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Hepatic posttranscriptional network comprised ofCCR4–NOT deadenylase and FGF21 maintains systemicmetabolic homeostasisMasahiro Moritaa,b,c,1,2, Nadeem Siddiquid,e,1, Sakie Katsumuraa,b, Christopher Rouyad,e, Ola Larssonf,Takeshi Nagashimag, Bahareh Hekmatnejadh,i, Akinori Takahashij, Hiroshi Kiyonarik, Mengwei Zanga,b,René St-Arnaudh,i, Yuichi Oikel, Vincent Giguèred,e,m, Ivan Topisirovicd,m,n, Mariko Okada-Hatakeyamag,o,Tadashi Yamamotoj,2, and Nahum Sonenbergd,e,2
aDepartment of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229; bBarshop Institute for Longevity andAging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229; cInstitute of Resource Development and Analysis,Kumamoto University, 860-0811 Kumamoto, Japan; dDepartment of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; eGoodman CancerResearch Centre, McGill University, Montreal, QC H3A 1A3, Canada; fDepartment of Oncology-Pathology, Scilifelab, Karolinska Institutet, SE-171 76Stockholm, Sweden; gLaboratory for Integrated Cellular Systems, RIKEN Center for Integrative Medical Sciences, Yokohama, 230-0045 Kanagawa, Japan;hResearch Centre, Shriners Hospital for Children–Canada, Montreal, QC H4A 0A9, Canada; iDepartment of Human Genetics, McGill University, Montreal, QCH3A 2T5, Canada; jCell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 904-0495 Okinawa, Japan; kLaboratoriesfor Animal Resource Development and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, 650-0047 Hyogo, Japan; lDepartment ofMolecular Genetics, Graduate School of Medical Sciences, Kumamoto University, 860-8556 Kumamoto, Japan; mGerald Bronfman Department of Oncology,McGill University, Montreal, QC H2W 1S6, Canada; nLady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGillUniversity, Montreal, QC H3T 1E2, Canada; and oLaboratory of Cell Systems, Institute for Protein Research, Osaka University, Suita, 565-0871 Osaka, Japan
Contributed by Nahum Sonenberg, February 24, 2019 (sent for review September 17, 2018; reviewed by Jack D. Keene and David J. Mangelsdorf)
Whole-body metabolic homeostasis is tightly controlled by hormone-like factors with systemic or paracrine effects that are derivedfrom nonendocrine organs, including adipose tissue (adipokines)and liver (hepatokines). Fibroblast growth factor 21 (FGF21) is ahormone-like protein, which is emerging as a major regulator ofwhole-body metabolism and has therapeutic potential for treatingmetabolic syndrome. However, the mechanisms that control FGF21levels are not fully understood. Herein, we demonstrate that FGF21production in the liver is regulated via a posttranscriptional networkconsisting of the CCR4–NOT deadenylase complex and RNA-bindingprotein tristetraprolin (TTP). In response to nutrient uptake, CCR4–NOT cooperates with TTP to degrade AU-rich mRNAs that encodepivotal metabolic regulators, including FGF21. Disruption of CCR4–NOT activity in the liver, by deletion of the catalytic subunit CNOT6L,increases serum FGF21 levels, which ameliorates diet-induced meta-bolic disorders and enhances energy expenditure without disruptingbone homeostasis. Taken together, our study describes a hepaticCCR4–NOT/FGF21 axis as a hitherto unrecognized systemic regula-tor of metabolism and suggests that hepatic CCR4–NOT may serveas a target for devising therapeutic strategies in metabolic syndromeand related morbidities.
