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J. Anim. Sci. 2005. 83:117-123
© 2005 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Nutrient restriction differentially modulates the mammalian target of rapamycin signaling and the ubiquitin-proteasome system in skeletal muscle of cows and their fetuses1

M. Du2, M. J. Zhu, W. J. Means, B. W. Hess and S. P. Ford

Department of Animal Science, University of Wyoming, Laramie 82071


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Literature Cited
 
The mammalian target of rapamycin (mTOR) signaling controls nutrient-stimulated protein synthesis in skeletal muscle, whereas ubiquitin-proteasome systems control the degradation of myofibrillar proteins. The objective of this study was to elucidate the effect of nutrient restriction on the mTOR signaling and ubiquitin-proteasome system in the skeletal muscle of cows and their fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet that provided 100% nutrient requirements or a nutrient-restricted diet at 68.1% of NEm and 86.7% of metabolizable protein requirement. Cows were slaughtered on 125 d of gestation, and the LM of both cows and fetuses was sampled for the measurement of mTOR, ribosomal protein S6, adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein ubiquitylation. When comparing the muscle samples from nutrient-restricted and control cows and their fetuses, no difference was observed for the content of mTOR and ribosomal protein S6, but the phosphorylation of mTOR at Ser2448 and ribosomal protein S6 at Ser235/336 were greater (P < 0.05) in control muscle than in muscle from nutrient-restricted animals. Because the phosphorylation of mTOR and ribosomal protein S6 upregulates translation, these results showed that nutrient restriction inhibits protein synthesis in muscle. The activity of AMPK in the muscle of nutrient-restricted cows was significantly lower (P = 0.05) than that of control cows. The protein ubiquitylation, however, was greater (P < 0.05) in the muscle from nutrient-restricted cows, showing accelerated protein degradation. No difference in the protein ubiquitylation was detected for fetal muscle. Data suggested that the decreased protein synthesis and promoted protein degradation resulted in muscle atrophy of pregnant cows, but not in fetal muscle. Results of this study show that in response to nutrient restriction, protein degradation was differentially regulated between cow and fetal muscle. The atrophy of cow muscle during nutrient deficiency may involve the enhanced degradation of muscle proteins.

Key Words: Cow • Fetus • Mammalian Target of Rapamycin • Nutrient Restriction • Skeletal Muscle • Ubiquitin


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Literature Cited
 
Skeletal muscle fibers have no ability to proliferate. Postnatal growth depends on a special pool of cells (called satellite cells) that proliferate, synthesize myofibrillar proteins, and fuse with existing muscle fibers, resulting in an increase of myofibrils in muscle fibers (Morgan and Partridge, 2003Go). However, myofibrils are also degraded, which decreases the number of myofibrils (Goll et al., 2003Go). Muscle mass is maintained through this constant turnover of myofibrils. Fasting induces a dramatic loss of muscle mass (Du et al., 2004Go). It has been suggested that ubiquitin-proteasome systems are involved in the degradation of myofibrillar protein induced by nutrient deprivation and cachexia (Costelli and Baccino, 2003Go; Lee et al., 2004Go; Tisdale, 2004Go). The mammalian target of rapamycin (mTOR) signaling pathway controls the nutrient-stimulated protein synthesis in muscle through phosphorylation of key factors involving protein synthesis (Bodine et al., 2001Go; Bolster et al., 2003Go; Sakamoto et al., 2003Go). Its activity is downregulated in cells when the supply of nutrients, especially AA, is insufficient (Nave et al., 1999Go; Bodine et al., 2001Go). We observed that malnutrition induced skeletal muscle atrophy in pregnant cows (Du et al., 2004Go), which could be due both to a decrease in protein synthesis and an enhancement in protein degradation. However, the effect of nutrient restriction on the synthesis of myofibrillar proteins is largely unclear. Moreover, knowledge about the effects of nutrient restriction on the synthesis and degradation of myofibrillar protein in fetal muscle, which is important in that the number of muscle fibers in adult muscle is determined by prenatal muscle development, is lacking. The objective of the current study was to evaluate the activity of mTOR signaling and ubiquitin-proteasome system in the skeletal muscle of both cows and fetuses following global nutrient restriction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Literature Cited
 
