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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:315-322 (2006)
© 2006 The Gerontological Society of America

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

Antonello E. Rigamonti, Sara M. Bonomo, Diego Scanniffio, Silvano G. Cella and Eugenio E. Müller

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

Address correspondence to Eugenio E. Müller, MD, PhD, Department of Medical Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milan, Italy. E-mail: eugenio.muller{at}unimi.it


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
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 peptides, 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.

AGING is associated with impairment in the ability of an organism to maintain energy balance in response to homeostatic perturbations. The reasons for this loss of regulation of energy balance are multifactorial, though one of the key factors appears to be a loss of appetite, termed "the anorexia of aging" (1,2). In humans, the anorexia of aging too is caused by numerous factors, including sensory impairment, social isolation, and psychological and physiologic factors; disease states further magnify it (3).

Clinical studies have shown that healthy older people fail to respond to over- or underfeeding with the compensatory changes in eating common to younger people. Hence, elderly people report less hunger after an overnight fast, and, reciprocally, a greater degree of satiation to meals than do the young people. This apparent insensitivity to metabolic cues can lead to inappropriate weight loss in response to acute or chronic illness or other stressors, resulting in greater morbidity and mortality in the geriatric population (4).

A circuitry of neurons in the arcuate nucleus (ARC) integrates blood-borne peripheral metabolic signals and regulates appetite and energy expenditure through actions on distal effector neurons. This circuit consists of neurons coexpressing the anabolic neuropeptides agouti-related peptide (AGRP) and neuropeptide Y (NPY), which synapse on and inhibit the firing of neurons coexpressing the catabolic neuropeptides, alpha-melanocyte stimulating hormone ({alpha}-MSH), derived from pro-opiomelanocortin (POMC), or cocaine-amphetamine regulated transcript (CART) (5). The AGRP/NPY and POMC/CART neurons synapse on each other; this complex neuronal network responds to many hormonal and metabolic signals and may be altered in the anorexia of aging (6).

Compared to young animals, aged male rats fail to increase food intake after a 72-hour fast, and are slow in regaining the lost body weight upon refeeding. This inability to maintain body weight is associated with an age-related deficiency of NPY messenger RNA (mRNA) and a blunted fasting-induced increase in NPY gene expression (7). With aging, ARC AGRP gene expression decreases, and CART mRNA increases, though POMC mRNA does not change (8). This altered neuropeptide gene expression profile may underlie the blunting of the counterregulatory response to fasting, likely mediated by leptin, an anorexigenic peptide produced by the adipose tissue (9).

Aging and obesity would produce leptin resistance, which attenuates the anorexigenic effects of exogenous leptin and leads to hyperleptinemia as the system strives towards homeostasis (10,11); thus the relatively hyperleptinemic state of aged animals likely blunts the sensitivity of the hypothalamic energy regulatory system, decreasing appetite even during episodes of negative energy balance (stress, acute or chronic illness) (12,13).

Recent evidence has implicated ghrelin, an orexigenic peptide produced by the gastric fundus, functionally related to growth hormone secretagogues (GHS), as a key regulator of food intake and energy balance (14). The orexigenic effect of ghrelin is mainly exerted via activation of NPY/AGRP neurons (15), though the involvement of other neuropeptides, particularly of the POMC system, cannot be ruled out.

To our knowledge, the ability of GHS/ghrelin to increase food intake of aged animals has not been investigated so far. Hence, the aim of the present study was to determine the orexigenic effect of GHS in young and old rats and dogs, alone or with molsidomine, a donor of nitric oxide (NO), as food intake stimulated by GHS/ghrelin is partially mediated by NO (16,17). Moreover, mRNA levels of a wide series of anabolic and catabolic peptides, receptors, and enzymes (i.e., ghrelin, GHS receptor [GHS-R], NPY, NPY receptors 1 and 5 [Y1-R and Y5-R], AGRP, POMC, CART, melanocortin receptor 4 [MCR-4], leptin, leptin receptor long, melanocortin concentrating hormone [MCH], pre-pro-orexin [PRE-PRO-OX], and neuronal and inducible NO-synthases [nNOS and iNOS, respectively]) were measured by reverse transcription–polymerase chain reaction (RT–PCR) in the whole hypothalamus of young and old rats, and related, whenever possible, to the behavioral response to the GHS.


