical immunosensors for the determination of the anorexigen biomarker peptide YY (PYY) is reported by using as scaffolds screen printed gold electrodes modified either by oxidative grafting of p-aminobenzoic acid (p-ABA) or by assembling of a 4-mercaptobenzoic acid (4-MBA) SAM. Covalent immobilization of capture antibodies on the surface-confined carboxyl groups was carried out by EDC/NHSS chemistry, and competitive immunoassays between target PYY and Biotin-PYY were implemented. Upon labeling with alkaline phosphatase (AP)-streptavidin conjugate and 1-naphtyl phosphate addition, differential pulse voltammograms recorded between -0.2 and +0.7 V were used as analytical readout. All the steps involved in the functionalization of the electrodes and the preparation of the immunosensors were monitored by electrochemical impedance spectroscopy. The calibration plot for PYY using the AP-Strept-Biotin-PYY(PYY)-anti-PYY-Phe-N-SPAuE immunosensor provided a linear current vs. log [PYY] plot extending between 10-6 and 103 ng/mL PYY with a detection limit of 3 × 10-7 ng/mL. These analytical characteristics are remarkably better than those obtained with the immunosensor prepared with 4-MBA SAM-SPAuEs. The AP-Strept-Biotin-PYY(PYY)-anti-PYY-Phe-N-SPAuE immunosensor was used to analyze human serum and saliva samples spiked with PYY at concentrations fitting with normal levels in these biological fluids.

Beta-glucans and cancer: The influence of inflammation and gut peptide.

Dietary ß-glucans are soluble fibers with potentially health-promoting effects. Gut peptides are important signals in the regulation of energy and glucose homeostasis. This article reviews the effects of different enriched ß-glucan food consumption on immune responses, inflammation, gut hormone and cancer. Gut hormones are influenced by enriched ß-glucan food consumption and levels of such peptide as YY, ghrelin, glucagon-like peptide 1 and 2 in humans influence serum glucose concentration as well as innate and adaptive immunity. Cancer cell development is also regulated by obesity and glucose dishomeostasy that are influenced by ß-glucan food consumption that in turn regulated gut hormones.

Cross-talk between innate cytokines and the pancreatic polypeptide family in acute pancreatitis.

BACKGROUND AND AIM: Low-grade inflammation persists in patients with acute pancreatitis (AP) after hospital discharge, and is linked to metabolic disorders. Neuropeptide Y (NPY) is well recognized as an important mediator of inflammation in these patients but the role of the other two structurally similar peptides, pancreatic polypeptide (PP) and peptide YY (PYY), in inflammation has been sparsely investigated. The aim was to investigate the association between PYY, PP, NPY and circulating levels of innate cytokines in patients after AP. METHODS: Fasting blood samples were collected to measure PYY (ng/mL), PP (ng/mL), NPY (pg/mL), interleukin-6 (IL-6) (ng/mL), monocyte chemoattractant protein (MCP) 1 (ng/mL), and tumour necrosis factor (TNF) α (ng/mL). Modified Poisson regression analysis and linear regression analyses were conducted. Age, sex, ethnicity, obesity, diabetes, aetiology, time from 1st attack of AP, recurrence, severity, physical activity, and smoking were adjusted for in several statistical models. P<0.05 was considered statistically significant. RESULTS: A total of 93 patients were recruited. Peptide YY was significantly associated (p<0.001) with IL-6, MCP-1, and TNFα in the unadjusted and all adjusted models. Pancreatic polypeptide was significantly associated (p<0.001) with IL-6, MCP-1, and TNFα in the unadjusted and at least one adjusted model. Peptide YY and PP together contributed 22.2%, 72.7%, and 34.6% to the variance of IL-6, MCP-1, and TNFα, respectively. Neuropeptide Y was not significantly associated with any of the three cytokines. CONCLUSIONS: Peptide YY and PP are associated with circulating innate pro-inflammatory cytokines in patients after AP and cumulatively contribute to nearly half of the variance of IL-6, MCP-1, and TNFα. Future research is warranted to investigate the signaling pathways that underlie these associations.

