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Nutrient Metabolism

Whole-Body and Hindlimb Protein Breakdown Are Differentially Altered by Feeding in Neonatal Piglets¹

M. Carole Thivierge, Jill A. Bush^*, Agus Suryawan^*, Hanh V. Nguyen^*, Renan A. Orellana^*, Douglas G. Burrin^*, Farook Jahoor^* and Teresa A. Davis^*,2

^* U.S. Department of Agriculture/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 and Nutraceutical and Functional Food Institute/Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval, Quebec, Canada, G1K 7P4

²To whom correspondence should be addressed. E-mail: tdavis{at}bcm.tmc.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The high rate of muscle protein accretion in neonates is sustained by the marked increase in muscle protein synthesis in response to feeding. Little is known about the role of proteolysis in the regulation of protein accretion in response to feeding during the neonatal period. To determine the feeding-induced response of protein breakdown at the whole-body level and in the hindlimb of neonates, 10- and 28-d-old piglets that had been food deprived overnight were infused (7 h) with [1-¹³C]phenylalanine and [ring-²H₄]tyrosine during an initial food deprivation period (3 h), followed by a feeding period (4 h). During feeding, endogenous flux of phenylalanine decreased (P < 0.01) in both the whole body and the hindlimb. Feeding reduced (P < 0.01) whole-body proteolysis but increased hindlimb proteolysis (P = 0.04), suggesting that tissues other than the hindlimb are involved in the reduction in whole-body proteolysis during feeding. Overnight food deprivation resulted in a net mobilization of phenylalanine from whole-body proteins (P < 0.01) but not hindlimb proteins. These responses were unaffected by age. The results suggest that the hindlimb requires a continuous supply of free amino acids to sustain the high rate of muscle protein turnover in neonates and that adaptive mechanisms provide free amino acids to sustain skeletal muscle protein accretion in early postnatal life when the amino acid supply is limited.

KEY WORDS: • phenylalanine hydroxylation • phenylalanine kinetics • neonate • skeletal muscle • stable isotopes

The protein synthetic capacity of newborns is high and enables their rapid rate of growth. Investigations have highlighted some of the mechanisms that underlie this phenomenon. Immature skeletal muscles have a high ribosome content, which provides the machinery required to sustain a high rate of protein gain (1). Such regulation is transitory during the neonatal period and decreases with age. The mechanisms behind the neonatal regulation of protein synthesis were investigated in neonatal piglets and rats, and both models provide congruent findings that the high synthesis rate of protein in neonates is due in part to an enhanced response of protein synthesis to feeding (2–4). Our studies using a novel hyperinsulinemic-euglycemic-euaminoacidemic clamp technique further showed that that this elevated response to feeding in neonates is mediated by enhanced sensitivity and responsiveness of muscle protein synthesis to insulin (5–7). Further exploration revealed that the feeding-induced activation of the insulin receptor and downstream intermediates of the phosphatidylinositol-3 kinase/mammalian target of rapamycin signaling pathway leading to translation initiation are elevated in young neonates (8–10). The enhanced activation of the insulin signaling proteins leading to translation initiation likely contributes to the efficient utilization of nutrients for growth and rapid gain in protein mass in the skeletal muscle of the neonate.

Molecular aspects of proteolysis involve disassembly of myofibrillar proteins by the calcium-dependent calpain system, which is followed by the ubiquitination of the myofibrillar proteins with 4 ubiquitin moieties (11,12). Polyubiquitinated proteins are efficiently degraded by the proteasome complex. These latter degradation regulatory mechanisms occur primarily in muscle, whereas lysosomal degradation dominates liver proteolysis (11–13). Although recent studies delineated the molecular regulatory aspects of protein degradation, little is known about the regulation of proteolysis at the physiologic level. Protein breakdown is of particular interest in neonates because their protein accretion rate is high due to their high rate of protein turnover, which results from the balance between synthesis and proteolysis.

