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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:B274-B279 (2000)
© 2000 The Gerontological Society of America

Effects of Aging and Dietary Restriction on mRNA Levels of Receptors for Growth Hormone–Releasing Hormone and Somatostatin in the Rat Pituitary

Isao Shimokawaa, Kurumi Yanagiharaa, Yoshikazu Higamia, Tomoaki Okimotoa, Masato Tomitaa, Takayoshi Ikedaa and Shuko Leeb

a Department of Pathology, Nagasaki University School of Medicine, Nagasaki City, Japan
b Department of Physiology, University of Texas Health Science Center at San Antonio

Isao Shimokawa, Department of Pathology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki City 852-8523, Japan E-mail: shimo{at}net.nagasaki-u.ac.jp.

Decision Editor: Jay Roberts, PhD


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Aging impairs and dietary restriction may modulate pituitary response to growth hormone (GH)–releasing hormone (GHRH) and somatostatin (SRIH) for GH secretion. Using the semiquantitative reverse-transcription polymerase chain reaction method, we analyzed the mRNA levels of the GHRH receptor (grfr) and SRIH receptor subtype 2 (sstr2) and subtype 5 (sstr5) in anterior pituitaries of male rats fed ad libitum or 30% dietary restricted. Aging reduced the mRNA levels of these receptors in a slightly different manner. The levels of grfr progressively decreased between 6 and 24 months, whereas those of sstr2 and sstr5 declined after 16 months. Dietary restriction did not diminish the aging-dependent changes, although it slightly augmented the levels of grfr, but not sstr2 and sstr5. The present results suggest that the aging-dependent impairment in pituitary response for GH secretion could result mostly from a decline in grfr rather than relative increase of sstrs. Although DR could slightly enhance the pituitary sensitivity to GHRH, the antiaging action may be minor at the level of gene expression.

AGING blunts the pituitary response to hypothalamic growth hormone (GH)–releasing hormone (GHRH) for GH secretion (1)(2)(3) or sensitizes it to somatostatin (SRIH) (4)(5) or both. The binding of GHRH or SRIH to specific cell-surface receptors initiates the actions of each peptide. Scatchard analyses with radiolabeled ligands in pituitary plasma membranes have demonstrated an aging-dependent reduction in the density of the binding sites of both GHRH (6) and SRIH-14 (4). The density of the binding sites, and thereby the density of specific receptors, is modulated by the rates of gene transcription and translation as well as the degradation of receptor proteins. The aging-dependent changes in these factors relating to the receptors, however, have not yet been fully elucidated.

At present, one gene that encodes specific receptors for GHRH, grfr (7), and five genes that encode six different subtypes of SRIH receptors, sstr1, 2A, 2B, and 3–5 (8), have been cloned. Of these subtypes of SRIH receptors, sstr2 and sstr5 are primarily involved in the inhibition of GH release from the pituitary gland in rats (8). The sstr5 subtype exhibits a preferential affinity for SRIH-28 (9), a more potent inhibitor in GH release than SRIH-14 (10). Sonntag and colleagues (11) have demonstrated that hypothalamic tissues sliced from aged rat brains secrete more SRIH-28 than do tissues from young rats in vitro. Our previous study has also shown an aging-dependent increment in the somatotroph response to SRIH-28 (5). These studies suggest the possible importance of SRIH-28 and sstr5 in the aging-dependent increment in the SRIH-associated inhibition of GH secretion.

Restriction of dietary intake without malnutrition, referred to as dietary restriction (DR), in laboratory rodents slows most aging processes compared with those of counterparts fed ad libitum throughout life (12). One such effect includes modulation of the hypothalamic–pituitary axis for GH secretion (5)(13). The pulsatile secretion of GH, which attenuates in aging animals (14), is observed in aging DR rats (13). The age-related increase in the sensitivity of somatotrophs to SRIH-28 is partially inhibited by dietary restriction (5).

