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.

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.

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.

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.

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.

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.

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.

Effects of electromagnetic fields and gender on neurotransmitters and amino acids in rats.

Epidemiological studies have linked electromagnetic field (EMF) exposure to certain forms of cancer, however only limited laboratory evidence supports a connection between EMF and biological effects. In the present study we exposed male and female rats to low level, 1000 milli-Gauss (mGs), direct current EMF generated with Helmholtz coils for 1 mo or 4 mo. The effects of these EMF exposures on regional brain neurotransmitter metabolism and circulating amino acid concentrations were determined. After 1 mo of EMF exposure the concentration of serotonin was elevated in the hypothalamus of male rats. Levels of the dopamine metabolite, 3-methoxytyramine, were increased in the corpus striatum of male and female rats that were exposed to EMF for 1 mo. Hypothalamic concentration of norepinephrine was elevated in both groups of male rats, as compared to respective female groups, but was not affected by EMF. Similarly, levels of tyrosine were increased in hypothalamus, corpus striatum and nucleus accumbens of male rats, but were not affected by EMF exposure. Following 4 mo of EMF exposure, no significant effect of EMF was observed. Significant sex differences in plasma amino acid concentrations were observed in both studies, with female rats exhibiting decreases in a majority of the amino acids measured. These results are suggestive that short-term exposure may cause small alterations in neurotransmitter metabolism and in circulating amino acids, which dissipate when exposure duration is increased.

Refractory hypothalamic adenylate cyclase in anorectic tumor-bearing rats: implications for NPY-induced feeding.

Although isoproterenol stimulated adenylate cyclase activity in hypothalamic membranes taken from freely-feeding, food-restricted or nonanorectic tumor-bearing rats, the response was greatly reduced in anorectic tumor-bearing rats. The addition of NPY to the membrane preparation inhibited adenylate cyclase activity in hypothalamus taken from freely-feeding and food-restricted rats, but NPY-inhibitory activity was significantly reduced in both groups of tumor-bearing rats. These results suggest that cyclic AMP formation is refractory in anorectic tumor-bearing rats, and that NPY-induced inhibition of hypothalamic adenylate cyclase is reduced in tumor-bearing rats prior to the onset of significant anorexia. Therefore, NPY-induced feeding may be reduced in tumor-bearing organisms due to a dysfunction in the cyclic AMP second messenger system.

Neuropeptide Y and the development of cancer anorexia.

OBJECTIVE: The authors determined whether radioligand binding of neuropeptide Y (NPY) to hypothalamus taken from nonanorectic and anorectic tumor-bearing rats was altered as compared with similar tissue taken from freely-feeding and food-restricted control rats. SUMMARY BACKGROUND DATA: Previous results indicate that tumor-bearing rats exhibit a refractory feeding response to NPY, the most potent feeding stimulus known. Additional studies indicate that the concentration of NPY in the hypothalamus of anorectic tumor-bearing rats is decreased as compared with freely-feeding or food-restricted control rats. METHODS: Because these observations of decreased response to exogenous peptide in the presence of decreased endogenous levels suggest an alteration in hypothalamic NPY receptors, this study investigated binding of 125I-NPY to hypothalamic membranes of tumor-bearing and control rats. RESULTS: Determinations of receptor affinity for NPY (half maximal concentration for displacement) indicated a 20-fold decrease in affinity with the development of anorexia, which changed to an 80-fold decrease during severe anorexia. Receptor density, as indicated by specific binding, exhibited only a 30% decrease, even during severe anorexia. CONCLUSIONS: These results suggest major alterations in NPY receptor mechanisms in experimental cancer anorexia, with receptor affinity being decreased progressively as the rats become more anorectic. The absence of a compensatory up-regulation in receptor density in the presence of decreased endogenous NPY concentrations indicate dysfunction in receptor regulatory mechanisms. This receptor aberration may be the central nervous system basis for the etiology of cancer anorexia.

Anorectic and neurochemical effects of pituitary adenylate cyclase activating polypeptide in rats.

Pretreatment of rats with intrahypothalamic injections of pituitary adenylate cyclase activating peptide (PACAP) 10 min prior to the injection of neuropeptide Y (NPY) significantly reduced food and water intake during the 4-h measurement period. Intrahypothalamic injection of PACAP in schedule-fed rats also reduced food and water intake for 2 h. A smaller 1-h reduction of water intake was observed in water-deprived rats, suggesting that the anticonsummatory effects of PACAP were primarily against food intake. PACAP treatment did not alter hypothalamic concentration of NPY, nor were neurotransmitters, precursors, or metabolites altered substantially in corpus striatum or nucleus accumbens regions. These results demonstrate primary anorectic effects of intrahypothalamic injection of PACAP. The demonstration of these anorectic effects may suggest a role of cyclic AMP activation and inhibition in the control of satiety and hunger.

[D-TRP32]neuropeptide Y: a competitive antagonist of NPY in rat hypothalamus.

