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FoxO1 Protein Cooperates with ATF4 Protein in Osteoblaststo Control Glucose Homeostasis*□S

Received for publication, July 14, 2011, and in revised form, January 6, 2012 Published, JBC Papers in Press, February 1, 2012, DOI 10.1074/jbc.M111.282897

Aruna Kode‡, Ioanna Mosialou‡, Barbara C. Silva‡, Sneha Joshi‡, Mathieu Ferron§, Marie Therese Rached‡1,and Stavroula Kousteni‡¶2

From the ‡Department of Medicine, Division of Endocrinology and the §Department of Genetics and Development, College ofPhysicians and Surgeons, Columbia University, New York, New York 10032 and the ¶Department of Physiology and CellularBiophysics, College of Physicians and Surgeons, New York, New York 10032

Background: The skeleton regulates glucose metabolism and energy expenditure.Results: Two transcription factors interact to regulate the activity of an osteoblast-secreted hormone favoring energymetabolism.Conclusion: The skeleton utilizes an intricate transcriptional machinery to maintain energy homeostasis.Significance: Transcription factor-mediated regulation of energy metabolism by the skeleton has potential applications indiseases of abnormal glucose metabolism.

The Forkhead transcription factor FoxO1 inhibits through itsexpression in osteoblasts �-cell proliferation, insulin secretion,and sensitivity. At least part of the FoxO1 metabolic functionsresult from its ability to suppress the activity of osteocalcin, anosteoblast-derived hormone favoring glucose metabolism andenergy expenditure. In searching formechanismsmediating themetabolic actions of FoxO1, we focused on ATF4, because thistranscription factor also affects glucose metabolism throughits expression in osteoblasts. We show here that FoxO1 co-lo-calizes with ATF4 in the osteoblast nucleus, and physicallyinteracts with and promotes the transcriptional activity ofATF4. Genetic experiments demonstrate that FoxO1 and ATF4cooperate to increase glucose levels and decrease glucose toler-ance. These effects result from a synergistic effect of the twotranscription factors to suppress the activity of osteocalcinthrough up-regulating expression of the phosphatase catalyzingosteocalcin inactivation. As a result, insulin production by�-cells and insulin signaling in the muscle, liver and white adi-pose tissue are compromised and fat weight increases by theFoxO1/ATF4 interaction. Taken together these observationsdemonstrate that FoxO1 and ATF4 cooperate in osteoblasts toregulate glucose homeostasis.

FoxO1, one of the four FoxO isoforms of the Forkhead familyof transcription factors, is highly expressed in insulin-respon-sive tissues, including pancreas, liver, skeletal muscle, and adi-pose tissue. In all these tissues FoxO1 orchestrates the tran-scriptional cascades regulating glucose metabolism, in part bybeing a major target of insulin signaling. In most cells insulinsignaling favors FoxO1 phosphorylation; this results in FoxO1

nuclear exclusion, thereby preventing its transcriptional activ-ity. In itsmost recently discoveredmode of action in the controlof energy metabolism, FoxO1 was shown to act as a transcrip-tional link between the skeleton and the pancreas as well asinsulin target tissues by regulating the novel endocrine functionof the skeleton in energy homeostasis (1–5). Indeed, through itsexpression in osteoblasts FoxO1 decreases �-cell proliferationand function, resulting in a decrease in insulin secretion (1). Italso suppresses insulin sensitivity in insulin-target tissues suchas adipose tissue, the liver, and the muscle. These effects com-promise glucose metabolism and increase blood glucose levels.This function of FoxO1 is due to its ability to promote carbox-ylation and inactivation of osteocalcin, an osteoblast-secretedhormone that favors, insulin secretion, and sensitivity andenergy expenditure (1). Adding another level of complexity tothis function in a feedback mode of regulation, FoxO1 is also atarget of insulin signaling in osteoblasts (3). Insulin suppressesthe activity of osteoblastic FoxO1, thus, promoting osteocalcinbioactivity.In the context of whole body physiology it is remarkable that

the exact same transcriptional mediator of insulin actions in allperipheral insulin-sensitive target organs also regulates themetabolic activity of osteocalcin and its insulin-up-regulatingas well as insulin-sensitizing functions. This property estab-lishes FoxO1 as a common unifying link of insulin signalingamong all glucose-regulating organs. At the same time it raisesthe question of how such a ubiquitously expressed transcrip-tion factor could fulfill in osteoblasts a function that it does notfulfill in other cell types; that is, to affect the glucose-regulatingfunction of other organs. To address this question we searchedfor osteoblast-specific or osteoblast-enriched transcription fac-tors that could be effectors or co-regulators of FoxO1 signalingin its metabolic functions in osteoblasts.ATF4 is a transcription factor that accumulates predomi-

nantly in osteoblasts and acts through them to affect glucosemetabolism and insulin sensitivity (6). Analysis ofAtf4�/�miceshowed that these animals had a metabolic phenotype similarto that of mice lacking FoxO1 in osteoblasts and characterized

* This work was supported, in whole or in part, by National Institutes of HealthGrants R01-AR055931, 3-R01-AR055931-02S1, and P01-AG032959.

□S This article contains supplemental Table S1.1 Present address: MRC Clinical Sciences Center, Imperial College London

Hammersmith Campus, Du Cane Road, London W12 0NN, U.K.2 To whom correspondence should be addressed: The Russ Berrie Medical

Sciences Pavilion, 1150 Saint Nicholas Ave., Rm. 411, New York, NY 10032.Tel.: 212-851-5223; Fax: 212-851-5225; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 12, pp. 8757–8768, March 16, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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by enhanced insulin secretion and insulin sensitivity in the liver,fat, and muscle. Thus, we examined whether FoxO1-mediatedregulation of glucose homeostasis occurs through its interac-tion with ATF4. Here we show that FoxO1 engages in a func-tional complex with ATF4 in osteoblasts. In this complex thetwo transcription factors synergize to regulate glucose meta-bolism, insulin production, and insulin sensitivity.

