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ANIMAL NUTRITION |
* Schools of Animal Studies and Veterinary Science, University of Queensland, St. Lucia 4072, Brisbane, Australia; and
Department of Animal and Poultry Science, University of Guelph, Guelph,Ontario, N1G 2W1, Canada
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: age • intake • protein synthesis • ribosome transit time • sheep
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Anthony et al., 2002; Davis et al., 2002). Translational elongation is also a target through eukaryotic elongation factor (eEF)-2 (Wang et al., 2001). However, in growth of an animal to maturity, it is the long-term effect of diet that is of primary interest, and there has been no study of the effect of a chronically elevated or restricted nutrient supply on the rates of translation initiation. In the one study of elongation of which we are aware, ribosomal transit time (RTT; the inverse of the elongation rate) in liver was increased 2-fold during prolonged restricted intake in rats (Merry and Holehan, 1991).
The decline in fractional synthesis rate (FSR) of proteins in tissues of rats as they age has been associated with a greater RTT (Khasigov and Nikolaev, 1987; Merry and Holehan, 1991) and eEF-1 abundance (Castañeda et al., 1986; Rattan et al., 1991). However, decreases in eIF-2 abundance (Castañeda et al., 1986; Kimball et al., 1992, 2004) and polysome size (Murthy, 1966; Zomzely et al., 1971) provide evidence that initiation controls the declining FSR.
To investigate the relative roles of initiation and elongation regulation in control of FSR, we exploited the established effects of intake and stage of maturity (El Haj et al., 1986; Attaix and Arnal, 1987; Lobley, 1993) on protein synthesis. Protein FSR, RTT, and number of active ribosomes were measured in ewe lambs at 3 levels of intake and in mature ewes. To our knowledge, this is the first report of RTT in ruminant tissues and the first attempt to discriminate initiation from elongation in chronic effects on protein synthesis.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The Animal Care Committee at the University of Queensland approved all animal procedures described in this study. The sheep were White Suffolk x Poll Dorset:Border Leicester/Merino (3:1). Twenty-four ewe lambs (5 to 8 mo old, 24.8 ± 0.74 kg) and 8 ewes (>24 mo, 57 to 60 kg) were trained to metabolism pens and fed a pelleted alfalfa diet (21% CP, 2.01 Mcal of ME/ kg; DM basis) supplied hourly from automatic feeders. Ewe lambs were allocated randomly to 3 intake levels of approximately maintenance (M), 1.5M, and 2M. To study the effect of age, a group of mature ewes was fed at 2M. Although mature ewes are often fed to maintain BW, the 2M intake level was chosen for this age comparison so that protein FSR would be stimulated nutritionally in both groups.
Animals were fed as allocated for 10 d before N balance was determined for 7 d. Feces, urine, and feed refusals for all animals were collected and weighed. Collection occurred through gravity separators. Urine was acidified at the point of collection with sulfuric acid. A 10% sample of urine was taken and bulked within animal, bulked feces were frozen, and bulk urine stored at 4°C. Feed was sampled daily and bulked. On the last day of collection, bulked samples were subsampled for determination of DM by drying at 70°C for 48 h in a forced-air oven, and N by rapid combustion (Leco Australia Pty. Ltd., Castle Hill, Australia).
Catheters, Infusion, and Sampling Procedures
On the last collection day for N balance, 1 animal chosen at random from each treatment group was catheterized in the left jugular vein under local anesthetic (2% lidocaine, Troy Laboratories, Smithfield, Australia). Four animals were catheterized and slaughtered each day over the next 8 d for a total of 8 animals per treatment. Animals were left at least 18 h after catheter placement before beginning the protein synthesis study.
Flooding dose solutions were prepared for lambs as 4 g of L-Phe (>98%, Sigma-Aldrich, Castle Hill, Australia) in 120 mL of sterile saline (9 g/L) with 700 µCi of L-[Ring-2,6-3H]-Phe (>95%, 50 Ci/mmol, Sigma-Aldrich). For ewes, 5 g of Phe was dissolved in 150 mL of saline with the same amount of isotope. All solutions were sterile filtered through 0.2-µm nylon membrane filters (Alltech Bioscience, Deerfield, IL).