CCR4–NOT | deadenylase | FGF21 | hepatokine | metabolic syndrome
The mRNA poly (A) tail plays an essential role in post-transcriptional regulation of gene expression by affecting
mRNA decay and translation (1–3). Deadenylation is the rate-limitingstep in mRNA degradation that, together with transcription, de-termines steady-state mRNA levels (4). mRNA deadenylation isprimarily catalyzed by the CCR4–NOT complex, a multisubunitprotein machinery composed of the CCR4 (CNOT6L/CNOT6)deadenylase, the CNOT1 scaffold protein, and several regu-latory proteins (CNOT2–CNOT11) (5–7).Direct recruitment of the CCR4–NOT complex to target
mRNAs destined for deadenylation and decay is mediated by sev-eral RNA-binding proteins (RBPs), including tristetraprolin (TTP),Nanos2, and Roquin (8–13). In addition, posttranscriptional si-lencing by miRNAs occurs through association of the CCR4–NOTcomplex with the miRNA-induced silencing complex (miRISC)(14–16). The selectivity of mRNA deadenylation is controlled bycis-acting mRNA elements to which CCR4–NOT-associated RBPsand miRISC bind (17, 18). Previous structural and biochemi-cal studies have provided mechanistic models for the selective
CCR4–NOT-dependent deadenylation by RBPs and the miRISC(18). However, the composition and function of CCR4–NOTcontaining messenger ribonucleoprotein (mRNP) complexes inphysiological and pathological states remain obscure (19).The CCR4–NOT complex has been implicated in the develop-
ment of metabolic diseases (20–24). These disorders, including di-abetes, steatosis, hyperlipidemia, and obesity, are major worldwide
Significance
The mRNA poly(A) tail controls gene expression at post-transcriptional levels, including mRNA degradation and trans-lation. Here, we show that a hitherto unknown hepaticposttranscriptional network centered on the CCR4–NOT dead-enylase plays a seminal role in regulating FGF21 expression andits effects on systemic metabolism. A genome-wide search forCNOT6L-associated mRNAs unveiled the mechanism wherebyCNOT6L selectively degrades a subset of mRNAs encoding met-abolic factors, including FGF21. Disruption of CCR4–NOT dead-enylase activity, by targeting its catalytic subunit CNOT6L, leadsto an increase in FGF21 levels, which is paralleled by a dramaticimprovement of metabolic syndrome. Overall, our findingsdescribe a new paradigm in regulation of whole-body metab-olism, whereby a hepatic posttranscriptional network governssystemic metabolic regulation via FGF21.
Author contributions: M.M., N. Siddiqui, T.Y., and N. Sonenberg designed research; M.M.,N. Siddiqui, S.K., C.R., B.H., A.T., and H.K. performed research; M.M., O.L., H.K., M.Z.,R.S.-A., Y.O., V.G., and M.O.-H. contributed new reagents/analytic tools; M.M., O.L.,T.N., and B.H. analyzed data; and M.M., N. Siddiqui, S.K., C.R., O.L., I.T., M.O.-H., T.Y.,and N. Sonenberg wrote the paper.
Reviewers: J.D.K., Duke University; and D.J.M., The University of Texas SouthwesternMedical Center.
The authors declare no conflict of interest.
Published under the PNAS license.
Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no.GSE62365).1M.M. and N. Siddiqui contributed equally to this work.2To whom correspondence may be addressed. Email: [email protected], [email protected], or [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1816023116/-/DCSupplemental.
Published online March 29, 2019.
