The lymphocyte activation gene (LAG) -3 is a member of the immunoglobulin super-family that is selectively transcribed in human activated T and NK cells. In this work, the possibility that LAG-3 expression by human CD4+ T cells was preferentially related to one or another phenotype of cytokine secretion was investigated. Surface LAG-3 expression correlated with IFN-gamma, but not IL-4, production in antigen-stimulated T cells and it was up-regulated by IL-12. Most activated CD4+ T cell clones with established Th1 or Th0 profiles of cytokine secretion expressed LAG-3 on their surface, whereas the great majority of Th2 clones showed neither surface LAG-3 nor LAG-3 mRNA expression. After activation, the majority of CD4+ T cell clones also released soluble LAG-3-related peptides, and such a release correlated positively with the production of IFN-gamma and inversely with the production of IL-4. Thus, LAG-3 expression by activated CD4+ human T cells appear to be preferentially associated with the differentiation/activation pathway leading to the production of IFN-gamma.

Introduction Vasoactive Intestinal Peptide (VIP) is a 28-amino acid neuropeptide that belongs to the secretin-glucagon superfamily of peptides and has 68% homology with PACAP. VIP is abundantly expressed in the central and peripheral nervous system and in the gastrointestinal tract, where it exercises several physiological functions. Previously, it has been reported that VIP regulates feeding behavior centrally in different species of vertebrates such as goldfishes, chicken, and rodents. Additional studies are necessary to analyze the role of endogenous VIP on the regulation of appetite/satiety together with feeding behavior, metabolic hormone release, body mass composition and energy balance. Aims To elucidate the physiological pathways by which VIP regulates appetite/satiety, feeding behavior, metabolic hormones and body mass composition. Methods VIP deficient (VIP −/−) and age-matched wild-type (WT) littermates were weekly monitored from 5 to 22 weeks of age using a whole body composition EchoMRI analyzer. Food intake and feeding behavior were analyzed using the BioDAQ automated monitoring system. Plasma levels of metabolic hormones including active-ghrelin, GLP-1, leptin, PYY, pancreatic polypeptide (PP), adiponectin, and insulin were measured in fasting as well as in postprandial conditions. Results The genetic lack of VIP led to a significant reduction of body weight and fat mass and to an increase of lean mass as the mice aged. Additionally, VIP−/− mice had a disrupted pattern of circadian feeding behavior resulting in an abolished regular nocturnal/diurnal feeding. These changes were associated with an altered secretion of adiponectin, GLP-1, leptin, PYY and insulin in VIP−/− mice. Our data demonstrates that endogenous VIP is involved in the control of appetite/satiety, feeding behavior, body mass composition and in the secretion of six different key regulatory metabolic hormones. Conclusions Our data show that endogenous VIP is involved in the control of appetite/satiety, feeding behavior, body mass composition and in the secretion of six key regulatory metabolic hormones. VIP plays a key role in the regulation of body weight and mass composition phenotype by significantly enhancing body weight and fat mass accumulation. Therefore, VIP signaling is critical for the modulation of appetite/satiety and body mass phenotype and is suggested to be a target for future treatment of obesity.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to regulate gastric acid secretion and intestinal motility. In the present study, the pattern of distribution of PACAP and PACAP type 1 receptor (PAC1) immunoreactivities were examined in the rat stomach and distal colon using a specific polyclonal antibody raised against rat/human PAC1. Western blot of the membrane preparations of NIH/3T3 cells transfected with the human PAC1 obtained by using rabbit polyclonal anti-PAC1 antibody showed a protein band with a molecular mass of approximately 50 kDa. NIH/3T3 cells transfected with the human PAC1 and incubated with the anti-PAC1 antibody displayed surface cell-type immunoreactivity, which was internalized following ligand exposure. In gastric or colonic longitudinal muscle/myenteric plexus (LMMP) whole mount preparations as well as cryostat sections, PACAP immunoreactivity was observed in cell bodies within the myenteric ganglia and nerve fibers in the muscle layers and mucosa. PAC1 immunoreactivity was confined mainly on the surface of the nerve cells. PACAP and PAC1 immunoreactivities showed a similar pattern of distribution in gastric and colonic tissues. Adjacent sections or LMMP whole mount preparations labeled with protein gene product 9.5 (PGP 9.5) revealed the neuronal identity of myenteric cells bearing PAC1. The neuronal localization of PACAP and PAC1 receptors supports their role in the neural regulation of gastric acid secretion and gastrointestinal motor function.

