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Endocrinology Vol. 148, No. 3 1323-1329
Copyright © 2007 by The Endocrine Society

Ghrelin and Growth Hormone Secretagogue Receptor Expression in Mice during Aging

Yuxiang Sun, Jose Manuel Garcia and Roy G. Smith

Huffington Center on Aging, Department of Molecular and Cellular Biology (Y.S., R.G.S.); Division of Endocrinology, Department of Medicine (J.M.G., R.G.S.); and Michael E. DeBakey Houston Veterans Affairs Medical Center (J.M.G.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Yuxiang Sun, Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, M320, Houston, TX 77030. E-mail: yuxiangs{at}bcm.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In well-nourished humans, GH and IGF-I decline during aging, and the responsiveness of the GH axis to exogenous ghrelin is attenuated with age. Intriguingly, the GH/IGF-I axis is rejuvenated by chronic treatment with the ghrelin mimetic MK-0677, resulting in improvements in body composition, suggesting that frail elderly subjects might benefit from treatment with ghrelin and ghrelin mimetics. Mouse models are widely used to study the effects of ghrelin, but the impact of age on the ghrelin pathway is unclear. In this study, total and active ghrelin peptides were measured in plasma, and ghrelin mRNA was quantitated in brain tissue from different aged C57BL/6J mice. Surprisingly, plasma levels of ghrelin peptide slightly increased with age; ghrelin mRNA levels were similar in brains from mice aged 2, 6, 12, and 28 months but higher in mice aged 18 and 24 months. The tissue distribution of Ghsr1a mRNA (ghrelin receptor) was also characterized, and pituitary and brain exhibited the highest levels of expression. In the pituitary gland, the highest concentration of Ghsr1a mRNA was observed at age 1–2 months, it was lower at 6 months, and remained unchanged for up to 30 months of age. This result is consistent with the finding that GH release in response to exogenous ghrelin was not significantly different in mice aged 7–30 months. In the brain, Ghsr1a mRNA levels remained stable during aging. Hence, in C57BL/6J male mice, aging is not associated with changes in circulating ghrelin levels or changes in ghrelin receptor expression in the pituitary gland and brain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CHARACTERIZATION AND cloning of the GH secretagogue receptor (GHS-R) followed the development of small-molecule GH secretagogues (1, 2, 3). Two distinct mRNAs were identified, one encoding a full-length G protein-coupled receptor (GHS-R1a) with seven transmembrane domains (TMs) and the other a truncated form (GHS-R1b) lacking TM6 and TM7. The cloning of GHS-R1a subsequently led to the discovery of an endogenous agonist called ghrelin that was isolated from the stomach (4). Like the synthetic GHS-R1a agonists, ghrelin stimulates GH release and appetite (4, 5, 6, 7), and our data in Ghsr–/– mice show unambiguously that these properties are dependent upon expression of the GHS-R (8).

The concentration of ghrelin in plasma increases in humans during fasting and declines after a meal (9). Indeed, ghrelin mRNA levels in the stomach of rats increase during a 48-h fast and decrease after feeding (10). Ghrelin is structurally unusual because it occurs naturally as an n-octanoylated peptide, and the octanoyl moiety on serine-3 of the 28-amino-acid peptide is essential for activation of GHS-R1a (4). Both the octanoylated peptide (active ghrelin) and desacyl-ghrelin (inactive) are present in serum. Intriguingly, intracerebroventricular administration of desacyl-ghrelin stimulates food intake by a pathway that is not mediated by the GHS-R (11). The ratio of active ghrelin to total ghrelin peptide varies in conditions such as cancer-induced cachexia, obesity, and renal failure (12, 13, 14), suggesting that this ratio is an indicator of energy balance (13).

