Laboratoire d'Etude du Métabolisme Azoté, Institut National de la Recherche Agronomique, Centre de Clermont-Ferrand-Theix, 63122 Saint Genès Champanelle, France
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ABSTRACT |
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Sarcopenia could result from the inability of an older individual to recover muscle lost during catabolic periods. To test this hypothesis, we compared the capacity of 5-day-refed 12- and 24-mo-old rats to recover muscle mass lost after 10 days without food. We measured gastrocnemius and liver protein synthesis with the flooding-dose method and also measured nitrogen balance, 3-methylhistidine excretion, and the gene expression of components of proteolytic pathways in muscle comparing fed, starved, and refed rats at each age. We show that 24-mo-old rats had an altered capacity to recover muscle proteins. Muscle protein synthesis, inhibited during starvation, returned to control values during refeeding in both age groups. The lower recovery in 24-mo-old rats was related to a lack of inhibition of muscle proteolysis during refeeding. The level of gene expression of components of the proteolytic pathways did not account for the variations in muscle proteolysis at both ages. In conclusion, this study highlights the role of muscle proteolysis in the lower recovery of muscle protein mass lost during catabolic periods.
sarcopenia; aging; proteolysis; flooding dose; 3-methylhistidine
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INTRODUCTION |
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IN HUMANS, alterations in muscle protein metabolism occur during the aging process, leading to a loss of muscle protein or sarcopenia. This sarcopenia is responsible for decreased mobility in elderly people and also reduces their ability to cope with nutritional, infectious, or traumatic stresses (32). Thus it is important to understand the mechanisms of sarcopenia to slow its development.
Sarcopenia results from an imbalance between muscle protein synthesis and degradation. This imbalance occurs over an extended period of time [years in humans and months in rodents where an age-related loss of muscle proteins has also been described at the level of hindlimb muscles (16)]. Thus very slight variations in muscle protein balance each day over a long period could easily account for the amount of protein lost. These small variations will probably be difficult to detect. In rats, no change with age in postabsorptive muscle protein synthesis rates has been detected (24). In humans, total, myofibrillar, and mitochondrial vastus lateralis muscle protein synthesis rates were reported to be lower in old vs. young adults in the postabsorptive state (27, 29, 31). However, it is likely that the age-related loss of muscle proteins also results from a succession of more intense catabolic periods (infections, trauma, and anorexia). Sarcopenia could result from an altered capacity of older individuals to recover muscle proteins lost during catabolic periods. Indeed, refed, undernourished patients aged over 65 yr have been shown to have a lower fat-free mass recovery than those under 65 yr (14). This lower recovery is probably related to a lower stimulation of protein synthesis and/or a lower inhibition of proteolysis during refeeding; these changes are likely to be more easily detected than those involved in the gradual decrease in muscle protein mass (they are probably similar).
Thus, to test if the age-related loss of muscle protein could be related to a lower recovery of muscle mass lost during catabolic periods, we compared the capacity of 12- and 24-mo-old rats to recover from a 10-day starvation-induced loss of muscle proteins. Animals were refed for 5 days, and we analyzed how variations in muscle protein synthesis (measured in vivo with the flooding-dose method) and muscle proteolysis (derived from 3-methylhistidine urinary excretion and gene expression of several components of proteolytic pathways) could explain variations in muscle protein mass.
We have shown that there is a decrease in the ability of 24-mo-old rats to recover muscle mass lost after a catabolic event and that this decrease was related more to a dysregulation of muscle proteolysis than to a lack of stimulation of muscle protein synthesis during refeeding.