EXPERIMENTAL PROCEDURES

Mice—All the protocols and experiments were conductedaccording to the guidelines of the Institute of ComparativeMedicine, Columbia University. Generation of FoxO1fl/fl,�1(I)Collagen-Cre (�1(I)Col-Cre), and Atf4�/� mice has previ-ously been reported (7–10). Mice with osteoblast specific dele-tion of FoxO1 (FoxO1osb�/�) were generated by crossingFoxO1fl/fl mice with transgenic mice expressing Cre under thecontrol of the osteoblast-specific collagen type 1A1 promoter(�1(I) Collagen-Cre) as previously described (1). Genotypingwas performed at weaning stage by PCR analysis of genomicDNA. In each experiment the mice used were of the samegenetic background as they were all littermates. In all experi-ments data presented were obtained from male animals.Histological Analysis of Pancreatic Islets, White Adipose Tis-

sue, and Liver Sections—Histological analysis was performed aspreviously described (1). Briefly, fat and pancreata were col-lected, fixed overnight in 10% neutral formalin solution,embedded in paraffin, sectioned at 4 �m, and stained withhematoxylin and eosin. Pancreatic sections were immuno-stained for� cells using guinea pig anti-swine insulin polyclonalantibody (Dako). �-Cell proliferation was assessed using anantibody recognizing Ki67 antigen, the prototypic cell cycle-related nuclear protein expressed by proliferating cells in allphases of the active cell cycle.�-Cell area represents the surfacepositive for insulin immunostaining divided by the total pan-creatic surface. �-Cell mass was calculated as �-cell area mul-tiplied by pancreatic weight. Livers were cryoembedded, sec-tioned at 5 �m, and stained with Oil red O (Crystalgen).Cell Cultures—Primary osteoblasts were prepared from cal-

varia of 5-day-old pups as previously described (2) and werecultured in fresh�MEMand 10%FBS. TheOB-6 bonemarrow-derived osteoblastic cell line has been described (11) and wascultured under the same conditions as calvaria.Transient Transfections and Luciferase Assays—Cos-7 cells

were seeded in 48-well plates at a density of 104 cells/well. Oneday after plating, cells were transfectedwith Lipofectamine Plus(Invitrogen) according to themanufacturer’s protocol.We car-ried out co-transfections of the FoxO1 or ATF4 expressionplasmids with either a FoxO-reporter construct or anOsteocal-cin-reporter construct or an ESP-reporter construct (50 ng).The Esp-luc, which bears mutations in the FoxO1 and ATF4site, was generated by site-directed mutagenesis using theQuikChange site-directedmutagenesis kit (Stratagene) accord-ing to the manufacturer’s instructions. Nucleotide substitu-tions at positions 3, 4, and 5 of these elements are indicated initalic: Foxo1, 5�-TGGGGTT-3� (WT: TGTTTTT) and ATF4,5�-ACGGAA-3� (WT: ACATCA). The total amount of DNAwas adjusted to 270 ng/well with pCMV5 or pcDNA controlvectors. Transfection was stopped by adding 20% FBS. Lucifer-

ase assays were carried out using the Dual Luciferase ReporterAssay System (Promega), and luciferase activity was quantifiedusing Fluostar Omega. pRL-CMV Renilla luciferase controlvector (20 ng) (Promega) was cotransfected as an internalstandard to normalize for transfection efficiency. Normalizedluciferase activity is presented as -fold induction over the emptyvector control (EV,3 considered 1). All experiments wererepeated at least twice.Western Blotting and Immunoprecipitation—Osteoblasts or

bone extracts (50 �g) from wild type and FoxO1osb�/� orAtf4�/� mice were analyzed on a SDS-polyacrylamide gel,transferred to a PVDF membrane, and immunoblotted withpFoxO1 (Cell Signaling), FoxO1, or ATF4 primary antibodies(Santa Cruz Biotechnology, Inc.). For immunoprecipitation,100 �g of bone/cell lysates protein were incubated with 2 �g ofspecific antibodies and 20 �l of protein A/G-agarose beads(Santa Cruz) overnight at 4 °C on a rotating device. To excludeDNA-mediated effects, co-immunoprecipitation was per-formed in the presence of ethidium bromide (50 �g/ml). Alter-natively, cell extracts were treated with benzonase (10 units/reaction) for 30 min. Image J software was used for gel bandquantitative densitometric analysis.Protein Interaction Analysis—GST-FoxO1 (aa 1–300, 290–

655) constructs and His-ATF4 (aa 1–151, 110–121, 186–349)constructs have been described (9, 12, 13). Full-length GST-FoxO1 construct was obtained from Addgene. The GST andHis fusion proteins were expressed in Escherichia coli strainBL21pLys by isopropyl thiogalactose induction. Extracts of theGST fusion protein-transformed cells were coupled with gluta-thione-Sepharose beads. After SDS-PAGE andCoomassie Bril-liant Blue staining for monitoring coupling efficiency, GSTfusion protein-bound beads were incubated with purified Hisfusion proteins overnight, washed extensively, and eluted byboiling in SDS-PAGE loading buffer. Bound proteins were visu-alized by Western blot using anti-His antibody.Immunocytochemistry—Primary osteoblasts were grown on

8-well chamber slides and treated with either vehicle or 100 �M

H2O2 for an hour. After a treatment period cells were fixedwithice-cold methanol for 5 min at �20 °C. After washing with 1�PBS, cells were blocked with 10% normal goat serum in PBS for1 h and then incubated overnight with indicated primary anti-bodies (1:200 dilution in 1% goat serum in PBS) in a humidifiedchamber at 4 °C. Samples were washed with 1� PBS and incu-bated with CY3-conjugated (red) and CY2-conjugated (green)secondary antibodies (Jackson ImmunoResearch) for 1 h atroom temperature to visualize FoxO1 and ATF4, respectively.Cells were counterstained with DAPI to show the nuclear mor-phology. Images were acquired with aNikon 80i EclipseMicro-scope using a Retiga digital camera.Real-timeQuantitative PCRAnalysis—DNase I (Invitrogen)-

treated RNAwas reverse-transcribed at 42 °C with SuperScriptII (Invitrogen). The expression of all the genes wasmeasured byreal-time quantitative PCR with the SYBR Green master mixusing �-actin as endogenous control with 1 cycle at 95 °C for 10

3 The abbreviations used are: EV, empty vector control; PPAR, peroxisomeproliferator-activated receptor; EV, empty vector; aa, amino acids.