   METHODS
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 Methods
Results
 Discussion
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Studies in Rats
Animals.-- Ten young (2-month-old; 275–300 g) and seven old (26-month-old; 800–1000 g) male Sprague Dawley rats (Charles River, Calco, Italy) were housed individually under controlled conditions (24°C, 60% humidity, artificial light from 7 AM to 7 PM) in a specific-pathogen-free environment. Standard dry diet (Mucedola S.p.A., Milan, Italy) and water were available ad libitum. Old rats, which survived from a huge colony of young animals, were apparently in healthy condition.

All experimental protocols, authorized by the Committee on Animal Care and Use of the University of Milan, met the Italian guidelines for Use of Laboratory Animals, which conform with the European Communities Directive of November 1986 (86/609/EEC).

Experimental design.-- To evaluate the orexigenic effect of GHS, different doses (80, 160, 320 µg/kg, sc) of EP51216, a GHS agonist we had previously investigated (18) (Europeptides, Argenteuil, France), or saline 0.9% (1 ml/kg, sc) were administered at 9 AM to overnight-fed young and aged rats. EP51216 (GAB-DTrp-(2Me-DTrp(2Me)-Trp(2Me)-Lys-NH2) has efficacy and potency close to those of hexarelin, and binds to GHS heart receptors; it does not interact with other neuropeptides (NPY, AGRP, and {alpha}-MSH) (personal communication, Dr. R. Deghenghi; Europeptides, 2000).

EP51216 (80 µg/kg) was also tested in combination with molsidomine (20 mg/kg, ip; Sigma-Aldrich, Milan, Italy), a NO donor drug (17) given 1 hour prior to the GHS peptide. A group of rats treated with molsidomine only was not used because the drug is inactive on food intake when administered alone in this species (17). Intracerebroventricular administration was ruled out as unfeasible because of the reduced number of animals.

After drug administration, all rats were put in their cages with a preweighed amount of food, and their food intake was carefully monitored every hour for 6 hours. Food intake was normalized to rat body weight (b.wt.).

Food-intake experiments were performed on different days with at least a 3-day interval between each administered dose, and with best efforts to avoid background noise and other stressful events. At the end of feeding experiments, after a wash-out period of about 15 days, rats were killed by decapitation, when they were in a fed state; the whole hypothalamus was then quickly dissected, later put in 300 µl of RNA (Celbio S.p.A., Milan, Italy), and stored at –20°C until processed for RNA extraction.

Total RNA isolation and RT–PCR assay.-- Total RNA was extracted from the hypothalamus according to the single-step acid guanidinium thiocyanate–phenol–chloroform extraction method. Integrity of extracted RNA was checked by electrophoresis with a 1% agarose gel containing ethidium bromide. First, strand complementary DNA (cDNA) was synthesized following standard procedures using 1 µg of total RNA extracted from each sample, with oligo dT18 (Gibco Life Technologies, Milan, Italy) and 100 U of Moloney murine leukemia virus reverse transcriptase (M-MuLV; Gibco Life Technologies). Reverse transcription was performed at 37°C for 50 minutes followed by an initial denaturation at 70°C for 15 minutes.

