Journal of Gerontology: BIOLOGICAL SCIENCES Copyright 2006 by The Gerontological Society of America 2006, Vol. 61A, No. 4, 315–322

Orexigenic Effects of a Secretagogue and Nitric Oxide in Aged Rats and Dogs: Correlation With the Hypothalamic Expression of Some Neuropeptidergic/Receptorial Effectors

Mediating Food Intake Downloaded from https://academic.oup.com/biomedgerontology/article/61/4/315/607480 by guest on 29 September 2021

Antonello E. Rigamonti, Sara M. Bonomo, Diego Scanniffio, Silvano G. Cella, and Eugenio E. Mu¨ller

Department of Medical Pharmacology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Italy.

Hypothalamic neurochemical alterations in mammals underlie disturbances of food intake. There is scarce information on these topics in elderly persons; therefore, the aims of the present study were: (i) to evaluate the orexigenic effects of a growth hormone secretagogue, administered to young and old rats and dogs, alone or in combination with molsidomine, a donor of nitric oxide and (ii) to evaluate by reverse transcription–polymerase chain reaction in the whole hypothalamus of young and old rats messenger RNA levels of a wide number of anabolic and catabolic , receptors, and enzymes involved in the control of feeding behavior, relating the detected titers, whenever possible, to the feeding responses to growth hormone secretagogue. In all, the results obtained strengthen the proposition that, in the hypothalamus of old rats, anti-anorexigenic compensatory mechanisms are operative, aimed at maintaining a ‘‘normal’’ feeding pattern. Thus, the occurrence of a primary, age-related alteration in the feeding mechanisms is unlikely.

GING is associated with impairment in the ability of AGRP/NPY and POMC/CART neurons synapse on each Aan organism to maintain energy balance in response to other; this complex neuronal network responds to many homeostatic perturbations. The reasons for this loss of hormonal and metabolic signals and may be altered in the regulation of energy balance are multifactorial, though one anorexia of aging (6). of the key factors appears to be a loss of appetite, termed Compared to young animals, aged male rats fail to ‘‘the anorexia of aging’’ (1,2). In humans, the anorexia of increase food intake after a 72-hour fast, and are slow in aging too is caused by numerous factors, including sensory regaining the lost body weight upon refeeding. This inability impairment, social isolation, and psychological and physi- to maintain body weight is associated with an age-related ologic factors; disease states further magnify it (3). deficiency of NPY messenger RNA (mRNA) and a blunted Clinical studies have shown that healthy older people fail fasting-induced increase in NPY gene expression (7). With to respond to over- or underfeeding with the compensatory aging, ARC AGRP gene expression decreases, and CART changes in eating common to younger people. Hence, mRNA increases, though POMC mRNA does not change elderly people report less hunger after an overnight fast, and, (8). This altered gene expression profile may reciprocally, a greater degree of satiation to meals than do underlie the blunting of the counterregulatory response to the young people. This apparent insensitivity to metabolic fasting, likely mediated by , an anorexigenic cues can lead to inappropriate weight loss in response to produced by the adipose tissue (9). acute or chronic illness or other stressors, resulting in greater Aging and obesity would produce leptin resistance, which morbidity and mortality in the geriatric population (4). attenuates the anorexigenic effects of exogenous leptin and A circuitry of neurons in the arcuate nucleus (ARC) leads to hyperleptinemia as the system strives towards integrates blood-borne peripheral metabolic signals and homeostasis (10,11); thus the relatively hyperleptinemic regulates appetite and energy expenditure through actions state of aged animals likely blunts the sensitivity of the on distal effector neurons. This circuit consists of neurons hypothalamic energy regulatory system, decreasing appetite coexpressing the anabolic agouti-related pep- even during episodes of negative energy balance (stress, tide (AGRP) and (NPY), which synapse on acute or chronic illness) (12,13). and inhibit the firing of neurons coexpressing the catabolic Recent evidence has implicated ghrelin, an orexigenic neuropeptides, alpha-melanocyte stimulating hormone peptide produced by the gastric fundus, functionally related (a-MSH), derived from pro-opiomelanocortin (POMC), or to growth hormone secretagogues (GHS), as a key regulator cocaine-amphetamine regulated transcript (CART) (5). The of food intake and energy balance (14). The orexigenic effect

315 316 RIGAMONTI ET AL.