Aging is associated with reduced body weight, muscle mass, and bone density; aging is also associated with a decrease in appetite and lean body mass (15, 16). Both rodent and human studies have shown that aging is correlated with a decrease in GH and IGF-I (17, 18, 19). The mechanisms leading to age-dependent attenuation of the GH/IGF-I axis and changes in body composition are unknown; however, clinical studies in elderly humans demonstrated that chronic daily administration of the ghrelin mimetic MK-0677 restored the pulse amplitude of episodic GH release and serum IGF-I levels to those of young adults (18). Treatment for 12–18 months was accompanied by increased bone density at the femoral neck (20). Furthermore, elderly patients treated with MK-0677 for 6 months after repair of hip fracture showed greater improvement relative to placebo in lower-extremity functional performance measures and in the ability to live independently (21). In old mice, chronic administration of a ghrelin mimetic partially restored cellularity of the thymus and increased resistance to tumor growth (22). These reports show that ghrelin mimetics rescue different aspects of the aging phenotype, which led to our hypothesis that aging is associated with reduced production of ghrelin or deficits in endogenous ghrelin signaling.

Studies to determine whether ghrelin levels change during aging have been inconclusive. For example, Rigamonti et al. (23) reported a decline in fasting ghrelin levels in elderly humans compared with young controls, but Sturm et al. (24) found that ghrelin levels were the same in young and elderly females. The opposing conclusions could be explained by differences in body mass index. In the Rigamonti study, body mass indexes were different between the two groups, whereas, in the Sturm study, the different aged groups were matched according to body weight. However, in both studies, the authors measured total ghrelin peptide and not active ghrelin levels. Similarly, rodent studies have produced inconclusive results. Kappeler et al. (25) showed an increase in total ghrelin peptide levels in Wistar and Lou C/Jall rats with aging. Liu et al. (26) found no correlation between total ghrelin in serum and age in C57BL/6 mice up to 6 months of age and a decline in gastric ghrelin mRNA levels at 19 months. To address the discrepancies regarding changes in ghrelin signaling during aging, we measured plasma active and total ghrelin levels and tissue ghrelin mRNA in C57BL6/J mice during aging.

The dose dependence of ghrelin and ghrelin mimetic-induced GH release is attenuated during aging in humans (18, 27, 28, 29, 30). This rightward shift in the dose-response curve could be explained by reduced levels of the ghrelin receptor (GHS-R) or less efficient signal transduction. In situ hybridization using nonoverlapping oligonucleotides for detection of GHS-R mRNA expression and RNase protection assays showed that, besides being expressed in the pituitary and the brain (including specific areas of the hypothalamus, midbrain, and hippocampus), low levels of GHS-R mRNA are found in the pancreas (31, 32). Although Ghsr mRNA expression has been reported to occur broadly in peripheral tissues of rodents, in many of these studies, the primers selected for RT-PCR analysis were not designed to discriminate between mRNAs encoding GHS-R1a and GHS-R1b, which is important because only the former is activated by ghrelin and ghrelin mimetics. GHS-R1a is a full-length functional G-protein-coupled receptor requiring mRNA derived from coding exons 1 and 2, whereas GHS-R1b lacks TM6 and -7 encoded by exon 2. Therefore, we performed real-time RT-PCR analysis to quantitate Ghsr1a mRNA in tissues from mice and used Ghsr–/– mice as an additional control for PCR artifacts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and materials
Different-age male C57BL6/J mice were purchased from the National Institute on Aging (Bethesda, MD). All mice were bred from the same colony, and each experimental group of mice was shipped to Baylor College of Medicine on the same day. The Ghsr–/– mice were generated as previously described (8); 6-wk-old N10 male littermate pairs were used in the real-time RT-PCR analysis to measure tissue distribution. The animals were maintained in standard vivarium housing conditions at 20 C. The mice were individually housed for at least 2 wk before the experiments were initiated. The animals were inspected upon dissection; the ones with obvious abnormality (tumors, enlarged organs, etc.) were eliminated from the experiments. All procedures used in animal experiments were approved by Institutional Animal Care and Use Committee of Baylor College of Medicine.

