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Endocrinology Vol. 139, No. 3 1314-1320
Copyright © 1998 by The Endocrine Society

ARTICLES

Growth Hormone Gene Expression and Secretion in Aging Rats Is Age Dependent and Not Age-Associated Weight Increase Related1

B. Velasco, L. Cacicedo, J. Escalada, J. Lopez-Fernandez and F. Sanchez-Franco

Servicio de Endocrinología, CIC, Instituto de Salud Carlos III (B.V., J.E., F.S-F.), C/Sinesio Delgado, 10-12, Madrid 28029, Spain; and Servicio de Endocrinología, Hospital Ramón y Cajal (L.C., J.L.-F.), Ctra. Colmenar Km 9.0, Madrid 28034, Spain

Address all correspondence and requests for reprints to: F. Sanchez-Franco, Centro de Investigacions Clinicas de Salud Instituto Carlos III, Servicio de Endocrinologia, C/Sinesio Delgado, 10, Madrid 28029, Spain.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH secretion declines with age in rats and humans and a reduction in GH gene expression has been demonstrated in aging rats. GH secretion also diminishes in obesity; thus, the aim of this study was to determine whether GH decrease in aging rats is due to body weight gain or to aging. Three groups of male Wistar rats of different ages were studied (young, 3 months; middle-aged, 11 months; old, 27 months). The middle-aged group was established on a statistical analysis and corresponded to the youngest age at which body weight was not significantly different from the old (27 month) group. Thus, by using this group as control for comparison with animals with the same weight and an older age, the effects due to aging itself could be determined. Body weight (g, mean ±SD) 3 months: 361 ± 5.6; 11 months: 713 ± 39; 27 months: 635 ± 38. In comparison with 3-month-old rats, the 11-month-old animals showed no difference in pituitary GH messenger RNA (mRNA) accumulation and pituitary and serum IR-GH levels. Similarly IGF-I.a, IGF-I.b mRNA transcripts and IGFBP-3 mRNA accumulation in the liver showed no significant differences between the two groups. On the contrary, when the 27-month-old rats were compared with the 11-month-old animals, lower levels of pituitary GH mRNA and serum and pituitary IR-GH were found. Pituitary GH mRNA decreased 37.5 ± 7.7% P < 0.001, pituitary IR-GH content diminished (5.2 ± 3.4 vs. 55 ± 10.7 ng/mg of protein, P < 0.001) and serum IR-GH decreased (3.5 ± 1.8 vs. 12.5 ± 4.2 ng/ml, P < 0.01). Liver IGF-I.a and IGF-I.b mRNA transcripts accumulation and serum IGF-I were significantly diminished. IGF-I.b mRNA accumulation decreased 35.8 ± 1.2% P < 0.05 and IGF-I.a 36 ± 5.6% P < 0.05; serum IR-IGF-I levels diminished (759 ± 152 vs. 1327 ± 67 ng/ml, P < 0.05). Liver IGFBP-3 mRNA accumulation decreased 79 ± 4.2% P < 0.001.

These results indicate that the decrease in GH gene expression and secretion, as well as the expression of genes induced by GH such as IGF-I and IGFBP-3, is due to aging and not to the increase in body weight that takes place with aging.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AGING is associated with both a relative accumulation of body fat and a reduction in GH secretion in rats (1, 2) and in humans (3, 4, 5, 6). GH secretion in the male rat is pulsatile in nature and is governed by an endogenous ultradian rhythm of approximately 3.3 h (7, 8). In old male rats, GH secretion is depressed, and this is associated with diminished pulsatile release of GH. The amplitude and duration of the pulses decrease, but the periodicity appears to be similar to that present in younger animals (2). Also, obesity is associated with a decline in GH secretion, which reverts when body weight decreases. Insulin-like growth factor-I (IGF-I) is an important regulator of animal growth and is believed to mediate many of the endocrine functions of GH (9). The liver has the greatest abundance of IGF-I messenger RNA (mRNA), and hepatic synthesis of IGF-I could account for the known turnover of this peptide in the circulation (10). Total hepatic IGF-I gene expression is regulated mainly by GH and the nutritional status in a complex and unelucidated way that links diet and growth (9, 11, 12).

