Induction of glycogenolysis in cultured Ewing's sarcoma cells by dopamine and β-adrenergic agonists

Summary This study describes hormonal regulation of glycogen metabolism in Ewing's sarcoma cells. ³H-Glycogen synthesized in cultured Ewing's sarcoma WE-68 cells from ³H-glucose was hydrolyzed in a concentration-dependent manner by various catecholamines. The order of potency for the glycogenolytic effects of catecholamines was isoproterenol dopamine > norepinephrine > epinephrine. The concentrations giving half-maximal effectiveness (EC₅₀) were about 2×10^-8 M, 3×10^-8 M, 8×10^-8 M, and 5×10^-7 M for isoproterenol, dopamine, norepinephrine, and epinephrine, respectively. Higher concentrations of each of the catecholamines were necessary to elicit EC₅₀ stimulation of cyclic AMP production in Ewing's sarcoma cells. Glycogenolysis induced by dopamine was blocked by chlorpromazine, a dopamine D₁-receptor antagonist, but not by haloperidol, a dopamine D₂-receptor antagonist. The glycogenolytic action of norepinephrine was markedly reduced by propranolol, a -adrenoreceptor antagonist, and was not affected by yohimbine, an -adrenoreceptor antagonist. In addition, chlorpromazine also antagonized the glycogenolytic response to norepinephrine. Dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, and the diterpene forskolin were also found to induce ³H-glycogen hydrolysis. Our data indicate that cate-cholamines exert their glycogenolytic effects in Ewing's sarcoma cells by stimulation of cyclic AMP formation via -adrenergic recepetors and dopamine D₁-receptors.     

NPY modulates PYY function in the regulation of energy balance and glucose homeostasis

Aims: Both the neuronal‐derived neuropeptide Y (NPY) and the gut hormone peptide YY (PYY) have been implicated in the regulation of energy balance and glucose homeostasis. However, despite similar affinities for the same Y receptors, the co‐ordinated actions of these two peptides in energy and glucose homeostasis remain largely unknown. Methods: To investigate the mechanisms and possible interactions between PYY with NPY in the regulation of these processes, we utilized NPY/PYY single and double mutant mouse models and examined parameters of energy balance and glucose homeostasis. Results: PYY^?/? mice exhibited increased fasting‐induced food intake, enhanced fasting and oral glucose‐induced serum insulin levels, and an impaired insulin tolerance, ? changes not observed in NPY^?/? mice. Interestingly, whereas PYY deficiency‐induced impairment in insulin tolerance remained in NPY^?/?PYY^?/? mice, effects of PYY deficiency on fasting‐induced food intake and serum insulin concentrations at baseline and after the oral glucose bolus were absent in NPY^?/?PYY^?/? mice, suggesting that NPY signalling may be required for PYY's action on insulin secretion and fasting‐induced hyperphagia. Moreover, NPY^?/?PYY^?/?, but not NPY^?/? or PYY^?/? mice had significantly decreased daily food intake, indicating interactive control by NPY and PYY on spontaneous food intake. Furthermore, both NPY^?/? and PYY^?/? mice showed significantly reduced respiratory exchange ratio during the light phase, with no additive effects observed in NPY^?/?PYY^?/? mice, indicating that NPY and PYY may regulate oxidative fuel selection via partly shared mechanisms. Overall, physical activity and energy expenditure, however, are not significantly altered by NPY and PYY single or double deficiencies. Conclusions: These findings show significant and diverse interactions between NPY and PYY signalling in the regulation of different aspects of energy balance and glucose homeostasis.     

Interferonγ and tumour necrosis factorα induce a synergistic antiproliferative response in human Ewing's sarcoma cells in vitro

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon (rhIFN) and tumour necrosis factor (rhTNF) on cell proliferation and survival and to characterize IFN and TNF receptor expression. Incorporation of [³H]thymidine into cells was inhibited by rhIFN after 24 h of incubation. Half-maximal inhibition was observed with 10–80 U/ml rhIFN. A maximal effect (50%–70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFN. The influence of rhTNF on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [³H]thymidine incorporation was not affected by rhTNF up to 2000 U/ml after 96 h of incubation, where-as in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNF. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFN and rhTNF after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1, IL-1, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophagecolony-stimulating factor, tested alone and in combination with rhIFN and rhTNF, had no influence on cell proliferation. Binding studies in the cell lines with¹²⁵I-rhIFN revealed a dissociation constant (K _d ) of 160–306 pM and approximately 8000–13500 receptors/cell. Binding experiments with¹²⁵I-rhTNF indicated 430–1250 receptors/cell withK _d ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNF are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNF may be useful in the design of novel strategies in Ewing's sarcoma therapy.     