Studies with human infants suggest that neonates are more resistant than adults to a reduction in protein breakdown, likely because free amino acids are constantly required in the free cellular pool to sustain the constant and rapid protein remodeling (14–16). To date, all investigations on the regulatory role of protein degradation on protein anabolism in neonates were conducted at the whole-body level, and no data exist specifically on skeletal muscle. Because changes in proteolysis can significantly alter protein stores, it is of both physiologic and clinical interest to better define the role of skeletal muscle proteolysis in the regulation of muscle protein metabolism in newborns.

To assess the role of proteolysis in the regulation of protein metabolism, whole-body and hindlimb protein kinetics were investigated in neonatal piglets. Because neonates are mainly in the fed state, the study was conducted during both overnight food deprivation and feeding to duplicate the normal physiologic states.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and trials. In the current study, 2 experiments were performed. The primary experiment was designed to explore the role of proteolysis in the regulation of postprandial protein accretion in skeletal muscle during neonatal life by way of in vivo tracer kinetic methodology in a piglet model. Repeated measurements comparing the effect of overnight food deprivation and feeding on proteolysis were carried out in an incomplete block design comparing 2 age groups of 28-d-old piglets and 10-d-old piglets (n = 7). Phenylalanine was chosen as the amino acid tracer because this amino acid is not oxidized within skeletal muscle, and its appearance in plasma originates solely from absorption and breakdown. Phenylalanine hydroxylation to tyrosine was quantified in the whole-body kinetic model to account for the loss of tracer through the hydroxylation pathway. A secondary pilot experiment was previously conducted in 28-d-old piglets (n = 3) to determine whether 2 amino acids labeled with stable isotopes were recycled back into the plasma when the duration of the tracer infusion lasted for 7 h. The experimental procedures in the pilot experiment were exactly the same as those in the primary experiment. The studies were approved by the Animal Care and Use Committee of Baylor College of Medicine and were conducted in accordance with the NRC guidelines.

    Experimental design: main experiment. Crossbred sows (Yorkshire x Landrace x Hampshire x Duroc; Agriculture Headquarters; Texas Department of Criminal Justice) were housed in lactation crates in individual environmentally controlled rooms. They were fed a commercial diet (Purina Lab Porcine Diet; 1.5–1.9 kg/d) and had free access to water. After farrowing, piglets remained with the sow until either 3 or 21 d of age and were not given supplemental creep feeding. On d 3 or 21 after birth, the surgical preparations were carried out in 3-d-old (n = 7) and 21-d-old (n = 7) piglets after overnight food deprivation. A feeding catheter was placed in the duodenum, a venous catheter for tracer infusion was placed in a jugular vein, and blood sampling catheters were placed in a carotid artery and the inferior vena cava. A perivascular flow probe was placed around the caudal aorta. The piglets were introduced to a commercial milk replacer (Litter Life, Merrick’s) 24 h after the surgery. The milk replacer was consumed ad libitum (minimum 6.25% body weight) (17) 72 h after the surgery and the piglets were fed this milk replacer until measurements were carried out at 10 (3.5 ± 0.4 kg body weight) and 28 d of age (6.6 ± 0.9 kg body weight).

    Experimental design: tracer recycling pilot experiment. Management procedures comparable to those used in the main experiment were carried out in the pilot experiment. Briefly, a similar crossbred sow was housed in a lactation crate under similar conditions with the same feeding management. After farrowing, piglets remained with the sow until 21 d of age. The surgical procedures were performed on 3 piglets at 21 d of age, preceded by overnight food deprivation of 16 h. The same catheter and flow probe arrangement was used in the pilot study as in the main experiment. Feeding management was exactly the same as in the main experiment.

    Surgical procedures. Surgery was carried out under sterile conditions; the preparation of the piglets and the anesthesia were similar to those previously described by Wray-Cahen et al. (6) and Bush et al. (18). The tip of the vena cava catheter was placed caudally to the renal vein.

    Isotopes. The tracers, L-[1-¹³C]phenylalanine, L-[1-¹³C]tyrosine, NaH¹³CO₃ (99 atom % excess, Cambridge Isotopes Laboratories), L-[ring-²H₄]tyrosine (98 atom % excess, Cambridge Isotopes Laboratories), and NaH¹⁴CO₃ (Cambridge Isotope Laboratories) were dissolved in physiological saline.

