Use of RNA Interference-mediated Gene Silencing and Adenoviral Overexpression to Elucidate the Roles of AKT/Protein Kinase B Isoforms in Insulin Actions
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m302094200
ISSN1083-351X
AutoresTakashi Katome, Toshiyuki Obata, Rie Matsushima, Norihisa Masuyama, Lewis C. Cantley, Yukiko Gotoh, Kazuhiro Kishi, Hiroshi Shiota, Yousuke Ebina,
Tópico(s)Pancreatic function and diabetes
ResumoInsulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms. Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms. The Akt (also referred as protein kinase B (PKB) 1The abbreviations used are: PKB, protein kinase B; CHO, Chinese hamster ovary; GLUT4, glucose transporter 4; 2-DG, 2-deoxyglucose; siRNA, short interference-RNA; RNAi, RNA interference; PKC, protein kinase C; PI3-kinase, phosphoinositide 3′-kinase; GSK3, glycogen synthase kinase 3; Erk, extracellular signal-regulated kinase; CMV, cytomegalovirus; FOXO, Forkhead box group O; PH, pleckstrin homology; dsRNA, double-stranded RNA; DN, dominant negative; CA, constitutively active; pfu, plaque-forming units; MOI, multiplicity of infection; PBS, phosphate-buffered saline; GFP, green fluorescent protein; HA, hemagglutinin; IGF, insulin-like growth factor.1The abbreviations used are: PKB, protein kinase B; CHO, Chinese hamster ovary; GLUT4, glucose transporter 4; 2-DG, 2-deoxyglucose; siRNA, short interference-RNA; RNAi, RNA interference; PKC, protein kinase C; PI3-kinase, phosphoinositide 3′-kinase; GSK3, glycogen synthase kinase 3; Erk, extracellular signal-regulated kinase; CMV, cytomegalovirus; FOXO, Forkhead box group O; PH, pleckstrin homology; dsRNA, double-stranded RNA; DN, dominant negative; CA, constitutively active; pfu, plaque-forming units; MOI, multiplicity of infection; PBS, phosphate-buffered saline; GFP, green fluorescent protein; HA, hemagglutinin; IGF, insulin-like growth factor.) was initially found to be an acute transforming component of the AKT8 virus isolated from a murine T cell lymphoma (1Staal S.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5034-5037Crossref PubMed Scopus (640) Google Scholar, 2Staal S.P. Hartley J.W. J. Exp. Med. 1988; 167: 1259-1264Crossref PubMed Scopus (82) Google Scholar). Its putative cellular homologue, Akt (c-Akt), encodes a serine/threonine protein kinase (3Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (787) Google Scholar) whose catalytic domain, located in the carboxyl terminus of the protein, is closely related to that of protein kinase C (PKC) and protein kinase A (3Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (787) Google Scholar, 4Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (387) Google Scholar). The kinase activity of Akt is stimulated by a variety of growth factors including insulin (5Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1298) Google Scholar). Recent extensive investigation revealed that Akt plays crucial roles in various cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism through phosphorylation of target molecules (6Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (967) Google Scholar). Following insulin stimulation, insulin-receptor substrate proteins are phosphorylated, after which phosphoinositide 3-kinase (PI3-kinase) is activated. The pleckstrin homology (PH) domain of Akt binds to the lipid products of PI3-kinase and thereby mediates recruitment to the membrane in response to PI3-kinase activation (5Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1298) Google Scholar, 7Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar). The translocation of Akt allows for phosphorylation at Thr308 by another Ser/Thr protein kinase, 3-phosphoinositide-dependent protein kinase 1 (8Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar, 9Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar) and at Ser473 by Akt itself (autophosphorylation) (10Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar) or an as yet unidentified protein kinase (11Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1389) Google Scholar, 12Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1034) Google Scholar). With regard to insulin action, Akt was shown to phosphorylate and inhibit glycogen synthase kinase 3 (GSK3) and subsequently promote glycogen synthesis in response to insulin (13Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4337) Google Scholar). However, regarding another important insulin action, which is the stimulation of glucose uptake by the GLUT4 translocation, the role of Akt remained controversial (14Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 15Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (500) Google Scholar). In addition, although the participation of atypical protein kinase C (i.e. PKCλ and PKCξ), which is also thought to be activated by phosphoinositides and 3-phosphoinositide-dependent protein kinase 1, in glucose uptake in 3T3-L1 adipocytes has been proposed (16Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar), the opposite result has also been reported in the case of the same cell type (17Tsuru M. Katagiri H. Asano T. Yamada T. Ohno S. Ogihara T. Oka Y. Am. J. Physiol. 2002; 283: E338-E345Crossref PubMed Scopus (39) Google Scholar). Thus, the role of atypical PKC also seems to be controversial. An adenoviral gene transfer technique now in common use is thought to be a powerful strategy to introduce high levels of transgene expression into mammalian cells (18He 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 (3236) Google Scholar). The technique facilitates expression of inactive Akt in cells to overcome endogenous Akt activity, dominant negatively (14Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 15Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (500) Google Scholar). According to other authors who have examined the role of Akt in glucose metabolism (14Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 15Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (500) Google Scholar), Akt-2A (Akt-T308A/S473A) and Akt-AAA (Akt-K179A/T308A/S473A) have been used. We used Akt-MAA (Akt-K179M/T308A/S473A) in the present study, since we considered that Met (for Lys179; so-called catalytic Lys) has a relatively large side chain and is likely to impair kinase activity by occupying the ATP-binding pocket more efficiently compared with Ala as well as losing a positive charge for the stabilization of ATP binding and a hydrogen bond with the Glu in helix C. RNA interference (RNAi) is a process whereby double-stranded RNA (dsRNA) induces the degradation of cognate mRNA for prevention of the expression of alien genes. Originally, the RNAi phenomenon was discovered in Caenorhabditis elegans; however, recent studies revealed that such phenomena are evolutionarily conserved from plants to mammals (19Fire A. Trends Genet. 1999; 15: 358-363Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 20Tuschl T. Zamore P.D. Lehmann R. Bartel D.P. Sharp P.A. Genes Dev. 1999; 13: 3191-3197Crossref PubMed Scopus (687) Google Scholar, 21Sharp P.A. Genes Dev. 1999; 13: 139-141Crossref PubMed Scopus (262) Google Scholar, 22Nishikura K. Cell. 2001; 107: 415-418Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 23Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8107) Google Scholar). According to recent data, duplexes of 21-nucleotide short interference RNA (siRNA) with 2-nucleotide 3′-overhangs are the most efficient triggers of sequence-specific mRNA degradation (24Elbashir S.M. Lendeckel W. Tuschl T. Genes Dev. 2001; 15: 188-200Crossref PubMed Scopus (2686) Google Scholar, 25Elbashir S.M. Martinez J. Patkaniowska A. Lendeckel W. Tuschl T. EMBO J. 2001; 20: 6877-6888Crossref PubMed Scopus (1199) Google Scholar). One fascinating aspect of RNAi is its extreme efficiency, since only a few trigger dsRNA molecules introduced into the cells suffice to inactivate a continuously transcribed cognate target mRNA for long periods of time (22Nishikura K. Cell. 2001; 107: 415-418Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Although the introduction of dsRNA into mammalian cells has been known to cause RNA-dependent