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• W2099051331 abstract "Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85α, p85β, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85α is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85α-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85α, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85α in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85α or p85β, respectively (p85α +/– and p85β–/–). Wild type and p85β–/– mice developed in vivo insulin resistance and demonstrated overexpression of p85α and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85α+/–mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85α expression. These findings demonstrated the importance of increased p85α in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85α as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle. Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85α, p85β, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85α is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85α-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85α, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85α in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85α or p85β, respectively (p85α +/– and p85β–/–). Wild type and p85β–/– mice developed in vivo insulin resistance and demonstrated overexpression of p85α and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85α+/–mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85α expression. These findings demonstrated the importance of increased p85α in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85α as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle. Insulin resistance is a common feature associated with growth hormone excess; however, the cellular mechanism underlying insulin resistance remains elusive. We previously demonstrated that transgenic mice overexpressing human placental growth hormone (TG-hPGH), 2The abbreviations used are: TGtransgenicGHgrowth hormonerrGHrecombinant rat GHhPGHhuman placental growth hormoneIGFinsulin-like growth factorPIphosphatidylinositolLIDliver-specific IGF-1 gene deletionIRSinsulin receptor substrateGAGH-releasing hormone antagonistWTwild type 2The abbreviations used are: TGtransgenicGHgrowth hormonerrGHrecombinant rat GHhPGHhuman placental growth hormoneIGFinsulin-like growth factorPIphosphatidylinositolLIDliver-specific IGF-1 gene deletionIRSinsulin receptor substrateGAGH-releasing hormone antagonistWTwild type at levels comparable with the third trimester of pregnancy, were severely insulin-resistant and display increased amounts of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) in skeletal muscle (1Barbour L.A. Shao J. Qiao L. Leitner W. Anderson M. Friedman J.E. Draznin B. Endocrinology. 2004; 145: 1144-1150Crossref PubMed Scopus (136) Google Scholar).Several recent studies have suggested that a disrupted balance between the levels of the PI 3-kinase subunits may alter insulin-stimulated PI 3-kinase activity (2Ueki K. Fruman D.A. Brachmann S.M. Tseng Y.H. Cantley L.C. Kahn C.R. Mol. Cell. Biol. 2002; 22: 965-977Crossref PubMed Scopus (217) Google Scholar, 3Terauchi Y. Tsuji Y. Satoh S. Minoura H. Murakami K. Okuno A. Inukai K. Asano T. Kaburagi Y. Ueki K. Nakajima H. Hanafusa T. Matsuzawa Y. Sekihara H. Yin Y. Barrett J.C. Oda H. Ishikawa T. Akanuma Y. Komuro I. Suzuki M. Yamamura K-I. Kodama T. Suzuki H. Koyasu S. Aizawa S. Tobe K. Fukui Y. Yazaki Y. Kadowaki T. Nat. Genet. 1999; 21: 230-235Crossref PubMed Scopus (346) Google Scholar, 4Ueki K. Algenstaedt P. Mauvais-Jarvis F. Kahn C.R. Mol. Cell. Biol. 2000; 20: 8035-8046Crossref PubMed Scopus (127) Google Scholar, 5Mauvais-Jarvis F. Ueki K. Fruman D.A. Hirshman M.F. Sakamoto K. Goodyear L.J. Iannacone M. Accili D. Cantley L.C. Kahn C.R. J. Clin. Investig. 