Food intake inhibition and reduction in body weight gain in rats treated with GI264879A, a non-selective NPY-Y1 receptor antagonist.

Neuropeptide Y has been proposed to play a major role in the hypothalamic regulation of feeding behavior through the activation of specific, central NPY receptor(s). In an effort to design small molecule antagonists of NPY receptors, we have synthesized a series of substituted dipeptides based on defined pharmacophores, previously identified by us and others as essential for the interaction with the peptide receptors. GI264879A behaves as a functional antagonist of Y1 receptors while displaying no binding selectivity for the different NPY receptor subtypes. We demonstrate here that administration of GI264879A to rats causes a significant decrease in food intake and body weight partly through a mechanism dependent on the integrity of the vagus nerve.

Glucagon-like peptide-2 stimulates gut mucosal growth and immune response in burned rats.

Major burn trauma often leads to reduced gut barrier function, immunosuppression, and increased bacterial translocation. We hypothesized that treatments that maintain normal gut after burn trauma will also reduce immunosuppression and bacterial translocation. Recent studies suggest that treatment with glucagon-like peptide-2 (GLP-2), which is synthesized in the intestine and released after food intake, elicits mucosal hyperplasia in the small intestine of rodents and prevents parenteral nutrition-induced gut hypoplasia. Therefore, we determined whether GLP-2 would prevent loss of gut integrity after major burn trauma. Osmotic minipumps were implanted into the peritoneum of 22 adult, male, Sprague-Dawley rats to infuse saline (10 microl/hr; n = 14) or GLP-2 (1 microg/hr; n = 8). On the next day 8 saline-infused and 8 GLP-2-infused rats were subjected to a 25 sec duration 30% BSA open flame burn, with the remaining rats serving as sham-burn controls. Five days after burn, all rats were killed. Gut protein was assessed, and immunosuppression was estimated by the mitogenic response of cultured splenocytes to phytohemagglutinin, pokeweed, and concanavalin A. Bacterial translocation was determined by culturing the mesenteric lymph nodes. Although protein content was significantly decreased in the ileum of burned rats treated with saline, the burned rats treated with GLP-2 exhibited significant increases in protein levels in duodenum, jejunum. and ileum. Colon protein was not affected by GLP-2 infusion. Saline-treated burned rats also exhibited immunosuppression, as suggested by significantly decreased responses to each of the mitogens. Infusion of GLP-2 normalized the response by the burned rats to each of the mitogens. Lymph nodes taken from sham rats exhibited no colony forming units, whereas in both of the burn groups, 50% of the cultures were positive. However, more aggressive colonization may have occurred in the saline-infused burned rats as compared with the GLP-2-infused burned rats (81 +/- 63 vs 3 +/- 2 colony forming units). These results suggest that GLP-2 may stimulate gut mucosa and reduce immunosuppression in burned rats. However, there does not seem to be a statistically significant positive effect of GLP-2 on bacterial translocation. Thus, improving small intestine mucosa may increase immunity while being ineffective against bacterial translocation.

Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells.

Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF(2alpha), Y(1) and Y(5) NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 microM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 microM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.

Antagonism of NPY-induced feeding by pretreatment with cyclic AMP response element binding protein antisense oligonucleotide.

Although second messenger systems subserving neuropeptide Y (NPY)-mediated behaviors have been identified for a variety of receptors in several tissues, downstream signaling events are not well known. The nuclear binding protein, cyclic AMP response element binding protein (CREB) appears to be a transcription factor that is activated following injection of NPY into rat hypothalamus. To allow determination of the functional nature of CREB mediation of NPY-induced feeding, injection cannulae were implanted into the perifornical hypothalamus of 18 rats. Treatment of seven rats with CREB antisense oligonucleotide (15 ug) significantly antagonized NPY feeding for up to one week after treatment, while similar injections of CREB sense oligonucleotide (15 ug) had no significant effect on NPY-induced feeding. Two weeks after the antisense oligonucleotide treatment, feeding was once again elicited by the injection of NPY. Hypothalamic CREB protein was also reduced significantly two days after the CREB antisense oligonucleotide treatment. These results suggest that activation of CREB, probably through phosphorylation, may be a necessary event for the signal transduction of NPY stimulation into feeding behavior.

Structure-activity studies including a Psi(CH(2)-NH) scan of peptide YY (PYY) active site, PYY(22-36), for interaction with rat intestinal PYY receptors: development of analogues with potent in vivo activity in the intestine.

