Abstract
The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, α-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 μmol·kg−1·h−1 for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) and eIF4G, lower (P < 0.04) abundance of the inactive 4E-BP1·eIF4E complex, and greater (P < 0.05) active eIF4G·eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P < 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC.
Introduction
Skeletal muscle protein synthesis is stimulated when the circulating concentration of the branched-chain amino acid, leucine, is increased within the physiological postprandial range (1–3). However, the other branched-chain amino acids, valine and isoleucine, were not able to stimulate protein synthesis in skeletal muscles containing primarily fast-twitch glycolytic fibers or those containing primarily slow-twitch oxidative fibers, as well as cardiac muscle at physiological concentrations (2). These findings suggest that leucine specifically induces the protein synthetic response in skeletal muscle to the postprandial increase in circulating amino acids. Furthermore, we recently reported that the ability of leucine to stimulate protein synthesis in skeletal muscle is dependent on the availability of other essential amino acids (3). Leucine can act as a nutrient signal to initiate translation initiation via phosphorylation (i.e. activation) of the 70-kDa ribosomal protein S6 kinase, ribosomal protein S6, and eukaryotic initiation factor (eIF)10 4E binding protein-1 (4E-BP1), resulting in a reduction of the inactive 4E-BP1·eIF4E complex and a concomitant increase in the formation of the active eIF4G·eIF4E complex, which can be enhanced by phosphorylation of eIF4G within the active complex (1–3). The mechanisms involved in leucine-mediated activation of translation initiation have been reviewed elsewhere (4–6).
Now that leucine is well recognized as a nutrient signal that stimulates protein synthesis in skeletal muscle via the activation of translation initiation factors, it is important to understand the mechanisms by which leucine exerts its anabolic effect. More specifically, we need to discern the effects of leucine itself from the effects of its metabolites on protein synthesis. The first step in leucine catabolism is the reversible transamination of leucine to α-ketoisocaproic acid (KIC), a reaction catalyzed by branched-chain aminotransferase isoenzymes [cytosolic and mitochondrial (BCATm)]. Branched-chain aminotransferase isoenzyme (cytosolic) is mainly found in neural tissue (7), whereas BCATm is expressed in all tissues, except in liver, and skeletal muscle constitutes the main reservoir of BCATm in the body (8). The next step in leucine catabolism is the rate-limiting and irreversible oxidative decarboxylation of KIC to isovaleryl-CoA, a reaction catalyzed by the branched-chain α-keto acid dehydrogenase (BCKD) enzyme complex. The BCKD complex consists of 3 enzymes (i.e. E1, E2, and E3) and is inactivated when BCKD kinase phosphorylates Ser-293 in the E1-α subunit (9,10). In vitro, KIC inhibits BCKD kinase activity, which results in the activation of BCKD (11).
Low-protein diets are regularly used in patients suffering from chronic kidney disease to decrease N intake and reduce the metabolic workload of liver and kidneys (12–14). Supplementation of very low-protein diets with keto acids is now being used to ameliorate the loss of residual renal function and to improve blood metabolites (13,15). Work conducted in cultured adipocytes (16,17) and chronic or supraphysiological administration in adult rats (18,19) have suggested that KIC (the α-keto acid of leucine) and norleucine (an aliphatic leucine analogue that does not charge leucyl-tRNA) can stimulate protein synthesis and/or the activation of translation initiation factors. Because of the potential for KIC or norleucine to serve as a nutritional adjuvant to also maintain muscle mass without increasing N intake in patients suffering from chronic renal disease, we wished to evaluate the effect of physiological doses of leucine, KIC, and norleucine on protein synthesis rates and the activation of indices of translation in skeletal muscle and, for comparison, liver of young pigs.
Methods
Pigs and housing.
Four multiparous crossbred sows (Yorkshire × Landrace × Hampshire × Duroc; Agriculture Headquarters, Texas Department of Criminal Justice, Huntsville, TX) were housed, fed, and managed as previously described (1,2). After birth, piglets resided with the sow and were not given supplemental creep feed. Piglets were studied at 5.8 ± 0.2 d of age weighing 1.9 ± 0.1 kg. Four days before the infusion studies, piglets were anesthetized and indwelling catheters were surgically inserted into the left jugular vein and left carotid artery using sterile techniques as described previously (2). Piglets were returned to the sow after surgery until studied. The Animal Care and Use Committee of Baylor College of Medicine approved all experimental procedures. This study was conducted according to the NRC's Guide for the Care and Use of Laboratory Animals.
Treatments and infusion.
Piglets were feed-deprived for 12–14 h prior to infusion and placed in a sling restraint system. The carotid catheter was used to infuse saline, leucine, KIC, or norleucine, and l[4-3H]phenylalanine into the arch of aorta and the jugular catheter was used to obtain blood samples from the precava vein. Pigs were randomly assigned to 1 of 4 treatments consisting of infusion of saline, leucine, KIC, or norleucine for 60 min. Infusion was initiated with a 10-min primed dose of 148 μmol·kg−1 for leucine, KIC, and norleucine followed by a constant infusion of 400 μmol·kg−1·h−1. During the priming period, saline-infused pigs received an equal volume of saline as those receiving leucine. This infusion protocol has previously increased plasma leucine to within the postprandial physiological range (20,1,2).
