United States Department of Agriculture/Agriculture Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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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.
neonate; insulin signaling; insulin sensitivity; protein synthesis
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INTRODUCTION |
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THE ENHANCED ACTIVATION of the insulin-signaling pathway leading to translation initiation in skeletal muscle of the neonate after food consumption has an important role in the enhanced responsiveness of muscle protein synthesis to feeding in the neonate (13, 16, 26, 27, 37). We have shown that the feeding-induced activation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and phosphatidylinositol 3-kinase (PI 3-kinase), as well as the abundance of the IR, is enhanced in skeletal muscle of the neonatal pig and decreases markedly with development. This developmental decline in the feeding-induced activation of early insulin-signaling components parallels the developmental decline in the feeding-induced activation of downstream signaling proteins, leading to the stimulation of protein synthesis in skeletal muscle and the developmental decrease in the ability of insulin to stimulate muscle protein synthesis. However, the molecular mechanism that regulates the developmental decline in the insulin sensitivity of skeletal muscle in neonatal pigs has not been elucidated.
When insulin binds to its receptor, it induces autophosphorylation of the receptor on its tyrosine residues, followed by the activation of its tyrosine kinase. The activated insulin receptor binds to its substrates, such as IRS proteins, resulting in the phosphorylation of their tyrosine residues (44). IRS activation triggers the activation of downstream signaling molecules, such as PI 3-kinase, leading to the stimulation of the metabolic effects of insulin, including protein synthesis (32). The accumulated evidence indicates that, in the early steps of the insulin-signaling pathway, tyrosine phosphorylation is essential for the activation of insulin signaling (39).
Protein-tyrosine-phosphatase 1B (PTP1B), a nontransmembrane phosphotyrosine phosphatase, has been shown to attenuate insulin signaling by catalyzing the dephosphorylation of the IR and IRS-1 (9, 17). Early studies suggest that PTP1B blocks insulin-induced S6 peptide phosphorylation and inhibits insulin-induced maturation of Xenopus oocytes (10, 41). Intracellular delivery of neutralizing antibodies against PTP1B augments IR and IRS-1 phosphorylation and, conversely, overexpression of PTP1B in transfected cells inhibits IR and IRS-1 phosphorylation (4, 24). Recent studies show that PTP1B-deficient mice exhibit hypersensitivity to insulin, suggesting that PTP1B is the major tyrosine phosphatase that regulates IR sensitivity (18, 28). PTP1B is abundantly expressed in insulin-sensitive tissues such as muscle and liver, and its activity is an important regulator of the sensitivity of the insulin-signaling pathway (2, 30, 33).
Mechanistic studies of PTP1B's action suggest that interaction between PTP1B and the IR is important for catalyzing IR dephosphorylation (5, 11, 12, 23, 36, 43). Receptor tyrosine kinases appear to undergo internalization and form complexes with PTP1B, thereby removing phosphate groups from tyrosine residues (23). Studies further suggest that the phosphorylated and activated IR transiently binds and phosphorylates tyrosine residues of PTP1B, thereby increasing its catalytic activity (5, 12). Catalytically active PTP1B can then interact with and dephosphorylate the IR, thus attenuating insulin action. Finally, PTP1B undergoes auto-dephosphorylation and returns to its inactive basal state (12). There is some evidence that the steady-state phosphotyrosine content of IRS-1 is also regulated by PTP1B (21, 35). In vitro, PTP1B, IRS-1, and Grb2 form a ternary complex, resulting in the dephosphorylation of IRS-1. Therefore, the interaction between Grb2 and PTP1B, with subsequent effects on IRS-1 dephosphorylation, may also have an important role in the regulation of the postreceptor insulin-signaling pathway (21).
In the present study, we examined whether PTP1B activation in skeletal muscle of neonatal pigs changes with development by measuring the activity of PTP1B in skeletal muscle of 7- and 26-day-old pigs in the fasted and fed states. Furthermore, we also studied the mechanisms involved in PTP1B activation by measuring the association of PTP1B with the IR, the tyrosine phosphorylation of PTP1B, and the association of PTP1B with Grb2 adaptor protein. For comparison, similar measurements were also made in liver, an organ in which insulin does not stimulate protein synthesis (14, 15) and which shows no developmental decline in insulin signaling (37).