of ghrelin is mainly exerted via activation of NPY/AGRP After drug administration, all rats were put in their cages neurons (15), though the involvement of other neuropep- with a preweighed amount of food, and their food intake tides, particularly of the POMC system, cannot be ruled out. was carefully monitored every hour for 6 hours. Food intake To our knowledge, the ability of GHS/ghrelin to increase was normalized to rat body weight (b.wt.). food intake of aged animals has not been investigated so far. Food-intake experiments were performed on different Hence, the aim of the present study was to determine the days with at least a 3-day interval between each adminis- orexigenic effect of GHS in young and old rats and dogs, tered dose, and with best efforts to avoid background noise alone or with molsidomine, a donor of nitric oxide (NO), as and other stressful events. At the end of feeding experi- food intake stimulated by GHS/ghrelin is partially mediated ments, after a wash-out period of about 15 days, rats were by NO (16,17). Moreover, mRNA levels of a wide series of killed by decapitation, when they were in a fed state; the Downloaded from https://academic.oup.com/biomedgerontology/article/61/4/315/607480 by guest on 29 September 2021 anabolic and catabolic peptides, receptors, and enzymes whole hypothalamus was then quickly dissected, later put in (i.e., ghrelin, GHS receptor [GHS-R], NPY, NPY receptors 1 300 ll of RNA (Celbio S.p.A., Milan, Italy), and stored at and 5 [Y1-R and Y5-R], AGRP, POMC, CART, melano- 208C until processed for RNA extraction. cortin receptor 4 [MCR-4], leptin, long, melanocortin concentrating hormone [MCH], pre-pro- Total RNA isolation and RT–PCR assay.—Total RNA [PRE-PRO-OX], and neuronal and inducible NO-synthases was extracted from the hypothalamus according to the single- [nNOS and iNOS, respectively]) were measured by reverse step acid guanidinium thiocyanate–phenol–chloroform transcription–polymerase chain reaction (RT–PCR) in the extraction method. Integrity of extracted RNA was checked whole hypothalamus of young and old rats, and related, by electrophoresis with a 1% agarose gel containing ethid- whenever possible, to the behavioral response to the GHS. ium bromide. First, strand complementary DNA (cDNA) was synthesized following standard procedures using 1 lg of total RNA extracted from each sample, with oligo dT18 METHODS (Gibco Life Technologies, Milan, Italy) and 100 U of Moloney murine leukemia virus reverse transcriptase (M-MuLV; Studies in Rats Gibco Life Technologies). Reverse transcription was per- formed at 378C for 50 minutes followed by an initial de- Animals.—Ten young (2-month-old; 275–300 g) and seven naturation at 708C for 15 minutes. old (26-month-old; 800–1000 g) male Sprague Dawley PCR amplification was performed with 2.5 U of Taq rats (Charles River, Calco, Italy) were housed individually polymerase (Euro Taq; Celbio S.p.A.) and with synthetic under controlled conditions (248C, 60% humidity, artifi- gene-specific primers (Gibco Life Technologies) (Table 1). cial light from 7 AM to 7 PM) in a specific-pathogen-free PCR was performed with a thermal cycler (Thermo Hybayd; environment. Standard dry diet (Mucedola S.p.A., Milan, Celbio S.p.A.) for a number of cycles that varied according Italy) and water were available ad libitum. Old rats, which to the primer used (Table 1) under the following conditions: survived from a huge colony of young animals, were ap- denaturation at 958C for 30 seconds, annealing at a specific parently in healthy condition. temperature in relation to the primer used (Table 1), and All experimental protocols, authorized by the Committee extension at 728C for 30 seconds. Electrophoretic analysis on Animal Care and Use of the University of Milan, met the of DNA fragments was performed on a 2% agarose gel Italian guidelines for Use of Laboratory Animals, which containing ethidium bromide, and was visualized by ultra- conform with the European Communities Directive of violet-induced fluorescence; this method showed that PCR November 1986 (86/609/EEC). at these cycles was linear. To normalize these results, the glyceraldehyde-6-phosphate dehydrogenase (GAPDH) gene Experimental design.—To evaluate the orexigenic effect (Table 1), was amplified. Conditions used were 30 seconds of GHS, different doses (80, 160, 320 lg/kg, sc) of at 958C, 60 seconds at 538C, and 30 seconds at 728C for EP51216, a GHS agonist we had previously investigated 30 cycles. Negative control of the PCR was made by omit- (18) (Europeptides, Argenteuil, France), or saline 0.9% (1 ting the DNA from the mixture. The intensity of each band ml/kg, sc) were administered at 9 AM to overnight-fed young was quantified using a densitometer. The resulting densities and aged rats. EP51216 (GAB-DTrp-(2Me-DTrp(2Me)- of each gene were expressed relative to the corresponding Trp(2Me)-Lys-NH2) has efficacy and potency close to those densities of the GAPDH bands from the same RNA sample. of hexarelin, and binds to GHS heart receptors; it does not interact with other neuropeptides (NPY, AGRP, and a- Studies in Dogs MSH) (personal communication, Dr. R. Deghenghi; Eu- ropeptides, 2000). Animals.—Six old (10–14 years old; 9–13 kg b.wt.; two EP51216 (80 lg/kg) was also tested in combination with male and four female) and seven young (4–6 years old; molsidomine (20 mg/kg, ip; Sigma-Aldrich, Milan, Italy), a 13–15 kg b.wt.; all male) well-trained beagle dogs were NO donor drug (17) given 1 hour prior to the GHS peptide. used. They were exercised routinely and were fed normal A group of rats treated with molsidomine only was not dry food (Diete standard; Charles River, Calco, Italy), once used because the drug is inactive on food intake when daily, at 4:00 PM, with water available ad libitum. Dogs were administered alone in this species (17). Intracerebroven- on a 12-hour light/dark regimen, with lights on at 7:00 AM. tricular administration was ruled out as unfeasible because At the beginning of the study, the body weights of dogs of the reduced number of animals. were stable and they were free of any observable disease. ANOREXIA OF AGING AND NEUROPEPTIDES 317