To avoid a spike in plasma Phe or insulin due to rapid injection, flooding dose solutions were injected slowly through 0.2-µm inline filters into the left jugular catheter over a 10-min period (Southorn et al., 1992). Blood samples (10 mL) were collected from the right jugular vein by syringe at 10, 20, and 38 min after the end of infusion.
Muscle biopsies (20 to 50 mg) were taken with a scalpel under local anesthesia (2% lidocaine, Troy Laboratories) 15 min after completion of injection from the left semimembranosus and 30 min after injection from the right semimembranosus. Biopsy samples were rinsed immediately in cold saline (9 g/L), placed in labeled aluminum pouches, and frozen by immersion in liquid N2. Biopsy samples were stored at –70°C until analysis.
Animals were killed by injection of sodium pentobarbitone (0.5 mL/kg) through the jugular catheter 40 min after cessation of isotope infusion. Tissue samples were excised and frozen within 6 min in liquid N2 in the following order: left semimembranosus, skin, LM, liver, duodenum, rumen, and right semimembranosus. Samples were transported to the laboratory on dry ice and stored at –70°C.
Sample Analysis
For measurement of protein and RNA content of tissues, and free and protein-bound Phe-specific radioactivities, duplicate 0.5-g samples of tissue were homogenized in 9 mL of 0.6 M perchloric acid. The supernatant after centrifugation at 8,500 x g for 5 min was stored at –20°C for free Phe-specific radioactivity determination (freepool). The washed pellet was incubated in 8 mL of 0.3 M KOH for 90 min at 37°C, from which 1 mL was retained for protein analysis (Lowry et al., 1951), and protein was precipitated from the remainder with successive perchloric acid washings and centrifugation at 1,000 x g for 10 min. Supernatants from these centrifugations were combined, and RNA content was calculated (Munro and Fleck, 1966). Protein in pellets was hydrolyzed by the method of Lobley et al. (1990).
Phenylalanine-specific activities were analyzed by HPLC according to Cohen and Strydom (1988) and Hagan et al. (1993). A 3-mL aliquot of hydrolysate was diluted to 15 mL with water, and a 1-mL aliquot of freepool was diluted to 3 mL with water. To each sample, 50 µL of 2.5 nM norleucine and 50 µL of 2.5 nM 
-amino-n-butyric acid were added as internal standards. Samples were loaded onto cation exchange resin columns and the eluant was collected, freeze-dried, dissolved, derivatized, and separated by HPLC to collect the phenylalanine fraction, which was counted to determine specific activity.
For measurement of DNA content of tissues and nascent and total protein radioactivity, samples of muscle and other tissues (0.5 and 0.75 g, respectively) were homogenized over ice in 3.5 mL of 0.3 M sucrose. The homogenate was strained through sterile gauze to remove large particulate matter. An aliquot of 0.4 mL was stored at –20°C for DNA analysis according to Labarca and Paigen (1980), 2 aliquots of 0.4 mL were frozen for total protein radioactivity, and 1 mL was analyzed immediately for nascent protein radioactivity according to Scornik (1974).
While on ice, nascent chain aliquots were diluted with 1 mL of 0.1 M Tris-HCl and 2 mM magnesium acetate (pH 7.6) and supernatants were centrifuged sequentially through 0.3 mL of 0.5 M sucrose with 1 mM magnesium acetate at 900 x g for 15 min, 0.3 mL of 4% sodium deoxycholate in water for 15 min at 4,000 x g, and 7 mL of 1 M sucrose with 1 mM magnesium acetate at 15,000 x g for 2.5 h at 2°C. The final pellet was resuspended in 1 mL of 0.3 M KOH with 0.5 mL of BSA (400 µg/mL). For determination of nascent and total protein radioactivity, 2 mL of 10% trichloroacetic acid was added to 0.65 mL of the resuspended pellet or 0.4 mL of the strained homogenate, respectively, and tubes were incubated at 90°C for 30 min. After centrifugation for 10 min at 1,000 x g, pellets were suspended in 2 mL of 10% trichloroacetic acid and centrifuged at 1,000 x g for 10 min, and then 2 mL of ethanol:chloroform:ether (2:2:1) at 1,000 x g for 10 min. The final pellet was suspended in 1 mL of diethyl ether, evaporated to dryness overnight, digested in 1 mL of Solvable (Packard, Meridian, CT), transferred to a scintillation vial containing 10 mL of scintillant (Optiphase HiSafe 3, The Australian Chromatography Company, Brisbane, Australia), left in the dark for 24 h, and counted (Packard 1900CA, Packard Instrument Co., Downers Grove, IL).