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EXPERIMENTAL PROCEDURES |
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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 and were not given supplemental creep feed. Piglets from 4 litters, weighing ~2 and 8 kg, were studied at 7 (n = 6) and 26 day of age (n = 6), respectively.
Pigs were either fasted for 18 h or fed after an 18-h fast by two gavage administrations of 30 ml/kg body weight of porcine mature milk (University of Nebraska, Lincoln, NE) at 60-min intervals. Pigs were killed after the 18-h fast or 30 min after the second gavage feeding, and samples of longissimus dorsi muscle and liver were rinsed in ice-cold saline and rapidly frozen. The protocol, which we have previously described (16, 26, 37), 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-phosphotyrosine (PY) antibody and Grb2 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PTP1B antibody was purchased from BD Transduction Laboratories (San Diego, CA). 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).
Preparation of tissue extracts. Samples for immunoblotting were prepared according to Goodyear et al. (22). Briefly, samples of frozen muscle and liver were pulverized in liquid nitrogen and homogenized in ice-cold buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM NaPP, 2 mM Na3VO4, 1 mM MgCl2, 1 mM CaCl2, 10 mM NaF, 5 mM Na-EDTA, 2 mM PMSF, 5 µg/ml leupeptin, 1% NP-40, and 10% glycerol]. 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 using the bicinchoninic acid assay (Pierce, Rockford, IL). Supernatants were used to determine PTP1B content and immunoprecipitation.
Immunoprecipitation.
To determine the tyrosine phosphorylation of PTP1B and the association
of PTP1B with the IR and Grb2, protein samples from tissue extract
preparations were immunoprecipitated with anti-mouse PY, anti-IR, and
anti-Grb2 antibody, respectively. The immunoprecipitation procedure was
conducted according to Fox et al. (19). Briefly, equal
amounts of protein samples (500 µg protein in 500 µl buffer) were
incubated by gently rocking overnight at 4°C with 20 µl of primary
antibody (PY, IR, or Grb2 antibody), 230 µl PBS, and 12.5 µl Triton
X-100. The next day, 500 µl of secondary antibody (BioMag goat
anti-mouse IgG or goat anti-rabbit IgG linked to magnetic beads) were
added. After 1 h of incubation at 4°C, the samples 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% -mercaptoethanol (pH 7.4)] by
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
1 h at 4°C. The beads
were captured using the magnetic rack and 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%
-mercaptoethanol (pH 7.4)]. The captured beads were
resuspended in 100 µl of 1× sample buffer [2% SDS, 100 mM
Tris · HCl (pH 6.8), 5%
-mercaptoethanol, 12% (vol/vol) glycerol, and 0.02% (wt/vol) bromophenol blue], boiled
for 5 min, and stored at
80°C until electrophoresis.
Western blot analysis. To measure PTP1B abundance, equal amounts of samples were subjected to SDS-PAGE (8% wt/vol), as described by Laemmli (with a Mini-PROTEAN II electrophoresis system, Bio-Rad Laboratories). 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 with a Bio-Rad model 1000/500 power supply. A polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA) was activated in 20% methanol for 1 min. The proteins were then transferred to the membrane in 25 mM Tris, 192 mM glycine, and 20% methanol (vol/vol; pH 8.3) at 100 V and 250 mA 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 PTP1B antibody for 1 h on a rocking platform 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, NY) before exposure onto Kodak-X-Omat film. The blots were quantified by computerized densitometry (Molecular Dynamics Pharmacia, Piscataway, NY). To measure PTP1B tyrosine phosphorylation, the association of PTP1B with IR, and the association of PTP1B with Grb2, equal amounts of the respective immunoprecipitant (PY, IR, and Grb2) were subjected to SDS-PAGE followed by immunoblotting, as described in Western blot analysis, with PTP1B as the primary antibody.
