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• W2097068854 abstract "Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. The p70 ribosomal protein S6 kinase 1 (S6K1) 2The abbreviations used are: S6K1, p70 ribosomal protein S6 kinase 1; mTOR, mammalian target of rapamycin; TSC, tuberous sclerosis complex; PI 3-kinase, phosphoinositide-3-kinase; PI(3)P, phosphatidylinositol 3-phosphate; DMEM, Dulbecco's modified Eagle's medium; siRNA, short interference-RNA; pfu, plaque-forming units; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; GFP, green fluorescent protein; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; RNAi, RNA interference; MOPS, 4-morpholinepropanesulfonic acid. 2The abbreviations used are: S6K1, p70 ribosomal protein S6 kinase 1; mTOR, mammalian target of rapamycin; TSC, tuberous sclerosis complex; PI 3-kinase, phosphoinositide-3-kinase; PI(3)P, phosphatidylinositol 3-phosphate; DMEM, Dulbecco's modified Eagle's medium; siRNA, short interference-RNA; pfu, plaque-forming units; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; GFP, green fluorescent protein; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; RNAi, RNA interference; MOPS, 4-morpholinepropanesulfonic acid. participates in a variety of intracellular signaling events, including mRNA translation, gene transcription, and cell cycle control (1Proud C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar, 2Volarevic S. Thomas G. Prog. Nucleic Acids Res. Mol. Biol. 2001; 65: 101-127Crossref PubMed Google Scholar, 3Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (595) Google Scholar). Insulin, and several nutrients such as amino acids, are known to increase the phosphorylation and activation of S6K1 (4Patti M.E. Brambilla E. Luzi L. Landaker E.J. Kahn C.R. J. Clin. Investig. 1998; 101: 1519-1529Crossref PubMed Google Scholar). A negative feedback loop involving S6K1 phosphorylation of IRS1 serves to limit insulin signaling (5Tremblay F. Marette A. J. Biol. Chem. 2001; 276: 38052-38060Abstract Full Text Full Text PDF PubMed Google Scholar, 6Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Nature. 2004; 431: 200-205Crossref PubMed Scopus (1330) Google Scholar). Thus, the absence of S6K1 results in an enhancement of insulin sensitivity and a reduction of body fat in mice (6Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Nature. 2004; 431: 200-205Crossref PubMed Scopus (1330) Google Scholar). Conversely, S6K1 activity is elevated in several tissues of animal models of obesity, and/or type 2 diabetes, and correlates with increased insulin resistance (6Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Nature. 2004; 431: 200-205Crossref PubMed Scopus (1330) Google Scholar). Both insulin and amino acid induction of S6K1 activity are mediated by mTOR, an evolutionarily conserved serine/threonine kinase that is the mammalian target of rapamycin (7Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1087) Google Scholar, 8Jonassen A.K. Mjos O.D. Sack M.N. Biochem. Biophys. Res. Commun. 2004; 315: 160-165Crossref PubMed Scopus (41) Google Scholar). mTOR is found in a complex (mTORC1) with raptor and mLST8 and is activated by the small GTPase Rheb (9Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1377) Google Scholar, 10McMahon L.P. Yue W. Santen R.J. Lawrence Jr., J.C. Mol. Endocrinol. 2005; 19: 175-183Crossref PubMed Scopus (47) Google Scholar). The tuberous sclerosis proteins TSC1 and TSC2 maintain Rheb in the inactive GDP-bound state because of the Rheb-GAP enzyme activity of TSC2. Activation of mTOR by insulin is associated with phosphorylation of TSC2, dissociation of the Rheb-TSC1-TSC2 complex, and relief of Rheb repression (11Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (703) Google Scholar, 12Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar, 13Cai S.L. Tee A.R. Short J.D. Bergeron J.M. Kim J. Shen J. Guo R. Johnson C.L. Kiguchi K. Walker C.L. J. Cell Biol. 2006; 173: 279-289Crossref PubMed Scopus (269) Google Scholar). One of the key enzymes responsible for phosphorylation of TSC2 by insulin is the serine/threonine kinase Akt (14Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1866) Google Scholar, 15Aoki M. Blazek E. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 136-141Crossref PubMed Scopus (283) Google Scholar), which is activated in a class I phosphoinositide 3-kinase (PI 3-kinase)-dependent manner (16Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (964) Google Scholar, 17Saltiel A.R. Cell. 2001; 104: 517-529Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). In contrast, nutrients such as amino acids activate the mTOR-S6K1 pathway independently of the insulin pathway, via the class III PI 3-kinase hVps34 (18Hinault C. Mothe-Satney I. Gautier N. Lawrence Jr., J.C. Van Obberghen E. FASEB J. 2004; 18: 1894-1896Crossref PubMed Scopus (74) Google Scholar, 19Nobukuni T. Joaquin M. Roccio M. Dann S.G. Kim S.Y. Gulati P. Byfield M.P. Backer J.M. Natt F. Bos J.L. Zwartkruis F.J. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14238-14243Crossref PubMed Scopus (608) Google Scholar). Unlike insulin, amino acids cause GTP loading on Rheb in a TSC2-independent manner suggesting a parallel pathway leading to mTOR activation. This nutrient-regulated pathway is essential for Akt/TSC2/mTOR signaling, however, because inhibition of hVps34 or its product PI(3)P prevents the insulin stimulation of S6K1 (19Nobukuni T. Joaquin M. Roccio M. Dann S.G. Kim S.Y. Gulati P. Byfield M.P. Backer J.M. Natt F. Bos J.L. Zwartkruis F.J. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14238-14243Crossref PubMed Scopus (608) Google Scholar). Recently, Du et al. (21Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (698) Google Scholar) identified TRB3, a mammalian homolog of Drosophila tribbles, also called NIPK (neuronal cell death-inducible protein kinase) (20Mayumi-Matsuda K. Kojima S. Suzuki H. Sakata T. Biochem. Biophys. Res. Commun. 1999; 258: 260-264Crossref PubMed Scopus (60) Google Scholar), as an endogenous inhibitor of Akt. TRB3 binds to unphosphorylated Akt and prevents its activation. Expression of TRB3 mRNA is induced in the liver by fasting and is elevated in db/db mice. Adenoviral expression of TRB3 impairs insulin-mediated glycogen synthesis and prevents inhibition of gluconeogenesis, thus increasing blood glucose levels (21Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (698) Google Scholar, 22Koo S.H. Satoh H. Herzig S. Lee C.H. Hedrick S. Kulkarni R. Evans R.M. Olefsky J. Montminy M. Nat. Med. 2004; 10: 530-534Crossref PubMed Scopus (472) Google Scholar). These observations have led to the suggestion that TRB3 elevation contributes to the development of insulin resistance. In support of this notion, knockdown of TRB3 improves glucose tolerance in C57BL/6 mice, and TRB3 overexpression reverses the insulin-sensitive phenotype of PGC1-deficient mice (22Koo S.H. Satoh H. Herzig S. Lee C.H. Hedrick S. Kulkarni R. Evans R.M. Olefsky J. Montminy M. Nat. Med. 2004; 10: 530-534Crossref PubMed Scopus (472) Google Scholar). However, TRB3-mediated inhibition of Akt may also inhibit insulin-induced S6K1 activation and protein synthesis. Inhibition of S6K1 is associated with insulin sensitization because of negative feedback on IRS1 (6Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Nature. 2004; 431: 200-205Crossref PubMed Scopus (1330) Google Scholar), so TRB3 might mitigate insulin resistance. In this study, we investigated the role of TRB3 in nutrient- and insulin-induced activation of S6K1, both in vitro and in vivo. We find that TRB3 is a potent inhibitor of insulin-stimulated, but not nutrient-stimulated, S6K1 activation. TRB3 protein levels are constitutively elevated in db/db mice and are not responsive to nutrients or insulin. Consequently, Akt signaling is severely compromised, but surprisingly, insulin activation of S6K1 is normal in db/db mice despite the elevated TRB3. Materials—[γ-32P]ATP was purchased from Amersham Biosciences. Phosphospecific rabbit polyclonal antibodies against p-Akt (Thr-380 and Ser-473), p-TSC2/tuberin (Thr-1462), p-mTOR (Ser-2448), p-S6K1 (Thr-389), p-S6K1 (Thr-421/Ser-424), p-S6 ribosomal protein (Ser-235/236), p-4E-BP1 (Ser-65), and p-GSK3α/β (Ser-21/9) and antibodies