Tissue protein synthesis in vivo.
Fractional rates of protein synthesis were measured using a 10 mL·kg−1 body weight flooding dose (1.5 mmol·kg−1 body weight) of l[4-3H]phenylalanine (18.5 MBq·kg−1 body weight; Amersham Biosciences) injected 30 min before ending the infusion as previously described (21). Pigs were killed at the end of the infusion and tissue samples were obtained from the longissimus dorsi, a muscle containing primarily fast-twitch glycolytic fibers, as well as the liver. Tissue samples were collected and immediately frozen in liquid nitrogen and stored at −70°C until analyzed, as previously described (22).
Plasma and tissue free amino acids, plasma branched-chain α-keto acids, and plasma insulin.
Blood samples were collected at 0, 30, and 60 min after the start of the infusion in heparinized tubes, centrifuged at 3000 × g for 1 min at room temperature, and stored at −70°C until analyzed. The concentrations of individual amino acids from frozen plasma samples and tissue homogenates were measured with an HPLC method (PICO-TAG reverse-phase column, Waters) as previously described (20). Plasma α-keto acids were derivatized with o-phenylenediamine, separated using gradient elution from a Spherisorb ODS2 column (250 mm × 4.6 mm, 5 μmol; Waters), and quantified by fluorescence emission at 410 nm, with excitation at 350 nm, as previously described (23). Plasma radioimmunoreactive insulin concentrations were measured using a RIA kit (Linco).
Protein immunoblot analysis.
Fresh tissue samples were homogenized, diluted in sample buffer (24), boiled for 10 min, cooled to room temperature, frozen in liquid nitrogen, and stored at −70°C until protein immunoblot analyses as previously described (2). Aliquots of homogenates were subjected to protein immunoblot analysis using rabbit polyclonal antibodies that recognize total 4E-BP1 (Bethyl Laboratories). Proteins were electrophoretically separated in polyacrylamide gels (24). For each assay, all tissue samples were analyzed together in triple-wide gels (C.B.S. Scientific C.) to eliminate inter-assay variation. Proteins were transferred to a polyvinylidene difluoride membrane (BioRad) and incubated with appropriate antibodies (all from Cell Signaling Technology, unless otherwise indicated). Blots were developed using an enhanced chemiluminescence kit (ECL, Amersham), visualized using ChemiDocIt (UVP), and analyzed with LabWorks Image Acquisition and Analysis Software (UVP). Site-specific phosphorylation and total protein content were determined.
The 4E-BP1·eIF4E and eIF4E·eIF4G complexes were immunoprecipitated overnight at 4°C using an anti-eIF4E monoclonal antibody (25) from aliquots of fresh muscle tissue homogenates as previously described (26,2). Immunoprecipitates were subjected to protein immunoblot analysis, as described above, using a rabbit polyclonal anti-4E-BP1 antibody (Bethyl Laboratories), the aforementioned monoclonal anti-eIF4E antibody, a rabbit polyclonal anti-eIF4G antibody (Novus Biologicals), and a rabbit polyclonal antibody that recognizes site-specific phosphorylation of eIF4G at Ser-1108.
BCKD complex activity.
Tissue extraction and BCKD activity were carried out as described previously (27). BCKD complex was extracted from −70°C frozen muscle and liver samples and then precipitated using 9% polyethylene glycol. BCKD activity was determined by a radioactive assay that measures 14CO2 release from α-keto [1-14C]isovalerate. Total BCKD complex activity was measured after activation of a separate aliquot of the same samples with 2 mmol of MnCl and λ-phosphatase. The activity state of BCKD is expressed as the ratio of actual activity before activation over total activity obtained after activation with phosphatase. A unit of activity was defined as 1 μmol CO2 formed/min at 30°C.
Calculations and statistics.
Fractional rates of protein synthesis (Ks, percentage of protein mass synthesized in a day) were calculated as: Ks (%/d) = [(Sb/Sa) × (1440/t)] × 100, where Sb (dpm·min−1) is the specific radioactivity of the protein-bound phenylalanine, Sa (dpm·min−1) is the specific radioactivity of the tissue free phenylalanine at the time of tissue collection and the linear regression of the blood specific radioactivity of the pig at 5, 15, and 30 min against time, and t is the time of labeling in min.
The pig was considered the experimental unit. ANOVA was performed using the General Linear Model procedure of SAS (release 8.02, SAS Institute) for randomized complete-block design (28) to test the effect of treatment on fractional rates of protein synthesis, the activation of translation initiation factors, tissue free amino acids, plasma branched-chain keto acids, and BCKD activity. An ANOVA for repeated measurements was used to analyze the concentration of plasma amino acids. Least squares means were compared using a t test and Fisher adjustment in SAS. Data are presented as least squared means ± SE.
