A leucine-supplemented diet restores the defective postprandial inhibition of proteasome-dependent proteolysis in aged rat skeletal muscle - PubMed (original) (raw)

Comparative Study

A leucine-supplemented diet restores the defective postprandial inhibition of proteasome-dependent proteolysis in aged rat skeletal muscle

Lydie Combaret et al. J Physiol. 2005.

Abstract

We tested the hypothesis that skeletal muscle ubiquitin-proteasome-dependent proteolysis is dysregulated in ageing in response to feeding. In Experiment 1 we measured rates of proteasome-dependent proteolysis in incubated muscles from 8- and 22-month-old rats, proteasome activities, and rates of ubiquitination, in the postprandial and postabsorptive states. Peptidase activities of the proteasome decreased in the postabsorptive state in 22-month-old rats compared with 8-month-old animals, while the rate of ubiquitination was not altered. Furthermore, the down-regulation of in vitro proteasome-dependent proteolysis that prevailed in the postprandial state in 8-month-old rats was defective in 22-month-old rats. Next, we tested the hypothesis that the ingestion of a 5% leucine-supplemented diet may correct this defect. Leucine supplementation restored the postprandial inhibition of in vitro proteasome-dependent proteolysis in 22-month-old animals, by down-regulating both rates of ubiquitination and proteasome activities. In Experiment 2, we verified that dietary leucine supplementation had long-lasting effects by comparing 8- and 22-month-old rats that were fed either a leucine-supplemented diet or an alanine-supplemented diet for 10 days. The inhibited in vitro proteolysis was maintained in the postprandial state in the 22-month-old rats fed the leucine-supplemented diet. Moreover, elevated mRNA levels for ubiquitin, 14-kDa ubiquitin-conjugating enzyme E2, and C2 and X subunits of the 20S proteasome that were characteristic of aged muscle were totally suppressed in 22-month-old animals chronically fed the leucine-supplemented diet, demonstrating an in vivo effect. Thus the defective postprandial down-regulation of in vitro proteasome-dependent proteolysis in 22-month-old rats was restored in animals chronically fed a leucine-supplemented diet.

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Figures

Figure 1

Figure 1. Skeletal muscle proteasome-dependent proteolysis after feeding 8- and 22-month-old animals

Rats from Experiment 1 were overnight starved (PA), or fed during 1 h either a alanine-supplemented (PP) or a leucine-supplemented meal (Leu). Proteasome-dependent proteolysis was measured as indicated in Methods in incubated epitrochlearis muscles harvested in the PA state or 2 h after meal ingestion. Values are means ±

s.e.m.

(vertical bars) for 9–10 animals and are expressed as a percentage of 8-month-old PA. Columns with different letters are significantly different from each other as assessed by ANOVA (P < 0.05).

Figure 2

Figure 2. Chymotrypsin-like (A), and trypsin-like (B) peptidase activities of the proteasome in pooled extensor digitorum longus muscles (n = 7–10 rats) from 8- and 22-month-old rats

Animals from Experiment 1 were overnight starved (PA) or fed during 1 h (PP or Leu) as described in Fig. 1 legend. Data represent the slopes of best fit of arbitrary fluorescence units released from Suc-LLVY-AMC (chymotrypsin-like activity) or Boc-LRR-AMC (trypsin-like activity) versus time. Data are expressed as percentage of 8-month-old PA and bars denote standard errors of the slopes. Columns with different letters are significantly different by comparing the slopes of best fit (P < 0.05).

Figure 3

Figure 3. Ubiquitination rates in pooled extensor digitorum longus muscles (n = 9–11 rats) from 8- and 22-month-old rats

The formation of high molecular weight [125I]Ub conjugates (HMWC) in extensor digitorum longus muscle extracts was followed by autoradiography (A). Animals from Experiment 1 were overnight starved (PA) or fed during 1 h (PP or Leu) as described in Fig. 1 legend. B, the comparison of ubiquitination rates (e.g. the slopes of best fit of cpm bound to HMWC following gel excision versus time). Data are expressed as percentage of 8-month-old PA and bars denote standard errors of the slopes. Columns with different letters are significantly different by comparing the slopes of best fit (P < 0.05).

Figure 4

Figure 4. Total and non-lysosomal Ca2+-independent proteolysis in 22-month-old rats chronically fed an alanine- or leucine-supplemented diet

Rats from Experiment 2 received a diet supplemented with either alanine or leucine for 10 days. The day of the experiment they were overnight starved (PA Ala or PA Leu) or fed during 1 h either diet (PP Ala or PP Leu). Rates of total proteolysis and non-lysosomal Ca2+-independent proteolysis were measured in incubated epitrochlearis muscles as described in Methods. Values are means ±

s.e.m.

(vertical bars) for 9–10 animals and are expressed in nmol of Tyr (mg protein)−1 h−1. *P < 0.05 versus PA Leu as assessed by ANOVA.

Figure 5

Figure 5. mRNA levels for genes encoding components of the Ub–proteasome pathway in 8- and 22-month-old rats chronically fed an alanine- or leucine-supplemented diet

Eight- and 22-month-old rats from Experiment 2 received for 10 days a diet supplemented with either alanine (A) or leucine (B). The day of the experiment animals were overnight starved, extensor digitorum longus muscles were harvested, and Northern blots for Ub, the 14-kDa E2, and subunits C2 and X of the 20S proteasome were performed as described in Methods. Hybridization signals were quantified and normalized using the corresponding 18S rRNA signals to correct for uneven unloading. Both transcripts for Ub and the lower transcript 14-kDa E2 were quantified. Values are means ±

s.e.m.

(vertical bars) for n = 5–7 rats, and are expressed as percentage of 8-month-old rats fed the Ala-supplemented diet. Representative Northern blots are also shown. *P < 0.05 versus 8-month-old rats by the unpaired Student's t test.

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