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

Agus Suryawan, Hanh V. Nguyen, Jill A. Bush, and Teresa A. Davis

United States Department of Agriculture/Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 synthesis; growth; insulin; muscle; liver


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DIETARY AMINO ACIDS are efficiently deposited as body protein in the neonate (17, 34). This high efficiency is associated with a marked increase in protein synthesis after feeding (11, 13, 14, 16, 20), a response that declines with development. The postprandial rise in protein synthesis and the developmental decline in this response are more pronounced in skeletal muscle than in visceral tissues (5, 6, 11, 13, 16). Moreover, in muscle, the developmental decline in this response parallels the marked fall in the nutrient-induced activation of translation initiation factors involved in the binding of mRNA to the 40S ribosomal subunit (18). Thus, in muscle of the 7-day-old pig, feeding increases the phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1), resulting in a dissociation of the inactive 4E-BP1 · eIF4E complex and association of the active eIF4E · eIF4G complex. All of these changes are blunted with development.

Insulin likely plays a key role in the regulation of growth in the neonate. The feeding-induced stimulation of skeletal muscle protein synthesis in neonatal pigs can be completely reproduced by the infusion of insulin, even when glucose and amino acids are maintained at fasting levels (48). This response to insulin falls in exact parallel to the response to feeding and suggests that the developmental changes in the response to feeding reflect a change in insulin-mediated signals that stimulate translation. This hypothesis is supported by our recent findings of a developmental decline in the postprandial activation of the insulin-signaling proteins, ribosomal protein S6 kinase (S6K1) and protein kinase B (PKB), which are just upstream of translation initiation (Kimball SR, Farrell PA, Nguyen HV, Jefferson LS, and Davis TA, unpublished observations). However, the role of the early steps in the insulin-signaling pathway in the feeding-induced activation of translation initiation, and subsequent stimulation of protein synthesis, in neonatal pigs has not been elucidated.

The signaling cascade is initiated by insulin on binding to its receptor (45). This leads to activation of insulin receptor tyrosine kinase and the subsequent phosphorylation of several cytosolic substrates, primarily insulin receptor substrate (IRS)-1 (41) and IRS-2 (42). Although IRS-1 and IRS-2 are structurally similar, recent research suggests that they are differentially expressed and mediate distinct responses. IRS-1 and IRS-2 function as "docking proteins," transmitting insulin signals to several proteins that contain Src-homology 2 (SH2) domains (39, 40) including phosphatidylinositol (PI) 3-kinase, which catalyzes the phosphorylation of PI. The p85 subunit of this enzyme associates with phosphorylated IRS-1 and IRS-2, which leads to stimulation of its enzyme activity (2). The activation of PI 3-kinase triggers the downstream signaling pathway that leads to various insulin-stimulated biological responses, including glucose and amino acid transport, glycogen synthesis, and protein synthesis (22).

Feeding and developmental stage both appear to affect the early steps in the insulin-signaling pathway. Feeding stimulates the tyrosine phosphorylation of the insulin receptor and IRS-1 in rat (28) and chicken (21) liver, but not in chick muscle (21). The insulin-stimulated tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2, as well as the association of PI 3-kinase with IRS-1 in both muscle and liver, is greater in the young adult than in the elderly rat (8, 9).

Studies using intact animals and cell lines suggest that early events in the insulin-signaling pathway may play an important role in the regulation of growth and protein synthesis. Lack of the insulin receptor gene is associated with profound growth retardation in humans (44). Anti-insulin receptor antibodies in embryonic chicks (25) and null mutations of the insulin receptor and IRS-1 in mice (30, 46, 49) are associated with low body weight (25, 30, 46) and muscle protein synthesis (49). Insulin receptor tyrosine kinase activity is required for insulin-stimulated IRS-1 and PI 3-kinase activation in transgenic mice (10), and activation of IRS-1 and PI 3-kinase is required for insulin-stimulated protein synthesis in 32D cell lines (35). Mutations of IRS-1 and IRS-2 genes in mice indicate that insulin action is largely mediated by IRS-1 in skeletal muscle and IRS-2 in liver (30) and that IRS-1 plays a more critical role than IRS-2 in regulating growth (46).