Table 1. Reverse Transcription–Polymerase Chain Reaction Primer Sets and Conditions

Annealing Temperature/No. Gene Primer Sequence of Cycles Product Length Ghrelin Sense: 59-TTGAGCCCAGAGCACCAGAAA-39 67.18C/40 348 bp Antisense: 59-AGTTGCAGAGGAGGCAGAAGCT-39 GHS-R Sense: 59-AGCGCTACTTGCCCATC-39 51.28C/35 288 bp Antisense: 59-CCGATGAGACTGTAGAG-39 AGRP Sense: 59-GTTCCCAGGTCTAAGTCTGAA-39 57.58C/30 205 bp

Antisense: 59-TGAAGAAGCGGCAGTAGCAC-39 Downloaded from https://academic.oup.com/biomedgerontology/article/61/4/315/607480 by guest on 29 September 2021 NPY Sense: 59-ATCACCAGACAGAGATATGGC-39 56.48C/30 108 bp Antisense: 59-AGGGTCTTCAAGCCTTGTTCT-39 Y1-R Sense: 59-GACCATGTGCAAACTGAATC-39 55.58C/35 314 bp Antisense 59-AGAAGAGTCGTGTAAGACAG-39 Y5-R Sense: 59-AGCCATGTGCCATATCATGC-39 57.58C/35 337 bp Antisense: 59-GAGGCAGGATATACTGCACT-39 nNOS Sense: 59-CAGGCTAAGAACAAGGGC-39 54.98C/35 345 bp Antisense: 59-TGGACTCAGATCTAAGGC-39 iNOS Sense: 59-CTTCAGGTATGCGGTATTGG-39 52.68C/38 351 bp Antisense: 59-CATGGTGAACACGTTCTTGG-39 MCH Sense: 59-GATATAGTATTTAATACATTCAGG-39 57.68C/27 400 bp Antisense: 59-GCAGGTATCAGACTTGCCAACAGG-39 MCR-4 Sense: 59-GGACGCCCAGAGCTTCACCGTG-39 68.08C/35 376 bp Antisense: 59-CTGTCGGATGGTACCCGTGCCC-39 PRE-PRO-OX Sense: 59-CGGATTGCCTCTCCCTGAGC-39 59.38C/38 397 bp Antisense: 59-CTAAAGCGGTGGCGGTTGC-39 Leptin Sense: 59-GACTTCATTCCTGGGCTCCACC-39 59.28C/35 235 bp Antisense: 59-CCCGAAGCTTCCAGGACACC-39 OB-RL Sense: 59-GCTGTGCAGTCACTCAGTGC-39 56.38C/35 674 bp Antisense: 59-AGCACTGTTACACTGGTCAC-39 POMC Sense: 59-GGCCTTTCCCCTAGAGTTCAA-39 56.68C/35 70 bp Antisense: 59-GCTCCAGGACTTGCTCCAAG-39 CART Sense: 59-GCCAAGTCCCCATGTGTGAC-39 55.88C/35 129 bp Antisense: 59-CACCCCTTCACAAGCACTTCA-39 GAPDH Sense: 59-GCCATCAACGACCCCTTCATTG-39 53.08C/30 500–600 bp Antisense: 59-TGCCAGTGAGCTTCCCGTTC-39 Note: GHS-R ¼ GHS receptor; AGRP ¼ peptide related to agouti; NPY ¼ neuropeptide Y; Y1-R and Y5-R ¼ NPY receptors subtypes 1 and 5; nNOS ¼ neuronal NO synthase; iNOS ¼ inducible NO synthase; MCH ¼ melanin-concentrating hormone; MCR-4 ¼ subtype 4; PRE-PRO-OX ¼ pre-pro-orexin; OB-RL ¼ leptin receptor subtype long; POMC ¼ pro-opiomelanocortin; CART ¼ cocaine- and amphetamine-regulated transcript; GAPDH ¼ glyceraldehyde-3- phosphate dehydrogenase.