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MATERIALS AND METHODS |
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Animals and experimental design. These experiments were performed in accordance with current legislation on animal experimentation in France. We used male Sprague-Dawley rats (obtained from Iffa Credo, Lyon, France) aged 12 or 24 mo at the end of the experimentation. They were maintained in individual metabolism cages at 22°C with a 12:12-h light-dark cycle (lights on from 9 PM to 9 AM) with free access to water; they were weighed daily and were offered a semiliquid casein-based diet containing 18.7 g protein/100 g dry matter, as described previously (23). The animals had free access to food between 9 AM and 5 PM. Food consumption was recorded daily by the measurement of the amounts of dry matter offered and remaining. After a 15-day acclimatization period (weight and food consumption were stable), starvation was initiated. Our aim was to induce a significant loss of muscle proteins to be able to analyze recovery. We first used 4-day starvation, but no significant loss occurred (23). We had to use 10-day starvation to obtain a significant but moderate (20%) loss of muscle proteins. Food deprivation was well tolerated by 12- and 24-mo-old rats, and there was no mortality related to food deprivation.
Six groups of animals were used: 1) in 12-mo-old rats, we used control fed rats (n = 6; fed), 10-day-starved rats (n = 6; starvation), and 10-day-starved 5-day-refed rats (n = 5; refed); and 2) in 24-mo-old rats, we used similar groups: fed (n = 6), starved (n = 5), and refed (n = 6).
Measurements of in vivo protein synthesis rates. Protein synthesis rates were measured according to the flooding-dose method (10), which reduces uncertainty over the labeling of the tracer amino acid in the precursor pool for protein synthesis. The method was adapted for mature rats as described previously (23): each rat was injected subcutaneously with a flooding dose of valine (300 µmol/100 g body wt) to flood the precursor pools, with 50% L-[1-13C]valine (Mass-Trace, Woburn, MA). The incorporation time was 40 min. There was a <10% difference between free valine enrichments in plasma, gastrocnemius, and liver (88-89%, 84-87%, and 78-85% of injected valine, respectively). We also demonstrated that free valine enrichment in plasma reached 89% of the plateau value within 5 min after injection and remained stable for more than 45 min (23). These measurements were made between 1:30 PM and 4:30 PM in fed or refed rats (postprandial state) and between 9 AM and 11 AM in starved rats.
A lethal intraperitoneal injection of pentobarbital sodium (Sanofi,
Libourne, France) was given just before killing. As soon as anesthesia
occurred, the rats were bled. The gastrocnemius, soleus, and tibialis
anterior of both hind legs were quickly excised. The liver was cut into
small pieces, dipped in cold saline to remove the blood, and wiped. The
digestive tract was emptied, cleaned, and dried. All tissues were
weighed, frozen in liquid nitrogen, and stored at 20°C or
80°C until analysis.
Free and protein-bound valine enrichment and RNA and protein content were measured in the liver and gastrocnemius as described previously (23).
Nitrogen balance, 3-methylhistidine, and creatinine
excretion. Urine and feces were collected, weighed
daily, and kept frozen at 20°C until analysis. Urine was
collected in tubes containing 5 ml of 1 M HCl. For analysis, urine and
feces samples from 1) the 5 days
before starvation, 2) the first 4 days of starvation, 3) the last 6 days of starvation, and 4) the first
4 days of renutrition were pooled. Food and feces samples were
freeze-dried and ground before analysis. The nitrogen content in urine,
food, and feces was measured with the Kjeldhal method, and the nitrogen
balance was calculated as the difference between ingested nitrogen and nitrogen excreted in urine and feces. The urine 3-methylhistidine content was measured by liquid chromatography with an automatic amino
acid analyzer after deproteinization with 20% trichloroacetic acid and
hydrolysis with 6 M HCl at 110°C for 17 h. The urine creatinine
content was obtained after reaction with picric acid and measurement of
absorbency at 492 nm (kit, Merck, Nogent sur Marne, France).
3-Methylhistidine excretion was then calculated per 100 g initial body
weight or per milligrams of creatinine.
Northern blot analysis. To further
elucidate the role of proteolysis in the adaptation to starvation and
refeeding, we examined the expression of the mRNAs encoding proteinases
and cofactors involved in lysosomal (cathepsin D),
Ca2+-dependent (m-calpain), and
ubiquitin-proteasome-dependent (ubiquitin, 14-kDa ubiquitin carrier
protein E2, and C2 and C8 proteasome subunits] proteolytic
pathways. Total RNA was extracted from 0.25-0.35 g of
tibialis anterior muscle with the Chomczynski and Sacchi (7) method.
