mTORC1 Activation Regulates β-Cell Mass and Proliferation by Modulation of Cyclin D2 Synthesis and Stability
2009; Elsevier BV; Volume: 284; Issue: 12 Linguagem: Inglês
10.1074/jbc.m807458200
ISSN1083-351X
AutoresNorman Bálcazar, Aruna Sathyamurthy, Lynda Elghazi, Aaron Gould, Aaron Weiss, Ichiro Shiojima, Kenneth Walsh, Ernesto Bernal‐Mizrachi,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoGrowth factors, insulin signaling, and nutrients are important regulators of β-cell mass and function. The events linking these signals to the regulation of β-cell mass are not completely understood. The mTOR pathway integrates signals from growth factors and nutrients. Here, we evaluated the role of the mTOR/raptor (mTORC1) signaling in proliferative conditions induced by controlled activation of Akt signaling. These experiments show that the mTORC1 is a major regulator of β-cell cycle progression by modulation of cyclin D2, D3, and Cdk4 activity. The regulation of cell cycle progression by mTORC1 signaling resulted from modulation of the synthesis and stability of cyclin D2, a critical regulator of β-cell cycle, proliferation, and mass. These studies provide novel insights into the regulation of cell cycle by the mTORC1, provide a mechanism for the antiproliferative effects of rapamycin, and imply that the use of rapamycin could negatively impact the success of islet transplantation and the adaptation of β-cells to insulin resistance. Growth factors, insulin signaling, and nutrients are important regulators of β-cell mass and function. The events linking these signals to the regulation of β-cell mass are not completely understood. The mTOR pathway integrates signals from growth factors and nutrients. Here, we evaluated the role of the mTOR/raptor (mTORC1) signaling in proliferative conditions induced by controlled activation of Akt signaling. These experiments show that the mTORC1 is a major regulator of β-cell cycle progression by modulation of cyclin D2, D3, and Cdk4 activity. The regulation of cell cycle progression by mTORC1 signaling resulted from modulation of the synthesis and stability of cyclin D2, a critical regulator of β-cell cycle, proliferation, and mass. These studies provide novel insights into the regulation of cell cycle by the mTORC1, provide a mechanism for the antiproliferative effects of rapamycin, and imply that the use of rapamycin could negatively impact the success of islet transplantation and the adaptation of β-cells to insulin resistance. The defects that result in diabetes are diverse, but the loss of pancreatic β-cell mass is a critical determinant for the development of this disease (1Kahn S.E. J. Clin. Endocrinol. Metab. 2001; 86: 4047-4058Crossref PubMed Scopus (583) Google Scholar, 2White M.F. Am. J. Physiol. 2002; 283: E413-E422Crossref PubMed Scopus (44) Google Scholar). The capacity for β-cells to expand in response to insulin resistance is required to maintain glucose homeostasis and prevent type 2 diabetes. Pancreatic β-cell mass is regulated by a dynamic balance of neogenesis, proliferation, hypertrophy, and apoptosis (3Bonner-Weir S. J. Mol. Endocrinol. 2000; 24: 297-302Crossref PubMed Scopus (262) Google Scholar). In particular, β-cell proliferation (determined by the number of mature β-cells entering the cell cycle) has a major role in the maintenance of β-cell mass in adult life and after proliferative stimuli (4Dor Y. Brown J. Martinez O.I. Melton D.A. Nature. 2004; 429: 41-46Crossref PubMed Scopus (1914) Google Scholar). Although there has been much research showing the role of β-cell mass in diabetes, there is a lack of knowledge pertaining to how β-cells enter the cell cycle, proliferate, and increase mass. In pancreatic β-cells, glucose, amino acids, and growth factors have been shown to induce G1-S progression (5Kwon G. Marshall C.A. Liu H. Pappan K.L. Remedi M.S. McDaniel M.L. J. Biol. 