PM. PACAP intraperitoneal treatment suppresses appetite and food intake via PAC1 receptor in mice by inhibiting ghrelin and increasing GLP-1 and leptin. Am J Physiol Gastrointest Liver Physiol 309: G816 -G825, 2015. First published September 3, 2015 doi:10.1152/ajpgi.00190.2015.-Pituitary adenylate cyclase-activating peptide (PACAP) is expressed within the gastroenteric system, where it has profound physiological effects. PACAP was shown to regulate food intake and thermogenesis centrally; however, PACAP peripheral regulation of appetite and feeding behavior is unknown. Therefore, we studied PACAP's effect on appetite and food intake control by analyzing feeding behavior and metabolic hormones in PAC1-deficient (PAC1Ϫ/Ϫ) and age-matched wild-type (WT) mice intraperitoneally injected with PACAP 1-38 or PACAP 1-27 before the dark phase of feeding. Food intake and feeding behavior were analyzed using the BioDAQ system. Active ghrelin, glucagon-like peptide-1 (GLP-1), leptin, peptide YY, pancreatic polypeptide, and insulin were measured following PACAP 1-38 administration in fasted WT mice. PACAP 1-38/PACAP1-27 injected into WT mice significantly decreased in a dose-dependent manner cumulative food intake and reduced bout and meal feeding parameters. Conversely, PACAP 1-38 injected into PAC1Ϫ/Ϫ mice failed to significantly change food intake. Importantly, PACAP1-38 reduced plasma levels of active ghrelin compared with vehicle in WT mice. In PAC1Ϫ/Ϫ mice, fasting levels of active ghrelin, GLP-1, insulin, and leptin and postprandial levels of active ghrelin and insulin were significantly altered compared with levels in WT mice. Therefore, PAC1 is a novel regulator of appetite/satiety. PACAP 1-38/PACAP1-27 significantly reduced appetite and food intake through PAC1. In PAC1Ϫ/Ϫ mice, the regulation of anorexigenic/orexigenic hormones was abolished, whereas active ghrelin remained elevated even postprandially. PACAP significantly reduced active ghrelin in fasting conditions. These results establish a role for PACAP via PAC1 in the peripheral regulation of appetite/satiety and suggest future studies to explore a therapeutic use of PACAP or PAC1 agonists for obesity treatment.pituitary adenylate cyclase-activating peptide; PAC1 receptor; appetite; ghrelin; GLP-1; leptin PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) was originally isolated from the ovine hypothalamus and is highly conserved among vertebrates (1). PACAP, because of its protein structure, particularly in the first 27 amino acids, belongs to the secretin-glucagon superfamily of hormones, which includes vasoactive intestinal peptide (VIP), gastric inhibitory peptide, glucagon-like peptide (GLP)-1 and GLP-2, growth hormone-releasing hormone, peptide histidine methionine, peptide histidine isoleucine, and exendins (34). PACAP exists in two variant forms: PACAP 1-27 and the COOHterminally extended form PACAP 1-38 that originates from the same precursor and stimulates adenylate cyclase activity in pituitary cells (23,35). PACAP functions through its P...

