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Pregnancy |
Center for the Study of Fetal Programming,3 Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071
Center for Women's Health Research,4 New York University Medical School, New York, New York 10016
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During prenatal muscle development, primary myofibers are formed, followed by the formation of secondary myofibers [3]. Primary myofibers have peripherally located myofibrils surrounding an axial core of nuclei and cytoplasm [3, 4]. The secondary myofibers are derived from muscle precursor cells, which are initially maintained in a proliferating, undifferentiated state [4]. Those precursor cells differentiate into myoblasts and fuse to form secondary myofibers parallel to primary myofibers [3]. Because the availability of nutrients influences the proliferation of cells, we hypothesized that the number of secondary fibers in prenatal muscles is influenced by maternal nutrients and that maternal nutrient restriction would have long-term consequences on muscle growth and development. Results of numerous studies have suggested that maternal undernutrition influences the physiology and development of fetuses, with long-term consequences that predispose to certain diseases, such as diabetes, obesity, and cardiovascular diseases, during postnatal life [5].
The mammalian target of rapamycin (mTOR) signaling pathway is critical both for sensing nutrient availability and for nutrient-stimulated muscle growth [6–9]. The downstream targets of mTOR are proteins that control mRNA translation [9]. Inhibition of mTOR leads to the arrest of cells in the G1 phase of the cell cycle [10, 11]. The mTOR signaling plays an essential role in skeletal myogenesis [12]. Thus, we hypothesized that mTOR plays a significant role in the control of altered prenatal development during maternal nutrient deprivation.
Protein degradation is important for regulating muscle mass. To our knowledge, no information about the influence of maternal nutrient restriction on protein degradation in fetal muscle is available. The calpain system and the ubiquitin-proteasome system play crucial roles in the control of muscle protein degradation. In a previous study of pregnant cows, we observed that calpastatin expression in fetal muscle was influenced by nutrient availability [13]. Thus, the calpastatin and ubiquitin systems may influence protein degradation in fetal skeletal muscle. Hence, the objective of the present study was to evaluate the effect of maternal nutrient restriction on the development of fetal skeletal muscle and the possible role of mTOR signaling, as well as the calpastatin and ubiquitin systems, in this process.
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MATERIALS AND METHODS |
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All animal procedures were approved by the University of Wyoming Animal Care and Use Committee. The detailed description of the procedures has been published previously [14]. Ten ewes of mixed breed were weighed on Day 20 of gestation so that individual diets could be calculated on a metabolic body-weight basis (weight0.75). The diet consisted of a pelleted beet pulp (79.7% total digestible nutrients, 93.5% dry matter, and 10.0% crude protein). Rations were delivered on a dry-matter basis to meet the total digestible nutrients required for maintenance of an early pregnant ewe (National Research Council [NRC] requirements) [15]. A mineral-vitamin mixture (51.43% sodium triphosphate, 47.62% potassium chloride, 0.39% zinc oxide, 0.06% cobalt acetate, and 0.50% ADE vitamin premix [8 000 000 IU of vitamin A, 800 000 IU of vitamin D3, and 400 000 IU of vitamin E per pound; amount of ADE vitamin premix was formulated to meet the vitamin A requirements] was included with the beet pulp pellets to meet requirements. On Day 21 of gestation, all ewes were placed in individual pens and fed control rations. On Day 28, five ewes were randomly assigned to a control-fed group (100% NRC requirements) and five to a nutrient-restricted group (50% NRC requirements) after equalizing for weight and body condition score. Body condition score can be used to estimate the energy reserve available to ewes [16]. Beginning on Day 28 of gestation and continuing at 7-day intervals, ewes were weighed and rations adjusted upward for weight gain and downward for weight loss. Ewes were killed on Day 78 of gestation, and fetuses were weighed. Fetuses were weighed again after removing their internal organs, and this weight was recorded as the empty body weight. Following weighing, a fetal muscle sample was immediately taken from the longissimus dorsi muscle on the right side at the level of the 12th rib. Muscle samples were snap-frozen in liquid nitrogen and then stored at –80°C until analyzed. Two ewes in both control-fed and nutrient-restricted groups carried twins; only one fetus of each twin pair was chosen for further analysis. All analyses were performed within 12 wk of fetal tissue collection.