Real-time quantitative RT-PCR analysis
Mice were fasted for 24 h and then killed for tissue isolation. Total RNA was isolated using the Trizol method. The coding sequence and chromosome contiguous sequence for each gene of interest were acquired through the NCBI public database and used to map exon-exon junctions. Probes were designed to transcend a junction to reduce the impact of potential genomic DNA contamination in RNA samples. Gene-specific primers and TaqMan probes were designed with Primer Express 2.0 (Applied Biosystems, Foster City, CA) using default settings (primer melting temperature was 58–60 C, and probe melting temperature was 10 C higher; GC content = 30–80%; length was 9–40 nucleotides). The primers/probes were as follows: Ghsr forward primer 5'-GGACCAGAACCACAAACAGACA-3', reverse primer 5'-CAGCAGAGGATGAAAGCAAACA-3', and TaqMan probe 5'-6FAM-TGAAGATGCTTGCTGTGGTG-MGB-3'; and ghrelin forward primer 5'-GGCAGGCTCCAGCTTCCT-3', reverse primer 5'-CTGGTGGCTTCTTGGATTCC-3', and TaqMan probe 5'-6FAM-AAAGCCCAGCAGAGAA-MGB-3'.

Assays were mixed at final concentrations of 900 nM primer and 250 nM probe. RT was performed on 1 µg total RNA with random primers, using TaqMan RT reagents (Applied Biosystems), as recommended by the manufacturer. One tenth of the RT reaction was used as a template for the subsequent PCR, which was carried out in triplicate. The reaction consisted of TaqMan universal master mix, template cDNA, and target assay mix as described above. Thermal cycling was carried out with an ABI prism 7000 sequence detection system (Applied Biosystems) under factory defaults (50 C for 2 min, 95 C for 10 min, and 40 cycles at 95 C for 15 sec and 60 C for 1 min). Threshold cycle number (CT) was defined as fluorescence values, generated by cleavage of the probe, exceeding baseline. For the absolute quantitation of Ghsr1a transcript copy, numbers were quantified as a comparison of measured CT values for each reaction compared with a standard curve generated from plasmid clone of Ghsr1a cDNA. The Ghsr1a cDNA transcripts were generated using MEGAScript T7 and MEGAClear (Ambion, Inc., Austin, TX). Copy number was then extrapolated from the curve of known transcript values with the ABI Prism 7000 software (Applied Biosystems). For the relative quantitation of ghrelin, the {Delta}{Delta}CT method was used. GAPDH and 18S RNA were used as internal controls.

Hormone assays
Mice were fasted for 24 h, and blood was drawn from the tail and collected in EDTA-containing tubes and kept at 4 C during processing. Samples were centrifuged at 3000 rpm for 30 min; hydrochloric acid and phenylmethylsulfonyl fluoride were immediately added to the plasma. Samples were aliquoted into polypropylene vials and stored at –80 C until assayed. Total plasma ghrelin levels were measured by RIA using kits purchased from LINCO Research (St. Charles, MO). This RIA kit uses a polyclonal antibody raised in rabbit against both octanoylated and des-octanoylated ghrelin and [125I]ghrelin as the tracer. The lower limit of detection was 80 pg/ml, and the intraassay coefficient of variation was 4%. Active ghrelin levels were measured by another commercially available RIA kit from LINCO Research. This RIA kit uses an antibody raised in guinea pig against octanoylated ghrelin and [125I]octanoylated ghrelin as the tracer. This assay has been found to be highly specific for active ghrelin with less than 0.1% cross-reactivity for des-octanoyl ghrelin, and no cross-reactivity with ghrelin 14–28, motilin-related peptide, leptin, insulin, glucagon, or glucagon-like peptide 7–36. The lower detection limit was 10 pg/ml. The intraassay coefficient of variation was 5.3%. For all hormones, plasma from an equal number of animals from each group was included in each assay. Ghrelin activity was defined as active ghrelin/total ghrelin peptide expressed as a percentage level [(active ghrelin x 100)/total ghrelin].