Old female dogs were out of reproductive age. All was monitored every hour for 6 hours. Food intake was experiments were carried out in conscious animals. Before normalized to dog b.wt. Because no consistent differences in the experiments, which were scheduled in a randomized food intake between male and female dogs were observed in order, animals were kept at rest in the laboratory for at least the different experiments, data were pooled. 1 hour. Protocols previously authorized by the Committee on Animal Care and Use of the University of Milan were Statistical Analysis strictly followed. Six-hour cumulative food intake was calculated and expressed (in g/kg b.wt.) as means 6 standard error of the mean (SEM). Rats’ hypothalamic mRNA levels of the Experimental design.—All experiments were performed different neuropeptides and receptors were expressed as following an overnight fast. To evaluate the orexigenic means 6 SEM of density ratios. effect of GHS in young and aged dogs, all animals were Statistical evaluation of the results of food-intake experi- administered EP51216 (500 lg/kg, sc) or saline (0.1 ml/kg, ments and of neuropeptide and receptor mRNA levels was sc), at 9 AM. EP51216 (or saline) was also tested associated performed by using the Student–Newman–Keuls test. with molsidomine (2 mg/kg, iv), which was given 1 hour Values of p , .05 were taken to be statistically significant. prior to the peptide. Preliminary studies had shown that, in dogs, molsidomine doses up to 2 mg/kg were ineffective to enhance EP51216-induced GH release, whereas higher RESULTS doses induced severe adverse effects (hypotension). There- fore, the 2 mg/kg dose of the compound was used in the Studies in Rats experiments. Baseline food intake in young and old rats was not After drug administration, all dogs were put in their boxes statistically different (means 6 SEM of 6-hour evaluation: with a preweighed amount of food, and their food intake 0.66 6 0.30 g/kg b.wt. vs 0.70 6 0.50 g/kg b.wt., p ¼ 318 RIGAMONTI ET AL.

Table 2. Densitometric Analysis of Polymerase Chain Reaction of Neuropeptides and Receptors in the Hypothalamus of Old and Young Rats

Gene Old Young Ghrelin 0.18 6 0.03 0.19 6 0.03 Leptin 0.18 6 0.01 0.19 6 0.01 OB-RL 0.30 6 0.09 0.18 6 0.03 GHS-R 0.53 6 0.03 0.65 6 0.03* AGRP 0.34 6 0.01 0.27 6 0.01*