Animals
All animal procedures were approved by the University of Wyoming Animal Care and Use Committee. From d 31 to 125 of gestation, 20 Angus x Gelbvieh rotationally crossed cows (initial BW = 576.8 ± 7.7 kg; BCS = 5.4 ± 0.1) were blocked by BW and fed one of two dietary treatments. Ten cows were allotted to a control diet consisting of native grass hay (12.1% CP and 70.7% IVDMD, DM basis) fortified with vitamins and minerals fed at NRC (1996)Go recommendations for a nonlactating, mature cow to gain 0.72 kg/d during the first 120 d of gestation. The other 10 cows were allotted to a nutrient-restricted diet, which consisted of feeding half the vitamins and minerals of the control diet and millet straw (9.9% CP and 54.5% IVDMD, DM basis) to provide 68.1% of the NEm and 86.7% of the metabolizable protein requirements during the first 120 d of gestation (NRC, 1996Go). This diet formulation resulted in a global nutrient restriction for nutrient-restricted cows, including deficiency in protein, energy, minerals and vitamins. Cows were housed individually. During the trial period, control cows increased in BW by 4.2%, whereas nutrient-restricted cows lost 7.1% of initial BW. Ten pregnant cows from each dietary treatment group were slaughtered at the University of Wyoming Meat Laboratory on d 125 of gestation (Du et al., 2004Go).

Within 30 min of exsanguination, a sample was removed from the LM of the right side of the split carcass at the 12th rib and snap-frozen in liquid N. The sample was then stored at –80°C until analysis. The carcasses were split, kept at 4°C for 24 h, and the LM area was measured at the cutting surface at the 12th rib of the left side of carcasses.

Immediately following exsanguination of pregnant cows, fetuses were removed from the uteri and weighted after removing internal organs. A sample was removed from the LM of the right side of fetuses at 12th rib and snap-frozen in liquid N and then stored at –80°C until analysis. The fetal muscle samples were taken within 10 min from cow death.

Preparation of Skeletal Muscle Homogenate
Muscle samples (0.1 g) were homogenized in a polytron homogenizer (7-mm-diameter generator) with 5 volumes of ice-cold lysis buffer (50 mM HEPES, pH 7.4; 2% SDS; 1% NP-40; 10% glycerol; 2 mM Na3VO4; 100 mM NaF; 1% protease inhibitor cocktail [Sigma, St. Louis, MO]) and centrifuged at 12,000 x g for 5 min. The protein content of supernatant was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA).

Antibodies
Polyclonal anti-mTOR, antiribosomal protein S6, and antiubiquitin antibodies, and phospho-specific antibodies for mTOR (Ser2448) and ribosomal protein S6 (Ser235/236) were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Immunoblotting
Each muscle homogenate containing equal amount of proteins was mixed with a same volume of 2x standard SDS sample loading buffer. A 5 to 20% acrylamide gradient gel was used for SDS-PAGE separation of proteins. A Hoefer mini-gel system was used for casting gels and running electrophoresis. Following electrophoresis, the proteins on the gel were transferred to nitro-cellulose membrane in a transfer buffer containing 20 mM Tris-base, 192 mM glycine, 0.1% SDS, and 20% methanol.

Membranes were incubated in a blocking solution consisting of 5% nonfat dry milk in 0.1% Tween-20, 50 mM Tris-HCl (pH7.6), and 150 mM NaCl (TBS/T) for 1 h. Then, membranes were incubated overnight in primary antibodies with proper dilution in TBS/T with 1% nonfat dry milk. At the end of the primary antibody incubation, the membranes were washed three times for 5 min each with 20 mL of TBS/T. After that, membranes were incubated with horseradish peroxidase-conjugated monkey-anti-mouse secondary antibody (1:2,000) for 1 h in TBS/T with gentle agitation. Following three 10-min washes, membranes were visualized using ECL Western blotting reagents (Amersham Bioscience, Piscataway, NJ) and exposure to film (MR, Kodak, Rochester, NY). The density of bands was quantified by using an Imager Scanner II and ImageQuant TL software (Amershan Bioscience), and a relative density of bands for samples from nutrient-restricted vs. control animals was reported (Du et al., 2004Go). To decrease the variation between blots, an equal number of samples from either nutrient-restricted or control animals was loaded in the same gel. The densities of bands among different blots were normalized by using the average band density in each blot. The identification of bands was done manually. For the quantification of protein ubiquitylation, the density of the whole lanes was measured.