PTP1B activity assay. The in vitro PTP1B activity was assayed according to a procedure previously described by Taghibiglou et al. (38). Briefly, samples of frozen skeletal muscle and liver were pulverized in liquid nitrogen and homogenized in ice-cold solubilization buffer (PBS containing 1% NP-40, 1% deoxycholate, 5 mM EDTA, 1 mM EGTA, 2 mM PMSF, and 0.1 mM leupeptin). The lysates were centrifuged for 10 min at 4°C in a microcentrifuge. The supernatants were collected for immunoprecipitation. Before immunoprecipitation, the supernatants were subjected to preclearing with nonimmune serum and protein A/G Plus Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 15 min at 4°C. Equal amounts of protein samples (750 µg of total protein) were subjected to immunoprecipitation with anti-PTP1B antibody (BD Transduction Laboratories) at 4°C overnight. PTP1B immunocomplexes were precipitated with protein A/G Plus Agarose at 4°C for an additional 2 h. Immunoprecipitates were washed in PTP assay buffer [100 mM HEPES (pH 7.6), 2 mM EDTA, 1 mM DTT, 150 mM NaCl, and 0.5 mg/ml BSA]. The pp60c-src COOH-terminal phosphoregulatory peptide (TSTEPQpYQPGENL; Biomol, Plymouth Meeting, PA) was added to a final concentration of 200 µM in a total reaction volume of 60 µl in PTP1B assay buffer. The sample mixtures were incubated for 1 h at 30°C. After the reaction, 40-µl aliquots were placed into 96-well plates, and 100 µl of Biomol Green reagent were added to each sample. After incubation for 30 min at room temperature, the absorbance was measured at 630 nm.
Statistics. Analysis of variance was used to assess the effect of development and food intake. Probability values of <0.05 were considered statistically significant. Data are presented as means ± SE.
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RESULTS |
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To determine whether there was an effect of development on the
PTP1B activity in skeletal muscle and liver, equal amounts of protein
were subjected to immunoblotting using an anti-PTP1B antibody followed
by an enzyme activity assay. The activity of PTP1B in skeletal muscle
of 7-day-old pigs was significantly (P < 0.05) lower
than that in 26-day-old pigs (Fig.
1A). PTP1B activity in liver
did not differ between 7- and 26-day-old pigs (Fig. 1B). In
both tissues, there was no difference in PTP1B activity between fasted
and fed groups.
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To evaluate the molecular mechanism for the developmental change in
PTP1B activation in skeletal muscle of neonatal pigs, we determined
PTP1B protein abundance, PTP1B association with IR, PTP1B tyrosine
phosphorylation state, and PTP1B association with Grb2. The results
showed that there was no difference between 7- and 26-day-old pigs in
PTP1B protein abundance in skeletal muscle (Fig.
2A). In the liver, PTP1B
protein abundance was significantly lower (P < 0.05)
in 7- than in 26-day-old pigs (Fig. 2B). Feeding did not
alter PTP1B abundance in either tissue.
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To determine whether the lower PTP1B activity in muscle of 7-day-old
pigs was due to a lower association of PTP1B with IR, equal amounts of
protein extracts from skeletal muscle and liver were subjected to
immunoprecipitation with an anti-IR -subunit antibody.
After SDS-PAGE and electrotransfer, polyvinylidene difluoride (PVDF)
membranes were incubated with anti-PTP1B antibody. Figure 3 shows that the association of PTP1B
with IR in muscle was significantly lower in 7-day-old compared with
26-day-old pigs. In contrast, there was no difference between the age
groups in the association of PTP1B with IR in liver. In both tissues,
the association of PTP1B with IR was not different between fasted and
fed groups.
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Recent studies indicate that PTP1B undergoes tyrosine phosphorylation
after insulin stimulation and that this is positively correlated with
enzyme activity (8, 12). To determine the effect of
development on PTP1B tyrosine phosphorylation, equal amounts of
skeletal muscle and liver extracts were immunoprecipitated with anti-PY
antibody followed by immunoblotting with anti-PTP1B antibody. Figure
4 shows that PTP1B tyrosine
phosphorylation in muscle was significantly lower in 7- than in
26-day-old pigs. PTP1B tyrosine phosphorylation levels in muscle of
fasted and fed pigs were similar. The PTP1B tyrosine phosphorylation in
liver was undetectable (data not shown).
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Because recent studies (21, 35) indicate that the binding
of Grb2 adaptor protein with PTP1B is important in forming the PTP1B-Grb2-IRS-1 ternary complex, followed by dephosphorylation of
IRS-1, we evaluated the effect of development on the association of
PTP1B with the Grb2 adaptor protein. Equal amounts of skeletal muscle
and liver extracts were immunoprecipitated with anti-Grb2 antibody,
followed by immunoblotting with an anti-PTP1B antibody. Figure
5 shows that Grb2 association with PTP1B
in muscle was significantly lower in 7- compared with 26-day-old pigs.
