Evidence for an alteration of plasma and liver proteins response to dexamethasone in aging rats

Mechanisms of Ageing and Development 122 (2001) 105 – 120 www.elsevier.com/locate/mechagedev

Evidence for an alteration of plasma and liver proteins response to dexamethasone in aging rats Isabelle Savary b,*, Elisabeth Debras a, Dominique Dardevet a, Fabienne Rambourdin a, Marie-Paule Vasson c, Christiane Obled a, Jean Grizard a a Unite´ d’Etude du Me´tabolisme Azote´, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand Theix, 63122 St Gene`s Champanelle, France b Unite´ de Recherche sur les Herbi6ores, Equipe Nutriments et Me´tabolismes, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand Theix, 63122 St Gene`s Champanelle, France c Laboratoire de Biochimie, Biologie Mole´culaire et Nutrition, Faculte´ de Pharmacie, Uni6ersite´ d’Au6ergne, 63000 Clermont Ferrand, France

Received 18 March 2000; received in revised form 16 September 2000; accepted 8 October 2000

Abstract The aim of this study was carried out to analyse the liver and plasma proteins response to dexamethasone in adult (6–8 months) and old (24 months) rats in order to ascertain the involvement of glucocorticoids in the aging process. The animals received dexamethasone (Dex) for 5 or 6 days. As Dex decreased food intake, all groups were pair fed to dexamethasone-treated old rats. The synthesis of mixed plasma and liver proteins (assessed by a flooding dose of [13C] valine) was similarly greatly improved in adult and old rats after Dex treatment. However, the level of mixed plasma proteins was only slightly increased. When specific plasma proteins were assessed, a similar increase in the concentration of albumin and alpha1 acid glycoprotein was observed in adult and old rats. By contrast, fibrinogen decreased to a greater extend in old rats and alpha2 macroglobulin became undetectable in old animals. It was concluded that the response of plasma and liver proteins to Dex was altered in old rats and may contribute to the pathogenesis of several diseases which occur during aging. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Rats; Glucocorticoids; Dexamethasone; Plasma proteins; Protein metabolism * Corresponding author. Tel.: +33-47-3624732; fax: +33-47-3624639. E-mail address: [email protected] (I. Savary). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 2 4 - 4

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1. Introduction Current theories propose that aging consists of many intrinsic processes which are characterized by a progressive decline in tissue functions. These age-associated characteristics occur in the absence of disease. However, evidence support the concept that the toxic response due to environmental factors enhance the characteristics of aging. Glucocorticoids are crucial hormones involved in the response to stressful situations. Moreover, they have been demonstrated to be involved in the aging process since adrenalectomy attenuates the development of age-specific effects (Landfield et al., 1981). Correlated impairments in the neuroendocrine system (e.g. hippocampus) and elevated glucocorticoid levels accelerate these impairments (Sapolsky et al., 1986). Sapolsky et al. (1986) proposed the glucocorticoid cascade hypothesis of aging which is based on the concept that neurons in the hippocampus containing a high density of steroid-hormone receptors are involved in the regulation of both glucocorticoid secretion (through negative feed back) and action (see Masoro, 1995 for a review). Decreases in the number of these receptors during aging are consistent with the increase in basal glucocorticoid secretion observed in many studies and the apparently greater responses to glucocorticoid excess in some variables (see Mobbs, 1996 for a review). Moreover, the fact that the return of circulating corticosterone to basal level is attenuated in old, stressed rats due to a decrease in the metabolic clearance of the hormone may also increase glucocorticoid action in old subjects (Sapolsky et al., 1983). The effects of glucocorticoids on the organism are numerous and include muscle wasting (Savary et al., 1998) along with liver hypertrophy and acute phase plasma protein synthesis (Odedra et al., 1983; Pedersen et al., 1989). These proteins are predominantly synthesized in the liver. Their synthesis can be mediated by a direct action of glucocorticoids on hepatocytes (especially on rodents: direct effect of dexamethasone on fibrinogen and albumin mRNA levels in hepatoma Fao cells (Andus et al., 1988)) but much more frequently through interaction with cytokines, growth factors and catecholamines (Benedikt et al., 1995). However, in some cases, the steroids can supress the cytokine production by inhibiting the T cell immunity (Almawi et al., 1996), which gives us a picture of the actual role of the glucocorticoids on the regulation of the acute phase proteins synthesis much more complex. The biological functions of acute phase proteins which are generally stimulated (positive) but can be also inhibited (negative) during the inflammatory status are numerous. These functions can be divided into three major categories: (1) Participation in host adaptation or defense (fibrinogen: role in hemostasis, tissue repair and wound healing; alpha1 acid glycoprotein: cell repair; C reactive protein: elimination of foreign pathogens and damaged cells); (2) Inhibition of serine proteinases (alpha2 macroglobulin alpha 1 antitrypsin); and (3) Transport function with antioxidant activity (albumin: transport of some amino acids, zinc and fatty acids; ceruloplasmin: transport of copper and antioxidant activity). A typical acute phase protein response can be demonstrated during inflammation (Kushner and Mackiewicz, 1993).

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We hypothesized that the plasma and liver proteins response to glucocorticoids may be associated with the decline in tissue function that occurs during aging. Indeed, there were alterations in the plasma concentration of acute phase proteins in aged subjects in a basal state (Caswell et al., 1993) (albumin falls whereas C reactive protein increases with age) or after lipopolysaccharide-induced inflammation (Kushner and Mackiewicz, 1993). For instance, the basal levels of some acute phase proteins have been shown to be modified during aging: a1 antitrypsine and albumin mRNA were decreased in mice (Post et al., 1991). Changes in acute phase proteins concentrations have also been recorded in aged humans: (1) increase for fibrinogen, C reactive protein and a1AGP (Milman et al., 1988; Ross et al., 1992; Caswell et al., 1993; Ballou et al., 1996); (2) no alteration for a1AGP (Caswell et al., 1993); or (3) decrease for albumin (Klonoff-Cohen et al., 1992; Caswell et al., 1993). In addition, some regulation of acute phase protein gene expression are markedly altered during aging (e.g. IL6 cytokines) (Wei et al., 1992). For a better understanding of the association of glucocorticoids, acute phase proteins and aging, we investigated the effect of dexamethasone on the concentration of plasma albumin, alpha 1 acid glycoprotein, fibrinogen and a2M and the synthesis of mixed plasma and liver proteins in adult and old rats.

