Peripheral injection of growth hormone stimulates protein intake in aged male and female Lou rats

Christelle Veyrat-Durebex1, Pierrette Gaudreau2, Veronique Coxam3, Nathalie Gaumet3, and Josette Alliot1

1 Psychophysiologie et Neuroendocrinologie, Complexe scientifique des cézeaux, Université Blaise Pascal, 63177 Aubière; 3 Métabolisme minéral, Institut National de la Recherche Agronomique Clermont-Theix, 63122 Ceyrat, France; and 2 Neuroendocrinologie du vieillissement, Centre Hospitalier de l'Université de Montréal, Pavillon Notre-Dame, Montréal, Québec, Canada H2L 4M1


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is well established that growth hormone (GH) induces growth rate and food efficiency and stimulates protein accretion in young mammals. Senescence is characterized by metabolic and hormonal disorders, particularly a decrease in protein turnover, which could be correlated to a decrease in GH and insulin-like growth factor I (IGF-I) secretion. We have shown that body weight, protein intake, and IGF-I plasma levels are greatly decreased with aging in Lou/C rats, particularly in males. In order to specify the GH effect on protein intake during aging, males and females (6, 19, and 24 mo) placed on a self-selection regimen were injected daily with a physiological dose of human GH (0.023 mg/rat sc). No GH effect on caloric intake was observed. Nevertheless, GH treatment stimulated body weight in older rats. It also increased protein intake in females and older males (19-24 mo). This stimulating effect was positively correlated with the degree of weight loss in senescent rats, suggesting that the decrease in protein intake observed with aging could be a marker of senescence.

aging; protein selection; body weight


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GROWTH HORMONE (GH) is an essential factor of somatic growth. It induces insulin-like growth factor I (IGF-I) production, which also mediates its anabolic action. GH stimulates protein synthesis and participates in carbohydrate, fat, and mineral metabolisms. With aging, a decrease in GH secretion leading to a decrease of IGF-I circulating levels has been observed in humans (19) as in rats (20, 28, 29). It was then suggested that the reduction in the ability to synthesize protein with age could be due to a decline in GH secretion. Indeed, protein synthesis in skeletal muscles of old ad libitum- fed rats can be restored by GH treatment (26). Serum IGF-I concentration depends on the nutritional state of animals and decreases during energy or protein deficiency (7, 23). Maiter et al. (18) showed that serum IGF-I was lower in rats fed a 5% protein diet than in rats fed a 15% one.

Previous studies performed on Lou/C rats showed that senescence in males is characterized by a body weight loss from 16 to 18 mo of age, whereas females succeeded in maintaining their weight up to 28 mo (32). A skeletal muscular atrophy was reported during aging (2). A concurrent reduction in daily protein intake was observed in males, and a recent study also showed a decrease in IGF-I blood level in males as in females (2). It has been reported in the literature that peripheral injections of GH increased body weight gain and stimulated food intake (3, 10, 24). More recently, Roberts et al. (24) showed that rats treated daily by GH selected a diet higher in protein.

In the first experiment, we studied the relationships between the decrease of both IGF-I secretion and protein intake during aging. In addition, because the Lou/C strain does not develop obesity with age, we characterized the evolution of lean and fat body mass with an analysis of carcass composition .

The aim of the second experiment was to determine if human GH (hGH) treatment is able to increase protein intake in aged rats. We daily injected male and female Lou/C rats with a physiological dose of hGH (0.023 mg · rat-1 · day-1; Ref. 3). Effects on body weight and regimen composition were studied with a macronutrients self-selection paradigm (32).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Diets

Our experiment was carried out with male and female Lou/C rats, an inbred strain of Wistar origin, which came from the University of Louvain (H. Bazin, Ref. 4) and was bred in the laboratory for 10 yr. Those rats exhibit a lighter body weight with no development of obesity with age and show an increased longevity compared with more usual strains of rats (32, 33).