In liver, there was no difference between 7- and 26-day-old pigs in the association of Grb2 with PTP1B. In both tissues, there was no difference in the association of Grb2 with PTP1B between fasted and fed
pigs.
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DISCUSSION |
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Recent studies from our laboratory have shown an enhanced activation of the insulin-signaling pathway leading to translation initiation in skeletal muscle of the neonatal pig after food consumption (16, 26, 37). This feeding-induced activation of the IR, IRS-I, PI 3-kinase, and protein kinase B decreases with development in parallel with the developmental decline in the feeding-induced activation of translation initiation factors that regulate the binding of mRNA to 43S ribosomal complex. These developmental changes also parallel the developmental changes in the ability of feeding and insulin infusion to stimulate protein synthesis and are specific to skeletal muscle (13, 15). Because of the profound developmental decline in an initial step in the insulin-signaling cascade, i.e., the tyrosine phosphorylation of the IR (37), it was crucial that we ascertain the mechanism involved in this developmental decline in IR activation. We hypothesized that the activation of PTP1B, a major protein tyrosine phosphatase that regulates the phosphorylation of the IR and IRS-I, increases with development in muscle of neonatal pigs.
Several studies have demonstrated that a reduction in PTP1B activity correlates with an enhanced insulin sensitivity (8). For example, enhanced insulin sensitivity after weight loss is closely correlated with a reduction in the abundance of PTP1B (1). In the current study, we found that PTP1B activity was 30% lower in skeletal muscle of 7- compared with 26-day-old pigs, which is consistent with the previously reported increased tyrosine phosphorylation of the IR and the high insulin sensitivity of protein synthesis in skeletal muscle of 7- compared with 26-day-old pigs (14, 37, 46). Importantly, no developmental change in PTP1B activation in liver was detected, which is consistent with the previously reported lack of developmental change in insulin sensitivity in the liver (14, 37). There were no differences in PTP1B activity between fasted and fed pigs in the current study, although studies in intact cells have shown that insulin increases PTP1B activity (25, 35). Because the PTP1B activation is transient (23), we may have missed the peak level of insulin-stimulated PTP1B activity by measuring PTP1B activity 90 min after feeding. It has been demonstrated recently that basal PTP1B activity in obese Zucker rats is higher than that in lean Zucker rats and that PTP1B activity is positively correlated with insulin resistance (34). The results of the current study suggest that the basal level of PTP1B activity increases from 7 to 26 days in piglets and that this is associated with a developmental decline in insulin sensitivity.
The results of the current study also show that PTP1B protein abundance
in skeletal muscle did not change with development. In the liver,
however, PTP1B protein abundance was lower in 7- compared with
26-day-old pigs, even though there were no differences in PTP1B
activity in liver of both groups. PTP1B protein abundance is generally
positively correlated with PTP1B activity (40, 42).
However, a study using hepatoma cell culture indicated that treatment
with TNF- did not change PTP1B protein abundance but reduced PTP1B
activity by 66% (3). Furthermore, Bleyle et al.
(7) suggested that the level of PTP1B abundance is not the
primary determinant in its modulation of IR kinase activity. Thus the
binding of PTP1B to the IR may be more important than PTP1B abundance
in assessing the enzyme activity. Our results also indicate that there
are tissue-specific factor(s) that regulate the activation of PTP1B.
A direct interaction between the activated IR and PTP1B that leads to an increase in PTP1B tyrosine phosphorylation and PTP1B activation in intact cells has been reported (12). Here, we found that the lower activity of PTP1B in muscle of 7- compared with 26-day-old pigs was paralleled by a reduced interaction between PTP1B and the IR and a lower tyrosine phosphorylation of PTP1B in muscle of 7- compared with 26-day-old pigs. In contrast, there was no developmental change in the interaction between PTP1B and the IR in liver. We also found that, in both tissues, there were no differences in the PTP1B-IR complexes between fasted and fed pigs. Recently, Mur et al. (35) demonstrated in cell culture that insulin stimulates the interaction of PTP1B with the IR. The lack of feeding-induced formation of PTP1B-IR complexes in the current study (fasted vs. fed pigs) is probably due to the time point that we selected for killing the pigs (1.5 h after feeding). Indeed, Haj et al. (23) found that activated receptor tyrosine kinases become associated with PTP1B by 30 min after growth factor stimulation. PTP1B rapidly removes the phosphate groups from the tyrosine residues of the receptors, and the receptors are sent back either to the plasma membrane or to degradative lysosomes (23).