2. Materials and methods

2.1. Animals These studies were performed in accordance with current legislation on animal experiments in France. Adult (6–8 months) and old (24 months) male Sprague Dawley rats were purchased from Iffa-Credo (L= Arbresle, France) and housed under controlled environmental conditions (temperature 22°C; 12 h dark period starting at 18:00 h). Rats had free access to a commercial laboratory chow (UAR, Epinay sur Orges, france) (g/kg: protein 220, fat 40, cellulose 40, carbohydrate 520, minerals 60, water 120, vitamin A, cholecalciferol and vitamin E) and water before the experiments were performed. Both adult and old rats were randomly divided into a control and a Dexamethasone- (Dex) treated group. (adult rats: n = 24 Dex-treated and n =24 controls; old rats: n = 15 Dex-treated and n= 15 controls). Dex (a synthetic glucocorticoid analogue that does not bind to plasma binding proteins) was given daily (at 09.00 h) in the drinking water. Dex concentration was adjusted every day on the basis of drinking water intake the day before. Adult and old animals received throughout the treatment period 543 (se 101) and 564 (se 68) mg/kg per day, respectively. As Dex has been reported to decrease food intake, all groups were pair-fed to the group that had the lowest food intake (i.e. Dex-treated old rats). As a consequence, the effect observed is a consequence of Dex treatment and caloric restriction. Dex was given for 5 days in old rats but for 6 days in adult rats as previously decribed in other studies (Dardevet et al., 1995, 1998; Savary et al., 1998; Dardevet et al., 1999). Rats were allowed to recover for either 3 (R + 3) or 7 (R+7) days. The animals become very sensitive to environmental factors at

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the end of the Dex treatment (and animal handling becomes particularly awkward). As a consequence the administration of Dex in the drinking water was prefered to an intravenous or intraperitoneal infusion.

2.2. Measurement of protein synthesis rates, killing and sampling Protein synthesis rates were measured in vivo using the flooding dose method (Garlick et al., 1980), which reduces uncertainty over the labelling of the tracer amino acid in the precursor pool for protein synthesis. Briefly, 50 min before euthanasia, each rat was injected subcutaneously (time 0) with a flooding dose of valine (300 mmol/100 g body weight) to flood the precursor pools. L-valine [1-13C] (99 atoms% 13C, Mass Trace, Woburn, MA, USA) was used as a tracer and the enrichment of the flooding dose was 50 atoms percent excess (APE). In preliminary experiments, Mosoni et al. (1995) verified that intratissular enrichment was similar in different organs and remained nearly constant during incorporation time. General anaesthesia was induced by subcutaneous injection of pentobarbital sodium (Sanofi, Libourne, France, 6 mg/kg body weight) 5 min before killing. Rats were exsanguinated and blood was collected in heparinized tubes and centrifuged. Plasma was collected and frozen. Liver was quickly excised, weighted and frozen in liquid nitrogen within 5 min after exsanguination.

2.3. Analytical methods Frozen livers were powdered under liquid nitrogen. A portion (about 1 g) of this frozen powder or 1 ml of plasma were homogenized in 7 volumes of ice-cold 0.6 M trichloracetic acid (TCA) to extract tissue free amino acids. The acid soluble fraction containing free amino acids was separated from the protein precipitate by centrifugation (10 000 g for 15 min). Proteins were resuspended in TCA and centrifuged. The acid soluble fractions were combined. TCA was removed on a column of cation-exchange resin (Amberlite AG50X8, 100–200 mesh, H+ form, Bio-Rad, Richmond, CA, USA). Amino acids were eluted with 4 M NH4OH; after evaporation, amino acids were dried and resuspended in 0.1 M-HCl for tissue valine enrichment determination which was performed by GC–mass spectrometry, with a HP 5972 organic mass spectrometer quadrupole coupled to a HP 5890 GC (Hewlett Packard, Les Ulis, France). Valine was measured as the tertiary butyldimethylsilyl derivative under electron impact ionisation. The ion m/z 288 and 289 were monitored by selective ion monitoring to determine the [13C] valine enrichment. The plasma and liver protein precipitates were further washed twice with 0.6 M-TCA and once in 0.2 M-perchloric acid. The liver protein precipitates were dissolved in 0.1 M-NaOH and incubated 1 h at 37°C. A portion was used to measure tissue protein content according to Smith et al. (1985) by colorimetric reaction with bicinchoninic acid (Pierce, Rockford, IL, USA). Liver proteins dissolved in 0.1 M-NaOH were precipitated with 0.4 volumes of 2 M-perchloric acid, stored overnight at 4°C, and centrifuged. The supernatant fraction was used to measure the RNA content by the method of Manchester and Harris (1968).

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A portion of the liver and plasma protein containing approximately 4 mmol valine was hydrolysed in 6 M-HCl for 48 h at 110°C and dried. Valine was measured as N-acetyl-propyl-amino acid derivative. The ratio 13CO2:12CO2 was measured with a gas isotope ratio spectrometer coupled with a GC (Isochrom II, Fisons, Manchester, UK). Plasma Insulin was determined by direct radioimmunoassay with commercial kit (ERIA Diagnostic Pasteur, Sanofi, France). Blood glucose was assessed enzymatically using glucose oxidase (EC 1.1.3.4; GOD-PAP method, Cobas Roche, Neuilly sur Seine, France).