PRE-PRO-OX 0.23 6 0.04 0.25 6 0.03 Downloaded from https://academic.oup.com/biomedgerontology/article/61/4/315/607480 by guest on 29 September 2021 NPY 0.33 6 0.01 0.27 6 0.02* Y-1R 0.26 6 0.01 0.47 6 0.11* Y-5R 0.48 6 0.16 0.39 6 0.07 nNOS 0.34 6 0.06 0.25 6 0.03 iNOS 0.35 6 0.07 0.23 6 0.01* MCH 0.33 6 0.06 0.45 6 0.05 CART 0.44 6 0.01 0.48 6 0.03 POMC 0.41 6 0.02 0.52 6 0.02* Figure 1. Six-hour cumulative food intake (g/b.wt. 3 1000) in old and young MCR-4 0.29 6 0.04 0.38 6 0.02* rats after administration of saline (0.1 ml/kg, sc) or different doses of EP51216 Notes: Values are expressed as specific gene expression related to GAPDH (80, 160, 320 lg/kg, sc). * , .05 vs old rats. Values are means 6 standard p expression 6 standard error of the mean. error of the mean. See text for details. *p , .05 vs. old rats. OB-RL ¼ leptin receptor subtype long; GHS-R ¼ GHS receptor; AGRP ¼ peptide related to agouti; NPY ¼ neuropeptide Y; Y1-R and Y5-R ¼ NPY not significant [NS], respectively) (Figure 2). EP51216 receptors subtypes 1 and 5; nNOS ¼ neuronal NO synthase; iNOS ¼ inducible stimulated food intake in both young and old rats, and its NO synthase; MCH ¼ melanin-concentrating hormone; MCR-4 ¼ melanocortin orexigenic activity was dose-dependent and was higher in receptor subtype 4; PRE-PRO-OX ¼ pre-pro-orexin; POMC ¼ pro-opiomela- young than in aged animals (Figure 1). These results were nocortin; CART ¼ cocaine- and amphetamine-regulated transcript; GAPDH ¼ consonant with the finding that the mRNA levels of GHS-R glyceraldehyde-3-phosphate dehydrogenase. were significantly lower in old than in young rats (0.53 6 0.03 vs 0.65 6 0.03, respectively, p , .05) (Table 2), 0.02, p , .05) (Table 2), but no significant difference in the though ghrelin and leptin gene expression were superimpos- hypothalamic expression of CART and MCH was evident able in either animal group (ghrelin: 0.18 6 0.03 vs 0.19 6 between the two age groups (CART: 0.44 6 0.01 vs 0.03, p ¼ NS; leptin: 0.18 6 0.01 vs 0.19 6 0.01, p ¼ NS), 0.48 6 0.03, p ¼ NS; MCH: 0.33 6 0.1 vs 0.45 6 0.05, and the same was true for leptin receptor long (0.30 6 0.09 p ¼ NS) (Table 2). vs 0.18 6 0.03, p ¼ NS) (Table 2). Molsidomine markedly enhanced the orexigenic effect of GHS in both young and aged rats, though the cumulative effect on feeding was lower in old (1.17 6 0.70 g/kg b.wt. vs 3.00 6 1.10 g/kg b.wt.) than in young rats (4.26 6 0.60 g/kg b.wt. vs 7.50 6 1.00 g/kg b.wt.) (Figure 2). The percent increment of the behavioral response was, however, similar in young and old rats. No statistically significant difference was present in the expression of nNOS in old versus young rats (0.25 6 0.03 vs 0.34 6 0.06, respectively; p ¼ NS) (Table 2) whereas, in contrast with nNOS, the hypothalamic expression of iNOS was significantly higher in old than in young rats (0.35 6 0.07 vs 0.23 6 0.01, p , .05) (Table 2). Evaluation of mRNA levels of hypothalamic anabolic and catabolic peptides related to the control of food intake and some receptors disclosed higher levels in old than in young rats for both NPY and AGRP (0.33 6 0.01 vs 0.27 6 0.02, p , .05; 0.34 6 0.01 vs 0.27 6 0.01, p , .05, respectively) (Table 2), whereas hypothalamic expressions of Y1-R and MCR-4 were significantly lower in old than young animals (Y1-R: 0.26 6 0.01 vs 0.47 6 0.1, p , .05; MCR-4: 0.29 6 0.04 vs 0.38 6 0.02, p , .05) (Table 2). No age- related difference was present for Y5-R (0.48 6 0.16 vs Figure 2. Six-hour cumulative food intake (g/b.wt. 3 1000) in old and young 0.39 6 0.07, p NS) and PRE-PRO-OX (0.23 6 0.04 vs rats treated with: saline (0.1 ml/kg, ip) þ saline (0.1 ml/kg, sc); saline (0.1 ml/kg, ¼ ip) þ EP51216 (80 lg/kg, sc); and molsidomine (20 mg/kg, ip) þ EP51216 (80 0.25 6 0.03, p ¼ NS) (Table 2). POMC mRNA levels were lg/kg, sc). *p , .05 vs saline þ saline. **p , .05 vs saline þ EP51216. Values lower in old than in young rats (0.41 6 0.02 vs 0.52 6 are means 6 standard error of the mean. See text for details. ANOREXIA OF AGING AND NEUROPEPTIDES 319