Specific 125I Neuropeptide Y Binding to Intact Cultured Bovine Adrenal Medulla Capillary Endothelial Cells

Objective: This study involved the pharmacological detection and characterization of binding sites for the neuromodulator neuropeptide Y (NPY) in an in vitro preparation of capillary endothelial cells derived from bovine adrenal medulla. Methods: Equilibrium binding assays were conducted on intact cells with ¹²⁵I Bolton-Hunter labeled NPY (¹²⁵I-BH-NPY). The specificity of the high-affinity binding site was evaluated in competition experiments with cold NPY, (Leu³¹, Pro³⁴)NPY (a Y₁ receptor ligand, Y₁RL), NPY_13–36 (a Y₂ receptor ligand, Y₂RL), and two other members of the pancreatic polypeptide-fold (PP-fold) family: peptide YY (PYY) and avian pancreatic polypeptide (APP). Forskolin-stimulated adenylate cyclase activity was assessed to detect the participation of this second messenger pathway in the neuromodulator action at the studied cell preparation. Results: Nonlinear regression analysis of the binding data indicated the existence of high-affinity binding sites with an equilibrium dissociation constant (K_d) value of 39.00 ± 12.84 nM and a maximal binding (B_max) of 489.89 ± 155.49 fmol/10⁶ cells (mean ± SE, n = 6). NPY, Y₁RL, and PYY displayed a concentration that inhibits the specific binding by 50% IC₅₀ (nM) values of 4.06 ± 1.66 (n = 4), 2.94 ± 0.75 (n = 5), and 18.36 ± 10.36 (n = 3), respectively. APP and Y₂RL were unable to compete with ¹²⁵I-NPY in the concentration range 0.001–1 μM. Further evaluation of second messenger pathways suggested that NPY binding sites in this model are coupled to the inhibition of adenylate cyclase. NPY significantly inhibited the forskolin-stimulated adenosine cyclic 3′,5′-(hydrogen phosphate) (cAMP) accumulation with a maximal effect of 37.03 ± 6.28%, n = 5 and an IC₅₀ of 5.96 ± 1.87 nM. The Y₁RL produced a comparable response (IC₅₀ = 5.35 ± 1.39 nM, n = 4; maximal inhibition of 61.05 ± 13.03%) and Y₂LR had no detectable effect at a similar concentration range. Conclusions: The results demonstrate the existence of a Y₁ receptor in the adrenal medulla capillary endothelial cells, which may be relevant to the postjunctional effect of NPY on this gland.     

Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells

Summary Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. ¹²⁵I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro³⁴]-NPY > NPY 3–36 > > NPY 13–36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits ¹²⁵I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (G_i or G_o) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7–36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through G_i to the inhibition of adenylyl cyclase. [Diabetologia (1998) 41: 1482–1491] Received: 10 November 1997 and in final revised form: 16 June 1998     

Type I and type II insulin-like growth factor receptors and their function in human Ewing's sarcoma cells

Summary Binding studies using recombinant human¹²⁵I-labelled insulin-like growth factor I ([¹²⁵I]IGF-I) revealed IGF-I receptors in three Ewing's sarcoma cell lines withK _d ranging from 74×10^–12 M to 100×10^–12 M andB _max=36–63 fmol/mg cell protein. [¹²⁵I]IGF-I binding was displaced by IGF-I, IGF-II and insulin with IC₅₀ values of 1.5 nM, 6.3 nM and 0.7 M respectively. Recombinant human [¹²⁵I]IGF-II radioligand-binding assays in the cell lines disclosed specific binding sites for IGF-II withK _d=(110–175)×10^–12 M andB _max varying from 21 fmol/mg to 72 fmol/mg cell protein. Neither IGF-I nor insulin displaced [¹²⁵I]IGF-II binding. IGF-I was found to increase basal glucose transport by maximally 1.5 times with EC₅₀=0.9 nM IGF-I. The efficacy and potency of IGF-II on glucose uptake were comparable to those of IGF-I whereas insulin was ineffective. IGF-I and IGF-II also provoked stimulation of glycogen synthesis in Ewing's sarcoma cells. The maximal glycogenic response was reached at 0.01 M IGF-I and 0.1 M IGF-II, the EC₅₀ value being approximately 1 nM IGF-I and 2 nM IGF-II. Insulin did not significantly influence glycogen formation. IGF-I and IGF-II but not insulin increased DNA synthesis in Ewing's sarcoma cells. The maximal mitogenic response was obtained with 10 nM IGF-I or IGF-II with an EC₅₀ value of about 0.7 nM for both peptides. -IR-3, a monoclonal antibody specific for the IGF type I receptor, effectively blocked IGF-I- and IGF-II-mediated metabolic responses. In conclusion, the data show that IGF-I and IGF-II induce rapid and longterm biological responses in Ewing's sarcoma cells exclusively through interaction with IGF type I receptors.     