2.4. Plasma proteins measurement Plasma proteins were measured by single radial diffusion using goat anti-rat fibrinogen and albumin anti-bodies (Cappel, Turnhout, Belgium) and rabbit antirat a2-macroglobulin and a1-acid glycoprotein antibodies raised in our laboratory. Standard curves were constructed using purified rat a1-acid glycoprotein (Sigma), albumin (Cappel), a pool of plasma from infected animals titrated for fibrinogen by nephelometry (Biodirect, Les Ulis, France), and rat a2-macroglobulin purified to homogeneity using Blue Sepharose CL6B (Pharmacia, Saint Quentin, Yvelines, France) and Ultragel AcA22 (BioSepra, Villeneuve la Garenne, France) according to the method described by Virca et al. (1978).

2.5. Calculations 2.5.1. For li6er The liver fractional synthesis rates (FSR, % per day) were calculated according to the method described by Garlick et al. (1980): FSR= 100× (EP− EN)/(EA × t) where t is the incorporation time (50 min), expressed in days, EP is the enrichment of protein-bound valine at the time of killing, EN the natural enrichment of protein bound-valine and EA the enrichment of liver free valine. EP, EN and EA were expressed in APE by reference to the natural enrichment of valine obtained from Sigma Chemical Company (Saint Louis, MO, USA). Absolute synthesis rates (ASR) (mg per day) were calculated by multiplying FSR by total tissue protein content. Ribosomal capacity was estimated as the ratio of RNA: protein (mg RNA/mg protein) because most of the RNA in tissues is ribosomal. Ribosomal efficiency was calculated as the ratio ASR: total RNA (mg protein per day per mg RNA). 2.5.2. For plasma proteins The fractional plasma synthesis rates were calculated according to the same method described for liver with the following exception: The incorporation time was corrected by the time needed for secretion of plasma proteins (i.e. 26 min). This time was determined in pilot experiment using old rats under control or DEX conditions killed 15 – 40 min after a flooding dose of valine. Our results were similar to what obtained in experiments previously made on young rats (Obled et al., 1997;

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unpublished results): No detectable effect of age or hormonal treatment could be demonstrated on secretion time of plasma.

2.6. Statistical methods The values are given as means with the residual standard error (RSE). All data were analysed separately for each age group by ANOVA (Statistical Analysis Systems, version 6.01, 1987; SAS Institute Inc., Cary, NC, USA) according to a two-way factorial model. The main effects tested were the treatment (T) and the stage at which animals were killed (S). The T× S interaction was included in the model. In case of significant S or T× S effects, means were compared using Student’s t-test.

3. Results

3.1. Animal characteristics As previously described (Savary et al., 1998), food intake was maintained at similar levels in all groups during both Dex treatment and the recovery period. Thus, the differences between groups did not originate from different intakes. The weights of adult rats were 588 (se 12) g and 568 (se 12) g for Dex-treated animals and pair fed groups, respectively at the beginning of the treatment, but were 471 (se 11) g and 501 (se 12) g after 6 days of treatment. At the beginning of the treatment, the weights of old rats were 636 (se 15) g and 644 (se 17) g for Dex treated animals and pair fed groups, respectively but after 5 days of treatment the weights were 537 (se 13) g and 594 (se 6) g. The decrease in food intake induced a weight loss in all rats (control and Dex treated) however, the loss of body weight was greater in Dex treated animals. Dex treatment induced similar body weight losses in old and adult rats -117 (se 17) g and -99 (se 20) g, respectively.

3.2. Corticosterone le6els (Table 1) The basal levels of corticosterone were higher in old animals compared to adult animals (130.1915.6 vs 233.69 21.9 ng/ml plasma for control old and adult animals, respectively at the end of the Dex treatment). Similar results were found between Con adult and old rats taken at R+ 3 or R+ 7. The Dex treatment induced a dramatic decrease in corticosterone levels in adult and old animals (8.19 1.1 and 7.1 9 2.3 ng/ml plasma at the end of the treatment for Dex treated adult and old rats, respectively). The corticosterone concentration increased progressively during the recovery period in previously Dex treated animals (125.1 9 26.4 and 149.3 9 28.1 ng/ml plasma at R + 7 for Dex treated adult and old animals, respectively).

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3.3. Li6er Liver weight showed a similar increase after Dex treatment in adult and old rats (+ 44.1% (Con: 12.3; Dex: 17.9 g), + 45.6% (Con 13.6; Dex 19.5 g) vs. their control, P B 0.01 for each group) (Table 2). During the recovery period, liver weight were more rapidly normalized in adult than in old rats (at 3 and 7 days, respectively). The protein content tended to increase after Dex treatment in old animals but only significantly in adult rats. As a result, protein concentration decreased in the liver after Dex treatment in adult and old rats (−18.2% (Con: 220; Dex: 180 mg/g), − 21.6% (Con: 215; Dex: 169 mg/g) vs. control, respectively, PB 0.01). Protein concentrations were rapidly normalized (at 3 days) in all animals during the recovery period. Dexamethasone also increased fractional protein synthesis rates in the liver (Fig. 1) of both adult and old rats ( + 16.5% (Con: 40.42; Dex: 47.08% per day) vs. control P B0.01 for adult rats; + 18.8% (Con: 43.48; Dex: 51.66% per day) vs. control P B0.01 for old rats). The effect of Dex did not persist in adult and old rats during the entire recovery period. The increase in liver protein synthesis after Dex treatment was also apparent using absolute synthesis rate (+ 36.9% (Con: 1.10; Dex: 1.51 g protein per day) vs. control PB 0.01 in adult rats, + 35.1% (Con: 1.24; Dex: 1.68 g protein per day) vs. control PB 0.01 in old rats) (Table 3). In this case, no residual effect of Dex on ASR was noticed during the recovery period in old rats whereas a residual effect was noted in adult rats at R+ 3. The increase in liver protein synthesis under Dex was mainly a reflection of the increase in RNA efficiency of the liver in adult rats (+17.6% (Con: 10.2; Dex: 12.0 mg protein/mg RNA per day) vs. control P B 0.01); the ribosomal efficiency was not highly altered for both age groups during the recovery period.