Results
Plasma and tissue free amino acids, plasma branched-chain keto acids, and plasma insulin.
Separate administration of leucine and KIC increased (P < 0.0001) plasma leucine after 60 min of infusion (Table 1). A higher (P = 0.0001) plasma concentration of leucine was achieved when pigs were infused with leucine compared with KIC. An elevation (P < 0.0001) in plasma leucine was observed as early as 30 min after the initiation of KIC infusion compared with saline controls. Whereas plasma leucine concentration increased from 30 to 60 min (P = 0.0003) of infusion in leucine-treated pigs, the plasma leucine concentration remained unchanged from 30 to 60 min of infusion in KIC-treated pigs. Slope-ratio analysis by orthogonal contrast of the slopes obtained from multiple-linear regression curves of plasma amino acid concentration compared with time of infusion was performed for all treatments. Infusion of leucine linearly increased (P < 0.05) glutamine and threonine in plasma compared with saline infusion. Administration of KIC caused a linear reduction (P < 0.02) in plasma alanine, isoleucine, and tryptophan compared with saline infusion. Norleucine infusion caused a linear reduction (P < 0.04) in plasma glycine and tryptophan and a trend for an increase in plasma threonine (P = 0.08) compared with saline treatment. Compared with saline-infused controls, the plasma concentration of KIC increased (P = 0.0002) with the infusion of leucine and KIC. However, infusion of KIC resulted in a higher (P = 0.005) plasma KIC compared with leucine infusion (Table 2). Infusion of norleucine decreased plasma α-ketoisovalerate (KIV; the α-keto acid of valine; P = 0.05) and α-keto-β-methylvalerate (KMV; the α-keto acid of isoleucine; P = 0.03) concentrations, whereas leucine infusion reduced (P = 0.02) plasma KMV levels compared with saline-infused controls. Plasma insulin concentrations following the infusion of leucine, KIC, and norleucine did not differ from baseline values (Table 1).
TABLE 1.
Plasma concentrations of free amino acids and insulin in young pigs at baseline and after 60 min of infusion with saline, leucine, KIC, or norleucine1
| Treatment |
||||||
|---|---|---|---|---|---|---|
| Item | Baseline2 | Saline | Leucine | KIC | Norleucine | SE |
| μmol/L | ||||||
| Alanine | 331 ± 29a | 325a,b | 215a,b | 180b | 213a,b | 60 |
| Arginine | 83.3 ± 4.6a | 65.1a,b | 66.7a,b | 57.6b | 68.0a,b | 9.3 |
| Asparagine | 86.8 ± 4.4a | 67.5b | 61.1b | 76.8a,b | 76.5a,b | 8.9 |
| Aspartate | 12.7 ± 1.1 | 12.3 | 12.7 | 11.3 | 8.9 | 2.2 |
| Glutamate | 129 ± 13 | 107 | 165 | 134 | 141 | 26 |
| Glutamine | 276 ± 12a,b | 228b | 316a | 259a,b | 254a,b | 23 |
| Glycine | 931 ± 39a | 686b | 844a,b | 765a,b | 887a,b | 77 |
| Histidine | 49.0 ± 3.6a | 29.9b | 30.6b | 36.4a,b | 35.1a,b | 7.1 |
| Isoleucine | 109 ± 6a | 110a | 106a | 48b | 92a | 18 |
| Leucine | 122 ± 7c | 123c | 487a | 319b | 77d | 14 |
| Lysine | 148 ± 9a | 130a,b | 133a,b | 96b | 130a,b | 19 |
| Methionine | 46.0 ± 2.9a | 31.7b | 39.3a,b | 31.0b | 34.6a,b | 6.4 |
| Norleucine | n.d.4 | n.d. | n.d. | n.d. | 558 | 5 |
| Phenylalanine3 | 101 ± 3 | 102 | 90 | 95 | 100 | 6 |
| Serine | 147 ± 7 | 120 | 130 | 127 | 136 | 15 |
| Threonine | 199 ± 14a | 143a,b | 214a | 133b | 183a,b | 31 |
| Tryptophan | 19.3 ± 0.9a | 19.8a | 15.4a,b | 10.5b | 15.0b | 2.0 |
| Tyrosine | 70.4 ± 3.2 | 67.1 | 73.0 | 66.0 | 71.1 | 6.2 |
| Valine | 234 ± 9a | 233a | 233a | 169b | 214a | 20 |
| Insulin, pmol/L | 17.3 ± 0.6 | 17.9 | 16.2 | 17.1 | 16.4 | 1.1 |
Data are means and pooled SEM, n = 7–8 per treatment group unless noted otherwise. Labeled means in a row with superscripts without a common letter differ, P < 0.05.
Data are means ± SE of all pigs before infusion regardless of assigned treatment.
Measured after 30 min of infusion.
n.d., not detected.
TABLE 2.