They were housed in pairs in plastic cages and maintained at 22 ± 1°C on a 12:12-h light-dark cycle (light off at 2000). They had free access to self-selection diets as previously described (33). Animals simultaneously had the choice between separate sources of the three macronutrients: protein (93% casein, metabolizable energy 3.12 kcal/g), fat (91% lard, 2% sunflower oil, 7.88 kcal/g), and carbohydrate (85% starch, 8% sucrose, 3.34 kcal/g; Table 1). Cellulose, vitamins, and mineral supplements were added to each of the macronutrient diets. The three 30.0 ± 2 g food-filled cups were anchored in the cage. The physical form was semisolid for fat diet and powder for protein and carbohydrate diets. This protocol widely used in the laboratory permitted us to previously determine the effect of age on macronutrient intakes (32, 33).

                              
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Table 1.   Composition of self-selection diets

Animals were given 3 wk of adaptation to the diets, with the last week (days 0-6) (P0) used as control. Body weight and food intake were recorded twice daily (at 0800 and 1800). Water was ad libitum.

Experiment 1

Procedure. We first tried to establish relationships between body weight and body composition changes during aging. For this, carcass composition was studied in 6 male and 6 female rats aged 4, 7, 10, 19, 22, and 29 mo.

In order to determine serum total IGF-I concentrations with aging, male and female rats aged 6 (n = 12), 18 (n = 8), 24 (n = 8), and 34 mo (n = 5) that were submitted to self-selection diets since 5 wk were used. Self-selected regimens and IGF-I concentrations were compared.

Body composition. Animals were anesthetized with pentobarbital sodium (30 mg/kg ip). Whole body lean and fat mass and bone mineral concentration were determined by dual-energy X-ray absorptiometry measurements on a Hologic QDR-4500A X-ray densitometer (Hologic France, Massy, France; Refs. 6, 25).

IGF-I RIAs. After animals were killed by decapitation, blood samples were drawn in heparinized tubes and centrifuged. Plasma was immediately frozen in dry ice and stored at -30°C until RIA.

IGF-I concentrations were measured after acid-ethanol extraction with a commercial RIA kit (Diagnostic Systems Laboratories, Webster, TX). The sensitivity of the assays was 21 ng/ml. All samples were determined in duplicate in a single assay. The intra-assay coefficient of variation was 6.1%.

Experiment 2

Procedure. The aim was to determine the effect of GH treatment on feeding pattern of male and female rats. The study was carried out with 40 male and 35 female Lou/C rats randomized into three groups of age: 6 mo old (16 males and 16 females), 19 mo old (12 males and 8 females), and 24 mo old (12 males and 8 females).

Treatment. GH was synthetic hGH (2.6 U/mg) provided by Eli Lilly (Indianapolis, IN). It was reconstituted from lyophilized form (12 mg) to a concentration of 60 mU/0.25 ml (0.023 mg/0.25 ml) by the addition of sterile physiological saline 0.9% NaCl. Treatment was administered daily between 0800 and 0900 via subcutaneous injection with a 25-gauge, 5/8-in hypodermic needle when animals were weighed.

The 6-mo-old rats were divided into an experimental (10 males and 10 females) and a control (6 males and 6 females) group.

On days 7-12, experimental animals aged 6, 19, and 24 mo were injected with vehicle (physiological saline, 0.9% NaCl) (P1). From day 13 to day 20, GH (60 mU/rat, 0.25-ml injection in 0.9% NaCl) was administered (P2). Then vehicle was injected from day 21 to day 27 (P3). The 6-mo-old control group received the vehicle throughout the experiment.

Statistics

All data are given as means ± SE. Statistical analysis used ANOVA for repeated measures. A Fisher's protected least significant differences test for a posteriori multiple means comparison was performed on each group of sex and age.