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) is a heptahelical, G protein-coupled receptor that has been shown to be expressed by non-squamous lung cancer and breast cancer cell lines, and to be coupled to the growth of these tumors. We have previously shown that PACAP and its receptor, PAC1, are expressed in rat colonic tissue. In this study, we used polyclonal antibodies directed against the COOH terminal of PAC1, as well as fluorescently labeled PACAP, Fluor-PACAP, to demonstrate the expression of PAC1 on HCT8 human colonic tumor cells, using FACS analysis and confocal laser scanning microscopy. Similarly, anti-PACAP polyclonal antibodies were used to confirm the expression of PACAP hormone by this cell line. We then investigated the signal transduction properties of PAC1 in these tumor cells. PACAP-38 elevated intracellular cAMP levels in a dose-dependent manner, with a half-maximal (EC(50)) stimulation of approximately 3 nM. In addition, PACAP-38 stimulation caused an increase in cytosolic Ca(2+) concentration [Ca(2+)](i), which was partially inhibited by the PACAP antagonist, PACAP-(6-38). Finally, we studied the potential role of PACAP upon the growth of these tumor cells. We found that PACAP-38, but not VIP, increased the number of viable HCT8 cells, as measured by MTT activity. We also demonstrated that HCT8 cells expressed the Fas receptor (Fas-R/CD95), which was subsequently down-regulated upon activation with PACAP-38, further suggesting a possible role for PACAP in the growth and survival of these tumor cells. These data indicate that HCT8 human colon tumor cells express PAC1 and produce PACAP hormone. Furthermore, PAC1 activation is coupled to adenylate cyclase, increase cytosolic [Ca(2+)](i), and cellular proliferation. Therefore, PACAP is capable of increasing the number of viable cells and regulating Fas-R expression in a human colonic cancer cell line, suggesting that PACAP might play a role in the regulation of colon cancer growth and modulation of T lymphocyte anti-tumoral response via the Fas-R/Fas-L apoptotic pathway.

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) receptor is a G proteincoupled receptor and class II receptor member. The receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues (Tr406), 53 residues (Tr416), 49 residues (Tr420), 44 residues (Tr424), and 37 residues (Tr433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radioligand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. 125 It has been previously demonstrated for receptors belonging to the class I heptahelical receptor family that specific intracellular amino acid residues are critical for internalization and coupling to guanine nucleotide-binding proteins (G proteins). These cytoplasmic domains have been partially characterized for the ␣-and ␤-adrenergic receptors (1, 2), the muscarinic receptors (3), and for rhodopsin (4). Studies of the ␤ 2 -adrenergic (2) and muscarinic receptors (3) have suggested that substitution, deletion, and/or insertion of residues in the third intracellular loop affect the ability of these receptors to couple to G proteins.At present, the role of the cytoplasmic domains for members of the class II family for receptor coupling to intracellular G proteins has not been fully explored. Based on studies with receptors belonging to the class I receptor family, we speculated that amino acids in either the third intracellular loop or the C terminus of the pituitary adenylate cyclase-activating polypeptide receptor (PAC1), 1 may be important for coupling to G proteins (5). Furthermore, the C terminus has been proposed to be necessary for the formation of a functional receptor-G protein complex and to result in heterodimerization for some GPCRs such as type B receptor for GABA and GABA B -like orphan receptor (6).Pituitary adenylate cyclase-activating polypeptide (PACAP) hormone was originally isolated from bovine hypothalamus and is one of the most recently identified peptides in the VIP/ secretin/glucagon family (7). The cloning of the rat and human PAC1 cDNA and genes identified a receptor protein composed of ϳ468 amino acids containing seven transmembrane domains that are characteristic of G protein-coupled receptors (5, 8). The PAC1 belongs to the class II GPCR superfamily that includes VPAC1 (the classical VIP1 receptor), VPAC2 (VIP2 receptor), secretin, glucagon, and calcitonin receptors (9); these molecules can be distinguished on the basis of their relative affinities for the agonists PACAP-27, PACAP-38, VIP, and helodermin (10). The cloning of the rat and human PAC1 gene have identified two unique exons that can be alternatively spliced yielding different third intracellular loops. The four major splice variants are named Null, Hip, Hop,8), and they have been shown to be differentially expressed in different tissues and are able to couple with different efficacies and potencies to phospholipase C (PLC) and adenylyl cyclase (AC) (5,8). Therefore, the expression of splice varian...