The current study tested the hypothesis that the feeding-induced changes in the level and phosphorylation of the insulin receptor, IRS-1, and IRS-2, as well as the associations of IRS-1 and IRS-2 with PI 3-kinase, decrease with development in skeletal muscle in parallel with the previously observed developmental decline in the feeding-induced activation of translation initiation factors (18) and protein synthesis (11) in skeletal muscle. For comparison, we examined the developmental changes in the liver, an organ in which insulin does not increase protein synthesis (12) and which shows no decline in its response to feeding during early postnatal development (11).


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals. Eight crossbred (Landrace × Yorkshire × Duroc × Hampshire) pregnant sows (Agriculture Headquarters, Texas Department of Criminal Justice, Huntsville, TX) were housed in lactation crates in individual, environmentally controlled rooms 2 wk before farrowing. Sows were fed a commercial diet (5084, PMI Feeds, Richmond, IN) and provided water ad libitum. After farrowing, piglets remained with the sow but were not allowed access to the sow's diet. A total of 24 piglets from four litters, weighing ~2 and 8 kg, were studied at 7 and 26 days of age, respectively.

Pigs within each litter were randomly assigned to one of two treatment groups (n = 6 per age group per treatment group), and were either 1) fasted for 18 h or 2) fed for 1.5 h after an 18-h fast. Water was provided throughout the fasting period. Pigs that were fed after the 18-h fast were given two gavage administrations of 30 ml/kg body wt of mature porcine milk (University of Nebraska, Lincoln, NE) at 60-min intervals. Pigs in the fasting group were killed after 18 h of fasting, and pigs in the fed group were killed 30 min after the second gavage feeding. Samples of longissimus dorsi muscle and liver were rinsed in ice-cold saline and rapidly frozen. The protocol was approved by the Animal Care and Use Committee of Baylor College of Medicine and was conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

Materials. BioMag goat anti-mouse IgG and goat anti-rabbit IgG magnetic beads were obtained from Polysciences (Warrington, PA), and the magnetic sample rack was from Promega (Madison, WI). Reagents for SDS-PAGE were from Bio-Rad Laboratories (Richmond, CA). The protein assay kit was purchased from Pierce (Rockford, IL). Anti-insulin receptor (beta -subunit) antibody, anti-phosphotyrosine (PY) antibody, and anti-IRS-1 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IRS-2 antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-PI 3-kinase (p85) was purchased from Mbl International (Watertown, MA). The enhanced chemiluminescence Western blotting detection kit (ECL-Plus) was obtained from Amersham (Arlington Heights, IL). Other chemicals and reagents were from Sigma Chemical (St. Louis, MO).

Insulin receptor content. To determine insulin receptor abundance, the samples were prepared as described by Burrin et al. (7), with slight modification as follows. The samples (±1 g) of frozen muscle and liver were pulverized in liquid nitrogen and homogenized in a buffer (buffer A) containing (in mM) 50 HEPES, pH 7.4, 150 NaCl, 10 sodium pyrophosphate, 2 Na3VO4, 1 MgCl2, 1 CaCl2, 10 NaF, 5 Na-EDTA, and 2 phenylmethylsulfonyl fluoride and 25 µg/ml leupeptin. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the resulting supernatant was centrifuged at 100,000 g for 1 h at 4°C. The 100,000-g pellet was solubilized in 3-5 ml of buffer A containing 1% Triton X-100 overnight at 4°C. The protein concentration of the solubilized membrane-containing fraction was determined using bicinchoninic acid (BCA; Pierce) by the method of Smith et al. (38).