PTP1B forms a protein complex through the proline-rich sequence of its COOH terminus with SH3 domain-containing adaptor proteins, including Grb2 (31). Recent evidence suggests that Grb2, a well-known docking protein for IRS-1, mediates the association of IRS-1 with PTP1B, leading to IRS-1 dephosphorylation (21, 35). The formation of a ternary protein complex consisting of PTP1B, Grb2, and IRS-1 has been suggested to have a crucial role in the dephosphorylation of IRS-1 by PTP1B. The results of this study are consistent with this hypothesis and show that the association between PTP1B and Grb2 in skeletal muscle was lower in muscle of 7- compared with 26-day-old-pigs, with no developmental change in liver. Although we were unable to detect the formation of a ternary protein complex consisting of PTP1B, Grb2, and IRS-1 in vivo, in vitro studies that detected ternary complex formation used inactivated PTP1B that binds IRS-1 strongly (21). In our studies, PTP1B catalytic activity may still be active; therefore transient formation of a ternary complex could not be detected. Nevertheless, several studies have shown that Grb2 associates with SH2 domain-containing PTPases in a variety of cell types (29, 40, 45), suggesting that Grb2 has an important cellular role in the regulation of PTPase activity. The results of the current study support this hypothesis.
The available data indicate that there are a number of other PTPases,
including PTP1-, LAR, and SHP2 (9, 20). Therefore, the
contribution of these phosphatases in the regulation of insulin sensitivity in the neonate cannot be ruled out. Moreover, there are
other tissue-specific functions of PTP1B in addition to being the
regulator of insulin signaling. Recently, PTP1B has been identified as
the major PTP that dephosphorylates and activates c-Src in several
human breast cancer cell lines (6). PTP1B is also capable of antagonizing signaling by the EGF receptor by directly
dephosphorylating the EGF receptor tyrosine kinase (31).
Nevertheless, in this study, we provide evidence indicating the
involvement of PTP1B activation in the increased insulin sensitivity in
skeletal muscle of neonatal pigs.
Even though there is an abundance of experimental evidence indicating that PTP1B acts as a negative regulator of insulin signaling, direct interaction of PTP1B with the IR, which is crucial for dephosphorylation of the activated IR, has been documented only in cultured cell systems or in vitro studies (5, 12, 21, 35, 36, 43). For example, with use of brown adipocyte culture, the direct interaction between wild-type PTP1B and the IR was demonstrated in insulin-stimulated cells (35). Our current study, for the first time, showed evidence of the direct interaction between PTPIB and the IR in the intact animal. Furthermore, to the best of our knowledge, developmental changes in the activation of PTP1B, which may have a significant role in regulating the enhanced insulin sensitivity in the neonate, have not been previously studied.
It is surprising that we did not observe feeding-induced changes in PTP1B activity, PTP1B-IR interaction, PTP1B-Grb2 interaction, and tyrosine phosphorylation of PTP1B. However, the results of this study showed that the basal level of the activation of PTP1B was lower in 7- compared with 26-day-old-pigs and that this was associated with higher insulin sensitivity in skeletal muscle. We suggest that this study is comparable to a rodent study (34), which showed that basal PTP1B activity in obese Zucker rats is significantly higher than that in lean Zucker rats and that PTP1B activity is positively correlated with insulin resistance.
Perspectives. In the present study, we showed that the activity of PTP1B was lower in skeletal muscle of 7- compared with 26-day-old pigs. This lower activity was associated with a reduced association of PTP1B with the IR and Grb2 and decreased PTP1B tyrosine phosphorylation. Furthermore, these developmental changes appear to be tissue specific, because we did not find differences between age groups in PTP1B activation in liver. This developmental increase in skeletal muscle in the activity of PTP1B, which functions to dephosphorylate the activated IR, was consistent with our previously reported developmental decrease in IR phosphorylation (37). These changes were also consistent with a developmental decrease in the activation of downstream insulin-signaling proteins, i.e., IRS-1, PI 3-kinase, and protein kinase B (26, 37), translation initiation factors (16, 26), protein synthesis in skeletal muscle (13), and the ability of insulin to stimulate skeletal muscle protein synthesis (46).