3.4. Total plasma proteins Dex treatment significantly increased plasma protein content of adult rats (+13.2% (Con: 61.1; Dex: 69.2 g/l) vs. control, PB 0.01) but not in old rats. This increase in protein content in adult rats did not persist during the recovery period (Table 4). Table 1 Effect of Dexamethasone on corticosterone levels in the plasma of adult and old ratsa Corticosterone (ng/ml)

DEX

R+3

R+7

Adult

Control Dex

130.1 915.6 8.1 9 1.1

138.6 929.5 47.9 97.0

109.1 9 13.5 125.1 926.4

Old

Control Dex

233.6 921.9 7.1 92.3

177.8 915.2 24.3 911.7

232.3 926.0 149.3 928.1

a

Corticosterone levels (ng/ml plasma) in of adult and old rats given dexamethasone (Dex) in their drinking water for 6 and 5 days, respectively, and in pair-fed controls; animals were killed at the end of the treatment (DEX) or allowed to recover for 3 (R+3) or 7 (R+7) days.

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Table 2 Effect of dexamethasone on weight, protein content and protein concentration in liver of adult and old ratsa ANOVA: PB DEX Weight (g) Adult Control Dex Old Control Dex Protein content (g) Adult Control Dex Old Control Dex

12.3 17.9 13.6 19.5

R+3

(n=9)b (n= 9)** (n=7) (n= 7)**

2.72 3.21* 2.87 3.25

Protein concentration (mg/g) Adult Control 220 Dex 180** Old Control 215 Dex 169**

9.2 11.4 14.2 13.2

RSE

R+7

(n =7) (n =7) (n = 4) (n =4)

2.06 2.62* 3.04 2.74 224 231 213 208

12.9 13.8 15.3 14.6

(n = 8) (n = 8) (n =4) (n = 4)

2.73 2.92 3.33 3.15 212 213 216 218

T

S

T×S

2.31

0.01

0.01

0.05

2.47

NS

0.05

0.01

0.49

0.01

0.01

NS

0.40

NS

NS

NS

10.8

0.01

0.01

0.01

15.8

0.01

0.01

0.01

a Weight, protein content and protein concentration in liver of adult and old rats given dexamethasone (Dex) in their drinking water for 6 and 5 days, respectively, and in pair-fed controls; animals were killed at the end of the treatment (DEX) or allowed to recover for 3 (R+3) or 7 (R+7) days. Mean values were significantly different from those of the control group at the same stage, *PB0.05, **PB0.01. b the values for n given for weight are the same for protein content and protein concentration at the same age of slaughter in the same treatment group.

Dexamethasone increased total plasma protein synthesis in both adult and old rats (+103.1% (Con: 36.8; Dex: 74.7%/day) vs. control PB 0.01 in adult rats and +62.6% (Con: 36.9; Dex: 60.0) vs. control PB0.01 in old rats) (Fig. 2). This increase tended to be lower in old than in adult rats.This effect also occured during the recovery period in adult rats (+ 43.9% (Con: 40.2; Dex: 57.8% per day) vs control at R+ 3 P B 0.01, and + 22.0% (Con: 48.8; Dex: 59.6) vs control at R+ 7 PB0.05). In contrast, it was only apparent at R+ 3 in old rats (+ 96.8% (Con: 28.4; Dex: 55.8%/day) vs. control PB 0.01).

3.5. Indi6idual plasma proteins (Table 4) 3.5.1. Albumin Dex induced an increase in plasma albumin concentrations in both age groups ( + 50.7% (Con: 21.7; Dex: 32.7 mg/ml) PB 0.01, + 37.2% (Con: 19.1; Dex: 26.2 mg/ml) P B 0.05 vs. control for adult and old rats, respectively). Plasma albumin was normalized during the recovery period in all animals.

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3.5.2. a1 Acid glycoprotein (a1AGP) A large increase in a1AGP was observed during Dex treatment in all rats (8–9 fold increase P B0.01). The concentrations of a1AGP in Dex treated rats decreased progressively during the recovery period and values at R+ 7 were only significantly higher in Dex treated old rats compared to control rats (due to a high value for a Dex treated old rat at R+7). 3.5.3. Fibrinogen Dex treatment decreased fibrinogen levels but this decrease was only significant in old rats ( −45.5% (Con: 4.65; Dex: 2.54 mg/ml) vs. control, PB 0.01). Fibrinogen levels were at control values during the recovery period in both groups. 3.5.4. a2 Macroglobulin (a2M) Dex treatment showed an increase in plasma a2M in adult rats ( +183% (Con: 27.1; Dex: 75.6 mg/ml) vs. control, PB 0.01).Even if a2M remained higher in Dex treated animals during the recovery period, the difference between Dex and Con animals was no more significant. In contrast, a2M was not detectable (concentrations were below 17 mg/ml) in old control and Dex-treated rats.

4. Discussion Alterations of the adaptive responsiveness to hormonal and other biochemical stimuli are characteristic of aged animals. The consequences of these alterations can include a reduced ability to respond to stress (Yoshikawa, 1984; Mosoni et al.,

Fig. 1. Fractional synthesis rates of liver proteins in adult and old rats given dexamethasone (black bars) in their drinking water for 6 and 5 days, respectively, and in pair-fed controls (open bars); The bars indicate mean values 9SE; animals were killed at the end of the treatment (DEX) or allowed to recover for 3 (R + 3) or 7 (R + 7) days.

