Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada
Submitted 5 May 2004 ; accepted in final form 19 October 2004
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ABSTRACT |
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ATP-ubiquitin proteasome proteolytic pathway; muscle free amino acids; muscle RNA, DNA, and protein concentration
The capacity to mobilize body protein from skeletal muscle is an important resource for lactation (1012, 30) that enables the dam to care for and nourish her offspring, and at least temporarily minimizes the maternal commitment to forage for food. Muscle protein mobilization is an adaptive response that enables a high level of milk production under conditions of poor nutrient supply (2, 21). Litter growth is largely maintained even when feed is restricted to 50% of ad libitum intake (36). Maternal protein mobilization is related to overall milk protein production and is proportional to the number of offspring suckled (5). Protein mobilization is also finely tuned in proportion to the dietary protein supply. At constant litter size, larger discrepancies between dietary protein intake and the demand for milk synthesis result in a greater impetus for protein mobilization (10, 11, 21, 45). Maternal protein loss cannot continue unabated and is attenuated if total protein mobilization exceeds a certain proportion of total body protein mass (10, 11) to prevent excessive depletion. Finally, protein mobilization must be sharply curtailed when weaning of the offspring occurs and milk production ceases.
Our research was motivated by a desire to understand the alterations in skeletal muscle protein metabolism that permit a highly controlled diversion of muscle protein reserves toward milk production. Protein synthesis in skeletal muscle is highly regulated, and a fall in the rate of this process is suggested to contribute to protein mobilization during starvation (24, 26) and in lactation (6, 8, 34, 39). However, the regulation of protein catabolism in lactating animals remains unclear, and the participation of skeletal muscle proteolysis is especially poorly understood. We hypothesized that, at constant litter size in lactating pigs, greater protein restriction would result in a larger proteolytic response.
To explore potential mechanisms by which loss of maternal protein might be orchestrated, we developed a biopsy technique to collect triceps muscle samples in late gestation (day 107) and midlactation (days 912) and within 3 h of weaning from the same animals. Samples were taken at all three time points from pigs that lost 7, 9, or 16% of their body protein in lactation under conditions of differential dietary protein supply (10). Muscle RNA, DNA, protein, the expression of genes involved in skeletal muscle proteolysis, and muscle free amino acids were measured.
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MATERIALS AND METHODS |
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The experimental design, housing, and management of gilts during gestation and lactation, and their reproductive and lactational performance, are described in Clowes et al. (10). In brief, upon parturition, first-parity sows (Camborough, PIC; Acme, AB, Canada) were allocated to be fed divergent levels of total lysine (50, 35, and 24 ± 1.0 g/day) and dietary protein (878, 647, and 491 ± 19 g/day). These dietary treatments induced sows to lose approximately 7, 9, or 16% of their body protein, as a percentage of their protein mass at parturition, in lactation and to be in a more negative lysine balance in the first 20 days of lactation (16, 27, and 42 g/day). At least 7 sows were allocated to each treatment: 8 sows to the 7% body protein loss treatment, 7 sows to the 9% body protein loss treatment, and 10 sows to the 16% body protein loss treatment. Losses of back fat (1.3 ± 0.29 mm) during lactation were small and not different among treatments, because sows were fed a similar energy level (61 ± 2.0 MJ metabolizable energy/day).
In late gestation (day 107) and midlactation (days 912), a muscle sample was collected from alternate sides of each sow by biopsy. On day 23 of lactation, within a few minutes of slaughter, a third muscle sample was collected 23 h after weaning. Samples were immediately trimmed of connective and adipose tissue, frozen in liquid nitrogen, and stored at 70°C until analyzed for RNA, DNA, protein, free amino acids, and mRNA expression of several components of the ubiquitin-proteasome proteolytic system by Northern hybridization analysis. To ensure that sows were fasted for 16 h before collection, feed was removed from sows at 1800 (24-h clock) on the evening before surgery and slaughter. The long head of the triceps brachii muscle was chosen because it is a mixed fiber type muscle in the pig (27, 40) and thus represents the main source of mobilizable protein in the body (16).