Immunoprecipitation of insulin receptor protein. To determine insulin receptor abundance and tyrosine phosphorylation, protein samples from membrane preparations were immunoprecipitated with anti-insulin receptor-beta antibody. The immunoprecipitation procedure was conducted as described by Fox et al. (23). Briefly, equal amounts of membrane protein (500 µg protein in 500 µl buffer) were incubated overnight at 4°C with anti-insulin receptor-beta antibody in 2.5% Triton X-100/PBS. BioMag goat anti-mouse IgG or goat anti-rabbit IgG magnetic beads (1 ml/tube) were washed three times in low-salt buffer (20 mM Tris, 150 mM NaCl, 5 mM disodium EDTA, 0.5% Triton X-100, and 0.1% beta -mercaptoethanol, pH 7.4) with the use of a magnetic sample rack and were resuspended in 500 µl of low-salt buffer containing 1% dry skim milk. Each sample was added to 500 µl of resuspended beads and rocked for at least 1 h at 4°C. The beads were captured using a magnetic rack and were washed twice in low-salt buffer and once in high-salt buffer (50 mM Tris, 500 mM NaCl, 5 mM disodium EDTA, 1% Triton X-100, 0.6% sodium deoxycholate, 0.1% SDS, and 0.04% beta -mercaptoethanol, pH 7.4). The captured beads were resuspended in 100 µl 1× sample buffer [2% SDS, 100 mM Tris · HCl, pH 6.8, 5% beta -mercaptoethanol, 12% (vol/vol) glycerol, and 0.02% (wt/vol) Bromphenol blue], boiled for 5 min, and stored at -80°C until electrophoresis.

Preparation of tissue extracts. Samples for immunoblotting were prepared as described by Goodyear et al. (27). Briefly, samples of frozen muscle and liver were pulverized and homogenized in ice-cold buffer A with 1% IGEPAL and 10% glycerol added. The homogenate was incubated for 45 min at 4°C with gentle mixing and then centrifuged at 35,000 g for 1 h at 4°C. The supernatant was collected, and an aliquot was assayed for protein concentration with BCA (37). Supernatants were used to determine IRS-1 and IRS-2 abundance, tyrosine phosphorylation of insulin receptor, IRS-1, and IRS-2, and the association of PI 3-kinase (p85) with IRS-1 and IRS-2.

Immunoprecipitation of PY, IRS-1 and IRS-2 proteins. To determine the abundance and tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2 and the association of PI 3-kinase (p85) with IRS-1 and IRS-2, protein samples from tissue extract preparations were immunoprecipitated with anti-mouse PY, anti-human IRS-1, or anti-human IRS-2 antibodies. The immunoprecipitation procedure was similar to that described for the insulin receptor.

Western blot analysis. Equal amounts of protein were subjected to SDS-PAGE (8% wt/vol; Mini-PROTEAN II electrophoresis system, Bio-Rad), as described by Laemmli (33). Electrophoretic separation was carried out in 1% SDS, 25 mM Tris, and 200 mM glycine (pH 8.4) at 200 V for 45-60 min at room temperature. The proteins were then transferred to an activated polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA) in 25 mM Tris, 192 mM glycine, and 20% methanol (vol/vol; pH 8.3) for 1 h at 4°C. The membrane was incubated for 1 h at room temperature in a Tris-buffered saline-Tween 20 solution (TBS-T) containing 10 mM Tris, 0.5 M NaCl, and 0.5% Tween 20, pH 7.4, with 5% (wt/vol) nonfat dried milk. After the blocking step, the membrane was incubated with one primary antibody (anti-insulin receptor, IRS-1, IRS-2, PI 3-kinase/p85, or PY) for 1 h and washed four times in rinsing solution. Membranes were then incubated with secondary antibody (horseradish peroxidase-conjugated IgG fraction of goat anti-rabbit IgG or goat anti-mouse IgG) diluted 1:2,000 in TBS-T. The membranes were then washed in TBS-T three times for 10 min and developed with an enhanced chemiluminescence detection kit (ECL-Plus, Amersham Pharmacia, Piscataway, NJ) before exposure onto Kodak-X-Omat film. The blots were quantified by computerized densitometry (Molecular Dynamics, Sunnyvale, CA).