2002; 109: 141-149Crossref PubMed Scopus (217) Google Scholar, 6Ueki K. Fruman D.A. Uballe C.M. Fasshauer M. Klein J. Asano T. Cantley L.C. Kahn C.R. J. Biol. Chem. 2003; 278: 48453-48466Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). This enzyme consists of a regulatory subunit, p85, and a catalytic subunit, p110 (7Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). Normally, the regulatory subunit exists in stoichiometric excess to the catalytic one, resulting in a pool of free p85 monomers not associated with the p110 catalytic subunit. The p85 monomers bind to phosphorylated IRS proteins, blocking access to p85-p110 heterodimers. Thus, there exists a balance between the free p85 monomer and the p85-p110 heterodimer with the latter being responsible for the PI 3-kinase activity. Increases or decreases in expression of p85 shift this balance in favor of either free p85 or p85-p110 complexes (3Terauchi Y. Tsuji Y. Satoh S. Minoura H. Murakami K. Okuno A. Inukai K. Asano T. Kaburagi Y. Ueki K. Nakajima H. Hanafusa T. Matsuzawa Y. Sekihara H. Yin Y. Barrett J.C. Oda H. Ishikawa T. Akanuma Y. Komuro I. Suzuki M. Yamamura K-I. Kodama T. Suzuki H. Koyasu S. Aizawa S. Tobe K. Fukui Y. Yazaki Y. Kadowaki T. Nat. Genet. 1999; 21: 230-235Crossref PubMed Scopus (346) Google Scholar, 4Ueki K. Algenstaedt P. Mauvais-Jarvis F. Kahn C.R. Mol. Cell. Biol. 2000; 20: 8035-8046Crossref PubMed Scopus (127) Google Scholar, 5Mauvais-Jarvis F. Ueki K. Fruman D.A. Hirshman M.F. Sakamoto K. Goodyear L.J. Iannacone M. Accili D. Cantley L.C. Kahn C.R. J. Clin. Investig. 2002; 109: 141-149Crossref PubMed Scopus (217) Google Scholar, 6Ueki K. Fruman D.A. Uballe C.M. Fasshauer M. Klein J. Asano T. Cantley L.C. Kahn C.R. J. Biol. Chem. 2003; 278: 48453-48466Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Because the monomer and the heterodimer compete for the same binding sites on the IRS proteins, an imbalance could cause either increased or decreased PI 3-kinase activity.In the present study, we found that excess p85 regulatory subunits in TG-hPGH muscle was accounted for by a specific increase in the free p85α isoform. To elucidate whether the increase in p85α was mediated by GH or IGF-1, we selected an insulin-resistant knock-out mouse with a liver-specific deletion of IGF-1 and with compensatory high GH levels (8Yakar S. Liu J.-L. Fernandez A.M. Wu Y. Schally A.V. Frystyk J. Chernausek S.D. Mejia W. LeRoith D. Diabetes. 2001; 50: 1110-1118Crossref PubMed Scopus (301) Google Scholar, 9Yakar S. Setser J. Zhao H. Stannard B. Haluzik M. Glatt V. Bouxsein M.L. Kopchick J.J. LeRoith D. J. Clin. Investig. 2004; 113: 96-105Crossref PubMed Scopus (200) Google Scholar). Increased skeletal muscle p85α in liver-specific IGF-1 gene deletion (LID) mice was observed and was reversible along with insulin resistance with the administration of a growth hormone-releasing hormone antagonist. Finally, to confirm the role of p85α in the genesis of GH-induced insulin resistance, we took advantage of insulin-sensitive mice with either heterozygous deletions of p85α or a complete knockout of p85β (5Mauvais-Jarvis F. Ueki K. Fruman D.A. Hirshman M.F. Sakamoto K. Goodyear L.J. Iannacone M. Accili D. Cantley L.C. Kahn C.R. J. Clin. Investig. 2002; 109: 141-149Crossref PubMed Scopus (217) Google Scholar, 10Ueki K. Yballe C.M. Brachmann S.M. Vicent D. Watt J.M. Kahn C.R. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 419-424Crossref PubMed Scopus (197) Google Scholar, 11Lamia K.A. Peroni O.D. Kim Y.