KIC, KIV, and KMV concentrations in plasma of young pigs after 60 min of infusion with saline, leucine, KIC, or norleucine1
| Treatment |
|||||
|---|---|---|---|---|---|
| α-Keto acid | Saline | Leucine | KIC | Norleucine | SE2 |
| μmol/L | |||||
| KIC | 23.8c | 67.2b | 96.5a | 12.4c | 6.8 |
| KIV | 9.5a | 7.1a,b | 10.3a | 6.0b | 1.2 |
| KMV | 28.1a | 16.8b | 21.4a,b | 17.3b | 3.2 |
Data are means per treatment, n = 7–8. Labeled means in a row without a common letter differ, P < 0.05.
Pooled SE of treatment groups.
Administration of leucine or KIC resulted in a greater (P < 0.0001) free intracellular leucine concentration in longissimus dorsi compared with that in pigs infused with saline or norleucine (Table 3). However, intracellular leucine was higher (P = 0.001) in pigs infused with leucine than those infused with KIC. Treatment with both leucine and KIC induced lower (P < 0.05) intracellular concentrations of several essential and nonessential amino acids than in saline infused controls (Table 3).
TABLE 3.
Free intracellular concentrations of amino acids in skeletal muscle of young pigs after 60 min of infusion with saline, leucine, KIC, or norleucine1
| Treatment |
|||||
|---|---|---|---|---|---|
| Amino acid | Saline | Leucine | KIC | Norleucine | SE2 |
| μmol/L | |||||
| Alanine | 260.9a | 150.7b | 127.8b | 147.3b | 26.1 |
| Arginine | 308.3 | 292.7 | 347.8 | 283.3 | 30.1 |
| Aspartate + asparagine | 173.2a | 131.4b | 144.6a,b | 149.6a,b | 14.2 |
| Glutamate + glutamine | 1,270.1 | 1,364.0 | 1,383.1 | 1,353.5 | 62.3 |
| Glycine | 503.2 | 509.4 | 541.1 | 539.2 | 49.9 |
| Histidine | 12.7a,b | 11.5a,b | 16.7a | 10.5b | 2.0 |
| Isoleucine | 29.1 | 26.5 | 28.7 | 28.9 | 1.8 |
| Leucine | 12.4c | 41.2a | 33.5b | 9.4c | 1.5 |
| Lysine | 14.1a | 8.0b | 6.6b | 14.3a | 1.7 |
| Methionine | 6.0 | 3.7 | 5.3 | 5.5 | 0.8 |
| Norleucine | n.d. | n.d. | n.d. | 42.2 | 1.2 |
| Phenylalanine | 123.1 | 120.4 | 116.0 | 117.8 | 2.7 |
| Proline | 35.3 | 32.6 | 25.5 | 33.5 | 3.8 |
| Serine | 93.2a | 71.9b | 90.9a,b | 77.8a,b | 7.3 |
| Threonine | 158.1a,b | 158.9a,b | 142.0b | 164.4a | 7.9 |
| Tryptophan | 1.96a | 1.30b,c | 1.10c | 1.63a,b | 0.2 |
| Tyrosine | 40.1 | 39.1 | 28.0 | 33.1 | 8.1 |
| Valine | 21.9a | 17.4a,b | 15.0b | 18.6a,b | 1.7 |
Data are means per treatment, n = 7–8. Labeled means in a row with superscripts without a common letter differ, P < 0.05.
Pooled SE of treatment groups.
BCKD activity.
In longissimus dorsi muscle, actual BCKD activity was not detected and total BCKD activity was not affected by treatment (Table 4). The lack of detection of actual BCKD activity precluded the calculation of the activation state in skeletal muscle and suggested that actual activity is very low in this muscle. Actual and total activity in liver was higher (P < 0.0001) compared with muscle for all treatments. Hepatic actual BCKD activity was numerically higher and total activity was numerically lower in all treatments compared with saline-infused pigs. The combination of these numeric differences resulted in a higher (P < 0.02) hepatic activity state for all treatments compared with saline-infused pigs.
TABLE 4.
Activity of BCKD in liver and skeletal muscle of young pigs after 60 min of infusion with saline, leucine, KIC, or norleucine1
| Treatment |
|||||
|---|---|---|---|---|---|
| Tissue | Saline | Leucine | KIC | Norleucine | SE2 |
| Liver | mU/g | ||||
| Actual | 115.0 | 158.5 | 144.7 | 156.7 | 16.6 |
| Total | 197.0 | 160.9 | 144.0 | 176.0 | 23.5 |
| State3 | 67.9b | 103.2a | 100.9a | 99.0a | 8.4 |
| Muscle | |||||
| Actual | n.d. | n.d. | n.d. | n.d. | |
| Total | 7.5 | 6.4 | 6.8 | 6.6 | 1.2 |
| State3 | — | — | — | — | — |
Data are means per treatment, n = 6–8. Labeled means in a row with superscripts without a common letter differ, P < 0.02.
Pooled SE of treatment groups.
Activity state expressed as a percentage of actual activity over total activity.
Translation initiation factors.
