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Departments of Medicine (L.A.B., B.D.), Obstetrics and Gynecology (L.A.B.), and Pediatrics (J.S., L.Q., M.A., J.E.F.), University of Colorado Health Sciences Center, and Research Service of the Denver Veterans Affairs Medical Center (W.L., B.D.), Denver, Colorado 80262
Address all correspondence and requests for reprints to: Linda A Barbour, M.D., University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B-198, Denver, Colorado 80262. E-mail: lynn.barbour{at}uchsc.edu.
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regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase; P < 0.001), yet a reduced ability of insulin to stimulate IRS-1-associated PI 3-kinase activity (P < 0.05). Although the levels of the p110 catalytic subunit protein of PI 3-kinase and IRS-1 were unchanged in the TG mice, insulin’s ability to stimulate p110 association with IRS-1 was markedly reduced (P < 0.0001). We demonstrate a unique mechanism of insulin resistance and suggest that hPGH may contribute to the insulin resistance of normal pregnancy by increasing the expression of the p85 monomer, which competes in a dominant negative fashion with the p85-p110 heterodimer for binding to IRS-1 protein. |
Introduction |
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Recently, we demonstrated that human placental GH (hPGH), expressed in transgenic (TG) mice at levels comparable to those in the third trimester of human pregnancy, can cause severe total body insulin resistance (5). hPGH is a product of the hPGH variant gene and member of the GH family that differs from pituitary GH by 13 amino acids (6, 7). It is not regulated by GHRH and is secreted tonically from the placenta throughout gestation. It has the same affinity for the GH receptor and almost completely replaces pituitary GH in the maternal circulation by 20 wk of pregnancy (7). hPGH does not cross the placenta and appears to regulate the maternal levels of IGF-I that parallels the increasing levels of hPGH after 20 wk of pregnancy (3).
We previously characterized TG mice that overexpressed hPGH at levels similar to those measured in the third trimester of human pregnancy (5). These mice displayed severe insulin resistance with overt hyperinsulinemia to maintain normoglycemia. The insulin levels in TG mice were 4- to 7-fold greater than those in their wild-type (WT) littermates (fasting and postprandial, respectively), and glucose disposal, as measured by an insulin challenge test, was markedly reduced.
The present study was designed to evaluate the mechanisms by which hPGH can cause insulin resistance in these TG mice overexpressing hPGH. We examined the expression, phosphorylation, and activity of several signaling intermediates involved in the metabolic action of insulin in skeletal muscle as well as the effect of hPGH on the insulin-stimulated translocation of GLUT-4 to the plasma membrane. Our results indicate that hPGH mediates insulin resistance through a unique action by specifically increasing the expression of the p85 regulatory unit of phosphatidylinositol 3-kinase (PI 3-kinase), resulting in a marked reduction in insulin receptor substrate-1 (IRS-1)-associated PI 3-kinase activity.
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Materials and Methods |
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). Free fatty acid levels were not different in the two groups of mice. Furthermore, we found these animals to weigh nearly twice as much as their littermates, but their percent fat mass was slightly, but statistically, lower (15%) than that of WT mice. The hPGH levels were 92.25 ng/ml in TG mice compared with undetectable in the WT mice, and IGF-I levels were increased in TG mice (5). Mean fasting insulin levels were 1.57 ± 0.22 compared with 0.38 ± 0.07 ng/ml (P < 0.001) in TG and WT mice, respectively, and both groups were normoglycemic at 16 wk of age.
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Materials
Regular insulin was purchased from Novo Nordisk (Princeton, NJ). BSA and the protease inhibitors, aprotinin and leupeptin, were purchased from Roche (Indianapolis, IN). Polyclonal antibodies to the insulin receptor (IR) and p110 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to IRS-1, agarose-conjugated IRS-1, and p85 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). GLUT-4 antibody was obtained from Chemicon International (Temecula, CA). Ser307-IRS-1 antibody was supplied by Dr. Morris White (Joslin Laboratory, Boston, MA). Secondary horseradish peroxidase-conjugated antibody, protein A-Sepharose, and chemiluminescence reagents (ECL kit) were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). AG resins, polyvinylidene difluoride membranes, PAGE gel equipment, and protein assay kits were purchased from Bio-Rad Laboratories (Hercules, CA). [
-32P]ATP was obtained from NEN (Boston, MA).
