Release of PYY from pig intestinal mucosa; luminal and neural regulation

The localization, molecular nature and secretion of Peptide YY (PYY), a putative gut hormone belonging to the Pancreatic Polypeptide family of peptides, was studied in pigs. Immunoreactive PYY was identified in a population of endocrine cells in the mucosal epithelium of the pig ileum. Release of PYY was observed in isolated perfused pig ileum in response to luminal stimulation with glucose and vascular administration of the neuropeptide gastrin-releasing peptide (GRP). Electrical stimulation of the vagus nerve supply to the distal small intestine in intact anaesthetized pigs resulted in release of PYY into the circulation. Stimulation of the splanchnic nerves did not affect the basal release of PYY. PYY-immunoreactivity extracted from ileal tissue or released to plasma or perfusate from the ileum was indistinguishable from synthetic porcine PYY by gel filtration and reverse phase HPLC. It is concluded that the secretion of PYY in the pig ileum may be regulated not only by nutritional luminal factors, but also by postsynaptic parasympathetic nerves.     

Meal-induced peptide tyrosine tyrosine inhibition of pancreatic secretion in the rat

In the present studies we examined the distribution, release, and biological actions of peptide tyrosine tyrosine (PYY) in the rat. The concentration and distribution of PYY was highest in the ileum and colon as determined by both radioimmunoassay of rat tissue extracts and immunocytochemistry. An ultrastructural comparison of rat and dog colonic PYY cells revealed a bipolar distribution of peptide-containing secretory granules in both species. Serum PYY and pancreatic exocrine secretory responses were monitored after presentation of a meal to meal-trained rats (n = 12). A significant increase in PYY concentrations was not observed until 120 min after meal presentation, a delayed response similar to that previously observed in the dog. PYY responses were also observed in rats after perfusion of the intestine at the level of the duodenum and ileum with an 80 mOsm micellar solution of sodium oleate. Duodenal instillations of the fatty acids resulted in a maximum PYY response after 120 min, whereas rats subject to ileal perfusion of fat exhibited maximum PYY release within the first hour. In other experiments, infusion of exogenous PYY at 100 pmol.kg-1.h-1, which reproduced plasma PYY levels observed after a meal and perfusion of the gut with fat, significantly inhibited CCK-stimulated bile pancreatic volume (P less than 0.02), protein (P less than 0.01), and amylase (P less than 0.01) output. These studies demonstrate a bipolar distribution of PYY-containing secretory granules in cells of the jejunal, ileal, and colonic mucosa, and show that PYY is released in response to a meal in amounts sufficient to inhibit cholecystokinin-stimulated pancreatic secretion. Evidence is presented that PYY may mediate the delayed inhibition of pancreatic secretion that is observed in the rat after ingestion of a meal.     

Peptide YY induces nerve-mediated responses in the guinea pig intestine.

The actions of peptide YY (PYY) were studied in longitudinal organ-bath preparations of the guinea pig intestine. PYY induced concentration-dependent (10(-9)-5 x 10(-8) M) relaxations of tissue from the duodenum, jejunum, ileum, and colon. These responses were unaffected by adrenergic blockade and atropine treatment but could be prevented by tetrodotoxin. The pharmacology of PYY actions in segments of the small and large intestine indicated the involvement of intrinsic nonadrenergic, noncholinergic inhibitory neurones in the relaxation response to this peptide. All tissues could be made tachyphylactic to PYY without affecting their ability to respond to the direct acting muscle relaxants ATP or papaverine. Moreover, nicotinic ganglion stimulated relaxations and cholinergic nerve-mediated contractions were also unaffected. These results show applied PYY to have potent neurogenic actions in the guinea pig intestine with some similarities to PYY actions in the rat intestine.     