In longissimus dorsi muscle, separate infusion of leucine and KIC equally stimulated the phosphorylation of the γ-form of 4E-BP1 (P < 0.006) compared with saline-infused pigs (Fig. 1A). This higher phosphorylation of 4E-BP1 was associated with a reduction (P < 0.04) in the inactive 4E-BP1·eIF4E complex (Fig. 1B) and concomitant increases in the active eIF4G·eIF4E complex (P < 0.05; Fig. 2A) and phosphorylation of eIF4G at Ser-1108 in the active eIF4G·eIF4E complex (P < 0.03; Fig. 2B) in both leucine- and KIC-infused pigs compared with saline controls. Administration of norleucine did not alter in muscle the phosphorylation of the γ-form of 4E-BP1, inactive 4E-BP1·eIF4E complex abundance, active eIF4G·eIF4E complex formation, or the phosphorylation of eIF4G within the active eIF4G·eIF4E complex compared with control saline-infused pigs. In the case of the liver, there was no effect of treatment on the phosphorylation of the γ-form of 4E-BP1, the abundance of the inactive 4E-BP1·eIF4E complex and the active eIF4G·eIF4E complex, or the phosphorylation of eIF4G at Ser-1108 within the active eIF4G·eIF4E complex (Supplemental Figs. 1 and 2).
FIGURE 1 .
Phosphorylation of 4E-BP1, percent of γ-form (A), and association of 4E-BP1 with eIF4E (B) in skeletal muscle of young pigs after 60 min of infusion with saline (Sal), leucine (Leu), KIC, or norleucine (Nleu). Values are means ± pooled SE, n = 7–8 per treatment group. Within each graph, means without a common letter differ, P < 0.04.
FIGURE 2 .
Association of eIF4G with eIF4E (A) and phosphorylation of eIF4G at Ser-1108 (P-Ser-1108) associated with eIF4E (B) in skeletal muscle of young pigs after 60 min of infusion with saline (Sal), leucine (Leu), KIC, or norleucine (Nleu). Values are means ± pooled SE, n = 7–8 per treatment group. Within each graph, means without a common letter differ, P < 0.05.
Protein synthesis.
Infusion of leucine or KIC resulted in a greater (P < 0.003) global fractional protein synthesis rate in longissimus dorsi muscle compared with that in saline-infused pigs (Fig. 3). Skeletal muscle protein synthesis rates did not differ in KIC- and leucine-infused pigs. Conversely, administration of norleucine had no effect on skeletal muscle protein synthesis compared with saline controls. Global fractional protein synthesis rates in liver were not affected by treatment (Supplemental Fig. 3).
FIGURE 3 .
Fractional rates of protein synthesis in skeletal muscle of young pigs after 60 min of infusion with saline (Sal), leucine (Leu), KIC, or norleucine (Nleu). Values are means ± pooled SE, n = 7–8 per treatment group. Within each graph, means without a common letter differ, P < 0.004.
Discussion
After a meal, the postprandial rise in insulin and amino acids independently stimulates protein synthesis in skeletal muscle (29–31). In the case of amino acids, we have previously demonstrated in young pigs that the branched-chain amino acid, leucine, and not isoleucine or valine, can stimulate protein synthesis in muscles containing primarily fast-twitch glycolytic fibers and those containing primarily slow-twitch oxidative fibers (1–3). These findings suggest that leucine specifically induces the protein synthetic response in skeletal muscle to the postprandial increase in circulating amino acids. Therefore, in the current study, we examined the ability of the leucine metabolite, KIC, and analogue, norleucine, to stimulate skeletal muscle protein synthesis in growing pigs.
Infusion of a primed dose followed by a constant infusion of 400 μmol·kg−1·h−1 of leucine for 60 min resulted in an elevation in plasma leucine within the postprandial range [i.e. 2- to 4-fold of food-deprived levels (20)], which is in agreement with previous reports from our group (1–3). Plasma concentrations of essential amino acids did not change, consistent with our previous reports of minimal changes in plasma concentrations of essential amino acids during a short-term (i.e. 60-min) leucine infusion (1,3). Free intracellular concentrations of lysine and tryptophan in longissimus dorsi muscle fell, however. The elevation of KIC in plasma of leucine-infused pigs suggests that BCATm catalyzed transamination initially exceeds oxidative capacity in muscle and other tissues. In addition, the elevation in the plasma concentration of leucine 30 min after the start of the KIC infusion indicates a rapid transamination of KIC into leucine by BCATm. The rise in KIC in plasma is likely coming from skeletal muscle where the low BCKD activity may promote KIC release over oxidation. Note that pig liver has a 20-fold greater oxidative capacity than skeletal muscle, which is very similar to what is observed in rodents (8). Furthermore, the rise in KIC did not increase skeletal muscle BCKD activity. Contrary to the minimal changes in essential amino acids observed during leucine treatment, infusion of KIC reduced the plasma concentration of the essential amino acids, isoleucine, tryptophan, and valine, and the nonessential amino acid, alanine, compared with saline infusion. Moreover, free intracellular concentrations of lysine, tryptophan, valine, and alanine in longissimus dorsi muscle also declined. According to our previous findings, we do not expect an impairment in protein synthesis until plasma concentrations of all essential amino acids drop to ∼50% of overnight feed-deprived levels, which was not the case in this study (3). Infusion of norleucine resulted in an increase in its plasma and skeletal muscle concentrations. Collectively, results from plasma and tissue free intracellular amino acids, and plasma branched-chain α-keto acids indicate that short-term treatment with KIC results in faster and more profound changes in the amino acid pool compared with leucine infusion. Thus, muscle transamination possibly explains significant reductions in plasma urea reported in patients suffering from chronic kidney disease undergoing dialysis and consuming a very low-protein diet supplemented with keto acids (15,12). Furthermore, administration of KIC was more efficient than leucine in reducing plasma amino acid concentrations, which is interpreted to suggest increased amino acid incorporation into muscle protein and likely lower levels of plasma urea.