Acute insulin stimulation in vivo and tissue collection
The mice were fasted for 6 h and anesthetized with ketamine (150 mg/kg) and acepromazine (5 mg/kg), abdominal cavities were opened, and portal veins were exposed. Approximately 300 mg gastrocnemius muscle from one hindlimb were rapidly removed and frozen immediately in liquid nitrogen. An insulin bolus of 10 U/kg body weight was then injected into the portal vein as described previously (9). Five minutes after injection, the gastrocnemius muscle from the opposite limb was excised and frozen immediately. The samples were stored at -80 C until analysis.
Western blotting of IR, IRS-1, p85, and p110
To determine the total protein levels of IR, IRS-1, p85, and p110, 75 µg muscle tissue homogenate protein were treated with Laemmli sample buffer, boiled for 5 min, resolved on a 7% denaturing SDS-PAGE gel, and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk (Bio-Rad Laboratories) in Tris-buffered saline-Tween 20 (TBS-T) for 1 h at room temperature. The membrane was washed three times with TBS-T and probed with a monoclonal anti-IR (1:1000 dilution in TBS-T), anti-IRS-1 (1:1000 dilution in TBS-T), anti-p85 antibody (1:2000 dilution in TBS-T), or anti-p110 antibody (1:500 dilution in TBS-T). Transfer and washing conditions were described previously (9). The bands were visualized with enhanced chemiluminescence and exposed to Kodak Biomax films (Eastman Kodak Co., Rochester, NY). The specific bands were quantitated using a Gel-Doc density scanner and Quantity One software (Bio-Rad Laboratories).
Phosphorylation of IR and IRS-1
To determine the Ser307 phosphorylation of IRS-1, 75-µg protein (using BSA as a standard) samples were subjected to 7% SDS-PAGE. After transferring and blocking, the membrane was incubated with anti-IRS-1-Ser307 antibody (1:1000 in TBS-T with 1% BSA overnight at 4 C). The membrane was detected as described above. Tyrosine phosphorylation of IR was determined by incubating 500 mg protein from the muscle homogenate overnight at 4 C with antiphosphotyrosine antibody (5 mg AB/8 mg protein) in 1 ml immunoprecipitation buffer containing 2% Triton X-100, 300 mmol/liter NaCl, 20 mmol/liter Tris-HCl, 2 mmol/liter EDTA, 1 mmol/liter EGTA, 0.4 mmol phenylmethylsulfonylfluoride, 0.4 mmol/liter sodium vanadate, and 1% Nonidet P-40. After immunoprecipitation, the samples were mixed with 50 ml protein A-Sepharose (10% solution) for 4 h at 4 C, and the immunoprecipitate was washed in 1 ml immunoprecipitation buffer, followed by centrifugation at 500 x g for 1 min at 4 C; this was repeated four times. The washed precipitate was mixed with Laemmli sample buffer (50 ml), boiled for 5 min, centrifuged for 5 min at 500 x g, and subjected to 7% SDS-PAGE. After transferring and blocking, the membrane was incubated with anti-IR or anti-IRS-1 antibodies. Bands were visualized by the previously described methodology.
Association of IRS-1 with p85 and p110
Immunoprecipitation of IRS-1 was performed by washing anti-IRS-1 agarose-conjugated beads (Upstate Biotechnology, Inc.) with PBS three times for 1 min each time at 14K rpm. A 400-µg lysate of protein was added to anti-IRS-1 agarose beads (10 µg anti-IRS-1), and the samples were rotated overnight at 4 C. The next morning, the samples were microfuged for 10 min at 4000 rpm at 4 C. The supernatant was removed, and the pelleted beads were washed with 600 ml ice-cold PBS for 2 min and microfuged three times. Pellets were speed-vacuumed to dryness, and 20 µl 2x Laemmli buffer were added to each sample. After boiling 3 min, samples were subjected to 7% SDS-PAGE. After transfer, the membrane was blocked overnight at 4 C with 5% milk in TBST. The membrane was rinsed three times for 20 min each time with TBST. A 20-ml quantity of a 1:1000 dilution of p85, p110, or IRS-1 in 5% milk/TBST was rocked overnight at 4 C, and the bands were visualized as described above.