    Infusions. The isotope infusions were performed after overnight food deprivation of 16 h according to the flow chart shown in Figure 1. At 15 min before time 0 (time 0 is the onset of the tracer infusions), the blood flow rate was measured continuously (Transonic System); 30 min before time 0, 3 background blood samples were taken from the artery and the vena cava every 10 min. At time 0, body pools were primed with [1-¹³C]phenylalanine (22 µmol/kg), [1-¹³C]tyrosine (3.3 µmol/kg), and [ring-²H₄]tyrosine (12 µmol/kg in the pilot experiment, adjusted to 6 µmol/kg in the main experiment). Simultaneously, the body bicarbonate pool was primed with 6 µmol/kg of NaH¹³CO₃. Thereafter, continuous infusions of [1-¹³C]phenylalanine [22 µmol/(kg · h)] and [ring-²H₄]tyrosine [12 µmol/(kg · h) in the pilot experiment and 6 µmol/(kg · h) in the main experiment] were initiated and maintained for 7 h. At time 1 h, a primed (0.75 µCi/kg) continuous infusion [1.0 µCi/(kg · h)] using NaH¹⁴CO₃ was initiated and maintained over the next 2 h to measure CO₂ production during the food deprivation period. At time 3 h, the NaH¹⁴CO₃ infusion was stopped and feeding started after a bolus equivalent to 0.5-h infusion of the elemental diet was given orally. An intraduodenal infusion of an elemental diet [10 mL/(kg · h)] (19) providing dextrose (104 g/L), lipids (21 g/L; Intralipid, Baxter Healthcare), a complete amino acid mixture (55 g/L; Ajinomoto), electrolytes, trace minerals, and vitamins sufficient to meet or exceed the requirements for neonatal pigs [215 kcal/(kg · d), 13 g amino acid/(kg · d), 240 mL/(kg · d) fluid intake] was started and maintained for the next 4 h. After 5 h, a primed (0.75 µCi/kg) continuous infusion of NaH¹⁴CO₃ [1.0 µCi/(kg · h)] was initiated and maintained over the next 2 h to measure CO₂ production in the fed state. Breath and blood samples were taken every 30 min over the last 2 h of the food deprivation and the feeding periods (4 samples/nutritional status). Breath samples for determination of ¹³CO₂ enrichments were collected from the pigs using a 1-way valve breath bag and transferred directly into a sterile vacutainer. The collected expired air was injected into a sterile 10-mL vacutainer for subsequent analysis of isotopic enrichment via isotopic ratio MS (IRMS).³ At time 7 h, the infusions were stopped and the piglets were killed by an i.v. lethal injection (pentobarbital sodium; 50 mg/kg). Immediately, longissimus dorsi skeletal muscle was rapidly sampled, frozen in liquid nitrogen, and stored at –70°C until subsequent analyses for fractional protein synthesis rate of the feeding period.

View larger version (18K): [in this window] [in a new window]   FIGURE 1 Flow chart of tracer infusions and blood sampling during the pilot and main infusion studies in 10- and 28-d-old pigs. Time 0 priming doses: L-[1-13C]phenylalanine, 22 µmol/kg; L-[ring-2H4]tyrosine, pilot study 12 µmol/kg and main study 6 µmol/kg; L-[1-13C]tyrosine, 3.3 µmol/kg; NaH13CO3, 6 µmol/kg. Continuous infusions: L-[1-13C]phenylalanine, 22 µmol/(kg · h); L-[ring-2H4]tyrosine, pilot study 12 µmol/(kg · h) and main study 6 µmol/(kg · h); dotted arrows indicate collection of background breath and blood samples; dashed arrows indicate NaH14CO3 priming dose (0.75 µCi/kg) and onset of continuous infusion [1.0 µCi/(kg · h)]; solid arrows indicate collection of treatment breath and blood samples.