Membrane Stripping and Reprobing
For reprobing the membrane, membranes were incubated in a stripping solution (100 mM 2-mercaptoethanol; 2% SDS; 62.5 mM Tris-Cl, pH 6.7) at 50°C for 30 min with shaking. Then, the membranes were thoroughly washed with TBS/T and blocked with 5% nonfat dry milk in TBS/T. Membranes were ready for immunoblotting as described above.

Measurement of AMPK Activity
The AMPK activity was measured as previously reported (Davies et al., 1989Go). Briefly, muscle (0.1 g) was minced with scissors and homogenized in ice-cold 0.25 M mannitol, 0.05 M Tris/HCl (pH 7.8), 1 mM EDTA, 1 mM ethylene glycol-bis(2-amino ethylether)-N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, 50 mM NaF, and 5 mM sodium pyrophosphate, plus 1% protease inhibitor cocktail (Sigma). The muscle homogenate was centrifuged at 13,000 x g for 5 min at 4°C. The supernatant (5µL) was incubated for 10 min at 37°C in 40 mM HEPES, 0.2 mM SAMS peptide (His-Met-Arg-Ser-Ala-Met-Ser-Gly-Leu-His-Leu-Val-Lys-Arg-Arg, In-vitrogen, Carlsbad, CA), 0.2 mM adenosine 5'-monophosphate (AMP), 80 mM NaCl, 8% (wt/vol) glycerol, 0.8 mM EDTA, 0.8 mM dithiothreitol, 5 mM MgCl2, and 0.2 mM ATP + 2 µCi [32P]ATP (pH 7.0) in a final volume of 25 µL. An aliquot (20 µL) was removed and spotted on a 2 x 2 cm piece of Whatman P81 filter paper (Whatman, Inc., Clifton, NJ). The remaining [32P]ATP was removed with three washes of 1% phosphoric acid and once with acetone. The filter paper was air-dried, and radioactivity was quantified after immersing the filter paper in 3 mL of Scintiverse (Fisher Scientific, Hanover Park, IL). The activity of AMPK was defined as the catalysis of phosphate incorporation (nM) into the peptide per minute, per milligram of muscle protein.

Statistical Analyses
Data were analyzed as a complete randomized design using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). The relative density of bands, AMPK activity, carcass weight, and cross-sectional area of LM were analyzed. The differences in the mean values were compared by the Tukey’s multiple comparison, and mean values and standard deviation were reported (P < 0.05).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Literature Cited
 
The rate of protein translation is tightly regulated by the availability of nutrients. The mTOR is the main kinase involved in the sensing of nutritional status in cells and coordinates it with the protein synthesis (Bodine et al., 2001Go; Bolster et al., 2003Go; Sakamoto et al., 2003Go). The mTOR kinase controls translation by phosphorylating eukaryotic initiation factor-4E binding protein-1 and ribosomal protein S6 kinase (S6K) (Hara et al., 2002Go). Unphosphorylated eukaryotic initiation factor-4E binding protein-1 binds to the eukaryotic initiation factor-4E (eIF4E) and prevents it from binding to eIF4G, thereby inhibiting the formation of the active translation complex (Kimball et al., 1997Go, 2002Go; Hara et al., 2002Go). The S6K is activated by phosphorylation and phosphorylates ribosomal protein S6, which drives translation of a small family of abundant transcripts that encode primarily ribosomal proteins and components of the translational apparatus (Jefferies et al., 1997Go; Schmelzle and Hall, 2000Go). Thus, activation of mTOR upregulates the translational machinery and promotes protein translation (Schmelzle and Hall, 2000Go).