A new endogenous form of PYY isolated from canine ileum: Gly-extended PYY(1-36)

We purified and identified the peptide YY (PYY) forms present and determined their levels from a portion of the canine ileum directly adjacent to the cecum by a new extraction method designed to prevent and evaluate degradation of endogenous peptides. We used three reverse phase chromatography steps with radioimmunoassay of fractions for PYY-like-immunoreactivity (PYY-LI). The purified fractions underwent intact protein/peptide mass spectrometry identification and sequencing (i.e. "top-down" MS analysis). This analysis confirmed the identity of a new form of PYY, PYY(1-36)-Gly, which co-elutes with PYY(1-36)-NH(2) through all three of separation steps used. The PYY(1-36)-Gly form represents approximately 20% of the total PYY found in this region of the canine intestine. In addition, we also found that the PYY(3-36)-NH(2) form represents 6% of the total PYY in the canine ileo-cecal junction. The physiological implication of the Gly-extended form of PYY(1-36) warrants further investigation.     

PYY immunoneutralization does not alter lipid-induced inhibition of gastric emptying in rats

PYY is released from the distal ileum by fat and may be involved in mediating lipid-induced inhibition of gastric acid secretion and intestinal motility. The role of PYY in intestinal lipid-induced inhibition of gastric emptying in awake rats was investigated using a specific polyclonal antibody raised against PYY. METHODS: Gastric emptying of liquids was measured in awake rats fitted with a Thomas gastric cannula. Intralipid (total dose 50 or 100 mg) was perfused for 10 min (0.05 ml/min) into a duodenal (n = 11) or mid-intestinal cannula (60 cm from Ligament of Treitz; n = 8), and gastric emptying was measured over the 5-10 min period. Gastric emptying was measured 15 min after IP injection of PYY (1 nmol/rat). PYY antibody (20 mg) or a control antibody (anti-KLH; keyhole limpet hemocyanin) was injected ip 8-12 h before experiments. RESULTS: Exogenous PYY (1 nmol) inhibited gastric emptying and administration of PYY antibody blocked this response. Perfusion of lipid (50 and 100 mg) into the proximal intestine produced a 46% and 66% inhibition of gastric emptying respectively. Inhibition of gastric emptying in response to 50 mg lipid in the proximal small intestine was unaffected by administration of PYY antibody but was abolished by administration of the CCK A receptor antagonist devazepide (0.1 mg/kg ip). Perfusion of lipid into the distal intestine (50 and 100 mg) inhibited gastric emptying by 10% and 32% respectively. Inhibition of gastric emptying in response to 100 mg lipid in the distal intestine was unaffected by PYY antibody. CONCLUSIONS: Lipid perfused into either the proximal or distal intestine inhibits gastric emptying via a PYY-independent mechanism. CCK is involved in proximal lipid induced inhibition of gastric emptying.     

Release of distal gut peptide YY (PYY) by fat in proximal gut depends on CCK

We tested the hypothesis that the release of PYY by fat confined to the proximal small intestine is dependent on CCK. Using a multi-fistulated model, plasma PYY levels were compared in 6 dogs after 60 mM oleate was perfused into the proximal one-half of the small intestine following i.v. administration of saline or devazepide, a CCK-A antagonist. Plasma PYY increased with fat (P < 0. 05), but plasma PYY level was lower following devazepide at 60 min and 90 min (P < 0.05). We conclude that CCK serves as a foregut signal linking fat in the proximal gut with the release of distal gut PYY.     

The role of PYY in feeding regulation

Peptide YY (PYY), a 36-amino-acid peptide, is secreted primarily from L-cells residing in the intestinal mucosa of the ileum and large intestine. PYY, which belongs to a family of peptides including neuropeptide Y (NPY) and pancreatic polypeptide, is released into the circulation as PYY(1-36) and PYY(3-36); the latter is the major form of PYY in gut mucosal endocrine cells and throughout the circulation. Plasma PYY levels begin to rise within 15 min after starting to eat and plateau within approximately 90 min, remaining elevated for up to 6 h. Exogenous administration of PYY(3-36) reduces energy intake and body weight in both humans and animals. Via Y2 receptors, the satiety signal mediated by PYY inhibits NPY neurons and activates pro-opiomelanocortin neurons within the hypothalamic arcuate nucleus. Peripheral PYY(3-36) binds Y2 receptors on vagal afferent terminals to transmit the satiety signal to the brain. PYY(3-36) may have therapeutic potential in human obesity.     