Plasma IGF-I was measured by a mouse/rat IGF-I RIA kit (Diagnostic Systems Laboratories, Inc., Webster, TX). To measure the GH response to ghrelin, mice were injected ip with pentobarbital (50 mg/kg body weight); 15 min later, 100 µl physiological saline with 0.5 mg/kg ghrelin or GHRH (Phoenix Pharmaceuticals, Inc., Belmont, CA), was injected ip. Blood was collected by tail bleeding at 0, 5, and 15 min after ghrelin injection. GH was measured in plasma samples using a rat GH enzyme immunoassay kit (91% cross-reactivity with mouse) (ALPCO Diagnostics, Salem, NH).

Data analysis
Data are presented in all figures as mean ± SEM. The number of subjects is indicated by n. In all experiments, we have at least three animals in each age group. Most experiments were repeated with different groups of animals of the same age. The figures shown are representative. Statistical significance between the groups was evaluated by ANOVA. Correlations between plasma ghrelin levels and age were obtained by nonparametric Spearman’s correlations when the data were not normally distributed. Statistical analysis was performed using either SigmaStat 3.0 or SPSS version 12.00 software for Windows (SPSS Inc., Chicago, IL). P < 0.05 was considered as statistically significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma ghrelin peptide levels in C57BL/6J mice at different age
The plasma levels of active ghrelin and total ghrelin were similar in male (n = 45) and female (n = 25) animals; hence, the data are presented together. Total ghrelin peptide levels positively correlated with age (Fig. 1AGo; r = 0.49; P ≤ 0.001), and a correlation for active ghrelin did not quite reach statistical significance (Fig. 1BGo; r = 0.23; P = 0.059). There was no correlation between ghrelin activity (active ghrelin/total ghrelin peptide x 100) and age (Fig. 1CGo; r = –0.14; P = 0.23).


Figure 1
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  		FIG. 1. Total ghrelin peptide, active ghrelin, and ghrelin activity in plasma from aging mice. C57BL mice were fasted for 24 h before bleeding; n = 70 (45 males and 25 females). Correlation is expressed by r value, and statistical significance is expressed by P value. A, Total ghrelin; B, active ghrelin; C, ghrelin activity = active ghrelin x 100/total ghrelin peptide.

 
Ghrelin gene expression in brains of different-age C57BL/6J mice
Plasma concentrations of ghrelin do not necessarily reflect ghrelin action on target tissues because RT-PCR studies in human tissues show that ghrelin mRNA is produced locally in most peripheral tissues as well as in the brain (33). Receptors for ghrelin are highly localized to specific centers of the brain involved in regulating GH release, appetite, and cognitive function; therefore, age-dependent changes in local ghrelin expression could have an impact on function in the central nervous system. Using ghrelin-knockout mice as RT-PCR specificity controls, we determined that 40 cycles was optimal for specific amplification of ghrelin mRNA. Ghrelin mRNA levels were measured in brains from young (2–3 months), middle-aged (12–18 months), and old (24 and 28 months) mice by quantitative RT-PCR. Brain ghrelin mRNA expression was similar in the 2-, 6-, 12-, and 28-month-old groups but was significantly higher in mice aged 18 and 24 months (Fig. 2Go).


Figure 2
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  		FIG. 2. Changes in expression of ghrelin mRNA in brains of C57BL/6J mice during aging. RNA was isolated from the brains of 2-, 6-, 12-, 18-, 24-, and 28-month-old male mice and subjected to real-time RT-PCR (n = 3 per group; *, P < 0.05, 12 vs. 18 months and 24 vs. 28 months).

 
Tissue distribution of Ghsr1a mRNA
We performed quantitative RT-PCR analysis to investigate the tissue distribution of Ghsr1a mRNA in mice. The PCR probes were designed to prime at exon boundaries, and tissues from Ghsr–/– mice were used as specificity controls. We determined that specific amplification of Ghsr1a mRNA was detected at 40 cycles. The highest concentration of Ghsr1a mRNA was found in the pituitary and brain. Markedly lower levels of Ghsr1a mRNA were detected in the heart, thymus, lung, adrenal, small intestine, spleen, pancreas, and kidney (Fig. 3Go). The expression levels are extraordinarily low. To give some perspective, the Scatchard analysis on rat pituitary cell membranes showed that the concentration of GHS-R binding sites was 2.3 fmol/mg protein (34). Additionally, it is important to note that Ghsr1a mRNA was not detected in liver, epididymal fat, brown fat, skeletal muscle, stomach, thyroid, salivary gland, bladder, and skin.