Immunoblotting
Polyclonal anti-mTOR and antiribosomal protein S6 antibodies as well as phospho-specific antibodies for mTOR (Ser2448) and ribosomal protein S6 (Ser235/236) were purchased from Cell Signaling Technology, Inc. (Beverly, MA).
Muscle (0.1 g) was minced with scissors and homogenized in a polytron homogenizer (7-mm diameter generator) with 400 µl of an ice-cold buffer containing 137 mM NaCl, 50 mM Hepes, 1 mM MgCl2, 1 mM CaCl2, 1% NP-40, 10% glycerol, 2 mM PMSF, 10 mM sodium pyrophosphate, 2.5 mM EDTA, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, 2 mM Na3VO4, and 100 mM NaF, pH 7.4. The protein content of lysates was determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA) [13].
Each muscle homogenate was mixed with a same volume of 2x standard SDS sample loading buffer. A Hoefer minigel system was used for casting gels and running electrophoresis. Two identical, 5–20% gradient gels were used for SDS-PAGE separation of proteins, and the same amount of protein from each muscle sample (150 µg) was loaded on both gels, which were run under the same condition. Following electrophoresis, the proteins on the gels were transferred to nitrocellulose membranes in a transfer buffer containing 20 mM Tris-base, 192 mM glycine, 0.1% SDS, and 20% methanol [13]. One membrane was used for the immunoblotting detection of total mTOR and S6, and the other membrane was used for detecting phosphorylated mTOR and S6.
Membranes were incubated in a blocking solution consisting of 5% nonfat dry milk in TBS/T (0.1% Tween-20, 50 mM Tris-HCl [pH 7.6], and 150 mM NaCl) 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 time with 20 ml of TBS/T. After that, membranes were incubated with horseradish peroxidase-conjugated monkey-anti-mouse secondary antibody (1:2000) for 1 h in TBS/T with gentle agitation. Following three 10-min washes, membranes were visualized using enhanced chemiluminescence Western blotting reagents (Amersham Bioscience) and exposure to film (MR; Eastman Kodak, Rochester, NY). The density of bands was quantified by using an Imager Scanner II and ImageQuant TL software [17, 18].
Measurement of AMP-Activated Protein Kinase Activity
The AMP-activated protein kinase (AMPK) activity was measured as previously reported [19]. Briefly, muscle (0.1 g) was minced with scissors and homogenized in 400 µl of a ice-cold buffer containing 0.25 M mannitol, 0.05 M Tris/HCl (pH 7.8), 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 50 mM NaF, 5 mM sodium pyrophosphate, and 1% protease-inhibitor cocktail (Sigma, St. Louis, MO). The muscle homogenate was centrifuged for 5 min at 13 000 x g at 4°C. The supernatant (5 µl) 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; Invitrogen), 0.2 mM AMP, 80 mM NaCl, 8% (w/v) glycerol, 0.8 mM EDTA, 0.8 mM DTT, 5 mM MgCl2, 0.2 mM ATP, and 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. The remaining [32P]ATP was removed with three washes of 1% phosphoric acid and one wash 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 coefficient of variation for AMPK analysis was 2.11%.