    Amino acid isotopic enrichments. Plasma phenylalanine and tyrosine were separated on a cation exchange resin column (AG-W50 resin, Bio-Rad) and converted to their n-propyl ester heptafluorobutyramide derivative according to the method of Reeds et al. (20). Because of the sensitivity of deuterated tyrosine derivatives to heat, plasma tyrosine was derivatized at 30°C for 2 h. The recovery of the tracers was always verified by linear regression (x = molar ratio and y = isotopic ratio).

GC-MS was carried out on a model HP 6890 gas chromatograph (Hewlett Packard) coupled with a quadrupole mass spectrometer model 5973 mass selective detector operating in the negative chemical ionization mode. Selective ion monitoring was carried out at m/z 383, 384 for phenylalanine and 595, 596, 599 for tyrosine. Breath ¹³CO₂ enrichment was determined on an IRMS (ThermoQuest Finnigan Delta Plus XL, Thermo Finnigan MAT GmbH) monitoring ion masses 44, 45.

    Muscle processing for isotopic enrichment. The enrichments of phenylalanine from muscle proteins and from the protein-free extracts of muscle were measured by GC-IRMS. Frozen muscle (100 mg) was homogenized in 2 mL of ice-cold trichloroacetic acid (TCA 10%; wt/v). The TCA-insoluble precipitates were hydrolyzed with 6 mol/L hydrochloric acid solution at 110°C for 24 h. Phenylalanine released from muscle proteins and from the cellular free pool was converted to its N-acetyl-propyl derivatives, separated by GC, combusted, and the carbon isotopic ratios were measured on an IRMS (Finnigan Delta Plus XL) by monitoring ions at m/z 44, 45.

    Blood specific radioactivity. To calculate whole-body CO₂ production, blood samples were processed to determine the specific radioactivity (SRA)³ of total blood CO₂. Briefly, 0.5 mL of fresh whole blood was added to 0.1 mL of 1 mol/L sodium bicarbonate in a sterile vacutainer. Both products were injected through the cap and kept on ice. The amount of blood was weighed precisely for the calculations. Immediately after the kinetic measurement period, the samples were processed by the addition of 5 mL of methanol injected through the cap of the vacutainer, mixed gently, and centrifuged at 1500 x g for 20 min at 4°C. The supernatant was quickly poured off into a 30-mL scintillation vial, 10 mL of scintillation Cocktail (Scintillation cocktail 3a70, RPI Research Products International) was added, and the samples were counted.

    Calculations. Total whole-body fluxes of phenylalanine and tyrosine were calculated from the dilution of [1-¹³C]phenylalanine and [ring-²H₄]tyrosine according to the following equation:

where Q_{Phe or Tyr} is the total turnover rate of phenylalanine or tyrosine in µmol/(kg · h); I_{Phe or Tyr} is the tracer infusion rates in µmol/(kg · h); E_i is the enrichment of either [1-¹³C]phenylalanine or [ring-²H₄]tyrosine in the infusates expressed as the tracer:tracee ratio; and E_p is the plasma enrichment of individual tracer at plateau expressed as the tracer:tracee ratio. Endogenous fluxes were obtained by subtracting the tracer amino acid infused in the starved experiment and the dietary plus tracer amino acid infused in the fed experiment (21).

Phenylalanine hydroxylation to tyrosine was calculated according to the model of Clark and Bier (22) with the modification of Thompson et al. (23) for labeled phenylalanine infusion:

where Q_{Phe to Tyr} is the hydroxylation of phenylalanine to tyrosine in µmol/(kg · h); Q_{Tyr or Phe} is the total tyrosine or phenylalanine flux calculated from the tracer infusion; E_1–13CTyr is the arterial enrichment of [1-¹³C]tyrosine derived from the [1-¹³C]phenylalanine tracer; and E_Phe is the arterial enrichment of phenylalanine at plateau.

Oxidative metabolism of phenylalanine was calculated as follows:

CO₂ production calculation:

where I_NaH14CO3 is the bicarbonate infusion rate in µmol/(kg · h); SRA ¹⁴CO_2-infusate is the specific radioactivity (dpm) per mL of the infusate; and SRA ¹⁴CO_2-blood is the specific radioactivity (dpm) per mL in blood. A previous study showed that ¹³C isotopic enrichment in breath CO₂ is similar to blood CO₂ (18). In the current study, SRA in blood CO₂ was then used for the calculation of CO₂ production given the simplicity of laboratory processing.