Muscle Biopsy
A preliminary experiment was conducted with four animals (196 kg body wt and 17 mm back fat depth at parturition) to develop a muscle biopsy technique that could be undertaken in the animal unit rather than in the surgery room to 1) minimize the additional handling, transport, and environmental change stresses on the sow, 2) reduce the time during which the suckling litter and sow are apart, and 3) determine the most appropriate time to collect samples. The surgery was conducted in the sows individual farrowing pen, under general anesthetic, as described by Clowes et al. (12) with minor modifications. Samples (24 g) were surgically collected from the long head of the triceps brachii muscle, and this procedure took 10 min. Alternate sides of the animal were used for sampling in late gestation and midlactation.
The surgery proved minimally disruptive to the sow and had little impact on the piglets. The piglets were only separated from the sow for the duration of the surgery and recovery period, which comprised one suckling bout (4060 min), and immediately commenced suckling once placed back on the sow. Sow body temperatures were not elevated postsurgery, feed intake in lactation was only slightly reduced at 13 days postsurgery, and body weight losses were small (5 kg) and in the range for first-parity sows in our herd not subjected to surgery. Respectable litter growth rates (2.36 ± 0.23 kg/day) were achieved.
Analyses
Before analysis, individual muscle samples were pulverized in a mortar and pestle in liquid nitrogen and stored at 70°C until analysis.
Muscle free amino acids. About 25 mg of powdered muscle tissue were homogenized in 1 ml of 3% (wt/vol) trichloroacetic acid (Sigma Diagnostics, St. Louis, MO). The mixture was centrifuged at 2,800 g, and the supernatant fraction containing tissue free amino acids was analyzed by HPLC using orthopthaldialdehyde derivatization and ethanolamine as the internal standard (38).
Muscle RNA, DNA, and protein. Muscle RNA, DNA and protein were quantified, in the same sample, using a modification of the Schmidt-Thannhauser procedure (32). In brief, 250 mg of frozen powdered muscle tissue were incubated on ice for 10 min with 4 ml of 2% (wt/vol) perchloric acid (PCA) to precipitate out the protein and nucleotides. The precipitate was centrifuged for 15 min at 2,800 g and 4°C and washed with 4 ml of 2% PCA at 4°C. The pellet was resuspended in 4 ml of 0.3 N NaOH and incubated at 37°C for 1 h, and the alkaline digest was cooled on ice. One hundred microliters of digest were removed, diluted with distilled, deionized H2O to make a 0.1 N NaOH alkaline digest, and stored at 20°C until bicinchoninic acid (BCA) assay microtiter plate protein quantification (Pierce, Rockford, IL).
Ice-cold 12% PCA (2 ml) was added to the remaining alkaline digest, incubated on ice for 10 min, and centrifuged for 10 min, and the RNA containing supernatant was collected. The pellet was washed in 4 ml of 2% PCA and centrifuged, and the washings and pellet were collected separately. The acid-soluble supernatant plus washings was diluted with H2O to make a 1% PCA solution to spectrophotometrically quantify the RNA [see Ref. 3; RNA (µg/ml) = (32.9 A260 6.11 A232) x dilution factor, where A260 and A232 are absorption at 260 and 232 nm, respectively]. The acid-insoluble precipitate was incubated in 10% PCA at 70°C for 1 h (13), and the DNA was quantified using the diphenylamine method (7, 15).
Northern hybridization. Analysis was conducted as described by Adegoke et al. (1), with minor modifications. Total RNA was extracted from 200300 mg of muscle tissue, using TRIzol (GIBCO BRL-Life Technologies, Frederick, MD; Ref. 12). Ethidium bromide (1.7 µg) was added to the sample buffer of each sample, and samples were made up to the same volume using sterile, distilled, deionized H2O. Muscle RNA to be hybridized with the cDNA probes [14-kDa ubiquitin-conjugating enzyme (14-kDa E2) and the C9 subunit of the 20S proteasome] was transferred to a nitrocellulose membrane (Nitropure; Micro Separations, Westborough, MA), and RNA to be hybridized with the riboprobe for ubiquitin was transferred to a nylon membrane (Zeta-Probe; Bio-Rad Laboratories, Hercules, CA) and fixed onto the membranes by baking, under vacuum, for 2 h at 80°C. All blots were exposed to X-ray film (Kodak BioMax MR; Eastman Kodak, Rochester, NY) and evaluated quantitatively with a GS-670 Imaging Densitometer (Bio-Rad Laboratories). The densitometric scans were normalized to the fluorescence level of the 18S ribosomal RNA ethidium bromide band. The amount of 18S rRNA loaded did not vary among treatments (P = 0.98) or time points (P = 0.62), and there was no treatment x time interaction (P = 0.76), establishing consistent RNA gel loading. After stripping, all membranes were reprobed with a 32P-labeled cDNA fragment encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Concentration curves, of increasing amounts of total RNA from lactating sow muscle, were run to determine the amount of total RNA to load for each probe. For example, muscle mRNA expression of both 14-kDa E2 transcripts increased linearly (r2 > 0.95) from 5 to 20 µg of total RNA (Fig. 1). Muscle mRNA expression of both ubiquitin transcripts plateaued between 5 and 10 µg of total RNA. Therefore, 15 µg of total RNA were loaded for the 14-kDa E2 hybridization and 3 µg for the ubiquitin hybridization. Muscle C9 mRNA expression increased linearly (r2 = 0.998) from 15 to 42 µg of total RNA (Fig. 1); thus 30 µg of total RNA were loaded onto a 1.2% agarose-formaldehyde gel for this hybridization. The design of the main experiment entailed the comparison of three animal treatments and three time points per animal, for a total of 75 samples. To accommodate this sample number, five gels and blots were simultaneously run for each mRNA measured. Each of the three animal treatments was represented within each blot, and all three time points were tested on the same blot for each sow.