Peptide YY (PYY) is a gut hormone that inhibits secretion and promotes absorption and growth in the intestinal epithelium. We have performed structure-activity studies with the active site, N-alpha-Ac-PYY(22-36)-NH(2), for interaction with intestinal PYY receptors. Investigation of aromatic substitutions at position 27 resulted in analogues that exhibited potent in vitro antisecretory potencies with N-alpha-Ac-[Trp(27)]PYY(22-36)-NH(2) exhibiting even greater potency than intact PYY. In vivo studies in dogs revealed that this analogue also promoted intestinal absorption of water and electrolytes during continuous intravenous and intraluminal infusion. Investigations carried out to identify features that would enhance stability revealed that incorporation of Trp(30) increased affinity for PYY receptors. A "CH(2)-NH" scan revealed that incorporation of reduced bonds at position 28-29 or 35-36 imparted greater receptor affinity. In general, disubstituted analogues designed based on the results of single substitutions exhibited good receptor affinity with N-alpha-Ac-[Trp(27),CH(2)-NH(35-36)]PYY(22-36)-NH(2) having the greatest affinity (IC(50) = 0.28 nM). Conservative multiple substitutions with Nle-->Leu and Nva-->Val also imparted good affinity. An analogue designed to encompass most of the favored substitutions, N-alpha-Ac-[Nle(24,28),Trp(30),Nva(31), CH(2)-NH(35-36)]PYY(22-36)-NH(2), exhibited a proabsorptive effect in dogs comparable to, but longer lasting than, that of intact hormone. Selected analogues also exhibited good antisecretory potencies in rats with N-alpha-Ac-[Trp(30)]PYY(22-36)-NH(2) being even more potent than PYY. However, the potencies did not correlate well with the PYY receptor affinity or the proabsorptive potencies in dogs. These differences could be due to species effects and/or the involvement of multiple receptors and neuronal elements in controlling the in vivo activity of PYY compounds. PYY(22-36) analogues exhibited good affinity for neuronal Y2 receptors but poor affinity for Y1 receptors. Also, crucial analogues in this series hardly bound to Y4 and Y5 receptors. In summary, we have developed PYY(22-36) analogues which, via interacting with intestinal PYY receptors, promoted potent and long-lasting proabsorptive and antisecretory effects in in vivo models. These compounds or analogues based on them may have useful clinical application in treating malabsorptive disorders observed under a variety of conditions.

Maintaining gut integrity during parenteral nutrition of tumor-bearing rats: effects of glucagon-like peptide 2.

Maintaining tumor-bearing rats on total parenteral nutrition (TPN) for eight days significantly reduced mass, protein, and DNA in small intestine and colon. Coinfusion of glucagon-like peptide 2 (GLP-2) significantly increased each of these variables in the duodenum, jejunum, and ileum, but not in the colon. Histological analysis of tissue revealed normal mucosa thickness and villus height in the small intestine of GLP-2-treated rats, whereas non-treated rats maintained on TPN exhibited villus shortening and thinning of the mucosa. Compared with TPN alone, no significant effects of GLP-2 were noted on tumor growth, liver weight, or heart weight. Coinfusion of GLP-2 with TPN had no significant effect on TPN-associated immunosuppression, as measured by mitogen-induced proliferation of cultured splenocytes. Although translocation of bacteria to the mesenteric lymph nodes appeared to be reduced in GLP-2-treated rats, the difference between groups was not statistically significant. These results suggest that hormonal alterations may be more important than an absence of luminal nutrition in TPN-associated mucosa changes in tumor-bearing rats. Additionally, maintenance of gut integrity during TPN does not appear to be a sufficient condition for the avoidance of the negative sequelae associated with this route of supplemental nutrition.

NPY messenger RNA is increased in medial hypothalamus of anorectic tumor-bearing rats.

Previous investigations suggest that neuropeptide Y (NPY) feeding mechanisms and corticotropin releasing factor (CRF) are altered in anorectic tumor-bearing (TB) rats. To better determine the relationship of NPY and CRF synthesis to cancer anorexia we measured mRNA for these peptides in medial and lateral hypothalamus of TB and control rats. NPY and CRF mRNA were reliably detected by Northern blot analysis only in medial hypothalamus, where NPY message was elevated significantly in TB rats. CRF mRNA tended to be reduced in both pair-fed (PF) and TB rats, but did not reach statistical significance. Concentrations of NPY or CRF were not altered significantly in either the lateral or medial hypothalamus of TB or PF rats. These results suggest that the transcription of NPY is elevated in PF rats and is increased further in anorectic TB rats. The lack of significant increases in levels of peptides may be related to dilution, due to measuring a relatively large block of hypothalamic tissue. Alternatively, translation of the signal for NPY production may be inhibited, or degradation of peptide levels may be increased.