Rat and human IGF-I genes contain six exons. The mature protein is encoded by exons 3 and 4, and the last one also contains the N-terminal portion of the so-called E peptide, which appears in the unprocessed hormone. Two molecular mechanisms of control of IGF-I gene expression have been described in rats and humans: one is the existence of several start sites for transcription in leader exons 1 and 2 encoding for different 5'-untranslated regions and amino-terminal extensions of putative IGF-I signal peptides (13). The second molecular mechanism is the alternative splicing of the primary IGF-I transcript involving exons 5 and 6, resulting in two different E peptides depending on whether exon 4 is spliced to exon 5 (Eb region; IGF-I.b) or exon 6 (Ea region; IGF-I.a) (14). E-peptides have been implicated in IGF-I mRNA stability (15) and some authors suggest a specific role for the Eb mRNA in directing IGF-I to the circulation based on its postnatal presence mainly in the liver and its higher responsiveness to the administration of GH in hypophysectomized rats (16).

Decreased serum IGF-I in dietary energy or protein restriction correlates with reduced steady-state levels of hepatic IGF-I mRNA (17, 18, 19, 20). Normal or elevated serum levels of IGF-I have also been reported in obese adults (21) and in the obese Zucker rat, an animal model of obesity and hyperinsulinemia and low GH (22). The observation that serum IGF-I changes in response to modifications in the dietary intake suggests that IGF-I concentration might serve as an index of nutritional status.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of homologous proteins that are able to bind the insulin-like growth factors; IGFBP-3 binds 90% of circulating IGF-I. Most of the IGFBPs, mainly IGFBP-3, are regulated by the nutritional status and GH, in parallel with the total circulating IGF-I levels (20). It has not been established whether the decline of the GH-IGF-I axis activity that occurs with aging is due primarily to advanced age or is secondary to adiposity changes that take place in older animals. To clarify this fact, in this study rats of 3, 11, and 27 months are evaluated for the relationship between age/body weight and the activity of GH/IGF-I axis. The 3-month-old rats differ from the old group both in age and weight, whereas the 11-month-old group shared the same body weight increase with the old rats and differed only in its age.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
Male Wistar rats were obtained from the University of Granada (Granada, Spain) and Criffa (Charles River, Barcelona, Spain). Upon arrival, animals were housed in a specific pathogen-free facility and kept on a 12-h light, 12-h dark cycle. Water and food were available ad libitum to all animals. Three different age groups of ten male Wistar rats were studied: 3-month-old (young adult group), 11-month-old (middle-aged group), and 27-month-old (old group). The middle-aged group (11 months old) was established as a younger age with the same weight as the old group based on statistical analysis. The inclusion was the youngest adult age animals at which the did not differ in weight from the old group. To determine the two points that define the interval while maximizing the probability of finding rats fulfilling these criteria, the optimum age/weight ratio in the selected conditions was established under the following statistical criteria: a) from the growth and weight increase curve of the male Wistar rats, the inflection point at which the natural aging process continues without a significant increase in weight taking place; and b) from this inflection point and through ROC curve models (23), the weight that defines the optimal interval that accomplishes the mentioned age/weight requirements is calculated (Fig. 1Go).



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  		Figure 1. The growth curve of the male Wistar rats is shown in the upper panel. The middle-aged group that corresponds to 50 weeks of age was established by statistical methods. Mean body weights of the young (3 months), middle-aged (11 months), and old (27 months) rats are represented in the lower panel. The values represent the mean ± SE, *, P < 0.05 and **, P < 0.001 vs. 3-month-old rats in the same experimental condition

 
I. To determine the inflection point, data from the growth curve of the follow-up study developed by Charles River España S.A were used (24). In a sample of 50 rats observed from birth to the 125th week of age, in which weight was studied as the dependent variable and age as the independent variable, the inflection point was determined as the one corresponding to the 50th week (11 months), when a mean weight of 570 g (CI:520–605) was reached. This point corresponds with an age at which weight increases proportionally to age. From then on, while the aging physiological process occurs weight increase is almost null. II. Optimum age range: From the study published by Charles River España S.A with statistical distribution N(516.59, 111.65), the observed values correspond to the mean values of 32 distributions that relate the age/weight variables, and take as reference the previously identified inflection point, a distribution of male Wistar rats of medium age n = 20, µ = 581.30, and s = 18.8 is obtained.