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One of the major mechanisms by which nutrient and growth factors regulate mTOR activity involves the tuberous sclerosis complex 2 (TSC2) 2The abbreviations used are: TSC, tuberous sclerosis complex; S6K, ribosomal S6 kinase; dox, doxycycline; Cdk, cyclin-dependent kinase; DT, double transgenic; ST, single transgenic; GST, glutathione S-transferase; GFP, green fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Rap, rapamycin. gene product (tuberin) as well as TSC1 (hamartin) and the small G protein Ras homolog enriched in brain. Phosphorylation of TSC2 by the serine-threonine kinase AKT induces mTOR signaling by derepressing the TSC2 GTPase-activating protein activity toward Ras homolog enriched in brain, (9Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (716) Google Scholar, 10Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. 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The mammalian mTORC1 contains Raptor and the G protein β-subunit-like protein (GβL). mTORC1 activates key regulators of protein translation; ribosomal S6 kinase (S6K), eukaryote initiation factor 4E-binding protein 1, and eukaryote initiation factor 4E (16Harris T. Lawrence J.C. Science STKE. 2003; 212: 1-17Google Scholar). The mTORC2 complex includes mTOR and rictor and is insensitive to rapamycin (14Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2157) Google Scholar, 15Jacinto E. Loewith R. Schmidt A. Lin S. Ruegg M.A. Hall A. Hall M.N. Nat. Cell Biol. 2004; 6: 1122-1128Crossref PubMed Scopus (1687) Google Scholar). This complex is potentially important for the regulation of β-cell mass and function, because it is responsible for the phosphorylation/activation of Akt on Ser473 (17Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5255) Google Scholar). Evidence for the importance of mTOR signaling on the modulation of β-cell mass and proliferation in vivo comes from genetically modified mice. Decreased β-cell mass and hyperglycemia in mice deficient for S6K and mutant for ribosomal protein S6 provide evidence for the importance of this pathway in these processes (18Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar, 19Um 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 (1367) Google Scholar, 20Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (472) Google Scholar). Moreover, activation of mTOR signaling by conditional deletion of TSC2 in β-cells induces β-cell proliferation and hypertrophy (21Shigeyama Y.K.T. Kido Y. Hashimoto N. Asahara S.I. Matsuda T. Takeda A. Inoue T. Shibutani Y. Koyanagi M. Uchida T. Inoue M. Hino O. Kasuga M. Noda T. Mol. Cell. Biol. 2008; 28: 2971-2979Crossref PubMed Scopus (143) Google Scholar, 22Rachdi L. Balcazar N. Osorio-Duque F. Elghazi L. Weiss A. Gould A. Chang-Chen K.J. Gambello M.J. Bernal-Mizrachi E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9250-9255Crossref PubMed Scopus (160) Google Scholar) The contribution and potential role for mTOR signaling and the mTORC complexes in β-cell mass and function have yet to be adequately explored. The current experiments delineate some of the molecular mechanisms involved in β-cell G1-S transition by the mTOR arm of Akt signaling. In these studies, the hypothesis that the mTORC1 (mTOR/Raptor) is a major regulator of β-cell cycle progression and mass in vivo was tested. To test this hypothesis, we studied the effects of inhibition of the mTORC1 complex under proliferative conditions induced by controlled activation of Akt signaling in β-cells. To activate Akt signaling in a controlled fashion, we developed a doxycycline (dox)-inducible mouse model. This animal model allowed us to induce β-cell proliferation and mass without disturbing peripheral tissues. These studies showed that the mTORC1 complex mediates the regulation of cell cycle in β-cells in vivo and does so by activation of cyclin-dependent kinase-4 (Cdk4). The regulation of cell cycle progression by mTORC1 signaling resulted from modulation of the synthesis and stability of cyclin D2, a critical regulator of β-cell cycle, proliferation, and mass. These studies indicate that the mTORC1 is major component relating proliferative signals induced by nutrients and growth factors and uncover the molecular mechanisms implicated in the regulation of β-cell cycle by this signaling pathway. Mice-RIP-rtTA mice express the reverse tetracycline transactivator under the control of the rat insulin II gene and are in C57Bl/6 (B6)/CBA background (23Milo-Landesman D. Surana M. Berkovich I. Compagni A. Christofori G. Fleischer N. Efrat S. Cell Transplant. 