GLP-1 and GIP are colocalized in a subset of endocrine cells in the small intestine

BACKGROUND: The incretin hormones GIP and GLP-1 are thought to be produced in separate endocrine cells located in the proximal and distal ends of the mammalian small intestine, respectively. METHODS AND RESULTS: Using double immunohistochemistry and in situ hybridization, we found that GLP-1 was colocalized with either GIP or PYY in endocrine cells of the porcine, rat, and human small intestines, whereas GIP and PYY were rarely colocalized. Thus, of all the cells staining positively for either GLP-1, GIP, or both, 55-75% were GLP-1 and GIP double-stained in the mid-small intestine. Concentrations of extractable GIP and PYY were highest in the midjejunum [154 (95-167) and 141 (67-158) pmol/g, median and range, respectively], whereas GLP-1 concentrations were highest in the ileum [92 (80-207) pmol/l], but GLP-1, GIP, and PYY immunoreactive cells were found throughout the porcine small intestine. CONCLUSIONS: Our results provide a morphological basis to suggest simultaneous, rather than sequential, secretion of these hormones by postprandial luminal stimulation.     

Cellular localization, half-life, and secretion of peptide YY

Tissue and plasma concentration of peptide YY (PYY) were measured by means of a radioimmunoassay (RIA) developed in our laboratory, using a specific PYY antiserum generated in New Zealand white rabbits against synthetic PYY, and dextran-coated charcoal to terminate the assay. Cellular localization of PYY was studied immunohistochemically using the peroxidase-antiperoxidase (PAP) technique. The highest tissue concentration of PYY was found in the mucosa of the terminal ileum and colon. PYY-containing secretory granules were primarily found in the basal pole of open-type endocrine cells. Basal plasma concentration of PYY was 70 +/- 9 pg/ml and rose to 357 +/- 30 pg/ml during the IV administration of PYY at 400 pmol/kg-h. A significant correlation was found (r = 0.94, p less than 0.05) between dose of PYY (12.5, 25, 50, 100, 200, 400 pmol/kg-h, IV) and plasma concentration of PYY. The calculated half-life of PYY in plasma was 8.3 +/- 1.9 minutes. Plasma concentration of PYY during the intraduodenal administration of sodium oleate (150 +/- 20 pg/ml) or long-chain triglyceride (187 +/- 37 pg/ml) was similar to plasma concentration of PYY obtained during the IV administration of PYY at 100 pmol/kg-h. Plasma concentration of PYY raised (126 +/- 10 pg/ml) after the administration of bombesin (400 pmol/kg-h, IV). Bile enhanced release of PYY. The present study suggests a hormonal role for PYY.     

In vitro effects of Clostridium perfringens type D epsilon toxin on water and ion transport in ovine and caprine intestine

Clostridium perfringens type D produces enterotoxaemia in sheep, goats and other animals. The disease is caused by C. perfringens epsilon toxin, and while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by sheep than goat intestine. In an attempt to clarify this, we examined the in vitro effects of epsilon toxin on sheep and goat intestine. Pieces of intestinal mucosa from recently slaughtered animals were mounted in a modified Ussing-type chamber where net water flux (J(w)), short-circuit current (I(sc)) and tissue conductance (G(t)) were simultaneously recorded. After 70 min of incubation with epsilon toxin a reduction in absorptive J(w) and an increase in I(sc) and G(t) were observed in colonic tissues of both sheep and goats, but no alterations were registered in the ileum of either species. These in vitro results show that epsilon toxin affects the transport function of the colonic mucosa but it does not seem to produce any transport alteration in the ileum mucosa.     