From this distribution, the points xia and xiß that correspond with the weights among which the rats with greater weights are found, are calculated with error a = ß = 0.05 and a' = 1-ß (P < 0.05), where xia = µ - (ta · s) and xiß = µ -(ta' · s), when ta = 1.960 and ta' = 1.645. The points obtained correspond to the weights xia = 581-(1.96 x 18.8) = 544.15 and xiß = 581-(1.645 x 18.8) = 550.37 g. It can be considered that with a statistical significance of 95% (P < 0.05), rats weighing less than 544.15 g. (xia) will be younger but will not have yet reached their maximum weight; the interval 544.15 - 550.37 defines the weight range with the greatest probability of rats being adult and having the maximum weight; and lastly, rats weighing more than 550.37 g. (xiß) will have a higher probability of being in the normal physiological process of aging with no significant body weight modification. Therefore, the body weight (g, mean ± SE) of animals were: for the 3-month-old group 361 ± 5.6; for the 11-month-old group 713 ± 39 and for the 27 month group 635 ± 38.

Animals were killed by decapitation and then blood was collected in 5% EDTA tubes. Plasma was stored at -20 C until assayed for immunoreactive GH e IGF-I. For mRNA measurement, the liver and the pituitary were removed under sterile conditions, rapidly frozen in dry ice and stored at -80 C until used. Half of the pituitary was used for RIA determination.

RIAs
Immunoreactive rat serum and pituitary GH (IR-GH) concentrations were determined using the National Pituitary Hormone Distribution Program rat hormone kit (NIAMDD, Bethesda, MD) with a sensitivity limit of 0.8 µg/liter. Plasma IGF-I was measured using a commercial RIA (Nichols Institute, San Juan Capistrano, CA) after acid ethanol extraction. Glucose was assayed in plasma by the glucose oxidase method using a commercial kit (ITC Diagnostics). Serum insulin was measured using a commercial kit (Coat-a-Count Insulin, Diagnostics Products Corp., Los Angeles, CA). Serum free fatty acids (FFA) were determined by an enzymatic kit (NEFAC, Wako Chemicals, Neuss, Germany). Serum triglyceride was measured using the lipase method with an enzymatic kit (ITC Diagnostics).

All these parameters were quantitated in serum and pituitary extracts from individual rats, and all samples, when compared, were analyzed in the same assay to avoid interassay variations.

RNA probes
IGF-I. One construct of 376 bp from the rat IGF-I complementary DNA (cDNA), containing part of the A domain, the entire D and E domains, and part of the 3'-untranslated region was generated to simultaneously quantify mRNAs with (IGF-I.b: 376 bp) and without (IGF-I.a: 224 bp) the 52-bp insert present in the E domain of some IGF-I mRNAs. For the protection assay, this template was linearized with HindIII and transcribed with T7 RNA polymerase (25).

GH. The rat GH probe was a fragment linearized with HindIII of the plasmid p-rGH-1 (26, 27).

Cyclophilin. The rat cyclophilin cDNA was a 132-bp fragment linearized with APAI and transcribed to generate the antisense probe with SP6 polymerase following previously described methods (28).

IGFBP-3. The rat IGFBP-3 probe corresponded to nucleotides of rat cDNA clone described by Albinston et al. (29) and transcribed with T7 polymerase following previously described methods.

GHr. The rat GHr probe was transcribed from a 900-bp BgII fragment of a rat GHr cDNA corresponding to the region encoding the signal peptide, the extracellular domain, the transmembrane domain, and a portion of the intracellular domain (30). This template was linearized with BamHI and transcribed with T7 RNA polymerase to generate a 445-bp antisense RNA probe. This probe yielded two protected bands when hybridized to total liver RNA, a 439-bp band corresponding to the GHr mRNA, and a 290-bp band corresponding to the alternately spliced mRNA which, in the rat, encodes the GHBP.

Ribonuclease protection assay
Liver RNA was extracted using the Chomczynski and Sacchi method (31). In the ribonuclease protection assay, total RNA from a pool of three individual rats were hybridized overnight with approximately 600.000 cpm of labeled antisense rat IGF-I at 45 C. The hybridization solution contained 75% (vol./vol.) formamide, 80 mM Tris-HCl, pH 7.6, 4 mM EDTA, 1.6 M NaCl, and 0.4% SDS. After hybridization, samples were digested using RNase A (40 µg/ml) and RNase T1 (2 µg/ml) for 1 h at 30 C. Protected hybrids were isolated by ethanol precipitation after phenol-chloroform extraction and separated according to size on an 8% polyacrylamide/8 M urea denaturing gel. Gels were exposed to x-ray film (Kodak, Cambridge, UK) at -80 C for 24–36 h. Quantitation of the intensities of the autoradiography bands corresponding to protected hybrids was done by densitometric scanning using Adobe-Photoshop 2.0 and NIH-Image 1.47 programs (Macintosh). All samples were hybridized at the same time with cyclophilin to correct for the differences in gel loading.