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Table 3 Effect of dexamethasone on ribosomal capacity, absolute synthesis rate and ribosomal efficiency in liver of adult and old ratsa ANOVA: PB DEX

R+3

R+7

RSE

T

S

TxS

2.64

NS

0.01

NS

5.77

NS

0.05

NS

0.24

0.01

0.01

NS

0.21

0.05

0.05

NS

1.06

0.01

NS

0.05

1.56

NS

NS

NS

Ribosomal capacity (mg RNA/mg protein) Adult Old

Control Dex Control Dex

Absolute synthesis rate Adult Control Dex Old Control Dex

39.9 39.6 42.7 45.4

(n=9)b (n=9) (n=7) (n=7)

40.5 38.7 36.3 39.7

(n = 7) (n =7) (n = 4) (n = 4)

(g protein per day) 1.10 0.81 1.51** 1.11* 1.24 1.20 1.68** 1.22

Ribosomal efficiency (mg protein/mg RNA per day) Adult Control 10.2 9.7 Dex 12.0** 10.8* Old Control 10.4 11.0 Dex 11.6 11.3

42.1 44.5 36.6 37.6

(n =8) (n =8) (n =4) (n =4)

1.21 1.34 1.24 1.32 10.6 10.3 10.2 11.2

a Ribosomal capacity, absolute synthesis rate and ribosomal efficiency of liver proteins in adult and old rats given dexamethasone (Dex) in their drinking water for 6 and 5 days, respectively, and in pair-fed controls; animals were killed at the end of the treatment (DEX) or allowed to recover for 3 (R+3) or 7 (R+7) days. Mean values were significantly different from those of the control group at the same stage, *PB0.05, **PB0.01. b the values for n given for ribosomal capacity are the same for absolute synthesis rate and ribosomal efficiency at the same age of slaughter in the same treatment group.

1995). For example, alterations in the glucocorticoid induction of many hepatic enzymes (e.g tyrosine aminotransferase and mitochondrial malate dehydrogenase) have been observed in aged animals (Kalimi et al., 1983). We also reported an altered effect of glucocorticoids on the expression of some proteolytic genes in aged epitrochlearis muscle (Dardevet et al., 1995). Expression of the 14-kDa ubiquitin-conjugating enzyme E2, which is involved in protein ubiquitylation, and of subunits of the 20 S proteasome (the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates) was not enhanced by glucocorticoids in aged rats (Dardevet et al., 1995). In contrast, we also reported that glutamine synthetase responsiveness to excessive glucocorticoids was not modified during aging in either tibialis anterior or soleus muscles (Meynial-Denis et al., 1996). These findings clearly indicate that glucocorticoid action is not systematically impaired in aged animals. However, due to the design of our study, it is necessary to remember that all the animals are dietary restricted. The liver is very sensitive to any kind of alteration of food intake (Breuille´ et al., 1998). Consequently, the effect observed in our animals

Table 4 Effect of dexamathasone on concentration of total protein, albumin, a1 acid glycoprotein, a2 macroglobulin and fibrinogen in plasma of adult and old ratsa

DEX Total plasma proteins (g/l) Adult Old Albumin (mg/ml) Adult Old a1 AGP (mg/ml) Adult Old a2 Macroglobulin (mg/ml) Adult Old Fibrinogen (mg/ml) Adult Old

(n =9)b (n =9)** (n =7) (n = 7)

Control Dex Control Dex

61.1 69.2 66.0 71.1

Control Dex Control Dex

21.7 32.7** 19.1 26.2**

R+3

R+7

56.7 57.4 67.7 64.0

60.9 58.5 64.4 62.0

(n = 7) (n =7) (n =4) (n = 4)

(n=8) (n= 8) (n= 4) (n= 4)

23.3 20.7 21.3 18.9

20.8 18.7 18.5 14.7

Control Dex Control Dex

40.3 376** 54.7 441**

49.8 174** 52.8 159

90.6 61.8 57.0 283**

Control Dex Control Dex

27.1 75.6** ND ND

22.1 32.6 ND ND

31.8 37.4 ND ND

Control Dex Control Dex

4.05 2.95 4.65 2.54**

3.56 3.65 3.12 3.03

4.46 5.13 4.01 4.19

RSE

T

S

T×S

4.77

NS

0.01

0.01

7.83

NS

NS

NS

4.58

NS

0.01

0.01

4.55

NS

0.05

0.05

0.01

0.01

0.01

.01

0.05

0.05

0.01

0.05

0.05

1.36

NS

0.05

NS

1.04

NS

NS

0.05

48.0 125

22.9

115

a Concentration of total proteins, albumin, a1 acid glycoprotein, a2 macroglobulin and fibrinogen in plasma of adult and old rats given dexamethasone (Dex) in their drinking water for 6 and 5 days, respectively, and in pair-fed controls; animals were killed at the end of the treatment (DEX) or allowed to recover for 3 (R+3) or 7 (R+7) days. Mean values were significantly different from those of the control group at the same stage, * PB0.05, ** PB0.01. ND: not detectable. b the values for n given for total plasma protein are the same for albumin, a1AGP, a2 macroglobulin and fibrinogen at the same age of slaughter in the same treatment group.