protein kinase-mediated apoptosis, the siRNA (e.g. 21-nucleotide dsRNA) has been shown not to cause such an effect (24Elbashir S.M. Lendeckel W. Tuschl T. Genes Dev. 2001; 15: 188-200Crossref PubMed Scopus (2686) Google Scholar). This important observation revealed new possibilities whereby one could prove gene function in mammalian cells. In attempts to elucidate the roles of Akt in insulin actions, we used both adenoviral overexpression and RNAi in both CHO cells and 3T3-L1 adipocytes. Since it is unclear which isoforms of Akt are responsible for each insulin action, we investigated the role of each isoform in insulin actions using a specific RNAi against each Akt isoform. Materials—[γ-32P]ATP and d-[6-3H]glucose were purchased from Amersham Biosciences. 2-Deoxy-d-[1,2-3H]glucose was from Moravek (Brea, CA). Phosphospecific antibodies against pAkt (Ser(P)473), pErk-1/2 (Thr(P)202/Tyr(P)204), and pGSK3 (Ser(P)9/Ser(P)21) were from Cell Signaling (Beverly, MA). Anti-insulin receptor, anti-14-3-3, anti-PKCξ, anti-phosphotyrosine (PY-99), and anti-Akt1-specific antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Erk-1/2, anti-GSK3α/β, anti-Akt2-specific, and anti-Akt3-specific antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). An anti-PKCλ antibody and anti-Akt antibody were obtained using rabbits immunized against synthetic peptides (CTMHPDHTQTVIPYNPSS and CVDSERRPHFPQFSYSASGTA, respectively) (26Wang L. Hayashi H. Kishi K. Huang L. Hagi A. Tamaoka K. Hawkins P.T. Ebina Y. Biochem. J. 2000; 345: 543-555Crossref PubMed Scopus (21) Google Scholar). The anti-Akt antibody used for in vitro kinase assay and immunoblotting was confirmed to recognize all isoforms of Akt. The anti-HA antibody used was also from an immunized rabbit, as described (27Noda S. Kishi K. Yuasa T. Hayashi H. Ohnishi T. Miyata I. Nishitani H. Ebina Y. J. Med. Invest. 2000; 47: 47-55PubMed Google Scholar). A synthetic peptide for in vitro protein kinase assay of Akt, AKTide-2T (ARKRERTYSFGHAA), was as described (28Obata T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). All other reagents, including pork insulin and wortmannin, were of analytical grade from Sigma or Nacalai (Kyoto, Japan). Synthetic siRNA and Its Preparation—All DNA/RNA chimeric oligonucleotides were purchased from Hokkaido System Science (Sapporo, Japan) and annealed to be double-stranded, as described (23Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8107) Google Scholar). The sequences of control siRNA against GFP are as follows: 5′-CUGGAGUUGUCCCAAUUCCTT-3′ and 5′-AGAAUUGGGACAACUCCAGTT-3′ (the 2-nucleotide overhanging of 2′-deoxythymidine is indicated as TT and denoted by underlines) (23Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8107) Google Scholar). The sequences of all other specific siRNAs against Akt are indicated in Fig. 3, A and C, and Fig. 6A.Fig. 6Isoform-specific gene silencing of Akt by RNA interference. A, the sequences of synthetic siRNA duplexes targeting each Akt isoform are shown. B, the indicated siRNAs (0.5 μg/24 wells) were co-transfected with either HA-Akt1–3, HA-tagged serum- and glucocorticoid-inducible protein kinase, or PKCλ or PKCξ expression vectors into COS-7 cells on a 24-well plate, and after 48 h of the transfection, these cells were harvested, and the obtained total cell lysates were analyzed by immunoblotting using either anti-HA antibody or specific antibodies. Data are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Plasmids—HA-tagged rat Akt2 and Akt3 were PCR-amplified using a rat brain cDNA library as a template and the following oligonucleotides as primers: 5′-GGACTAGTGCCATGTACCCATACGATGTGCCAGATTACGCCAATGAGGTATCTGTCATCAAG-3′ (HA tag denoted by underline)/5′-GGATCGATCACTCTCGGATGCTGGCTGAAGTAGGAGAACTG-3′ (stop codon is denoted in boldface type) and 5′-GGACTAGTGCCACCATGTACCCATACGATGTGCCAGATTACGCCAGCGATGTTACCATCGTTAAAG-3′ (the region of HA tag denoted by underline)/5′-GGATCGATTATAGTGGACACTTTTCAGGTGGTGTTATTG (stop codon is denoted in boldface type), respectively. Amplified PCR fragments were subcloned into pCR2.1-TOPO (Invitrogen) using the TA cloning method according to the manufacturer's protocol, their DNA sequences were confirmed, and they were subcloned into pCX2 (EcoRI site) and pEGFP-C1 (CloneTech; NheI and HindIII sites; enhanced green fluorescence protein clone was removed), respectively. Mammalian expression constructs encoding HA-tagged rat Akt1 wild type and rat Akt1-2A (T308A/S473A) were kindly provided by Dr. W. Ogawa (Kobe University, Kobe, Japan) (14Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar). The dominant negative form of rat Akt-MAA (Akt-MAA: K179M/T308A/S473A) was made using QuikChange kits (Stratagene) with rat Akt1-2A (as a template) and the following oligonucleotides: 5′-cgctactatgccatgatgatcctcaagaaggagg-3′/5′-cctccttcttgaggatcatcatggcatagtagcg-3′. Constitutively active Akt was generated by inserting an oligonucleotide duplex encoding the c-Src myristoylation signal (MGSSKSKPKDPSQRRS, 5′-GATCCACCATGGGGAGCAGCAAGAGCAAGCCCAAGGACCCCAGCCAGCGCA-3′, 5′-GATCTGCGCTGGCTGGGGTCCTTGGGCTTGCTCTTGCTGCTCCCCATGGTG-3′) upstream of human Akt1. The oligo-DNA duplex was initially subcloned into pCR-blunt (Invitrogen) at BamHI and BglII sites, and then a fragment of human Akt1 (29Masuyama N. Oishi K. Mori Y. Ueno T. Takahama Y. Gotoh Y. J. Biol. Chem. 2001; 276: 32799-32805Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), digested with BamHI, was subcloned into the vector that had been digested with BglII. Finally, myr-Akt1 was digested with BamHI and subcloned into pAdTrackCMV, an adenovirus transfer vector, for adenovirus generation. FLAG-tagged FKHRL1 was kindly provided by Dr. M. Greenberg (Harvard Medical School, Boston, MA) (30Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5381) Google Scholar). BP1-luc was provided by Dr. T. Unterman (31Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). pRL-SV40 vector was from Promega (Madison, WI). Mammalian expression plasmids for PKCλ and PKCξ were kindly provided by Dr. S. Ohno (Yokohama City University, Yokohama, Japan) (17Tsuru M. Katagiri H. Asano T. Yamada T. Ohno S. Ogihara T. Oka Y. Am. J. Physiol. 2002; 283: E338-E345Crossref PubMed Scopus (39) Google Scholar). Cell Culture—COS-7 cells and Chinese hamster ovary (CHO) cells stably expressing Myc-tagged rat glucose transporter 4 (GLUT4myc) were maintained in either Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal calf serum (Invitrogen) or F-12 culture medium (Biological Industries, Kibbutz Beit Haemek, Israel) with 10% fetal calf serum, respectively (32Kanai F. Nishioka Y. Hayashi H. Kamohara S. Todaka M. Ebina Y. J. Biol. Chem. 1993; 268: 14523-14526Abstract Full Text PDF PubMed Google Scholar). A 3T3-L1 fibroblast was maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% calf serum (Invitrogen) and differentiated into 3T3-L1 adipocytes stably expressing GLUT4myc (3T3-L1-GLUT4myc), as described (32Kanai F. Nishioka Y. Hayashi H. Kamohara S. Todaka M. Ebina Y. J. Biol. Chem. 1993; 268: 14523-14526Abstract Full Text PDF PubMed Google Scholar). More than 90% of the cells expressed the adipocyte phenotype (i.e. lipid droplets). Human brain tumor-derived U251-MG cells were provided by the Health Science Research Resources Bank of Japan (Sen-nan, Osaka, Japan) and were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum as above. Preparation of Recombinant Adenovirus—The recombinant adenovirus encoding either dominant-negative (DN) Akt (Akt-MAA: K179M/T308A/S473A) or constitutively active (CA) Akt (myr-Akt1) was generated using the AdEasy system, as described (18He 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 (3236) Google Scholar). The recombinant adenoviruses were amplified in 293 cells. The 50% tissue culture infectious dose (TCID50) was determined as pfu/ml, and either CHO cells or 3T3-L1 adipocytes were infected at the multiplicities of infection (MOI; pfu/cell), as indicated. Adenoviral Gene Transduction—CHO-GLUT4myc cells or 3T3-L1 adipocytes (the 8th day after initiation of differentiation) were incubated with OPTI-MEM (Invitrogen) containing the indicated MOI of adenovirus for 2 h