In this study, a physiological increase in plasma leucine, as a result of infusion of either leucine or KIC, resulted in the phosphorylation of 4E-BP1 with a concomitant reduction in the inactive 4E-BP1·eIF4E complex and increases in the active eIF4G·eIF4E complex and the phosphorylation of eIF4G within the active complex in skeletal muscle. These changes in translation initiation activation are in agreement with our previous reports (1–3,32) and can be interpreted to suggest that independent administration of leucine and KIC fully activated this arm of translation initiation in skeletal muscle but not in liver. Furthermore, the activation of 4E-BP1 and the reduction of the inactive 4E-BP1·eIF4E complex in skeletal muscle were similar between leucine and KIC treatments despite the lower free intracellular concentration of KIC-derived leucine. In a previous study conducted with feed-deprived rats, an oral gavage of KIC resulted in a 5.4-fold increase in plasma leucine and concomitant phosphorylation of 4E-BP1 in skeletal muscle without affecting its phosphorylation state in liver (19). Similarly, administration of KIC to cultured adipocytes resulted in an increase in 4E-BP1 phosphorylation (16). The effects of leucine and KIC on the phosphorylation of 4E-BP1, the inactive 4E-BP1·eIF4E complex, the active eIF4G·eIF4E complex, and phosphorylation of eIF4G within the active eIF4G·eIF4E complex observed in this study mirror the effects of growth factors, such as insulin and IGF-I, on the mammalian target of rapamycin signaling leading to protein synthesis (2,4,3,5). Results from this study and others (16,19) confirm the ability of KIC to activate translation and protein synthesis initiation in an apparent mTOR-mediated mechanism. Furthermore, the stimulatory effects of leucine and KIC on muscle protein synthesis appear to be independent of insulin. Our short-term, equimolar, and parenteral administration of norleucine, compared with leucine, did not stimulate the activation of translation initiation factors in skeletal muscle despite the increase in free intracellular norleucine in skeletal muscle. These results are in contrast to those obtained in cultured adipocytes (16) and skeletal muscle of growing rats (33), where large doses of norleucine markedly increased the phosphorylation of 4E-BP1. Differing results among these studies may reflect the different analogue concentrations achieved.
In neonates, skeletal muscle protein synthesis is highly responsive to anabolic stimuli such as feeding, amino acids, insulin, and leucine (22,29,2). Results from this work indicate that leucine nearly doubled the fractional rates of skeletal muscle protein synthesis compared with controls but had no effect in the liver. More importantly, the protein synthetic response of skeletal muscle to KIC did not differ from that obtained with leucine, despite the lower plasma (65.5%) and intracellular (81.3%) concentrations of leucine obtained with KIC treatment compared with leucine infusion. Thus, KIC could be used as an efficient nitrogen-free stimulator of muscle protein synthesis in patients affected with chronic illnesses who are consuming low-protein diets. However, this potential therapeutic use must be thoroughly evaluated, because the efficiency of KIC to replace dietary leucine has been previously reported to be highly dependent on dietary protein concentration (34). Because KIC is freely transaminated to leucine, this process could be a significant contributor to the reductions in plasma urea reported in patients suffering from chronic kidney disease consuming a very low-protein diet supplemented with keto acids (15,12). In addition to the potential muscle protein effect, inclusion of keto acids in low-protein diets have significantly contributed to the maintenance of residual renal function in patients undergoing dialysis (15,13).
The results from this study demonstrate the ability of KIC to stimulate skeletal muscle protein synthesis in young pigs to levels comparable to leucine infusion (1–3), parenteral administration of a complete amino acid solution (30), or meal feeding (35). Furthermore, KIC increased protein synthesis with significantly lower plasma and intracellular concentrations of leucine. In conclusion, KIC can potentially be used to stimulate muscle protein synthesis when parenteral administration of nitrogenous compounds may not be advisable.
Supplementary Material
Acknowledgments
We thank J. Fleming for technical assistance, J. C. Stubblefield for care of animals, and L. F. Weiser for secretarial assistance. J.E., A.S., S.M.H., and T.A.D. designed the research; J.E., J.W.F., A.S., H.V.N., and C.G.H. conducted the research; J.E., A.S., S.M.H., and T.A.D. analyzed the data; J.E., A.S., S.M.H., and T.A.D. wrote the paper; and T.A.D. had primary responsibility for the final content. All authors read and approved the final manuscript.