Peptide YY attenuates transcription factor activity in tumor necrosis factor-alpha-induced pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a disease characterized by inflammation. Nuclear factor (NF)-kappaB, Smad proteins, and the steroid hormone family peroxisome proliferator-activated receptors (PPARs) are involved in regulation of gene transcription during the disease process. Peptide YY (PYY), a gastrointestinal hormone, inhibits NF-kappaB translocation to acinar nuclei in tumor necrosis factor (TNF)-alpha-induced AP. We investigated TNF-alpha induction of Smad proteins, PPARalpha/gamma, and NF-kappaB by TNF-alpha, and hypothesized that PYY would attenuate this effect. STUDY DESIGN: Rat acinar cells were treated with recombinant TNF-alpha (200 ng/mL). PYY (3 to 36) was added at 500 pM at 30 minutes after TNF-alpha treatment until cell harvest at 2 hours. Western blot analysis and intracellular staining of the p65 subunit of NF-kappaB were performed. NF-kappaB, Smad3/4, and PPARalpha/gamma binding activities were determined by protein/DNA array analysis and verified by electrophoretic-mobility shift assay and densitometry. RESULTS: Cellular localization of NF-kappaB p65 showed nuclear staining within 2 hours, with controls stained in the cytoplasm. With PYY, p65 stained in the cytoplasm. Nuclear p65 was increased significantly (p < 0.05) by TNF-alpha at 2 hours and PYY reduced it. Array analysis revealed upregulation of NF-kappaB, PPARalpha/gamma, and Smad3/4 with TNF-alpha. TNF-alpha stimulated NF-kappaB activation sevenfold, and binding was enhanced (p < 0.05). PYY reduced NF-kappaB binding to control levels. PPAR binding increased 51% after TNF-alpha treatment and was reduced to 33% with PYY. Smad3/4 binding was increased (p < 0.05) above controls with TNF-alpha and PYY reduced it by 40%. CONCLUSIONS: TNF-alpha increases early nuclear translocation of the p65 subunit of NF-kappaB in acinar cells. Exposure to TNF-alpha activates transcription factors NF-kappaB, Smad3/4, and PPARalpha/gamma. PYY reduces this activation. Treatment with PYY may have therapeutic potential in improving AP.

Gastric protective effect of peripheral PYY through PYY preferring receptors in anesthetized rats.

The influence of intravenous peptide YY (PYY) on the gastric injury induced by 45% ethanol was investigated in urethane-anesthetized rats. PYY (25, 75, 125, and 250 pmol x kg(-1) x h(-1)) significantly reduced gastric lesions by 36, 59, 40, and 38%, respectively. Antibody against ratPYY (2 mg/rat) injected intravenously completely prevented the gastroprotective effect of intravenous PYY (75 pmol x kg(-1) x h(-1)), whereas injected intracisternally (460 microg/20 microl), it significantly prevented intracisternal PYY (24 pmol/rat)-induced 58% reduction of ethanol lesions but not that induced by intravenous PYY. Vagotomy did not influence the gastroprotective effect of intravenous PYY. The Y(1)/"PYY-preferring" receptor agonist [Pro(34)]PYY (75 pmol x kg(-1) x h(-1) iv) significantly decreased ethanol-induced gastric lesions by 82%, whereas [Leu(31), Pro(34)]NPY, a Y(1)/Y(3) agonist, and PYY-(3-36), a Y(2) agonist, had no effect. These data indicate that PYY-infused intravenously at doses reported to mimic postprandial peak blood levels prevents ethanol-induced gastric injury through vagal independent pathways and PYY-preferring receptors.

Morphological changes in pancreatic islets of KATP channel-deficient mice: the involvement of KATP channels in the survival of insulin cells and the maintenance of islet architecture.