Reciprocal changes in hypothalamic receptor binding and circulating leptin in anorectic tumor-bearing rats.

Although reduced biological activity of the obese gene product, leptin, has been associated with obesity, little information is available concerning leptin alterations during anorexia. Therefore, we measured circulating leptin concentrations and hypothalamic leptin binding in anorectic tumor-bearing and pair-fed control rats. Plasma concentrations of leptin decreased in tumor-bearing rats early in the course of tumor growth, and fell to nearly non-detectable levels during severe anorexia. The pair-fed control rats that ate the same amount of food as did the anorectic tumor-bearing rats exhibited a 50% decrease in plasma leptin concentration. Concentrations of free fatty acids were elevated in both tumor-bearing and pair-fed groups, while circulating levels of triglycerides were increased only in anorectic tumor-bearing rats. Leptin receptor density was doubled in the hypothalamus of tumor bearing rats, while binding affinity was decreased by 50%. These results suggest that peripheral leptin production is down-regulated, perhaps due to increased lipolysis in tumor-bearing rats. It appears that hypothalamic leptin systems up-regulate receptor numbers in response to decreased blood leptin level, however, the decrease in binding affinity may compensate for these alterations. Therefore, the influence of leptin on hypothalamic neuropeptide Y feeding systems may be minimal in anorectic tumor-bearing rats.

WRYamide, a NPY-based tripeptide that antagonizes feeding in rats.

Modifications of (D-Trp32) neuropeptide Y (NPY) led to the development of potential peptide-based lower molecular weight (500-800 Da) NPY feeding antagonists. One compound, WRYamide (N-Ac-Trp-Arg-Tyr-NH2), blocked NPY-induced feeding for 1 to 4 h when injected intrahypothalamically (i.h.t.) at 1 to 40 microgram. Schedule-induced feeding was also antagonized for up to 24 h by 20 microgram of WRYamide, i.h.t. Injection of 2.5 mg/kg (1 mg/rat) of WRYamide, i.v., also reduced significantly schedule-induced feeding for 4 h. A conditioned taste aversion could not be classically conditioned to saccharin using WRYamide as the unconditioned stimulus. These results may lead to the development of systemically active anti-obesity drugs.

Reduction of gut hypoplasia and cachexia in tumor-bearing rats maintained on total parenteral nutrition and treated with peptide YY and clenbuterol.

Prevention of gut hypoplasia associated with total parenteral nutrition (TPN) was investigated in 67 adult male Fisher 344 rats. Mass and protein content of the small intestine was reduced by 31% and 39%, respectively, after 7 d of TPN in tumor-bearing (TB) rats. Coinfusing peptide YY (PYY; 1 nmol.kg-1.h-1) and treating the rats with the anabolic beta-adrenergic agonist, clenbuterol (CLE; 2 mg.kg-1.d-1), resulted in significant savings in small intestine weight (26% increase) and protein (42% increase). Although the colon also exhibited a significant decrease in mass (31%), none of the treatment combinations were effective in this region of the gut. Histologic analysis of ileum suggested that the additive effects of PYY and CLE were due to differential effects of these compounds on mucosal and muscular tissues, respectively. This combination of treatments also resulted in significant savings (30% increase) in gastrocnemius protein, suggesting a reduction in the cachectic response. These results suggest that TPN-induced gut hypoplasia and cancer cachexia may be reduced by the proper combination of nutritional, hormonal, and pharmacologic treatments. In addition, the anabolic effects of various treatments may be additive to counteract TPN-induced gut atrophy.

Prevention of parenteral nutrition-induced gut hypoplasia by coinfusion of glucagon-like peptide-2.

Maintaining rats on total parenteral nutrition (TPN) for 6 days significantly reduced mass (-34%), protein (-32%), and DNA (-35%) in small intestine and colon (29-37% decrease). Coinfusion of glucagon-like peptide-2 (GLP-2) normalized each of these variables in duodenum, jejunum, and ileum, but not in colon. Histological analysis of tissue revealed normal mucosa thickness and villus height in small intestine of GLP-2-treated rats, whereas nontreated rats maintained on TPN exhibited villus shortening (-30%) and thinning (-23%) of mucosa. These results suggest that hormonal alterations may be more important than an absence of luminal nutrition in TPN-associated mucosal changes. Additionally, GLP-2 normalization of gut mucosa permits accurate assessment of the influence of reversal of hypoplasia on gut barrier function.