Together, these results suggest that the increased insulin sensitivity in skeletal muscle of 7- compared with 26-day-old pigs is due, at least in part, to reduced PTP1B activation. The enhanced activation of the insulin-signaling pathway leading to translation initiation in skeletal muscle of the neonate likely has an important role in determining the high rates of protein synthesis in skeletal muscle and the more efficient use of dietary amino acids for growth in the neonate. ![]() |
ACKNOWLEDGEMENTS |
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We thank D. Burrin for helpful discussion, H. Nguyen and W. Liu for laboratory assistance, J. Cunningham and F. Biggs for care of animals, L. Loddeke for editorial review, and J. Croom for secretarial assistance.
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FOOTNOTES |
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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. The 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.
September 11, 2002;10.1152/ajpendo.00210.2002
Received 13 May 2002; accepted in final form 5 September 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahmad, F,
Azevedo JL,
Cortright R,
Dohm GL,
and
Goldstein BJ.
Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes.
J Clin Invest
100:
449-458,
1997
2.
Ahmad, F,
and
Goldstein BJ.
Alterations in specific protein-tyrosine phosphatases accompany insulin resistance of streptozotocin diabetes.
Am J Physiol Endocrinol Metab
268:
E932-E940,
1995
3.
Ahmad, F,
and
Goldstein BJ.
Effect of tumor necrosis factor-alpha on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases.
J Cell Biochem
64:
117-127,
1997[ISI][Medline].
4.
Ahmad, F,
Li PM,
Meyerovitch J,
and
Goldstein BJ.
Osmotic loading of neutralizing antibodies demonstrates a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway.
J Biol Chem
270:
20503-20508,
1995
5.
Bandyopadhyay, D,
Kusari A,
Kenner KA,
Liu F,
Chernoff J,
Gustafson TA,
and
Kusari J.
Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin.
J Biol Chem
272:
1639-1645,
1997
6.
Bjorge, JD,
Pang A,
and
Fujita DJ.
Identification of protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines.
J Biol Chem
275:
41439-41446,
2000
7.
Bleyle, LA,
Peng Y,
Ellis C,
and
Mooney RA.
Dissociation of PTPase levels from their modulation of insulin receptor signal transduction.
Cell Signal
11:
719-725,
1999[ISI][Medline].
8.
Byon, JC,
Kusari AB,
and
Kusari J.
Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction.
Mol Cell Biochem
182:
101-108,
1998[ISI][Medline].
9.
Calera, MR,
Vallega G,
and
Pilch PF.
Dynamics of protein-tyrosine phosphatases in rat adipocytes.
J Biol Chem
275:
6308-6312,
2000
10.
Cicirelli, MF,
Tonks NK,
Diltz CD,
Weiel JE,
Fischer EH,
and
Krebs EG.
Microinjection of a protein-tyrosine-phosphatase inhibits insulin action in Xenopus oocytes.
Proc Natl Acad Sci USA
87:
5514-5518,
1990[Abstract].
11.
Dadke, SS,
Kusari J,
and
Chernoff J.
Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region.
J Biol Chem
275:
23642-23647,
2000
12.
Dadke, SS,
Kusari A,
and
Kusari J.
Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor.
Mol Cell Biochem
221:
147-154,
2001[ISI][Medline].
13.
Davis, TA,
Burrin DG,
Fiorotto ML,
and
Nguyen HV.
Protein synthesis in skeletal muscle and jejunum is more responsive to feeding in 7- than in 26-day-old pigs.
Am J Physiol Endocrinol Metab
270:
E802-E809,
1996
14.
Davis, TA,
Fiorotto ML,
Beckett PR,
Burrin DG,
Reeds PJ,
Wray-Cahen D,
and
Nguyen HV.
Differential effects of insulin on peripheral and visceral tissue protein synthesis in neonatal pigs.
Am J Physiol Endocrinol Metab
280:
E770-E779,
2001
15.