www.pnas.org/cgi/doi/10.1073/pnas.1816023116 PNAS | April 16, 2019 | vol. 116 | no. 16 | 7973–7981
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health problems causally associated with dysregulation of meta-bolic homeostasis. Whole-body metabolic homeostasis is closelycontrolled in a systemic or paracrine manner by hormone-likefactors secreted from nonendocrine organs, such as adiposetissue (adipokines) and liver (hepatokines) (25, 26). Hormone-like proteins can either enhance [e.g., fibroblast growth factor 21(FGF21) and leptin] or impair (e.g., resistin and selenoprotein P)energy metabolism (26, 27). However, there are no studies thatdirectly link the deadenylase activity of CCR4–NOT to hormone-like proteins and metabolic disorders.Here, we identified target mRNAs associated with the
CNOT6L deadenylase subunit of the CCR4–NOT complex inthe liver by performing RNA immunoprecipitation followed bymicroarray analysis (RIP-CHIP). We demonstrate that, in re-sponse to feeding, the CCR4–NOT/TTP complex targets theAU-rich mRNA encoding the hepatokine FGF21, which allevi-ates diet-induced metabolic disorders (28–31). Deletion of theCnot6l gene in mice decreased susceptibility to diet-inducedmetabolic disorders, such as obesity, steatosis, and hyperlipid-emia in a deadenylase activity-dependent manner. We found thatthe observed metabolic disorders can largely be explained byCNOT6L-dependent control of Fgf21 mRNA decay. Thus, weconclude that the CNOT6L deadenylase targets a subset ofmRNAs, including Fgf21, to control whole-body metabolism.Our findings show that CNOT6L plays a major role in regulation ofFGF21 levels, thus providing unprecedented evidence that CNOT6Lmay serve as a therapeutic target to treat metabolic diseases.
ResultsRIP-CHIP Identifies CNOT6L-Associated mRNAs That Contain AU-RichElements and Encode for Metabolic Regulators. The CCR4–NOTcomplex is a multisubunit protein machinery composed of theCCR4 (CNOT6L/CNOT6) deadenylase, the CNOT1 scaffoldprotein, and several regulatory proteins (CNOT2–CNOT11) (5),and has been implicated in metabolic disorders (20–23). Mam-mals have two paralogs of the CCR4 deadenylase gene, Cnot6and Cnot6l (32, 33). CNOT6L is highly expressed in metaboli-cally active tissues, such as the liver and adipose tissue, whereasCNOT6 is predominantly expressed in testis and thymus (SIAppendix, Fig. S1 A and B). To investigate the role of CCR4–NOT in regulation of metabolism, we first identified bona fideendogenous mRNA targets of the CCR4–NOT deadenylase inthe liver. This was achieved by immunopurifying CCR4–NOT-associated mRNP complexes and analyzing their mRNA contenton a transcriptome-wide scale using microarrays (Fig. 1A). He-patocytes derived from mice lacking Cnot6l (Cnot6l−/−) wereused as a control (SI Appendix, Fig. S1 C–F). We identified 195CNOT6L-associated mRNAs (Dataset S1), then searched con-sensus sequences among 3′UTRs of 195 mRNAs (Dataset S2).These mRNAs were significantly enriched for AU-rich elements(AREs) within their 3′UTR (Fig. 1B, SI Appendix, Fig. S2A, andDataset S2). AREs generally destabilize mRNAs and are foundwithin the 3′UTR of mRNAs, such as cytokines and growthfactors that respond to acute external stimuli (34, 35). We clas-sified 195 mRNAs according to their biological functions(Fig. 1C). CNOT6L-associated mRNAs exhibited enrichmentfor those encoding metabolic factors (27% of 195 genes, P =2.0 × 10−3) (Fig. 1C). We selected 12 mRNAs among thosemRNAs encoding metabolism-related proteins according to theP values and those that were associated with known metabolicfunctions, and validated the interactions between CNOT6L and12 of these mRNAs using qRT-PCR (SI Appendix, Fig. S2 Band C). We found that of the 12 selected mRNAs, 5 containedAREs, and that the stability and steady-state levels of thesemRNAs, such as Fgf21, Ankrd1, Socs3, and Foxk2, were in-creased in Cnot6l−/− hepatocytes (Fig. 1 D and E and SI Ap-pendix, Fig. S2D). These results suggest that CNOT6L decreases
the stability of mRNAs that contain AREs and are enriched inthose encoding metabolic regulators.