Supported in part by NIH grants AR-44474 (T.A. Davis) and by the USDA/Agricultural Research Service (USDA/ARS) under Cooperative Agreement no. 6250510000-33 (T.A. Davis). This research was also supported in part by NIH Training grant T32 HD-07445. This work is a publication of the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine. The contents of this publication do not necessarily reflect the views or politics of the USDA, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
Author disclosures: J. Escobar, J. W. Frank, A. Suryawan, H. V. Nguyen, C. G. Van Horn, S. M. Hutson, and T. D. Davis, no conflicts of interest.
Supplemental Figures 1–3 are available with the online posting of this paper at jn.nutrition.org.
Abbreviations used: BCATm, branched-chain aminotransferase isoenzymes (mitochondrial); BCKD, branched-chain α-keto acid dehydrogenase; 4E-BP1, 4E binding protein-1; eIF, eukaryotic initiation factor; KIC, α-ketoisocaproic acid; KIV, α-ketoisovalerate; KMV, α-keto-β-methylvalerate.
References
- 1.Escobar J, Frank JW, Suryawan A, Nguyen HV, Kimball SR, Jefferson LS, Davis TA. Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation. Am J Physiol Endocrinol Metab. 2005;288:E914–21. [DOI] [PubMed] [Google Scholar]
- 2.Escobar J, Frank JW, Suryawan A, Nguyen HV, Kimball SR, Jefferson LS, Davis TA. Regulation of cardiac and skeletal muscle protein synthesis by individual branched-chain amino acids in neonatal pigs. Am J Physiol Endocrinol Metab. 2006;290:E612–21. [DOI] [PubMed] [Google Scholar]
- 3.Escobar J, Frank JW, Suryawan A, Nguyen HV, Davis TA. Amino acid availability and age affect the leucine stimulation of protein synthesis and eIF4F formation in muscle. Am J Physiol Endocrinol Metab. 2007;293:E1615–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Kimball SR, Jefferson LS. Signaling pathways and molecular mechanisms through which branched-chain amino acids mediate translational control of protein synthesis. J Nutr. 2006;136:S227–31. [DOI] [PubMed] [Google Scholar]
- 5.Vary TC, Lynch CJ. Nutrient signaling components controlling protein synthesis in striated muscle. J Nutr. 2007;137:1835–43. [DOI] [PubMed] [Google Scholar]
- 6.Proud CG. Amino acids and mTOR signalling in anabolic function. Biochem Soc Trans. 2007;35:1187–90. [DOI] [PubMed] [Google Scholar]
- 7.Hutson SM, Fenstermacher D, Mahar C. Role of mitochondrial transamination in branched chain amino acid metabolism. J Biol Chem. 1988;263:3618–25. [PubMed] [Google Scholar]
- 8.Suryawan A, Hawes JW, Harris RA, Shimomura Y, Jenkins AE, Hutson SM. A molecular model of human branched-chain amino acid metabolism. Am J Clin Nutr. 1998;68:72–81. [DOI] [PubMed] [Google Scholar]
- 9.Harris RA, Paxton R, Powell SM, Goodwin GW, Kuntz MJ, Han AC. Regulation of branched-chain alpha-ketoacid dehydrogenase complex by covalent modification. Adv Enzyme Regul. 1986;25:219–37. [DOI] [PubMed] [Google Scholar]
- 10.Lynch CJ, Halle B, Fujii H, Vary TC, Wallin R, Damuni Z, Hutson SM. Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR. Am J Physiol Endocrinol Metab. 2003;285:E854–63. [DOI] [PubMed] [Google Scholar]
- 11.Lau KS, Fatania HR, Randle PJ. Regulation of the branched chain 2-oxoacid dehydrogenase kinase reaction. FEBS Lett. 1982;144:57–62. [DOI] [PubMed] [Google Scholar]
- 12.Mircescu G, Garneata L, Stancu SH, Capusa C. Effects of a supplemented hypoproteic diet in chronic kidney disease. J Ren Nutr. 2007;17:179–88. [DOI] [PubMed] [Google Scholar]
- 13.Jiang N, Qian J, Sun W, Lin A, Cao L, Wang Q, Ni Z, Wan Y, Linholm B, et al. Better preservation of residual renal function in peritoneal dialysis patients treated with a low-protein diet supplemented with keto acids: a prospective, randomized trial. Nephrol Dial Transplant. 2009;24:2551–8. [DOI] [PubMed] [Google Scholar]
- 14.Chauveau P, Couzi L, Vendrely B, de Précigout V, Combe C, Fouque D, Aparicio M. Long-term outcome on renal replacement therapy in patients who previously received a keto acid-supplemented very-low-protein diet. Am J Clin Nutr. 2009;90:969–74. [DOI] [PubMed] [Google Scholar]
- 15.Feiten SF, Draibe SA, Watanabe R, Duenhas MR, Baxmann AC, Nerbass FB, Cuppari L. Short-term effects of a very-low-protein diet supplemented with ketoacids in nondialyzed chronic kidney disease patients. Eur J Clin Nutr. 2005;59:129–36. [DOI] [PubMed] [Google Scholar]