2001; 10: 645-650Crossref PubMed Scopus (71) Google Scholar). The tetOAkt1 mice have been previously described (24Shiojima I. Sato K. Izumiya Y. Schiekofer S. Ito M. Liao R. Colucci W.S. Walsh K. J. Clin. Investig. 2005; 115: 2108-2118Crossref PubMed Scopus (759) Google Scholar) and contain the myristoylated AKT 1 gene under the regulation of tetracycline-responsive element. Generation and phenotypic characterization of myr-Akt mice have been previously described elsewhere in detail (24Shiojima I. Sato K. Izumiya Y. Schiekofer S. Ito M. Liao R. Colucci W.S. Walsh K. J. Clin. Investig. 2005; 115: 2108-2118Crossref PubMed Scopus (759) Google Scholar). Double transgenic RIP-rtTA/tetOAkt1 mice (DT) were obtained by crossing RIP-rtTA with tetOAkt1. The single transgenic (ST) was used as control and included RIP-rtTA mice. Experiments were performed in 2-month-old males. Control and experimental animals were on comparably mixed background. Doxycycline treatment was performed by adding 2 mg/ml doxycycline to the drinking water. Mice overexpressing a constitutively active form of Akt under the control of the rat insulin promoter (caAktRIP) have been previously described (25Bernal-Mizrachi E. Wen W. Stahlhut S. Welling C.M. Permutt M.A. J. Clin. Investig. 2001; 108: 1631-1638Crossref PubMed Scopus (344) Google Scholar). All of the procedures were performed in accordance with the Washington University Animal Studies Committee. Islet and MIN6 Cell Culture-MIN6 cells were stably infected with a lentivirus containing a constitutively active Akt mutant (MIN6-caAkt) or GFP (MIN6-GFP) as control. These lines were maintained as described previously (26Bernal-Mizrachi E. Wice B. Inoue H. Permutt M.A. J. Biol. Chem. 2000; 275: 25681-25689Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). For experiments with rapamycin, MIN6 cells were cultured with rapamycin for 16 h. The in vitro experiments in islets were performed in islets from ST or DT mice treated with vehicle or dox in the drinking water for 3 weeks. After isolation, the islets were cultured in medium with vehicle (ST) or dox (DT) for 40 h. Rapamycin (50 nm) or vehicle was added to the medium for the last 16 h of culture before harvesting. This experimental protocol reproducibly inhibit mTORC1 complex in vitro. These experimental conditions were used for all of the islet experiments except for the studies in Fig. 2C. Islet Isolation and Western Blot Analysis-Islet isolation was accomplished by collagenase digestion as described previously (25Bernal-Mizrachi E. Wen W. Stahlhut S. Welling C.M. Permutt M.A. J. Clin. Investig. 2001; 108: 1631-1638Crossref PubMed Scopus (344) Google Scholar). The following morning after isolation, the islets were hand picked and treated with 2 μg/μl doxycycline and/or 50 nm rapamycin for 16 h as indicated in the figures and results. Isolated islets were lysed in a buffer containing 0.3% CHAPS, 150 mm NaCl, 5 mm EDTA, 10 mm Tris, pH 8.0, and protease and phosphatase inhibitors (Roche Applied Science). Islet lysates were subjected to immunoblotting using the following antibodies: Akt, pGSK3 α/β, p70S6K (phospho-p70 S6K Thr 389) phospho-S6 ribosomal protein (Ser235/236), cyclin D1, cyclin D3, and p27 were from Cell Signaling (Beverly, MA), and cyclin D2 was obtained from Lab Vision Corporation (Fremont, CA). Cdk4 was from Santa Cruz Biotechnology (Santa Cruz, CA), and α-tubulin was from Sigma. Secondary horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG were from Cell Signaling. Immunohistochemistry, Islet Morphometry, and Analysis of Proliferation and Apoptosis-Pancreatic tissue was fixed overnight in 3.7% formalin solution and embedded in paraffin using standard techniques. Immunostaining for insulin was done as describe previously (25Bernal-Mizrachi E. Wen W. Stahlhut S. Welling C.M. Permutt M.A. J. Clin. Investig. 