Reversal of tumor-induced biochemical abnormalities by insulin treatment in rats.

In F344 rats bearing transplantable 3-methylcholanthrene (CAS: 56-49-5)-induced sarcomas, plasma concentrations of immunoreactive insulin were decreased following the development of mild or severe anorexia. Plasma levels of immunoreactive glucagon and lactate were elevated in severely anorectic tumor-bearing (TB) rats, while plasma glucose concentrations remained normal. Both groups of TB rats exhibited decreased plasma levels of serine, glutamine, citrulline, and tryptophan and increased concentrations of alanine. Plasma levels of proline and phenylalanine were also elevated in the severely anorectic TB rats. In a second experiment, 7 daily treatments with insulin corrected the anorexia for 6 days and increased body weights of TB rats. Plasma concentrations of lactate and immunoreactive glucagon were decreased, and the abnormal plasma concentrations of glutamine, proline, analine, and phenylalanine were altered toward normal following the insulin treatments. Therefore, these data are consistent with insulin treatments benefiting the TB host by increasing feeding, increasing body weight, reducing tumor glycolysis and metabolism, reducing gluconeogenesis, and reducing host catabolism, while not stimulating tumor growth. Thus insulin therapy may have potential benefits in cancer treatment by shifting glucose metabolism toward the host and away from the tumor.

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.

Autoanalgesia: opiate and non-opiate mechanisms.

Autoanalgesia (behaviorally-activated antinociception) was elicited by lesion-induced hyperemotionality or the classical conditioning of fear to the environmental stimuli associated with measuring antinociception. Both hyperemotionality and antinociception exhibited parallel decline in septal-lesioned rats with daily handling and in VMH-lesioned rats following treatment with diazepam. Autoanalgesia elicited by conditioned fear was blocked by spinal cord transection but not by diazepam. Although opiate binding experiments suggested the involvement of endorphins as mediators of autoanalgesia, hypophysectomy, morphine tolerance or very high doses of opiate antagonists failed to reduce the antinociception. Electrolytic lesions of the nucleus raphe magnus, a descending serotonergic system, did cause a significant reduction in autoanalgesia. Therefore, endorphin systems may be activated by the stress involved in autoanalgesic paradigms as a parallel system, whose functional integrity is not necessary for the expression of behaviorally-induced antinociception.

Changes in brain amines associated with cancer anorexia.

Analysis of indole amine metabolism within acute (Walker 256 carcinosarcoma) and chronic (methycholanthrene-induced sarcoma) animal models of cancer anorexia demonstrated elevated levels of plasma free tryptophan, whole brain tryptophan, serotonin and 5-hydroxyindoleacetic acid in anorectic tumor-bearing rats. Whole brain levels of catecholamines were not changed within either tumor line. Regional central nervous system determination of tryptophan metabolism in rats bearing Walker 256 tumors revealed elevated tryptophan in the hypothalamus, corpus striatum, mesencephalon, diencephalon, cerebellum and cortex, increased serotonin in the diencephalon and cerebellum and elevated 5-hydroxyindoleacetic acid in the diencephalon, hippocampus, pons-medulla, cerebellum and cortex. Although tryptophan was significantly increased only in the corpus striatum and diencephalon of the more chronic methycholanthrene tumor model, serotonin concentration was elevated in the corpus striatum, diencephalon, hippocampus, pons-medulla, cerebellum and cortex, while levels of 5-hydroxyindoleacetic acid were significantly increased in all these areas as well as in the mesencephalon. Since similar changes in indole activity were not observed in pair-fed control rats, it is concluded that the elevated serotonin and 5-hydroxyindoleacetic acid levels in tumor-bearing rats did not result from undernutrition alone. Assay of regional catecholamines revealed few food-relevant changes, with norepinephrine being elevated in the corpus striatum and decreased in the pons-medulla of tumor-bearing rats. Therefore, these experiments suggest that the increased serotonin metabolism observed in tumor-bearing rats may be involved in the etiology of the anorexia of cancer.

Analysis of the interaction of naltrexone with eating following adrenergic and cholinergic stimulation of the hypothalamus.

The anoretic potency of the narcotic antagonist drug nalthrexone, was investigated against eating elicited by the perifornical hypothalamic injection of 24.0 nmol of norepinephrine or 6.0 nmol of carbachol. Although eating following administration of norepinephrine was not significantly affected by pretreatment with naltrexone, carbachol-induced eating and drinking were dramatically attenuated by doses of naltrexone as small as 0.25 mg/kg. These data suggest the existence of an opiate link in these cholinergically-mediated behaviors.

[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.

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.

A comparison of nicotine and structurally related compounds as discriminative stimuli.