Hemodynamic and clinical effects of oral levodopa in children with congestive heart failure.

OBJECTIVES: This study was undertaken to evaluate the safety, efficacy and pharmacodynamic variables of oral levodopa in pediatric patients with congestive heart failure refractory to standard therapy. BACKGROUND: Therapeutic options for children with congestive cardiomyopathies are limited to digoxin, diuretic agents and angiotensin-converting enzyme inhibitors. Previous work in adults with congestive heart failure has shown a short-term effectiveness of levodopa and improvement of cardiac function. METHODS: Baseline two-dimensional and M-mode echocardiography, surface electrocardiography, Holter monitoring and exercise testing, when applicable, were performed. Levodopa was administered in a dose escalation scale from 8 mg/kg body weight per dose to 20 mg/kg per dose over 3 days with concomitant metoclopramide and pyridoxine. Catecholamine levels at initiation of the trial and throughout dose escalation were measured, with echocardiographic and electrocardiographic correlation. After 24-h drug washout, cardiac catheterization was performed both before and after administration of levodopa. RESULTS: Between February 1992 and December 1995, nine children (age 10 +/- 1.7 years, weight 27.8 +/- 4.3 kg) were enrolled in this study. At cardiac catheterization, serum dopamine levels rose from 108.5 +/- 59.2 pg/ml to 1,375.8 +/- 567.9 pg/ml (p = 0.03) at 100 +/- 14.8 min after levodopa administration without a significant change in serum norepinephrine or epinephrine levels. Paralleling these increases, there were significant changes in the cardiac index (1.7 +/- 0.3 to 3.2 +/- 0.7 liters/min per m2), stroke volume index (16.1 +/- 3.2 to 31.2 +/- 7.0 ml/m2 per min), oxygen consumption (138.6 +/- 24.4 to 188.3 +/- 30.8 ml/min per m2) and systemic vascular resistance (36.8 +/- 8 to 21.9 +/- 5.5 indexed Wood's units; all p < 0.01). There was a significant reversal of the daily fluid volume output/input ratio from 0.8 +/- 0.1 to 1.2 +/- 0.1 (p < 0.01). Levodopa administration was complicated by hypertension or tachycardia, or both, requiring a dose reduction in three patients, and by significant gastrointestinal distress in one. There was sustained symptomatic improvement a median of 19.5 months after drug initiation in seven of the patients. CONCLUSIONS: These preliminary data support the hemodynamic value of oral levodopa in the treatment of severe congestive heart failure in children.

Induction of endotoxin tolerance in rat bone marrow cells by in vivo infusion of tumor necrosis factor.

OBJECTIVE: To determine in a rat model whether a low-dose infusion of tumor necrosis factor (TNF) affects the production of the inflammatory cytokines TNF and interleukin (IL)-6, the immunosuppressive factor prostaglandin E2 (PGE2), and complement component C3 (C3) by isolated bone marrow-adherent and -nonadherent cells, cultured in the presence of lipopolysaccharide, a component of bacterial endotoxin. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of a university medical center. SUBJECTS: Sprague-Dawley rats (n = 18), 250 to 275 g. INTERVENTIONS: Animals received a continuous infusion of one of the following three treatments for 4 days: a) TNF in saline containing bovine serum albumin; b) saline containing bovine serum albumin; and c) saline alone. MEASUREMENTS AND MAIN RESULTS: After infusion, isolated bone marrow cells were cultured for 1 day and 3 days, with and without lipopolysaccharide (1 microgram/mL); culture supernatants were assayed for TNF, IL-6, PGE2, and C3. TNF infusion caused a decrease in the in vitro production of TNF, IL-6, and PGE2 by the lipopolysaccharide-stimulated adherent and nonadherent bone marrow cells. This tolerance to lipopolysaccharide stimulation was present after both 1 day and 3 days of culture. TNF infusion caused an increase in C3 production by the nonadherent cells. The production of TNF by adherent cells from saline-infused or bovine serum albumin-infused animals (controls) was greater in 3-day cultures compared with 1-day cultures, whereas the production of IL-6 and PGE2 was less. CONCLUSIONS: These results indicate that TNF infusion caused cells in the bone marrow to be tolerant to lipopolysaccharide stimulation or that TNF infusion programmed the cells to become tolerant to lipopolysaccharide stimulation on differentiation and/or maturation. The results also indicate that bone marrow cells may be regulated by TNF (probably indirectly) at different phases of maturation and/or differentiation with respect to the production of different mediators. Although TNF is considered to be an inflammatory cytokine, at low concentrations it may be an important down-regulator of the inflammatory response.