2001; 108: 1631-1638Crossref PubMed Scopus (344) Google Scholar). Immunofluorescence for phospho-pS6 ribosomal protein (Ser235/236) (Cell Signaling) and insulin was performed as described previously (25Bernal-Mizrachi E. Wen W. Stahlhut S. Welling C.M. Permutt M.A. J. Clin. Investig. 2001; 108: 1631-1638Crossref PubMed Scopus (344) Google Scholar). Assessment of β-cell mass was performed by point counting morphometry from five insulin-stained sections (4 μm) separated by 200-μm National Institutes of Health ImageJ software (v1.3.8x) as described previously (27Fatrai S. Elghazi L. Balcazar N. Cras-Meneur C. Krits I. Kiyokawa H. Bernal-Mizrachi E. Diabetes. 2006; 55: 318-325Crossref PubMed Scopus (166) Google Scholar, 28Girish V. Vijayalakshmi A. Indian J. Cancer. 2004; 41: 47PubMed Google Scholar). Proliferation was performed in insulin-and Ki67-stained sections (Novocastra, Burlingame, CA) from ST and DT mice. Proliferating cells were identified by co-staining for Ki67 and insulin. Apoptosis was determined in pancreatic sections stained for insulin and cleaved-caspase 3 (Cell Signaling). Co-staining for insulin and cleaved-caspase 3 identified apoptotic cells. At least 1000 insulin-stained cells were counted for each animal. Quantitative Reverse Transcription-PCR-Total RNA was isolated using RNeasy (Qiagen). cDNA was synthesized using random hexamers and reverse transcribed with Superscript II (Invitrogen) according to the manufacturer's protocol. Real time PCR was performed on ABI 7000 sequence detection system using TaqMan gene expression assays (Applied Biosystem, Foster City, CA). Primers from cell cycle components were purchased from Applied Biosystem with reference numbers p21 (Mn 00432448), p27 (Mm 00432359), Cdk4 (Mm 01273583), Ccnd1 (Mm 00432359), Ccnd2 (Mm 00438071), and Ccnd3 (Mm 01273583). In Vitro Cdk4 Kinase Assays-In vitro Cdk4 activity were performed as described previously (29Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1025) Google Scholar). Four-week-old ST and DT mice were placed on vehicle or dox treatment in their drinking water for 3 weeks. After isolation, DT islets were cultured for 40 h in medium containing dox (2 μg/ml). Rapamycin (50 nm) or vehicle was added for 16 h before harvesting. Lysates from islets or MIN6 cells were immunoprecipitated using anti-Cdk4 antibody (Santa Cruz Biotechnology) and 50 μl of protein G-Sepharose beads (Sigma-Aldrich). The final kinase reaction was carried out in 50 mmol/liter HEPES, pH 7.5, 10 mmol/liter MgCl2, 1 mmol/liter dithiothreitol, 2.5 mmol/liter EGTA 10 mmol/liter glycerophosphate, 0.1 mmol/liter Na2VO4, 1 mmol/liter NaF, 5 μmol/liter ATP, 6 mCi/reaction of [γ-32P]ATP (Amersham Biosciences), and GST-Rb 769–921 (Santa Cruz Biotechnology). The samples were incubated at 30 °C for 30 min and separated by polyacrylamide gel electrophoresis. The amount of 32P-labeled GST-Rb was visualized and quantified by autoradiography using a PhosphorImager. The levels of immunoprecipitated Cdk4 were used as loading control. In Vitro Kinase Assay for Akt-Akt kinase activity was measured using the Akt kinase assay kit from Cell Signaling. Four-week-old ST and DT mice were placed on vehicle or dox treatment in their drinking water for 3 weeks. After isolation, ST and DT islets were cultured for 40 h with vehicle or dox (2 μg/ml), respectively. Rapamycin (50 nm) or vehicle was added for the last 16 h before harvesting. Resuspended immobilized Akt antibody slurry (20 μl) was added to 100 μg of lysates to selectively immunoprecipitate Akt by gentle rocking 2 h at 4 °C. The pellet was washed twice with 500 μl of 1× lysis buffer and twice with 500 μl of 1× kinase buffer (25 mm Tris, pH 7.5, 