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min followed by 40 cycles at 95 °C for 30 s and 60 °C for 1 min.The primer sequences are given in supplemental Table 1.Electrophoretic Mobility Shift Assay (EMSA)—30-Mer com-

plementary oligonucleotides spanning the ATF4 and theFOXO1 binding site of the murine Esp promoter and firstintron or mutated ATF4 site and the FOXO1 site were used toperform gel shift assays. The wild type sequence of the oligonu-cleotides used for ATF4 site was 5�-AGCATCCTGCCAACA-TCACCAAGAACCGGT-3� and for FOXO1 site was 5�-CAT-TCCCACGCATGTTTTTCTCACCCGTTC-3�, The mutatedsequence (shown in lowercase) for ATF4 site was 5�-AGCAT-CCTGCCAACggaACCAGAACCGGT-3� and for FOXO1site was 5�-CATTCCCACGCATGgggTTCTCACCCGTTC-3�.Overlapping oligonucleotide strands were heat-denatured andannealed overnight. 50 ng of duplex oligonucleotides was5�-end labeled with [�-32P]ATP and T4 polynucleotide kinase(Promega) and purified using a Micro Bio-Spin P-30 column(Bio-Rad). The eluted probewas used for EMSA.Aliquots of thenuclear preparations from murine primary calvaria cells (5 �gof protein) were incubated for 20 min at 27 °C with 2 �g ofpoly(dI-dC) with or without unlabeled specific or nonspecificDNA competitor or ATF4 and FOXO1 antibodies (Santa Cruz)in binding buffer followed by the addition of the labeled oligo-nucleotide probe and incubation for 30 min at 27 °C. The sam-ples were separated by electrophoresis on a 6% nondenaturingpolyacrylamide gel and analyzed by using Typhoon Phosphor-Imager (Molecular Dynamics).Metabolic Studies—An intraperitoneal glucose tolerance test

was performed by administering 2 g of glucose per kg of bodyweight intraperitoneally after an overnight fast. Blood glucosewas monitored using blood glucose strips (Diabetes Associa-tion) and theAccu-Check glucometer. For the insulin tolerancetest, mice were fasted from 4 to 6 h and injected intraperitone-ally with insulin (0.5 units/kg of body weight), and blood glu-cose levels were measured at the indicated times. Insulin toler-ance test data are presented as percentage of initial bloodglucose concentration. Body composition was measured withNMR (Bruker Optics).Physiological Assays—Sera were collected by heart puncture

from mice in the fed state. Blood was kept on ice for 15 minbefore centrifugation for 15 min at maximum speed. Insulin(Mercodia) was measured by enzyme-linked immunosorbentassay (ELISA). Serum osteocalcin was measured by an immu-noradiometric assay (Immutopics) according to the manufac-turer’s instructions. A triple ELISA system was used for quan-tification of mouse total, carboxylated, and uncarboxylatedosteocalcin as described previously (14). Thismethodmeasuresthe exact serumconcentration ofGLU,GLA13, and total osteo-calcin and distinguishes between completely decarboxylatedosteocalcin (GLU-OCN) and the osteocalcin decarboxylatedon Glu-13, a residue that is critical for in vivo activation ofosteocalcin.Statistical Analyses—Results are given as the means � S.E.

Statistical analyses were performed using unpaired two-tailedStudent’s t or a oneway analysis of variance (Student-Newman-Keuls) for more than two groups.

RESULTS

An interaction between FoxO1 and ATF4 was demonstratedby a co-immunoprecipitation experiment showing that the twotranscription factors physically associate in osteoblasts as wellas in bone (Fig. 1,A andB). This interactionwasDNA-indepen-dent as demonstrated by the lack of any effect of ethidium bro-mide or benzonase on the FoxO1/ATF4 complex (Fig. 1C). Wealso defined the binding interface between FoxO1 andATF4 byusing deletion mutants of each protein. For FoxO1 we usedGST-tagged full-length or two GST-tagged fragments thatcarry either the N-terminal domain, aa 1–300 containing theForkhead domain, or the C-terminal domain, aa 290–655 con-taining the transactivation domain of FoxO1 (Kitamura et al.(12), Puigserver et al. (13)). For ATF4 (Yang et al. 9) we used 3His-tagged fragments that carry the transactivation domain 1(aa 1–151) or the kinase-inducible domain (aa 110–221) or theDNA-binding and leucine zipper domains (aa 186–349). TheDNA binding domain of ATF4 interacted with the C-terminal,transactivation domain of FoxO1 (Fig. 1D). In addition, full-length FoxO1, similar to the transactivation domain of FoxO1,interacted with the DNA binding domain of ATF4 (Fig. 1E).These results establish that FoxO1 and ATF4 physically inter-act through their transactivation and DNA binding domains,respectively.Immunohistochemical analysis in primary osteoblasts con-

firmed that FoxO1 and ATF4 co-localize in the nucleus (Fig.1F). However, both proteins can also be found in the cytoplasmas they are known to shuttle between the two subcellular com-partments. In the absence of any stimuli from growth factors orstress-related stimulus, the two transcription factors are pre-dominantly located in the cytoplasm. Growth factor or stresssignals stimulate their translocation to the nucleus, where theyactively operate to initiate transcriptional events that promotemultiple cell functions. Indeed, stressing the cells by treatmentwith H2O2 induced nuclear accumulation and colocalization ofFoxO1 and ATF4 in the nucleus (Fig. 1G).We reasoned that if FoxO1 and ATF4 participate in a func-

tional complex, their interactionwould occur at either the tran-scriptional or the protein activation level. Therefore, we firstexamined whether one transcription factor affects the expres-sion of the other. Atf4 expression and protein levels were notaltered in FoxO1-deficient osteoblasts (Fig. 1, H and J). Simi-larly, ATF4 inactivation had no effect on gene expression orprotein levels of FoxO1 in osteoblasts (Fig. 1, I and Ks), indicat-ing that transcriptional events were not involved in the interac-tion between FoxO1 and ATF4. In contrast, either transcrip-tion factor could transactivate the other. ATF4 stimulatedFoxO1 activity asmeasured on a FoxO1 reporter construct car-rying FoxO binding sites from the IGF-1 promoter (Fig. 1L).Because this reporter construct does not contain anATF4bind-ing site, the ability of ATF4 to enhance FoxO1 activity on thispromoter suggests that ATF4 enhances FoxO1 activity withoutdirectly binding to its promoter. In support of this observation,phosphorylation of FoxO1 at Ser-256 was increased in thebones of Atf4�/� mice (Fig. 1K). Because FoxO1 phosphoryla-tion at Ser-258 indicates FoxO1 inactivation, this result sug-gests that FoxO1 activity is decreased in Atf4�/� bones. Simi-