PCR amplification was performed with 2.5 U of Taq polymerase (Euro Taq; Celbio S.p.A.) and with synthetic gene-specific primers (Gibco Life Technologies) (Table 1). PCR was performed with a thermal cycler (Thermo Hybayd; Celbio S.p.A.) for a number of cycles that varied according to the primer used (Table 1) under the following conditions: denaturation at 95°C for 30 seconds, annealing at a specific temperature in relation to the primer used (Table 1), and extension at 72°C for 30 seconds. Electrophoretic analysis of DNA fragments was performed on a 2% agarose gel containing ethidium bromide, and was visualized by ultraviolet-induced fluorescence; this method showed that PCR at these cycles was linear. To normalize these results, the glyceraldehyde-6-phosphate dehydrogenase (GAPDH) gene (Table 1), was amplified. Conditions used were 30 seconds at 95°C, 60 seconds at 53°C, and 30 seconds at 72°C for 30 cycles. Negative control of the PCR was made by omitting the DNA from the mixture. The intensity of each band was quantified using a densitometer. The resulting densities of each gene were expressed relative to the corresponding densities of the GAPDH bands from the same RNA sample.


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

 
Studies in Dogs
Animals.-- Six old (10–14 years old; 9–13 kg b.wt.; two male and four female) and seven young (4–6 years old; 13–15 kg b.wt.; all male) well-trained beagle dogs were used. They were exercised routinely and were fed normal dry food (Diete standard; Charles River, Calco, Italy), once daily, at 4:00 PM, with water available ad libitum. Dogs were on a 12-hour light/dark regimen, with lights on at 7:00 AM. At the beginning of the study, the body weights of dogs were stable and they were free of any observable disease. Old female dogs were out of reproductive age. All experiments were carried out in conscious animals. Before the experiments, which were scheduled in a randomized order, animals were kept at rest in the laboratory for at least 1 hour. Protocols previously authorized by the Committee on Animal Care and Use of the University of Milan were strictly followed.

Experimental design.-- All experiments were performed following an overnight fast. To evaluate the orexigenic effect of GHS in young and aged dogs, all animals were administered EP51216 (500 µg/kg, sc) or saline (0.1 ml/kg, sc), at 9 AM. EP51216 (or saline) was also tested associated with molsidomine (2 mg/kg, iv), which was given 1 hour 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 doses induced severe adverse effects (hypotension). Therefore, the 2 mg/kg dose of the compound was used in the experiments.

After drug administration, all dogs were put in their boxes with a preweighed amount of food, and their food intake was monitored every hour for 6 hours. Food intake was normalized to dog b.wt. Because no consistent differences in food intake between male and female dogs were observed in the different experiments, data were pooled.

Statistical Analysis
Six-hour cumulative food intake was calculated and expressed (in g/kg b.wt.) as means ± standard error of the mean (SEM). Rats' hypothalamic mRNA levels of the different neuropeptides and receptors were expressed as means ± SEM of density ratios.

Statistical evaluation of the results of food-intake experiments and of neuropeptide and receptor mRNA levels was performed by using the Student–Newman–Keuls test. Values of p <.05 were taken to be statistically significant.


   RESULTS
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 Results
Discussion
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Studies in Rats
Baseline food intake in young and old rats was not statistically different (means ± SEM of 6-hour evaluation: 0.66 ± 0.30 g/kg b.wt. vs 0.70 ± 0.50 g/kg b.wt., p = not significant [NS], respectively) (Figure 2). EP51216 stimulated food intake in both young and old rats, and its orexigenic activity was dose-dependent and was higher in young than in aged animals (Figure 1). These results were consonant with the finding that the mRNA levels of GHS-R were significantly lower in old than in young rats (0.53 ± 0.03 vs 0.65 ± 0.03, respectively, p <.05) (Table 2), though ghrelin and leptin gene expression were superimposable in either animal group (ghrelin: 0.18 ± 0.03 vs 0.19 ± 0.03, p = NS; leptin: 0.18 ± 0.01 vs 0.19 ± 0.01, p = NS), and the same was true for leptin receptor long (0.30 ± 0.09 vs 0.18 ± 0.03, p = NS) (Table 2).