Neuropeptide Y treatment and food deprivation increase cyclic AMP response element-binding in rat hypothalamus.

Intrahypothalamic (IHT) administration of neuropeptide Y (NPY) induces a robust feeding response in rats. We have shown previously that NPY-induced feeding is mediated by a pertussis-toxin-sensitive G protein in rats. NPY receptors are coupled to cAMP and Ca2+. Because these second messengers are known to activate cAMP response element binding proteins, (CREB), cAMP response element modulators, or activating transcription factor 1, we investigated the involvement of these transcription factors in NPY-induced feeding in rats. Compared with control injections of cerebrospinal fluid (1 microl), IHT administration of NPY increased cAMP response element (CRE) binding to rat hypothalamic nuclear extracts in a time-dependent manner, as detected by an electrophoretic mobility shift assay. In contrast, IHT administration of the anorectic neuropeptide, pituitary adenylate cyclase activating polypeptide, strongly inhibited the CRE binding. Food deprivation for 48 hr also increased CRE binding, whereas 8 hr of refeeding normalized CRE activity. Preincubation of the hypothalamic nuclear extracts of NPY-treated and unfed rats with antibody specific to CREB blocked CRE binding, whereas preincubation with phosphoCREB antibody retarded the migration of CRE-protein complex, indicating that phosphoCREB is involved in this process. Consistently, immunohistochemical studies with food-deprived rats showed an intense phosphoCREB signal in the paraventricular nuclei and ventromedial hypothalamus in comparison to rats fed ad libitum. Hypothalamic calcium/calmodulin-dependent protein kinase II activity was also increased by IHT-NPY. These results suggest that calcium/calmodulin-dependent protein kinase II induced phosphorylation of CREB may be involved in regulating feeding behavior induced by NPY.

Immunostimulation following fish oil-based parenteral nutrition in tumor-bearing rats.

Tumor-bearing (TB) and control rats were maintained for four to seven days on total parenteral nutrition (TPN) in which the lipid component (11.1% of total calories) was Intralipid or fish oil. Although no TB rats maintained on standard Intralipid-based TPN died prematurely in these experiments, one-third of the TB rats maintained on fish oil-based TPN died of bleeding complications by Day 4 of TPN infusion. The bleeding appeared to be due to the particle size in the fish oil emulsion, because a subsequent study employing a better emulsified TPN with smaller lipid particles exhibited fewer bleeding problems. Tumor growth, skeletal muscle atrophy, and gut hypoplasia were not reduced in the TB group maintained on fish oil-based TPN. However, immunosuppression observed in the Intralipid-based TPN group, as measured by mitogen response of cultured splenocytes, was significantly improved in the rats maintained on fish oil-based TPN. Levels of eicosapentaenoic acid were reduced in all rats receiving TPN. Although the hypothesized antitumor and anticachexia effects of fish oil administration were not realized, significant improvement in immunosuppression was noted. These results suggest that intravenously administered fish oil may have potential positive effects for the host.

Assessment of feeding response of tumor-bearing rats to hypothalamic injection and infusion of neuropeptide Y.

Tumor-bearing rats exhibited significant decreases in 1- to 4-h intake of rat chow following the intrahypothalamic injection of 2 micrograms neuropeptide Y (NPY). This refractory feeding response was present prior to the onset of anorexia and became more severe as anorexia worsened. The constant infusion of NPY (125 ng/h) into the perifornical hypothalamus of TB and control rats elicited increased feeding for only 2 days. Because chromatography revealed minipump NPY to be intact after 10 infusion days, downregulation of NPY receptors may have occurred. Daily injection of increasing doses of NPY stimulated ad lib feeding in non-TB rats, while having no effect on TB rats. Desensitization to NPY-induced feeding following daily injections of the peptide was suggested by the loss of feeding response to a dose (500 ng) of NPY that increased food intake prior to the daily NPY treatments. These results suggest that hypothalamic NPY feeding systems are refractory in TB rats, even before they exhibit anorexia. In addition, a rapid loss of the feeding response occurred in rats with constant infusion of NPY into hypothalamic tissue or with daily intrahypothalamic injections of the peptide, suggesting possible NPY receptor-mediated alterations. Therefore, control of obesity or anorexia through NPY feeding mechanisms may prove difficult due to rapid compensatory receptor changes.