TTP Recruits the CCR4–NOT Complex to ARE-Containing Target mRNAsDestined for Degradation. Because the CCR4–NOT complex di-rectly interacts with the ARE-binding protein, TTP (9, 11, 12, 36),we investigated whether TTP promotes CCR4–NOT-dependentdegradation of endogenous target mRNAs in hepatocytes. TheCCR4–NOT complex subunits (CNOT6L, CNOT1, CNOT3,and CNOT7) were precipitated with TTP from a hepatocyteextract (Fig. 2A). TTP directly binds to CNOT1 via a conservedphenylalanine (F319) (9). Mutation of F319 to alanine (F319A)dramatically reduced the interaction between TTP and theCCR4–NOT complex subunits (Fig. 2A). Moreover, CNOT6Lcoimmunoprecipitated with TTP in hepatocytes (Fig. 2B). De-pletion of TTP (Fig. 2C and SI Appendix, Fig. S3A), but notanother CCR4–NOT-associated protein, Roquin (SI Appendix,Fig. S3 B–D), impaired the binding of CNOT6L to ARE-containing mRNAs (Fig. 2D), and enhanced their stability andsteady-state levels (Fig. 2 E and F). These results demonstratethat TTP mediates the interaction of the CCR4–NOT complexwith ARE-containing mRNAs encoding several pivotal meta-bolic regulators to promote their degradation.
The CCR4–NOT/TTP Complex Degrades the Hepatokine Fgf21 mRNA inResponse to Feeding. Fgf21 mRNA contains canonical AREs in its3′UTR (SI Appendix, Fig. S3 E and F) and was dramaticallyenriched (P = 4.7 × 10−5) in CNOT6L-immunoprecipitatedmaterial (Dataset S1). Fgf21 mRNA levels were increased inCnot6l−/− (Fig. 1E) and TTP-depleted hepatocytes (Fig. 2F).This correlated with longer Fgf21 mRNA half-life in Cnot6l−/−
and TTP-depleted hepatocytes (T1/2, ∼40 min) vs. wild-type andcontrol (T1/2, ∼25 min), respectively (Fig. 3 A and B). Luciferasereporter assays confirmed that the 3′UTR is required for CCR4–NOT/TTP-mediated degradation of Fgf21 mRNA (Fig. 3 Cand D). The CCR4–NOT/TTP complex also induced dead-enylation of an Fgf21 3′UTR reporter mRNA (Fig. 3E).These data demonstrate that the CCR4–NOT/TTP complexpromotes deadenylation and degradation of Fgf21 mRNA viainteraction with its 3′UTR (Fig. 3F).Fgf21 transcription is stimulated by peroxisome proliferator-
activated receptor-α (PPAR-α) under fasting conditions, leadingto hepatic lipid oxidation, triglyceride clearance, and ketogenesis(37–39). In contrast, Fgf21 mRNA is rapidly suppressed 2 h afterrefeeding by an unknown mechanism (37, 39). Serum FGF21 andhepatic Fgf21 mRNA levels were ∼2.5-fold higher under fastingand refeeding conditions in Cnot6l−/− compared with wild-typemice (Fig. 3 G and H). CNOT6L deficiency partially rescuedFgf21 mRNA degradation by refeeding (Fig. 3I), indicating thatreduction in serum FGF21 levels upon refeeding is mediated atleast in part by the CCR4–NOT complex. Consistently, refeedingstimulated CNOT6L deadenylase activity in the liver (Fig. 3J andSI Appendix, Fig. S3G). These results demonstrate that theCCR4–NOT/TTP complex controls hepatic FGF21 productionby inducing degradation of Fgf21 mRNA after feeding.