Calculations and Statistical Analysis
Tissue protein FSR were calculated using the tissue intracellular pool as the precursor pool (McNurlan et al., 1979):
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where SAh was the specific radioactivity of Phe in the hydrolysates, SAf was the specific radioactivity of the precursor pool, and t was incorporation time of 50 min, including injection time. Previous work has shown that within 2 min of starting the slow injection, plasma-specific radioactivity reaches a plateau (Southorn et al., 1992), so the 10-min injection time was considered part of the incorporation time. For a 50-min curve, this assumption may result in an underestimation of true FSR by less than 5% but does not affect treatment comparisons.
Translational efficiency (KRNA) was calculated as FSR/Cs, where Cs was total RNA:protein in the tissue (Lobley et al., 1994).
Ribosomal transit time was calculated according to Scornik (1974) as
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where RAn was the radioactivity in nascent chains, RAt was the activity in total protein, and t was 50 min.
Rates of initiation and elongation interact to determine the number of ribosomes bound to and actively translating mRNA according to:
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At steady state, the protein synthesis rate (mg of protein/min) is equal to the number of initiation events per minute times the average molecular weight of the proteins synthesized. Thus, the number of active ribosomes per gram of RNA was calculated according to the above relationship, assuming that the average molecular weight of newly synthesized proteins was 50,000 Da:
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Because the active ribosome number is presented per unit of total RNA, and approximately 80% of cellular RNA is ribosomal, the number reflects the proportion of ribosomes actively involved in translation, not unlike the proportion of ribosomes as polysomes presented by others (Palmiter, 1972; Scornik, 1974; Pérez-Sala et al., 1987).
Effects of level of intake of lambs and of age on observed variables were analyzed in 2 separate ANOVA, each according to the model yij = µ + ai + 
ij), where µ was overall mean, ai was the ith effect of intake level (i = 1 to 3) or age at 2M intake (i = 1 to 2), and ij was the random error distributed as N(0, 
2). Linear and quadratic contrasts between intake levels were estimated.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Total protein radioactivity in muscle biopsies increased linearly over time (r2 = 0.91 ± 0.16) for all 32 animals. The ratio of plasma to injectate-specific radioactivity at 38 min was 0.82 ± 0.02. Plasma-specific radioactivities of Phe measured at 10, 20, and 38 min after infusion were not different (P > 0.25) from each other for any treatment. Ratios of tissue intracellular specific radioactivity at slaughter to plasma-specific radioactivity at 38 min for lambs were: liver 0.86 ± 0.03; skin 0.94 ± 0.04; duodenum 0.88 ± 0.04; semimembranosus 0.95 ± 0.01; rumen 0.84 ± 0.11; and LM 0.93 ± 0.08. An intracellular to plasma ratio >0.75 has previously been accepted as a satisfactory flood level (McNurlan et al., 1979).
Intake, Digestibility, and N Balance
Dry matter intake differed (P < 0.05) between intake levels but not (P > 0.50) with age [M = 53.9 g/(d·kg0.75); 1.5M = 75.1 g/(d·kg0.75); 2M lambs = 92.4 g/(d·kg0.75); and 2M ewes = 91.7 g/(d·kg0.75)]. Dry matter digestibility did not differ (P > 0.50), giving estimated ME intake of 110, 151, 186, and 182 kcal of ME/(d·kg0.75) for M, 1.5 M, 2M lambs, and 2M ewes, respectively. Ewes had an average s.c. thickness at the 12th rib of 16.8 mm (SEM 0.7 mm) which indicates that the ewes were all at a similar stage of body condition. Subcutaneous fat thickness was not measured in lambs.