Release of peptide YY by fat in the proximal but not distal gut depends on an atropine-sensitive cholinergic pathway

Peptide YY (PYY) is released when PYY cells in short term culture are exposed to fat suggesting that this peptide may be released by fat in the distal gut without neural stimulation. PYY is also released by fat in the proximal 1/2 of small intestine. To test the hypothesis that the release of PYY by fat in the proximal but not distal intestine may depend on an atropine-sensitive, cholinergic pathway, PYY levels were compared in four dogs equipped with duodenal and mid-intestinal fistulas when 60 mM oleate was perfused into either the proximal (between fistulas) or distal (beyond mid-intestinal fistula) 1/2 of gut at 2 ml/min for 120 min with intravenous administration of saline or atropine. We found that, when fat was confined to the proximal 1/2 of the intestine, PYY release was reduced following intravenous atropine when compared with saline (p<0.01). However, when fat was confined to the distal 1/2 of the intestine, PYY release was not affected by the intravenous atropine. We conclude that PYY release by fat in the proximal but not distal intestine depends on an atropine-sensitive, cholinergic pathway.     

Distribution of peptide YY in the gastrointestinal tract of the rat, dog, and monkey

The objective of this study was to characterize the distribution and concentration of peptide YY (PYY) in the gastrointestinal tract of the rat, dog, and monkey. In the rat, the greatest concentration of PYY was detected in the ileum and colon. The concentrations of PYY in the ileum and colon were 72 +/- 49 and 768 +/- 180 ng/g tissue, respectively. In the dog, PYY was found primarily in extracts of the mucosal layer of the ileum and colon, with smaller amounts in the distal jejunum. The concentration of PYY in the mucosal layers of the canine distal jejunum was 113 +/- 25 ng/g tissue, proximal jejunum 302 +/- 56, mid jejunum 507 +/- 151, distal ileum 691 +/- 184, and colon 1706 +/- 774 ng/g tissue. In the monkey gastrointestinal tract, PYY was detected predominantly in mucosal extracts of the jejunum, ileum, and colon. The concentration of PYY in the mucosal layer extract of the jejunum was 92 +/- 23, ileum 615 +/- 127, and colon 1013 +/- 243 ng/g tissue.     

Multiple regulation of peptide YY secretion in the digestive tract

In the last two decades, multiple aspects of the peptide YY (PYY) secretion have been investigated. Besides fat and fatty acids, many luminal nutrients in the distal intestine appear to induce PYY release. Some studies have shown that bile acid, but not nutrients, plays a crucial role in the regulation of PYY secretion. Moreover, chyme in the proximal intestine also regulates the peptide release by indirect action through humoral and neuronal factors. Gastrin, cholecystokinin, and the vagus nerve are major candidates for mediators of these indirect actions. Several growth factors have been shown to regulate PYY synthesis in mucosa of the distal intestine. This review is aimed at presenting an overview of these recent studies on PYY secretion in the distal intestine.     

Catheterization of intestinal loops in ruminants does not adversely affect loop function

Catheterized intestinal loops may be a valuable model to elucidate key components of the host response to various treatments within the small intestine of ruminants. We examined whether catheterizing ileal loops in sheep affected the overall health of animals and intestinal function, whether a bacterial treatment could be introduced into the loops through the catheters, and whether broad-spectrum antibiotics could sterilize the loops. Escherichia coli cells transformed to express the GFP gene were introduced readily into the loops through the catheters, and GFP E. coli cells were localized within the injected loops. Catheterized loops, interspaces, and intact ileum exhibited no abnormalities in tissue appearance or electrical resistance. Expression of the IFNγ, IL1α, IL4, IL6, IL12p40, IL18, TGFβ1, and TNFα cytokine genes did not differ significantly among the intact ileum, catheterized loops, and interspaces, nor did the expression of the gene for inducible nitric oxide synthase. Broad-spectrum antibiotics administered during surgery did not sterilize the loops or interspaces and did not substantively change the composition of the microbiota. However, antibiotics reduced the overall number of bacterial cells within the loop and the relative abundance of community constituents. We concluded that catheterization of intestinal loops did not adversely affect health or loop function in sheep. Furthermore, allowing animals to recover fully from surgery and to clear pharmaceuticals will remove any confounding effects due to these factors, making catheterized intestinal loops a feasible model for studying host responses in ruminants.     