Resistance to Diet-Induced Obesity, Enhanced Energy Expenditure,and Improved Insulin Sensitivity in Cnot6l−/− Mice. FGF21 pro-motes weight and lipid reduction, and delays development ofdiabetes (29, 31). To examine the impact of hepatic CCR4–NOTon FGF21 levels in a physiological context pertinent to the devel-opment of metabolic disorders, we analyzed Cnot6l−/− mice, whichare viable and fertile and age without gross abnormalities (SI Ap-pendix, Fig. S4A). The mice are leaner and protected from high-fatdiet (HFD)-induced obesity compared with wild-type mice (Fig. 4Aand SI Appendix, Fig. S4 A and B). Cnot6l deficiency amelioratedHFD-induced hyperglycemia, hyperlipidemia, and hyperinsulinemia(Fig. 4 B–D and SI Appendix, Fig. S4 C–E). Furthermore, serum
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levels of ketone body β-hydroxybutyrate were increased in Cnot6l−/−
mice (Fig. 4E and SI Appendix, Fig. S4F), suggesting enhancementin fatty-acid oxidation. Finally, Cnot6l deletion significantlyimproved HFD-induced glucose tolerance and alleviated insulinresistance compared with control wild-type mice that exhibitedtypical metabolic syndrome phenotypes (Fig. 4 F and G). We nextmeasured whole-body energy metabolism and found that loss ofCnot6l resulted in a significant increase in oxygen consumption(Fig. 4 H and I and SI Appendix, Fig. S4 G and H). There were nodifferences in food intake or locomotor activity, but increased heatproduction and decreased respiratory exchange ratio were ob-served (Fig. 4 J–M and SI Appendix, Fig. S4 I–L).Cnot6l−/− mice on an HFD exhibited significantly reduced weight
of liver, white adipose tissue (WAT), and brown adipose tissue(BAT) compared with wild-type mice (SI Appendix, Fig. S5A).While an HFD-induced hepatic steatosis in wild-type mice,
Cnot6l−/− liver appeared smaller (∼25%) than control (Fig. 5 Aand B). Smaller lipid droplets and lower hepatic triglycerideaccumulation were observed in Cnot6l−/− liver on an HFDcompared with control (Fig. 5 C and D). Moreover, Cnot6l de-letion led to alterations in expression of genes relevant to energyexpenditure and fat metabolism, such as Pgc-1α, Scd1, and Cd36mRNAs in the liver (Fig. 5E and SI Appendix, Fig. S5B). Inagreement with the enhanced energy expenditure in Cnot6l−/−
mice, less fat accumulation and smaller adipocytes were ob-served in Cnot6l−/− BATs (Fig. 5 F–H). Cnot6l ablation engen-dered expression of Ucp1 and Pgc-1α, which are induced byFGF21 treatment (40–43), in BAT and subcutaneous WAT(sWAT) (Fig. 5 I and J and SI Appendix, Fig. S5 C and D) (40–43).Consequently, the weight and adipocyte size of sWAT and epi-didymal WAT (eWAT) were significantly decreased in Cnot6l−/−
compared with control mice (Fig. 5 K–N). These results indicate
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Fig. 1. RIP-CHIP identified CNOT6L-associated mRNAs, many of which contain AREs and encode metabolic regulators. (A) Schematic diagram of RIP-CHIP inwild-type and Cnot6l−/− primary hepatocytes. Primary hepatocytes were isolated from wild-type and Cnot6l−/− livers. Each sample was divided into two fortotal RNA extraction (upper arrows) and anti-CNOT6L immunoprecipitation followed by RNA extraction (lower arrows). Input and immunoprecipitated (IPed)RNA were hybridized to separate microarrays. Cnot6l−/− hepatocytes were used as a negative control. (B) Percentage of nontarget mRNAs containing AREs(blue bar) and CNOT6L-target mRNAs containing AREs (red bar). The bar graph was generated from Dataset S1. (C) Functional classification of 195 CNOT6L-associated mRNAs using the Panther Classification System. (D) Stability of the indicated mRNAs in wild-type and Cnot6l−/− hepatocytes. Hepatocytes wereincubated with actinomycin D for the indicated times. Levels of the indicated mRNAs were determined by RT-qPCR, normalized to the level of Hprt mRNA,expressed as percent change of the initial mRNA level, and plotted semilogarithmically. n = 3 per group. (E) Input levels of the indicated mRNAs in wild-typeand Cnot6l−/− hepatocytes were determined by RT-qPCR. n = 3 per group. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test. Arepresentative experiment of two independent experiments (each carried out in triplicate) is presented.