Nonparametric Wilcoxon's rank-sum test was performed when we have compared high protein (HP) and low protein (LP) eaters.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1

Body weight and carcass composition. As previously observed (33), male and female Lou/C rats still grew between 4 and 7 mo [F(5, 60) = 7.1, P < 0.0001]. Analysis of carcass composition showed that modifications of body weight were highly correlated to the lean body mass (r2 = 1, P < 0.0001; Fig. 1): intergroup variations observed in mature and old rats' body weight (from 7 to 22 mo) were mainly linked to variations in lean body mass.


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Fig. 1.   Whole body weight black-triangle, lean (light gray bars) and fat (dark gray bars) body mass, and percentage of fat  in 4-, 7-, 10-, 19-, 22-, and 29-mo-old male and female rats. Values are means ± SE. * P < 0.05 vs. 22 mo [Fisher's protected least significant differences (PLSD) a posteriori test].

At 29 mo, body weight loss was due to a decrease in lean body mass for males (P < 0.02) and in fat body mass for females (P < 0.01). The main characteristic of Lou/C strain was no significant changes in percentage of fat in whole body weight throughout life.

Daily caloric intake, protein selection, and serum IGF-I concentrations. We found the already observed effect of sex on daily caloric intake [F(1, 25) = 16.6, P < 0.0004]. Males ingested more calories than females. ANOVA showed an interaction of sex × age [F(3, 25) = 3.4, P < 0.04]. Oldest males decreased 24-h caloric intake, whereas females increased it. Sexual differences observed from 6 to 24 mo of age then disappeared (Fig. 2A).


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Fig. 2.   Total caloric intake (A; kcal), percentage of protein intake (B; kcal), and serum insulin-like growth factor I (IGF-I) concentrations (C) of 6-, 18-, 24-, and 34-mo-old male and female rats. Values are means ± SE. * P < 0.05 vs. 6 mo. dagger  P < 0.05 vs. 24 mo (Fisher's PLSD a posteriori test).

An effect of sex was found on protein intake [F(1, 25) = 4.2, P < 0.05]. Females selected more protein than males. However, protein intake decreased with age in both sex groups [F(3, 25) = 8.8, P < 0.0004]. From 18 mo of age, males greatly decreased the protein part of caloric intake (P < 0.01) from 18.2% at 6 mo to 8% at 18, 24, and 34 mo (Fig. 2B). This decrease was observed in females between 24 and 34 mo (P < 0.02).

A main effect of sex also appeared on plasma levels of IGF-I [F(1, 24) = 9.8, P < 0.005]. It was lower in young females than in young males. A decrease in IGF-I concentrations between 6 and 18 mo in both male and female rats was observed [F(3, 24) = 22.6, P < 0.0001; Fig. 2C].

Older rats could be separated into two groups: HP eaters when rats selected >10% of 24-h caloric intake as protein and LP eaters when it was <10%. Aging is related to a decrease in percentage of HP eaters, a decrease in protein intake, and reduction of IGF-I blood levels (Table 2). IGF-I blood levels differed between HP and LP eater rats: high protein intake was related with higher IGF-I concentrations (Table 2).

                              
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Table 2.   Effects of age and initial protein intake on percentage of protein in 24-h caloric intake and on IGF-I blood concentrations

We can conclude that senescence in Lou/C rats is characterized by a body weight loss, mainly due to a decrease in lean body mass. Aging is also accompanied by a decreased protein intake concomitant to an alteration of the hypothalamo-adenohypophysial-soma axis, shown by the decrease of IGF-I blood levels.

Experiment 2: Effect of hGH Treatment

Effect on body weight. At 6 mo, only male Lou/C rats gained weight during the 6 days of saline injections (days 7-12; 0.6 ± 0.1 g/day). GH had no effect on the growth of males but it induced a significant higher body weight gain in treated females compared both with control females (Table 3) and with body weight before treatment. In both sex groups, the end of treatment was followed by an inhibition of growth.