Twenty micrograms of total RNA were then electrophoresed in 1% agarose
gels containing formaldehyde and electrophoretically transferred to a
nylon membrane (GeneScreen, NEN, Boston, MA). RNA was covalently bound
to the membrane by ultraviolet cross-linking. The membranes were
hybridized with cDNA probes encoding human cathepsin D and m-calpain,
chicken polyubiquitin, and rat 14-kDa ubiquitin-conjugating enzyme E2,
and C2 and C8 proteasome subunits as described previously (28).
Hybridizations were conducted overnight at 65°C with
[32P]cDNA fragments
labeled by random priming. After being washed at the same temperature,
the membranes were autoradiographed at 80°C with
intensifying screens on Hyperfilm-MP (Amersham). Autoradiographic signals were quantified by digital image processing and analysis (National Institutes of Health Image 1.54) and were normalized with the
corresponding 18S rRNA signals to correct for uneven loading.
Statistics. Results are given as means ± SE. Two-way ANOVA was performed to discriminate between the effect of age (12- or 24-mo-old rats) and the effect of nutritional state (fed, starved, and refed). The evolution of the nitrogen balance and 3-methylhistidine excretion during starvation and refeeding was analyzed in refed 12- and 24-mo-old animals by repeated-measures ANOVA. When ANOVA detected a significant effect, means were compared by the Student's t-test. The nonparametric Wilcoxon's test was used to compare means for mRNA level measurements. When not otherwise stated, the significance level was set at 0.05. These analyses were performed with the SAS computer program.
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RESULTS |
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Food intake, body, and tissue weights.
At the end of the acclimatization period and before any nutritional
treatment, food intake was 20.4 ± 0.5, 18.1 ± 0.9, and 19.6 ± 0.7 g dry matter in the three groups of 12-mo-old rats (to be
fed, to be starved, and to be refed, respectively) and 17.4 ± 1.0, 18.1 ± 0.7, and 18.7 ± 0.6 g dry matter in the three groups of
24-mo-old rats (to be fed, to be starved, and to be refed,
respectively). There was a tendency for a lower food intake in
24-mo-old rats compared with 12-mo-old rats
(P = 0.06). After food deprivation and
during the first 2 days of refeeding, food intake was lower in refed rats than in control rats in both age groups and then became higher than in control rats (Table 1). The total
amount of dry matter consumed during the first 4 days of refeeding was
the same in all groups. Food intake recorded in fed and refed rats
during the day of killing was lower than on the previous day because the feeding period was shorter (6 vs. 8 h). Food intake on this day was
also higher in refed rats than in control fed rats. Food intake during
refeeding was similar for 12- and 24-mo-old rats.
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At the end of the acclimatization period and before any nutritional treatment, body weights were 698 ± 36, 662 ± 27, and 655 ± 28 g in the three groups of 12-mo-old rats (to be fed, to be starved, and to be refed, respectively) and 686 ± 28, 727 ± 40, and 672 ± 18 g in the three groups of 24-mo-old rats (to be fed, to be starved, and to be refed, respectively). There were no significant differences between groups. After 10 days without food, 12-mo-old rats lost 20% body weight and 24-mo-old rats lost 19%. Refeeding for 5 days produced a 10-15% increase in body weight (15.0 ± 1.4% in 12-mo-old rats; 10.1 ± 0.8% in 24-mo-old rats).