The activity of mTOR is controlled by phosphorylation. Phosphorylation at Ser2448 activates mTOR, whereas phosphorylation at Thr2446 inhibits its activity (Cheng et al., 2004Go). The phosphorylation of these two sites are mutually exclusive (Cheng et al., 2004Go). Using an antibody specific to the Ser2448 phosphorylation of mTOR, a significant difference was observed in the phosphorylation of mTOR between muscles from nutrient-restricted and control cows and their fetuses. No significant difference in the overall content of mTOR was detected (Figure 1Go). These results indicated that nutrient restriction down-regulated mTOR signalling. It is largely unclear, however, how the cells sense the availability of nutrients and phosphorylate mTOR kinase. It was reported that Ser2448 phosphorylation is via protein kinase B, which can be stimulated by insulin and certain growth factors, whereas phosphorylation at Thr2446 is accomplished by AMPK, a kinase sensing energy status in cells (Cheng et al., 2004Go). Because AMPK is activated by AMP inside cells, nutrient restriction is expected to increase the content of AMP and thereby activate AMPK. To confirm this, the activity of AMPK was analyzed (Figure 2Go); however, an unexpected finding was that the activity of AMPK in the skeletal muscle of nutrient-restricted cows was significantly less (P ≤ 0.05) than that of control cows (Figure 2Go). This is intriguing. A possible explanation is due to feedback inhibition. The AMPK in nutrient-restricted animals is constantly activated, but this activation results in feedback inhibition. In support of this notion, a point mutation, Arg200 to Gln, in the AMPK {gamma}3 subunit was found in Hampshire pigs, which resulted in a constitutively active AMPK and led to feedback inhibition of AMPK (Milan et al., 2000Go). The AMPK activity in pigs carrying this mutation is only one-third of the activity of normal pigs (Milan et al., 2000Go; Andersson, 2003Go). For the fetal muscle, however, no difference in AMPK activity was detected (Figure 2Go). Another possibility is that the AMPK is not involved in the regulation of mTOR signaling in skeletal muscle during this long-term nutrient restriction. In cell culture studies, activated AMPK can activate eukaryotic elongation factor 2 (eEF2) kinase, which phosphorylates and inactivates eEF2 and thus downregulates protein synthesis (Horman et al., 2002Go; Browne et al., 2004Go). Phosphorylation of mTOR at Thr2446 by AMPK downregulates mTOR signaling (Cheng et al., 2004Go). These short-term cell culture studies showed a negative effect of AMPK on mTOR signaling; however, in this long-term nutrient restriction study, a counteractive effect of AMPK on mTOR signaling was not observed.



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  		Figure 1. Mammalian target of rapamycin (mTOR) phosphorylation in cow and fetal muscles. Muscle proteins were separated by SDS-PAGE and transferred to membranes for immunoblotting. Phospho-specific mTOR antibody (Ser2448) was used to detect phospho-mTOR. Membranes then were stripped and re-probed with mTOR antibody. The LM from cows and their fetuses of either control (Con) or nutrient-restricted (NR) treatment (n = 10 per treatment) were used. Asterisks indicate that there was less (P < 0.05) phospho-mTOR in muscle from NR cows and fetuses than in control cows and fetuses.

 


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  		Figure 2. Activity of adenosine 5'-monophosphate-activated protein kinase (AMPK) in cow and fetal muscles. The longissimus muscles from cows and their fetuses of either control (Con) or nutrient-restricted (NR) treatment (n = 10 per treatment) were used. The AMPK activity was measured through the specific phosphorylation of SAMS (His-Met-Arg-Ser-Ala-Met-Ser-Gly-Leu-His-Leu-Val-Lys-Arg-Arg) peptide by AMPK. The activity of AMPK was expressed as the incorporation of phosphate (nM) into peptide per minute, per milligram of total muscle proteins. The asterisk indicates that there was less (P < 0.05) AMPK activity in muscle from NR cows than from control cows.

 
The mTOR can directly phosphorylate S6K and S6K phosphorylates S6 (Jefferies et al., 1997Go; Bodine et al., 2001Go). Similar with mTOR, a lower ratio of ribosomal protein S6 was phosphorylated in nutrient-restricted cows and their fetuses compared with that of control cows (Figure 3Go). The hypophosphorylation of ribosomal protein S6 in nutrient-restricted animals downregulates the translational machinery (Jefferies et al., 1997Go; Schmelzle and Hall, 2000Go), which should result in the inhibition of protein synthesis in the skeletal muscle of nutrient-restricted pregnant cows and their fetuses.



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  		Figure 3. Ribosomal protein S6 phosphorylation in cow and fetal muscles. Muscle proteins were separated by SDS-PAGE and transferred to membrane for immunoblotting. Phospho-specific ribosomal protein S6 antibody (Ser235/236) was used to detect phospho-S6. Then, membranes were stripped and re-probed by ribosomal protein S6 antibody. The LM from cows and their fetuses of either the control (Con) or nutrient-restricted (NR) treatment (n = 10 per treatment) were used. The asterisk indicates that there was less (P < 0.05) phosphor-S6 in muscle from NR cows and fetuses than from control cows and fetuses.