Figure 3
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  		FIG. 3. Tissue distribution of Ghsr1a mRNA. Representative results of real-time RT-PCR analysis of tissues from 6-wk-old N10 littermate male mice pairs (Ghsr+/+ and Ghsr–/–). Ghsr-1a mRNA was not detected in liver, epididymal fat, brown fat, skeletal muscle, stomach, thyroid, salivary gland, bladder, and skin of Ghsr+/+ mice. Ghsr-1a mRNA levels in the same tissues tested above from Ghsr–/– mice were all undetectable after 40 amplification cycles. The knockout (KO) data are in the figure just to show that there was no detectable expression in any of the tissues from Ghsr–/– mice.

 
Ghsr1a mRNA in pituitary glands and brains of different-age C57BL6/J mice
To assess whether changes in Ghsr expression occurred during aging, we measured Ghsr1a mRNA concentrations in the pituitary gland and brain by quantitative RT-PCR. Three independent groups of C57BL6/J male mice were selected, and all showed similar trends of Ghsr1a expression levels. The pooled data are shown in Fig. 4Go. In the brain, there was no significant difference with age. In the pituitary gland, the highest level was detected at 1–2 months of age. By 6 months of age, the level had declined; there were variable levels of expression in pituitary from 6–30 months of age, but no significant difference was detected among these groups.


Figure 4
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  		FIG. 4. Ghsr1a mRNA expression in the pituitary gland and brain of 1- to 30-month-old male C57BL/6J mice. Ghsr1a mRNA was quantitated by real-time RT-PCR; n = 7–12 for each age group. The pituitary expression in 1- to 2-month-old mice was significantly higher than that of other ages (P < 0.001). There were variable levels of expression in pituitary from 6 to 30 months of age, but they were not significantly different between groups in the brain (P > 0.05).

 
Body weight and plasma IGF-I level
MK-0677 activates GHS-R, enhancing the GH/IGF-I pathway, which may be beneficial during aging (2). In humans, GH and IGF-I levels gradually decline with age, and there is a significant decrease, even in middle age, when compared with young individuals (35). We compared body weights and plasma IGF-I levels in C57BL/6J male mice aged 3, 6, 8, 11, 12, 17, 18, 23, 24, and 29 months. Unexpectedly, beyond 3 months of age, body weights remained constant, and plasma IGF-I levels did not decrease (data not shown).

GH response to acute ghrelin injection
In humans, the magnitude of the stimulatory effect of ghrelin mimetics and ghrelin on GH release is attenuated in elderly subjects, although the attenuation may be overcome by increasing the dose (27, 28, 29). Acute administration of ghrelin to wild-type animals stimulates GH release. We showed that ghrelin’s effect on GH is mediated by GHS-R1a by demonstrating that in contrast to wild-type mice, Ghsr–/– mice were refractory to ghrelin stimulation of GH release (8). To address whether ghrelin-induced GH release is attenuated in C57BL6/J mice during aging, we treated anesthetized male mice of different ages with ghrelin and measured GH in plasma at 0, 5, and 15 min after ip ghrelin injection. Consistent with the lack of age-related differences in Ghsr1a expression in the pituitary gland in these mice (Fig. 4Go), GH release in response to exogenous ghrelin did not change as a function of age (Fig. 5Go).


Figure 5
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  		FIG. 5. Magnitude of ghrelin-induced GH release does not change during aging in C57BL/6J mice. Male mice aged 7, 12, 16, and 30 months were anesthetized (ip) with pentobarbital. Fifteen minutes later, 100 µl physiological saline with 10 µg ghrelin was injected ip. Blood was collected by tail bleeding at 0, 5, and 15 min after ghrelin injection to measure GH levels. There was no statistically significant difference between any of the groups (P > 0.05).