Histochemical Examination
Muscle samples were fixed in 4% (w/v) paraformaldehyde in a phosphate buffer (0.12 M, pH 7.4), embedded in paraffin, and cut into sections (thickness, 10 µm). Sections were rehydrated using a series of incubations in xylene and ethanol solutions and then stained with hematoxylin-eosin for standard light microscopy. Sections were also blocked with 10% serum, incubated with antimyogenin antibody (1:200 dilution, F5D monoclonal antibody; DSHB, Iowa City, IA), and then incubated with fluorescein isothiocyanate-conjugated secondary antibodies (Amersham Biosciences). The sections were examined using a laser-induced fluorescence confocal microscope (Confocal Microscopy Facility, Microscopic Core Facility, University of Wyoming) [13]. The number of primary and secondary myofibers were counted on 10 different microscopic fields of each sample, and a ratio of secondary to primary myofibers was reported. Only those fasciculi for which primary and secondary myofibers were clearly differentiated were counted. The area of 10 fasciculi for each muscle sample was quantified using the ImageJ 1.30v software (National Institutes of Health, Bethesda, MD).
Statistical Analysis
Data were analyzed as a complete randomized design using the general linear model of the Statistical Analysis System. The relative density of bands, AMPK activity, total fetal weight, carcass weight, and ratio of secondary to primary myofibers were analyzed. The differences in the mean values were compared by Tukey multiple comparison. Data are presented as the mean and SD (P < 0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Calpastain content in fetal muscle from nutrient-restricted ewes was 18% lower than that in fetal muscle from control-fed ewes, but this difference was not significant (Fig. 4). No difference in protein ubiquitylation was detected between the two groups (Fig. 5). These results indicate that maternal nutrient restriction did not cause a significant change in myofiber protein degradation.
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Decreased activation of mTOR signaling in the fetuses from nutrient-restricted ewes would be expected to reduce skeletal muscle protein synthesis. This was confirmed by a significantly lower carcass weight for fetuses from nutrient restricted ewes (Fig. 6). No significant difference in fetal weight was observed, either between twins and singles or between males and females (data not shown). To show that reduction in fetal carcass weight was related to the retarded muscle development in fetuses from nutrient-restricted ewes, the histochemical structure of fetal muscle was examined.
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It is difficult to identify myofibers and to differentiate primary and secondary myofibers directly during light microscopy (Fig. 7A). Myofibers were clearly identified and the primary and secondary myofibers differentiated using immunostaining with a myogenin monoclonal antibody and observation under a fluorescent microscope (Fig. 7B). The primary myofibers are large, with a nucleus located at the center of a fasciculus (a single bundle of muscle fibers) that appeared as a dark spot during fluorescent microscopy. Each primary myofiber was surrounded by secondary myofibers, which are smaller in size and contained a peripherally located nucleus (Fig. 7). The numbers of secondary and primary myofibers of fetal muscle from both groups were counted, and the ratio was calculated (Fig. 8). A significantly lower ratio of secondary to primary myofibers was observed in fetal muscle from nutrient-restricted ewes (Fig. 8). Furthermore, the overall sizes of muscle fasciculi were smaller for fetal muscle from nutrient-restricted ewes (P < 0.05) (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The activity of mTOR is facilitated by phosphorylation at Ser2448 [20]. In a recent study, mTOR was reported to be inhibited by phosphorylation at Thr2446 [25]. The phosphorylations at Ser2448 and Thr2446 are mutually exclusive; the increase in the phosphorylation at Ser2448 decreases the phosphorylation at Thr2446 [25]. Using an antibody specific to the phosphorylation of mTOR at Ser2448, we detected that the phosphorylation of mTOR was reduced in the fetal muscle from nutrient-restricted ewes. This change would be expected to reduce mTOR activity during nutrient restriction. The reduction of phosphorylation of ribosomal protein S6, a downstream effector of mTOR, supports this view.
The mTOR phosphorylation at Ser2448 is mediated by AMPK [25]. Thus, the activity of AMPK was further analyzed. A heterotrimeric enzyme containing 
, ß, and 
subunits, AMPK is a critical regulator of energy metabolism [26]. It is activated by an increase in the ratio of AMP to ATP in muscle cells and by phosphorylation at Thr172 [27]. It also is crucial for sensing the energy status of cells. Maternal nutrient restriction would tend to increase the AMP content in muscle cells and, thus, to activate AMPK. However, no significant difference in AMPK activity was detected. A possible reason for this lack of difference might be the fact that this level of maternal nutrient restriction did not impair the level of energy delivery severely enough to activate the AMPK activity in fetal muscle.