Statistics. Analysis of variance (general linear modeling) was used to assess the effects of feeding, age, and their interaction. If there was an interaction between feeding and age, Student's t-test was used to test for differences between treatment groups. Probability values of <0.05 were considered statistically significant. Data are presented as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin receptor, IRS-1, and IRS-2 abundance in muscle and liver. To determine whether there were effects of feeding and development on the relative amount of insulin receptor in skeletal muscle and liver, equal amounts of membrane protein were subjected to immunoblotting using antibody to the beta -subunit of the insulin receptor. Insulin receptor abundance in skeletal muscle was twofold higher in 7- than in 26-day-old pigs (P < 0.05; Fig. 1). Insulin receptor abundance in liver was unaffected by age. Feeding did not alter insulin receptor abundance in either skeletal muscle or liver of 7- and 26-day-old pigs.


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Fig. 1.   Insulin receptor (IR), IR substrate (IRS)-1, and IRS-2 protein abundance in skeletal muscle and liver of fasted (F) and fed (R) pigs at 7 (7d) and 26 days (26d) of age. Muscles and livers were homogenized as described in EXPERIMENTAL PROCEDURES. Equal amounts of protein were subjected to SDS-PAGE followed by immunoblot analysis with anti-IRbeta , anti-IRS-1, and anti-IRS-2 antibodies. A: protein expression of IR in muscle and liver membrane preparation as determined by immunoblot analysis. B: representative immunoblot of IRS-1 protein content. C: representative immunoblot of IRS-2 protein content. Results are means ± SE arbitrary densitometric units (n = 6 animals/age/treatment). Feeding did not have any effect on the abundance of IR, IRS-1, or IRS-2. IR abundance in muscle, but not in liver, was higher in 7- than in 26-day-old pigs (P < 0.05).

To evaluate the effects of feeding and development on the relative amount of IRS-1 and IRS-2 in skeletal muscle and liver, equal amounts of total soluble protein were subjected to immunoblotting using anti-IRS-1 or -2 antibodies, respectively. The results show that the abundance of both IRS-1 and IRS-2 in skeletal muscle and liver was unaffected by feeding and development (Fig. 1).

Insulin receptor, IRS-1, and IRS-2 phosphorylation in muscle and liver. The effects of feeding and development on tyrosine phosphorylation of the insulin receptor beta -subunit, IRS-1, and IRS-2 were determined by subjecting an equal amount of protein extracts from skeletal muscle and liver to immunoprecipitation with an anti-phosphotyrosine (alpha PY) antibody. After SDS-PAGE and electrotransfer, PVDF membrane was incubated with anti-insulin receptor beta -subunit antibody, anti-IRS-1, or anti-IRS-2 antibodies. The amount of tyrosine-phosphorylated insulin receptor beta -subunit in both skeletal muscle and liver was increased by feeding (P < 0.001 and P < 0.05, respectively; Fig. 2). The feeding-induced stimulation of insulin receptor phosphorylation in skeletal muscle was greater (P < 0.05) in 7-day-old (16-fold) than in 26-day-old pigs (4-fold). In contrast, the feeding-induced phosphorylation of the insulin receptor in liver did not differ between 7- and 26-day-old pigs (both 2-fold). The baseline fasting level of phosphorylation of the insulin receptor appeared to be lower, and the magnitude of the response to feeding appeared to be higher, in skeletal muscle than in liver.


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Fig. 2.   IR, IRS-1, and IRS-2 tyrosine phoshorylation in skeletal muscle and liver of fasted and fed pigs at 7 and 26 days of age. Muscles and livers were homogenized as described in EXPERIMENTAL PROCEDURES. Equal amounts of protein were immunoprecipitated with anti-phosphotyrosine (PY) antibody, and samples were subjected to immunoblot analysis using anti-IRbeta , anti-IRS-1, and anti-IRS-2 antibodies. A: representative immunoblot of IR tyrosine phosphorylation. B: representative immunoblot of IRS-1 tyrosine phosphorylation. C: representative immunoblot of IRS-2 tyrosine phosphorylation. Results are means ± SE arbitrary densitometric units (n = 6/age/treatment). Feeding increased the tyrosine phosphorylation of IR, IRS-1, and IRS-2 in muscle (P < 0.001) and liver (P < 0.05), respectively. IR, IRS-1, and IRS-2 tyrosine phosphorylation in muscle, but not in liver, was higher in 7- than in 26-day-old pigs (P < 0.05).