IRS-1-associated PI 3-kinase activity
The level of IRS-1-associated PI 3-kinase activity was determined in muscle extracts after immunoprecipitation with IRS-1 antibody overnight at 4 C (400 µg muscle protein/4 µg antibody), followed by incubation with protein A-Sepharose overnight. The immunoprecipitation complex was spun at 14,000 x g for 10 min, followed by washing three times with isotonic PBS containing 1% Nonidet P-40, twice in 0.5 M LiCl2/100 mM Tris-HCl (pH 7.6), and twice with 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 1 mM EDTA. The pellets were resuspended in 50 µl of the final wash buffer, and 10 µl 100 mM MgCl2 were added along with 10 µl of a mixture containing 0.5 mg/liter -phosphatidylinositol (Sigma-Aldrich Corp., St. Louis, MO) in 10 mM Tris/1 mM EGTA, and the tube was sonicated for 20 sec. To start the PI 3-kinase reaction, 10 µl ATP mix containing 100 mM MgCl2, 10 mM Tris (pH 7.5), 0.55 mM ATP, and 1 mCi/ml [-32P]ATP were added for 10 min at room temperature. The reaction was stopped with 20 µl 8 N HCl, and 5 min later 160 µl CHCl3:MeOH were added (1:1). The phases were separated by centrifugation, and the lower organic phase was removed, lyophilized to dryness, and resuspended in 15 µl ethanol. Five microliters of the product were then loaded on to a silica gel thin layer chromatography plate precoated in 1% potassium oxalate. The lipids were resolved in CHCl3/MEOH/H2O/NH4OH (60:47:11.3:2), dried, and visualized by autoradiography. The images were quantified using a Kodak Dynamic phosphorimager, and results (in duplicate) were expressed as the percent stimulation over basal (arbitrary units) relative to the WT mice.
GLUT-4 translocation to the plasma membrane (PM)
Approximately 0.3 g muscle was pulverized in liquid nitrogen and placed in 2 ml ice-cold homogenization buffer [5 ml 0.25 M sucrose, 20 ml 0.5 mM EDTA (pH 8.0), 1 ml 50 mM HEPES (pH 7.7), 40 ml 2 mg/ml aprotinin, 20 ml 5 mg/ml leupeptin, 100 ml 0.2 mM PMSF, 60 ml 3 mM dithiothreitol, and 13.7 ml ddH2O) for each sample studied (four WT and four TG mice). The whole homogenate was transferred to a glass vial and homogenized using a motorized Teflon pestle. Each homogenate sample was then centrifuged for 10 min at 1,200 x g, and the supernatant was transferred to a 15-ml conical tube. To further increase the yield of plasma membrane, the pellet was resuspended in 1 ml homogenization buffer, homogenized, and centrifuged a second time at 1,200 x g. The supernatant from this second homogenized sample was added to the supernatant in the 15-ml conical tube, and the pellet that contained the nuclei was discarded. The supernatant fractions were centrifuged for 15 min at 9,000 x g to remove the lysosomes and peroxisomes. Pellets were discarded, and supernatants were transferred to polypropylene tubes and centrifuged at 25,000 x g. The pellet containing the Golgi and sarcoplasmic reticulum was discarded, and the supernatant was transferred to ultracentrifuge tubes and subjected to 100,000 x g centrifugation for 90 min to separate the remaining cytosolic fraction and to further enrich the plasma membranes. The pellets containing the plasma membranes were resuspended in 200 ml pellet resuspension buffer [0.25 M sucrose, 50 mM HEPES (pH 7.7), 100 mM KCl, 5 mM MgCl2, and protease inhibitors/dithiothreitol in ddH2O] and frozen at -80 C until analysis. For Western analysis, 75 µg plasma membrane protein was subjected to 12% SDS-PAGE using rabbit anti-GLUT-4 (1:1,000 dilution in 5% milk-TBS) according to the methods outlined above.
Statistical analysis
A two-sample t test for independent samples with equal variances were used for all comparisons. The results were represented as the mean ± SEM. P < 0.05 was considered significant.
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), there was only a minimal and insignificant change in the PM fraction in the TG mice. The amount of GLUT-4 present in the PM fraction after insulin injection in TG mice was approximately 5-fold less than that in WT mice (P = 0.05), confirming insulin resistance in mice expressing hPGH.
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, and p110 subunits of PI 3-kinase in skeletal muscle of WT and TG mice. The total IR protein levels were modestly, but statistically significantly, decreased by 19% (P < 0.01) in TG mice, but IRS-1 levels were not different between the groups of mice (Fig. 2). Most notable was a significant increase in the basal levels of p85 (Fig. 2), a regulatory subunit of the PI 3-kinase (P < 0.001). In contrast, p110 levels, the catalytic subunit of PI 3-kinase, were unchanged.
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). In contrast, insulin-stimulated, IRS-1-associated PI 3-kinase activity was significantly reduced in TG compared with WT mice (P < 0.05).
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and p110 association with IRS-1 protein, we determined whether insulin could stimulate binding of the PI 3-kinase heterodimer (p85-p110) to IRS-1. In the basal state, the association of p85 with IRS-1 was increased about 2-fold in TG compared with WT mice (Fig. 5A; P < 0.001), consistent with increased p85 expression in muscle of the TG mice. However, after insulin stimulation, there was a significant increase in the association of p85 with IRS-1 in the WT mice, but no further increase in TG mice (P < 0.001).
