Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.     

Deptor knockdown enhances mTOR Activity and protein synthesis in myocytes and ameliorates disuse muscle atrophy

Deptor is an mTOR binding protein that affects cell metabolism. We hypothesized that knockdown (KD) of Deptor in C2C12 myocytes will increase protein synthesis via stimulating mTOR-S6K1 signaling. Deptor KD was achieved using lentiviral particles containing short hairpin (sh)RNA targeting the mouse Deptor mRNA sequence, and control cells were transfected with a scrambled control shRNA. KD reduced Deptor mRNA and protein content by 90%, which increased phosphorylation of mTOR kinase substrates, 4E-BP1 and S6K1, and concomitantly increased protein synthesis. Deptor KD myoblasts were both larger in diameter and exhibited an increased mean cell volume. Deptor KD increased the percentage of cells in the S phase, coincident with an increased phosphorylation (S807/S811) of retinoblastoma protein (pRb) that is critical for the G(1) to S phase transition. Deptor KD did not appear to alter basal apoptosis or autophagy, as evidenced by the lack of change for cleaved caspase-3 and light chain (LC)3B, respectively. Deptor KD increased proliferation rate and enhanced myotube formation. Finally, in vivo Deptor KD (~50% reduction) by electroporation into gastrocnemius of C57/BL6 mice did not alter weight or protein synthesis in control muscle. However, Deptor KD prevented atrophy produced by 3 d of hindlimb immobilization, at least in part by increasing protein synthesis. Thus, our data support the hypothesis that Deptor is an important regulator of protein metabolism in myocytes and demonstrate that decreasing Deptor expression in vivo is sufficient to ameliorate muscle atrophy.     

Amino acids and insulin are regulators of muscle protein synthesis in neonatal pigs

The stage of development between birth and weaning in mammals is a period of very rapid growth that is crucial for the long-term well-being of the animal. The rate of protein deposition in neonatal animals is very high because dietary protein is efficiently utilized to increase body protein mass. Our studies in neonatal pigs have shown that this high efficiency of protein deposition is largely due to the marked increase in protein synthesis after feeding, and this response is particularly profound in the skeletal muscle. The enhanced stimulation of muscle protein synthesis in neonates after feeding is independently mediated by the rise in insulin and amino acids and this response declines with age. Intracellular signaling components that respond to the postprandial rise in amino acids and insulin have been identified and their activation has been shown to be elevated in skeletal muscle of neonatal pigs after a meal and to decrease with development. The enhanced activation of these components in the amino acid and insulin signaling pathways in neonatal muscle contributes to the high rate of muscle protein synthesis and rapid gain in skeletal muscle mass in newborn pigs, which are essential determinants of efficient growth during development.     

Glutamine supplementation promotes anaplerosis but not oxidative energy delivery in human skeletal muscle

The aims of the present study were twofold: first to investigate whether TCA cycle intermediate (TCAI) pool expansion at the onset of moderate-intensity exercise in human skeletal muscle could be enhanced independently of pyruvate availability by ingestion of glutamine or ornithine alpha-ketoglutarate, and second, if it was, whether this modification of TCAI pool expansion had any effect on oxidative energy status during subsequent exercise. Seven males cycled for 10 min at approximately 70% maximal O2) uptake 1 h after consuming either an artificially sweetened placebo (5 ml/kg body wt solution, CON), 0.125 g/kg body wt L-(+)-ornithine alpha-ketoglutarate dissolved in 5 ml/kg body wt solution (OKG), or 0.125 g/kg body wt L-glutamine dissolved in 5 ml/kg body wt solution (GLN). Vastus lateralis muscle was biopsied 1 h postsupplement and after 10 min of exercise. The sum of four measured TCAI (SigmaTCAI; citrate, malate, fumarate, and succinate, approximately 85% of total TCAI pool) was not different between conditions 1 h postsupplement. However, after 10 min of exercise, SigmaTCAI (mmol/kg dry muscle) was greater in the GLN condition (4.90 +/- 0.61) than in the CON condition (3.74 +/- 0.38, P < 0.05) and the OKG condition (3.85 +/- 0.28). After 10 min of exercise, muscle phosphocreatine (PCr) content was significantly reduced (P < 0.05) in all conditions, but there was no significant difference between conditions. We conclude that the ingestion of glutamine increased TCAI pool size after 10 min of exercise most probably because of the entry of glutamine carbon at the level of alpha-ketoglutarate. However, this increased expansion in the TCAI pool did not appear to increase oxidative energy production, because there was no sparing of PCr during exercise.     

Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation

Protein synthesis in skeletal muscle of adult rats increases in response to oral gavage of supraphysiological doses of leucine. However, the effect on protein synthesis of a physiological rise in plasma leucine has not been investigated in neonates, an anabolic population highly sensitive to amino acids and insulin. Therefore, in the current study, fasted pigs were infused intra-arterially with leucine (0, 200, or 400 micromol.kg(-1).h(-1)), and protein synthesis was measured after 60 or 120 min. Protein synthesis was increased in muscle, but not in liver, at 60 min. At 120 min, however, protein synthesis returned to baseline levels in muscle but was reduced below baseline values in liver. The increase in protein synthesis in muscle was associated with increased plasma leucine of 1.5- to 3-fold and no change in plasma insulin. Leucine infusion for 120 min reduced plasma essential amino acid levels. Phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein-1 (4E-BP1), ribosomal protein (rp) S6 kinase, and rpS6 was increased, and the amount of eIF4E associated with its repressor 4E-BP1 was reduced after 60 and 120 min of leucine infusion. No change in these biomarkers of mRNA translation was observed in liver. Thus a physiological increase in plasma leucine stimulates protein synthesis in skeletal muscle of neonatal pigs in association with increased eIF4E availability for eIF4F assembly. This response appears to be insulin independent, substrate dependent, and tissue specific. The results suggest that the branched-chain amino acid leucine can act as a nutrient signal to stimulate protein synthesis in skeletal muscle of neonates.     

Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs

Infusion of physiological levels of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates. To determine whether insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonates, insulin secretion was blocked with somatostatin in fasted 7-day-old pigs (n = 8-12/group) while glucose and glucagon were maintained at fasting levels and insulin was infused to simulate either less than fasting, fasting, intermediate, or fed insulin levels. At each dose of insulin, amino acids were clamped at either the fasting or fed level; at the highest insulin dose, amino acids were also reduced to less than fasting levels. Skeletal muscle protein synthesis was measured using a flooding dose of l-[4-(3)H]phenylalanine. Hyperinsulinemia increased protein synthesis in skeletal muscle during hypoaminoacidemia and euaminoacidemia. Hyperaminoacidemia increased muscle protein synthesis during hypoinsulinemia and euinsulinemia. There was a dose-response effect of both insulin and amino acids on muscle protein synthesis. At each insulin dose, hyperaminoacidemia increased muscle protein synthesis. The effects of insulin and amino acids on muscle protein synthesis were largely additive until maximal rates of protein synthesis were achieved. Amino acids enhanced basal protein synthesis rates but did not enhance the sensitivity or responsiveness of muscle protein synthesis to insulin. The results suggest that insulin and amino acids independently stimulate protein synthesis in skeletal muscle of the neonate.     

Leucine or carbohydrate supplementation reduces AMPK and eEF2 phosphorylation and extends postprandial muscle protein synthesis in rats

Muscle protein synthesis (MPS) increases after consumption of a protein-containing meal but returns to baseline values within 3 h despite continued elevations of plasma amino acids and mammalian target of rapamycin (mTORC1) signaling. This study evaluated the potential for supplemental leucine (Leu), carbohydrates (CHO), or both to prolong elevated MPS after a meal. Male Sprague-Dawley rats (～270 g) trained to consume three meals daily were food deprived for 12 h, and then blood and gastrocnemius muscle were collected 0, 90, or 180 min after a standard 4-g test meal (20% whey protein). At 135 min postmeal, rats were orally administered 2.63 g of CHO, 270 mg of Leu, both, or water (sham control). Following test meal consumption, MPS peaked at 90 min and then returned to basal (time 0) rates at 180 min, although ribosomal protein S6 kinase and eIF4E-binding protein-1 phosphorylation remained elevated. In contrast, rats administered Leu and/or CHO supplements at 135 min postmeal maintained peak MPS through 180 min. MPS was inversely associated with the phosphorylation states of translation elongation factor 2, the "cellular energy sensor" adenosine monophosphate-activated protein kinase-α (AMPKα) and its substrate acetyl-CoA carboxylase, and increases in the ratio of AMP/ATP. We conclude that the incongruity between MPS and mTORC1 at 180 min reflects a block in translation elongation due to reduced cellular energy. Administering Leu or CHO supplements ～2 h after a meal maintains cellular energy status and extends the postprandial duration of MPS.     