Protein degradation in muscle is largely controlled by the calpain and ubiquitin-proteasome systems. The calpain system is mainly comprised of calpain I, calpain II, and calpastatin. We have shown that calpastatin is slightly down-regulated in the skeletal muscle of pregnant cows when their diet was similarly nutrient restricted by 50% and up-regulated in the fetuses [13]. In the present sheep study, maternal nutrient restriction did not alter fetal muscle calpastatin. Furthermore, no difference in the ubiquitylation of proteins in the fetal muscle of control-fed and nutrient-restricted animals was detected. These data suggest that little or no difference exists in protein degradation between control-fed and nutrient-restricted fetuses. This is consistent with a former report concerning tumor-bearing pregnant rats, in which significant muscle wasting was observed in the mother but not in the fetus [28]. Thus, maternal nutrient restriction appears to have a greater effect in the inhibition of protein accretion than in the degradation of existing protein in fetal muscle. As a result, the total weight and empty body weight of fetuses from nutrient-restricted ewes were reduced, which is consistent with retarded development of both the skeletal muscle and the skeleton of fetuses.
Muscle cells at this stage of development expresses myogenin [29, 30]; we immunostained those tissue sections with antimyogenin antibody to clearly identify myofibers. Identification is difficult with conventional hematoxylin-eosin stain. During fluorescent microscopy, muscle cells were easily identified. Differentiation of primary and secondary myofibers also was facilitated by the presence of a centrally localized nucleus in primary myofibers and a central location of the fibers themselves within fasciculi. Maternal nutrient restriction significantly reduced the ratio of secondary to primary muscle fibers, and the size of fasciculi was smaller in the muscle of fetuses from nutrient-restricted ewes. This finding agrees with reports concerning other species. In the pig, undernutrition in utero results in low birth weight and decreased number of muscle fibers. The progeny of undernourished sows had a significantly lower ratio of secondary to primary myofibers [31]. In guinea pig fetuses, a similar reduction in fiber number was observed in fetuses that were undernourished in utero [32]. The difference in the number of secondary myofibers is related to the differences in mTOR signaling. Activation of mTOR increases the expression of cyclin D1, CDK1, and CDC25A, and it promotes the G1 cell-cycle progression [33]. The decrease in mTOR signaling in the muscle of fetuses from nutrient-restricted ewes is consistent with decreased proliferation of progenitor cells and myoblasts and reduction in the number of secondary myofibers in fetal muscle.
The reduction in the number of myofibers in fetal skeletal muscle following maternal nutrient restriction will likely result in physiological consequences during postnatal life. Skeletal muscle is the primary tissue for the utilization of glucose and fatty acids [34, 35]. As a result, reduced muscle mass will decrease the metabolism of glucose and fatty acids in response to insulin stimuli and, thus, will predispose offspring of nutrient-restricted mothers to diabetes and obesity. Human infants who are small at birth have a greater risk for type 2 diabetes [36–38]. Decreased muscle mass is a major factor in low birth weight [36, 39].
In conclusion, mTOR signaling in fetal muscle was down-regulated following maternal nutrient restriction. This down-regulation of mTOR signaling was associated with a reduced ratio of secondary to primary muscle fibers in the muscle of fetuses from nutrient-restricted ewes. Critical factors that regulate degradation of proteins in fetal muscle were not significantly affected by the level of nutrient restriction imposed. Because the number of muscle fibers is determined by prenatal development, the reduced number of muscle fibers in fetuses exposed to undernutrition will likely have long-term implications for postnatal health.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Min Du, Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071. FAX: 307 766 2355; mindu{at}uwyo.edu
Received: 21 July 2004.
First decision: 6 August 2004.
Accepted: 11 August 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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