Thus, in the hypothalamus of old rats, mapping of gene expression of neuropeptides, receptors, and enzymes, dif- ferently involved in the control of feeding, was sought to be of interest. Another goal was then to correlate, whenever possible, this hypothalamic mapping to the consummatory response induced in old rats and dogs, and their young counterparts, by an analog of GHS, which belongs to a family of synthetic GHS and orexigenic peptides, whose prototype is the natural gastric-derived peptide, ghrelin (14). Downloaded from https://academic.oup.com/biomedgerontology/article/61/4/315/607480 by guest on 29 September 2021 In the present study, the mRNA levels of GHS-R were significantly lower in old than in young rats, though hypo- thalamic ghrelin expression was superimposable in both animal groups. It is noteworthy that this age-associated decrease in hypothalamic expression of GHS-R has also been reported in humans (21), and likely accounts for the blunted GH response to GHS present in elderly persons (22–24), even if Englander and colleagues (25) have shown that GH release in response to exogenous ghrelin is en- hanced in the aged rat. In the hypothalamus, ghrelin is expressed in only few neurons adjacent to the third ventricle, located between the Figure 3. Six-hour cumulative food intake (g/kg b.wt.) in old and young dogs dorsal, ventral, paraventricular, and arcuate nuclei (26). treated with: saline (0.1 ml/kg, iv) þ saline (0.1 ml/kg, sc); molsidomine (2 mg/ Thus, the low hypothalamic mRNA of this peptide might kg, iv) þ saline (0.1 ml/kg, sc); saline (0.1 ml/kg, iv) þ EP51216 (500 lg/kg, sc); explain our inability of pinpointing a statistically significant and molsidomine (2 mg/kg, iv) þ EP51216 (500 lg/kg, sc). *p , .05 vs saline þ saline in old dogs. **p , .05 vs saline þ saline in old dogs. Values are means 6 difference between young and old rats. standard error of the mean. See text for details. Alternatively, as hypothalamic ghrelin has been proposed to control energy homeostasis, whereas circulating ghrelin Studies in Dogs would be mainly implicated in stimulation of appetite and Baseline food intake in young and old dogs (means 6 food intake (27), a different, though complementary, role of SEM of 6-hour evaluation) was statistically different, young central and peripheral ghrelin may not be ruled out. In this animals eating much more (21.00 6 2.30 g/kg b.wt.) context, a predominant impairment of the gastric synthesis/ (Figure 3) than old dogs (3.60 6 1.40 g/kg b.wt., p , .05). release component of the peptide would account for the EP51216 markedly stimulated food intake in old dogs decreased plasma ghrelin levels, as also reported in elderly (9.60 6 1.60 g/kg b.wt. vs 3.60 6 1.40 g/kg b.wt., p , .05) persons (28), though an increased stomach ghrelin produc- (Figure 3), but failed to do so in young dogs (22.10 6 2.00 tion and secretion has also been reported in the aged rat (29). g/kg b.wt. vs 21.00 6 2.30 g/kg b.wt., p ¼ NS). Baseline food intake in young and old rats was not Molsidomine did not modify either baseline food intake statistically different. These results, however, have to be (3.51 6 1.80 g/kg b.wt. vs 3.60 6 1.40 g/kg b.wt., 22.30 6 considered in the experimental context of a short-term 1.60 g/kg b.wt. vs 21.00 6 2.30 g/kg b.wt., respectively, diurnal evaluation of food intake, when rodents, in contrast p ¼ NS) or the orexigenic activity of GHS in aged and with the nocturnal behavior, eat less. Young dogs, instead, young dogs (10.04 6 1.92 g/kg b.wt. vs 9.60 6 1.60 g/kg eat once daily and, especially, more than old dogs; thus, the b.wt., 23.20 6 1.10 g/kg b.wt. vs 22.10 6 2.00 g/kg b.wt., feeding conditions of young and old humans are more respectively, p ¼ NS) (Figure 3). properly represented by young and old dogs. EP51216 stimulated food intake in both young and old rats, and, in line with the decrease of hypothalamic expres- DISCUSSION sion of GHS-R in the latter, its orexigenic activity was lower The complexity of the neural regulation of food intake in in old than in young animals. Similarly, in old dogs, food mammals is now unanimously recognized. Blood-borne intake was markedly stimulated by EP51216, which, how- peripheral metabolic signals, originated especially from ever, failed to do so in young dogs, whose voracious ap- lipid stores and the gastrointestinal tract, regulate appetite petite was likely maximal under baseline conditions. and energy expenditure via effector neurons mainly located The major neuronal populations thought to be responsible in the mediobasal hypothalamus (19). for integrating the peripheral ghrelin-mediated feeding signal Up to now, the functional organization of the central con- are the orexigenic NPY/AGRP neurons (stimulation) and the trol of food intake and the periphery-brain interactions have anorexigenic POMC/CART neurons (inhibition) (14). been better characterized in young than in old animals, and In the ARC, GHS-R mRNA is mainly expressed by NPY the same is true for the human counterpart (1,2,20). It would neurons (30,31), a finding consonant with the initial obser- seem that the anorexia or hyporexia of elderly persons vation by Dickson and Luckman (32) of c-fos induction in recognizes many factors, e.g., sensory impairment, social NPY neurons following GHS administration. Based on this isolation, decline of physiological functions, diseases (20). notion, the marked reduction of hypothalamic expression of 320 RIGAMONTI ET AL.