Neuropeptide Y (NPY) is a potent orexigenic peptide. Structure-activity studies have revealed that nearly the entire sequence of NPY is required to elicit feeding responses. Therefore, in order to develop antagonistic peptides for NPY-induced feeding, we synthesized full-length analogs of NPY, substituting D-Trp in the C-terminal receptor binding region, and screened their activity in rat hypothalamus. Although [D-Trp36]NPY and [D-Trp34]NPY inhibited isoproterenol-stimulated hypothalamic membrane adenylate cyclase activity, [D-Trp32]NPY exhibited no intrinsic activity. Furthermore, [D-Trp32]NPY inhibited [125I]NPY binding to rat hypothalamic membranes with a potency comparable to that of NPY. The presence of 30 and 300 nM concentrations of [D-Trp32]NPY shifted the inhibitory dose-response curve of NPY on isoproterenol-stimulated hypothalamic membrane adenylate cyclase activity parallel to the right with comparable KB values. Moreover, in vivo experiments in rats revealed that [D-Trp32]NPY (10 micrograms) significantly attenuated the 1-h feeding response induced by NPY (1 microgram). Several other substitutions at position 32 including 2-D-Nal resulted in agonist activity, suggesting that there are strict structural requirements to induce the antagonistic property in NPY. These findings confirm that [D-Trp32]NPY is a competitive antagonist of NPY in both in vitro and in vivo systems. Analogs based on [D-Trp32]NPY may have potential clinical application, since NPY has been implicated in the pathophysiology of a number of feeding disorders including obesity, anorexia, and bulimia.

Hypothalamic concentration and release of neuropeptide Y into microdialysates is reduced in anorectic tumor-bearing rats.

Hypothalamic concentration of neuropeptide Y was decreased significantly in anorectic tumor-bearing rats, while NPY level was increased significantly in matched carcass weight control rats as compared with freely-feeding controls. In vivo microdialysis of the perifornical hypothalamic area of tumor-bearing rats prior to the development of anorexia revealed no alteration in NPY in dialysates. Following the development of anorexia, however, tumor-bearing rats exhibited significant reduction in NPY concentration in dialysates as compared with either matched carcass weight or freely-feeding control group. These results suggest that hypothalamic NPY concentration and release are decreased selectively in anorectic tumor-bearing rats. Since NPY also elicits less feeding in tumor-bearing rats, dysfunction of hypothalamic NPY feeding mechanisms may be of primary importance in cancer anorexia.

Possible role of neuropeptide Y in experimental cancer anorexia.

The efficacy of NPY to elicit feeding in TB rats was reduced prior to the onset of overt anorexia, with the feeding response decreasing further as anorexia developed. Hypothalamic concentration of NPY was reduced in TB rats, with the magnitude of the decrease paralleling the degree of anorexia. Binding affinity of NPY to hypothalamic membranes taken from TB rats suggested decreased binding affinity with no change in receptor number. Infusing ammonium salts at a concentration and rate necessary to increase blood ammonia levels to the degree observed in TB rats, produced anorexia and decreased NPY feeding. These results suggest that NPY feeding systems are abnormal in TB rats and that hyperammonemia may be of primary importance in this dysfunction.

Insulin reverses ammonia-induced anorexia and experimental cancer anorexia.

Previous experiments suggest that experimental cancer-induced anorexia is associated with hyperammonemia and that daily injections of insulin may attenuate the anorexia for several days. In the present study, we determined whether similar daily insulin treatments would correct anorexia induced by the infusion of ammonium salts and compared this feeding response with that of insulin-treated tumor-bearing (TB) rats. Daily treatment of control and anorectic TB rats with systemically administered insulin for six days increased feeding in all control rats and 40% of the TB rats. All insulin-treated groups exhibited equal degrees of hypoglycemia irrespective of anorexia. Basal concentrations of lactate and glucagon were elevated in saline-treated TB rats. Plasma lactate levels were normalized by insulin treatment, whereas glucagon was normalized only in the TB rats that fed to insulin and increased further in TB rats that did not feed to insulin. Elevated hypothalamic tyrosine was reduced in insulin-treated TB rats that ate, and 5-hydroxy-indoleacetic acid was increased further when the rats did not eat. Insulin also blocked anorexia resulting from the intravenous infusion of ammonium salts. Hypothalamic concentrations of tyrosine and tryptophan were increased by the ammonia infusion and reduced significantly in insulin-treated infused rats. These results indicate that insulin treatment can reverse experimental cancer-induced anorexia and hyperammonemia-induced anorexia. Neurochemical changes associated with these treatments are also similar, but not identical.

Anorexia following the systemic injection of amylin.

The intravenous injection of 100 micrograms/kg of rat amylin reduced food intake in schedule-fed rats for 1 h of an 8 h measurement period. Associated with this brief anorexia was a hyperglycemic response, observed 30 min after a subsequent amylin administration. Determination of neurochemical alterations revealed increased concentration of serotonin in the hypothalamus and decreased level of the dopamine metabolite, 3-methoxytyramine, in the corpus striatum. Since similar neurochemical alterations were observed following the systemic injection of glucose, both the neurochemical changes and anorexia following intravenous amylin treatment may be secondary to hyperglycemia.