SRA of CO₂ in blood:

where SRA CO_2-blood is the specific radioactivity in blood CO₂ in dpm/mmol; SRA_-blood is blood specific radioactivity in dpm/mL of blood; [CO₂] concentration in blood is the concentration of CO₂ in blood expressed in mmol/mL.

Oxidation rate of phenylalanine:

where E_13CO2-breath is the isotopic enrichment of expired CO₂ and E_phe-ppool is the isotopic enrichment of arterial plasma phenylalanine.

The steady-state equation of total whole-body flux of phenylalanine:

where Q_Phe is the entry rate of unlabeled phenylalanine into the central plasma pool equating phenylalanine absorbed [i.e., duodenal administration rate of phenylalanine; I_diet in mmol/(kg · h)] and phenylalanine appearance from protein breakdown (PB) in mmol/(kg · h). The disposal rate of phenylalanine from the plasma pool is represented by its disappearance from the plasma for protein synthesis (PS) in mmol/(kg · h) and its irreversible loss through oxidation. Protein synthesis and breakdown were derived from the steady-state flux equation.

Hindlimb movements of the tracer were calculated according to the tracer:mass balance technique. The hindlimb kinetic model is schematized in the publication of Bush et al. (18). Then,

where HLQ_Phe expresses hindlimb phenylalanine flux; A_phe and V_phe represent the arterial and venous concentration of phenylalanine in plasma; E_A-Phe and E_V-Phe represent the isotopic enrichment of phenylalanine in arterial and venous hindlimb plasma; E_phe-ppool is the phenylalanine isotopic enrichment of the protein synthesis precursor pool in the hindlimb; and venous plasma from the vena cava is assumed to be representative of the free intracellular phenylalanine isotopic enrichment. Because phenylalanine is not oxidized in the hindlimb (24) and its hydroxylation to tyrosine occurs in the liver and the kidneys (25), hindlimb phenylalanine flux represents its disappearance for protein synthesis (HLPS).

Hindlimb flux:

Hindlimb protein breakdown was derived from the hindlimb flux equation:

Fractional synthesis rate (FSR) of muscle was calculated according to the precursor:product technique of free and bound phenylalanine pools in muscle. This method assumes a linear incorporation of the tracer into muscle protein when the cellular free pool enrichment is constant after several hours of infusion (26). The percentage of protein mass synthesized in a day was calculated as:

where E_bound is the isotopic enrichment of the protein bound phenylalanine; E_free is the isotopic enrichment of the precursor free pool; t is the time of labeling in minutes. The isotopic enrichment at baseline was assumed to be the same as plasma phenylalanine (26). Tissue free phenylalanine was shown to be a valid measure of tissue precursor pool aminoacyl-tRNA (27).

    Statistical analyses. The effects of feeding on dependent variables measured were subjected to an ANOVA. The statistical design was repeated measurements comparing 2 nutritional states in 2 groups of piglets, 10-d-old piglets (n = 5) and 28-d-old piglets (n = 6), according to the GLM procedure of SAS (28). This design allowed the comparison between nutritional states, age effects, and their interaction on the dependent variables. The statistical model was as follows, including the overall mean (µ) and residual error (e) associated with ijkl observations:

The error term for testing the effect of age was derived from the variance of the piglets within each age group. Type III sum of squares of the variance analysis were interpreted. Least-square means are given in tables with pooled SEM and differences were considered significant at P 0.05; 0.05 < P 0.10 was considered to be a tendency. Regressions were performed using Proc Reg in the GLM procedures of SAS. Type I sum of squares was interpreted (28). Student’s t test was performed to measure the difference of the mean for muscle FSR with SD presented.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Tracer recycling. A pilot experiment was conducted to determine whether a 7-h period of tracer infusions would generate recycling of [1-¹³C]phenylalanine and [ring-²H₅]tyrosine from body stores back into the plasma central compartment, which would invalidate the steady-state flux equation. Blood sampling was carried out every 30 min during the 7-h period to allow continuous monitoring of variations in enrichments. Arterial isotopic enrichments of [1-¹³C]phenylalanine and [ring-²H₄]tyrosine during the last 3 time points of the food deprivation and the feeding periods suggested steady-state enrichments in these different pools (Table 1). Feeding the elemental diet reduced (–30%) plasma [1-¹³C]phenylalanine isotopic enrichments, which appeared to reach steady state 60 min after the initiation of the elemental diet. Isotopic enrichment of the [ring-²H₄]tyrosine was reduced by 29% during the first 2 h of the feeding period compared with the food deprivation period and by 35% over the last 2 h of the feeding period. These successive dilutions in [ring-²H₄]tyrosine were likely a consequence of 1) a dilution by the onset of the administration of tyrosine in the elemental diet and 2) a dilution due to unlabeled tyrosine appearing from phenylalanine hydroxylation. To further evaluate the variations in isotopic enrichments, regression assessments were performed using the last 3 data points of the food deprivation and feeding periods. The probabilities of these regression analyses revealed that the slopes did not differ from 0 and/or that the variations observed were within the biological noise (CV < 6%; Table 1).

    Hindlimb metabolism. Plasma flow to the hindlimb was increased by age and feeding state and was increased more by feeding in older piglets (Table 4). Similarly, net flux of phenylalanine, as well as protein synthesis, increased with feeding but increased more in 28-d-old piglets than in 10-d-old piglets. In contrast to whole-body proteolysis, constitutive protein breakdown in the hindlimb increased with feeding and there was no effect of age. Protein accretion was positive in both food-deprived and fed piglets and tended to increase more in 28- than in 10-d-old piglets. FSR of skeletal muscle protein decreased by 65% between 10 and 28 d of age in fed piglets (Table 4).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

    Tracer recycling. Whole-body kinetic studies involve systemic tracer infusion, and plasma is considered to be a central compartment, which is the result of variations in individual pools of the body (30). Such a compartmental model is usually interpreted as a single-pool model, and we assume that the tracer is not recycled from intermediate pools of the body back into plasma. With such a model, we assume that the infusion of the tracer is the sole entry of the tracer into the central pool, and the resulting arterial isotopic enrichment derives from the dilution of the tracer infused by unlabeled tracee coming from absorption, breakdown, or other metabolic pathways. Such an assumption allows us to calculate whole-body flux. In the current proposal, recycling was investigated in a pilot experiment to determine whether there was a return of labeled amino acids into the plasma in neonates during a 7-h tracer infusion. We found no evidence of recycling, and the variations in isotopic enrichments observed were within the biological noise. Previous investigation on the topic showed that recycling is detectable during a 24-h (31) and 18-h infusion period in humans (32). However, when the tracers are administrated over a 4-h period, no return of the tracer is seen (31). The results observed in the current study suggest that a 7-h tracer infusion is appropriate in neonatal piglets.