Despite the aging- or diet-induced alterations in the hypothalamus–pituitary axis for GH secretion, it has not yet been determined whether these factors affect the gene expression of grfr, sstr2, or sstr5 in the pituitary gland. In this study, therefore, we quantified the mRNA levels of grfr, sstr2, and sstr5 in the pituitary gland of male F344 rats fed ad libitum throughout life or dietarily restricted from 6 weeks of age by using the reverse transcriptase–polymerase chain reaction (RT-PCR) method. In particular, we tested the following hypotheses: in the anterior pituitary of aged rats, (i) the mRNA level of grfr is decreased, (ii) the mRNA levels of sstr2 and sstr5 may decline, but the rates of reduction, especially that of sstr5, are less than that of grfr, and (iii) DR diminishes these aging-dependent changes.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Experimental Animals
We have fully described the animal care and dietary regimen elsewhere (15). Briefly, male F344 rats, purchased at 4 weeks of age from Charles River Laboratory Japan (Yokohama, Japan), were housed separately and maintained in a barrier facility (temperature, 22°C ± 1°C; 12:12-hour light: dark cycle with lights on at 0500) in the Laboratory Animal Center at Nagasaki University School of Medicine. Until 6 weeks of age, all rats received food ad libitum (Charles River Formula-1, Oriental Yeast Co., Ltd., Tokyo). A modified alternate-day feeding for DR was started at 6 weeks of age. A group of dietarily restricted rats (group DR) was provided approximately 160% of the mean daily intake of control rats fed ad libitum (group AL) 30 minutes before the lights were turned off on only Monday, Wednesday, and Friday; this procedure reduced the weekly dietary intake in group DR to 70% of that of group AL. The regimen yielded a 2-day cycle in the pattern of diet intake in group DR (15); for example, the dietary intake of rats sacrificed on Wednesday morning was less than that of rats sacrificed on Thursday morning. Preliminary experiments, however, detected no significant differences in the mRNA levels measured in this study based on the diet-intake pattern (data not shown). Anterior pituitaries removed from rats sacrificed between 0900 and 1200 at 6, 16, and 24 months of age were subjected to the present study. The numbers of rats examined were the following: (N): AL, 6 months (10 rats); DR, 6 months (10 rats); AL, 16 months (5 rats); DR, 16 months (4 rats); AL, 24 months (4 rats); and DR, 24 months (10 rats).

RT-PCR
Total RNAs were isolated from the anterior pituitaries by the guanidinum thiocyanate/acid phenol method (16) by use of Isogen (Nippon Gene, Toyama, Japan). The RNA concentration was determined by OD260. The recovered amounts of RNA were as follows: AL, 6 months, 5.1 ± 0.9; DR, 6 months, 4.5 ± 0.5; AL, 16 months, 4.0 ± 0.4; DR, 16 months, 4.6 ± 0.9; AL, 24 months, 5.8 ± 1.6; DR, 24 months, 5.0 ± 1.2 [µg/mg protein, mean ± standard deviation (SD), n = 3–10]. The amounts of RNA recovered did not differ among age groups or diet groups. Using a mixture of the total RNA extracted from the anterior pituitaries of 3–6-month old rats, we performed preliminary studies to set optimal conditions for the present quantitative study. According to the protocol provided by the manufacturer (Perkin-Elmer/Cetus), the total RNA (0.125–1.0 µg) was reverse transcribed into first-strand cDNA by 100 pmol of random hexamer oligonucleotide primers, 20 units of ribonuclease (RNase) inhibitor, 100 units of murine leukemia virus (MuLV) reverse transcriptase, and the appropriate buffer (a final concentration of 3-mM MgCl2, 1x PCR buffer II, 1 mM each deoxynucleoside triphosphate [dNTP]) in a volume of 20 µl. The mixture was incubated for 10 minutes at 25°C, 60 minutes at 42°C, 5 minutes at 99°C, 5 minutes at 5°C, and then stored at -30°C until PCR amplifications.