 
To address whether lower doses would unveil minor differences, we have tested 10 and 50% of the dose used. The stimulatory effects of different doses were indistinguishable, showing that there was no difference between young and old mice (data not shown). Furthermore, we also tested the GH stimulatory effect of different doses of GHRH. There appears to be a dose response in the young mice but not in the old mice, and at a high dose of GHRH (0.5 mg/kg), the response of old mice appears to be modestly lower than that of young mice (P = 0.07, data not shown). These data further confirm that the GH stimulatory effect is modestly age dependent with GHRH but age independent with ghrelin.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The production of desacyl-ghrelin and the specific enzymes that regulate acylation and hydrolysis of ghrelin are unknown. Ghrelin decreases in obesity (13) and in renal failure (14) but increases in patients with cancer-induced cachexia (12). Whether age has a role in determining the ratio between octanoylated ghrelin (active ghrelin) and desacyl-ghrelin had not previously been determined in rodents. In this study, we showed that total ghrelin peptide in plasma slightly increased from 2 to 28 months of age (P < 0.001, Fig. 1AGo); active ghrelin also increased and almost reached statistical significance (P = 0.059, Fig. 1BGo). Our ghrelin results are consistent with those found in Wistar and Lou C/Jall rats where total ghrelin levels were higher in 24-month-old rats than in 3- and 12-month-old rats (25). Other investigators showed some changes in serum total ghrelin levels in C57BL/6J mice from age 3 d to 6 months (26). However, no detail measurements were made beyond 6 months of age, which is where we detected the increase in total and active ghrelin levels. In male Brown Norway rats, basal ghrelin levels are comparable between 4 and 25 months of age, but fasting-induced increases in ghrelin are less robust in aging rats (36). We found no correlation between ghrelin activity and age (Fig. 1CGo), suggesting that aging is not a significant determinant in the conversion of octanoylated ghrelin to desacyl-ghrelin. We also found that ghrelin activity is much lower in C57BL6/J mice than that in humans (12), suggesting that regulation of ghrelin production might be species dependent.

It is likely that circulating ghrelin levels are not a good reflection of ghrelin action on target tissues; ghrelin is made in many tissues, suggesting its role also as a paracrine or autocrine hormone. Therefore, we measured ghrelin mRNA in the brain. In mice aged from 2–28 month of age, ghrelin mRNA levels in brain did not decline (Fig. 2Go). The transient increase in brain ghrelin mRNA expression observed at 18 and 24 months may reflect a compensatory response to negative energy balance during aging.

Using Ghsr–/– mice as controls, we investigated the tissue distribution of Ghsr1a expression. Quantitative RT-PCR data clearly show that ghrelin receptor Ghsr1a mRNA is expressed at the highest levels in the pituitary gland and brain with extremely low levels of expression in peripheral tissues including the pancreas (Fig. 3Go). In liver, epididymal fat, brown fat, skeletal muscle, stomach, thyroid, salivary gland, bladder, and skin, we found no evidence of Ghsr1a expression. This is in agreement with our previous data and studies in human tissues (32, 33), which showed that GHS-R1a is not as widely expressed as GHS-R1b and ghrelin.

Pituitary and brain were selected to further study age-associated expression, because they express the highest levels of Ghsr1a. Ghsr1a mRNA levels in the pituitary gland were highest in 1- to 2-month-old mice with significantly lower and stable expression between 6 and 30 months of age (Fig. 4Go). It has been shown that pituitary Ghsr1a mRNA expression is maintained at similar levels in 3-, 12-, and 24-month-old Wistar rats but decreases in 24-month-old Lou C/Jall rats (25). In humans, the GH-releasing effects of GHS undergo marked age-related variations, increasing at puberty, being stable during adulthood, and decreasing during aging (30). It is not known whether this age-dependent attenuation is a result of reduced GHS-R expression or deficits in signaling. The high level of Ghsr1a expression detected in the 1- to 2-month-old mouse pituitary is likely associated with puberty. Therefore, our results in mice are consistent with humans at puberty and during adulthood, but there may be a discrepancy during aging.