GHS-R in old rats might involve NPY neurons. Hence, the levels in the mouse brain (40), an orexin resistance may not increase of hypothalamic expression of NPY in old rats is be ruled out in elderly persons. In our study, however, no interesting, but apparently paradoxical, unless it is inter- statistically significant difference was present in young preted to mean a compensatory homeostatic mechanism versus old rats in the hypothalamic expression of PRE-PRO- aimed to maintain a normal feeding pattern in aged animals. OX, the precursor of A and B. Concurrent increase of mRNA levels of AGRP in the same In contrast to orexin and ghrelin interactions, no direct animals supports this view. In this context, the blunted anatomical and functional relationships between ghrelin stimulation of food intake by GHS in old rats (and dogs) and MCH are known. In the present study, similar mRNA may be due to GHS inability to further stimulate NPY neu- levels of MCH in the hypothalamus of young and old rats rons, maximally activated in the anorexia of aging. Inferen- suggest a negligible role of this orexigenic peptide in the Downloaded from https://academic.oup.com/biomedgerontology/article/61/4/315/607480 by guest on 29 September 2021 tial support to our findings is the observation that NPY-CSF pathogenesis of the anorexia of aging. titers are increased in old women, although they are un- Leptin is a functional antagonist of ghrelin (41). Although altered in old men (33). adipose tissue leptin mRNA expression reportedly increases Presence in old rats of a reduced hypothalamic expression with age in rats (42) and mice (43), studies in rats (42) of Y1-R is intriguing, though receptor down-regulation by and pigs (44) did not find an increase in serum leptin titers enhanced NPY release cannot be ruled out. However, irre- with aging. Results from studies in humans are conflicting spective of any interpretation, this finding agrees with the (9,44–49). reported diminished orexigenic response to NPY, when in- To our knowledge, the present is the first report on leptin jected into the paraventricular nucleus (PVN) of old rats (34). mRNA levels in the hypothalamus of aged rats. As no Evidence has been provided for interactions in the NPY/ statistically significant difference was present between AGRP, ghrelin/GHS, and POMC systems in the central con- young and old rats, a minor role of (hypothalamic) leptin trol of feeding; hence, alterations of these integrated neural in the pathogenesis of anorexia of aging may be envisaged, circuitries might contribute to the anorexia of aging (20). though an age-related alteration of post-transductional Morphological and electrophysiological studies have dis- mechanisms of the leptin receptor may not be ruled out closed the existence of direct synaptic contacts between (leptin resistance?) (46). NPY terminals and neighboring POMC cell bodies and NO is a short-lived gas product produced endogenously dendrites, and in all support the notion that NPY exerts an by the action of endothelial, neuronal, and inducible NOS inhibitory tone over the anorexigenic POMC projections, on the amino acid L-arginine. A wealth of animal data most likely via a Y1-R-mediated effect (35). In our study, strongly support a role for NO as a transducer of the effects the increased hypothalamic expression of NPY/AGRP in of a number of hormones in the control of feeding, e.g., old rats might account for the reduced POMC mRNA levels, leptin, NPY, and also GHS/ghrelin (16,50). In our study, though we presently ignore the meaning of the reduction of molsidomine, an NO donor, markedly enhanced the hypothalamic expression of Y1-R. orexigenic effect of GHS in both young and aged rats, In the context of the POMC system, an additional mecha- though to a lesser extent in the latter. nism for the anorexia of aging rests on the increase