There was a linear increase (P < 0.05) in N balance for M, 1.5M, and 2M diets [0.62 ± 0.08, 1.01 ± 0.08, and 1.27 ± 0.06 g of N/(d·kg0.75), respectively]. The linear relationship of N balance to intake was (n = 24)
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There was a lesser (P < 0.05) N balance in 2M ewes [1.03 ± 0.12 g of N/(d·kg0.75)] than in 2M lambs.
Variables of Protein Synthesis
Level of feeding had no effect (P > 0.11) on RNA, DNA, or total protein content of tissues (Table 1) except in semimembranosus, where DNA content of semimembranosus decreased linearly (P = 0.02) between M and 2M and RNA:DNA increased (P = 0.02). Concentrations of RNA in skin (P < 0.001) and both skeletal muscles (P < 0.01) declined with increasing maturity. Ratios of RNA:DNA were not affected (P > 0.08) by age. The ratios of RNA:protein, on the other hand, were reduced in liver (P = 0.002), skin (P = 0.03), rumen (P = 0.04), and skeletal muscle (P < 0.05) of mature ewes compared with ewe lambs. Protein:DNA ratios in LM increased with age (P = 0.03). Protein:DNA ratio indicates cell size, RNA:DNA indicates protein synthetic capacity per cell, and RNA:protein indicates synthetic capacity on the same basis as FSR (i.e., per unit of protein).
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0.007) and KRNA (P 0.04) increased linearly with greater feed intake in all tissues except rumen (Table 2). All tissues from older animals exhibited lower FSR (P 0.10) but KRNA was not affected (P > 0.10).
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). They were constant (P > 0.50) across the 3 levels of intake for all tissues except rumen, where RTT increased linearly (P = 0.05). The greater efficiency of translation (KRNA) as intake increased combined with no change in RTT resulted in a linear increase in proportion of active ribosomes in liver (P = 0.008) and skin (P = 0.03), and a tendency for a linear increase in semimembranosus (P = 0.07). Ribosome transit time was delayed in skin (P = 0.02) and LM (P < 0.001) by maturity and tended to be delayed in duodenum (P = 0.08) and rumen (P = 0.10). In contrast, RTT in liver was lower in mature ewes compared with lambs (P = 0.05). The cellular proportion of active ribosomes was increased by maturity in skin (P = 0.004) and LM (P = 0.001) and depressed by maturity in liver (P = 0.04).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ribosomal transit time was calculated as the time to increase radioactivity in tissue protein by an amount equal to twice the activity of the nascent chains. Following a flooding dose of radiolabelled AA, nascent chain activity will increase rapidly to a plateau, and then activity in the total protein pool will increase linearly for some time. Based on the assumption that all the nascent polypeptide chains attached to active ribosomes are, on average, one-half the length of a finished polypeptide chain, the RTT is the length of time required to increase total protein activity by twice the nascent activity (Scornik, 1974). Consequently, RTT includes termination time in addition to elongation time. Muscle biopsies were taken at time intervals following isotope infusion to evaluate kinetics of accumulation of protein radioactivity. In all cases, accumulation was linear, indicating appropriate conditions for the calculation of RTT.
In the current study, RTT was measured for a variety of tissues, apparently for the first time in sheep. Previous estimates of RTT in mammalian tissues in vivo range from 58 to 204 s for rodent liver (Mathews and Haschemeyer, 1976; Khasigov and Nikolaev, 1987; Nagai and Ogata, 1990) and from 70 to 154 s for rodent brain (Khasigov and Nikolaev, 1987). Ribosomal transit time was 426 s in the brain of hibernating ground squirrels (Frerichs et al., 1998). Our estimates of RTT for sheep liver between 71 and 93 s are similar to those for normal, fed rat livers. Skeletal muscle displayed similar RTT, but skin and ruminal tissue were in the 150- to 300-s range and adult duodenum was greatest at 464 s.