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larly, forced expression of FoxO1 along with ATF4 alsoenhanced the transactivating ability of ATF4 as measured byOG2-Luc, a reporter construct carrying 147 bp of the osteocal-cin promoter fused to the luciferase gene. This construct con-tains one ATF4 binding site (9) (Fig. 1M).Improved GlucoseMetabolism in FoxO1osb�/�;Atf4�/� Mice—

Having established that the two transcription factors are involvedin a functional interaction, we asked whether ATF4 is involved inthe glucose-regulating action of FoxO1 through osteoblasts. As afirst approach to answering this question we compared the meta-bolic phenotype of mice lacking either FoxO1 or ATF4 in osteo-blasts (1, 6).Bothdeletionsdecreasedglucose levels, improvedglu-cose tolerance and insulin sensitivity, and were associated withincreased insulin secretion and high energy expenditure, indicat-ing that the two transcription factorsmay synergize onosteoblaststo regulate energy metabolism. We tested this hypothesis geneti-cally by using compound mutant mice lacking a single allele ofFoxO1 in osteoblasts and ATF4 (FoxO1osb�/�;Atf4�/�).

First we searched for an effect on blood glucose levels. FoxO1or ATF4 deletion led to low blood glucose levels at the randomfed state, whereas FoxO1 or ATF4 happloinsufficiency had aminimal or no effect on blood glucose (Fig. 2A). Happloinsuffi-ciency for both transcription factors resulted in a low glucosephenotype similar to that of knock-out animals. Similar to glu-cose levels, the ability to metabolize glucose was improved asseen by the improvement of glucose tolerance in FoxO1osb�/�;Atf4�/�mice versus FoxO1osb�/� orAtf4�/� andwild type con-trol animals (Fig. 2B).The lower glucose levels of ATF4 and FoxO1 double happlo-

insufficientmicewas associatedwith an increase in serum insu-lin levels to an extent similar as that of FoxO1osb�/� andAtf4�/� animals (Fig. 2C). Consistent with the increase in insu-lin levels, islet size, islet numbers, �-cell area, and �-cell masswere increased inAtf4�/�;FoxO1osb�/�mice (Fig. 2,D–G). Theincrease in insulin levels in the serum of FoxO1osb�/�;Atf4�/�

mutant mice was due to an increase in �-cell proliferation thatwas equal to the increase seen in mice lacking FoxO1 in osteo-blasts or lacking ATF4 (Fig. 2, H–I).

FoxO1 and ATF4 Function Synergistically to Control InsulinSensitivity—Low glucose levels and the improvement in glu-cose tolerance in the FoxO1osb�/�;Atf4�/�mice was not onlydue to increased insulin production. Indeed, in addition tohyperinsulinemia, insulin sensitivity, assessed by an insulin tol-erance test, was increased with double FoxO1 and Atf4 happlo-insufficiency as compared with FoxO1osb�/� or Atf4�/� miceorwild type littermates (Fig. 3A). Consistentwith higher insulinlevels and greater insulin sensitivity, the expression of the insulintarget gene Pgc1� was increased in the muscle of FoxO1osb�/�;Atf4�/� mice as compared with FoxO1osb�/�, Atf4�/�, andwild type animals (Fig. 3B). The expression of two Pgc1� targetgenes, Nrf1 andMcad, was also increased (Fig. 3, C and D).

To uncover the mechanism leading to increased insulin sen-sitivity in FoxO1osb�/�;Atf4�/� mice, we examined the expres-sion profile of various insulin-regulating adipokines. Expres-sion levels of the insulin-sensitizing hormone adiponectin (15)were increased by FoxO1 and Atf4 haploinsufficiency in osteo-blasts (Fig. 3E). In contrast, expression of the insulin-sensitizinghormone leptin (16) or expression of resistin, an adipokinemediating insulin resistance (17), was not affected inFoxO1osb�/�;Atf4�/� mice (Fig. 3, F and G). These observa-tions were consistent with the lack of any effect of FoxO1 orAtf4 deletion in leptin and resistin expression in FoxO1osb�/�

andAtf4�/� mice. Confirming the regulation of adiponectin bythe FoxO1/ATF4 interaction in osteoblasts and the increase ininsulin sensitivity, expression of the adiponectin targets acyl-CoA oxidase, peroxisome proliferator-activated receptor-�(Ppar�), and uncoupling protein 2 (Ucp2) was increased in themuscle of FoxO1osb�/�;Atf4�/� mice (Fig. 3, H–J).The up-regulation of insulin-sensitive genes Pgc1� and

Mcad and the adiponectin targets Ppar� andUcp2 in the mus-cle may indicate an increase in energy expenditure inFoxO1osb�/�;Atf4�/� mice. All four genes are involved in mito-chondrial biogenesis, and increased mitochondrial respirationincreases fatty acid oxidation, which leads to a decrease in lipo-genesis, increase in insulin sensitivity, and improved glucosetransport. More specifically, PPAR� is a key regulator of fatty