Additive actions of the cannabinoid and neuropeptide Y systems on adiposity and lipid oxidation

Aims: Energy homeostasis is regulated by a complex interaction of molecules and pathways, and new antiobesity treatments are likely to require multiple pharmacological targeting of anorexigenic or orexigenic pathways to achieve effective loss of excess body weight and adiposity. Cannabinoids, acting via the cannabinoid‐1 (CB1) receptor, and neuropeptide Y (NPY) are important modulators of feeding behaviour, energy metabolism and body composition. We investigated the interaction of CB1 and NPY in the regulation of energy homeostasis, hypothesizing that dual blockade of CB1 and NPY signalling will induce greater weight and/or fat loss than that induced by single blockade of either system alone. Methods: We studied the effects of the CB1 antagonist Rimonabant on food intake, body weight, body composition, energy metabolism and bone physiology in wild‐type (WT) and NPY knockout (NPY^?/?) mice. Rimonabant was administered orally at 10 mg/kg body weight twice per day for 3 weeks. Oral Rimonabant was delivered voluntarily to mice via a novel method enabling studies to be carried out in the absence of gavage‐induced stress. Results: Mice with dual blockade of CB1 and NPY signalling (Rimonabant‐treated NPY^?/? mice) exhibited greater reductions in body weight and adiposity than mice with single blockade of either system alone (Rimonabant‐treated WT or vehicle‐treated NPY^?/? mice). These changes occurred without loss of lean tissue mass or bone mass. Furthermore, Rimonabant‐treated NPY^?/? mice showed a lower respiratory exchange ratio than that seen in Rimonabant‐treated WT or vehicle‐treated NPY^?/? mice, suggesting that this additive effect of dual blockade of CB1 and NPY involves promotion of lipid oxidation. On the other hand, energy expenditure and physical activity were comparable amongst all treatment groups. Interestingly, Rimonabant similarly and transiently reduced spontaneous and fasting‐induced food intake in WT and NPY^?/? mice in the first hour after administration only, suggesting independent regulation of feeding by CB1 and NPY signalling. In contrast, Rimonabant increased serum corticosterone levels in WT mice, but this effect was not seen in NPY^?/? mice, indicating that NPY signalling may be required for effects of CB1 on the hypothalamo‐pituitary‐adrenal axis. Conclusions: Dual blockade of CB1 and NPY signalling leads to additive reductions in body weight and adiposity without concomitant loss of lean body mass or bone mass. An additive increase in lipid oxidation in dual CB1 and NPY blockade may contribute to the effect on adiposity. These findings open new avenues for more effective treatment of obesity via dual pharmacological manipulations of the CB1 and NPY systems.     

Mechanisms underlying the insulinostatic effect of peptide YY in mouse pancreatic islets

Summary Peptide YY is an insulinostatic peptide which is released into the circulation from the intestinal mucosa upon food intake. Peptide YY is also co-stored with glucagon in the secretory granules of the pancreatic alpha cells. We examined the mechanisms underlying the insulinostatic effect of peptide YY in isolated mouse pancreatic islets. We found that peptide YY (0.1 nmol/l-1 mol/l) inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from incubated isolated islets, with a maximal inhibition of approximately 70% observed at a dose of 1 nmol/ 1 (p<0.001). Also in perifused islets the peptide (1 nmol/l) inhibited insulin secretion in response to 11.1 mmol/l glucose (p<0.001). Furthermore, peptide YY inhibited glucose-stimulated cyclic AMP formation (by 67%, p<0.05), and insulin secretion stimulated by dibutyryl cyclic AMP (p<0.01). In contrast, the peptide was without effect both on the cytoplasmic Ca²⁺ concentration in dispersed mouse islet-cell suspensions as measured by the FURA 2-AM technique, and on insulin release in isolated islets, when stimulated by the protein kinase C-activator 12-O-tetradecanoyl phorbol 13-acetate. Finally, in pre-labelled perifused islets, peptide YY caused a small and transient increase in the ⁸⁶Rb⁺ efflux (p<0.001), but only in the absence of extracellular Ca²⁺. We conclude that peptide YY inhibits glucose-stimulated insulin secretion from isolated mouse islets by inhibiting two different steps in the cyclic AMP cascade, that is, both the accumulation and the action of the cyclic nucleotide. In contrast, the data suggest that protein kinase C, K⁺ channels, the cytoplasmic Ca²⁺ concentration or other processes directly regulating the exocytosis are not involved in the signal transduction underlying peptide YY-induced inhibition of insulin secretion.Abbreviations PYY Peptide YY - TPA 12-O-tetradecanoylphorbol 13-acetate     