To determine whether the developmental decline in the feeding-induced stimulation of insulin receptor phosphorylation in skeletal muscle was due to differences between 7- and 26-day-old pigs in insulin receptor abundance, equal amounts of membrane protein were immunoprecipitated with anti-insulin receptor-beta antibody, samples were subjected to immunoblot analysis with the use of anti-insulin receptor-beta or anti-PY antibodies, and the data for insulin receptor phosphorylation were normalized for insulin receptor concentration. Figure 3 shows that, even when normalized for insulin receptor content, the feeding-induced stimulation of insulin receptor phosphorylation in skeletal muscle was higher (P < 0.05) in 7- (28-fold) than in 26-day-old pigs (13-fold).


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Fig. 3.   IR tyrosine-phosphorylation normalized for IR abundance in skeletal muscle of fasted and fed pigs at 7 and 26 days of age. Membrane preparation was obtained as described in EXPERIMENTAL PROCEDURES. Equal amounts of membrane protein were immunoprecipitated with anti-IRbeta antibody, and samples were subjected to immunoblot analysis using anti-IRbeta or anti-PY antibodies. Presented is the representative immunoblot of IR content and IR tyrosine phosphorylation. Results are means ± SE arbitrary densitometric units (n = 6/age/treatment). Feeding markedly stimulated IR phosphorylation (P < 0.001). IR tyrosine phosphorylation in muscle was higher in 7- than in 26-day-old pigs (P < 0.05).

Figure 2 also shows representative immunoblots of the effect of feeding and development on the tyrosine phosphorylation of IRS-1 and IRS-2. Feeding increased tyrosine phosphorylation of IRS-1 and IRS-2 in skeletal muscle and liver. The feeding-induced stimulation of the phosphorylation of IRS-1 in skeletal muscle was greater (P < 0.05) in 7- (14-fold) than in 26-day-old pigs (8-fold). Similar developmental differences were observed for IRS-2 phosphorylation in muscle (21-fold at 7 days and 12-fold at 26 days, P < 0.05). Because IRS-1 and IRS-2 abundance was unaffected by age, developmental change in the phosphorylation of IRS-1 and IRS-2 in skeletal muscle was unrelated to IRS-1 and IRS-2 abundance. In the liver, there were no differences between 7- and 26-day-old pigs in the feeding-induced stimulation of the phosphorylation of both IRS-1 (2- vs. 2-fold) and IRS-2 (2- vs. 2-fold.) In skeletal muscle compared with liver, the baseline fasting level of phosphorylation of IRS-1 and IRS-2 appeared to be lower, and the magnitude of the response to feeding appeared to be higher.

Association of PI 3-kinase with IRS-1 and IRS-2 in muscle and liver. To determine the association of PI 3-kinase with IRS-1 and IRS-2 in response to feeding and development, equal amounts of skeletal muscle and liver extracts were immunoprecipitated with anti-IRS-1 or IRS-2 antibody, followed by immunoblotting with anti-PI 3-kinase (p85) antibody. The representative blots (Fig. 4) showed that feeding increased the association of PI 3-kinase (p85) with IRS-1 and IRS-2 in both skeletal muscle and liver. The feeding-induced stimulation of the association of PI 3-kinase (p85) with IRS-1 in skeletal muscle was greater (P < 0.05) in 7- than in 26-day-old pigs (9- vs. 5-fold). Similarly, the association of PI 3-kinase (p85) with IRS-2 in skeletal muscle was greater in 7- than in 26-day-old pigs (6- vs. 4-fold, P < 0.05) after eating. In the liver, there were no differences between 7- and 26-day-old pigs in the feeding-induced stimulation of the association of PI 3-kinase with both IRS-1 (3- vs. 3-fold) and IRS-2 (2- vs. 2-fold.) The baseline fasting level of association of PI 3-kinase with IRS-1 and IRS-2 appeared to be lower, and the magnitude of the response to feeding appeared to be higher in skeletal muscle than in liver.