    Whole-body phenylalanine metabolic pathways. In this study, feeding decreased the endogenous flux of phenylalanine, which was in accordance with a reduction of protein breakdown rate with feeding. Simultaneously, feeding was associated with an elevation in the overall regulatory processes related to the disappearance of phenylalanine from the plasma pool. These include irreversible losses of phenylalanine through hydroxylation to tyrosine, its oxidation to CO₂, and its incorporation into protein. Feeding increased the hydroxylation of phenylalanine to tyrosine in 28-d-old piglets (443%) to a greater extent than in 10-d-old piglets (146%). The age-related difference in the irreversible loss of phenylalanine toward hydroxylation to tyrosine raises a concern regarding the capacity of young neonates to acutely regulate phenylalanine hydroxylation in response to phenylalanine availability. Previous investigations on this topic suggested that human newborns can regulate phenylalanine hydroxylation in response to graded supplies of phenylalanine (14–16,33). Although a high phenylalanine intake increases hydroxylation of phenylalanine to tyrosine and phenylalanine retention (33), an increase in the urinary excretion of alternate catabolites of phenylalanine suggests that phenylalanine can be supplied in excess relative to the disposal capabilities (33). The observations of the current experiment suggest that phenylalanine may not be converted in amounts adequate to sustain tyrosine requirements. A significant linear relation existed between arterial phenylalanine concentration and its hydroxylation to tyrosine (r² = 0.91) over a wide range of phenylalanine concentrations (from 103 to 397 µmol/L). Although the regression relation was tight, the low slope of the regression reveals that for each unit of increment in arterial phenylalanine, 7.7% of that phenylalanine was diverted toward hydroxylation. Thus, phenylalanine hydroxylation may not supply tyrosine in appropriate amounts when tyrosine intake is limited. This conclusion was supported by the reduction in arterial concentration of tyrosine after feeding in 10-d-old piglets accompanied by an increased whole-body flux of tyrosine with feeding. When protein turnover is high, the relative rate of the catabolic pathway of phenylalanine, with the balance between tyrosine arterial supply and peripheral removal, may not be sufficient to meet tyrosine requirements in neonates. In the current study, tyrosine was potentially a limiting amino acid for protein synthesis. However, the protein synthesis response to feeding was substantial in young as well as in older neonates. The results suggest no detectable effect of tyrosine shortage over a 4-h period of enteral feeding.

The fractional oxidation and the absolute oxidation rates of phenylalanine were increased by feeding but were not further altered by age, suggesting that the oxidative capacity of neonatal piglets was not developmentally regulated. Instead, the results highlight the capacity of neonates to adjust phenylalanine oxidation in accordance with its arterial concentration, as seen in adults. Residue analysis of linear regression revealed that the arterial concentration of phenylalanine explains 61% of the variation in phenylalanine oxidation. Previous studies showed similar relations for lysine and leucine oxidation with their arterial concentration in fed but not in starved human subjects (r² = 0.55) (34,35). This suggests that other variables might be more appropriate to predict oxidation rates of amino acids when their arterial concentration is low.

Feeding increased the phenylalanine disposal rate for protein synthesis and this reached a numerically higher rate in 10-d-old piglets than in 28-d-old piglets. Simultaneously, feeding decreased whole-body proteolysis, as typically observed when insulin or amino acids were provided to adults (36–38) and newborns (15,39–41). Studies suggested that newborn babies or premature infants may be more resistant to depression of protein breakdown, likely due to their rapid and continual remodeling of muscle protein requiring constant amino acid availability at the cellular level (14–16). Whole-body proteolysis measured in the current study suggests a similar trend, with the younger piglets tending to be more resistant to a depression in protein breakdown in response to feeding.

    Hindlimb protein metabolism regulation. To determine the role of proteolysis in the regulation of muscle protein deposition in the hindlimb of neonatal piglets, we used a dual stable isotope tracer:mass balance technique. Hindlimb phenylalanine net flux was increased by feeding and this feeding effect was greater in 28- than in 10-d-old piglets. Phenylalanine disappearance from the plasma pool for protein synthesis followed a similar pattern. Because protein metabolism by the hindlimb represents that of individual muscles, skin, bone, and adipose tissues, we also measured the rate of incorporation of labeled phenylalanine into protein in the longissimus dorsi muscle, which contains primarily fast-twitch muscle fibers. Similar to the results of our previous studies (3), the fractional protein synthesis rates in the longissimus dorsi muscle measured during the feeding period decreased sharply with age from 15.4%/d in 10-d-old piglets to 5.6%/d in 28-d-old piglets (Table 4). The greater absolute rate of hindlimb protein synthesis in mmol/kg body weight measured in 28-d-old piglets compared with 10-d-old piglets likely reflects the difference in hindlimb composition between the age groups, with the hindlimb protein mass accounting for a higher proportion of total mass in older pigs than in younger pigs. In addition, the higher rate of blood flow in 28- than in 10-d-old pigs and the greater rise in blood flow with feeding in the older age group contributed to the higher absolute rate of hindlimb protein synthesis and its greater response to feeding in the older pigs. This increase in blood flow may be mediated by the vasodilator properties of insulin through a endothelium-derived nitric oxide–dependent mechanism (42,43).