The ATP-sensitive potassium channel (KATP channel) is an essential ion channel involved in glucose-induced insulin secretion. The KATP channel is composed of an inwardly rectifying potassium channel, Kir6.2, and the sulfonylurea receptor (SUR 1); in the pancreas it is reported to be shared by all endocrine cell types. A previous study by our research group showed that Kir 6.2-knockout mice lacked KATP channel activities and failed to secrete insulin in response to glucose, but displayed normal blood glucose levels and only mild impairment in glucose tolerance at younger ages. In some aged knockout mice, however, obesity and hyperglycemia were recognizable. The present study aimed to reveal morphological changes in pancreatic islets of Kir 6.2-knockout mice throughout life. At birth, there were no significant differences in the islet cell arrangement between the knockout mice and controls. At 14 postnatal weeks glucagon cells appeared in the central parts of islets, and this image became more pronounced with aging. In animals older than 50 weeks insulin cells decreased in numbers and intensity of insulin immunoreactivity; most islets in 70- and 80-week-old mice were predominantly composed of glucagon cells and peptide YY (PYY)-containing cells. Staining of serial sections and double staining of single sections from these old mice demonstrated the frequent coexpression of glucagon and PYY, which is a phenotype for the earliest progenitor cells of pancreatic endocrine cells. These findings suggest that the KATP channel is important for insulin cell survival and also regulates the differentiation of islet cells.

Increased intracellular content of enteroglucagon in proximal colon is related to intestinal adaptation to germ-free status.

Changes in the frequency of endocrine cells are evidence of intestinal adaptation to germ-free (GF) status. Not only the distribution of these cells along the intestine, but also the differences in intracellular content of these regulatory peptides may be explored to explain functional and structural aspects of GF intestinal adaptation. Focusing on the endocrine L-cells, we analyzed the intracellular content of enteroglucagon (EG) and peptide YY (PYY) throughout the intestine of the 14 GF and 14 conventional (CV) mice by using immunohistochemistry and the supra-optimal dilution technique. The percentage of EG-immunoreactive cells, but not of PYY-immunoreactive cells stained at supra-optimal dilution was significantly higher in the proximal colon of GF mice than in the CV counterparts (P < 0.05). Since the content of co-stored PYY did not differ between GF and CV mice, the higher content of EG was compatible with a selective cellular response. Moreover, in the cecum of GF mice, the density of EG-immunoreactive cells was significantly higher than that of PYY-immunoreactive cells (P < 0.05). These results are consistent with preferential production of EG by L-cells at the expense of PYY in the proximal colon and in the enlarged cecum of GF mice. In addition, they may reflect the dynamics of the GF intestinal epithelium and/or be correlated with the higher serum levels of these peptides. The role of endocrine cells needs to be better studied in human and other experimental adaptative conditions in order to elucidate the regulatory mechanisms of intestinal functions.

Peripheral PYY inhibits intracisternal TRH-induced gastric acid secretion by acting in the brain.

The site of action of peripheral peptide YY (PYY)-induced inhibition of vagally stimulated gastric acid secretion was studied using immunoneutralization with PYY antibody in urethan-anesthetized rats. Gastric acid secretion (59+/-7 micromol/90 min) stimulated by intracisternal injection of the stable thyrotropin-releasing hormone (TRH) analog RX-77368 (14 pmol/rat) was dose-dependently inhibited by 52%, 69%, and 83% by intravenous infusion of 0.25, 0.5, and 1.0 nmol. kg(-1) x h(-1) PYY, respectively. PYY or PYY(3-36) (2.4 pmol/rat) injected intracisternally also inhibited the acid response to intracisternal RX-77368 by 73% and 80%, respectively. Intravenous pretreatment with PYY antibody (4.5 mg/rat), which shows a 35% cross-reaction with PYY(3-36) by RIA, completely prevented the inhibitory effect of intravenously infused PYY (1 nmol x kg(-1) x h(-1)). When injected intracisternally, the PYY antibody (280 microg/rat) reversed intracisternal PYY (2.4 pmol)- and intravenous PYY (1 nmol x kg(-1) x h(-1))-induced inhibition of acid response to intracisternal RX-77368 by 64% and 93.5%, respectively. These results provide supporting evidence that peripheral PYY inhibits central vagal stimulation of gastric acid secretion through an action in the brain.

Co-localization of neuroendocrine hormones in the human fetal pancreas.