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monomer block an association of the p85-p110 heterodimer with IRS-1, resulting in lower PI 3-kinase activity (Fig. 5B
). To ensure that the amount of IRS-1 was immunoprecipitated equally in WT and TG samples, IRS-1 protein was determined in all IRS-1 immunoprecipitates (Fig. 5C). IRS-1 was equally immunoprecipitated in WT and TG samples. |
Discussion |
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regulatory subunit of this enzyme in skeletal muscle. The PI 3-kinase signaling pathway is activated by insulin binding to its cell surface receptor with immediate activation of tyrosine kinase activity and phosphorylation of the IR and IRS-1 proteins (10). Although we found a modest decrease in the number of IRs in TG mice, the most likely explanation for this observation is that the hyperinsulinemia present in these animals down-regulates the number of IRs. The fact that tyrosine and serine phosphorylation of IRS-1 was identical in WT and TG mice suggests that the mechanism of the hPGH-induced resistance does not involve impairment at the level of IRS-1. In contrast, a dramatic increase in the amount of p85 subunit of PI 3-kinase drew our attention to this signaling intermediate.
PI 3-kinase is composed of a regulatory subunit (p85) and a catalytic subunit (P110), which must form a heterodimer for PI 3-kinase activation to occur (11). This heterodimer then binds to tyrosine-phosphorylated IRS-1 (YXXM motif) using the SH2 domains of the p85 subunit. Here we demonstrate a decreased association of the p85-p110 heterodimer with IRS-1 after insulin stimulation despite an increased basal association of the p85 subunit with IRS-1. Our results suggest that excess p85 monomer can compete with the p85-p110 heterodimer for binding to IRS-1 in a dominant negative fashion, thereby causing a decrease in the IRS-1-associated PI 3-kinase activity and a subsequent reduction in GLUT-4 translocation to the plasma membrane. The latter has been clearly shown to be dependent upon normal PI 3-kinase activation (12).
Our hypothesis that increased expression of the p85 monomer could exert an inhibitory influence on PI 3-kinase activity is based on densitometry of Western blots, but is also supported by a number of publications in the literature. Terauchi and colleagues (13) first demonstrated that mice with a targeted heterozygous disruption of the gene encoding the p85 regulatory subunit of PI 3-kinase showed increased insulin sensitivity and hypoglycemia due to increased glucose transport in skeletal muscle and adipocytes. Ueki et al. (14) demonstrated a similar phenomenon in L6 myotubes, where increasing the levels of p85 inhibited both phosphotyrosine- and p110-associated PI 3-kinase activities. Mauvais-Jarvis and Kahn (15) also demonstrated that heterozygous deletion of the Pik3r1 gene coding for murine p85 and its splice variants actually improved insulin sensitivity in normal mice as well as in mice made insulin resistant by heterozygous deletions of IR or IRS-1. Mice with a deletion of p85 had lower fasting and postprandial glucose levels and a significant decrease in the incidence of diabetes when crossed with genetically insulin-resistant mice (15). Although our results are also consistent with an inhibitory role of p85, future experiments must address potential changes in other isoforms of p85 and p110 as well as the availability of free p85 in relationship to p85-p110 dimers. Insulin-resistant states may modify the expression of these isoforms differentially (16). There are three alternative p85 splice isoforms and four different p110 isoforms that could also be differentially regulated in insulin-resistant states. Any of these could play an additional role in uncoupling the p85 to p110 ratio. However, our finding of reduced PI 3-kinase activation is consistent with an inhibitory role for p85 given that phosphorylation of IRS-1 was unaffected, yet the excess p85 monomer prevented formation of the p85-p110 dimer. Thus, we postulate that a moderate increase in the expression of the p85 monomer resulted in altered stoichiometry of the p85-IRS complex compared with the p85/p110/IRS-1 complex necessary for PI 3-kinase activation. This may similarly apply to the insulin resistance of pregnancy and the insulin resistance induced by the expression of hPGH.