In contrast to whole-body protein breakdown, hindlimb proteolysis was increased by 33% in response to feeding. Enhancement in skeletal muscle proteolysis by feeding in neonates contrasts with previous studies in adult skeletal muscle. Feeding, BCAA, and insulin exert an antiproteolytic effect on whole-body protein degradation in mature subjects (37,44,45). This antiproteolytic effect of feeding amino acids or leucine is demonstrated by a reduction of plasma 3-methyl histidine, a specific marker of myofibrillar proteolysis (46,47). Skeletal muscle protein accretion in most cases results from reduced protein breakdown in adults (37,45,48,49). Studies in human newborns suggest that the ability to reduce proteolysis in response to parenteral nutrition increases with development (14). Furthermore, neonatal regulation of protein breakdown appears to be regulated primarily by the availability of free amino acids in the intracellular pool, and this regulation is not insulin dependent (15,16). Such a reduction in whole-body proteolysis in neonates is not elicited by the administration of nonprotein substrates such as glucose and lipids provided alone (50). Direct molecular regulation of muscle protein breakdown by amino acids through the dominating proteolytic pathway in muscle has not yet been reported. In the current in vivo experiment, whole-body and hindlimb protein synthesis were increased by 30–43 and 146–197%, respectively, after feeding. These data suggest that hindlimb protein synthesis is more sensitive to feeding than whole-body protein synthesis. This likely exacerbates the cellular depletion of free amino acids in the hindlimb and simultaneously enhances the resistance to reducing proteolysis that occurs in early life. This disposal of amino acids toward protein synthesis likely influences proteolysis by altering the cellular pool size of the amino acids. It must be emphasized that the previous investigations studied the regulation of whole-body protein degradation in human neonates, and the current study provides the first data specifically concerning the regulation of proteolysis in the hindlimb, a tissue bed composed primarily of skeletal muscle, during the neonatal period.

In summary, the current research showed that the high rate of whole-body protein accretion in response to feeding, accompanied by a postprandial increase in the concentration of insulin and amino acids, is driven by a sensitive response of protein synthesis to feeding and a tightly controlled rate of protein degradation in neonates. Young neonatal piglets tend to exhibit more resistance to a feeding-induced reduction in proteolysis than older neonates, consistent with the requirement for free amino acid availability to sustain a constant and rapid rate of protein remodeling typical of neonates. Interestingly, feeding increased proteolysis in the hindlimb, in contrast to that in the whole body, suggesting that other tissues, such as those in the splanchnic bed, are involved in the reduction in whole-body proteolysis in response to feeding. Together, these mechanisms maintain the high rate of protein turnover in skeletal muscle during food deprivation and the accelerated anabolic utilization of amino acids during feeding. These coordinated responses likely contribute to the efficient use of nutrients for growth and the rapid gain in protein mass in the skeletal muscle of neonates.

	ACKNOWLEDGMENTS

 
Appreciation is extended to M. L. Fiorotto, A. Sunehag, and D. M. Bier for helpful discussions. We thank W. Liu for technical assistance, J. Stubblefield for animal care, and L. Weiser for secretarial assistance.

	FOOTNOTES

 
¹ Funded in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grant R01-AR-44474 (T.A.D.) and the USDA/ARS under Cooperative Agreement no. 58-6250-6-001 (T.A.D.). M.C.T. was a recipient of a post-doctoral fellowship granted by the Natural Sciences and Engineering Research Council of Canada.

³ Abbreviations used: FSR, fractional synthesis rate; GLM, General Linear Models; HL, hindlimb; HLPB, hindlimb protein breakdown; HLPS, hindlimb protein synthesis; IRMS, isotope ratio MS; SRA, specific radioactivity; TCA, trichloroacetic acid.

Manuscript received 15 October 2004. Initial review completed 18 November 2004. Revision accepted 16 March 2005.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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