OBJECTIVE AND DESIGN: Co-localization of the four major pancreatic hormones, and also of islet amyloid polypeptide (IAPP), peptide tyrosine tyrosine (PYY), secretin and neurotensin, has been studied in the endocrine pancreas of human fetuses at 16, 18 and 22 weeks of gestation. METHODS: Double and triple immunofluorescence stainings have been used. RESULTS: All three fetal pancreata contained cells that showed insulin, glucagon, somatostatin, pancreatic polypeptide (PP), IAPP, secretin and PYY immunoreactivity. Neurotensin cells were found in the youngest fetus and gastric inhibitory polypeptide (GIP) in the two older fetuses. Co-localization of two hormones occurred in most of the endocrine cell types in the three fetuses examined, but three hormones occurred in only a few cells and especially in the youngest fetus. Somatostatin cells were the only cell type which was largely monohormonal. Our findings showed that there are two different co-localization patterns: insulin was co-localized mainly with IAPP and glucagon, while secretin and PYY occurred together with glucagon and PP. CONCLUSIONS: These data are the first to describe secretin and neurotensin in the fetal pancreas. Two different co-localization patterns could be distinguished: insulin, IAPP and glucagon, and glucagon, secretin, PP and PYY.

Intestinal fat-induced inhibition of meal-stimulated gastric acid secretion depends on CCK but not peptide YY.

Fat in small intestine decreases meal-stimulated gastric acid secretion and slows gastric emptying. CCK is a mediator of this inhibitory effect (an enterogastrone). Because intravenously administered peptide YY (PYY) inhibits acid secretion, endogenous PYY released by fat may also be an enterogastrone. Four dogs were equipped with gastric, duodenal, and midgut fistulas. PYY antibody (anti-PYY) at a dose of 0.5 mg/kg or CCK-A receptor antagonist (devazepide) at a dose of 0.1 mg/kg was administered alone or in combination 10 min before the proximal half of the gut was perfused with 60 mM oleate or buffer. Acid secretion and gastric emptying were measured. We found that 1) peptone-induced gastric acid secretion was inhibited by intestinal fat (P < 0.0001), 2) inhibition of acid secretion by intestinal fat was reversed by CCK-A receptor antagonist (P < 0.0001) but not by anti-PYY, and 3) slowing of gastric emptying by fat was reversed by CCK-A antagonist (P < 0. 05) but not by anti-PYY. We concluded that inhibition of peptone meal-induced gastric acid secretion and slowing of gastric emptying by intestinal fat depended on CCK but not on circulating PYY.

Distribution of enteroglucagon- and peptide YY-immunoreactive cells in the intestinal mucosa of germ-free and conventional mice.

There are evidences that microflora modulates endocrine cells in the gastrointestinal tract. In the present study we investigated the distribution of EG- and PYY-immunoreactive cells throughout the intestine of adult male NMRI conventional and germ-free mice. EG-immunoreactive cells were significantly more frequent in the proximal and middle colon than in the remainder of the intestine in both groups. In germ-free animals, these cells were more frequent in the cecum and less frequent in the distal ileum compared to conventional mice. PYY-immunoreactive cells were more frequent in the distal colon than in the remainder of the intestine in both groups, but they were significantly more frequent in the middle and distal colon of germ-free animals than in that of conventional counterparts. The number of EG-immunoreactive cells was 4.5-fold higher than the number of PYY-immunoreactive cells in the cecum of germ-free mice. The present results indicate the existence of an inverse gradient of EG- and PYY-immunoreactive cells along the colon, which is not significantly changed in the absence of a microflora. PYY production seems to be more significant in the distal colon. The cecum and the proximal portion of the colon are probably the regions of greatest functional importance for EG production, which is related to the microflora and probably to fermentation products, whether or not the effect of this peptide is trophic or antitrophic.

Neuropeptide Y and peptide YY immunoreactivities in the pancreas of various vertebrates.

NPY-like immunoreactivity was observed in nerve fibers and endocrine cells in pancreas of all species examined except the eel, which showed no NPY innervation. The density of NPY-positive nerve fibers was higher in mammals than in the lower vertebrates. These nerve fibers were distributed throughout the parenchyma, and were particularly associated with the pancreatic duct and vascular walls. In addition, the density of NPY-positive endocrine cells was found to be higher in lower vertebrates than mammals; in descending order: eel = turtle = chicken > bullfrog > mouse = rat = human > guinea pig = dog. These NPY-positive cells in the cel and certain mammals tended to be localized throughout the islet region, whereas in the turtle and chicken they were mainly scattered in the exocrine region. PYY-immunoreactivity was only present in the pancreatic endocrine cells of all species studied, and localized similarly to NPY. Thus these two peptides may play endocrine or paracrine roles in the regulation of islet hormone secretion in various vertebrate species.