Figure 02
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  		Figure 2. Six-hour cumulative food intake (g/b.wt. x 1000) in old and young rats treated with: saline (0.1 ml/kg, ip) + saline (0.1 ml/kg, sc); saline (0.1 ml/kg, ip) + EP51216 (80 µg/kg, sc); and molsidomine (20 mg/kg, ip) + EP51216 (80 µg/kg, sc). *p <.05 vs saline + saline. **p <.05 vs saline + EP51216. Values are means ± standard error of the mean. See text for details

 

Figure 01
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  		Figure 1. Six-hour cumulative food intake (g/b.wt. x 1000) in old and young rats after administration of saline (0.1 ml/kg, sc) or different doses of EP51216 (80, 160, 320 µg/kg, sc). *p <.05 vs old rats. Values are means ± standard error of the mean. See text for details

 

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

 
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 ± 0.70 g/kg b.wt. vs 3.00 ± 1.10 g/kg b.wt.) than in young rats (4.26 ± 0.60 g/kg b.wt. vs 7.50 ± 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 ± 0.03 vs 0.34 ± 0.06, respectively; p = NS) (Table 2) whereas, in contrast with nNOS, the hypothalamic expression of iNOS was significantly higher in old than in young rats (0.35 ± 0.07 vs 0.23 ± 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 ± 0.01 vs 0.27 ± 0.02, p <.05; 0.34 ± 0.01 vs 0.27 ± 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 ± 0.01 vs 0.47 ± 0.1, p <.05; MCR-4: 0.29 ± 0.04 vs 0.38 ± 0.02, p <.05) (Table 2). No age-related difference was present for Y5-R (0.48 ± 0.16 vs 0.39 ± 0.07, p = NS) and PRE-PRO-OX (0.23 ± 0.04 vs 0.25 ± 0.03, p = NS) (Table 2). POMC mRNA levels were lower in old than in young rats (0.41 ± 0.02 vs 0.52 ± 0.02, p <.05) (Table 2), but no significant difference in the hypothalamic expression of CART and MCH was evident between the two age groups (CART: 0.44 ± 0.01 vs 0.48 ± 0.03, p = NS; MCH: 0.33 ± 0.1 vs 0.45 ± 0.05, p = NS) (Table 2).

Studies in Dogs
Baseline food intake in young and old dogs (means ± SEM of 6-hour evaluation) was statistically different, young animals eating much more (21.00 ± 2.30 g/kg b.wt.) (Figure 3) than old dogs (3.60 ± 1.40 g/kg b.wt., p <.05). EP51216 markedly stimulated food intake in old dogs (9.60 ± 1.60 g/kg b.wt. vs 3.60 ± 1.40 g/kg b.wt., p <.05) (Figure 3), but failed to do so in young dogs (22.10 ± 2.00 g/kg b.wt. vs 21.00 ± 2.30 g/kg b.wt., p = NS). Molsidomine did not modify either baseline food intake (3.51 ± 1.80 g/kg b.wt. vs 3.60 ± 1.40 g/kg b.wt., 22.30 ± 1.60 g/kg b.wt. vs 21.00 ± 2.30 g/kg b.wt., respectively, p = NS) or the orexigenic activity of GHS in aged and young dogs (10.04 ± 1.92 g/kg b.wt. vs 9.60 ± 1.60 g/kg b.wt., 23.20 ± 1.10 g/kg b.wt. vs 22.10 ± 2.00 g/kg b.wt., respectively, p = NS) (Figure 3).


Figure 03
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  		Figure 3. Six-hour cumulative food intake (g/kg b.wt.) in old and young dogs treated with: saline (0.1 ml/kg, iv) + saline (0.1 ml/kg, sc); molsidomine (2 mg/kg, iv) + saline (0.1 ml/kg, sc); saline (0.1 ml/kg, iv) + EP51216 (500 µg/kg, sc); and molsidomine (2 mg/kg, iv) + EP51216 (500 µg/kg, sc). *p <.05 vs saline + saline in old dogs. **p <.05 vs saline + saline in old dogs. Values are means ± standard error of the mean. See text for details

 

   DISCUSSION
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 Methods
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 Discussion
References
 
The complexity of the neural regulation of food intake in mammals is now unanimously recognized. Blood-borne peripheral metabolic signals, originated especially from lipid stores and the gastrointestinal tract, regulate appetite and energy expenditure via effector neurons mainly located in the mediobasal hypothalamus (19).