A greater RTT implies either a greater average number of AA per protein molecule being synthesized or ribosomal delays or pauses along the mRNA during the process of elongation (Wolin and Walter, 1988). Differences between tissues in this experiment are likely due to the distribution of lengths of proteins specifically expressed, but differences between feed intake levels and animal ages are less likely to be reflective of a changing protein profile (Mathews and Haschemeyer, 1976; Khasigov and Nikolaev, 1987), and RTT is taken to represent the inverse of elongation rate.
Translational Regulation of Protein Synthesis
Study of eukaryotic protein translation has advanced to the stage that it is now possible to measure the cellular abundance and phosphorylation state of a plethora of initiation and elongation factors and their regulators. However, without a clear indication as to whether initiation, elongation, or some other factor such as synthetic capacity is responsible for the well-documented effects on FSR of level of feeding and stage of maturity (El Haj et al., 1986; Attaix and Arnal, 1987; Lobley, 1993), the number of translation participants to investigate as candidates to explain FSR control remains dauntingly large. Furthermore, the potential is high to find spurious changes in abundance or phosphorylation state that are not causative of the FSR effect. Thus, to provide direction in future study of the molecular mechanisms of chronic FSR control, we set out to discriminate initiation from elongation in the chronic effects of feed intake and age on FSR. To our knowledge, protein elongation rates have not been measured in farm species before.
In general, for all tissues except rumen, protein FSR was increased as the level of intake increased and was lower in adults, which is in agreement with previous reports (Attaix et al., 1988; Lobley, 1993). The effect of feed intake arose largely through changes in translational efficiency, similar to what has been reported for fed vs. fasted animals (Yoshizawa et al., 1995; Davis et al., 1996). We have shown for the first time that there was no effect on RTT of a prolonged difference in feed intake, suggesting that an upregulation of initiation rate was responsible for the increased proportion of ribosomes actively translating mRNA and the increased protein synthesis rate.
Elevated insulin and AA concentrations in blood following a meal stimulate changes in phosphorylation states, and thereby increase the activities of several components of the translation initiation apparatus in mammalian cells. The ribosomal protein p70 S6 kinase and eIF-4E binding protein 1 are two such components that have been implicated in elevated rates of global protein synthesis during absorption of a meal by young pigs and rats (Davis et al., 2000; Anthony et al., 2002). Insulin increased initiation rate in soleus muscle in vitro with no change in RTT (Monier and LeMarchand-Brustel, 1982), but Levenson et al. (1989) noted increased eEF-2 activity upon insulin stimulation of 3T3 cells. The p70 S6 kinase activated synergistically by insulin, AA, and glucose is known to stimulate eEF-2 activity by dephosphorylation (Wang et al., 2001) so an effect on elongation may be expected. Despite the putative mechanism of a feeding response, elongation has been studied little. Within hours of replenishment of lost nutrients by ingestion or infusion, the average number of ribosomes attached to each strand of mRNA in liver and muscle of growing rats and mice is increased (Kikuchi et al., 1986; Yoshizawa et al., 1995), and there is a report that RTT is reduced (Nagai and Ogata, 1990). In any case, an increase in polysome size indicates that initiation rate has increased to a greater extent than elongation rate and is therefore the more sensitive responder to cues of elevated nutrient supply. In our study of sheep, chronically elevated nutrient intake over a period of 3 wk had no effect on elongation rate, and translational efficiency was increased entirely through an effect on initiation rate.