-B. Rameh L.E. Kahn B.B. Cantley L.C. Mol. Cell. Biol. 2004; 24: 5080-5087Crossref PubMed Scopus (87) Google Scholar) and investigated the effect of GH injections on skeletal muscle p85α levels and IRS-1-associated PI 3-kinase activity. Animals with heterozygous disruption of p85α gene (p85α+/– mice) were unable to increase p85α expression and remained insulin-sensitive, whereas their wild type (WT) and p85β–/– counterparts responded to 3 days of GH administration with increases in p85α expression, reduction in insulin-stimulated IRS-1-associated PI 3-kinase activity and insensitivity to insulin. Together, these findings suggested that increased p85α is a potent negative regulator of insulin signaling in skeletal muscle and insulin resistance in vivo in response to growth hormone.EXPERIMENTAL PROCEDURESAnimals—TG mice that overexpress human placental growth hormone (TG-hPGH) driven by the metallothionine promoter have been described previously (1Barbour L.A. Shao J. Qiao L. Leitner W. Anderson M. Friedman J.E. Draznin B. Endocrinology. 2004; 145: 1144-1150Crossref PubMed Scopus (136) Google Scholar, 12Barbour L.A. Shao J. Qiao L. Pulawa L.K. Jensen D.R. Bartke A.B. Garrity M. Draznin B. Friedman J.E. Am. J. Obstet. Gynecol. 2002; 186: 512-517Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The TG-hPGH mice are extremely insulin-resistant when examined by glucose and insulin challenge tests. Their IGF-1 levels are elevated, but free fatty acids are not increased.Mice heterozygous for the Pik3r1 allele (p85α+/–) and homozygous Pik3r2 knock-out mice (p85β–/–) have been characterized previously in the laboratories of Drs. Kahn and Cantley (5Mauvais-Jarvis F. Ueki K. Fruman D.A. Hirshman M.F. Sakamoto K. Goodyear L.J. Iannacone M. Accili D. Cantley L.C. Kahn C.R. J. Clin. Investig. 2002; 109: 141-149Crossref PubMed Scopus (217) Google Scholar, 10Ueki K. Yballe C.M. Brachmann S.M. Vicent D. Watt J.M. Kahn C.R. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 419-424Crossref PubMed Scopus (197) Google Scholar, 11Lamia K.A. Peroni O.D. Kim Y.-B. Rameh L.E. Kahn B.B. Cantley L.C. Mol. Cell. Biol. 2004; 24: 5080-5087Crossref PubMed Scopus (87) Google Scholar). All mice were of mixed genetic background consisting of 129S and C57BL/6; therefore, C57BL/6 mice were used as WT controls.Skeletal muscle from mice with a LID was kindly provided by D. LeRoith, NIDDK, National Institutes of Health, Bethesda, MD and have been described previously (8Yakar S. Liu J.-L. Fernandez A.M. Wu Y. Schally A.V. Frystyk J. Chernausek S.D. Mejia W. LeRoith D. Diabetes. 2001; 50: 1110-1118Crossref PubMed Scopus (301) Google Scholar, 9Yakar S. Setser J. Zhao H. Stannard B. Haluzik M. Glatt V. Bouxsein M.L. Kopchick J.J. LeRoith D. J. Clin. Investig. 2004; 113: 96-105Crossref PubMed Scopus (200) Google Scholar). These mice show a marked reduction in circulating IGF-1, elevated GH levels, and severe insulin resistance, primarily at the level of skeletal muscle. Treatment with a GH-releasing hormone antagonist, MZ4-71 (GA), for 28 days effectively normalized GH levels and also restored insulin sensitivity (9Yakar S. Setser J. Zhao H. Stannard B. Haluzik M. Glatt V. Bouxsein M.L. Kopchick J.J. LeRoith D. J. Clin. Investig. 2004; 113: 96-105Crossref PubMed Scopus (200) Google Scholar).Four to six mice in each group were used in each comparison. All mice were studied at ∼14–18 weeks and were fed a normal mouse diet ad libitum. The guidelines for the care and treatment of the animals were approved by the Institutional Animal Care and Use Committee at the University of Colorado Health Sciences Center.Materials—Regular insulin was purchased from Novo Nordisc, Princeton, NJ. Bovine serum albumin and protease inhibitors aprotinin and leupeptin were purchased from Roche Applied Science. Antibodies to IRS-1 and total (pan) p85, and specific to the α component of p85 (p85α), were purchased from Upstate Biotechnology, Lake Placid, NY. Antibodies to p110 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary horseradish peroxidase-conjugated antibody, protein A-Sepharose, and chemiluminescence reagents (ECL kit) were obtained from Amersham