                              
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Table 3.   Effects of hGH treatment in 6-mo-old rats

As reported in Table 4, a stimulating effect of GH on body weight was seen in 19-mo-old males and females. The slight effect observed in the oldest groups during GH injections and the drop of body weight observed after the end of treatment seem to show an enduring GH effect in 24-mo-old rats.

                              
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Table 4.   Evolution of 19- and 24-mo-old male and female body wt during experiment

Effect on daily caloric intake. Table 5 gives daily caloric intake (kcal/24 h) during injections. There was no significant effect of GH treatment on food intake. Only the 6-mo-old male rats decreased their food intake.

                              
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Table 5.   Effect of hGH treatment on mean 24 h caloric intake

Effect on macronutrient selection. Relative protein intake during experiment is shown in Fig. 3. ANOVA showed a main effect of age [F(2, 100) = 12, P < 0.0001]. A significant decrease in protein intake was observed between 19 and 24 mo, more important in males (from 24.5 ± 5.5 to 11.3 ± 0.9%) than for females (from 17.5 ± 3.0 to 14.0 ± 2.9%). These results are in accordance with those previously obtained in the laboratory. We have shown a decrease of the protein part of the regimen to <10% from 12 to 16 mo in males and from 24 mo in females (33). We can notice that 19-mo-old males still selected a high percentage of protein (>24.4%). This was not observed in the 18-mo-old males of the first experiment. It could be due to the higher percentage of HP eater rats in this group (Table 6).


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Fig. 3.   Percentage of protein in daily intake (g). P1, vehicle injection (means of days 9-12); P2, growth hormone (GH) injection (means of days 15-20); P3, vehicle injection (days 24-27). Values are means ± SE. * P < 0.05 vs. P1. open circle  P < 0.05 vs. P2 (Fisher's PLSD a posteriori test).


                              
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Table 6.   Percentage of rats eating >10% of 24-h caloric intake as protein (in kcal)

ANOVA showed a main stimulating effect of GH on protein intake [F(2, 100) = 6.0, P < 0.005]. A posteriori analysis showed that this effect was more important in oldest rats (P < 0.05) and in all female groups (P < 0.01; Fig. 3).

Cessation of the treatment induced a decrease in protein intake, significant for the oldest males (P < 0.03).

When expressed as percent concentration scores of each macronutrient in the daily caloric intake, an increase of protein intake at the expense of the fat part of the regimen for 6- and 24-mo-old male rats was observed during treatment [F(1, 9) = 7.0, P < 0.05 and F(1, 9) = 7.3, P < 0.03, respectively]. No significant effects were observed on carbohydrate intake (Fig. 4).


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Fig. 4.   %Concentration scores of 3 macronutrients across daily energy intake (kcal). P1, vehicle injection (means of days 9-12); P2, GH injection (means of days 15-20); P3, vehicle injection (days 24-27). P, protein; F, fat; C, carbohydrate. Values are means ± SE. * P < 0.05 vs. P1. open circle  P < 0.05 vs. P2 (Fisher's PLSD a posteriori test).

At 19 mo, around the critical period when senescence takes place in Lou/C males, a great interindividual variability of GH effect was observed. There was a more important number of HP eater rats in this 19-mo-old group than in the 18-mo-old group studied in the first experiment.

The great body weight and protein intake variability suggested possible differences in the physiological age of older rats (19 and 24 mo). So we compared the effect of GH in HP and LP eater groups. As expected, GH induced a nonsignificant slight increase in protein intake in HP old as in young rats. Nevertheless, it induced a significant rise in protein intake in LP eaters (P < 0.05; Fig. 5).