Protein FSR in all tissues were lower in the adult 2M ewes compared with 2M lambs, but, in contrast to the feeding effect, changes in translational efficiency were responsible only in liver and duodenum. Fractional synthesis rate is a measure of protein synthetic rate per unit of protein, and skin, rumen, and skeletal muscle all exhibited lower synthetic capacity per unit of protein, as evidenced by a reduced RNA:protein ratio. Developmental decreases in both synthetic capacity (El Haj et al., 1986; Lobley, 1993; Martin et al., 1993) and translational efficiency (Blazejowski and Webster, 1983; El Haj et al., 1986; Kimball et al., 2004) have been observed previously. Interestingly, even though translational efficiency was affected little in sheep tissues, RTT was prolonged in all but liver and semimembranosus. Increases in RTT with advancing age have been previously reported in rat liver and brain (Khasigov and Nikolaev, 1987; Merry and Holehan, 1991) but have not been studied in other tissues or in other species. Of the 2 elongation factors in mammalian cells, eEF-1 has been shown to decline in amount and activity with age (Gabius et al., 1983; Castañeda et al., 1986; Rattan et al., 1991). Consequently, there has been the suggestion that this decline in eEF-1 expression causes the age-dependent decrease in protein synthetic rates (Rattan, 1996), but the issue remains contentious because decreases in polysome size do not bear out a role for elongation regulation (Murthy, 1966; Zomzely et al., 1971) and an assortment of initiation factors also decrease in concentration and activity (Kimball et al., 1992, 2004). Our results perhaps clarify the issue by showing that, although elongation rate decreased with age (an increase in RTT), the decrease in cellular RNA, which is mostly ribosomes, per unit of protein accounted for the decline in tissue FSR.
In fact, a change in elongation rate does not influence protein synthetic rate unless the initiation rate is somehow affected. Elongation acts merely as a delay between complete attachment of ribosomal subunits to the mRNA and release of the full polypeptide. At steady state, initiation rate is equal to polypeptide release rate; elongation rate dictates, under those conditions, how many ribosomes are strung out along a single mRNA strand. Consequently, inhibition of elongation with cycloheximide in vitro increases the number of attached ribosomes (Palmiter, 1972; Ku and Thomason, 1994). Similarly, the slowdown of elongation rate in liver during glucagon administration or in muscle bearing no weight was associated with increased polysome size (Ayuso-Parrilla et al., 1976; Ku and Thomason, 1994). Often, an increase in RTT is associated with no change or a decrease in polysome size (Nielsen and McConkey, 1980; Pérez-Sala et al., 1987; Frerichs et al., 1998), indicating that initiation rate has decreased by at least the same extent as elongation rate. Simulation of multiple ribosome binding and translocation along mRNA showed that polysome size was more sensitive to elongation rate than to initiation rate (Ku and Thomason, 1994), contrary to conventional interpretation of polysome profiles. Polysome size was not measured in our study, but an estimate of the proportion of ribosomes bound to mRNA was obtained and found to be elevated in skin and LM of adult ewes compared with the lambs. Thus, although the RNA:protein ratio was lower in many tissues of adult sheep and responsible for a decreased FSR, and hence initiation rate, a greater proportion of that RNA was actively translating mRNA, perhaps because of the longer RTT.
A circumstance under which elongation rate can influence initiation rate, and thereby exert some degree of control over protein synthetic rate, occurs when mRNA remains fully saturated with ribosomes (Palmiter, 1972). The increase in active ribosome proportion in old vs. young sheep suggests that mRNA was not saturated in the 2M lambs, and protein synthetic rate could be accelerated by a stimulation of initiation rate. This conclusion is borne out by the observation that an upregulation of initiation rate appeared to be responsible for the effect of feed intake on protein FSR in tissues.
We have measured, for the first time, rates of protein initiation and elongation in various tissues of growing sheep when daily DMI was elevated over a period of 3 wk, and in mature ewes vs. growing lambs. It was concluded that RTT was not influenced by intake but that the estimated proportion of ribosomes attached to and actively translating mRNA was increased, suggesting that initiation rate accounted for the greater FSR. The mechanism by which FSR was affected by a chronically elevated or restricted nutrient supply may involve the same initiation factors that have been implicated in short-term control via phosphorylation. Most tissues of mature ewes exhibited lower FSR compared with growing lambs because of reductions in synthetic capacity, not because of the efficiency of translation, even though RTT was prolonged. In this case, regulation of initiation factors was not likely to explain the lower FSR, whereas the study of elongation factors may shed light on the effects of maturity on polysome size.
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Footnotes |
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2 Corresponding author: d.poppi{at}uq.edu.au
Received for publication March 12, 2007. Accepted for publication May 6, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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