Antisecretory Mechanisms of Peptide YY in Rat Distal Colon

Peptide YY (PYY) is a potent regulator ofintestinal secretion. These studies investigated therole of Y1 and Y2 receptor subtypes in mediating theantisecretory effects of PYY on mucosa-submucosapreparations of rat distal colon. Addition of vasoactiveintestinal peptide (VIP) to these tissues resulted in a140 ± 18% increase in basal short-circuit current(I_sc) and the induction of Cl^-secretion. VIP-stimulated increases in I_sc were abolished bythe addition of each of PYY, (Pro³⁴)-PYY, aY1 receptor-selective agonist, and PYY-(3-36), anendogenous Y2 receptor-selective ligand. However, whentissue neural transmission was blocked with tetrodotoxin, neither PYY norits receptor subtype-selective analogs were able toinhibit VIP-stimulated increases in I_sc.These results suggest that in the rat distal colon, thesc antisecretory actions of PYY are mediated through acombination of Y1 and Y2 receptor subtypes or through anovel receptor subtype that is unable to discriminatebetween (Pro³⁴)-PYY and PYY-(3-36).     

Neuropeptide Y receptor genes are associated with alcohol dependence, alcohol withdrawal phenotypes, and cocaine dependence

Background: Several lines of evidence in both human and animal studies suggest that variation in neuropeptide Y (NPY) or its receptor genes (NPY1R, NPY2R and NPY5R) is associated with alcohol dependence as well as alcohol withdrawal symptoms. Additional studies suggest that cocaine may affect NPY expression. Methods: A total of 39 single nucleotide polymorphisms (SNPs) were genotyped across NPY and its 3 receptor genes in a sample of 1,923 subjects from 219 multiplex alcoholic families of European American descent recruited as part of the Collaborative Studies on the Genetics of Alcoholism (COGA) study. Family‐based association analysis was performed to test the primary hypothesis that variation in these genes is associated with alcohol dependence. Secondary analyses evaluated whether there was an association of these SNPs with symptoms of alcohol withdrawal, cocaine dependence, or comorbid alcohol and cocaine dependence. Results: Although variations in NPY itself were not associated with these phenotypes, variations in 2 NPY‐receptor genes were. SNPs in NPY2R provided significant evidence of association with alcohol dependence, alcohol withdrawal symptoms, comorbid alcohol and cocaine dependence, and cocaine dependence (all p < 0.03). Haplotype analyses strengthened the evidence for these phenotypes (global 0.0004 < p < 0.005). SNPs in NPY5R demonstrated significant association with alcohol withdrawal characterized by seizures (p < 0.05). Conclusion: These results indicate that sequence variations in NPY receptor genes are associated with alcohol dependence, particularly a severe subtype of alcohol dependence characterized by withdrawal symptoms, comorbid alcohol and cocaine dependence, and cocaine dependence.     