FIGURE 1. A functional interaction between FoxO1 and ATF4 in osteoblasts. A and B, immunoprecipitation (IP) and immunoblotting (WB) of FoxO1 andATF4 in nuclear extracts from primary osteoblasts and bones of WT mice is shown. The three lanes show binding in protein lysates from three individual mice.C, OB-6 cell lysates were coimmunoprecipitated with ATF4 and FoxO1 in the presence of vehicle (veh), ethidium bromide (EtBr) or after pretreatment withbenzonase (benz). Non-immune IgG was used as negative control. D, left panel, binding of GST-FoxO1 and His-ATF4 in a cell-free system and mapping of theFoxO1 interaction domain is shown. Purified full-length His-ATF4 was incubated with glutathione-Sepharose beads coupled with GST or the indicted GST-FoxO1 fusion proteins. Bound proteins were analyzed by SDS-PAGE and immunoblotting using the anti-His antibody. A small aliquot of the GST-coupled beadswas subjected to SDS-PAGE and Coomassie Brilliant Blue staining (bottom panel). Right panel. binding of GST-FoxO1 and His-ATF4 in a cell-free system andmapping of the ATF4 interaction domain. Purified truncated fragments of His-ATF4 were incubated with glutathione-Sepharose beads coupled with GST or theGST-FoxO1 (aa 290 – 655) fusion protein. Bound proteins were analyzed by SDS-PAGE and immunoblotting using the anti-His antibody. E, left panel, binding ofGST-FoxO1 and His-ATF4 in a cell-free system and mapping of ATF4 interaction domain is shown. Purified truncated fragments of His-ATF4 were incubatedwith glutathione-Sepharose beads coupled with full-length GST-FoxO1 fusion protein. Bound proteins were analyzed by SDS-PAGE and immunoblotting usingthe anti-His antibody. A small aliquot of the GST-coupled beads was subjected to SDS-PAGE and Coomassie Brilliant Blue staining (right panel). F, shown isimmunohistochemical localization of FoxO1 and ATF4 in primary osteoblasts. Single cell images depict FoxO1, ATF4, DAPI, and a combination of ATF4 withDAPI or FoxO1 with ATF4 stainings. 40� magnification images show staining with the indicated antibodies. G, shown is immunohistochemical localization ofFoxO1 and ATF4 in primary osteoblasts treated with H2O2. 20� magnification images show multiple cells with nuclear co-localization of FoxO1 and ATF4.H, RT-PCR (real time PCR) analysis of Atf4 expression in the bone of WT and FoxO1osb

�/� mice (n � 5 mice/group) is shown. I, RT-PCR (real time-PCR) analysis ofFoxO1 expression in the bone of WT and Atf4�/� mice (n � 5 mice/group) is shown. J, immunoblotting analysis of protein levels of ATF4 in the bone of WT andFoxO1osb

�/� mice is shown. K, immunoblotting analysis of protein levels and phosphorylation of FoxO1 in the bone of WT and Atf4�/� mice is shown.L, co-transfection of FoxO1, ATF4, and FoxO-Luc reporter construct in COS-7 cells is shown. Results are presented as -fold induction over EV (EV � 1). *, p � 0.05versus FoxO-luc; #, p � 0.05 versus FoxO1/FoxO-luc). M, co-transfection of FoxO1, ATF4, and OG2-Luc reporter construct in COS-7 cells is shown. Results arepresented as -fold induction over EV (EV � 1). * p � 0.05 versus OG2-luc; #, p � 0.05 versus ATF4/OG2-Luc and versus FoxO1/OG2-Luc. In J and K the results showduplicate samples. The intensity of the bands was calculated by densitometry for each sample and corrected for loading by dividing with �-actin. The numbersbelow the lanes indicate ratio of phosphorylated or total FoxO1 or ATF4 versus �-actin signal.

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acid oxidation in skeletal muscle. Mcad is involved in the firststep of the mitochondrial �-oxidation of fatty acids. Deficiencyin MCAD is the most common inborn error observed in theprocessing of mitochondrial �-oxidation of fatty acids, and it isone of the most common inherited disorders of metabolism.The synergistic effect of FoxO1 and ATF4 in the expression ofthese genes suggests that the two transcription factors interactto regulate mitochondrial activity and metabolism in themuscle.In parallel with the improved glucose metabolism, gonadal

fat was decreased in mice happloinsufficient for FoxO1 and

Atf4, as compared with single heterozygous FoxO1osb�/� orAtf4�/� mice or wild type animals (Fig. 4A). The decrease ingonadal fat was similar to that seen in FoxO1osb�/� or Atf4�/�

mice. Total fat content was decreased (Fig. 4B), whereas leanmass and body weight were not affected in FoxO1osb�/�;Atf4�/�mice (Fig. 4,C andD). However, whereasAtf4�/�miceshow a decrease in fat content, lean mass, and body weight,FoxO1osb�/� mice showed no changes in any of the threeparameters. Therefore, regulation of total fat and muscle massappears to be independent of a synergistic interaction betweenthe two transcription factors and due to ATF4 deletion. It is

FIGURE 2. Improved glucose metabolism and increased insulin production in FoxO1osb�/�;Atf4�/� mice. A, blood glucose at random feeding is shown;

n � 8. B, glucose tolerance test; n � 8 mice/group. C, shown are serum insulin levels at random feeding is shown; n � 8. Insulin staining (D) and increased isletnumber (E), �-cell area (F), and �-cell mass (G) Ki67 immunostaining (H) shows larger islets and increased �-cell proliferation in the pancreas of FoxO1osb

�/�;Atf4�/�/� mice (I). n � 5 mice/group. In all panels bars indicate means � S.E. *, p � 0.05 versus WT; #, p � 0.05 versus FoxO1osb

�/�;Atf4�/� group. All mice were2 months of age.

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possible that these additional properties of ATF4 are due to thefact that it was deleted in all cell types rather than just osteo-blasts as was the case with FoxO1. Alternatively, ATF4 mayregulate distinct protein(s) in osteoblasts with additional met-abolic effects. Indeed, ablation of osteoblasts in amousemodel,has indicated that in addition to osteocalcin, other osteoblast-secreted proteins may confer the glucose-regulating propertiesof the skeleton (5). The decrease in gonadal fat weight demon-strated in FoxO1osb�/�;Atf4�/� mice occurred despite theirhyperinsulinemia and improved insulin sensitivity, suggesting adefect in adipogenesis that would be independent of insulinregulation. Indeed, adipocyte numbers were decreased by 23%in FoxO1osb�/�;Atf4�/� mice (Fig. 4E). In contrast, adipocytesize was increased despite the decrease in perigonadal fatweight further, suggesting that adipocyte differentiationmay becompromised in these mice (Fig. 4F).