During starvation, 12-mo-old rats lost 25% gastrocnemius muscle weight
(Fig. 1), and recovery was started but not
completed after 5 days (+7%). In 24-mo-old rats, loss was similar
(21%) but there was no recovery within 5 days. As previously
observed, muscle mass in the 24-mo-old rats was less than in the
12-mo-old rats. The effect of starvation was very marked in the liver
and in the digestive tract (Fig. 1). After 10 days without food, liver weight decreased by 49% in 12-mo-old rats and by 34% in 24-mo-old rats. Whatever the age, recovery was complete. At time of death, liver
wet weight was not different between 12- and 24-mo-old
rats. The results were similar in the digestive tract with
a 24% decrease in weight after starvation in 12-mo-old rats (19% in
24-mo-old rats), and there was complete recovery at both ages. The
weight of the digestive tract was greater in 24-mo-old rats than in
12-mo-old rats.
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Nitrogen balance. Because it was
possible to measure nitrogen balance continuously in the same rat, we
could take into account individual variability and follow the nitrogen
balance in 12- and 24-mo-old refed rats, which were successively fed,
starved, and refed. Nitrogen balance became negative during food
deprivation, reflecting a loss of total body proteins (Fig.
2). Refeeding induced a restoration of
positive nitrogen balance in both groups. However, nitrogen balance was
significantly lower during refeeding in 24-mo-old rats compared with
12-mo-old rats. Nitrogen balance became significantly higher during
refeeding than during the control period in 12-mo-old rats. We
confirmed this significantly lower recovery by calculating the total
amount of nitrogen regained during refeeding minus the amount lost
during food deprivation in each group (12-mo-old refed rats = 138 ± 111 mg N; 24-mo-old refed rats =
1,182 ± 175 mg N). It should be underlined that recovery in the intestine and the liver (Fig. 1) was complete in 24-mo-old rats. Thus it is likely
that it was muscle proteins that recovered less in 24-mo-old rats,
which is confirmed by the gastrocnemius muscle results (Fig. 1).
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Gastrocnemius protein metabolism.
Protein metabolism in the gastrocnemius muscle is shown in Table 2.
After 10 days without food, 12-mo-old rats lost 19% protein, which was
partially recovered after 5 days of refeeding, reaching 89% of the
control value. In 24-mo-old rats, there was
a 21% decrease in mean protein mass after starvation and there was no
recovery. Protein mass in 24-mo-old rats was less than in 12-mo-old
rats in all groups. Adult rats lost 25% of total RNA during
starvation, and there was no recovery (+3%). In 24-mo-old rats,
variations in total RNA during starvation and refeeding were not
significant. Capacity for protein synthesis, which is the ratio of
total RNA to total protein, was not modified by nutritional treatments
at both ages but was significantly greater in 24-mo-old rats than in
12-mo-old rats only in refed rats. Absolute protein synthesis rates
were markedly lower during starvation in 12-mo-old rats (57%)
and to a lesser extent in 24-mo-old rats (
35%). Refeeding
allowed a return to control values whatever the age. Absolute
synthesis rates, when similar at both ages except during starvation
when they were significantly higher in 24-mo-old rats. Ribosomal
efficiency, which is the amount of protein synthesized per unit RNA
and per day, was significantly decreased during starvation in 12-mo-old
rats (
37%) and tended to decrease in 24-mo-old rats (
13%). It was restored during refeeding at both ages. Ribosomal efficiency was significantly higher in 24-mo-old rats than in 12-mo-old
rats.
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Liver protein metabolism. Protein
metabolism in the liver is shown in Table 3. Total liver protein mass
was markedly lower in starved animals than in control animals for both
age groups (40% in 12-mo-old rats;
46% in 24-mo-old
rats. Recovery was good at both ages, but
in 24-mo-old rats, liver protein mass reached slightly higher values in
refed rats than in control rats (a significant interaction between age
and nutritional treatments was detected by ANOVA). A significant effect
of age on liver protein mass was also detected. Starvation markedly
affected liver total RNA (
40% in 12-mo-old rats;
52% in
24-mo-old rats). Refeeding induced a partial recovery at both ages.