against Akt, mTOR, S6K1, 4E-BP1, GSK3, and p44/42 mitogen-activated protein kinase (ERK1/ERK2) were from Cell Signaling (Beverly, MA). Antibodies against TRB3-(1–145) were purchased from Calbiochem. Analytical grade porcine insulin was from Sigma. Plasmids—Mammalian expression plasmids were used to express the following proteins: TRB3 (Dr. M. Montminy, Salk Institute for Biological Studies, La Jolla, CA); FLAG-tagged 4E-BP-1 (FLAG-4E-BP1) (Dr. K. L. Guan, University of Michigan Medical School, Ann Arbor, MI); and HA-tagged S6K1 (HA-S6K1) (Dr. J. Blenis, Harvard Medical School, Boston). Preparation of Recombinant Adenovirus—The recombinant adenoviruses encoding TRB3, or green fluorescent protein (GFP), were generated using the AdEasy system, as described previously (23He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3221) Google Scholar). The recombinant adenovirus was amplified in HEK293 cells, and the 50% tissue culture infectious dose (TCID50) was determined as pfu/ml (24Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Cell Culture and Recombinant Adenovirus Infection—HepG2 cells and HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Mouse hepatocytes were isolated from the livers of male ddY mice (25–35 g, 8–10 weeks) using collagenase perfusion (25Ichihara A. Nakamura T. Tanaka K. Mol. Cell. Biochem. 1982; 43: 145-160Crossref PubMed Scopus (94) Google Scholar) and were plated onto culture dishes. Prior to experimentation, hepatocytes were maintained in William's medium supplemented with 10% FBS, 1 nm dexamethasone, and 1 nm insulin. Cells were infected by adenovirus at the multiplicity of infection (m.o.i. pfu/cell) indicated. Preparation and Transfection of Synthetic siRNA—All DNA/RNA chimeric oligonucleotides were purchased from Qiagen (Valencia, CA). Double-stranded RNA duplexes, corresponding to amino acids 34–40 of human TRB3 (5′-CGAGCUCGAAGUGGGCCCC-3′), and control GFP were purified and transfected into human HepG2 hepatocytes using Lipofectamine 2000 reagent (Invitrogen). Cells were used 24–48 h following transfection. Stimulation of Liver Cells with Insulin, Pervanadate, Amino Acids, or Nutrients—Primary mouse hepatocytes, and HepG2 cells, were treated with insulin (100 nm) for the indicated times. In some experiments, primary mouse hepatocytes were treated for 15 min with pervanadate (100 μm), an inhibitor of tyrosine phosphatases that causes activation of Akt. For amino acid stimulation, subconfluent HepG2 cells were starved for 24 h in serum-free DMEM with 0.1% bovine serum albumin and then incubated in amino acid-free medium for 2 h. Cells were then incubated for 60 min with, or without, an isomolar mixture of amino acids. The concentrations of each amino acid used were based on levels from the portal vein of a fasted rat (26Blommaart E.F. Luiken J.J. Blommaart P.J. van Woerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). For nutrient stimulation, cells were washed and preincubated with PBS for 1 h and then incubated in either PBS or DMEM for 30 min. In some experiments, wortmannin (50 nm) or rapamycin (25 ng/ml) was added to the incubation medium 30 min prior to and during the stimulation with insulin, amino acids, or nutrients. Following stimulation, cells were washed once with PBS and then harvested. Cells were lysed, and the cell lysates were processed for immunoblotting or the S6K1 assay. Stimulation of Epitope-tagged S6K1 or 4E-BP1 by Nutrients in HepG2 Cells—HepG2 cells were transiently co-transfected with plasmids encoding HA-S6K1 and FLAG-4E-BP1, and then nutrient stimulation was conducted 48 h following transfection. Cell extracts were immunoprecipitated using 2 μg of anti-HA tag antibody (Sigma) or anti-FLAG tag antibody (Sigma) and protein A-Sepharose beads (Amersham Biosciences). The precipitated proteins were eluted from the beads with SDS sample buffer and analyzed by immunoblotting, using the antibodies indicated. Immunoblotting—The antibodies used for immunoblotting were as indicated, and signals were detected using horseradish peroxidase-mediated chemiluminescence (Amersham Biosciences), as described previously (24Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Assay of S6K1 Activity—S6K1 activity was measured using the p70S6 kinase assay kit following the manufacturer's protocol (Upstate Biotechnology, Inc., Lake Placid, NY). Briefly, cells were homogenized using ultrasonication in lysis buffer (10 nm Tris-HCl, pH 7.4, 100 mm NaCl, 1% Nonidet P-40, 1% Triton X-100, 50 mm NaF, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin), and the endogenous S6K1 in the supernatant was immunoprecipitated using 2 μg of anti-S6K1 antibody and protein A-Sepharose. The immunoprecipitates were resuspended in dilution buffer (20 mm MOPS at pH7.2, 25 mm β-glycerophosphate, 5 mm EGTA, 1 mm sodium orthovanadate, and 1 mm dithiothreitol). The kinase reaction was performed for each precipitated sample in an assay mixture containing 50 μm substrate peptide (AKRRRLSSLRA) and 0.2 μCi of [γ-32P] ATP. Following incubation for 10 min at 30 °C, aliquots were applied onto P81 phosphocellulose squares and washed, and radioactivity was measured using a scintillation counter. Animal Experiments—In vivo experiments were conducted using 8–9-week old-male C57BL/6 and db/db mice. The mice were housed under controlled light/dark (12/12 h) and temperature conditions. All procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Animal Subjects Committee of the University of California, San Diego. Mice were fed normal rodent chow (type MF, Oriental Yeast, Tokyo, Japan) and tap water ad libitum. Exogenous overexpression of TRB3 or GFP was induced in the livers of C57BL/6 mice via the injection of 5 × 108 pfu/ml of virus into the tail vein as described (27Miyake K. Ogawa W. Matsumoto M. Nakamura T. Sakaue H. Kasuga M. J. Clin. Investig. 2002; 110: 1483-1491Crossref PubMed Scopus (129) Google Scholar). Mice were euthanized 7 days following virus injection. During the course of the experiments, mice were fasted for 24 h with free access to water, and the fasting blood glucose was monitored. Mice were refed for the following 24 h, and blood glucose was again determined. This fasting-feeding protocol was repeated for 7 days. All mice were sacrificed for blood and liver collection at the end of the experiments. Blood samples were collected from the tail vein. Plasma was obtained by centrifugation of collected blood and assayed for insulin. Blood glucose and plasma insulin levels were determined as described previously (28Kanezaki Y. Obata T. Matsushima R. Minami A. Yuasa T. Kishi K. Bando Y. Uehara H. Izumi K. Mitani T. Matsumoto M. Takeshita Y. Nakaya Y. Matsumoto T. Ebina Y. Endocr. J. 2004; 51: 133-144Crossref PubMed Scopus (10) Google Scholar). Some of the mice were subjected to insulin injection (0.75 units/kg body weight, 10 min) prior to sacrifice. For glucose tolerance tests, mice fasted for 24 h were injected intraperitoneally with 2 g of glucose per kg of body weight. For insulin tolerance tests, mice fasted for 24 h were injected intraperitoneally with 0.75 units of insulin per kg of body weight. Blood glucose level was measured in tail vein blood collected at the designated times. Statistical Analysis—Data are expressed as means ± S.D. and were analyzed using analysis of variance and Bonferroni multiple comparison tests. p < 0.05 was accepted as statistically significant. TRB3 Inhibits Insulin-induced Activation of S6K1 Both in Vitro and in Vivo—TRB3 is an endogenous inhibitor of Akt (21Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (698) Google Scholar). Therefore, enhanced expression of TRB3 is expected to inhibit insulin-induced activation of S6K1 through the inhibition of Akt. Adenoviral overexpression of TRB3 in primary mouse hepatocytes inhibited insulin-stimulated S6K1 activity in a dose-dependent manner (Fig. 1A). TRB3 overexpression inhibited phosphorylation of Akt (Thr-308 and Ser-473), TSC2/tuberin (Thr-1462), and mTOR (Ser-2448), all of which are located upstream of S6K1 activation in the insulin signaling pathway, and also inhibited phosphorylation of the S6 ribosomal protein (Ser-235/236), a substrate of S6K1 (Fig. 1B). It should be noted that stimulation of endogenous S6K activity as measured by S6 