Biosciences. AG Resins, polyvinylidene difluoride membranes, PAGE gel equipment, and protein assay kits were from Bio-Rad. [γ-32P]ATP was obtained from PerkinElmer Life Sciences. Recombinant rat growth hormone (rrGH) was purchased from the National Hormone and Pituitary Program, Harbor-UCLA Medical Center (Los Angeles, CA).Insulin Tolerance Tests—Insulin tolerance tests were performed in p85α+/–, p85β–/–, and WT mice before and after GH injections (1 mg/kg subcutaneously twice daily for 3 days). Mice were fasted for 6 h and injected intraperitoneally with insulin (0.75 units/kg of body weight). Blood (5 μl) was collected from the tail vein at 0 and 60 min after insulin injection. Glucose measurements were performed with an Acucheck Advantage glucose meter.Acute Insulin Stimulation in Vivo and Tissue Collection—Mice were fasted for 6 h and anesthetized with ketamine (150 mg/kg) and acepromazine (5 mg/kg), and abdominal cavities were opened, and the inferior vena cava was exposed. Approximately 300 mg of gastrocnemius muscle from one hind limb was rapidly removed and frozen immediately in liquid nitrogen. An insulin bolus of 10 units/kg of body weight was then injected into the inferior vena cava vein as described previously (1Barbour L.A. Shao J. Qiao L. Leitner W. Anderson M. Friedman J.E. Draznin B. Endocrinology. 2004; 145: 1144-1150Crossref PubMed Scopus (136) Google Scholar). At 5 min 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 Total p85, p85α, and p110—Muscle tissue was homogenized, and protein was assayed as described previously (1Barbour L.A. Shao J. Qiao L. Leitner W. Anderson M. Friedman J.E. Draznin B. Endocrinology. 2004; 145: 1144-1150Crossref PubMed Scopus (136) Google Scholar). Membranes were blocked with 5% nonfat milk (Bio-Rad,) in TBS-T for 1 h at room temperature. The membrane was washed three times with TBS-T and probed with a polyclonal total p85 antibody (1:500 dilution), monoclonal p85α antibody (1:500 dilution), or polyclonal p110 antibody (1:250 dilution). Transfer and washing conditions were performed as described previously (1Barbour L.A. Shao J. Qiao L. Leitner W. Anderson M. Friedman J.E. Draznin B. Endocrinology. 2004; 145: 1144-1150Crossref PubMed Scopus (136) Google Scholar). The bands were visualized with enhanced chemiluminescence (ECL) and exposed to Kodak BIOMAX films (Eastman Kodak Co.). The specific bands were quantitated using a GEL-DOC density scanner and Quantity One software (Bio-Rad).Association of p85α with p110—Immunoprecipitation of p110 was carried out as described previously using polyclonal anti-p110 antibody (Santa Cruz Biotechnology) (1Barbour L.A. Shao J. Qiao L. Leitner W. Anderson M. Friedman J.E. Draznin B. Endocrinology. 2004; 145: 1144-1150Crossref PubMed Scopus (136) Google Scholar). Immunoblotting with either anti-p85α or anti-p110 antibodies was performed in the immunoprecipitates and supernatants.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 of muscle protein/4 μg of antibody) followed by incubation with protein A-Sepharose overnight, as described previously (1Barbour L.A. Shao J. Qiao L. Leitner W. Anderson M. Friedman J.E. Draznin B. Endocrinology. 2004; 145: 1144-1150Crossref PubMed Scopus (136) Google Scholar). The lipids were resolved by thin layer chromatography in CHCl3:MEOH:H20:NH4OH (60:47:11·3: 2), dried, and visualized by autoradiography. The images were quantified using a Kodak Dynamic phosphorimaging device.Cell Culture Experiments—3T3-L1 preadipocytes were grown to 60% confluence in growth medium (Dulbecco's modified Eagle's medium containing 5% glucose, 10% fetal calf serum, 50 μg/ml gentamicin, and 0.5 mm glutamine). After 24 h incubation with 1% fetal calf serum, cells were exposed to rrGH, either 500 or 2500 ng/ml for an additional 24 h. Cells were lysed, and expression of p85α was determined by Western blotting as described above.Statistics—Results are expressed as mean ± S.E. and compared using either paired or unpaired t test as indicated. p values of < 0.05 are considered statistically significant.RESULTSIn our initial experiments, we previously reported increases in the amounts of total p85 expressed in skeletal muscle of transgenic mice overexpressing hPGH (1Barbour L.A. Shao J. Qiao L. Leitner W. Anderson M. Friedman J.E. Draznin B. Endocrinology. 