Northern analysis
Total RNA was electrophoresed in a 1% agarose-0.66 M formaldehyde gel, followed by electrotransfer to nylon membrane (Nytran, Shleicher & Shuell, Keene, NH) and UV cross-linking (Hoefer Scientific Instruments, San Francisco, CA). Membranes were prehybridized and hybridized for 24 h at 65 C and 42 C for IGFBP-3 and GH probes, respectively, in 50% formamide with approximately 600.000 cpm/ml of [32P] uridine triphosphate (800 Ci/mmol) labeled antisense rat probe. Autodiagrams and quantitation of intensities were done as described above. Equal loading was confirmed, and data were expressed as arbitrary units after correction for hybridization with cyclophilin.

Statistical analysis
The statistical significance of differences between values was calculated by ANOVA. The difference was considered statistically significant when P values were below 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight and serum parameters
Some characteristics of the three groups of animals are shown in Table 1Go. A significant increment in body length is evident between the 3-month-old group and the 11-month-old and 27-month-old groups. Young rats have plasma insulin and glucose concentrations similar to those observed in middle-aged and old animals, which indicates that they are mature with regard to the preservation of glucose homeostasis. Middle-aged and old animals show significantly elevated triglyceride levels, a characteristic also observed in noninsulin-dependent diabetic patients and normoglycemic/hyperinsulinemic individuals (32) as well as in other rat strains in association with aging (33, 34). Although a relationship between triglyceride and free fatty acid concentrations has been previously shown (32), in our study free fatty acid levels in middle-aged and older rats are similar.


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  		Table 1. Characteristics of the animals

 
Serum IR-GH and plasma IR-IGF-I levels are shown in Table 1Go. Middle-aged rats show serum IR-GH levels similar to those of young animals. Serum IR-GH levels are reduced in the old rats group. This may be due to the high SE present in the young group due to pulsatility of GH secretion in young male rats. IR-IGF-I levels in the old rats are significantly decreased in comparison to the young (7.8 ± 7.3% P < 0.001) and middle-aged groups (35.4 ± 11% P < 0.05). The young group of animals has the same values as the middle-age group.

Pituitary IR-GH content and GH mRNA accumulation
To understand the mechanism of serum GH alterations in old rats, pituitary IR-GH content and GH mRNA levels were measured. As shown in Fig. 2AGo, pituitary IR-GH content is similar between young and middle-aged groups and both have significantly higher pituitary IR-GH content than the old animals (young vs. old 92.9 ± 7.3% P < 0.001 and middle-aged vs. old 89.0 ± 11.6% P < 0.001). A similar pattern of GH mRNA accumulation has been found in the three groups as shown in Fig. 2BGo; with similar levels in the young and middle-aged groups and significant decreases in the old group (young vs. old 37.5 ± 5.6% P < 0.001) (middle-aged vs. old 37.5 ± 7.7% P < 0.001). This profile of mRNA is parallel to that found for pituitary content and serum IR-GH, indicating that the potential mechanism of this alteration may be based on the decrease of GH gene expression, with the consequent modification of GH secretion.



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  		Figure 2. Pituitary rGH gene expression and pituitary IR-GH content in young (3 months), middle-aged (11 months), and old (27 months) rats. A, Pituitary IR-GH levels. B, GH mRNA levels. Two micrograms of total pituitary RNA were subjected to Northern blot using the GH probe described in Materials and Methods. After correction for cyclophilin levels, optical density units were adjusted so that the ratio obtained from pituitaries of 3-month-old rats equalled 100. The values represent the mean ± SE; ***, P < 0.001 vs. 3 m rats.

 
Liver IGF-I and IGFBP-3 mRNA levels
Another area of interest was to determine whether the age or the weight could modulate the liver IGF-I and IGFBP-3 gene expression. As expected, old rats present a decrease in both IGF-I transcripts (Fig. 3Go). IGF-I.b mRNA accumulation diminishes with age: young vs. old P < 0.001, and middle-aged vs. old P < 0.05. The amount of hepatic IGF-I.a mRNA also decreases in direct relation with age: young vs. old P < 0.001 and middle-aged vs. old P < 0.05, and in a way unrelated to weight. No significant differences have been observed between young and middle-aged groups. According to these data, the IGF-I.b/total IGF-I ratio is significantly decreased in old rats (Fig. 3Go).