Even though the structure and regulation of hPGH differ from those of pituitary GH, both hormones bind the same receptors and could work in a similar fashion. The mechanism by which pituitary GH causes insulin resistance, as seen in acromegaly, has been examined using a TG mouse model (17). TG mice overexpressing pituitary GH demonstrated decreased expression of skeletal muscle IR and decreased tyrosine phosphorylation of IRS-1. Consistent with our data, pituitary GH overexpression was also associated with an increased abundance of the p85 subunit of PI 3-kinase and a decrease in insulin-stimulated PI 3-kinase activity. Similarly, the association of p85 to IRS-1 was increased in the basal state in mice overexpressing pituitary GH. However, unlike WT mice, which showed an increase in the association of p85 to IRS-1 after insulin stimulation, no such increase was noted in TG mice overexpressing pituitary GH (17). Thus, there may be a common mechanism between the action of pituitary and placental GH in triggering insulin resistance in skeletal muscle. The question of why normal tyrosine and serine/threonine phosphorylation of IRS-1 is not accompanied by even further increased association with p85 in TG mice in this and other studies remains enigmatic. However, this may be a function of the chronic hyperinsulinemia, hPGH, or both in these animals that causes increased p85 expression and prevents further recruitment to IRS-1.
Protein kinase B (Akt) as well as protein kinase C are downstream markers of PI-3 kinase activation. However, GH has been demonstrated to activate Akt and protein kinase C independently of insulin (18, 19, 20, 21). Thus, measurements of Akt activation cannot be used to assess insulin action in TG animals overexpressing hPGH, and the clearest indicator of insulin-induced PI 3-kinase activation is GLUT-4 translocation to the plasma membrane.
The insulin resistance of normal pregnancy is an essential physiological adaptation designed to restrict maternal glucose uptake to ensure that adequate nutrients are shunted to the growing fetus. This insulin resistance is thought to be mediated by placental hormones that reprogram the mother’s metabolism to divert a sufficient supply of glucose to meet fetal growth demands. Although hPL has classically been postulated to mediate this insulin resistance, hPL appears to play a more central role in stimulating insulin secretion by inducing the growth of pancreatic islets (4). Kirwan and colleagues (22) recently reported that in humans, circulating plasma TNF was produced by the placenta and correlated highly with in vivo insulin resistance during pregnancy, but hPGH was not studied. We demonstrated that hPGH is a likely candidate to mediate the insulin resistance of normal pregnancy (5). We found that TG mice overexpressing hPGH at levels similar to those found in the third trimester of human pregnancy (92 ng/ml) exhibited extreme insulin resistance characterized by fasting and postprandial hyperinsulinemia and decreased insulin-stimulated glucose disposal. These findings were the first to implicate hPGH as a metabolically active hormone capable of inducing the insulin resistance of pregnancy. Together these findings challenge the classic paradigm of hPL as the singular insulin resistance hormone of pregnancy and suggest PGH as a cause of insulin resistance in gestation. Our results agree with observations made by Friedman et al. (23) and Shao et al. (24) who studied the insulin resistance of human pregnancy. Friedman found that the skeletal muscle of healthy pregnant women in their third trimester of pregnancy manifested a nearly 2-fold increase in p85 levels. Although they did not report PI 3-kinase activity, there was a finding of reduced insulin-stimulated glucose transport (23), suggesting that increased p85 may have contributed to the insulin resistance.
In conclusion, we demonstrate that hPGH causes severe insulin resistance by specifically increasing the expression of the p85 subunit and subsequently affecting the ability of insulin to stimulate the association of the p85-p110 heterodimer with IRS-1, reducing PI 3-kinase insulin signaling. Although the effect by which hPGH selectively increases the p85 subunit and promotes insulin resistance may also involve the SOCS-1 (suppressors of cytokine signaling) and -6 or Janus kinase-2 pathways, which can cross-talk with insulin signaling (21, 25), the current data allow us to postulate that high levels of the p85 monomers may compete in a dominant negative fashion with the p85-p110 heterodimer for binding to tyrosine-phosphorylated IRS-1 proteins. We demonstrate a unique mechanism of insulin resistance in these mice and suggest that hPGH may contribute to the insulin resistance of normal pregnancy secondary to its effect on p85 expression and its interference with PI 3-kinase activity in skeletal muscle (Fig. 6).
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Acknowledgments |
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Footnotes |
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Abbreviations: hPGH, Human placental GH; hPL, human placental lactogen; IR, insulin receptor; IRS-1, insulin receptor substrate-1; PI 3-kinase, phosphatidylinositol 3-kinase; PM, plasma membrane; TBS-T, Tris-buffered saline-Tween 20; TG, transgenic; WT, wild-type.
Received September 29, 2003.
Accepted for publication November 11, 2003.
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  M. H. Vickers, S. Gilmour, A. Gertler, B. H. Breier, K. Tunny, M. J. Waters, and P. D. Gluckman 20-kDa placental hGH-V has diminished diabetogenic and lactogenic activities compared with 22-kDa hGH-N while retaining antilipogenic activity Am J Physiol Endocrinol Metab, September 1, 2009; 297(3): E629 - E637. [Abstract] [Full Text] [PDF]
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