Up to now, the functional organization of the central control of food intake and the periphery-brain interactions have been better characterized in young than in old animals, and the same is true for the human counterpart (1,2,20). It would seem that the anorexia or hyporexia of elderly persons recognizes many factors, e.g., sensory impairment, social isolation, decline of physiological functions, diseases (20).

Thus, in the hypothalamus of old rats, mapping of gene expression of neuropeptides, receptors, and enzymes, differently 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).

In the present study, the mRNA levels of GHS-R were significantly lower in old than in young rats, though hypothalamic 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 enhanced in the aged rat.

In the hypothalamus, ghrelin is expressed in only few neurons adjacent to the third ventricle, located between the dorsal, ventral, paraventricular, and arcuate nuclei (26). Thus, the low hypothalamic mRNA of this peptide might explain our inability of pinpointing a statistically significant difference between young and old rats.

Alternatively, as hypothalamic ghrelin has been proposed to control energy homeostasis, whereas circulating ghrelin would be mainly implicated in stimulation of appetite and food intake (27), a different, though complementary, role of central and peripheral ghrelin may not be ruled out. In this context, a predominant impairment of the gastric synthesis/release component of the peptide would account for the decreased plasma ghrelin levels, as also reported in elderly persons (28), though an increased stomach ghrelin production and secretion has also been reported in the aged rat (29).

Baseline food intake in young and old rats was not statistically different. These results, however, have to be considered in the experimental context of a short-term diurnal evaluation of food intake, when rodents, in contrast with the nocturnal behavior, eat less. Young dogs, instead, eat once daily and, especially, more than old dogs; thus, the feeding conditions of young and old humans are more 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 expression of GHS-R in the latter, its orexigenic activity was lower in old than in young animals. Similarly, in old dogs, food intake was markedly stimulated by EP51216, which, however, failed to do so in young dogs, whose voracious appetite was likely maximal under baseline conditions.

The major neuronal populations thought to be responsible for integrating the peripheral ghrelin-mediated feeding signal are the orexigenic NPY/AGRP neurons (stimulation) and the anorexigenic POMC/CART neurons (inhibition) (14).

In the ARC, GHS-R mRNA is mainly expressed by NPY neurons (30,31), a finding consonant with the initial observation by Dickson and Luckman (32) of c-fos induction in NPY neurons following GHS administration. Based on this notion, the marked reduction of hypothalamic expression of GHS-R in old rats might involve NPY neurons. Hence, the increase of hypothalamic expression of NPY in old rats is interesting, but apparently paradoxical, unless it is interpreted to mean a compensatory homeostatic mechanism aimed to maintain a normal feeding pattern in aged animals. Concurrent increase of mRNA levels of AGRP in the same animals supports this view. In this context, the blunted stimulation of food intake by GHS in old rats (and dogs) may be due to GHS inability to further stimulate NPY neurons, maximally activated in the anorexia of aging. Inferential support to our findings is the observation that NPY-CSF titers are increased in old women, although they are unaltered in old men (33).

Presence in old rats of a reduced hypothalamic expression of Y1-R is intriguing, though receptor down-regulation by enhanced NPY release cannot be ruled out. However, irrespective of any interpretation, this finding agrees with the reported diminished orexigenic response to NPY, when injected into the paraventricular nucleus (PVN) of old rats (34).

Evidence has been provided for interactions in the NPY/AGRP, ghrelin/GHS, and POMC systems in the central control of feeding; hence, alterations of these integrated neural circuitries might contribute to the anorexia of aging (20).