Consistent with the decrease in gonadal fat weight, theexpression of the adipogenic gene C/EBP� and two lipolyticgenes perillipin and triglyceride lipase (Tgl), whose expressionis inhibited by insulin, was decreased in FoxO1osb�/�;Atf4�/�

mice as compared with FoxO1osb�/�, Atf4�/�, and wild typemice (Fig. 4, G–I). Expression of lipoprotein lipase (Lpl) wasunaffected (Fig. 4J). These molecular changes indicated thatwhereas both adipogenesis and lipolysis may be regulated by aFoxO1/ATF4 interaction in osteoblasts, lipogenesis and fattyacid uptake are probably not affected.Finally, we looked for a potential involvement of the FoxO1

and ATF4 synergism in the control of insulin signaling inanother major glucose-regulating organ, the liver. Suggesting arole for the FoxO1/ATF4 interaction, expression of the insulintarget FoxA2, which regulates lipogenesis and ketogenesis dur-ing fasting, was increased in FoxO1osb�/�;Atf4�/� mice as

FIGURE 3. Increased insulin sensitivity by FoxO1/ATF4 interaction in osteoblasts. A, an insulin tolerance test is shown. n � 8 mice/group. B–D, real-timePCR analysis of the insulin target genes Pgc1�, Nrf1, and Mcad in skeletal muscle is shown; n � 4 mice/group. Expression of adiponectin (E), leptin (F), andresistin (G) in gonadal fat is shown. n � 4 mice/group. Real-time PCR analysis of the adiponectin target genes acyl-CoA oxidase (H), Ppar� (I), and Ucp2 (J) inskeletal muscle is shown; n � 4 mice/group. In all panels bars indicate the means � S.E. *, p � 0.05 versus WT and #, p � 0.05 versus FoxO1osb

�/�;Atf4�/� group.All mice were 2 months of age.

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FIGURE 4. Increased fat metabolism and hepatic insulin sensitivity in FoxO1osb�/�;Atf4�/� mice. A, gonadal fat pad weight is shown. n � 8 mice/group.

Total fat content (B), body weight (C), and lean body mass (D) are shown; n � 8 mice/group. Histomorphometric analysis of white fat sections show adipocytenumbers (E) and adipocyte size (F); n � 5 mice/group. G–J, real-time PCR analysis of the insulin target genes C/EBP�, perillipin, Tgl, and Lpl in white fat, n � 4mice/group is shown. K–M, real-time PCR analysis of the insulin target genes FoxA2, G6Pase, and Pepck1 in the liver is shown; n � 4 mice/group. N, Oil red Ostaining in liver sections is shown. Images were taken at 60� magnification; arrows indicate lipid droplets. In all panels bars indicate the means � S.E.; *, p � 0.05versus WT and #, p � 0.05 versus FoxO1osb

�/�;Atf4�/� group. All mice were 2 months of age.

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compared with FoxO1osb�/�, Atf4�/� and wild type mice (Fig.4K). In contrast, expression of G6Pase and Pepck1 wasdecreased in FoxO1osb�/�;Atf4�/� mice as compared withFoxO1osb�/�, Atf4�/�, and wild type animals (Fig. 4, L andM).Consistent with the improved insulin sensitivity observed inFoxO1osb�/�;Atf4�/� mice, their liver fat content wasdecreased as compared with heterozygous FoxO1osb�/� orAtf4�/� mice (Fig. 4N). These results indicate that FoxO1 andATF4, through their interaction in osteoblasts, inhibit insulinsensitivity in the liver.FoxO1/ATF4 Interaction Suppresses Osteocalcin Activity—

To identify the molecular target that mediates the effects ofosteoblast-expressed FoxO1 and ATF4 to the pancreas and theother insulin sensitive target tissues, we focused on osteocalcinand examined whether the two transcription factors co-opera-tively affect osteocalcin expression or activity. Osteocalcinexpression in the bone and serum levels were not altered inFoxO1osb�/�;Atf4�/� mice as compared with single heterozy-gous animals or wild type littermates (Fig. 5, A and B). Asexpected, expression and serum levels of osteocalcin wereincreased inFoxO1osb�/�but decreased inAtf4�/�mice (Fig. 5,A and B). Consistent with these observations, in cotransfectionexperiments performed in Cos-7 cells, a FoxO1 expression vec-tor decreased the activity of a reporter construct containing a2.9-kb fragment of the osteocalcin promoter and first intronfused to the luciferase gene (Fig. 5C). On the other hand, ATF4stimulated the activity of the osteocalcin reporter construct(Fig. 5C). The osteocalcin reporter contains the previouslyidentified �1270 bp (TGTTTTG), -1074 bp (TGTTTT), and�250 bp (TGTTTGC) FoxO1 binding sites as well as an ATF4binding site at �75 bp (1, 9). However, transcriptional repres-sion by FoxO1 is rarely mediated through direct binding to anyof the known FoxO1 binding sites (18). Thus, we examinedwhether FoxO1 represses osteocalcin transcription throughinhibiting Runx2. We found that whereas Runx2 expressionwas not altered in the bones of FoxO1osb�/� mice (Fig. 5D), theability of Runx2 to activate osteocalcin transcription was inhib-ited by FoxO1 (Fig. 5E). The Runx2 binding site (�146 bp) ispresent in the reporter construct used.Because osteocalcin expression was not affected in

FoxO1osb�/�;Atf4�/� mice, we examined potential changes inits activity. Osteocalcin exists in a carboxylated or partiallyuncarboxylated (undercarboxylated) form. In its under/uncar-boxylated form, osteocalcin acts as a hormone to favor alto-

gether �-cell proliferation, insulin secretion, insulin sensitivity,and energy expenditure (2). We examined whether FoxO1 andATF4 regulate osteocalcin activity by measuring its degree ofcarboxylation in the serum. We found that 33% of osteocalcinpresent in the serum of FoxO1osb�/�;Atf4�/� mice was under-carboxylated (Fig. 5F), as measured by decarboxylation on Glu-13, a residue that has been shown to be critical for in vivo acti-vation of osteocalcin (14). In contrast, only 23% ofundercarboxylated osteocalcin was present in the serum ofFoxO1osb�/� or Atf4�/� or wild type mice. Undercarboxylatedosteocalcin was increased in FoxO1osb�/� and Atf4�/� mice tolevels similar to that of FoxO1osb�/�;Atf4�/� mice.