Neuropeptide Y stimulates autophagy in hypothalamic neurons

Aging is characterized by autophagy impairment that contributes to age-related disease aggravation. Moreover, it was described that the hypothalamus is a critical brain area for whole-body aging development and has impact on lifespan. Neuropeptide Y (NPY) is one of the major neuropeptides present in the hypothalamus, and it has been shown that, in aged animals, the hypothalamic NPY levels decrease. Because caloric restriction (CR) delays aging, at least in part, by stimulating autophagy, and also increases hypothalamic NPY levels, we hypothesized that NPY could have a relevant role on autophagy modulation in the hypothalamus. Therefore, the aim of this study was to investigate the role of NPY on autophagy in the hypothalamus. Using both hypothalamic neuronal in vitro models and mice overexpressing NPY in the hypothalamus, we observed that NPY stimulates autophagy in the hypothalamus. Mechanistically, in rodent hypothalamic neurons, NPY increases autophagy through the activation of NPY Y₁ and Y₅ receptors, and this effect is tightly associated with the concerted activation of PI3K, MEK/ERK, and PKA signaling pathways. Modulation of hypothalamic NPY levels may be considered a potential strategy to produce protective effects against hypothalamic impairments associated with age and to delay aging.Aging is associated with accumulation of specific cellular proteins within neurons, a pathologic hallmark of many neurodegenerative diseases. Because average human life expectancy has increased, but also the prevalence of cognitive decline and dementia, aging research is now focused in finding strategies that increase both lifespan and healthspan.Autophagy is a highly regulated intracellular process involved in the turnover of most cellular constituents and in the maintenance of cellular homeostasis (1, 2). It is well described that basal autophagic activity decreases with age, contributing to the accumulation of altered macromolecules (3). In addition, autophagy impairment contributes to different aspects of aging phenotype and to aggravation of age-related diseases (4).Caloric restriction (CR), the reduced intake of calories without malnutrition, extends lifespan of many organisms, from yeast to mammals, and delays the progression of age-related diseases, at least in part, by stimulating autophagy (5–8). One major neuroendocrine effect of CR is the increase of neuropeptide Y (NPY) in the hypothalamus (9–12). The hypothalamus has a key role in the control of body homeostasis, neuroendocrine outputs, and feeding behavior. Recently, it was described that this brain area is critical for the development of whole-body aging and has impact on lifespan (13, 14). In the hypothalamus, NPY is involved in the regulation of different physiological functions, such as regulation of food intake, body temperature, circadian rhythms, memory processing, and cognition (15–19). These diverse actions of NPY are mediated by G protein-coupled receptor subtypes named NPY Y₁, Y₂, Y₄, and/or Y₅ (20, 21), all of which have been reported to be present in the hypothalamus (22).Others showed that CR does not increase lifespan in NPY knockout mice (23), and aging decreases NPY levels in rodent hypothalamus (24–26). However, transgenic rats overexpressing hypothalamic NPY have improved stress resistance and increased mean lifespan (27). Although these observations suggest that NPY may play a relevant role in aging and lifespan, the effect of NPY on autophagy remains unknown.The aim of the present study was, therefore, to investigate the role of NPY, and NPY receptors, on autophagy regulation in the hypothalamus and the mechanisms underlying this process.     

Ewing's-sarcoma-associated HBA-71 tumor antigen represents a new differentiation marker of human thymocytes

Summary The monoclonal HBA-71 antibody recognizes a new human tumor-associated antigen of Ewing's sarcoma and peripheral neuroectodermal tumors, which is also expressed in some normal tissues, including thymus, islets of Langerhans, ependyme, adenohypophysis, Sertoli/Leydig and granulosa cells. Besides a tumor-specific reciprocal chromosomal translocation t (11:22), the expression of the HBA-71 antigen is the only marker which can be used for reliable differential diagnosis of these rare malignancies of childhood and adolescence among other small round cell tumors. The HBA-71 antigen is further characterized here by ultrastructural, functional and cell-matrix interaction studies. In immunohistochemical staining the HBA-71 reacted with the cell surface of human cortical thymocytes. The HBA-71 antigen was also found to be localized at the cell-surface glycocalyx of tumor cells using immunogold staining and electron microscopy. A panel of additional monoclonal antibodies with reactivity patterns similar to those of the HBA-71 antibody was obtained by immunization of mice with ES cell lines and boostering with thymocytes. The HBA-71 antibody triggers proliferation of thymocytes and to a lesser extent also stimulates peripheral mononuclear blood cells. Antibody-induced thymocyte cultures exhibit the phenotype of immature, CD3_low thymocytes with uniform and stable expression of the HBA-71 antigen. In contrast to the thymocytes the HBA-71 antibody has an inhibitory effect on the continuous growth of the HBA-71⁺ tumor cell lines. The HBA-71 antigen may be involved in the regulation to growth of the positive normal and malignant tissues. Positive modulation of the antigen expression was induced in Ewing's sarcoma cell lines in response to insulin, insulin-like growth factor I (IGF-I) and by interaction of the cells with the extracellular matrixAbbreviations used ES Ewing's sarcoma - PNET peripheral neuroectodermal tumors - FITC fluorescein-isothiocyanate - PMBC peripheral mononuclear blood cells Supported by Bürgermeisterfond der Stadt Wien, grant no. 703     

Vascular neuropeptide Y contributes to atherosclerotic plaque progression and perivascular mast cell activation

Aim

Neuropeptide Y is an abundantly expressed neurotransmitter capable of modulating both immune and metabolic responses related to the development of atherosclerosis. NPY receptors are expressed by a number of vascular wall cell types, among which mast cells. However, the direct effects of NPY on atherosclerotic plaque development and progression remain to be investigated. In this study we thus aimed to determine whether NPY is expressed in atherosclerotic plaques and to establish its role in atherosclerotic plaque development.