PCR amplifications of the first-strand cDNA product were also performed according to the manufacturer's protocol (Perkin-Elmer/Cetus). Each aliquot consisted of 15 µl of a PCR mixture (1.5-mM MgCl2, 1xPCR buffer II, 3.125-mU Ampli Taq), 5 µl of a RT reaction mixture, and 5-µl distilled water containing 1 µM of a set of primers. Primers for sstr2, which amplified sstr2A, the major splice variant of sstr2 (17), and sstr5 cDNAs were designed according to Raulf and colleagues (18) with a minor modification; those for GHRH cDNA followed Matsubara and colleagues (19). Histone H3.3 mRNA was amplified as an internal control (20). The primers used in this study and the expected sizes of the PCR products were as follows: sstr2 sense, 5'-TCATCAAGGTGAAGTCCTCTGG-3'; sstr2 antisense, 5'-AGATACTGGTTTGGAGGTCTCCA-3'; (PCR product size, 414 bp); sstr5 sense, 5'CCTTTCCTGGCCACGCAGAAC-3'; sstr5 antisense, 5'GGCCAGGTTGACGATGTTGAC-3' (PCR product size, 550 bp); grfr sense, 5'CAGCATCTCCATTGTAG-3'; grfr antisense, 5'-ACGTACCAGTGCATAGC-3' (PCR product size, 368 bp); H3.3 sense, 5'-GCAAGAGTGCGCCCTCTACTG-3'; and H3.3 antisense, 5'-GGCCTCACTTGCCTCCTGCAA-3' (PCR product size, 213 bp). The following temperature profile was used for all amplifications: an initial denaturing step at 94°C for 180 seconds and then 12–30 cycles at 94°C for 30 seconds, 62°C (58°C for grfr) for 60 seconds, and 72°C for 120 seconds, followed by a 7-minute incubation at 72°C. The amplified products (5 µl) were electrophoresed on a 2.5% agarose gel stained with ethidium bromide. Luminescent images of the products over an ultraviolet transilluminator were captured by a computer-assisted charge-coupled-device camera, and the images were quantified by IPLab Gel 1.5 (Signal Analytics Corporation, VA) software on a microcomputer. We also verified the amplified products by a sequence analysis.

We followed the concept of kinetic analysis for RT-PCR products to quantitate the levels of specific mRNA (21). In the preliminary experiments, the fluorescence values of RT-PCR products for H3.3-mRNA as a function of PCR cycles (12–30 cycles) for serial dilutions of the initial amount of RNA (0.125–1.0 µg) were measured. The rates of amplification were exponential between 14 and 20 cycles for the dilutions, 0.250–1.000 µg, and between 18 and 22 cycles for 0.125 µg of the total RNA, and the inclinations of these exponential portions appeared to be similar (Fig. 1). Therefore the initial amount of total RNA in each sample was adjusted to 0.5 µg and the PCR cycle for H3.3 was set to 18 to estimate an initial amount of H3.3-mRNA in each sample. Regression analysis by use of data at cycle 18 validated the linear relationship between the fluorescence values and the initial amount of RNA over the range of 0.125–1.000 µg (r > 0.99); this relationship implies that the present PCR conditions for H3.3 can measure the levels of H3.3 at the range between 1/4-fold and two-fold of the level at 3–6 months of age. By the same procedures, the optimum number of PCR cycles for grfr, sstr2, and sstr5 mRNA were set to 24 when the initial amount of total RNA was 0.500 µg.



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  		Figure 1. The ethidium bromide fluorescence intensity of polymerase chain reaction products as a function of polymerase chain reaction cycles in serial dilutions of the template RNA for H3.3.

 
Fig. 2 shows the correlation between the initial amount of RNA and the fluorescence intensity of RT-PCR products for each mRNA in three different runs. Regression analyses again validated the linear relationship between the two parameters for all the runs (r > 0.99), except grfr/2 (r = 0.977), although the inclination of the amplification curves differed in some of the runs. The results demonstrate that fluorescence values can be comparable in a single run, although they may not be between different runs.



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  		Figure 2. Correlation between the initial amount of RNA and the fluorescence intensity of reverse transcriptase–polymerase chain reaction products for (a) H3.3, (b) grfr, (c) sstr2, and (d) sstr5 in three different runs. The correlation coefficients (r) were > 0.99 in all the runs, except grfr/2 (r = 0.977).