Distribution of enteric neural peptide YY in the dog gastrointestinal tract

Various regions of the dog gastrointestinal tract were investigated for the distribution of peptide YY (PYY) neurons using immunocytochemistry and radioimmunoassay. PYY neurons that encircled non-PYY-immunoreactive neurons were mainly observed in the myenteric plexus from the stomach to the colon. There was more PYY-like immunoreactivity in the muscle layer of the stomach and ileum than in the other intestines. The results of high performance liquid chromatography revealed that neural PYY-immunoreactive substance is identical to authentic PYY. PYY was not localized in the cholinergic neurons. These results indicate that PYY, as a neuropeptide, is involved in the regulation of gastrointestinal function.     

Effect of CCK-1 receptor blockade on ghrelin and PYY secretion in men

Cholecystokinin (CCK), peptide YY (PYY), and ghrelin have been proposed to act as satiety hormones. CCK and PYY are stimulated during meal intake by the presence of nutrients in the small intestine, especially fat, whereas ghrelin is inhibited by eating. The sequence of events (fat intake followed by fat hydrolysis and CCK release) suggests that this process is crucial for triggering the effects. The aim of this study was therefore to investigate whether CCK mediated the effect of intraduodenal (ID) fat on ghrelin secretion and PYY release via CCK-1 receptors. Thirty-six male volunteers were studied in three consecutive, randomized, double-blind, cross-over studies: 1) 12 subjects received an ID fat infusion with or without 120 mg orlistat, an irreversible inhibitor of gastrointestinal lipases, compared with vehicle; 2) 12 subjects received ID long-chain fatty acids (LCF), ID medium-chain fatty acids (MCF), or ID vehicle; and 3) 12 subjects received ID LCF with and without the CCK-1 receptor antagonist dexloxiglumide (Dexlox) or ID vehicle plus intravenous saline (placebo). ID infusions were given for 180 min. The effects of these treatments on ghrelin concentrations and PYY release were quantified. Plasma hormone concentrations were measured in regular intervals by specific RIA systems. We found the following results. 1) ID fat induced a significant inhibition in ghrelin levels (P < 0.01) and a significant increase in PYY concentrations (P < 0.004). Inhibition of fat hydrolysis by orlistat abolished both effects. 2) LCF significantly inhibited ghrelin levels (P < 0.02) and stimulated PYY release (P < 0.008), whereas MCF were ineffective compared with controls. 3) Dexlox administration abolished the effect of LCF on ghrelin and on PYY. ID fat or LCF significantly stimulated plasma CCK (P < 0.006 and P < 0.004) compared with saline. MCF did not stimulate plasma CCK release. In summary, fat hydrolysis is essential to induce effects on ghrelin and PYY through the generation of LCF, whereas MCF are ineffective. Furthermore, LCF stimulated plasma CCK release, suggesting that peripheral CCK is the mediator of these actions. The CCK-1 receptor antagonist Dexlox abolished the effect of ID LCF, on both ghrelin and PYY. Generation of LCF through hydrolysis of fat is a critical step for fat-induced inhibition of ghrelin and stimulation of PYY in humans; the signal is mediated via CCK release and CCK-1 receptors.     