Ghsr1a mRNA expression in whole brain did not change up to 30 month of age, but a decrease in brain ghrelin mRNA expression was observed at 28 month of age (Figs. 2Go and 4Go). This result suggests that Ghsr1a expression in the brain is unchanged as a function of age. It is possible that Ghsr1a expression is differentially altered in specific brain centers and is masked by assaying whole brain. For example, in dwarf rats, Ghsr1a mRNA is up-regulated in the hypothalamus but down-regulated in the hippocampus (37). Indeed, these results support the notion that Ghsr1a expression is differentially regulated in different regions of the brain during aging.

In humans, GH pulse amplitude and plasma IGF-I levels show a clear linear decline with age, which can be restored to young adult levels by administration of a long-acting ghrelin mimetic; the dose dependence of the GH response to the mimetic is attenuated according to age (18, 28, 29, 30, 35). The data suggest that the expression levels of ghrelin and/or ghrelin receptor may decline during aging. However, in our studies with C57BL/6J male mice, plasma IGF-I levels did not show an age-associated decline. Furthermore, neither plasma ghrelin and brain ghrelin expression nor GHS-R expression in the pituitary gland and whole brain showed an age-correlated decline. Moreover, the GH response of the pituitary gland to ghrelin administration also did not appear to decline with age (Fig. 5Go).

In rats, the effect of aging on the GH/IGF-I axis has been shown to be dependent upon genetic background. Serum GH levels do not decline up to 28 months of age in Fischer 344 rats (38). Decreased amplitude of pulsatile GH secretion is observed beginning at 12 months in Wistar rats and not until 24 months in Lou C/Jall rats; the plasma IGF-I levels decrease with age in Lou C/Jall rats but not in Wistar rats (25). The data suggest that Lou C/Jall rats have delayed age-associated reduction in GH pulsatility accompanied by lower IGF-I than Wistar rats. To acutely evaluate GH levels, sequential blood samples have to be collected in 10-min intervals for at least 12 h, which is difficult to accomplish in mice.

One possible explanation for differences in sensitivity of the ghrelin/GH/IGF-I pathway during aging of mice and humans is metabolic responses associated with ambient temperature (39). Although mice require much higher metabolic rates to maintain basal body temperature, laboratory mice are routinely housed at an ambient temperature of 20–22 C, which is below the zone of thermoneutrality (29–33 C). In this relatively cold environment, mice may enter a condition known as torpor, wherein they consume more calories, use more oxygen, increase their blood pressure and heart rate, and increase activity of their thyroid axis to maintain basal body temperature (39). These effects are more evident in mice than in rats or humans; their greater surface area/body mass ratio could impact plasma GH and IGF-I levels (40, 41, 42).

In conclusion, our studies show that aging is not associated with reduced production of ghrelin in C57BL6/J male mice. Similarly, the levels of Ghsr1a mRNA in the pituitary and whole brain do not decline with age. Consistent with these observations, the sensitivity of the pituitary gland to exogenous ghrelin also appears to be unaffected by aging. Our results reinforce the need for careful consideration of metabolic differences, environmental conditions, genetic background, and species when attempting to extrapolate regulatory mechanisms from animal models to humans.


   Acknowledgments
 
We thank Dr. J. Russ Carmical for his expertise and input on TaqMan PCR assay design. We thank Hilda G. Kennedy and Linda M. Hicks for their excellent technical support and Michael R. Honig and Edith A. Gibson for their editorial assistance.


   Footnotes
 
This work was supported by National Institutes of Health Grants RO1AG18895 and RO1AG19230 (R.G.S.) and the Ted Nash Long Life Foundation (R.G.S.).

Disclosure Summary: Y.S. has nothing to disclose. J.M.G. is the principal investigator for a trial of a ghrelin agonist in cancer cachexia. R.G.S. is a paid advisory board member for Elixir Pharmaceuticals.

First Published Online December 7, 2006

Abbreviations: CT, Threshold cycle number; GHS-R, GH secretagogue receptor; TM, transmembrane domain.

Received June 13, 2006.

Accepted for publication November 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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