1 Of seven nicotine-like compounds tested as discriminative stimuli in the rat, only 3-pyridyl-methylpyrollidine (3-PMP) generalized to the stimulus effects of nicotine. 2 3-PMP caused equivalent nicotine-like responding at a dose (800 microgram/kg) approximately 4 times that used for the original nicotine discrimination (200 microgram/kg). The ED50 for 3-PMP was about 5 times that for nicotine. 3 Testing of the compounds as possible antagonists of the nicotine-elicited cue were negative. 4 The nicotine-like cue produced by an 800 microgram/kg injection of 3-PMP was effectively blocked by mecamylamine but not by hexamethonium or atropine. Thus, 3-PMP appears to produce generalization to the nicotine cue via action on central nicotinic-cholinoceptors as has been previously reported for the nicotine discriminative stimulus. 5 Mecamylamine blocked the stimulus-effects of 3-PMP (800 microgram/kg) and of nicotine (200 microgram/kg) with an ED50 of 0.32 and 0.20 microgram/kg respectively.

Effects of medial raphe and raphe magnus lesions on the analgesic activity of morphine and methadone.

The effects of lesions of the raphe nuclei on opiate-induced antinociception and brain serotonin (5-HT) levels were investigated. Lesions of the medial raphe nucleus effectively antagonized the analgesic effects of morphine, but not methadone, and lowered brain 5-HT. The decrement in analgesic activity of morphine was reversed by pretreatment with 5-hydroxytryptophan. Lesions of the raphe magnus, a descending 5-HT system, antagonized the analgesic potency of both morphine and methadone. These experiments indicate a differential effect of 5-HT manipulation on opiate-induced analgesia, suggesting a different mechanism of analgesic action for morphine and methadone.

Influence of total nitrogen, asparagine, and glutamine on MCA tumor growth in the Fischer 344 rat.

Previous studies from this laboratory have demonstrated that growth of the methylcholanthrene (MCA) sarcoma is dependent on total nitrogen substrate availability in vivo and on the specific amino acids asparagine and glutamine in vitro. This experiment determines whether these two phenomena can be used to selectively depress tumor growth and maintain host carcass. Sixty-two rats were inoculated with sarcoma and were infused for 10 days with isocaloric (60 kcal/day) TPN solutions at 100%, 16%, 10%, and 5% of normal nitrogen levels, either with (W) or isonitrogenously without (WO) the amino acids asparagine, glutamine, aspartic acid, and glutamic acid. W solutions contained 33% of these amino acids. Mean weights of 100 W tumors were significantly greater (p = 0.002) than all other groups. Total body weights minus tumor weights were similar in W versus WO animals at each rate of nitrogen infusion. Mean venous plasma concentrations of asparagine, aspartic acid, glutamine, and glutamic acid were similar in all eight groups. These data indicate that the same degree of tumor depression produced by nitrogen deprivation can also be produced by removal of asparagine, glutamine, and their precursors from nutrient solutions without adverse effects on carcass mass. The mechanisms involved are not readily explained by analysis of venous plasma amino acid concentrations.

Acivicin reduces tumor growth during total parenteral nutrition (TPN).

Glutamine appears to be an important substrate for tumor growth. Since tumor growth rate may be stimulated by total parenteral nutrition (TPN), we investigated the effect of the glutamine antimetabolite, acivicin, on methylcholanthrene sarcoma growth in rats maintained on TPN or on rat chow. Acivicin treatment significantly reduced tumor growth by 67% in rats receiving TPN and by 71% in rats maintained on chow. Carcass weights were significantly increased by TPN in both acivicin-treated and saline solution-treated tumor-bearing rats. Tumor-carcass ratios were significantly decreased in both groups of acivicin-treated tumor-bearing rats. Acivicin treatment or a similar approach may therefore be useful for stabilizing tumor growth in patients receiving TPN.

Reversal of cancer cachexia in rats by cimaterol and supplemental nutrition.

The anabolic beta 2-agonist cimaterol was used in conjunction with supplemental nutrition to reverse cancer-induced cachexia and malnutrition in tumor-bearing rats. Cimaterol was administered to tumor-bearing rats receiving total parenteral nutrition or enteral nutrition for 10 days, beginning 2 weeks after subcutaneous transplantation of methylcholanthrene sarcoma. A significant increase occurred in both muscle weight and muscle protein in animals receiving cimaterol in conjunction with either enteral or parenteral feeding, compared to food fed tumor-bearing animals. Muscle protein content was increased significantly by 16% in cimaterol-treated rats maintained on parenteral nutrition and by 11% in cimaterol-treated enterally fed rats compared with the respective tumor-bearing controls. Urinary concentrations of 3-methylhistidine, an estimation of muscle turnover or catabolism, were significantly reduced in both tumor-bearing groups treated with cimaterol compared to 3-methylhistidine levels of the untreated tumor-bearing groups. The anabolic effects of cimaterol were expressed in the presence of a large tumor burden resulting in reversal of muscle depletion and muscle breakdown regardless of the route of supplemental nutrition. Thus, beta 2-agonists may be considered as a possible therapy for cancer cachexia.