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Fig. 4.   The association of phosphatidylinositol (PI) 3-kinase (p85 subunit) with IRS-1 and IRS-2 in pig skeletal muscle and liver. Muscles or livers were homogenized as described in EXPERIMENTAL PROCEDURES. Equal amounts of protein were immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies, and samples were subjected to immunoblot analysis using anti-p85 of PI 3-kinase antibody. A: relative protein expression of p85alpha -subunit of PI 3-kinase associated with IRS-1 as determined by immunoblot analysis of muscle and liver lysates. B: relative protein expression of p85alpha -subunit of PI 3-kinase associated with IRS-2 as determined by immunoblot analysis of muscle and liver lysates. Results are means ± SE arbitrary densitometric units (n = 6/age/treatment). Feeding markedly stimulated the association of PI 3-kinase (p85) with IRS-1 and IRS-2 in muscle (P < 0.001) and liver (P < 0.05). The association of PI 3-kinase (p85) with IRS-1 and IRS-2 in muscle, but not in liver, was higher in 7- than in 26-day-old pigs (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of development on insulin receptor, IRS-1, and IRS-2 abundance. The rapid growth of skeletal muscle in the neonate is supported by an elevated rate of skeletal muscle protein synthesis (15, 19, 26, 50). To identify the mechanisms that regulate the high rate of protein deposition during early postnatal development, we have utilized the neonatal pig model (36). Pigs are studied at 7 days of age to avoid the complications of endocytosis of ingested protein in the newborn and at 26 days of age for comparison with a more advanced stage of development but before diet composition changes. We have demonstrated previously (11) that, in response to feeding, skeletal muscle protein synthesis increases from 15 to 24% per day at 7 days of age, but from only 4 to 6% per day at 26 days of age. By contrast, protein synthesis in liver increases after feeding from 76 to 93% per day at 7 days of age and from 63 to 72% per day at 26 days of age. Thus the overall developmental decline in protein synthesis is more pronounced in skeletal muscle than in liver (11). In addition, both the magnitude of the feeding-induced stimulation of protein synthesis and the developmental decline in this response to feeding are more profound in skeletal muscle than in liver. This stimulation of protein synthesis by feeding in muscle, but not in liver, of the 7- and 26-day-old pig can be reproduced by the infusion of insulin (12, 48). In the current study, we wished to determine whether the developmental changes in feeding-induced stimulation of skeletal muscle protein synthesis are associated with developmental changes in either the abundance or activation of early insulin-signaling components in skeletal muscle. For comparison, we also examined the effect of development on these signaling components in the liver, an organ in which the developmental changes in protein synthesis contrast sharply with those in skeletal muscle.

The results show that the insulin receptor concentration in skeletal muscle was twofold higher in 7- than in 26-day-old pigs. Studies have shown that insulin receptor abundance in muscle is also higher in suckling than in adult rats and dogs (1, 29). However, in those studies, age-related changes in insulin receptor abundance were confounded by changes in diet composition. In the current study, 7- and 26-day-old pigs were suckled until studied, and thus the results indicate that insulin receptor number is indeed developmentally regulated. However, neither feeding nor development altered the relative amount of IRS-1 and IRS-2 proteins in skeletal muscle of neonatal pigs, suggesting that the amount of IRS-1 and IRS-2 protein is not limiting for downstream signaling events, such as translation initiation, in neonatal skeletal muscle.

The developmental decline in insulin receptor abundance in skeletal muscle in the current study is consistent with our recent findings (Kimball SR, Farrell PA, Nguyen HV, Jefferson LS, and Davis TA, unpublished observations) of a developmental decline in the abundance in muscle of components of the insulin signal transduction pathway that are downstream of PI 3- kinase (4, 38), i.e., PKB and the protein kinase mammalian target of rapamycin (mTOR). In addition, the developmental changes in the abundance of these signaling proteins paralleled the developmental change in both the activity of translation initiation factors that regulate the binding of met-tRNA to the 40S ribosomal subunit complex, i.e., eIF2 (18), and the overall rate of muscle protein synthesis (11). These responses are consistent with the hypothesis that the reduction in insulin receptor abundance in skeletal muscle during early postnatal development contributes to the long-term changes in muscle protein synthesis that occur over the course of development.