2004; 145: 1144-1150Crossref PubMed Scopus (136) Google Scholar). Further analyses revealed that the increases in p85 levels were predominantly accounted for by significant increases in p85α-specific isoform (p < 0.005; Fig. 1), whereas the levels of p110 were unchanged (data not shown). We confirmed this 2–3-fold increase in the p85α isoform by immunoprecipitating and immunoblotting with a p85α-specific antibody (data not shown).The catalytic p110 subunit was depleted by triple immunoprecipitation from the muscle lysate (4Ueki K. Algenstaedt P. Mauvais-Jarvis F. Kahn C.R. Mol. Cell. Biol. 2000; 20: 8035-8046Crossref PubMed Scopus (127) Google Scholar, 5Mauvais-Jarvis F. Ueki K. Fruman D.A. Hirshman M.F. Sakamoto K. Goodyear L.J. Iannacone M. Accili D. Cantley L.C. Kahn C.R. J. Clin. Investig. 2002; 109: 141-149Crossref PubMed Scopus (217) Google Scholar, 6Ueki K. Fruman D.A. Uballe C.M. Fasshauer M. Klein J. Asano T. Cantley L.C. Kahn C.R. J. Biol. Chem. 2003; 278: 48453-48466Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and the levels of p110 and p85α were analyzed by immunoblotting the immunoprecipitate and the supernatant. The amount of p85α recovered in the immunoprecipitate reflects p85α bound to p110, whereas the amount of p85α in the supernatant denotes free p85α subunit, existing in excess of p110.The levels of p85α in the p110 immunoprecipitate were similar between the two groups of mice. However, the amount of free p85α (recovered in the supernatant) was significantly increased (p < 0.02; in the TG-hPGH mice (Fig. 2), confirming a substantially greater expression of p85α in the insulin-resistant TG-hPGH animals.FIGURE 2Excess free p85α in supernatant unbound to p110 in WT and TG-hPGH mice. The p110 catalytic unit was depleted by triple immunoprecipitation from the muscle lysate, and the levels of p85α were analyzed by Western blot of the immunoprecipitate (IP) and the supernatant (Sn). The amount of p85α recovered in the immunoprecipitate reflects p85α bound to p110, whereas the amount of p85α in the supernatant denotes free p85α subunit, existing in excess of p110. The upper panel depicts a representative blot. The values are mean ± S.E. of the scanning densitometry values and expressed in arbitrary units; *, p < 0.05; WT versus TG-hPGH mice in supernatant (4–6 in each group). IB, immunoblot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because TG-hPGH animals have elevated IGF-1 levels (8Yakar S. Liu J.-L. Fernandez A.M. Wu Y. Schally A.V. Frystyk J. Chernausek S.D. Mejia W. LeRoith D. Diabetes. 2001; 50: 1110-1118Crossref PubMed Scopus (301) Google Scholar, 9Yakar S. Setser J. Zhao H. Stannard B. Haluzik M. Glatt V. Bouxsein M.L. Kopchick J.J. LeRoith D. J. Clin. Investig. 2004; 113: 96-105Crossref PubMed Scopus (200) Google Scholar), which could potentially contribute to the p85α excess, we measured the expression of p85α in a different model of insulin resistance and GH excess. LID mice demonstrate profound insulin resistance, primarily at the level of skeletal muscle (8Yakar S. Liu J.-L. Fernandez A.M. Wu Y. Schally A.V. Frystyk J. Chernausek S.D. Mejia W. LeRoith D. Diabetes. 2001; 50: 1110-1118Crossref PubMed Scopus (301) Google Scholar). These animals display low circulating levels of IGF-1 and compensatory elevations of GH (8Yakar S. Liu J.