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ANOVA: PB

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is the result of both an action of food restriction and glucocorticoid treatment with an opposite effect of Dex and food restriction on protein metabolism in liver (Santidrian et al., 1981). This effect of a long term food restriction has been previously shown to have a beneficial effect on protein turnover in liver of old animals (Ward, 1992). Then, the food restriction applied to our adult and old rats might have a different effect with age. However, our animals undergo a short term food restriction and not a long term food restriction and we have no data concerning the effect of a short term food restriction on protein turnover in liver of adult and old animals. The measurement of corticosterone in the plasma of our rats show clearly an alteration of the basal levels of this hormone with age which is consistent with the findings coming from many studies (see Mobbs 1996 for a review). The Dex treatment induced a dramatic decrease in the corticosterone levels, which is one of the metabolic adaptations (with the increase in insulinemia) that the rats used to counteract the catabolic effect of dexamethasone. However, no difference in corticosterone levels was found between adult and old animals when Dex was administred. As a consequence, the difference of response to Dex observed between adult and old animals do not come from the corticosterone levels. The Dex levels in plasma has not been measured in this study. However, some unpublished results from Minet-Quinard et al. have shown that the metabolic clearance of Dex in adult and old animals was similar. Since the clearance of Dex is similar in adult and old rats, the Dex levels in our animals should be equal and different plasma levels of Dex can not explain the effects observed. As a consequence, a greater sensitivity of the tissues themselves to Dex and/or a higher resistance to anabolic hormones like insulin or IGF-1 can explain the greater catabolic effect of Dex in old animals compared to the adults. In our experiment, plasma albumin, a1AGP and a2M concentrations increased after glucocorticoid treatment in adult rats whereas plasma fibrinogen decreased.

Fig. 2. Fractional synthesis rates of plasma proteins in adult and old rats given dexamethasone (black bars) in their drinking water for 6 and 5 days, respectively, and in pair-fed controls (open bars); The bars indicate mean values 9SE; animals were killed at the end of the treatment (DEX) or allowed to recover for 3 (R + 3) or 7 (R + 7) days.

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These effects of glucocorticoids have already been observed in growing rats (Van Gool et al., 1984; Marinkovic et al., 1989). Our experiment further demonstrated that plasma albumin and a1AGP levels still respond normally to glucocorticoids in old rats. In contrast, plasma fibrinogen decreased more and a2M was always undetectable in old rats. Accordingly, the effect of glucocorticoids on mixed plasma protein concentrations was less important in old than in adult rats. Our experiment therefore provide further support to the hypothesis that the plasma and liver proteins response to an administration of Dex may be altered during aging. The consequence of fibrinogen and a2M alterations in the physiological processes of old Dex treated rats remains unknown. The involvement of fibrinogen in controlling hemostasis and fibrinolysis may be of particular importance to the response of the whole organism to injury (which is mediated in part by glucocorticoids). a2M is a potent inhibitor of serine proteases and could therefore inactivate these enzymes (Kushner and Mackiewicz, 1993). Defective acute phase protein response was also reported in old rats after inflammation, although the mechanisms involved and the effect on acute phase proteins was different from that observed with glucocorticoids. Indeed, glucocorticoids are only one of the numerous mediators which lead to an alteration of the acute phase protein response during inflammation. Inflammation has been shown to increase plasma a1AGP, a2M and fibrinogen concentrations (positive acute phase proteins) but to decrease albumin concentration (negative acute phase protein) in young rats (Kushner and Mackiewicz, 1993). These changes in plasma protein concentrations were accompanied by an increase in positive acute phase protein mRNA (a1AGP, a1 antitrypsin) and also by a decrease in negative acute phase protein mRNA (albumin) in the liver of young mice (Post et al., 1991). Aging decreased the ability of the a1AGP and albumin gene to respond to inflammation (Post et al., 1991). Dexamethasone significantly enhanced mixed plasma protein synthesis in adult and old rats alike. Response thus was expressed more by protein synthesis than by protein concentration. Similar results were obtained with albumin (the more abundant plasma protein) and fibrinogen in head trauma patients: albumin synthesis was greatly increased despite hypoalbuminaemia; fibrinogen concentration was 3 times higher in patients than in controls while fibrinogen ASR was 9 times higher in patients (Mansoor et al., 1997). Two hypotheses could account for these findings: (1) enhanced parallel degradation of albumin and fibrinogen; and (2) transcapillary escape of these proteins from the plasma compartment. Each of these hypotheses needs to be investigated further with regard to total and specific plasma proteins in Dex-treated rats. Plasma proteins are mainly synthesized in the liver. Increased liver protein synthesis was observed in both groups. Whether or not this increase was related to the proteins secreted remains to be verified. The method used in this study to measure the synthesis of constitutive proteins in the liver may not be fully adequate. Hepatic proteins may have been secreted by the liver at t= 50 min. Therefore, the liver protein synthesis figures reported here may be underestimating total hepatic protein synthesis and overestimating endogenous hepatic protein synthesis. It was