of hypo- The possibility that in rats a declining NO tone plays thalamic expression of AGRP. The latter colocalizes with a role in the pathogenesis of the anorexia of aging may be NPY in a subset of ARC-NPY neurons and acts in synergy ruled out. In fact, the percent increment of this response was with the inhibitory action of NPY on POMC neurons, being similar in young and old animals. In contrast, molsidomine the endogenous antagonist of MCR-3 and MCR-4 receptors, failed to enhance in old dogs GHS-induced feeding, though which transduce the anorexigenic effects of a-MSH (36). a low dose of the NO donor was used in these experiments. Reductions of POMC and MCR-4 mRNA levels present in In agreement with these observations, no statistical old rats of our study strengthen the purported existence of difference was observed in the expression of nNOS in the ‘‘anti-anorexigenic’’ compensatory mechanisms aimed at hypothalamus of old rats, though the small number of maintaining a ‘‘normal’’ feeding pattern in aging. animals might have prevented proper evaluation. Central administration of CART potently suppresses Although there is no unanimous consensus, there is feeding and blocks the increase in food intake induced by evidence that NOS levels and activity increase with aging in NPY; moreover, immunohistochemical findings would indi- rats in many sites, including the brain, kidney, and skeletal cate the existence of functional interactions between NPY muscle (51–55). Consistent with this, L-arginine analogs and CART in the control of food intake (37,38). However, have an enhanced inhibitory effect on kidney function of old in our study, the hypothalamic expression of CART was rats (56), and exert a greater inhibition of food intake in old similar in young and old rats, suggesting that the increased than in young mice (53). These inferential data suggest that NPY mRNA levels were divorced in aged rats from a declining NO tone is not a cause of the anorexia of aging, functional inhibition of CART neurons. but rather the latter in rodents is associated with an increase The interaction between ghrelin and orexins is well in NO tone, aimed at stimulating feeding to counteract other characterized. In fact, ghrelin-immunoreactive axonal ter- anorexigenic inputs (20). minals directly synapse with orexin-producing neurons and Supporting this proposition and in apparent contrast with anti-orexin immunoglobulin G attenuates ghrelin-induced nNOS results, in this study, hypothalamic expression of feeding (26). Interestingly, in humans, plasma orexin-A iNOS in aged rats was significantly higher than in young concentrations correlate with age (39) and, in view of the rats, thus providing experimental support to the intriguing reported age-related decline in 2 mRNA ‘‘nitric oxide hypothesis of aging’’ of McCann and col- ANOREXIA OF AGING AND NEUROPEPTIDES 321

leagues (57). According to this view, aging is associated ACKNOWLEDGMENTS with a progressive increase of iNOS levels in specific neu- This work was supported in part by Ministero Italiano dell’Universita`e roendocrine areas of the central nervous system, resulting in della Ricerca, (Fondo di Investimento per la Ricerca di Base, progetto an overproduction of toxic NO. The neurotoxic effects of RBNE01JKLF). NO would be exerted on neurons of the PVN and ARC, two Address correspondence to Eugenio E. Mu¨ller, MD, PhD, Department of Medical Pharmacology, University of Milan, via Vanvitelli 32, 20129 areas playing a key role in the control of food intake. The Milan, Italy. E-mail: [email protected] induction of iNOS in the feeding-regulating regions of the mediobasal hypothalamus would likely contribute to anorexia of aging and also to the age-related impairment REFERENCES of the neuroendocrine system, as somatotropic and gonad- 1. 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