Davis, TA,
Fiorotto ML,
Burrin DG,
Reeds PJ,
Nguyen HV,
Beckett PR,
Vann R,
and
O'Connor PMJ
Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs.
Am J Physiol Endocrinol Metab
282:
E880-E890,
2002
16.
Davis, TA,
Nguyen HV,
Suryawan A,
Bush JA,
Jefferson LS,
and
Kimball SR.
Developmental changes in the feeding-induced stimulation of translation initiation in muscle of neonatal pigs.
Am J Physiol Endocrinol Metab
279:
E1226-E1234,
2000
17.
Egawa, K,
Maegawa H,
Shimizu S,
Morino K,
Nishio Y,
Bryer-Ash M,
Cheung AT,
Kolls JK,
Kikkawa R,
and
Kashiwagi A.
Protein-tyrosine phosphatase-1B negatively regulates insulin signaling in L6 myocytes and Fao hepatoma cells.
J Biol Chem
276:
10207-10211,
2001
18.
Elchebly, M,
Payette P,
Michaliszyn E,
Cromlish W,
Collins S,
Loy AL,
Normandin D,
Cheng A,
Himms-Hagen J,
Chan CC,
Ramachandran C,
Gresser MJ,
Tremblay ML,
and
Kennedy BP.
Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene.
Science
283:
1544-1548,
1999
19.
Fox, HL,
Pham PT,
Kimball SR,
Jefferson LS,
and
Lynch CJ.
Amino acid effects on translational repressor 4E-BP1 are mediated primarily by L-leucine in isolated adipocytes.
Am J Physiol Cell Physiol
275:
C1232-C1238,
1998
20.
Goldstein, BJ,
Ahmad F,
Ding W,
Li PM,
and
Zhang WR.
Regulation of the insulin signaling pathway by cellular protein-tyrosine phosphatases.
Mol Cell Biochem
182:
91-99,
1998[ISI][Medline].
21.
Goldstein, BJ,
Bittner-Kowalczyk A,
White MF,
and
Harbeck M.
Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the Grb2 adaptor protein.
J Biol Chem
275:
4283-4289,
2000
22.
Goodyear, LJ,
Giorgino F,
Sherman LA,
Carey J,
Smith RJ,
and
Dohm GL.
Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects.
J Clin Invest
95:
2195-2204,
1995[ISI][Medline].
23.
Haj, FG,
Verveer PJ,
Squire A,
Neel BG,
and
Bastiaens PI.
Imaging sites of receptor dephosphorylation by PTP1B on the surface of the endoplasmic reticulum.
Science
295:
1708-1711,
2002
24.
Kenner, KA,
Anyanwu E,
Olefsky JM,
and
Kusari J.
Protein-tyrosine phosphatase 1B is a negative regulator of insulin- and insulin-like growth factor-I-stimulated signaling.
J Biol Chem
271:
19810-19816,
1996
25.
Kenner, KA,
Hill DE,
Olefsky JM,
and
Kusari J.
Regulation of protein tyrosine phosphatases by insulin and insulin-like growth factor I.
J Biol Chem
268:
25455-25462,
1993
26.
Kimball, SR,
Farrell PA,
Nguyen HV,
Jefferson LS,
and
Davis TA.
Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle.
Am J Physiol Endocrinol Metab
282:
E585-E592,
2002
27.
Kimball, SR,
Jefferson LS,
Nguyen HV,
Suryawan A,
Bush JA,
and
Davis TA.
Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process.
Am J Physiol Endocrinol Metab
279:
E1080-E1087,
2000
28.
Klaman, LD,
Boss O,
Peroni OD,
Kim JK,
Martino JL,
Zabolotny JM,
Moghal N,
Lubkin M,
Kim YB,
Sharpe AH,
Stricker-Krongrad A,
Shulman GI,
Neel BG,
and
Kahn BB.
Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice.
Mol Cell Biol
20:
5479-5489,
2000
29.
Kon-Kozlowski, M,
Pani G,
Pawson T,
and
Siminovitch KA.
The tyrosine phosphatase PTP1C associates with Vav, Grb2, and mSos1 in hematopoietic cells.
J Biol Chem
271:
3856-3862,
1996
30.
Kusari, J,
Kenner KA,
Suh KI,
Hill DE,
and
Henry RR.