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possible to compute the absolute rate of plasma protein synthesis in adult rats after Dex treatment. To perform these calculations, an average blood volume of 5.5% of body weight and 0.4 hematocrit were assumed for all animals. The synthesis values obtained were 5309 21 mg plasma proteins per day in Dex-treated animals and 271 920 mg plasma proteins per day in controls. The difference between the two groups was 259929 mg proteins per day. This difference was consistent with that between absolute synthesis rates in the liver of Dex and control rats (i.e. 4079 125 mg protein per day). The stimulation of hepatic protein synthesis observed in this experiment after Dex treatment was thus mainly due to the increase in exported protein synthesis. A similar conclusion could be drawn from the data of old Dex-treated and control rats. This is in accordance with a study by Millward et al. (1976) who demonstrated a small increase in fixed protein synthesis in the liver 2 days only after triamcinolone acetonide treatment. Glucocorticoids can induce the expression of only certain plasma and liver proteins in rat hepatocytes in vitro (e.g. albumin and a1AGP see Benedikt et al., 1995 for a review). Such results remains to be proved in human beings (no effect of Dex alone has been shown on serum amyloid A and C-reactive protein in human hepatoma cell lines Ganapathi et al., 1991). However, the plasma and liver protein synthesis alteration after a Dex treatment also appears to reflect interactions between other regulators, in certain cases. A major effect of glucocorticoids seems to be an enhancement of IL-6 (and to a lesser extend IL-1 type cytokines) effect on acute phase protein synthesis (Marinkovic et al., 1989; Fey et al., 1994). They could also antagonize the inhibitory effect of cytokines on albumin expression. Therefore, it seems unlikely that the impaired response to glucocorticoids in aged rats was due to impaired glucocorticoid action per se because albumin and a1AGP (potential specific targets of the hormone) responded normally. The impaired response to glucocorticoids in old rats could be the consequence of a modification of cytokine secretion and action. For instance, TNFa and IL-6 levels have been shown to be higher in old than in adult rodents (Daynes et al., 1993; Spaulding et al., 1997). Such is the case also for IL-1 and IL-6 in healthy elderly humans (Wei et al., 1992). IL-6 has also been suspected to be involved in the occurrence of a broad spectrum of age-related diseases (Ershler, 1993). Therefore, we speculate that the alteration of fibrinogen and a2M response to Dex could result from altered regulation of IL-6 as a result of the Dex treatment. These so-called type 2 acute phase proteins specifically respond to IL-6; dexamethasone was necessary to maximize the effect of IL-6 in this case (Benedikt et al., 1995). Some further experiments might clarify the real effect of Dex in association (or not) with cytokines and IL-6. To conclude, our study showed that the response of plasma and liver proteins to dexamethasone was greatly altered in aged rats. The alteration mainly involved the named ‘type 2 acute phase proteins’ (e.g. fibrinogen and a2M) which are known to be regulated by glucocorticoids via an IL-6 type cytokine action. In contrast, the direct targets of dexamethasone (albumin and a1AGP) responded normally. This suggests that the alteration of the protein response did not originate from glucocorticoid action per se but from cytokine dysregulation, especially the IL-6 type cytokine. The roles of acute phase proteins are numerous and interrelated and thus

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the physiological significance of altered fibrinogen and a2M concentrations in old rats remains to be established. This would be of particular interest due to the greater sensitivity to stressful situations, the emergence of a chronic inflammatory state and the various degenerative diseases which occur during aging.

References Almawi, W.Y., Beyhum, H.N., Rahme, A.A., Reider, M.J., 1996. Regulation of cytokine and cytokine receptor expression by glucocorticoids. J. Leukoc. Biol. 60 (5), 563 – 572. Andus, T., Geiger, T., Hirabo, T., Kishimoto, T., Heinrich, P.C., 1988. Action of recombinant human interleukin 6, interleukin 1( and tumor necrosis factor ( on the mRNA induction of acute-phase proteins, Eur. J. Immunol. 18, 739–746. Ballou, S.P., Lozanski, G.B., Hodder, S., Rzewnicki, D.L., Mion, L.C., Sipe, J.D., Ford, A.B., Kushner, I., 1996. Quantitative and qualitative alterations of acute phase proteins in healthy elderly persons. Age and Aging 25, 224–230. Benedikt, H.J., Pannen, M.D., Robotham, J.L., 1995. The acute-phase response. New Horizons 3, 183–197. Breuille´, D., Arnal, M., Rambourdin, F., Bayle, G., Levieux, D., Obled, C., 1998. Sustained modifications of protein metabolism in various tissues in rat model of long-lasting sepsis. Clin. Sci. 94, 413 – 423. Caswell, M., Pike, L.A., Bull, B.S., Stuart, J., 1993. Effect of patient age on tests of the acute-phase response. Arch. Pathol. Lab. Med. 117, 906 – 910. Dardevet, D., Sornet, C., Taillandier, D., Savary, I., Attaix, D., Grizard, J., 1995. Sensitivity and protein turnover response to glucocorticoids are different in skeletal muscle from adult and old rats. Lack of regulation of the ubiquitin-proteasome proteolytic pathway in aging. J. Clin. Invest. 96, 2113 – 2119. Dardevet, D., Sornet, C., Savary, I., Debras, E., Patureau-Mirand, P., Grizard, J., 1998. Glucocorticoid effects on insulin- and IGF-1- regulated muscle protein metabolism during aging. J. Endocrinol. 156 (1), 83–89. Dardevet, D., Sornet, C., Grizard, J., 1999. Glucocorticoid-induced insulin resistance of protein synthesis is dependent of the rapamycin-sensitive pathways in rat skeletal muscle. J. Endocrinol. 162 (1), 77 – 85. Daynes, R.A., Araneo, B.A., Ershler, W.B., Maloney, C., Li, G.Z., Ryu, S.Y., 1993. Altered regulation of IL-6 production with normal aging. J. Immunol. 150, 5219 – 5230. Ershler, W.B., 1993. Interleukin-6: a cytokine for gerontologists. J. Am. Geriatr. Soc. 41, 176 – 181. Fey, G.H., Hocke, G.M., Wilson, D.R., Ripperger, J.A., Juan, T.S.C., Cui, M.Z., Darlington, G.J., 1994. Cytokines and the acute phase response of the liver. In: Arias, M., Boyer, J.L., Fausto, N., Jakobi, W.B., Schachter, D.A., Shafritz, D.A. (Eds.), The liver: Biology and Pathobiology, Third edition. Raven Press, Ltd, New York, pp. 113 – 143. Ganapathi, M.K., Rzewnicki, D., Samols, D., Jiang, S.L., Kushner, I., 1991. Effect of combinations of cytokines and hormones on synthesis of serum amyloid A and C-reactive protein in Hep 3B cells. J. Immunol. 147 (4), 1261–1265. Garlick, P.J., McNurlan, M.A., Preedy, V.R., 1980. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H] phenylalanine. Biochem. J. 192, 719 – 723. Kalimi, M., Gupta, S., Hubbard, J., Greene, K., 1983. Glucocorticoid receptors in adult and senescent rat liver. Endocrinology 112, 341–347. Klonoff-Cohen, H., Barrett-Connor, E.L., Elderstein, S., 1992. Albumin levels as predictor of mortality in the healthy elderly. J. Clin. Epidemiol. 45, 207 – 212. Kushner, I., Mackiewicz, A., 1993. The acute phase response: an overview. In: Mackiewicz, A., Kushner, I., Baumann, H. (Eds.), Acute Phase Proteins. Molecular Biology, Biochemistry, and Clinical Applications. CRC Press, Inc., pp. 3– 21. Landfield, P.W., Baskin, R.K., Pitler, T.A., 1981. Brain-age correlates retardation by hormonal pharmacological manipulations. Science 214, 581 – 584. Manchester, K.L., Harris, E.J., 1968. Effect of denervation on the synthesis of ribonucleic acid and deoxyribonucleic acid in rat diaphragm muscle. Biochem. J. 108, 177 – 183.