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  		Figure 3. Liver IGF-I mRNA of young (3 months), middle-aged (11 months), and old (27 months) rats. Twenty micrograms of total liver RNA were subjected to solution hybridization/RNase protection assay using the antisense probes described in Materials and Methods. The positions of each protected fragment are indicated on the left. After correction for cyclophilin levels, optical density units were adjusted so that the ratio obtained from livers of 3-month-old rats equalled 100. Lane 1, Undigested IGF-I probe. Lane 2, IGF-I and cyclophilin probes after RNase A and T1 digestion. Lane 3, Molecular weight marker. Lane 4, Undigested cyclophilin probe. The lower panel shows IGF-I.b/total IGF-I ratio. Results are the mean ± SE (n = 4–5). *,P < 0.05; **, P < 0.01; ***,P < 0.001; #, P < 0.05 vs. 3 m.

 
The hepatic IGFBP-3 mRNA level is dramatically reduced in old animals: young vs. old P < 0.001 and middle-aged vs. old P < 0.0.001. Young and middle-aged animals show a similar accumulation of IGFBP-3 mRNA (Fig. 4Go).



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  		Figure 4. Liver IGFBP-3 mRNA of young (3 months), middle-aged (11 months), and old (27 months) rats. Twenty micrograms of total liver RNA were subjected to Northern blot using the IGFBP-3 probe described in Materials and Methods. After correction for cyclophilin levels, optical density units were adjusted so that the ratio obtained from livers of 3-month-old rats equalled 100. Results are the mean ± SE (n = 3). ***,P < 0.001 vs. 3-month-old rats.

 
GHr/GHBP mRNA levels
As shown in Fig. 5Go, neither weight nor age caused an alteration in GH receptor and GHBP mRNA levels.



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  		Figure 5. Liver GHr and GHBP mRNAs of young (3 months), middle-aged (11 months), and old (27-month-old) rats. Twenty micrograms of total liver RNA were subjected to solution hybridization/RNase protection assay using the antisense probes described in Materials and Methods. The positions of each protected fragment are indicated on the left. After correction for cyclophilin levels, optical density units were adjusted so that the ratio obtained from livers of 3-month-old rats equalled 100. Lane 1, Undigested IGF-I probe. Lane 2, IGF-I and cyclophilin probes after RNase A and T1 digestion. Lane 3, Molecular weight marker. Lane 4, Undigested cyclophilin probe. Results are the mean ± SE (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The effect of aging and/or age-associated obesity on the GH-IGF-I system is explored in this study. We define and assess three distinct ages: one young group used as control of age/weight and two other groups of equal weight but different ages. This design allows us to differentiate the effect of increased body weight or age on the GH-IGF-I axis. Based on the statistical analysis, we established one middle-aged group with body weight and length similar to that of the old group, thus eliminating the variable of the increase in body weight.

GH secretion declines during normal aging, resulting in lower IGF-I levels (35, 36, 37, 38, 39). In previous studies (27), we have demonstrated, comparing young and old animals, that the GH gene expression and GH secretion diminish with aging. In this study, we confirm that circulating levels of IR-GH, as well as pituitary GH mRNA levels, are decreased in the aging male rat. Additionally, we demonstrate that there are no differences in these parameters when young rats are compared with middle-aged male animals. Also, no differences are shown in pituitary IR-GH content between these two groups. However, middle-aged and old male rats, in spite of having a similar weight, show a significant difference in serum IR-GH levels, pituitary GH mRNA accumulation, and pituitary IR-GH content. These results indicate that the three levels of GH axis, such as pituitary mRNA accumulation and IR-GH content, as well as IR-GH secretion, diminish with aging and not with body weight increase. Regarding the circulating GH there is a limitation of the interpretation of its physiological significance because analysis of only one sample was done. The decrease in GH secretion rate, made evident by the decrease in GH amplitude or frequency of pulses (2) appears to be dependent of the lower GH content in the pituitary due to the decrease in GH gene expression, indicating that a transcriptional mechanism might account for the decrease in GH secretion related to aging. These data further support that in this stage, aging, GH regulation is mainly development dependent and not regulated by body weight or body composition modifications that occur with aging. Furthermore, this coincides with the concept that GH is regulated by different development stages in animals and humans, such as puberty and aging; puberty as a model of GH gene overexpression and secretion and aging as a model of relative or partial GH deficiency.