-L. Fernandez A.M. Wu Y. Schally A.V. Frystyk J. Chernausek S.D. Mejia W. LeRoith D. Diabetes. 2001; 50: 1110-1118Crossref PubMed Scopus (301) Google Scholar, 9Yakar S. Setser J. Zhao H. Stannard B. Haluzik M. Glatt V. Bouxsein M.L. Kopchick J.J. LeRoith D. J. Clin. Investig. 2004; 113: 96-105Crossref PubMed Scopus (200) Google Scholar). Furthermore, treatment of these animals with a GA for 28 days reduced their levels of GH and completely reversed their insulin resistance (9Yakar S. Setser J. Zhao H. Stannard B. Haluzik M. Glatt V. Bouxsein M.L. Kopchick J.J. LeRoith D. J. Clin. Investig. 2004; 113: 96-105Crossref PubMed Scopus (200) Google Scholar).We found that expression of p85α in skeletal muscle of LID mice was significantly higher (p < 0.01) than in WT mice (Fig. 3), suggesting a direct influence of GH. Moreover, treatment of LID mice with GA resulted in normalization of the p85α expression (Fig. 3) concomitant with a reversal of insulin resistance (9Yakar S. Setser J. Zhao H. Stannard B. Haluzik M. Glatt V. Bouxsein M.L. Kopchick J.J. LeRoith D. J. Clin. Investig. 2004; 113: 96-105Crossref PubMed Scopus (200) Google Scholar).FIGURE 3Expression of p85α in skeletal muscle of LID knock-out mice before and after administration of GA for 28 days. Equivalent amounts of protein isolated from the muscle of WT, LID, and LID+GA mice were subjected to SDS-PAGE gel and blotted with PI 3-kinase p85α antibody. Control animals treated with GA were also studied. A representative blot is shown in the upper panel. The values are mean ± S.E. of the scanning densitometry values (n = 4–6) and expressed in arbitrary units; *, p < 0.01; WT versus LID mice and LID+GA versus LID mice (n = 4–6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lastly, we performed a series of definitive experiments in animals lacking either p85α (heterozygotes p85α+/–) or p85β (homozygotes p85β–/–). These mice were studied before and after twice daily injections of GH for 3 days. Both p85α+/– and p85β–/– mice are more sensitive to insulin than the WT mice, as described previously (2Ueki K. Fruman D.A. Brachmann S.M. Tseng Y.H. Cantley L.C. Kahn C.R. Mol. Cell. Biol. 2002; 22: 965-977Crossref PubMed Scopus (217) Google Scholar, 5Mauvais-Jarvis F. Ueki K. Fruman D.A. Hirshman M.F. Sakamoto K. Goodyear L.J. Iannacone M. Accili D. Cantley L.C. Kahn C.R. J. Clin. Investig. 2002; 109: 141-149Crossref PubMed Scopus (217) Google Scholar, 10Ueki K. Yballe C.M. Brachmann S.M. Vicent D. Watt J.M. Kahn C.R. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 419-424Crossref PubMed Scopus (197) Google Scholar, 11Lamia K.A. Peroni O.D. Kim Y.-B. Rameh L.E. Kahn B.B. Cantley L.C. Mol. Cell. Biol. 2004; 24: 5080-5087Crossref PubMed Scopus (87) Google Scholar). Here we demonstrated that GH failed to induce insulin resistance in the p85α+/– mice while inducing insulin resistance in the WT and p85β–/– animals. Blood glucose levels were measured following an insulin challenge test before and after GH administration to document the development of insulin resistance, shown in Fig. 4. Although the WT and the p85β–/– mice became resistant to insulin after 3 days of GH injections, as noted by blunted blood glucose responses to insulin at 60 min (p < 0.005 versus pre-GH treatment), the p85α+/– mice remained highly sensitive to insulin.FIGURE 4Insulin tolerance tests in WT, p85α+/–, and p85β–/– before and after 3 days of administration of GH. Blood was collected from the tail vein at 0 and 60 min after insulin injection. The solid line indicates blood glucose values prior to GH injection; the dotted line indicates blood glucose values after GH injection. The values are mean ± S.E. of 4–6 animals/group expressed as mg/dl; *, p < 0.005 versus pretreatment with GH at 60 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The levels of p85α in skeletal muscle from the p85α+/– mice