Morphological and electrophysiological studies have disclosed the existence of direct synaptic contacts between NPY terminals and neighboring POMC cell bodies and dendrites, and in all support the notion that NPY exerts an inhibitory tone over the anorexigenic POMC projections, most likely via a Y1-R-mediated effect (35). In our study, the increased hypothalamic expression of NPY/AGRP in old rats might account for the reduced POMC mRNA levels, though we presently ignore the meaning of the reduction of hypothalamic expression of Y1-R.

In the context of the POMC system, an additional mechanism for the anorexia of aging rests on the increase of hypothalamic expression of AGRP. The latter colocalizes with NPY in a subset of ARC-NPY neurons and acts in synergy with the inhibitory action of NPY on POMC neurons, being the endogenous antagonist of MCR-3 and MCR-4 receptors, which transduce the anorexigenic effects of {alpha}-MSH (36). Reductions of POMC and MCR-4 mRNA levels present in old rats of our study strengthen the purported existence of "anti-anorexigenic" compensatory mechanisms aimed at maintaining a "normal" feeding pattern in aging.

Central administration of CART potently suppresses feeding and blocks the increase in food intake induced by NPY; moreover, immunohistochemical findings would indicate the existence of functional interactions between NPY and CART in the control of food intake (37,38). However, in our study, the hypothalamic expression of CART was similar in young and old rats, suggesting that the increased NPY mRNA levels were divorced in aged rats from functional inhibition of CART neurons.

The interaction between ghrelin and orexins is well characterized. In fact, ghrelin-immunoreactive axonal terminals directly synapse with orexin-producing neurons and anti-orexin immunoglobulin G attenuates ghrelin-induced feeding (26). Interestingly, in humans, plasma orexin-A concentrations correlate with age (39) and, in view of the reported age-related decline in orexin receptor 2 mRNA levels in the mouse brain (40), an orexin resistance may not be ruled out in elderly persons. In our study, however, no statistically significant difference was present in young versus old rats in the hypothalamic expression of PRE-PRO-OX, the precursor of orexins A and B.

In contrast to orexin and ghrelin interactions, no direct anatomical and functional relationships between ghrelin and MCH are known. In the present study, similar mRNA levels of MCH in the hypothalamus of young and old rats suggest a negligible role of this orexigenic peptide in the pathogenesis of the anorexia of aging.

Leptin is a functional antagonist of ghrelin (41). Although adipose tissue leptin mRNA expression reportedly increases with age in rats (42) and mice (43), studies in rats (42) and pigs (44) did not find an increase in serum leptin titers with aging. Results from studies in humans are conflicting (9,44–49).

To our knowledge, the present is the first report on leptin mRNA levels in the hypothalamus of aged rats. As no statistically significant difference was present between young and old rats, a minor role of (hypothalamic) leptin in the pathogenesis of anorexia of aging may be envisaged, though an age-related alteration of post-transductional mechanisms of the leptin receptor may not be ruled out (leptin resistance?) (46).

NO is a short-lived gas product produced endogenously by the action of endothelial, neuronal, and inducible NOS on the amino acid L-arginine. A wealth of animal data strongly support a role for NO as a transducer of the effects of a number of hormones in the control of feeding, e.g., leptin, NPY, and also GHS/ghrelin (16,50). In our study, molsidomine, an NO donor, markedly enhanced the orexigenic effect of GHS in both young and aged rats, though to a lesser extent in the latter.

The possibility that in rats a declining NO tone plays a role in the pathogenesis of the anorexia of aging may be ruled out. In fact, the percent increment of this response was similar in young and old animals. In contrast, molsidomine failed to enhance in old dogs GHS-induced feeding, though a low dose of the NO donor was used in these experiments. In agreement with these observations, no statistical difference was observed in the expression of nNOS in the hypothalamus of old rats, though the small number of animals might have prevented proper evaluation.