Total RNA was greater in 24-mo-old rats than in 12-mo-old rats. There
was no effect of age and nutritional treatment on the capacity for
protein synthesis. Liver protein absolute synthesis rates were markedly
lower in starved animals than in control animals (
58% in
12-mo-old rats;
52% in 24-mo-old rats). Refeeding restored
control values at both ages. Synthesis rates were greater in 24-mo-old
rats than in 12-mo-old rats. In 12-mo-old rats, liver ribosomal
efficiency was significantly lower in starved rats than in the other
groups. In 24-mo-old rats, ribosomal efficiency was significantly
higher in refed rats than in the other groups. ANOVA detected no effect
of age but a significant interaction between age and nutritional
treatment effect.
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3-Methylhistidine excretion.
3-Methylhistidine excretion is an index of skeletal muscle proteolysis,
although it also derives from the degradation of smooth muscles from
the splanchnic area. It can be expressed as a fractional rate, per
milligrams of creatinine, an index of muscle mass (Fig.
3, top),
or as an absolute rate, per body weight. Here, we used initial body
weight (Fig. 3, bottom) to take into
account variations in body weight during starvation. In
the same way as for nitrogen balance, we measured 3-methylhistidine and
creatinine excretion in refed animals. 3-Methylhistidine excretion tended to decrease during starvation, in particular during the first
few days. This decrease was significant only for 12-mo-old rats when
3-methylhistidine was expressed per 100 g initial body weight. During
refeeding, 3-methylhistidine excretion was either unchanged or
decreased compared with control values in 12-mo-old rats. In 24-mo-old
rats, regardless of the method of expression, 3-methylhistidine
excretion was always significantly higher during refeeding than during
the control period (the interaction between age and nutritional state
effects was significant). Thus the effect of refeeding on
3-methylhistidine excretion was significantly different in 12- and
24-mo-old rats. 3-Methylhistidine excretion adjusted to creatinine
excretion was significantly higher in 24-mo-old rats compared with
12-mo-old rats. This was not the case when 3-methylhistidine excretion
was adjusted to initial body weight. This is related to the lower
creatinine excretion in 24-mo-old rats, reflecting a loss of muscle
mass.
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Expression of components of proteolytic
pathways. There was no marked effect of nutritional
treatments on mRNA levels for various critical components of
proteolytic pathways (Fig. 4). These levels
tended to increase during starvation; the increases were significant
for ubiquitin, 14-kDa E2, m-calpain in 12-mo-old rats, and for
cathepsin D in 24- mo-old rats. Values obtained in refed rats were
similar to values obtained in control rats in all cases. Regarding
the effect of age (Fig. 5), there was often
a tendency for an increase with age, but this increase was significant
only for the C2 subunit of the 20 S proteasome.
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DISCUSSION |
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The loss of muscle protein observed during senescence could be related to an inability of older individuals to recover muscle mass lost during catabolic periods. To test this hypothesis, we analyzed how 12- and 24-mo-old rats could recover from a food deprivation-induced loss of muscle mass and examined whether variations in muscle protein synthesis and degradation could explain variations in muscle mass in both age groups.
Our data showed that the capacity to recover lost muscle mass is impaired in old rats: 1) 5 days of refeeding 10-day-starved 24-mo-old rats did not induce any increase in gastrocnemius muscle fresh weight and total protein. In 12-mo-old rats, gastrocnemius muscle weight and total protein increased during refeeding to a value not significantly different from control fed rats. 2) The amount of body protein recovered during refeeding, as reflected by nitrogen balance, was significantly and markedly higher in 12-mo-old rats than in 24-mo-old rats. However, recovery was the same at both ages in liver and digestive tract. Muscle mass represents the predominant proportion of body protein. Thus it seems very likely that the lower recovery of body proteins detected by nitrogen balance represents a lower recovery of body muscle protein.
To our knowledge, no studies have compared the recovery capacities of 12- and 24-mo-old rats after prolonged periods of food deprivation. However, using dietary protein depletion to induce a decrease in muscle mass, Carter and Lynch (5) showed that hindlimb muscle mass was still unchanged compared with depleted animals after 3 wk of protein refeeding in 24-mo-old rats, whereas it was significantly higher in 3-mo-old rats. An age-related impaired capacity to recover muscle mass lost during catabolic periods has also been described in humans (14).