120

I. Sa6ary et al. / Mechanisms of Ageing and De6elopment 122 (2001) 105–120

Mansoor, O., Cayol, M., Gachon, P., Boirie, Y., Schoeffler, P., Obled, C., Beaufrere, B., 1997. Albumin and fibrinogen syntheses increase while muscle protein synthesis decreases in head-injured patients. Am. J. Physiol. 273, E898–E902. Marinkovic, S., Jahreis, G.P., Wong, G.G., 1989. Baumann H. IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo. J. Immunol. 142, 808 – 812. Masoro, E.J., 1995. Glucocorticoids and aging. Aging 7, 407 – 413. Meynial-Denis, D., Mignon, M., Miri, A., Imbert, J., Aurousseau, E., Taillandier, D., Attaix, D., Arnal, M., Grizard, J., 1996. Glutamine synthetase induction by glucocorticoids is preserved in skeletal muscle of aged rats. Am. J. Physiol. 271, E1061 – E1066. Milman, N., Graudal, N., Andersen, H.G., 1988. Acute phase reactants in the elderly. Clin. Chim. Act. 176, 59–62. Millward, D.J., Nnanyelugo, D.O., Bates, P., Heard, C.R., 1976. Muscle and liver protein metabolism in rats treated with glucocorticoids. Proc. Nutr. Soc. 35 (1), 47A. Mobbs, C.V., 1996. Neuroendocrinology of aging. In: Schneider, E.L., Rowe, J.W., Johnson, T.E., Holbrool, N.J., Morrisson, J.H. (Eds.), Handbook of the Biology of Aging, 4th. Academic Press, San Diego, pp. 234–282. Mosoni, L., Valluy, M.C., Serrurier, B., Prugnaud, J., Obled, C., Guezennec, C.Y., Patureau Mirand, P., 1995. Altered response of protein synthesis to nutritional state and endurance training in old rats. Am. J. Physiol. 268, E238–E335. Odedra, B.R., Bates, P.C., Millward, D.J., 1983. Time course of the effect of catabolic doses of corticosterone on protein turnover in rat skeletal muscle and liver. Biochem. J. 214, 617 – 627. Pedersen, P., Hasselgren, P.O., Angeras, U., Hall-Angeras, M., Warner, B.W., LaFrance, R., Li, S., Fisher, J.E., 1989. Protein synthesis in liver following infusion of the catabolic hormones corticosterone, epinephrine, and glucagon in rats. Metabolism 38, 927 – 932. Post, D.J., Carter, K.C., Papaconstantinou, J., 1991. The effect of aging on constitutive mRNA levels and lipopolysaccharide inducibility of acute phase genes. Ann. N.Y. Acad. Sci. 621, 66 – 77. Ross, R.D., Frengley, J.D., Mion, L.C., Kushner, I., 1992. Elevated C-reactive protein in older people. J. Am. Geriat. Soc. 40, 104–105. Santidrian, S., Moreyra, M., Munro, H.N., Young, V.R., 1981. Effect of cortico1sterone and its route of administration on muscle protein breakdown, measured in vivo by urinary excretion of N tmethylhistidine in rats: Response to different levels of dietary portein and energy. Metabolism 30, 798–804. Sapolsky, R.M., Krey, L., McEwen, B.S., 1983. The adrenocortical stress-response in the aged male rat: impairment of recovery from stress. Exp. Gerontol. 18, 55 – 64. Sapolsky, R.M., Krey, L, McEwen, B.S., 1986. The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis. Endocr. Rev. 7, 284 – 301. Savary, I., Debras, E., Dardevet, D., Sornet, C., Capitan, P., Prugnaud, P., Patureau Mirand, P., Grizard, J., 1998. Effect of glucocorticoid excess on skeletal muscle and heart protein synthesis in adult and old rats. Brit. J. Nutr. 79, 297 – 304. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Spaulding, C.C., Walford, R.L., Effros, R.B., 1997. Calorie restriction inhibits the age-related dysregulation of the cytokines TNF-a and IL-6 in C3B10RF1 mice. Mech. Aging Dev. 93, 87 – 94. Van Gool, J., Boers, W., Sala, M., Ladiges, N.C.J.J., 1984. Glucocorticoids and catecholamines as mediators of acute-phase proteins, especially rat a-macrofoetoprotein. Biochem. J. 220, 125 – 132. Virca, G.D., Travis, J., Hall, P.K., Roberts, R.C., 1978. Purification of human a2-Macroglobulin by chromatography on Bibacron Blue Sepharose. Ann. Biochem. 89, 274 – 278. Ward, W.F., 1992. Alterations in liver protein turnover with age: effects of dietary restriction. Age and Nutrition 3, 212–216. Wei, J., Xu, H., Davies, J.L., Hemmings, G.P., 1992. Increase of plasma IL-6 concentration with age in healthy subjects. Life Sci. 51, 1953–1956. Yoshikawa, T.T., 1984. Aging and infectious diseases: state of the art. Gerontology 30, 275 – 278. .