In the liver, however, the abundance of insulin receptor, IRS-1, and IRS-2 did not change between 7 and 26 days of age, even though we had previously found a modest reduction in liver protein synthesis with development (11). This suggests that the developmental decline in liver protein synthesis is regulated by factors other than insulin receptor abundance. We have previously reported that the activation of a translation initiation factor, eIF2B, which regulates the binding of met-tRNA to the 40S ribosomal subunit, decreases with postnatal development in liver (18).

Effects of feeding and development on the activation of insulin receptor, IRS-1, IRS-2, and PI 3-kinase. Insulin binding is followed by autophosphorylation of tyrosine residues of the insulin receptor beta -subunit (45). This event greatly enhances its tyrosine kinase activity toward endogenous substrates such as IRS-1 and IRS-2 and is considered important for the biological effects of insulin. Insulin binding also activates PI 3-kinase by causing insulin receptor substrates to bind to the SH2 domain of the p85 subunit of PI 3-kinase (37, 40). In this study, feeding increased the tyrosine phosphorylation of the insulin receptor beta -subunit, IRS-1, and IRS-2, as well as the association of the p85 subunit of PI 3-kinase with both IRS-1 and IRS-2 in skeletal muscle. By contrast, feeding has no effect on the phosphorylation of the insulin receptor and IRS-1 in muscle of mature chickens (21).

Importantly, the feeding-induced activation of the insulin receptor, IRS-1, IRS-2, and PI 3-kinase in muscle was markedly greater in 7- than in 26-day-old pigs. Moreover, the phosphorylation of the insulin receptor, IRS-1, and IRS-2 in muscle was elevated in 7- compared with 26-day-old pigs, even when expressed relative to the amount of these signaling proteins present in the muscle. Studies in rat muscle have shown that the insulin-stimulated activation of insulin receptor, IRS-1, IRS-2, and PI 3-kinase decreases with aging (8, 9). Together, these results suggest that the feeding-induced activation of early insulin-signaling components in skeletal muscle declines progressively throughout life.

Studies largely in cell culture suggest that the stimulation of translation by anabolic agents such as insulin requires activation of PI 3-kinase, PKB, mTOR, and S6K1 (4, 38). Our recent findings of a developmental decline in the feeding-induced activation of PKB and S6K1 (Kimball SR, Farrell PA, Nguyen HV, Jefferson LS, and Davis TA, unpublished observations), together with our current findings, are suggestive of a developmental decline in the activation of the insulin-signaling pathway leading to translation initiation. The changes in the insulin-signaling pathway are paralleled by comparable changes in the feeding-induced activation of translation initiation factors that regulate the binding of mRNA to the 40S ribosomal subunit (18) and the feeding-induced stimulation of protein synthesis (11) in skeletal muscle. These changes include a developmental decline in the feeding-induced phosphorylation of 4E-BP1, dissociation of the inactive 4E-BP1 · eIF4E complex, and association of the active eIF4E · eIF4G complex (18). Inhibition of mTOR strongly attenuates both the feeding-induced assembly of the active eIF4E · eIF4G complex and S6K1 activation in neonatal pigs (31), further supporting the role of the PI 3-kinase/mTOR signal transduction pathway in the regulation of muscle protein synthesis in the neonate.