5 mm β-glycerol phosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, and 10 mm MgCl2). The immunoprecipitated pellet was then incubated with 40 μl of 1× kinase buffer supplemented with 200 μm ATP and 1 μg of GSK-3 fusion protein for 30 min at 30 °C allowing immunoprecipitated Akt to phosphorylate GSK-3. The reaction was terminated with 20 μl of 3× SDS sample buffer. The samples were boiled for 5 min and loaded on 12% SDS-PAGE gel. Band intensity was quantified using National Institutes of Health ImageJ software (v1.3.8x). Input protein was used as control for quantification of the Akt activity levels. Akt-induced phosphorylation of GSK-3 was detected by Western blotting using phospho-GSK-3/(Ser21/9) antibody. Pulse-Chase Analysis-Islets from four ST and four DT mice/time point were washed in Dulbecco's modified Eagle's medium without methionine and cysteine for 30 min at 37 °C. The islets were pulse-labeled for 30 min with [35S]Protein labeling mix (PerkinElmer Life Sciences) at 280 μCi total/reaction and then washed with warm phosphate-buffered saline before being placed in complete Dulbecco's modified Eagle's medium containing a 100-fold excess of unlabeled methionine for 30 and 60 min (chase). For the rapamycin experiments, the islets were preincubated with 50 nm rapamycin for 90 min before the pulse, and the treatment was continued during the pulse for 30 min. Islet lysates from the pulse and the chase conditions were immunoprecipitated using anti-cyclin D2 antibody. The immunoprecipitates from all the conditions were washed and separated by SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane. Immunoprecipitated proteins were visualized by autoradiography using a PhosphorImager (Amersham Biosciences). The pulse-label experiments in MIN6 cells were performed essentially using the same conditions described for the islet experiment. Immunoblotting for cyclin D2 was used as loading control. Quantitation of band intensity was performed as described for immunoblotting experiments. Metabolic Studies-Fasting blood samples were obtained after overnight fasting from the tail vein. Glucose was measured on whole blood using AccuChek II glucometer (Roche Applied Science). Glucose tolerance tests were performed in 18-h fasted animals by injecting glucose (2 mg/g) intraperitoneally as described previously (25Bernal-Mizrachi E. Wen W. Stahlhut S. Welling C.M. Permutt M.A. J. Clin. Investig. 2001; 108: 1631-1638Crossref PubMed Scopus (344) Google Scholar). Statistical Analysis-The quantitative data are presented as the means ± S.E. from at least three independent experiments, five mice, or 100 islets, unless indicated. We used the Student's t test to compare independent means. A p value of <0.05 was considered statistically significant. Development of an Inducible System to Activate Akt/mTOR Signaling in β-Cell-To determine the molecular mechanisms involved in β-cell G1-S transition by the mTOR arm of Akt signaling, we developed a dox-inducible system. DT mice were generated by crossing mice expressing the tetracycline reverse transactivator under the control of the rat insulin promoter (RIP-rTTA) with mice expressing constitutive active Akt under the control of the tetracycline operator (tetOcaAkt) (23Milo-Landesman D. Surana M. Berkovich I. Compagni A. Christofori G. Fleischer N. Efrat S. Cell Transplant. 2001; 10: 645-650Crossref PubMed Scopus (71) Google Scholar, 24Shiojima I. Sato K. Izumiya Y. Schiekofer S. Ito M. Liao R. Colucci W.S. Walsh K. J. Clin. Investig. 