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RESULTS |
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The capacity for protein synthesis (RNA-to-DNA ratio) decreased by 15% between late gestation and midlactation (P < 0.05) in all treatments and continued decreasing in sows that lost the most protein, to 68% of prepartum levels at weaning (Fig. 2A). The protein-to-DNA ratio also decreased (P < 0.001) between late gestation and weaning in all treatments (Fig. 2B). The size of this decline reflected (P < 0.10) the degree of protein lost in lactation and resulted in protein-to-DNA ratios at weaning that were, respectively, 84, 77, and 69% of prepartum levels in sows that lost 7, 9, and 16% of their protein mass at parturition. Muscle RNA concentrations decreased (P < 0.001) 1015% between late gestation and midlactation in all treatments. By the day of weaning, they increased to prepartum levels in sows that lost 7 and 9% of their body protein mass but remained low in sows that lost the most protein (Fig. 2C). Muscle DNA concentrations increased (P < 0.001) by
5% between late gestation and midlactation in all treatments and then increased to weaning in a manner that reflected the degree of protein lost in lactation (Fig. 2D). The increase was largest (P < 0.05) in sows that lost the most protein, resulting in the DNA concentration of these sows at weaning being 134% of prepartum levels.
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When equal amounts of total RNA from each animal and time point were studied, marked changes in GAPDH mRNA expression were observed over the course of lactation and by treatment (Fig. 3). GAPDH mRNA fell by nearly 50% (P < 0.001) between late gestation and midlactation and remained low until weaning. The overall GAPDH mRNA level was lowest (P < 0.01) in sows that lost the most protein in lactation. These differences precluded the use of GAPDH mRNA as a "housekeeping" gene to normalize gene expression. Therefore, the measured mRNA expression was corrected for slight differences in loading, using the relative fluorescence of the 18S rRNA ethidium bromide stain.
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Changes in Muscle Free Amino Acid Concentrations
The chromatographic procedure used resolved 19 amino acids. Because muscle free amino acid concentrations in late gestation did not differ among treatments, the amino acid data are presented as a percentage of prepartum values. The average concentrations of individual muscle free amino acids in late gestation are presented in Table 2. Muscle free amino acids were divided into two groups based on their pattern of change between late gestation and weaning. The first group consisted of six essential amino acids (EAA; leucine, isoleucine, valine, phenylalanine, lysine, and threonine). Methionine and histidine were not included in this group because a value was not obtained for every sow at every time point. Arginine was not included because its concentrations did not change over lactation. The second group consisted of seven nonessential amino acids (NEAA; alanine, glycine, serine, aspartic acid, asparagine, and glutamic acid). Glutamine was excluded from this group because it behaved differently from the other NEAA.
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The increase in muscle free NEAA concentrations between late gestation and midlactation (Fig. 5B) and in glutamine concentrations between late gestation and weaning (Fig. 5C) was proportional to the degree of protein mobilized by the sow in lactation. Increases in these amino acids were highest (P < 0.05) in sows that lost the most protein. The NEAA concentrations in midlactation were, respectively, 122, 131, and 146% of prepartum values, and the glutamine concentrations at weaning were, respectively, 205, 240, and 308% of prepartum values in sows that lost 7, 9, and 16% of their protein mass at parturition. The decrease in muscle free EAA concentrations between midlactation and weaning (such as isoleucine) was greatest (P < 0.05) in sows that lost the most protein (Fig. 5D). Similarly, muscle free valine concentrations at weaning were lowest (P < 0.05) in sows that had mobilized the most protein.