Dietary protein and lactose increase translation initiation factor activation and tissue protein synthesis in neonatal pigs

Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk diets containing three levels of protein (5, 15, and 25 g x kg body wt(-1) x day(-1)) and two levels of lactose (low = 11 and high = 23 g x kg body wt(-1) x day(-1)) from 1 to 6 days of age. On day 7, pigs were gavage fed after 4-h food deprivation, and tissue protein synthesis rates and biomarkers of mRNA translation were assessed. Piglet growth and protein synthesis rates in muscle and liver increased with dietary protein and plateaued at 15 g x kg body wt(-1) x day(-1) (P < 0.001). Growth tended to be greater in high-lactose-fed pigs (P = 0.07). Plasma insulin was lowest in pigs fed 5 g x kg body wt(-1) x day(-1) protein (P < 0.0001). Plasma branched-chain amino acids increased as protein intake increased (P < 0.0001). Muscle (P < 0.001) and liver (P < or = 0.001) ribosomal protein S6 kinase-1 and eIF4E-binding protein phosphorylation increased with protein intake and plateaued at 15 g x kg body wt(-1) x day(-1). The results indicate that growth and protein synthesis rates in neonatal pigs are influenced by dietary protein and lactose intake and might be mediated by plasma amino acids and insulin levels. However, feeding protein well above the piglet's requirement does not further stimulate the activation of translation initiation or protein synthesis in skeletal muscle and liver.     

Acute IGF-I infusion stimulates protein synthesis in skeletal muscle and other tissues of neonatal pigs

Studies have shown that protein synthesis in skeletal muscle of neonatal pigs is uniquely sensitive to a physiological rise in both insulin and amino acids. Protein synthesis in cardiac muscle, skin, and spleen is responsive to insulin but not amino acid stimulation, whereas in the liver, protein synthesis responds to amino acids but not insulin. To determine the response of protein synthesis to insulin-like growth factor I (IGF-I) in this model, overnight-fasted 7- and 26-day-old pigs were infused with IGF-I (0, 20, or 50 microg. kg(-1). h(-1)) to achieve levels within the physiological range, while amino acids and glucose were clamped at fasting levels. Because IGF-I infusion lowers circulating insulin levels, an additional group of high-dose IGF-I-infused pigs was also provided replacement insulin (10 ng. kg(-0.66). min(-1)). Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, low-dose IGF-I increased protein synthesis by 25-60% in various skeletal muscles as well as in cardiac muscle (+38%), skin (+24%), and spleen (+32%). The higher dose of IGF-I elicited no further increase in protein synthesis above that found with the low IGF-I dose. Insulin replacement did not alter the response of protein synthesis to IGF-I in any tissue. The IGF-I-induced increases in tissue protein synthesis decreased with development. IGF-I infusion, with or without insulin replacement, had no effect on protein synthesis in liver, jejunum, pancreas, or kidney. Thus the magnitude, tissue specificity, and developmental change in the response of protein synthesis to acute physiological increases in plasma IGF-I are similar to those previously observed for insulin. This study provides in vivo data indicating that circulating IGF-I and insulin act on the same signaling components to stimulate protein synthesis and that this response is highly sensitive to stimulation in skeletal muscle of the neonate.     

Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process

Protein synthesis is repressed in both skeletal muscle and liver after a short-term fast and is rapidly stimulated in response to feeding. Previous studies in rats and pigs have shown that the feeding-induced stimulation of protein synthesis is associated with activation of the 70-kDa ribosomal protein S6 kinase (S6K1) as well as enhanced binding of eukaryotic initiation factor eIF4E to eIF4G to form the active eIF4F complex. In cells in culture, hormones and nutrients regulate both of these events through a protein kinase termed the mammalian target of rapamycin (mTOR). In the present study, the involvement of mTOR in the feeding-induced stimulation of protein synthesis in skeletal muscle and liver was examined. Pigs at 7 days of age were fasted for 18 h, and then one-half of the animals were fed. In addition, one-half of the animals in each group were administered rapamycin (0.75 mg/kg) 2 h before feeding. The results reveal that treating 18-h fasted pigs with rapamycin, a specific inhibitor of mTOR, before feeding prevented the activation of S6K1 and the changes in eIF4F complex formation observed in skeletal muscle and liver after feeding. Rapamycin also ablated the feeding-induced stimulation of protein synthesis in liver. In contrast, in skeletal muscle, rapamycin attenuated, but did not prevent, the stimulation of protein synthesis in response to feeding. The results suggest that feeding stimulates hepatic protein synthesis through an mTOR-dependent process involving enhanced eIF4F complex formation and activation of S6K1. However, in skeletal muscle, these two processes may account for only part of the stimulation of protein synthesis, and thus additional steps may be involved in the response.     

Regulation of cardiac and skeletal muscle protein synthesis by individual branched-chain amino acids in neonatal pigs

Skeletal muscle grows at a very rapid rate in the neonatal pig, due in part to an enhanced sensitivity of protein synthesis to the postprandial rise in amino acids. An increase in leucine alone stimulates protein synthesis in skeletal muscle of the neonatal pig; however, the effect of isoleucine and valine has not been investigated in this experimental model. The left ventricular wall of the heart grows faster than the right ventricular wall during the first 10 days of postnatal life in the pig. Therefore, the effects of individual BCAA on protein synthesis in individual skeletal muscles and in the left and right ventricular walls were examined. Fasted pigs were infused with 0 or 400 micromol x kg(-1) x h(-1) leucine, isoleucine, or valine to raise individual BCAA to fed levels. Fractional rates of protein synthesis and indexes of translation initiation were measured after 60 min. Infusion of leucine increased (P < 0.05) phosphorylation of eukaryotic initiation factor (eIF)4E-binding protein-1 and increased (P < 0.05) the amount and phosphorylation of eIF4G associated with eIF4E in longissimus dorsi and masseter muscles and in both ventricular walls. Leucine increased (P < 0.05) the phosphorylation of ribosomal protein (rp)S6 kinase and rpS6 in longissimus dorsi and masseter but not in either ventricular wall. Leucine stimulated (P < 0.05) protein synthesis in longissimus dorsi, masseter, and the left ventricular wall. Isoleucine and valine did not increase translation initiation factor activation or protein synthesis rates in skeletal or cardiac muscles. The results suggest that the postprandial rise in leucine, but not isoleucine or valine, acts as a nutrient signal to stimulate protein synthesis in cardiac and skeletal muscles of neonates by increasing eIF4E availability for eIF4F complex assembly.     

Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mTOR-independent process

Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eIF4E.eIF4G complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eEF2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mTOR, S6K1, and 4E-BP1 and enhanced eIF4E.eIF4G formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mTOR-independent pathways.     

Leucine-enriched essential amino acid and carbohydrate ingestion following resistance exercise enhances mTOR signaling and protein synthesis in human muscle

We recently showed that resistance exercise and ingestion of essential amino acids with carbohydrate (EAA+CHO) can independently stimulate mammalian target of rapamycin (mTOR) signaling and muscle protein synthesis in humans. Providing an EAA+CHO solution postexercise can further increase muscle protein synthesis. Therefore, we hypothesized that enhanced mTOR signaling might be responsible for the greater muscle protein synthesis when leucine-enriched EAA+CHOs are ingested during postexercise recovery. Sixteen male subjects were randomized to one of two groups (control or EAA+CHO). The EAA+CHO group ingested the nutrient solution 1 h after resistance exercise. mTOR signaling was assessed by immunoblotting from repeated muscle biopsy samples. Mixed muscle fractional synthetic rate (FSR) was measured using stable isotope techniques. Muscle protein synthesis and 4E-BP1 phosphorylation during exercise were significantly reduced (P < 0.05). Postexercise FSR was elevated above baseline in both groups at 1 h but was even further elevated in the EAA+CHO group at 2 h postexercise (P < 0.05). Increased FSR was associated with enhanced phosphorylation of mTOR and S6K1 (P < 0.05). Akt phosphorylation was elevated at 1 h and returned to baseline by 2 h in the control group, but it remained elevated in the EAA+CHO group (P < 0.05). 4E-BP1 phosphorylation returned to baseline during recovery in control but became elevated when EAA+CHO was ingested (P < 0.05). eEF2 phosphorylation decreased at 1 and 2 h postexercise to a similar extent in both groups (P < 0.05). Our data suggest that enhanced activation of the mTOR signaling pathway is playing a role in the greater synthesis of muscle proteins when resistance exercise is followed by EAA+CHO ingestion.     