with tilting every 30 min; we then added the growth medium and incubated the preparation for 32 h followed by serum starvation with serum-free medium (i.e. F-12 or Dulbecco's modified Eagle's medium) for 16 h. Experiments were performed 48 h after the infection. Akt in Vitro Kinase Assay—Akt kinase activity was measured using AKTide-2T (28Obata T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) as a substrate, as described (33Shioi T. McMullen J.R. Kang P.M. Douglas P.S. Obata T. Franke T.F. Cantley L.C. Izumo S. Mol. Cell. Biol. 2002; 22: 2799-2809Crossref PubMed Scopus (446) Google Scholar). Cells seeded on either six-well or 24-well plates were incubated in serum-free medium for 16 h and then stimulated with 100 nm insulin for 10 min. Cells were washed once with PBS and lysed in solubilizing buffer containing 20 mm Tris-HCl (pH 7.5), 140 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 0.5 mm Na3VO4, 50 mm β-glycerophosphate, 20 mm Na4P2O7, 20 mm NaF, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. After centrifugation at 15,000 × g for 10 min at 4 °C, the obtained cell lysates were immunoprecipitated with the anti-Akt antibody (26Wang L. Hayashi H. Kishi K. Huang L. Hagi A. Tamaoka K. Hawkins P.T. Ebina Y. Biochem. J. 2000; 345: 543-555Crossref PubMed Scopus (21) Google Scholar). Immunoprecipitated enzyme was incubated in 30 μl of reaction mixture containing 20 mm HEPES-NaOH (pH 7.4), 100 mm NaCl, 10 mm NaF, 10 mm β-glycerophosphate, 0.1 mm Na3VO4, 10 mm MgCl2, 0.5 mm EGTA, 1 μm protein kinase inhibitor, 1 mm dithiothreitol, 5 μm cold ATP, 3 μCi of [γ-32P]ATP, and 50 μm AKTide-2T for 10 min at 37 °C. The reactions were terminated by adding 10 μl of 8 n HCl and spotted onto P81 phosphocellulose paper (Whatman, Rockland, MA) and then washed five times with 0.5% phosphoric acid. Radioactivity was measured using a liquid scintillation counter. GLUT4 Translocation—Insulin-stimulated GLUT4 translocation to plasma membrane was examined by determination of the exposure of Myc epitope (GLUT4myc) to the outer surface of plasma membrane in response to insulin, as described (34Kishi K. Muromoto N. Nakaya Y. Miyata I. Hagi A. Hayashi H. Ebina Y. Diabetes. 1998; 47: 550-558Crossref PubMed Scopus (148) Google Scholar). In brief, cells on 24-well plates were incubated in 500 μl of Krebs-Ringer-HEPES buffer for 20 min at 37 °C and then stimulated with 100 nm insulin for 10 min at 37 °C. After fixation with 2% paraformaldehyde, cells were washed with PBS and then incubated with 100 mm glycine/PBS for 15 min. After the blocking, cells were incubated with 300 μl of anti-Myc antibody (9E10; 1:1000 dilution) for 2 h, washed with PBS, and incubated with 300 μl of horseradish peroxidase-conjugated anti-mouse IgG (1:2000 dilution) at room temperature for 1 h. The wells were then washed with five times with PBS. GLUT4myc translocation was determined and analyzed by enhanced chemiluminescence and a Luminescencer-JNR luminometer (AB-2100; ATTO, Tokyo, Japan). 2-Deoxy-d-glucose Uptake—Insulin-stimulated 2-deoxy-d-glucose uptake was measured based on the determination of 2-deoxy-d-[1,2-3H]glucose incorporation into the cell, as described (34Kishi K. Muromoto N. Nakaya Y. Miyata I. Hagi A. Hayashi H. Ebina Y. Diabetes. 1998; 47: 550-558Crossref PubMed Scopus (148) Google Scholar). Glycogen Synthesis Assay—Insulin-stimulated glycogen synthesis was measured based on the determination of d-[6-3H]glucose incorporation into the glycogen fraction, as described (34Kishi K. Muromoto N. Nakaya Y. Miyata I. Hagi A. Hayashi H. Ebina Y. Diabetes. 1998; 47: 550-558Crossref PubMed Scopus (148) Google Scholar). Immunoblotting—For immunoblotting, we used the indicated antibodies, and the signals were detected using horseradish peroxidase-mediated chemiluminescence, as described (35Obata T. Maegawa H. Kashiwagi A. Pillay T.S. Kikkawa R. J Biochem. (Tokyo). 