VIP is highly expressed in the colon and regulates motility, vasodilatation, and sphincter relaxation. However, its role in the development and progress of colitis is still controversial. Our aim was to determine the participation of VIP on dextran sodium sulfate (DSS)-induced colonic mucosal inflammation using VIP−/− and WT mice treated with VIP antagonists. Colitis was induced in 32 adult VIP−/− and 14 age-matched WT litter-mates by giving 2.5 % DSS in the drinking water. DSS-treated WT mice were injected daily with VIP antagonists, VIPHyb (n=22), PG 97–269 (n=9), or vehicle (n=31). After euthanasia, colons were examined; colonic cytokines mRNA were quantified. VIP−/− mice were remarkably resistant to DSS-induced colitis compared to WT. Similarly, DSS-treated WT mice injected with VIPHyb (1 μM) or PG 97–269 (1 nM) had significantly reduced clinical signs of colitis. Furthermore, colonic expression of IL-1, TNF-α, and IL-6 was significantly lower in VIP−/− and VIPHyb or PG 97–269 compared to vehicle-treated WT. Genetic deletion of VIP or pharmacological inhibition of VIP receptors resulted in resistance to colitis. These data demonstrate a pro-inflammatory role for VIP in murine colitis and suggest that VIP antagonists may be an effective clinical treatment for human inflammatory bowel diseases.

High-protein diets (HPDs) recently have been used to obtain body weight and fat mass loss and expand muscle mass. Several studies have documented that HPDs reduce appetite and food intake. Our goal was to determine the long-term effects of an HPD on body weight, energy intake and expenditure, and metabolic hormones. Male C57BL/6 mice (8 wk old) were fed either an HPD (60% of energy as protein) or a control diet (CD; 20% of energy as protein) for 12 wk. Body composition and food intakes were determined, and plasma hormone concentrations were measured in mice after being fed and after overnight feed deprivation at several time points. HPD mice had significantly lower body weight (in means ± SEMs; 25.73 ± 1.49 compared with 32.5 ± 1.31 g; = 0.003) and fat mass (9.55% ± 1.24% compared with 15.78% ± 2.07%; = 0.05) during the first 6 wk compared with CD mice, and higher lean mass throughout the study starting at week 2 (85.45% ± 2.25% compared with 75.29% ± 1.90%; = 0.0001). Energy intake, total energy expenditure, and respiratory quotient were significantly lower in HPD compared with CD mice as shown by cumulative energy intake and eating rate. Water vapor was significantly higher in HPD mice during both dark and light phases. In HPD mice, concentrations of leptin [feed-deprived: 41.31 ± 11.60 compared with 3041 ± 683 pg/mL ( = 0.0004); postprandial: 112.5 ± 102.0 compared with 8273 ± 1415 pg/mL ( < 0.0001)] and glucagon-like peptide 1 (GLP-1) [feed-deprived: 5.664 ± 1.44 compared with 21.31 ± 1.26 pg/mL ( = <0.0001); postprandial: 6.54 ± 2.13 compared with 50.62 ± 11.93 pg/mL ( = 0.0037)] were significantly lower, whereas postprandial glucagon concentrations were higher than in CD-fed mice. In male mice, the 12-wk HPD resulted in short-term body weight and fat mass loss, but throughout the study preserved body lean mass and significantly reduced energy intake and expenditure as well as leptin and GLP-1 concentrations while elevating postprandial glucagon concentrations. This study suggests that long-term use of HPDs may be an effective strategy to decrease energy intake and expenditure and to maintain body lean mass.