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DISCUSSION |
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Direct measures of muscle protein synthesis and degradation were not made in our study. Classic techniques for measurement of these processes employing isotopically labeled amino acids would have been very difficult in the context studied here. Some of the limitations for research on protein turnover in large animals, and in swine in particular, are as follows. 1) Studies are cost prohibitive if sows and their progeny have to be killed and disposed of suitably, for tissue sampling or because of contamination with radioactivity, rather than returned to the research herd. 2) Studies using isotopes are especially costly because of large animal size. 3) Tracer recycling precludes making repeated measures in the same animals over time (14, 25). 4) Because of unique metabolic pathways, urinary 3-methylhistidine may not be used to follow muscle protein catabolism in this species. A further consideration is that the level of stress and disruption in the experimental setting may not be compatible with maintenance of normal maternal behavior, as the approaches for in situ muscle metabolism tend to be relatively invasive and may require venous and arterial cannulation/perfusion/biopsy. For all of these reasons, we opted for a variety of indirect indexes of muscle metabolism in our serial biopsies.
RNA-to-DNA ratio (capacity for protein synthesis) has been extensively reported on and related to determined rates of protein synthesis (29). Proteolytic processes in muscle appear to have a large degree of transcriptional regulation, and increases in mRNA expression of genes that encode proteins in the ATP-dependent proteolytic pathway (the cofactor ubiquitin, ubiquitin conjugation system enzymes, and the proteasome complex) generally correlate with protein levels and directly measured protein degradation rates (22, 37, 42, 43). Changes in the free amino acid pool of skeletal muscle reflect the net balance of several inputs (amino acid uptake, appearance from protein degradation) and outputs (protein synthesis, export) and may be considered a crude barometer of the net balance of amino acid appearance and loss in the tissue. Decreases in muscle free amino acid levels are often larger and more numerous than those in the plasma pool under conditions of muscle atrophy (17, 31, 41).
Changes in Muscle Variables Between Late Gestation and Lactation
RNA and gene expression. We provided evidence that upregulation of the ubiquitin-ATP-dependent proteolytic pathway occurs at the level of gene expression in the muscle of lactating sows that mobilize protein. The fall in total RNA and in GAPDH mRNA makes the increases in mRNA for ubiquitin, 14-kDa E2, and the C9 subunit of the 20S proteasome all the more striking and selective. The marked increase in the 1.2-kb 14-kDa E2 transcript agrees with other evidence that this transcript is tightly upregulated in various conditions of muscle atrophy in rats (reviewed in Refs. 4, 23).
This is the first time that this proteolytic pathway has been studied in the muscle of lactating animals. Upregulation of this pathway occurs in numerous conditions involving protein catabolism, including fasting, cancer, sepsis, diabetes, acidosis, burn injury, and muscle denervation, and, in all of these cases, protein degradation and mRNA are increased (4, 22, 23). The increase in mRNA expression of genes that encode proteins in the ubiquitin-ATP-dependent proteasome proteolytic pathway suggests that the rate of muscle protein degradation was higher in our sows during lactation than gestation. This inference is supported by several studies in the lactating first-parity sow. In early lactation (days 1 and 4), myofibrillar protein breakdown was at least 15% higher, and protein degradation was higher in sows fed <36 vs. 55 g lysine/day (20, 45). These differences were maintained until the end of lactation (days 15 and 20). In several studies in the lactating rat (34, 35) and dairy goat (39), where only protein synthesis was measured, a fall in protein synthesis was either not observed during lactation or was too small to account by itself for the net muscle protein loss. From these studies it can also be inferred that protein degradation rates rose, and Pine and colleagues (34, 35) suggested that the increase in protein degradation was quantitatively the more important effect.