Concordantly with the decrease in GH secretion, a parallel diminution of plasma IR-IGF-I and liver IGF-I mRNA is shown in these experiments. In agreement with previous studies, our data demonstrate a decrease in serum IGF-I values in old rats (40, 41), but we find no difference in this parameter between middle-aged and young controls. Although IGF-I gene is inducible by GH, some nutritional factors or body weight increase due to obesity can also potentiate its expression in the absence of parallel GH secretion rate alteration (42, 43, 44, 45). Previous studies performed in our laboratory in obese Zucker rats (46) have shown that these obese animals have normal or increased linear growth and normal or increased circulating IR-IGF-I in spite of a lower GH secretion rate. Therefore, the situation described in this study, of a decreased plasma IGF-I with no modifications in nutritional factors or in weight between the middle-aged and old animals, must be due to a significant physiological decline in GH secretion rate and not to weight, body composition, or nutritional alterations. In fact, in these two groups no significant differences were found in the weight to length ratio or in metabolic parameters such as serum glucose, insulin, serum triglyceride, and free fatty acids. For these reasons, we consider the experimental design used in this study more accurate than previous models that manipulate the diet of the animals (47).

The present work demonstrates the existence of an age-related decline in liver IGF-I gene expression and plasma IGF-I levels, in parallel with the GH gene expression and secretion. These data are in agreement with previous studies (27, 47, 48, 49), suggesting a relationship between hepatic IGF-I mRNA accumulation and IR-IGF-I peptide modifications in plasma, and at the same time hepatic IGF-I gene expression itself being correlated with GH secretion in aging. Additionally, modifications of the relative abundance of the hepatic a and b IGF-I mRNA transcripts were seen. This predominance of IGF-I.b mRNA, found by other authors in response to exogenous GH (25, 50), or in the younger animals in this study has no clear biological significance at this time.

In this study, a significant decrease in IGFBP-3 mRNA accumulation in old rats is shown, indicating, in accordance with previous studies (51), that IGFBP-3 mRNA accumulation is correlated with GH secretion in aging rats in a similar manner to hepatic IGF-I mRNA and serum IR-IGF-I levels. These results support the hypothesis that the decrease in the expression of genes induced by GH is again due to aging and not to nutritional factors or body composition modifications. Thus, considering these results together, it is possible to conclude that the transcriptional regulation of GH gene expression is the premiere event in the regulation of the GH-IGF-I-IGFBP-3 axis during aging as one stage of development, independent of body weight, body composition, or nutritional factors.

The presence of similar values of GHr and GHBP mRNA accumulation in the three experimental groups suggests that the expression of this gene is not influenced by age, GH secretion rate, or weight. In accordance with these data, other authors have demonstrated that the level of expression of this gene is not GH or IGF-I dependent (52, 53).

The implication of metabolic factors in the mechanism of the decline of GH secretion rate in the aging rat can be excluded by the data presented in this study. None of the factors, known to influence GH secretion, such as serum glucose or serum free fatty acids, are significantly different among the three groups. Additionally, fat mass increase, which regularly occurs with aging, can also be excluded to be a major cause of GH secretion decline of longevity, at least in the rat. This conclusion is based on the fact that fat mass is considered a major determinant of plasma free fatty acid levels (54, 55), and these are not significantly different among the different age groups. Serum triglyceride levels were elevated in the old rats in this study, compared with the young 3-month-old animals, in agreement with previous reports using Fisher-344 rats (34). However, its implication in the GH secretion decrease of senescence can also be excluded, as this difference did not exist between the old and middle-aged groups that had a significant difference in GH secretion and GH gene expression. The explanation for an age-dependent increase in serum triglyceride levels and not in free fatty acids remains undilucidated, particularly when measurements of body weight, fat mass, and epididymal fat have been shown to be highly correlated throughout the different ages of the rats (54). Alterations in peripheral insulin and insulin-resistance, which have been associated with the increases in body weight and fat mass that occur with aging in humans and rats (33, 34, 56, 57, 58, 59, 60), do not appear to be important participants in the mechanism of GH decline of senescence because plasma insulin and glucose levels are not significantly different among the three groups of animals.

In summary, we confirm that the activity of the GH-IGF-I axis declines with aging and suggest that this decrease is age and not age-associated weight or fat mass increase dependent.


   Acknowledgments
 
We thank Drs. E. Hernández, D. LeRoith, and S. Ojeda for providing the cDNAs necessary to generate the riboprobes and J. Veiga for his statistical support. The rat GH kit was provided by the National Hormone and Pituitary Program/NIDDK. We also thank Purificación Mota Nieto for her technical assistance.


   Footnotes
 
1 This work was supported by grants from INSALUD (Fondo de Investigácion Sanitania (FIS): 94/308 and FIS: 96/1574). Back

Received August 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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