2005; 115: 2108-2118Crossref PubMed Scopus (759) Google Scholar). The control group for these experiments included mice containing the RIP-rTTA transgene only, but similar results were observed when compared with tetOcaAkt (ST). Immunoblotting for Akt showed that islets from DT mice treated with dox for 6 h exhibited a slight increase in Akt levels and activity assessed by phosphorylation of GSK3β on Ser9 (Fig. 1A). Akt expression and levels of phospho-GSK3β were significantly increased in islets from DT mice treated with dox for 24 h (Fig. 1A). These results indicated that the expression of Akt1 transgene in β-cells is regulated in a DOX-dependent manner. To induce Akt in β-cells from adult mice, 12-week-old mice were given doxycycline in their drinking water for 20 weeks. Intraperitoneal glucose tolerance tests before dox treatment showed no difference in glucose tolerance between ST and DT mice (Fig. 1B). Improved glucose tolerance in DT mice was observed as early as after 6 weeks of dox treatment (supplemental Fig. S1). The improved glucose tolerance in DT mice was maintained after 20 weeks of dox treatment (Fig. 1C). Assessment of 6-h fasting glucose levels in ST and DT mice at different time points during dox treatment demonstrated that glucose levels in DT mice during doxycycline treatment were not different from those obtained from ST mice (Fig. 1D). Serum concentrations of insulin in DT mice increased after 2 weeks of dox administration (Fig. 1E). Compared with ST mice, insulin levels in DT mice remained elevated after 16 weeks of dox administration (Fig. 1E). The histology of the pancreas and quantitation of β-cell mass were assessed by islet morphometry. After 20 weeks of doxycicline treatment, β-cell mass was augmented more than 5-fold in DT mice compared with ST mice (Fig. 1F). β-Cell proliferation by determined Ki67 immunostaining in insulin-stained pancreatic sections showed a 3-fold increased in proliferative rate in DT mice (Fig. 1G). These results showed that the inducible system could be used as a powerful tool to study the molecular mechanisms involved in cell cycle progression and β-cell mass under proliferative conditions induced by Akt. Rapamycin Treatment Inhibits the Activation mTORC1 Signaling by Akt-The importance of the mTORC1 in the metabolic and morphologic phenotype observed in DT mice was assessed using the following experimental design (Fig. 2A): 1) 4-week-old ST and DT mice were placed on vehicle or dox treatment in their drinking water for 3 weeks. 2) After 3 weeks of dox treatment, rapamycin was injected intraperitoneally for 2 weeks in half of the ST and DT mice. The other half of the ST and DT mice continued vehicle and dox treatment (Fig. 2A). To assess the inhibition of mTOR signaling by rapamycin treatment in ST and DT mice, we performed immunofluorescence staining using anti-phospho-S6 ribosomal protein (Ser235/236) antibody (pS6rp) (Fig. 2B). DT mice exhibited a significant increase in pS6rp staining when compared with ST mice (Fig. 2B). Staining for pS6rp in the pancreas from rapamycin-treated ST and DT mice was completely absent, suggesting that the dose of rapamycin was effective in inhibiting mTOR activation by Akt (Fig. 2B). To complement these studies, we performed immunoblotting in islet lysates from these mice. After dox administration, Akt levels were higher in DT compared with ST mice (Fig. 2C). Rapamycin treatment had no effect on Akt levels from ST or DT mice (Fig. 2C). Similar to the results shown on Fig. 2B, levels for pS6rp were higher in DT than ST mice. Rapamycin treatment of ST and DT mice completely abolished the phosphorylation of S6rp (Fig. 2C). Rapamycin Treatment Partially Reverses the Akt-mediated Improvement in Carbohydrate Metabolism-Assessment of carbohydrate metabolism in ST and DT mice treated with vehicle or rapamycin was then performed. Before administration of dox, glucose tolerance in 4-week-old ST and DT mice was comparable (Fig. 3A). In contrast to ST mice, DT mice treated with dox for 3 weeks exhibited lower glucose levels at 30, 60, and 120 min after glucose injection (Fig. 3B). Glucose tolerance after rapamycin treatment showed impaired glucose tolerance in ST+Rap mice when compared with ST mice (Fig. 3C). Glucose levels at 30, 60, and 120 min in DT+Rap mice were higher than those of DT mice treated with vehicle (Fig. 3C). Glucose tolerance in DT+Rap mice showed that glucose levels at 30 and 60 min were comparable with those of ST+Rap (Fig. 3C). The