We observed a decrease in the capacity for protein synthesis in muscle between late gestation and midlactation. By the time the sow was weaned, this variable had changed further in a manner related to the degree of protein mobilized. This agrees with our previous observation of lower (20%) triceps muscle RNA-to-DNA ratio on day 25 of lactation in sows that had lost >10% of their body protein (12). These reductions in the capacity for protein synthesis may underlie a decrease in the rate of muscle protein synthesis. This inference is supported by data in the rat and dairy goat, in which the rate of muscle protein synthesis was measured by either a flooding or a tracer dose of [3H]phenylalanine or L-[35S]methionine. Compared with dry goats, no reduction in the fractional rate of muscle protein synthesis was observed in early lactation in goats that had mobilized a moderate amount of protein (
28 g/day; Ref. 6). However, a 30% reduction was observed in goats of a similar weight that had mobilized more protein (
57 g/day; Ref. 8). Similarly, the rate of muscle protein synthesis decreased only in rats that lost a large proportion of their body weight (20 vs. 5%) during lactation (34); a 25% reduction in the fractional and a >45% reduction in the absolute rates of muscle protein synthesis were observed. Changes in the rate of muscle protein synthesis may help provide control and sensitivity to the degree of protein mobilized in lactation.
The large reduction in muscle GAPDH mRNA between late gestation and lactation in our sows suggests that the glycolytic pathway in muscle was downregulated, at least at the level of gene expression. This is consistent with our recent observation that the common pattern of gene expression in diverse forms of muscle atrophy includes downregulation of multiple enzymes participating in muscle energy metabolism (22). The data could also suggest that the size of the glycolytic muscle fibers was reduced relative to the oxidative fibers. Glycolytic muscle fibers appear to be targeted for degradation in muscle undergoing protein mobilization (18), and this was reflected in a decrease in the activity of glycolytic enzymes such as GAPDH (19, 27).
Muscle free amino acids. The muscle free amino acid pool increased during early lactation for most amino acids. The pattern observed later in lactation was strikingly different, with increased levels of glutamine and a fall in the levels of threonine, leucine, and phenylalanine to below prepartum levels. These changes (accumulation or loss) reflect an imbalance between the inputs into the muscle free amino acid pool (rate of amino acid appearance from myofibrillar protein breakdown and uptake from blood) and its outputs (rates of protein synthesis and amino acid export out of the tissue into blood).
We observed a sharp rise in muscle free glutamine levels in sows toward the end of lactation, in a manner related to the degree of protein loss. These data suggest a relative oversupply of glutamine, and this is consistent with the observations that plasma glutamine concentrations also increase in later lactation (28) and that free glutamine concentrations increased by almost fourfold (831% of free amino acids) in sow milk over the course of lactation (44). Similarly, the relative depletion of several EAA in muscle suggests an overall inadequacy of EAA supply in the free amino acid pool in muscle in late lactation.
Changes in Muscle Metabolism on Loss of Divergent Degrees of Body Protein
By the end of lactation, sows on our study were calculated to have lost 16, 20, and 36% of their muscle mass present at parturition (9). However, the demand for milk continually increases as the litter grows, even after feed intake has reached maximum, so that rates of muscle protein mobilization increase as lactation progresses. This would occur to the greatest degree when dietary protein was most limited. All of the changes described above (RNA-to-DNA ratio, protease gene expression, and amino acid pools) were magnified in the animals on the highest dietary protein restriction. This makes sense, considering that the law of diminishing returns applies to mobilization of a fixed resource. To release the same total amount of amino acids per day, the fractional mobilization rate must increase progressively to compensate as muscle mass shrinks. Our data are consistent with a continuous attempt to keep raising the muscle mobilization rate, so that, during later lactation and in animals provided the lowest dietary protein intake, all of the observed effects were magnified (i.e., further upregulation of the ubiquitin-ATP-dependent proteasome proteolytic pathway and decrease in the capacity for protein synthesis).
The situation of diminishing returns described above may provide a mechanism whereby lactation becomes self-limiting. The fractional rate of muscle protein mobilization may simply have a maximal rate. Once this is attained, the overall daily rate of amino acid delivery from muscle will shrink with the size of the muscle protein mass to a point where milk production will decline for lack of substrate. This would be consistent with the fall in milk protein concentration seen in late lactation, which was most prominent in animals with the most restricted protein intake (10, 11). Maternal nitrogen balance became less negative, indicating a reduction in protein mobilization, and growth of the litter of piglets slowed. The highest degree of protein loss was also accompanied by a decline in ovarian function. These physiological changes to delay or prevent a further cycle of reproduction would also help preserve the remaining maternal protein mass.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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