Although there is no unanimous consensus, there is evidence that NOS levels and activity increase with aging in rats in many sites, including the brain, kidney, and skeletal muscle (51–55). Consistent with this, L-arginine analogs have an enhanced inhibitory effect on kidney function of old rats (56), and exert a greater inhibition of food intake in old than in young mice (53). These inferential data suggest that a declining NO tone is not a cause of the anorexia of aging, but rather the latter in rodents is associated with an increase in NO tone, aimed at stimulating feeding to counteract other anorexigenic inputs (20).

Supporting this proposition and in apparent contrast with nNOS results, in this study, hypothalamic expression of iNOS in aged rats was significantly higher than in young rats, thus providing experimental support to the intriguing "nitric oxide hypothesis of aging" of McCann and colleagues (57). According to this view, aging is associated with a progressive increase of iNOS levels in specific neuroendocrine areas of the central nervous system, resulting in an overproduction of toxic NO. The neurotoxic effects of NO would be exerted on neurons of the PVN and ARC, two areas playing a key role in the control of food intake. The 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 of the neuroendocrine system, as somatotropic and gonadotropic functions (58).

Before closing, limitations of the present study have to be considered. First, our data mostly refer to neuropeptide mRNA levels and not to protein content, and a dissociation between transcription and translation has been occasionally reported in the hypothalamus of old rodents (58); hence, our data cannot be extrapolated tout court to the content and function of the protein. Second, the expressions of neuropeptide and receptor genes were evaluated in the whole hypothalamus, and not in hypothalamic areas (ARC and PVN) where neuropeptides/receptors are selectively expressed. Third, most of the results presented refer to rats, whose mechanisms of control on food intake may differ from those of humans. Fourth, some results are difficult to explain on the basis of a "rational" interpretative scheme (e.g., hypothalamic expressions of leptin and POMC). Fifth, age-related adiposity might be responsible per se for some alterations of hypothalamic neuropeptidergic/receptor pattern in old animals. However, despite these and other limitations, it is our opinion that broadening of the present investigations is warranted.

Conclusion
Old animals may remain weight stable when food is freely available, but metabolic perturbations affect their ability to regulate energy balance. A possible explanation rests on the activation, by old animals, of a feeding drive (stimulation of orexigenic pathways, NPY/AGRP, and inhibition of anorexigenic pathways, POMC/MCR-4), which is necessary to maintain a normal food intake under basal conditions, but unable to fit energy balance under stressful conditions (i.e., fasting or illness), because of the maximal recruitment of the adaptive orexigenic systems. The compensatory response, however, would be extremely unpredictable for the (primary?) impairment of the ghrelin system (decrease of GHS-R expression).

There are presently few drugs capable of stimulating appetite in elderly persons (20). Corticosteroids, antidepressants, dopamine receptor blockers, and anabolic steroids have been used as appetite stimulants in elderly palliative-care patients and have induced some body weight gain, but primarily through heavy side effects (3). Ghrelin administration increases hunger sensation and food intake in healthy young persons (59), but no data are presently available on the elderly population. In the present work, GHS markedly stimulated feeding in old dogs, an animal species whose neuroendocrine regulation is similar to that of humans. These findings suggest that clinical trials, assessing the usefulness of ghrelin and/or GHS for the treatment of appetite-related disorders of elderly persons, may be meaningful.


   Acknowledgments
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This work was supported in part by Ministero Italiano dell'Università e della Ricerca, (Fondo di Investimento per la Ricerca di Base, progetto RBNE01JKLF).


   Footnotes
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Decision Editor: James R. Smith, PhD

Received April 20, 2005

Accepted August 26, 2005


   References
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  1. Clarkston WK, Pantano MM, Morley JE, Horowitz M, Littlefield JM, Burton FR. Evidence for the anorexia of ageing: gastrointestinal transit and hunger in healthy elderly vs. young adults. Am J Physiol. 1997;272:R243-R248.
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