 
In the present study, the RT reaction was performed all at once, and aliquots of the RT reaction mixture were subjected to the PCR reactions. The PCR reactions for one mRNA species were performed in one run; then the products of specific mRNA species and H3.3 in each sample were mixed and electrophoresed in a lane. The captured images were analyzed as described above.

Analysis of Data
As shown in Fig. 3 and Fig. 4, the levels of H3.3 did not significantly change according to aging or dietary restriction. Therefore the fluorescence intensities of grfr, sstr2, and sstr5 were normalized in relation to those of H3.3 for statistical analyses in the individual samples (Table 1 ). The fluorescence levels were expressed as the mean ± standard error of the mean (SEM); each mean value was the value relative to that in group AL at 6 months. In each receptor type, two-factor analysis of variance (ANOVA) was used to test the data for the main effects of age (Age) and diet (Diet) and the interaction between these two factors (Age x Diet). A post hoc test, Duncan's new multiple range test, was also performed when needed. Three-factor ANOVA was performed to analyze the data as a unit for the main effects of age, diet, and receptor (Receptor) types and the interaction between or among these factors (Age x Diet, Age x Receptor, Diet x Receptor, Age x Diet x Receptor); in particular, the interaction between the factors for receptor type and age or diet was noted to test whether the levels of each receptor type were modulated similarly according to aging or DR. The data were transformed to log values to stabilize the variance before these analyses. Statistical significance was accepted at p < .05. These statistical analyses were performed with a statistical computer software program, SuperANOVA for Macintosh (Abacus Concepts, Berkeley, CA). The power analysis for ANOVA was also performed by SAS (SAS Institute, Cary, NC), as no significant DR effect was noted in the levels of sstr2 and sstr5.



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  		Figure 3. The effects of aging and dietary restriction on grfr-mRNA levels in the rat anterior pituitary. The mRNA levels were quantified by the reverse transcriptase–polymerase chain reaction method. Representative cases (upper panel): lanes 1–3: group AL, 6 months; lanes 4–6, group AL, 24 months; lanes 7–9, group DR, 6 months; lanes 10–12, group DR, 24 months. Each bar represents the mean ± standard error of the mean of 4 to 10 rats (lower panel). AL: rats fed ad libitum throughout life. DR: rats fed 70% of the mean intake of group AL from 6 weeks of age. The statistical analyses are summarized in Table 1 .

 


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  		Figure 4. Effects of aging and dietary restriction on sstr2 and sstr5 mRNA levels in the rat anterior pituitary. Representative cases (upper panel): lanes 1–3, group AL, 6 months; lanes 4–6, group AL, 24 months; lanes 7–9, group DR, 6 months; lanes 10–12, group DR, 24 months. Each bar represents the mean ± standard error of the mean of 4 to 10 rats (lower panel). AL: rats fed ad libitum throughout life. DR: rats fed 70% of the mean intake of group AL from 6 weeks of age. The statistical analyses are summarized in Table 1 .

 

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  		Table 1. The Ratios of mRNA Levels

 

   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
The mRNA level of grfr progressively declined with advancing age in a similar fashion in both diet groups (Fig. 3, Table 1 ). The levels were slightly but significantly greater in group DR than group AL.

The mRNA levels of sstr2 and sstr5 declined between 16 and 24 months of age in both diet groups (Fig. 4, Table 1 ); there was no statistical difference between 6 and 16 months. No significant effect of dietary restriction was noted in the levels.

Three-factor ANOVA with regard to age-, diet-, and receptor-type effects on the mRNA levels showed significant effects of age and diet. There was no significant interaction between or among these factors, although the interaction between the age and the receptor effects was almost significant.

Power analyses for two-factor ANOVA were performed for the diet effect on sstr2 and sstr5 at {alpha} = 0.05 as follows: powers (1-ß) were over 0.86 for sstr2 and 0.98 for sstr5, and 0.99 for grfr.