Effects of peptide YY on intestinal blood flow distribution and motility in the dog

Previous investigators in our laboratory have demonstrated that peptide YY (PYY), a putative gut hormone, exerts a potent emetic effect when administered intravenously to conscious dogs. The current study was carried out to examine the effects of an emetic dose of PYY on cardiovascular status, splanchnic blood flow distribution (estimated using 15 micron microspheres) and small intestinal motility in anesthetized dogs. PYY, infused i.v. at a dose of 25 pmol/kg/min led to a localized significant reduction in small intestinal muscularis externa blood flow both 15 and 30 min after the start of PYY infusion in both jejunum and ileum. This decreased muscularis perfusion was not accompanied by any significant change in whole gut wall blood flow and was explained on the basis of an observed significant redistribution of blood flow away from the muscularis towards the mucosa/submucosa. Similar, although non-significant, effects of PYY on colonic blood flow distribution were also observed. Despite the effects on jejunum and ileum, PYY exerted minimal effects on duodenal blood flow. The decrease in ileal and jejunal muscularis blood flows was accompanied by a significant increase in the amplitude of intestinal contractions in these regions. Frequency of contractions was unaltered however. These results demonstrate that PYY infusion leads to concurrent changes in small intestinal blood flow and motility.     

Cholecystokinin and peptide YY are released by fat in either proximal or distal small intestine in dogs

We tested the hypothesis that the release of cholecystokinin (CCK) and peptide YY (PYY) may be independent of the region of the small intestine exposed to fat. In five dogs equipped with duodenal and midgut fistulas, the small intestine was compartmentalized so that fat was confined to either the proximal or distal one-half of the gut. Plasma CCK and PYY levels were measured by radioimmunoassay and compared by the square root of the area under the curve (sqrt AUC), representing the plasma peptide concentration over time. CCK was released similarly whether fat was delivered into the proximal (69.9+/-4.7 pM) or distal (71.0+/-5.5 pM) gut, but significantly more CCK (88.9+/-5.6 pM; p<0.05) was released when both the proximal and distal gut were perfused simultaneously with fat. PYY was released similarly whether fat was delivered into the proximal (34.9+/-2.6 pM) or distal (40.0+/-1.2 pM) gut or both (38.6+/-2.2 pM). We conclude that CCK and PYY are released by fat in either the proximal or distal one-half of the small intestine.     

Role of MGAT2 and DGAT1 in the release of gut peptides after triglyceride ingestion

Triglyceride ingestion releases gut peptides from enteroendocrine cells located in the intestinal epithelia and provides feedback regulations of gastrointestinal function. The precise mechanisms sensing lipids in the intestinal wall, however, are not well characterized. In the current study, we investigated the release of gut peptides following oral triglyceride loading in mice deficient for monoacylglycerol acyltransferase 2 (MGAT2KO) and diacylglycerol acyltransferase 1 (DGAT1KO), enzymes that sequentially re-synthesize triglyceride to secrete as chylomicron at the small intestine. In wild-type (Wt) mice, oral triglyceride loading resulted in hypertriglycemia. In addition, plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were significantly increased 30 min after triglyceride loading, before decaying in 2 h. In MGAT2KO and DGAT1KO mice, oral triglyceride loading did not result in hypertriglycemia and the increase in GIP was significantly suppressed in both KO mouse strains. In contrast, the increases in plasma GLP-1 and PYY in both KO mouse strains were comparable to Wt mice 30 min after triglyceride loading, however, they remained elevated in DGAT1KO mice even 2 h after triglyceride loading. In parallel to the changes in GLP-1 and PYY, gastric emptying was delayed after oral triglyceride loading in MGAT2KO mice comparably to Wt type mice and was further delayed in DGAT1KO mice. STC-1 and GLUTag, GLP-1-producing intestinal endocrine L-cell lines, displayed a significant level of DGAT1 activity but not MGAT activity. These findings suggest that synthesis and/or secretion of triglyceride-rich lipoproteins play an important role in the release of GIP. Moreover, DGAT1 may directly regulate the release of GLP-1 and PYY in L-cells.