Osteocalcin bioactivity can be regulated in a bimodal mech-anism of action. In the first mechanism osteocalcin activity isnegatively regulated by another gene expressed in osteoblasts,Esp. Protein-tyrosine phosphatase (OST-PTP), the product ofEsp, decreases osteocalcin bioactivity by favoring its carboxyl-ation (2). In the second mechanism bone resorption induces achange in the pH that spontaneously decarboxylates and acti-vates osteocalcin (3). Respective to the latter mechanism, wedid not detect any increases in osteoclast function inFoxO1osb�/�;Atf4�/� mice that would indicate an increase inbone resorption (Fig. 5G). However, expression of Esp wasreduced in the bone of FoxO1osb�/�;Atf4�/�mice as comparedwith heterozygous FoxO1osb�/� or Atf4�/� and to wild typemice (Fig. 5H). The mechanism of the stimulatory effect ofFoxO1 and ATF4 on Esp expression was explored further usingCos-7 cells. Transfection of either FoxO1 or ATF4 stimulatedthe activity of Esp as measured using an Esp reporter constructthat carries 722 bp of the promoter region and 1095 bp of thefirst intron and exon of the gene (region �722 to �1095) (Fig.5I). This region of Esp contains a FoxO1 binding site at 947 bpof the first intron of the gene and oneATF4 binding site presentat position �340 bp (1, 9). A combination of different amountsof FoxO1 and ATF4 showed a synergistic effect of the two tran-scription factors in up-regulating Esp-luc activity. Collectively,these observations suggested that down-regulation of Espexpression in FoxO1osb�/�;Atf4�/� as compared withFoxO1osb�/� orAtf4�/�mice could account for the decrease inosteocalcin carboxylation and the metabolic phenotype of theFoxO1;Atf4mutant mice.The synergistic mode of action of FoxO1 and ATF4 on Esp

expression was further examined. Site directed mutagenesiswas performed on each of the FoxO1 or ATF4 binding sites

FIGURE 5. FoxO1/ATF4-dependent regulation of osteocalcin expression and activity. A, serum osteocalcin levels in WT and FoxO1osb�/�;Atf4�/� mice, n �

5 mice/group are shown. B, real-time PCR analysis of osteocalcin gene expression in WT and FoxO1osb�/�;Atf4�/� bones is shown; n � 4 mice/group.

C, co-transfection analysis of ATF4, FoxO1, and Ocn-Luc reporter construct in COS-7 cells is shown. Results are presented as -fold induction over EV (EV � 1). *, p �0.05 versus Ocn-Luc; #, p � 0.05 versus ATF4/Ocn-Luc. D, real-time PCR analysis of Runx2 gene expression in bones of WT and FoxO1osb

�/� mice is shown; n �4 mice/group. E, co-transfection analysis of FoxO1, Runx2, and Ocn-Luc in COS-7 cells is shown. Results are presented as -fold induction over EV (EV � 1). *, p �0.05 versus Ocn-Luc; #, p � 0.05 versus Runx2/Ocn-Luc. F, shown are changes in undercarboxylated osteocalcin (osteocalcin decarboxylated on Glu-13) in serumof WT and FoxO1osb

�/�;Atf4�/� mice, n � 5 mice/group. Values are presented as % of total osteocalcin present in the serum. G, osteoclast function in WT andFoxO1osb

�/�;Atf4�/� mice is shown; n � 8 mice/group. H, real-time PCR analysis of Esp expression in femurs of WT and FoxO1osb�/�;Atf4�/� is shown; n � 4

mice/group. In all panels, bars indicate the means � S.E. *, p � 0.05 versus WT and #, p � 0.05 versus FoxO1osb�/�;Atf4�/� group. Mice were 2 months of age.

I, co-transfection analysis of FoxO1, ATF4, and Esp-Luc in COS-7 cells is shown. Results are presented as -fold induction over EV (EV � 1). *, p � 0.05 versusEsp-Luc; #, p � 0.05 versus FoxO1/Esp-Luc, 2XFoxO1/Esp-Luc ATF4/ESP-Luc, and 2XATF4/Esp-Luc. J and K, EMSA was performed using 32P-labeled oligonu-cleotides containing the ATF4 site (K) at �340 bp of the ESP promoter and the FoxO1 (L) site present at 947 bp of the first intron of the Esp gene. Nuclear extracts(NE, 5 �g) from primary osteoblasts were used as a source of ATF4 or FOXO1 protein. Preincubation was done with either ATF4 antibody or FOXO1 antibody.Similar analysis was also performed using mutant ATF4 site and mutant Foxo1 site as radiolabeled probes. Similar results were observed in at least threeindependent experiments. L, co-transfection analysis of FoxO1, ATF4, Esp-Luc, and Esp-Luc mutants in COS-7 cells are shown. Esp-Luc (FoxO1 mutant) andEsp-Luc (ATF4 mutant denote Esp-Luc reporter plasmids with mutated FoxO1 or ATF4 binding sites, respectively. Results are presented as -fold induction overEV (EV � 1). *, p � 0.05 versus Esp-Luc; #, p � 0.05 versus FoxO1/Esp-Luc and ATF4/ESP-Luc.

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present in the Esp promoter.Mutation of the ATF4 binding site(ACATCA) present at �340 bp of the Esp promoter abolishedbinding of ATF4 to Esp (Fig. 5J). Similarly, mutation of theTGTTTTT binding site of FoxO1 at 947 bp of the first intron ofthe Esp gene abolished binding of FoxO1 to Esp (Fig. 5K). Wefound that mutation of either the ATF4 or FoxO1 binding siteabolished activation of Esp by each transcription factor (Fig.5L). In addition, combination of FoxO1 and ATF4 also failed toinduce activation of Esp when either the ATF4 or FoxO1 bind-ing site was mutated. Thus, synergistic activation of Esp byATF4 and FoxO1 requires both factors to be bound simultane-ously to DNA. Collectively, these observations indicate thatFoxO1 and ATF4 interact in osteoblasts to control glucosehomeostasis by regulating the carboxylation of osteocalcinthrough regulating Esp expression.