- 16.Lynch CJ, Fox HL, Vary TC, Jefferson LS, Kimball SR. Regulation of amino acid-sensitive TOR signaling by leucine analogues in adipocytes. J Cell Biochem. 2000;77:234–51. [DOI] [PubMed] [Google Scholar]
- 17.Lynch CJ. Role of leucine in the regulation of mTOR by amino acids: revelations from structure-activity studies. J Nutr. 2001;131:S861–5. [DOI] [PubMed] [Google Scholar]
- 18.Lynch CJ, Hutson SM, Patson BJ, Vaval A, Vary TC. Tissue-specific effects of chronic dietary leucine and norleucine supplementation on protein synthesis in rats. Am J Physiol Endocrinol Metab. 2002;283:E824–35. [DOI] [PubMed] [Google Scholar]
- 19.Yoshizawa F, Sekizawa H, Hirayama S, Yamazaki Y, Nagasawa T, Sugahara K. Tissue-specific regulation of 4E–BP1 and S6K1 phosphorylation by alpha-ketoisocaproate. J Nutr Sci Vitaminol (Tokyo). 2004;50:56–60. [DOI] [PubMed] [Google Scholar]
- 20.Burrin DG, Davis TA, Ebner S, Schoknecht PA, Fiorotto ML, Reeds PJ, McAvoy S. Nutrient-independent and nutrient-dependent factors stimulate protein synthesis in colostrum-fed newborn pigs. Pediatr Res. 1995;37:593–9. [DOI] [PubMed] [Google Scholar]
- 21.Garlick PJ, McNurlan MA, Preedy VR. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine. Biochem J. 1980;192:719–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Davis TA, Fiorotto ML, Nguyen HV, Reeds PJ. Protein turnover in skeletal muscle of suckling rats. Am J Physiol. 1989;257:R1141–6. [DOI] [PubMed] [Google Scholar]
- 23.Pailla K, Blonde-Cynober F, Aussel C, De Bandt JP, Cynober L. Branched-chain keto-acids and pyruvate in blood: measurement by HPLC with fluorimetric detection and changes in older subjects. Clin Chem. 2000;46:848–53. [PubMed] [Google Scholar]
- 24.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5. [DOI] [PubMed] [Google Scholar]
- 25.Kimball SR, Horetsky RL, Jefferson LS. Signal transduction pathways involved in the regulation of protein synthesis by insulin in L6 myoblasts. Am J Physiol. 1998;274:C221–8. [DOI] [PubMed] [Google Scholar]
- 26.Lin TA, Kong X, Haystead TA, Pause A, Belsham G, Sonenberg N, Lawrence JC Jr. PHAS-I as a link between mitogen-activated protein kinase and translation initiation. Science. 1994;266:653–6. [DOI] [PubMed] [Google Scholar]
- 27.Nakai N, Kobayashi R, Popov KM, Harris RA, Shimomura Y. Determination of branched-chain alpha-keto acid dehydrogenase activity state and branched-chain alpha-keto acid dehydrogenase kinase activity and protein in mammalian tissues. Methods Enzymol. 2000;324:48–62. [DOI] [PubMed] [Google Scholar]
- 28.Kaps M, Lamberson WR. Biostatistics for animal science. Cambridge (MA): CABI Publishing; 2004.
- 29.Davis TA, Fiorotto ML, Burrin DG, Reeds PJ, Nguyen HV, Beckett PR, Vann RC, O'Connor PM. Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs. Am J Physiol Endocrinol Metab. 2002;282:E880–90. [DOI] [PubMed] [Google Scholar]
- 30.O'Connor PM, Bush JA, Suryawan A, Nguyen HV, Davis TA. Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs. Am J Physiol Endocrinol Metab. 2003. a;284:E110–9. [DOI] [PubMed] [Google Scholar]
- 31.O'Connor PM, Kimball SR, Suryawan A, Bush JA, Nguyen HV, Jefferson LS, Davis TA. Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs. Am J Physiol Endocrinol Metab. 2003. b;285:E40–53. [DOI] [PubMed] [Google Scholar]
- 32.Suryawan A, Jeyapalan AS, Orellana RA, Wilson FA, Nguyen HV, Davis TA. Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation. Am J Physiol Endocrinol Metab. 2008;295:E868–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Lynch CJ, Patson BJ, Anthony J, Vaval A, Jefferson LS, Vary TC. Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue. Am J Physiol Endocrinol Metab. 2002;283:E503–13. [DOI] [PubMed] [Google Scholar]
- 34.Kang CW, Tungsanga K, Walser M. Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis. Am J Clin Nutr. 1986;43:504–9. [DOI] [PubMed] [Google Scholar]
- 35.Davis TA, Fiorotto ML, Nguyen HV, Reeds PJ. Enhanced response of muscle protein synthesis and plasma insulin to food intake in suckled rats. Am J Physiol. 1993;265:R334–40. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.