This lower capacity of muscle mass recovery after fasting in 24-mo-old rats could be related to alterations either in muscle protein synthesis or in muscle protein degradation pathways. In our study, muscle protein synthesis was stimulated to a similar extent in 12- and 24-mo-old refed rats by 5 days of refeeding. This stimulation of muscle protein synthesis did not occur in the first hours of refeeding (23), and it is still possible that this stimulation occurred more slowly in 24-mo-old rats. However, based on protein measurements at day 5, it seems that the lower recovery of muscle proteins in 24-mo-old rats was not related to a lack of stimulation of muscle protein synthesis.
Because muscle protein mass is controlled by the balance between protein synthesis and degradation, if the lower recovery is not related to age-related differences in muscle protein synthesis, it must be related to protein degradation. Indeed, the muscle proteolysis response to refeeding, as reflected by 3-methylhistidine excretion, was significantly different in 12- and 24-mo-old rats.
3-Methylhistidine excretion represents the myofibrillar component of muscle protein degradation and may also reflect turnover of 3-methylhistidine in the splanchnic region; the proportion of urine 3-methylhistidine originating from skeletal muscle was estimated to be from 50% (22) to 85% (4). Thus it is only an index of muscle proteolysis. It can be expressed either per milligrams of creatinine, an index of muscle mass, or per body weight. Expressed in either way, it was significantly higher during the refeeding period than during the control period in 24-mo-old rats, thus preventing muscle mass recovery. On the contrary, in 12-mo-old rats it was decreased (when expressed per mg creatinine) or unchanged (when expressed per 100 g initial body wt). Thus the effect on 3-methylhistidine excretion of refeeding was significantly different in 12- and 24-mo-old rats.
The tendency of a lower 3-methylhistidine excretion per milligrams of creatinine in 12-mo-old rats could have been related to overeating during refeeding. However, overeating was similar in refed 12-mo-old rats and refed 24-mo-old rats. To our knowledge, no studies have compared the rate of 3-methylhistidine excretion in 12- and 24-mo-old rats during starvation refeeding. However, a decrease in 3-methylhistidine excretion during refeeding after fasting has been described in 320-g rats (26) and in adult women (15). Other studies with younger animals, less resistant to food deprivation, showed that refeeding allowed a return to control values after increased excretion during starvation (12, 25). Thus the results that we obtained are consistent with results obtained in other studies.
It must be noted that 3-methylhistidine excretion per milligrams of creatinine, which is equivalent to a fractional degradation rate, was higher in 24-mo-old rats than in 12-mo-old rats whatever the group. Similarly, fractional synthesis rates (data not shown) were higher in 24-mo-old rats than in 12-mo-old rats. We propose that these higher fractional rates are mainly due to the age-related loss of muscle mass (lower creatinine excretion and lower muscle protein mass). Similar results for fractional synthesis rates have been obtained previously (24). As discussed in this paper, it is not clear whether this represents a true increase in muscle protein turnover or if this is a bias due to the calculation. Both 3-methylhistidine excretion per initial body weight and gastrocnemius absolute synthesis rates were unchanged with age.
These results are not consistent with data obtained in humans where total, myofibrillar, and mitochondrial vastus lateralis muscle protein synthesis rates were reported to be lower in old vs. young adults in the postabsorptive state (27, 29, 31). This discrepancy could be related to a species difference. However, human measurements were performed with a small sample of the same muscle and need to be confirmed in other muscles or with different techniques. Indeed, most human studies have not been able to detect a decrease with age in whole body protein synthesis rates per kilogram fat-free mass (2).