Methods and results

NPY expression was seen to be increased up to 2-fold in unstable human endarterectomy plaques, as compared to stable plaques, and to be significantly upregulated during lesion progression in apoE^−/− mice. In apoE^−/− mice focal overexpression of NPY in the carotid artery significantly increased atherosclerotic plaque size compared to controls, while plaque composition was unaffected. Interestingly, perivascular mast cell activation was significantly higher in the NPY-overexpressing mice, suggesting that NPY may impact plaque progression in part via mast cell activation. Furthermore, in vitro NPY-induced murine mast cell activation resulted in the release of pro-atherogenic mediators including IL-6 and tryptase.

Conclusions

Our data show that NPY expression is increased during atherogenesis and in particular in unstable plaques. Furthermore, perivascular overexpression of NPY promoted plaque development and perivascular mast cell activation, suggestive of a role for NPY-induced mast cell activation in lesion progression.     

Effects of neuropeptide Y on collateral development in a swine model of chronic myocardial ischemia

We investigated the role of neuropeptide Y (NPY), abundant in the myocardial sympathetic nervous system and endothelial cells, in angiogenesis during chronic myocardial ischemia. Adult male Yorkshire swine underwent ameroid constrictor placement on the proximal left circumflex coronary artery. After 3 weeks, an osmotic pump was placed to deliver either placebo (control, n = 8) or NPY_3–36 (NPY, n = 8) to the collateral dependent region. Five weeks after pump placement, after cardiac catheterization and hemodynamic assessment, the heart was harvested for analysis. NPY treated animals demonstrated increased mean arterial pressures and improved left ventricular function (+ dP/dt). Cardiac catheterization demonstrated a significant increase in the blush score in the NPY group (p < 0.001). Blood flow to the ischemic myocardium was not different between groups at rest or during ventricular pacing. Immunohistochemical double staining for CD-31 and smooth muscle actin demonstrated an increase in capillary and arteriole formation in NPY treated animals (p = 0.02 and p < 0.001). Immunoblotting showed a significant upregulation of DPPIV (p = 0.009) and NPY receptors 1 (p = 0.008), 2 (p = 0.02) and 5 (p = 0.03) in the NPY treated group. Additionally, there was significant upregulation of VEGF (p = 0.04), eNOS (p = 0.014), phospho-eNOS (ser1177) (p = 0.02), and PDGF (p < 0.001) in NPY treated group. The anti-angiogenic factors endostatin and angiostatin were significantly decreased in NPY treated animals (endostatin, p = 0.03; angiostatin, p = 0.04). Exogenous NPY_3-36 resulted in improved myocardial function and increased angiogenesis and arteriogenesis by stimulating growth factor, pro-angiogenic receptor upregulation, and decreasing anti-angiogenic expression, but did not increase blood flow to the ischemic myocardium. NPY may act as a good adjunct to primary agents of therapeutic angiogenesis.     

Influence of Pro12Ala peroxisome proliferator-activated receptor γ2 polymorphism on glucose response to exercise training in type 2 diabetes

Aims/hypothesis Exercise training improves glycaemic control in some but not all individuals and little research has been done regarding genetic impact on the exercise training response in type 2 diabetes. The purpose of this study was to investigate the influence of the Pro¹²Ala variant of the peroxisome proliferator-activated receptor (PPAR) 2 gene on changes in fasting plasma glucose in response to exercise training.Methods The study population comprised 139 sedentary type 2 diabetic patients (age: 54.4±7.2; HbA₁c: 7.7±0.9%) who completed 3 months of supervised exercise training. The primary outcome variable in our analysis was the post-intervention change in blood glucose. Other assessments included measures of body composition, insulin sensitivity indices and maximal oxygen uptake (VO_2max).Results The frequency of the Ala allele was 8.3% and the genotypes were in Hardy–Weinberg equilibrium. At baseline, neither body composition variables (weight, BMI, waist circumference), glucose homeostasis variables (glucose, insulin, HbA₁c, homeostasis model assessment method) nor VO_2max were different between genotypes (wild-type: Pro¹²Pro n=117, Ala carriers: X¹²Ala n=22). The exercise-training intervention led to similar improvements in body composition and glucose homeostasis variables in both genotype groups (p<0.05). The change in fasting plasma glucose was significantly different between PPAR2 genotypes (–1.66 mmol/l vs –0.54 mmol/l, Ala carriers and wild-type, respectively) (p=0.034 unadjusted and p=0.089 including baseline glucose) and the significant association between genotype and glucose response remained after adjusting for statistically significant predictors (age, changes in insulin and BMI [p=0.015]) and including baseline glucose, insulin and BMI (p=0.031).Conclusions/interpretation These data suggest that the Pro¹²Ala polymorphism may influence the glycaemic response to exercise in type 2 diabetes.     