The potential mechanisms that underlie the developmental decline in the feeding-induced activation of the insulin-signaling pathway in skeletal muscle are intriguing. The modest developmental decline in postprandial insulin concentration [-17% (11)] seems unlikely to play a primary role in the marked developmental decline in the feeding-induced activation of the insulin receptor (-70%) and downstream signaling components. Moreover, when circulating insulin, amino acids, and glucose concentrations were maintained at similar levels in 7- and 26-day-old pigs using our novel hyperinsulinemic-euglycemic-euaminoacidemic clamps technique, we found that the insulin sensitivity and responsiveness of whole body amino acid, whole body glucose disposal, and muscle protein synthesis decrease with development (12, 47, 48). Whether these developmental changes in insulin action are due to differences in the relative proportion of classic insulin receptors to hybrid receptors [composed of insulin- and type I insulin-like growth factor alpha beta -hemireceptors (3)], insulin receptor isoforms [differing in 12 amino acids (24)], or tyrosine vs. serine/threonine phosphorylation of insulin receptor or signaling molecules (43) is open to speculation. However, the commonality of the developmental change in the biological responses to insulin suggests that the enhanced activation of early insulin-signaling components in muscle of the neonate after the consumption of a meal stimulates more signal downstream, leading to an enhanced activation of insulin-stimulated responses such as protein synthesis and glucose uptake.

In the liver, feeding increased the activation of the insulin receptor, IRS-1, IRS-2, and PI 3-kinase; however, these responses, in contrast to those in muscle, were unaffected by age. Moreover, the level of activation of these signaling proteins in the basal fasting condition was higher in liver than in skeletal muscle, and the magnitude of the responses to feeding was lower in liver than in skeletal muscle. This suggests that fasting, feeding, and development have a less-pronounced effect on the early steps in the insulin-signaling pathway in liver than in muscle. The results raise the question as to whether the modest increase in the phosphorylation of these proteins in response to feeding, from a high fasting phosphorylation level, will elicit a significant downstream response leading to an increase in protein synthesis. Although this conclusion is indeed speculative, it is consistent with our previous findings that insulin infusion does not stimulate liver protein synthesis in neonatal pigs, even when circulating amino acids are maintained at fasting levels (12). Thus it seems likely that the postprandial rise in some factor other than insulin activates translation initiation in the liver of the neonate.

Perspectives. Our observation that insulin receptor abundance in skeletal muscle decreased with development, in parallel with the overall developmental decline in muscle protein synthesis (11), supports the hypothesis that insulin receptor abundance plays a role in the developmental regulation of muscle protein synthesis. The feeding-induced activation of the insulin receptor, IRS-1, IRS-2, and PI 3-kinase in skeletal muscle decreased with development, in parallel with the developmental decline in the feeding-induced activation of downstream signaling proteins (Kimball SR, Farrell PA, Nguyen HV, Jefferson LS, and Davis TA, unpublished observations), translation initiation factors (18), and protein synthesis (11) in skeletal muscle and the ability of insulin to stimulate skeletal muscle protein synthesis (48). In contrast, insulin receptor abundance and the modest feeding-induced activation of the early insulin-signaling components in liver did not change with development. Together, these results support the hypothesis that the developmental decline in the response of muscle protein synthesis to food consumption results from a reduction in the capacity of the insulin-signaling pathway in muscle to transduce to the translational apparatus the stimulus provided by the postprandial rise in circulating insulin concentration. If supported by further studies, the results imply that the enhanced activation of the insulin-signaling pathway in skeletal muscle of the neonate after food consumption plays a crucial role in determining the high rate of protein deposition in skeletal muscle and the more efficient use of dietary amino acids for growth in the neonate.


    ACKNOWLEDGEMENTS

We thank M. Fiorotto, P. Reeds, and D. Burrin for helpful discussions, P. O'Connor and W. Liu for the laboratory assistance, J. Cunningham and F. Biggs for care of animals, and L. Loddeke for editorial review.


    FOOTNOTES

This work is a publication of the United States Department of Agriculture/Agricultural Research Service (USDA/ARS) Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX. This project has been funded in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grant R01-AR-44474 (T. A. Davis) and the USDA/ARS under Cooperative Agreement no. 58-6250-6-001 (T. A. Davis). The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products or organization imply endorsement by the US Government.

Address for reprint requests and other correspondence: T. A. Davis, USDA/ARS Children's Nutrition Research Center, Dept. of Pediatrics, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030 (E-mail: tdavis{at}bcm.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 November 2000; accepted in final form 11 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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