1998; 123: 813-820Crossref PubMed Scopus (22) Google Scholar). Luciferase Assay—The specific (against Akt; siAKTc) and nonspecific (against GFP; siGFP) synthetic 21-mer dsRNAs (total of 0.5 μg/well) were co-transfected with FKHRL1 (0.01 μg/well), BP1-luc (0.5 μg/well), and pRL-SV40 (0.1 μg/well) into either COS-7 cells or 3T3-L1 adipocytes plated on a 24-well plate using LipofectAMINE 2000 (Invitrogen). After 48 h of transfection, luciferase activity was measured using dual luciferase reporter assay kits (Promega, Madison, WI). A PI3-kinase inhibitor, LY294002 (100 μm), was also used for 1 h before cell harvest. Normalized relative luciferase activity (Photinus pyralis luciferase/Renilla reniformis luciferase activity) was expressed. The S.D. value of R. reniformis luciferase activity was within 5% of the average value. Statistical Analysis—The data are represented as mean ± S.E. p values were determined by Bonferroni-Dunncan's multiple comparison test, and a p < 0.05 value was considered to be statistically significant. Constitutively Active Akt Mimicked and Dominant Negative Akt Inhibited Insulin Actions in Both CHO Cells and 3T3-L1 Adipocytes—We generated recombinant adenovirus encoding either constitutively active Akt (CA-Akt; myr-AKT) or dominant negative Akt (DN-Akt; Akt-K179M/T308A/T473A) and examined effects of these Akt mutants on insulin actions, including GLUT4 translocation to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis. As shown in Figs. 1A and 2A, infection of CA-Akt adenovirus markedly enhanced Akt kinase activity in both CHO cells and 3T3-L1 adipocytes. In parallel, the infection mimicked effects of insulin on GLUT4 translocation, 2-DG uptake, and glycogen synthesis as shown in Figs. 1, B–D, and 2, B–D. On the other hand, the control adenovirus that encoded green fluorescent protein (GFP) had little effect on these actions as well as on Akt kinase activity. Interestingly, although CA-Akt enhanced glycogen synthesis in quiescent cells, the maximum rate of glycogen syntheses in insulin-stimulated cells was blunted in both CHO cells and 3T3-L1 adipocytes (Figs. 1D and 2D; see "Discussion").Fig. 2Effects of CA- and DN-AKT on insulin actions in 3T3-L1-GLUT4myc adipocytes. Cells were infected with the indicated MOI (pfu/cell) of adenovirus, and insulin-stimulated Akt kinase activity (A and E; the antibody used for immunoprecipitation can recognize all Akt isoforms), GLUT4 translocation (B and F), 2-DG uptake (C and G), and glycogen synthesis (D and H) were measured. Data are expressed by mean ± S.E. from 4–6 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control (GFP). For 2-DG uptake, 8.6 pmol/min/2 × 105 cells was regarded as 1 arbitrary unit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next examined the effect of DN-Akt on insulin actions in these cells. As shown in Figs. 1E and 2E, DN-Akt adenovirus efficiently inhibited insulin-stimulated Akt kinase activity and insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis in an MOI-dependent manner in both cell lines. In the case of 3T3-L1 adipocytes, although DN-Akt (at MOI 80) inhibited Akt activity almost completely, insulin-stimulated metabolic activities, including 2-DG uptake and glycogen synthesis, still remained, which suggests an additional pathway in 3T3-L1 adipocytes (see "Discussion"). Expression of DN-Akt had little effect on either phosphorylation of Erk-1/2 (by immunoblotting with phosphospecific antibody) or activation of PI3-kinase activity (by in vitro kinase assay) in insulin-stimulated cells (data not shown). RNAi-mediated Gene Silencing of Akt—We then carried out RNAi-mediated gene silencing using a synthetic 21-mer oligonucleotide RNA duplex (siRNA) to determine the requirement of Akt for insulin actions. We designed a pair of oligonucleotides corresponding to the sequence of Akt1 that matches maximally with the other two Akt isoforms (rat, mouse, and human) and named "siAKTc," as shown in Fig. 3A. There were a few mismatches within the corresponding sequences of rat Akt3, human Akt3, and mouse Akt2 and Akt3. COS-7 cells were then co-transfected with the siAKTc and Akt expression plasmid that also expressed GFP driven by an independent CMV promoter as w
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