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Fig. 5.   Percentage of protein intake in daily caloric intake (kcal). P0, before treatment (means of days 0-6); P2, GH injection (means of days 15-20) HP and LP, high and low protein eaters, respectively (19 and 24 mo). Values are means ± SE. * P < 0.05 vs. P0 (Wilcoxon's rank sum test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our first experiment confirmed that senescence in Lou/C rats is characterized by a decrease in protein intake concomitant with a decreased IGF-I secretion (2). This last result suggests an alteration of the hypothalamo-adenohypophysial-soma axis during aging. Diminished mean GH release in old rats could partially account for the reduction in protein synthetic processes and turnover reported to occur in aged mammals (12, 27) and could play a role in the decreased intake of protein. From 19 mo, we can separate Lou/C rats into HP or LP eaters. Intake of protein is then correlated to IGF-I blood concentrations. We could hypothesize that protein consumption is a marker of aging: physiological age could be different from chronological age.

Our second experiment showed that subcutaneous injection of hGH produced a stimulating effect on body weight of all groups except in still growing 6-mo-old males. However, in this group, cessation of the treatment induced a growth break. These results indicate that there is a functional hypothalamo-adenohypophysial-soma axis in young rats and that exogenous GH injections can still stimulate the negative feedback that regulates its secretion and possibly receptor densities. Differences observed between 6-mo-old males and females could be explained by the sexually dimorphic release of GH in young rats: in females, GH pulses are of lower amplitude and occur more irregularly and frequently than in males (21, 22). In this case, GH pulses could already have reached a maximal value in males. Because our strain does not develop obesity with age and body weight is highly correlated to lean body mass in mature and old rats, we hypothesized that GH effects on body weight could be due to the development of lean tissue. Such an hypothesis has to be confirmed in further studies, by directly studying the effect of GH on body composition.

In contrast to previous studies with pharmacological doses (from 7 µg to 5 mg · rat-1 · day-1) (10, 24), GH treatment did not stimulate daily food intake. Interestingly, GH induced an increased protein intake in all groups. As hypothesized, the stimulating effect was greater in old rats. It was also more pronounced in females than in males. We have shown that this effect depended on initial protein intake: GH restored a "young" protein intake in LP eater rats.

Two hypotheses can be made to explain the effect of GH on protein intake. First, GH exerts its anabolic effect by increasing the efficiency of ingested protein. It induces an increase in circulating amino acids, leading to the development of different tissues like muscular mass. Several data indicated that circulating amino acids may play a role in protein intake by interacting with brain neurotransmitters, such as serotonin and dopamine (17), and by regulating hypothalamic preproGH-releasing hormone mRNA contents (8, 9). Second, GH secretion is directly controlled by two hypothalamic peptides, GH-releasing hormone, which promotes GH synthesis and secretion, and somatostatin (somatotropin release-inhibiting hormone), which inhibits its release (5). Feifel and Vaccarino (15) reported that GHRH could increase food intake and more particularly protein consumption (14). Nevertheless, evidence of a reduced ability of GHRH to induce GH secretion during aging in rats (1, 11, 16) and humans (30, 31) has been documented. In male Sprague-Dawley rats, rat GH responsiveness to 0.4 and 1.6 µg/kg rat GHRH started to decline at the higher dose in 12-mo-old rats and was completely blunted at both doses of GHRH in 20-mo-old rats (13, 16). We now have to determine if the age-related changes in GH secretion of Lou/C rats could be due to either an hypothalamic dysfunction in the synthesis and/or release of GHRH or a reduced ability of GHRH to stimulate pituitary GH secretion. The effects of GHRH injection in Lou/C rats are currently under investigation for a comparison with GH effects.


    ACKNOWLEDGEMENTS

We wish to acknowledge Eli Lilly for providing hGH for this experiment.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Josette Alliot, Psychophysiologie et Neuroendocrinologie, EA 995, Complexe scientifique des cézeaux, Université Blaise Pascal, 63 177 Aubière Cedex, France (E-mail: alliot{at}cicsun.univ-bpclermont.fr).

Received 8 October 1998; accepted in final form 3 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 276(6):E1105-E1111
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