The effect of starvation on muscle protein metabolism was similar in 12- and 24-mo-old rats. Mobilization of muscle proteins was similar; muscle protein synthesis was inhibited, and 3-methylhistidine excretion tended to be reduced at both ages. Such a sparing of muscle during starvation through an inhibition of both protein synthesis and degradation has already been described (9, 11, 13, 18, 19, 23). Our results are characteristic of the effect of long-term fasting in moderately fat mature animals (fat content is 15% body wt in 12-mo-old rats, 20% body wt in 24-mo-old rats; unpublished data). There was no effect of starvation on the oxidative soleus protein mass (data not shown). This is consistent with results obtained previously (1, 23). Glycolytic or mixed muscles, which are predominant in the body, are more sensitive to starvation than oxidative muscle.
The increase in 3-methylhistidine excretion during refeeding in 24-mo-old rats was not related to a higher expression of mRNA encoding the components of proteolytic pathways (ubiquitin, C8, 14-kDa E2, m-calpain, and cathepsin D); it was unchanged or slightly lower in the refed group compared with the control group. Similarly, there was a small but significant increase for ubiquitin, 14-kDa E2, and m-calpain mRNA during starvation in 12-mo-old rats, whereas 3-methylhistidine excretion was unchanged or decreased in these animals. This could be related to the fact that 3-methylhistidine excretion reflected the mean of several days, whereas mRNA expression was measured on the 10th day of starvation and on the 5th day of refeeding. On the contrary, in young growing rats and 2-kg rabbits, there was a good correlation between the observed increase in protein degradation (measured in vitro or with 3-methylhistidine excretion) during starvation and variations of mRNA expression for proteasome subunits, ubiquitin, and 14-kDa E2 (21, 30) or for µ-calpain, m-calpain, cathepsin D, and proteasome C2 subunit (17). It is possible that the level of regulation of proteolysis may be different in older rats that are less sensitive to starvation. Indeed, Belkhou et al. (3) showed that muscle cathepsin B, H, and L activities fell during phase II of starvation during which protein degradation is inhibited in 375-g rats.
Liver protein mass and protein synthesis were markedly decreased during starvation and returned to fed values after refeeding at both ages. Overeating in refed rats probably helped the recovery. Similar results were obtained previously with 4-day starvation (23). With longer starvation (10 days), an additional 8% loss of protein and an additional 26% loss of RNA occurred. In studies with similar methodologies, a decrease in liver protein synthesis during starvation has also been obtained (6, 20). However, De Blaauw et al. (8) with the arteriovenous dilution model obtained an increase in liver protein synthesis of 112-h starved 200- to 300-g rats. The reasons for this discrepancy between incorporation studies and the arteriovenous dilution model are still unclear.
Recovery of liver protein was favored relative to muscle protein. In 24-mo-old rats, recovery of liver and muscle proteins seemed better and worse, respectively, than in 12-mo-old rats. Whether the greater recovery of liver proteins leads to the lesser recovery of muscle proteins in 24-mo-old rats by decreasing amino acid availability for muscle (or vice versa) remains an open question.
In conclusion, the capacity of muscle to recover proteins lost during starvation seems to be impaired in 24-mo-old rats. Although we cannot exclude the possibility of a slower stimulation of protein synthesis during refeeding, after 5 days, the lower recovery seems to be mainly due to a different regulation of muscle protein degradation, which is stimulated during refeeding. Further studies are necessary to understand the mechanisms responsible for this dysregulation of muscle protein degradation.
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ACKNOWLEDGEMENTS |
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We would like to thank Eveline Aurousseau and Daniel Taillandier for advice in the performance of Northern blots, Marcel Sallas and Christian Lafarge for help in animal care, and Hélène Lafarge and Danièle Bonin for help in the management of the bibliography.
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FOOTNOTES |
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L. Mosoni was supported by a grant from Institut Danone.
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: L. Mosoni, Unité d'Etude du Métabolisme Azoté, Institut National de la Recherche Agronomique, Theix, 63122 Saint Genès Champanelle, France (E-mail: mosoni{at}clermont.inra.fr).
Received 5 November 1998; accepted in final form 7 June 1999.
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