Resting energy expenditure of host and tumor is similar in rats with methylcholanthrene-induced sarcoma.

In the present study we assessed the resting energy expenditure of 30 free-feeding control and methylcholanthrene-induced sarcoma-bearing rats prior to and following surgical removal of the tumor. Tumor-bearing rats demonstrated carcass wasting and massive tumor growth. The resting energy expenditure data in our model suggest that neither the presence and growth of a tumor nor its removal significantly change resting energy expenditure beyond the normal range for non-tumor-bearing rats. We suggest that in the partition of energy costs between host and tumor, both carry a similar input, proportional to their relative weight, into the total combined resting energy expenditure of host and tumor.

Tests of adipsia and conditioned taste aversion following the intrahypothalamic injection of amylin.

Intrahypothalamic injection of amylin (AMY) was shown to reduce the intake of rat chow and water for 8 and 4 h, respectively, in schedule-fed rats. Amylin also reduced water intake to a much lesser degree in 24-h water-deprived rats. A test of the ability of AMY to form a conditioned taste aversion yielded no change in saccharin preference, as compared to controls treated with vehicle. These results suggest that although AMY has adipsic effects, the reduction in water is not of sufficient magnitude to cause the anorexia. In addition, the failure of AMY to support a conditioned taste aversion suggests that AMY does not cause anorexia by inducing malaise. Therefore, in addition to other metabolic effects, AMY may be involved in the control of food and water intake.

Cholecystokinin-induced hypophagia is not potentiated by cancer anorexia.

The interaction of cholecystokinin-induced hypophagia with cancer anorexia was investigated within both acute (Walker 256 carcinosarcoma) and chronic (methylcholanthrene-induced sarcoma) animal models of cancer anorexia. Cholecystokinin octapeptide (CCK8) effectively reduced feeding for at least one hour in both groups of rats. However, this peptide was no more effective in inducing hypophagia in tumor-bearing rats than in nontumor-bearing control rats when tested at a variety of doses (0.5, 5.0 and 50.0 microgram/kg; IP) both prior to and after the development of anorexia. Therefore, these data do not support a role of cholecystokinin in the mediation of experimental cancer anorexia, since no synergism of CCK8-induced hypophagia with the anorexia was observed.

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.

Pertussis toxin inhibits neuropeptide Y-induced feeding in rats.

Neuropeptide Y (NPY) is the most powerful peptide drug stimulating feeding in rats. Rats with paraventricular hypothalamic (PVH) cannulae were used to investigate the mechanisms involved in NPY-induced feeding. Consistent with previous reports, injection of 2 micrograms of NPY into the PVH significantly increased the cumulative food intake over 1-, 2- and 4-hr periods. Ad lib feeding decreased significantly two days after pertussis toxin (PT) administration, but recovered to nearly normal levels on the fourth day. PT had no immediate effect on NPY-induced feeding; however, four days after PT was injected NPY (2 micrograms) did not increase the food intake compared to control. In vitro investigations showed that isoproterenol-stimulated adenylate cyclase activity in the hypothalamus of control rats was inhibited by NPY. In PT-treated rats, however, no inhibition of cAMP production was observed. These results suggest that cAMP may mediate NPY-induced feeding and that a PT-sensitive G protein may be involved in this signal transduction.

Syntheses, structures and anorectic effects of human and rat amylin.

Amylin, a 37-residue polypeptide with a single disulfide bond originally isolated from the pancreas of type-II diabetic patients, has been shown to cause peripheral insulin resistance and to attenuate the inhibition of hepatic glucose output by insulin. We have also shown that amylin is present in the rat hypothalamus and that it inhibits food intake by rats. In order to further investigate the anorectic properties we synthesized both human and rat amylin by the solid phase method and purified to homogeneity in an overall yield of 10-20%. Structural analyses indicated that human amylin exhibited predominantly a beta-sheet structure at both acidic and alkaline pH, whereas no ordered structure was evident in the case of rat amylin. Intrahypothalamic injection of rat amylin resulted in a potent dose-dependent inhibitory effect on the food intake by rats adapted to eat their daily ration of food in an eight-hour period. Human amylin was less effective as an anorectic agent. Furthermore, rat amylin completely blocked the potent orexigenic effect of neuropeptide Y (NPY). These investigations show that there is a fundamental difference in the secondary structures of human and rat amylin and that rat amylin is a potent inhibitor of both basal and NPY-induced feeding by rats.

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.

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.