phosphorylation is greater than measurement of S6K activity in vitro. We ascribe this difference to the high basal activity associated with the in vitro kinase assay. Additionally, overexpression of TRB3 inhibited the insulin-induced phosphorylation of another mTOR target, the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which regulates the initiation of 5′-capped mRNA translation (29Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Phosphorylation of the major mTOR-regulated site (Ser-65) is dramatically inhibited (30Gingras A.C. Raught B. Gygi S.P. Niedzwiecka A. Miron M. Burley S.K. Polakiewicz R.D. Wyslouch-Cieszynska A. Aebersold R. Sonenberg N. Genes Dev. 2001; 15: 2852-2864Crossref PubMed Scopus (1161) Google Scholar) (Fig. 1B). In contrast, TRB3 had little effect on insulin-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 (Fig. 1B). Similar results are seen when cells are stimulated with sodium pervanadate (21Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (698) Google Scholar). The level of S6K1 phosphorylation at Thr-389, a site targeted by mTOR (31Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2329) Google Scholar), was elevated by pervanadate treatment (Fig. 1C). No phosphorylation of S6K1 on Thr-421/Ser-424 was observed (Fig. 1C). These are the rapamycin-insensitive phosphorylation sites in mouse hepatocytes (31Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2329) Google Scholar). Adenoviral overexpression of TRB3 greatly inhibited the phosphorylation of Thr-389 in response to pervanadate (Fig. 1C), in agreement with the insulin results. We examined the effect of RNA interference (RNAi) of TRB3 in HepG2 cells that are known to express endogenous TRB3 (21Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (698) Google Scholar). Knockdown of TRB3 enhanced basal S6K1 activity 1.2-fold (p < 0.01), and enhanced insulin-stimulated S6K1 activity 1.6-fold (p < 0.05) (Fig. 2A). Transfection with TRB3 RNAi caused a 70% reduction in endogenous TRB3 protein levels in HepG2 cells (Fig. 2B). We also examined the effects of TRB3 RNAi on the insulin-stimulated phosphorylation of the S6 ribosomal protein and 4E-BP1. Knockdown of TRB3 resulted in no effect on basal S6 and 4E-BP1 phosphorylation but increased the insulin-stimulated phosphorylation of S6 ribosomal protein and 4E-BP1 2.5-fold (Fig. 2, B and C). Thus, endogenous TRB3 provides tonic inhibition of insulin-stimulated S6K1 activity in hepatocytes. To investigate the effect of TRB3 on insulin stimulation of S6K1 in vivo, we infected adult male C57BL/6 mice with adenoviruses encoding TRB3 or GFP as control. TRB3-infected mice were treated with or without insulin (0.75 units/kg body weight) for 10 min, and the hepatic S6K1 activity was measured. As shown in Fig. 3A, adenoviral overexpression of TRB3 completely suppressed the insulin-induced hepatic S6K1 activation in mice. TRB3 also suppressed the insulin-stimulated phosphorylation of the endogenous substrate S6 protein (Fig. 3B). To determine the physiological effects of exogenous TRB3 overexpression, we examined body weight, food intake, and metabolic parameters in these animals. Control GFP-infected mice and TRB3-infected mice had similar increases in body weight over 7 days, although food consumption did not change (Table 1). TRB3 expression had no effect on either insulin or blood glucose level in the fasted state. Plasma insulin concentrations in the fed state were modestly increased in TRB3-infected mice (2.3 ng/ml) versus GFP-infected mice (1.6 ng/ml) on day 7 following viral infection. Blood glucose levels were also increased in the fed state (155 versus 114 mg/dl, n = 6). As shown in Fig. 4, A and B, overexpression of TRB3 elevated the blood glucose excursion during a glucose tolerance test and impaired the glucose lowering effect of insulin during an insulin tolerance test as has been published previously (21Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (698) Google Scholar). Given the results from both in vitro and in vivo studies, we conclude that TRB3 inhibits insulin signaling via the Akt/TSC2/mTOR/S6K1 pathway in the liver