 
Protein degradation in muscle is largely controlled by calpain and ubiquitin-proteasome systems (Goll et al., 1992Go, 2003Go; Costelli and Baccino, 2003Go). In our previous study, calpastatin content was downregulated in the skeletal muscle of nutrient-restricted cows, whereas it was upregulated in fetuses from nutrient-restricted animals (Du et al., 2004Go). In this study, ubiquitylation of proteins were analyzed by immunoblotting. As shown in Figure 4Go, numerous bands were detected on the blot. The detection of numerous bands was expected because muscle contains various proteins ubiquitylated by conjugating ubiquitins to those proteins. To examine the difference in the extent of ubiquitylation, the whole density of each lane was quantified and compared. For fetal muscle from nutrient-restricted and control cows, no significant difference in the protein ubiquitylation was detected (Figure 4Go). The cow muscle, however, a significant higher amount of ubiquitylated proteins was detected in nutrient-restricted samples, showing that nutrient restriction enhanced muscle protein degradation (Figure 4Go). It is reported that myofibrillar proteins are preferably degraded during nutrient restriction (Kadowaki et al., 1989Go; Wing et al., 1995Go). In this study, although a slight increase in the ubiquitylation of myosin heavy chain and several other unidentified proteins was observed, the overall pattern of ubiquitylation was relatively constant (Figure 4Go). This could be due to the fact that our nutrient restriction (68.1% NEm and 86.7% of metabolizable protein requirements) was much milder than those used in other food-deprivation and protein-restriction studies (Kadowaki et al., 1989Go; Wing et al., 1995Go).



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  		Figure 4. Content of ubiquitylated proteins in cow and fetal muscles. Muscle proteins were separated by SDS-PAGE and transferred to membranes for immunoblotting. Anti-ubiquitin antibody was used to detect ubiquitylated proteins. The LM from cows and their fetuses of either control (Con) or nutrient-restricted (NR) treatment (n = 10 per treatment) were used. MHC = myosin heavy chain. The asterisk indicates that the amount of ubiquitylated proteins was greater (P < 0.05) in muscle from NR cows than from control cows.

 
The upregulation of protein ubiquitylation is in agreement with previous reports that food deprivation enhances the expression of proteins associated with the ubiquitin-proteasome system (Jagoe et al., 2002Go; Costelli and Baccino, 2003Go). The ubiquitin-dependent proteolysis is upregulated during muscle atrophy (Costelli and Baccino, 2003Go). This result is also consistent with our earlier report that the content of calpastatin was down-regulated in nutrient-restricted animals, which resulted in a higher calpain activity and thus a greater turnover of muscle proteins (Du et al., 2004Go).

The accelerated protein degradation and decreased muscle protein synthesis might cause the muscle atrophy of nutrient-restricted cows (Table 1Go). The LM at the 12th rib for the control cow was 72 ± 9 cm2 vs. only 60 ± 7 cm2 for nutrient-restricted cow (P < 0.05; Table 1Go). For the fetal muscle, the BW of fetuses from the nutrient-restricted group was numerically lower than that of the control group, although this was not statistically significant (Table 1Go).


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  		Table 1. Longissimus muscle area of pregnant cows and carcass weight of fetuses
 
These results showed that there was a differential regulation in the protein degradation between cows and fetuses following nutrient restriction. Data indicate that nutrient restriction accelerated the protein degradation in the muscle of pregnant cows but not in fetal muscle. This is consistent with a previous study on tumor-bearing pregnant rats, where significant muscle wasting was observed in the mother, but with little or no influence on fetal growth (Carbo et al., 1998Go).

This study demonstrated that mTOR signaling was downregulated during the nutrient restriction phase for both cow and fetal muscles. This downregulation of mTOR signaling might be associated with the muscle atrophy in nutrient-restricted animals. The ubiquitylation of proteins was increased only for cow muscle under nutrient restriction. These results indicate that the accelerated degradation of proteins is only important for the atrophy of adult muscle, whereas decreasing protein synthesis might be the main reason for the growth retardation of fetal muscle due to maternal nutrient restriction.


   Footnotes
 
1 This work was supported by National Research Initiative Competitive Grants 2003-35206-12814 and 2004-35503-14792 from the USDA Cooperative State Research, Education, and Extension Service. Back

2 Correspondence—phone: 307-766-3429; fax: 307-766-2355; e-mail: mindu{at}uwyo.edu.

Received for publication June 27, 2004. Accepted for publication September 29, 2004.


   Literature Cited
Top
 Abstract
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 Materials and Methods
 Results and Discussion
 Literature Cited
 


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