Insulin stimulation of muscle protein synthesis in obese Zucker rats is not via a rapamycin-sensitive pathway

The obese Zucker rat is resistant to insulin for glucose disposal, but it is unknown whether this insulin resistance is accompanied by alterations of insulin-mediated muscle protein synthesis. We examined rates of muscle protein synthesis either with or without insulin in lean and obese Zucker rats with the use of a bilateral hindlimb preparation. Additional experiments examined insulin's effect on protein synthesis with or without rapamycin, an inhibitor of protein synthesis. Protein synthesis in red and white gastrocnemius was stimulated by insulin compared with control (no insulin) in obese (n = 10, P<0.05) but not in lean (n = 10, P>0.05) Zucker rats. In white gastrocnemius, rapamycin significantly reduced rates of protein synthesis compared with control in lean (n = 6) and obese (n = 6) rats; however, in red gastrocnemius, the attenuating effect of rapamycin occurred only in obese rats. The addition of insulin to rapamycin resulted in rates of synthesis that were similar to those for rapamycin alone for lean rats and to those for insulin alone (augmented) for obese rats in both tissues. Our results demonstrate that insulin enhances protein synthesis in muscle that is otherwise characterized as insulin resistant. Furthermore, rapamycin inhibits protein synthesis in muscle of obese Zucker rats; however, stimulation of protein synthesis by insulin is not via a rapamycin-sensitive pathway.     

Leucine ingestion promotes mTOR translocation to the periphery and enhances total and peripheral RPS6 phosphorylation in human skeletal muscle

Amino Acids - The activation of the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, by anabolic stimuli (such as muscle contraction or essential amino...     

Regulation of muscle protein degradation,not synthesis,by dietary leucine in rats fed a protein-deficient diet

The aim of this study was to elucidate the effects of long-term intake of leucine in dietary protein malnutrition on muscle protein synthesis and degradation. A reduction in muscle mass was suppressed by leucine-supplementation (1.5% leucine) in rats fed protein-free diet for 7 days. Furthermore, the rate of muscle protein degradation was decreased without an increase in muscle protein synthesis. In addition, to elucidate the mechanism involved in the suppressive effect of leucine, we measured the activities of degradation systems in muscle. Proteinase activity (calpain and proteasome) and ubiquitin ligase mRNA (Atrogin-1 and MuRF1) expression were not suppressed in animals fed a leucine-supplemented diet, whereas the autophagy marker, protein light chain 3 active form (LC3-II), expression was significantly decreased. These results suggest that the protein-free diet supplemented with leucine suppresses muscle protein degradation through inhibition of autophagy rather than protein synthesis.     

Developmental changes in the feeding-induced activation of the insulin-signaling pathway in neonatal pigs