and creates a state of insulin resistance.TABLE 1Metabolic parameters in adenoviral infected miceVirusWeightFood intakeInsulinGlucoseFedFastedFedFastedGFPTRB3GFPTRB3GFPTRB3GFPTRB3GFPTRB3GFPTRB3gg/dayng/mlmg/mlDay 117.8 ± 0.5617.4 ± 0.842.56 ± 0.232.68 ± 0.261.6 ± 0.451.6 ± 0.230.4 ± 0.150.6 ± 0.23108 ± 12.2110 ± 10.871 ± 9.274 ± 9.8Day 318.0 ± 0.6718.6 ± 0.652.68 ± 0.422.77 ± 0.381.5 ± 0.291.8 ± 0.430.5 ± 0.190.5 ± 0.23110 ± 10.6133 ± 11.4*74 ± 9.775 ± 7.8Day 518.8 ± 0.4518.9 ± 0.712.79 ± 0.242.59 ± 0.431.4 ± 0.322.1 ± 0.51*0.4 ± 0.130.6 ± 0.22105 ± 11.8150 ± 12.6*78 ± 7.282 ± 7.3Day 719.5 ± 0.8619.7 ± 0.992.76 ± 0.382.69 ± 0.561.6 ± 0.212.3 ± 0.43*0.5 ± 0.130.6 ± 0.24114 ± 10.4155 ± 13.3*81 ± 7.585 ± 8.3 Open table in a new tab FIGURE 4Effect of TRB3 overexpression on intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test in vivo. A, glucose tolerance test of mice infected with control GFP or TRB3 adenovirus. Mice were injected intraperitoneally with glucose (2 g/kg), and blood glucose levels were monitored at the intervals indicated. B, insulin tolerance test of mice infected with control GFP or TRB3 adenovirus. Mice were injected intraperitoneally with insulin (0.75 units/kg), and blood glucose levels were monitored at the intervals indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TRB3 Does Not Inhibit Nutrient-stimulated Activation of S6K1 in Vitro—Nutrient signaling to mTOR has been shown to be independent of the insulin-regulated Akt/TSC2 pathway. Therefore, we investigated whether overexpression of TRB3 affects nutrient-induced activation of S6K1. HepG2 cells infected with or without the TRB3-expressing adenovirus were stimulated with either the amino acid mixture (two times the normal culture levels) or DMEM culture medium. Both the amino acid mixture and the DMEM stimulated S6K1 activity 1.5-fold (p < 0.01; Fig. 5A). The addition of insulin together with the nutrients showed an additive effect (2.3-fold, p < 0.01). Rapamycin, an inhibitor of mTOR, strongly inhibited both the insulin and nutrient-stimulated S6K1 activity (Fig. 5A), confirming a central role for mTOR in the regulation of S6K1 activation. Wortmannin also inhibited the insulin-stimulated S6K1 activity, consistent with signaling through the class I PI 3-kinases but had no effect on nutrient-stimulated S6K1 (Fig. 5A). TRB3 overexpression blocked insulin-stimulated S6K1 as shown in vivo earlier but did not have a significant effect on the nutrient stimulation of S6K1. To determine whether the upstream kinase mTOR was activated normally, we measured the phosphorylation state of two mTOR targets S6K1 and 4E-BP1. In these experiments, we transfected HepG2 cells with tagged S6K1 and 4E-BP1 to show the effect of nutrient stimulation more clearly. The nutrient-induced phosphorylation of the exogenously expressed S6K1 and 4E-BP1 was weakly, but significantly, inhibited by overexpression of TRB3 (Fig. 5B). As shown in Fig. 5, C and D, S6K1 phosphorylation on Thr-389 was inhibited 28 ± 6.6%, and 4E-BP1 phosphorylation on Thr-65 was inhibited 15 ± 4.5%. We also examined the effect of RNAi knockdown of TRB3 on nutrient-stimulated activation of S6K1 in HepG2 cells. Although knockdown of TRB3 enhanced basal S6K1 activity, there was no enhancement in the nutrient stimulation (Fig. 6A). This was further verified by measuring the phosphorylation of the endogenous substrate S6. Knockdown of TRB3 had no effect on the nutrient-stimulated phosphorylation of S6 (Fig. 6, B and C). There was no effect on the phosphorylation of 4E-BP1 either indicating that mTOR activation was unaffected (Fig. 6D). These results suggest that nutrient stimulation of S6K1 is mediated by mTOR but" @default.
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• W2097068854 date "2006-10-01" @default.
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• W2097068854 title "Effect of TRB3 on Insulin and Nutrient-stimulated Hepatic p70 S6 Kinase Activity" @default.
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• W2097068854 doi "https://doi.org/10.1074/jbc.m511636200" @default.
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