Considering the ANOVA results, we concluded the following: (i) the mRNA levels of the three receptors declined with aging, but in a slightly different fashion; (ii) the levels of grfr progressively declined with aging, whereas those of sstr2 and sstr5 were reduced after 16 months; (iii) the diet effect was noted in only grfr.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The age-dependent decline in the grfr mRNA levels examined in this study seems to be in accord with results from Scatchard analyses of radiolabeled ligands in pituitary plasma membranes (6)(22). The high-affinity class of GHRH binding sites, which mainly mediates the biological actions of GHRH, is significantly reduced in pituitary membranes in aging rats (6). It has also been reported that the aging-dependent decline in the high-affinity class of binding sites may result from structural modifications, such as disulfide bond reduction, in the receptor (22). The decreased transcription rate of the grfr gene and a subsequent reduction in the renewal of grfr could simply be linked to a decline in the density of grfr and the blunt sensitivity of the pituitary gland to GHRH in aging animals; however, the reduced rate of renewal and thereby turnover of grfr may also facilitate the structural modifications by harmful physiological processes such as oxidation or glycoxidation.

The present study suggests that, with aging, the mRNA levels of sstr2 and sstr5 decline, but in a slightly different fashion from those of grfr. However, the levels of sstr5 declined to levels similar to those of grfr at 24 months, and the age-dependent reduction rate of sstr2 seems to be slightly less than that of grfr. A Scatchard analysis has also demonstrated an aging-related reduction in the binding sites of SRIH (4). It is unlikely, therefore, that the number of receptors for SRIH, especially sstr5, is relatively increased compared with that of grfr, resulting in the augmented sensitivity of pituitary cells to SRIH. Postreceptor mechanisms of SRIH-signal transduction may explain the aging-associated increase in pituitary response to SRIH.

The localization of grfr in pituitary cells remains to be investigated on a cellular basis; indirect evidence has suggested that grfr is predominantly expressed in pituitary cells in a somatotroph lineage (23)(24). Although sstr2 and sstr5 are expressed in all types of anterior pituitary cells (25)(26), a recent immunocytochemical study has shown that sstr5 is a predominant subtype in somatotrophs (27). Morphometric analyses have indicated an age-dependent, 40%–56% reduction in the cell density of immunostained somatotrophs (15)(28). Therefore the aging-related decline in the mRNA levels of grfr and sstr5 results, at least, partly from the reduction in the somatotroph cell density. On the other hand, the expression of sstr2 is not as predominant as that of grfr and sstr5 in somatotrophs (27); therefore the age-dependent decline in the mRNA levels of sstr2 does not seem to result only from the reduction in somatotroph cell density.

Based on a recent study in which a ligand binding assay was used (29), it was reported that long-term, but not short-term, DR protects the aging-related reduction in the high-affinity class of GHRH binding sites. The present study, however, has demonstrated no significant antiaging effects of DR on the mRNA levels of grfr, although grfr levels were slightly upregulated between 6 and 24 months. Dietary restriction may modulate posttranscriptional processes in the production of grfr.

The present study also demonstrated no statistically significant effects of dietary restriction on the mRNA levels of sstr2 or sstr5. Although our previous in vitro study (5) suggested that dietary restriction only partially protects the age-dependent increment in somatotroph response to SRIH, further analyses regarding this issue, particularly in vivo studies, will be needed.

In summary, the present study has provided one segment of basic data on the mechanisms causing the aging phenomena of pituitary response for GH secretion to the hypothalamic peptides. The declines in the mRNA levels of grfr and sstrs and subsequent decreases in the binding capacity do not necessarily imply parallel falls in the biological actions induced by the ligand–receptors interactions. The present study, however, suggests that the aging-dependent impairment in pituitary response for GH secretion could primarily result from a decline in grfr rather than an increased level of sstrs. Although DR may slightly enhance the pituitary sensitivity to GHRH, its effects on the aging-related alterations in the anterior pituitary could be minor. The impact of dietary restriction on hypothalamic neurons may be important to understanding its effects on the GH axis.


   Acknowledgments
 
We thank Yutaka Araki for his invaluable technical assistance. Appreciation is also extended to the staff of the Laboratory Animal Center of Nagasaki University School of Medicine for the maintenance of the rats used in the present study.

Received January 21, 1999

Accepted December 7, 1999


   References
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 Abstract
 Methods
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 Discussion
 References
 

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