In neonatal animals, feeding stimulates skeletal muscle protein synthesis, a response that declines with development. Both the magnitude of the feeding response and its developmental decline can be reproduced by insulin infusion, suggesting that an altered responsiveness to insulin is a primary determinant of the developmental decline in the stimulation of protein synthesis by feeding. In this study, 7- and 26-day-old pigs were either fasted overnight or fed porcine milk after an overnight fast. We examined the abundance and degree of tyrosine phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and IRS-2 in skeletal muscle and, for comparison, liver. We also evaluated the association of IRS-1 and IRS-2 with phosphatidylinositol 3-kinase (PI 3-kinase). The abundance of IR protein in muscle was twofold higher at 7 than at 26 days, but IRS-1 and IRS-2 abundances were similar in muscle of 7- and 26-day-old pigs. The feeding-induced phosphorylations were greater at 7 than at 26 days of age for IR (28- vs. 13-fold), IRS-1 (14- vs. 8-fold), and IRS-2 (21- vs. 12-fold) in muscle. The associations of IRS-1 and IRS-2 with PI 3-kinase were also increased by refeeding to a greater extent at 7 than at 26 days (9- vs. 5-fold and 6- vs. 4-fold, respectively). In liver, the abundance of IR, IRS-1, and IRS-2 was similar at 7 and 26 days of age. Feeding increased the activation of IR, IRS-1, IRS-2, and PI 3-kinase in liver only twofold, and these responses were unaffected by age. Thus our findings demonstrate that the feeding-induced activation of IR, IRS-1, IRS-2, and PI 3-kinase in skeletal muscle decreases with development. Further study is needed to ascertain whether the developmental decline in the feeding-induced activation of early insulin-signaling components contributes to the developmental decline in translation initiation in skeletal muscle.     

Protein-tyrosine-phosphatase 1B activation is regulated developmentally in muscle of neonatal pigs

The high activity of the insulin-signaling pathway contributes to the enhanced feeding-induced stimulation of translation initiation in skeletal muscle of neonatal pigs. Protein-tyrosine-phosphatase 1B (PTP1B) is a negative regulator of the tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate 1 (IRS-1). The activity of PTP1B is determined mainly by its association with IR and Grb2. We examined the level of PTP1B activity, PTP1B protein abundance, PTP1B tyrosine phosphorylation, and the association of PTP1B with IR and Grb2 in skeletal muscle and liver of fasted and fed 7- and 26-day-old pigs. PTP1B activity in skeletal muscle was lower (P < 0.05) in 7- compared with 26-day-old pigs but in liver was similar in the two age groups. PTP1B abundances were similar in muscle but lower (P < 0.05) in liver of 7- compared with 26-day-old pigs. PTP1B tyrosine phosphorylation in muscle was lower (P < 0.05) in 7- than in 26-day-old pigs. The associations of PTP1B with IR and with Grb2 were lower (P < 0.05) at 7 than at 26 days of age in muscle, but there were no age effects in liver. Finally, in both age groups, fasting did not have any effect on these parameters. These results indicate that basal PTP1B activation is developmentally regulated in skeletal muscle of neonatal pigs, consistent with the developmental changes in the activation of the insulin-signaling pathway reported previously. Reduced PTP1B activation in neonatal muscle likely contributes to the enhanced insulin sensitivity of skeletal muscle in neonatal pigs.     

Effect of a soy protein diet on protein and energy metabolism and organ development in protein-restricted growing pigs

Two feeding experiments were carried out with castrated male pigs weighing between 10 and 30 kg to study acute and persisting dietary effects on growth and on protein and energy metabolism in growing pigs. Pigs were fed semi-synthetic isoenergetic and isonitrogenous diets at 50% protein requirement with either soy protein isolate (SPI) or casein (CAS) as sole protein source. Intake of protein and ME amounted to 9% w/w and 1800 kJ x kg BW (-0.62) x d(-1) in Exp. 1, respectively, and 9% w/w and 1430 kJ x kg BW(-0.62) x d(-1) in Exp. 2. The CAS diet was supplemented by Lys, Met, Thr and Trp. In Exp. 1 (acute effects), 18 pigs received the CAS diet for 24 days (period 1); 9 pigs were then switched to a SPI diet whereas 9 pigs continued on the CAS diet for another 31 days (period 2). In Exp. 2, a third period of 31 days was added in which the SPI group was switched back to CAS diet. The control group was fed on the CAS diet throughout Exp. 2 (86 days). Altogether the majority of parameters were not affected neither comparing SPI with CAS in Exp. 1 nor inspecting possible persistence of effects in Exp. 2. In detail, in Exp. 1 SPI compared to CAS feeding resulted in a lower efficiency of protein utilisation and lower protein retention. Attendant upon the lower protein retention an increased energy retention as fat was only observed in tendency. SPI feeding caused a decreased body weight, thyroid weight and increased hepatic carbohydrate content that persisted after the diet was changed back to CAS (Exp. 2).