were ∼50% of those in the WT mice (Fig. 5). Although GH administration significantly increased p85α in the WT and p85β–/– animals (p < 0.05 and <0.01, respectively), it had no effect on p85α in the p85α+/– mice. As expected, insulin increased IRS-1-associated PI 3-kinase activity in all animals not exposed to GH (Fig. 6A, p < 0.01). Insulin-stimulated PI 3-kinase activity was somewhat greater in the p85α+/– mice than in two other groups. Moreover, whereas administration of GH blunted insulin-stimulated IRS-1-associated PI 3-kinase activity in the WT and p85β–/– animals, it failed to affect PI 3-kinase activity in the p85α+/– mice. In addition we measured the amount of p110 associated with IRS-1 in p85α+/– and p85β–/– mice versus controls treated with and without GH (Fig. 6B). As expected, we observed a reduced amount of p110 in the IRS-1 immunoprecipitates after GH treatment, confirming that p85α competes with the p85-p110 heterodimer for the IRS-1 binding sites in GH-treated animals. Notably, in p85α+/– mice, the GH effect is absent, mirroring the PI 3-kinase activity results, demonstrating that reducing the p85α levels restores insulin-stimulated p110 association with IRS-1.FIGURE 5Expression of p85α in WT, p85α+/–, and p85β–/– mice before and after GH treatment. Equivalent amounts of protein isolated from the muscle of experimental animals were subjected to SDS-PAGE gel and blotted with an antibody to p85α. A representative blot is shown in the upper panel. The values are mean ± S.E. of the scanning densitometry values expressed in arbitrary units; *, p < 0.05, and **, p < 0.01 versus pretreatment with GH (n = 4–6 in each group).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6PI 3-kinase activity in WT, p85α+/–, p85β–/–before and after GH treatment, with and without insulin. A, the level of IRS-1-associated PI 3-kinase activity was determined in muscle extracts as described under “Experimental Procedures.” PI3-P, PI 3-phosphate. B, the amount of IRS-1-associated p110 protein was determined in muscle extracts. The values are the mean ± S.E. of the scanning densitometry values expressed in arbitrary units; *, p < 0.01 insulin-stimulated versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that the effect of GH on p85α levels is direct, we examined the effect of rat recombinant GH on p85α levels in cultured 3T3-L1 preadipocytes (Fig. 7). GH treatment increased p85α protein expression in 3T3-L1 preadipocytes, demonstrating that GH directly increases p85α levels.FIGURE 7Effect of rrGH on p85α expression in 3T3-L1 preadipocytes. Cells were cultured to 60% confluence and challenged with rrGH, either 500 or 2500 ng/ml for 24 h. Results demonstrate a representative blot and a summary of three experiments. The values are expressed as the mean ± S.E. *, p < 0.01. control versus GH treated (2500 ng/ml).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe seminal finding of this investigation was that GH, whether endogenous or administered exogenously, induces in vivo insulin resistance by increasing the amount of skeletal muscle p85α monomers, unbound to its catalytic p110 subunit. Confirmation of this mechanism was illustrated in this study by critical observations in knock-out mice of opposing insulin sensitivity. Liver-specific IGF-1 knock-out mice, known to be insulin-resistant and to manifest high GH levels (8Yakar S. Liu J.-L. Fernandez A.M. Wu Y. Schally A.V. Frystyk J. Chernausek S.D. Mejia W. LeRoith D. Diabetes. 2001; 50: 1110-1118Crossref PubMed Scopus (30" @default.
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• W2099051331 title "Increased P85α Is a Potent Negative Regulator of Skeletal Muscle Insulin Signaling and Induces in Vivo Insulin Resistance Associated with Growth Hormone Excess" @default.
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