PYY Regulates Pancreatic Exocrine Secretion Through Multiple Receptors in the Awake Rat

Peptide YY (PYY) is one of several regulatory peptides reported to modulate pancreatic secretion. PYY circulates in two forms, PYY_1–36 and PYY_3–36, and binds to multiple receptor subtypes. We sought to determine if PYY_1–36 or PYY_3–36 regulates neurally mediated pancreatic secretion through the Y1, Y2, and/or Y5 receptor subtypes. Experiments were conducted in awake, surgically recovered rats. In order to determine the effects of the PYYs on basal pancreatic secretion, either PYY_1–36, [Pro³⁴] PYY_1–36 (a Y1/Y5 agonist), or PYY_3–36 (a Y2/Y5 agonist) were infused for 40 min at doses of 0, 12.5, 25, or 50 pmol/kg/hr while measuring pancreatic juice volume and protein. PYY_1–36 increased pancreatic protein secretion at 25 and 50 pmol/kg/hr (P < 0.05) in a dose-dependent manner (P < 0.001, R ² = 0.990). The Y2/Y5 receptor agonist PYY_3–36 significantly inhibited pancreatic juice volume and protein at 12.5 and 25 pmol/kg/hr, but stimulated protein secretion at higher doses (P < 0.001, R ² = 0.995). The Y1/Y5 agonist, [Pro³⁴] PYY_1–36, had no significant effect on basal pancreatic exocrine secretion. Therefore, PYY_1–36, PYY_3–36 and [Pro³⁴] PYY_1–36 produced different, dose-dependent changes on basal pancreatic exocrine secretion. Inhibition of pancreatic secretion by circulating PYY_1–36 and PYY_3–36 are primarily mediated by the Y2 receptor. Since [Pro³⁴] PYY_1–36 did not change pancreatic secretion, it can be concluded that circulating PYY_1–36 or PYY_3–36 does not modulate pancreatic secretion through the Y1 or Y5 receptors. Since the stimulatory effects of PYY_1–36 on pancreatic secretion could not be explained by the actions of PYY_3–36 or [Pro³⁴] PYY_1–36 on Y1 or Y2 receptors, and since PYY_1–36 fails to bind to Y3 or Y4 receptors, we also conclude that PYY_1–36 may stimulate pancreatic secretion in a dose-dependent mechanism through a PYY receptor subtype different from Y1, Y2, Y3, Y4 or Y5.     

DPP-IV inhibition enhances the antilipolytic action of NPY in human adipose tissue

Context: Dipeptidyl peptidase IV (DPP‐IV) inactivates the incretin hormone glucagon‐like peptide. It can also affect the orexigenic hormone neuropeptide Y (NPY_1–36) which is truncated by DPP‐IV to NPY_3–36, as a consequence NPY’s affinity changes from receptor Y1, which mediates the antilipolytic function of NPY, to other NPY receptors. Little is known whether DPP‐IV inhibitors for the treatment of type 2 diabetic (T2DM) patients could influence these pathways. Aims: To investigate the in vitro effects of NPY with DPP‐IV inhibition in isolated abdominal subcutaneous (AbdSc) adipocytes on fat metabolism, and assessment of NPY receptor and DPP‐IV expression in adipose tissue (AT). Methods: Ex vivo human AT was taken from women undergoing elective surgery (body mass index: 27.5 (mean ± s.d.) ± 5 kg/m², age: 43.7 ± 10 years, n = 36). Isolated AbdSc adipocytes were treated with human recombinant (rh)NPY (1–100 nM) with and without DPP‐IV inhibitor (1 M); glycerol release and tissue distribution of DPP‐IV, Y1 and Y5 messenger RNA (mRNA) were measured and compared between lean and obese subjects. Results and conclusion: rhNPY reduced glycerol release, an effect that was further enhanced by co‐incubation with a DPP‐IV inhibitor [control: 224 (mean ± s.e.) ± 37 μmol/l; NPY, 100 nM: 161 ± 27 μmol/l**; NPY 100 nM/DPP‐IV inhibitor, 1 M: 127 ± 14 μmol/l**; **p < 0.01, n = 14]. DPP‐IV was expressed in AbdSc AT and omental AT with relative DPP‐IV mRNA expression lower in AbdSc AT taken from obese [77 ± 6 signal units (SU)] vs. lean subjects (186 ± 29 SU*, n = 10). Y1 was predominantly expressed in fat and present in all fat depots but higher in obese subjects, particularly the AbdSc AT‐depot (obese: 1944 ± 111 SU vs. lean: 711 ± 112 SU**, n = 10). NPY appears to be regulated by AT‐derived DPP‐IV. DPP‐IV inhibitors augment the antilipolytic effect of NPY in AT. Further studies are required to show whether this explains the lack of weight loss in T2DM patients treated with DPP‐IV inhibitors.     