metabolic alterations induced by rapamycin treatment reversed after discontinuation of treatment (supplemental Fig. S2). These results indicate that the improvement in carbohydrate metabolism observed by activation of Akt in β-cells was partially reversed by the inhibition of the mTORC1. Assessment of islet morphometry at the end of the experimental protocol showed that the β-cell mass in ST and ST+Rap mice was comparable (Fig. 3D). In contrast, the β-cell mass in DT mice was higher than that of ST mice (Fig. 3D; p < 0.05). Rapamycin treatment of DT mice reduced β-cell mass to the levels found in ST and ST+Rap (Fig. 3D). Proliferation assessed by Ki67 staining demonstrated that ST and ST+Rap exhibited similar rates of proliferation (Fig. 3E). The proliferative rate observed in DT mice was 2-fold greater than that of ST mice (p < 0.05; Fig. 3E). The increased proliferative rate observed in DT mice was completely inhibited by rapamycin treatment (Fig. 3E). Assessment of apoptosis by cleaved caspase 3 staining demonstrated that the apoptotic rate was increased in DT mice when compared with ST mice (p < 0.05; Fig. 3F). The apoptotic rate in DT mice was reduced by rapamycin treatment (Fig. 3F). Rapamycin treatment had no effect on apoptosis in ST mice (Fig. 3F). Taken together, these experiments suggest that overexpression of Akt in β-cells induces β-cell mass by increased proliferation in an mTORC1-dependent mechanism. Rapamycin Treatment Had No Effect on mTORC2 and Akt Activity-Recent experiments suggest that the mTORC2 complex phosphorylates Akt on Ser473 (17Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5255) Google Scholar). Although the mTORC2 complex was initially described as rapamycin-insensitive, recent evidence suggests that rapamycin treatment can modulate mTORC2 activity in some systems (30Sarbassov D. Ali S. Sengupta S. Sheen J. Hsu P. Bagley A. Markhard A. Sabatini D.M. Mol. Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2191) Google Scholar, 31Zeng Z. dos Sarbassov D. Samudio I.J. Yee K.W. Munsell M.F. Ellen Jackson C. Giles F.J. Sabatini D.M. Andreeff M. Konopleva M. Blood. 2007; 109: 3509-3512Crossref PubMed Scopus (305) Google Scholar). To test whether the effects of rapamycin treatment in β-cell mass and proliferation resulted from inhibition of mTORC2/Akt signaling, we assessed the phosphorylation status of Akt and Akt kinase activity in islets from ST and DT mice treated with vehicle or rapamycin. Phosphorylation of Akt on Thr308 was increased in islets from DT mice (Fig. 4A). Rapamycin treatment had no effect on phosphorylation of Akt on Thr308 in islets lysates from ST and DT mice (Fig. 4A). Similarly, Akt phosphorylation on Ser473 was increased in DT mice compared with ST mice, and these changes were not affected by rapamycin (Fig. 4A). In vitro Akt kinase activity in islets from ST and DT mice showed increased Akt kinase activity in DT mice (Fig. 4B). Akt activity in ST or DT mice was not altered by rapamycin (Fig. 4B). Similar to islets from ST and DT mice, in vitro Akt kinase activity was increased in MIN6 cells stably transfected with a constitutively active mutant of Akt (MIN6-caAkt), and this activity was not altered by rapamycin treatment (Fig. 4C). Phosphorylation of endogenous GSK3α/β on Ser21/9 was increased in islet lysates from DT mice when compared with ST mice (4.2 ± 1.0, p < 0.05). In contrast to the in vitro Akt kinase activity, rapamycin treatment of DT islets inhibited the phosphorylation of GSK3α/β on Ser21/9, suggesting that a downstream target of mTOR could phosphorylate GSK3α/β on the same residue as Akt (Fig. 4D; p < 0.05). These experiments suggest that rapamycin treatment did not alter the phosphorylation status or activity of Akt in islets or cell lines overexpressing a constitutively active form of Akt. Rapamycin Treatment Inhibited Cdk4 Activity by Reducing Cyclin D2 and D3 Levels-The effect of rapamycin in β-cell proliferation in DT islets wa
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