Skeletal muscle protein tyrosine phosphatase activity and tyrosine phosphatase 1B protein content are associated with insulin action and resistance.
J Clin Invest
93:
1156-1162,
1994[ISI][Medline].
31.
Liu, F,
and
Chernoff J.
Protein tyrosine phosphatase 1B interacts with and is tyrosine phosphorylated by the epidermal growth factor receptor.
Biochem J
327:
139-145,
1997[ISI][Medline].
32.
Mendez, R,
Myers MG,
White MF,
and
Rhoads RE.
Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-1 phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase.
Mol Cell Biol
16:
2857-2864,
1996[Abstract].
33.
Meyerovitch, J,
Backer JM,
and
Kahn CR.
Hepatic phosphotyrosine phosphatase activity and its alterations in diabetic rats.
J Clin Invest
84:
976-983,
1989[ISI][Medline].
34.
Mohammad, A,
Wang J,
and
McNeill JH.
Bis(maltolato)oxovanadium(IV) inhibits the activity of PTP1B in Zucker rat skeletal muscle in vivo.
Mol Cell Biochem
229:
125-128,
2002[ISI][Medline].
35.
Mur, C,
Valverde AM,
Kahn CR,
and
Benito M.
Increased insulin sensitivity in IGF-I receptor-deficient brown adipocytes.
Diabetes
51:
743-754,
2002
36.
Seely, BL,
Staubs PA,
Reichart DR,
Berhanu P,
Milarski KL,
Saltiel AR,
Kusari J,
and
Olefsky JM.
Protein tyrosine phosphatase 1B interacts with the activated insulin receptor.
Diabetes
45:
1379-1385,
1996[Abstract].
37.
Suryawan, A,
Nguyen HV,
Bush JA,
and
Davis TA.
Developmental changes in the feeding-induced activation of the insulin-signaling pathway in neonatal pigs.
Am J Physiol Endocrinol Metab
281:
E908-E915,
2001
38.
Taghibiglou, C,
Rashid-Kolvear F,
Van Iderstine SC,
Le-Tien H,
Fantus IG,
Lewis GF,
and
Adeli K.
Hepatic very low density lipoprotein-ApoB overproduction is associated with attenuated hepatic insulin signaling and overexpression of protein-tyrosine phosphatase 1B in a fructose-fed hamster model of insulin resistance.
J Biol Chem
277:
793-803,
2002
39.
Taha, C,
and
Klip A.
The insulin signaling pathway.
J Membrane Biol
169:
1-12,
1999[ISI][Medline].
40.
Tauchi, T,
Feng GS,
Marshall MS,
Shen R,
Mantel C,
Pawson T,
and
Broxmeyer HE.
The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells.
J Biol Chem
269:
25206-25211,
1994
41.
Tonks, NK,
Cicirelli MF,
Diltz CD,
Krebs EG,
and
Fischer EH.
Effect of microinjection of a low-Mr human placenta protein tyrosine phosphatase on induction of meiotic cell division in Xenopus oocytes.
Mol Cell Biol
10:
458-463,
1990[ISI][Medline].
42.
Venable, CL,
Frevert EU,
Kim YB,
Fischer BM,
Kamatkar S,
Neel BG,
and
Kahn BB.
Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/protein kinase B activation.
J Biol Chem
275:
18318-18326,
2000
43.
Wang, XY,
Bergdahl K,
Heijbel A,
Liljebris C,
and
Bleasdale JE.
Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.
Mol Cell Endocrinol
173:
109-120,
2001[ISI][Medline].
44.
White, MF,
and
Kahn CR.
The insulin signaling system.
J Biol Chem
269:
1-4,
1994
45.
Wong, L,
and
Johnson GR.
Epidermal growth factor induces coupling of protein-tyrosine phosphatase 1D to Grb2 via the COOH-terminal domain Grb2.
J Biol Chem
271:
20981-20984,
1996
46.
Wray-Cahen, D,
Nguyen HV,
Burrin DG,
Beckett PR,
Fiorotto ML,
Reeds PJ,
Wester TJ,
and
Davis TA.
Response of skeletal muscle protein synthesis to insulin in suckling pigs decreases with development.
Am J Physiol Endocrinol Metab
275:
E602-E609,
1998