Terbutaline,A β2-Adrenoreceptor agonist,inhibits gastric acid secretion and stimulates release of peptide YY and gastric inhibitory polypeptide in dogs

Terbutaline, a ₂-adrenoreceptor agonist, inhibits pentagastrin-stimulated gastric acid secretion. The purpose of this study was to examine the effect of intravenous administration of terbutaline on plasma levels of peptide YY(PYY) and gastric inhibitory polypeptide (GIP), both of which are known to inhibit gastric acid secretion. Seven dogs with gastric and duodenal fistulas were given pentagastrin (1 g/kg/hr) intravenously for 150 min in combination with terbutaline (10 or 20 g/kg/hr) or saline during the 60- to 120-min period of pentagastrin infusion. Pentagastrin-stimulated gastric acid secretion was significantly (P <0.05) inhibited by intravenous administration of terbutaline. Terbutaline significantly increased plasma PYY levels, 24% in response to terbutaline at 10 g/kg/hr, and 59% at 20 g/kg/hr. Plasma GIP levels were also increased significantly, 24% with terbutaline at 10 g/kg/hr, and 39% at 20 g/kg/hr. Our data suggest that terbutaline-induced inhibition of pentagastrin-stimulated gastric acid secretion is mediated, at least in part, by the release of PYY and GIP. The adrenergic nervous system may influence gastric acid secretion through the release of PYY and GIP.Supported by grants from the National Institutes of Health (5 R37 DK 15241-17, PO1 DK 35608, and RO1 DK 37406), and the John Sealy Memorial Endowment Fund (2723).     

Effects of combined use of class I antiarrhythmic agents on Vmax of guinea-pig ventricular muscles

The effects of the combined use of class-I antiarrhythmic drugs on the resting potentials (RP), amplitude of action potential (AMP), and V _max of the action potential were investigated in guinea-pig ventricular papillary muscles that were superfused with oxygenated Krebs-Ringer solution at 35°C. Disopyramide (40 M) reduced V _max to 68.6 ± 3.1% (mean ± SE, n=5) of the control with minimal changes in RP and AMP when preparations were stimulated at 1 Hz. The addition of mexiletine (20 M) to the solution containing disopyramide (40 M) caused a minimal reduction of V _max (less than 5%) for the stimulation of 1 Hz, but a significant reduction of V _max (13% p<0.05) when=" stimulation=" was=" increased=" to=" 2=" hz.=" this=" amount=" of=" the=" reduction=" is=" compatible=" with=" that=" obtained=" by=" mexiletine=" alone,=" suggesting=" a=" simple=" additive=">⁺ channel inhibition by this drug combination. This additive effect was also observed in the recovery process of V _max from the use-dependent block induced by train stimuli at 1 Hz. Flecainide (5 M) reduced V _max to 58.6±13.3% (n=5). The addition of mexiletine to the superfusate with flecainide produced a further depression of 14±2.6% of V _max , even at 1 Hz. This depression was larger than that produced by mexiletine, suggesting a synergistic action of the two drugs on the Na⁺ channel. Such information about the interaction of the class I drug combinations with the Na⁺ channel may be clinically important.     

Gastrointestinal regulatory peptides in systemic sclerosis

Objective. Gastrointestinal involvement commonly occurs in systemic sclerosis (SSc), but its pathogenesis is not well understood. Since there is evidence of a defect in neurotransmitter release, we were interested in examining the relationship between gastrointestinal dysfunction and plasma concentrations of gastrointestinal regulatory peptides in patients with SSc. We studied 43 consecutive patients, 18 with diffuse and 25 with limited cutaneous disease. Methods. Levels of corticotropin-releasing hormone (CRH), gastrin, motilin, neuropeptide Y (NPY), and peptide YY (PYY) were determined by radioimmunoassay and high-performance liquid chromatography (HPLC). Results. Plasma concentrations of CRH, motilin, NPY, and PYY were significantly increased among SSc patients compared with healthy control subjects, and HPLC-characterization of motilin, NPY, and PYY showed a different pattern of fragments. No correlation was found between esophageal hypomotility and the concentration of peptide. Acid output did not correlate with gastrin levels, but was more often increased in patients with increased CRH and NPY values. Fat malabsorption, assessed by the triolein breath test, was more common among patients with increased motilin and PYY. Conclusion. This study shows that elevated peptide concentrations commonly occur in patients with SSc. Since regulatory peptides are involved in gastrointestinal motility, secretion, and absorption, further characterization of this neuroendocrine system may help in understanding the complex regulation of gastrointestinal dysfunction in SSc.