DISCUSSION

In this study we have shown that two transcription factors,FoxO1 and ATF4, interact in osteoblasts to control their endo-crine properties as regulators of glucose homeostasis. Osteo-blast-expressed FoxO1 and ATF4 cooperate to increase bloodglucose levels, trigger glucose intolerance and insulin insensi-tivity, and hinder insulin signaling in insulin-sensitive targettissues such as the muscle, liver, and white adipose tissue. Pan-creatic function is also affected as the FoxO1/ATF4 interactionin osteoblasts leads to suppression of �-cell proliferation with asubsequent decrease in insulin production.The hormonal mediator of the energy homeostatic proper-

ties of the skeleton is a protein specifically expressed by osteo-blasts, osteocalcin (2). Glucose metabolism, insulin sensitivity,and energy expenditure are all favored by osteocalcin as shownby genetic models of gain and lack of osteocalcin function. Themetabolic activity of osteocalcin is determined by its degree ofcarboxylation. Osteocalcin is carboxylated in three glutamicresidues in a vitamin K-dependent post-translational modifica-tion that confers to it high affinity to minerals present in bone.However, it is the undercarboxylated form of osteocalcin (theone in which Glu-13 is not carboxylated) that is metabolicallyactive on �-cells (14). Osteocalcin carboxylation is under thecontrol of insulin signaling in osteoblasts, which acting throughits receptor suppresses the expression of the anti-osteoclasto-genic cytokine osteoprotegerin (Opg) and as a result stimulatesbone resorption (3). In turn, the acidic environment of theresorptive lacunae promotes osteocalcin decarboxylation. Esp,protein-tyrosine phosphatase that is also expressed in osteo-blasts, acts as a substrate of the insulin receptor. Esp antago-nizes insulin signaling in osteoblast and by doing so promotescarboxylation and, therefore, osteocalcin inactivation. Con-firming the genetic studies, administration of uncarboxylatedosteocalcin to mice lowers blood glucose levels by increasinginsulin production and insulin sensitivity and favors white adi-pose tissue metabolism by enhancing energy expenditure (19).Our studies show that FoxO1 and ATF4 potently control theglucose-regulating properties of osteoblasts precisely by con-trolling osteocalcin activity, as their cooperative action sup-presses osteocalcin carboxylation. This effect is in line with anda consequence of their ability to concomitantly stimulateexpression of Esp by directly binding to the Esp promoter.

A calculation of the actual amounts of undercarboyxlatedosteocalcin in the different groups suggests that whereas wildtype, FoxO1osb�/�, and Atf4�/� mice have the same amount(30 ng/ml), active osteocalcin levels are increased in Atf4�/�;FoxO1osb�/� mice (42 ng/ml), very high in the FoxO1osb�/�

mice (90 ng/ml) but low in theAtF4�/� mice (23 ng/ml). Basedon this information, the levels of undercarboxylated osteocal-cin in these mice do not completely explain the improved met-abolic phenotype of FoxO1osb�/� and Atf4�/� animals. At thepresent time we can foresee two explanations for this apparentinconsistency. The first one is that the ratio of undercarboxy-lated to total osteocalcin is a more reliable marker of osteocal-cin activity than the total level of undercarboxylated osteocal-cin. This would imply that either carboxylated andundercarboxylated osteocalcin may have opposite effects onmetabolism or that total osteocalcin levels itself may affect glu-cose homeostasis. In support of this hypothesis are studies sug-gesting that total and carboxylated osteocalcin can both influ-ence glucose homeostasis in humans (20, 21). Alternatively, it ispossible that themetabolic phenotype of theAtf4 and FoxO1osbnull animals is not solely due to a change in osteocalcin levelsbut is the result of additional actions of these transcription fac-tors in osteoblasts. In support of the latter contention, recentevidence suggests that there are osteocalcin-independent influ-ences of osteoblasts on energy metabolism (5).The cooperativemode of interaction with another transcrip-

tion factor as ameans of regulating target protein activity is notcommon for the spectrum of FoxO1 functions. Indeed, mostfrequently FoxO1 has been shown to regulate the expression oftranscription factors that are required for cell differentiationand function. For example, in the adipose tissue FoxO1 sup-presses the expression of amaster adipogenic transcription fac-tor, PPAR�, through direct binding to the promoter. However,these effects are transcriptional and involve direct regulationof the expression of FoxO1 target transcription factors. Unlikethem, the interaction of FoxO1withATF4 occurs at the proteinlevel, is independent of changes in gene expression, and resultsin regulation of the activity of common transcriptional targetsshared by both transcriptional factors. A similar mode ofFoxO1 interactions with other transcription factors has beenreported in both the pancreas and white adipose tissue. In thelatter, FoxO1 physically interacts with C/EBP� to promoteexpression of adiponectin (22) but suppresses PPAR� activityby competitively inhibiting the formation of a PPAR�-RXRfunctional complex (23). In the pancreas FoxO1 negatively reg-ulates expression of Pdx1, which regulates �-cell developmentandmass by competing with the transcription factor FoxA2 forbinding to the Pdx1 promoter (24). This is one of the maineffects thatmediates the suppressive effect of pancreatic FoxO1on �-cell differentiation (25). A similar functional and physicalinteraction of FoxO1 with Notch1 is described in myoblasts,which leads to corepressor clearance from the Notch effectorCsl, leading to stabilization of a functional FoxO1-Notch1 com-plex (12).We reveal a mechanism by which two transcription factors,

the broadly expressed FoxO1 and the osteoblast-enrichedATF4, interact in osteoblasts to regulate glucose metabolismand insulin sensitivity. In view of FoxO1 biology, this mecha-

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nism of action may find a counterpart in the regulation of glu-cosemetabolism and insulin signaling in other glucose-regulat-ing organs where other transcription factors are involved in cellfate determination and function, such as �-cell development,myocyte formation, and hepatic cell function. Respective toskeletal biology, we show that FoxO1 andATF4 regulate energymetabolism at least in part in an osteocalcin-dependent man-ner. The fact that many recent clinical studies have suggestedthat osteocalcin is a marker of glucose tolerance (2, 4, 26–29)attributes to the FoxO1/ATF4 interaction a biological signifi-cance with potential applications in diseases of abnormal glu-cose metabolism.

Acknowledgments—We are grateful to Dr Timothy Townes for pro-viding Atf4�/� mice. We are also thankful to Charles Duncan andJayesh G. Shah for technical assistance. We are thankful to the histol-ogy and metabolic units facilities of the Diabetes and EndocrinologyResearch Center of Columbia University Medical Center (supportedby NIDDK, National Institutes of Health Grant DK063608-07) forhelp with histological analysis.

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FoxO1 Interacts with ATF4

8768 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 12 • MARCH 16, 2012

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Therese Rached and Stavroula KousteniAruna Kode, Ioanna Mosialou, Barbara C. Silva, Sneha Joshi, Mathieu Ferron, Marie

HomeostasisFoxO1 Protein Cooperates with ATF4 Protein in Osteoblasts to Control Glucose

doi: 10.1074/jbc.M111.282897 originally published online February 1, 20122012, 287:8757-8768.J. Biol. Chem. 

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