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that loss of CNOT6L activity dramatically improves HFD-inducedmetabolic disorders.
Hepatic CNOT6L Deadenylase Activity Systemically Controls LipidMetabolism, Steatosis, and Whole-Body Metabolism. To supportour observation that hepatic CNOT6L deadenylase activity playsan important role in metabolic regulation, we reintroduced awild-type or an inactive (E239A) CNOT6L mutant (33, 44) intoCnot6l−/− liver by adenoviral-mediated gene transduction (SIAppendix, Fig. S6A) and maintained the mice on an HFD for2 wk. Liver-specific expression of wild-type CNOT6L signifi-cantly increased liver weight and triglyceride content in Cnot6l−/−
mice (Fig. 6 A–C). Notably, CNOT6L partially reversed the re-sistance of Cnot6l−/− mice to HFD-induced obesity (Fig. 6D andSI Appendix, Fig. S6B). This demonstrates that the reduced
weight phenotype in Cnot6l−/− mice is attributed by the systemiceffects caused by the lack of CCR4–NOT liver activity. RestoringCNOT6L levels in the liver significantly decreased Ucp1 levels inBATs and eWAT (Fig. 6 E and F), increased the size of lipiddroplets in adipose tissues and eWAT weight (Fig. 6 C and G),and reversed serum insulin and blood glucose levels (Fig. 6 Hand I). In striking contrast, the E239A mutant of CNOT6L failedto reverse the phenotypes of Cnot6l−/− mice (Fig. 6 A–I and SIAppendix, Fig. S6 A and B). Taken together, these data highlightthe contribution of hepatic CNOT6L deadenylase activity inmetabolic disorders and whole-body energy homeostasis.
FGF21 Is the Mediator of CNOT6L-Dependent Hepatic Steatosis andObesity. To determine whether FGF21 mediates the effects of thehepatic CCR4–NOT/TTP complex on whole-body metabolism,
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Fig. 2. TTP recruits the CCR4–NOT complex to ARE-containing target mRNAs destined for degradation. (A) Interaction of the CCR4–NOT complex with WT ormutant (F319A) TTP protein in hepatocyte extracts. Western blots of the indicated proteins from recombinant maltose binding protein (MBP)-tagged TTPproteins immobilized on amylose beads and incubated with hepatocyte extracts. (B) Interaction of Flag-CNOT6L with HA-TTP in hepatocytes examined bycoimmunoprecipitation with anti-HA antibody, followed by Western blot with anti-Flag antibody. (C) Western blots of the indicated proteins in control andTTP knockdown (KD) hepatocytes. α-Tubulin and β-actin were used as loading controls. (D) Association of CNOT6L with the indicated mRNAs in the lysatesfrom control and TTP KD hepatocytes. Immunoprecipitated (IPed) and input RNAs were isolated from anti-CNOT6L immunoprecipitates and total cell lysates,respectively. Levels of IPed and input mRNAs were determined by RT-qPCR. IPed mRNA levels were normalized to those of input mRNA. n = 3 per group. (E)Stability of the indicated mRNAs in control and TTP KD hepatocytes. Hepatocytes were incubated with actinomycin D for the indicated times. Levels of theindicated mRNAs were determined by RT-qPCR, normalized to the level of Hprt mRNA, expressed as percent change of the initial mRNA level, and plottedsemilogarithmically. n = 3 per group. (F) Input levels of the indicated mRNAs in the lysates from control and TTP KD hepatocytes. n = 3 per group. Datarepresent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ND, not determined; Student’s t test. For D–F, a representative experiment of two independentexperiments (each carried out in triplicate) is presented.
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we depleted FGF21 (SI Appendix, Fig. S6C) using adenovirus-delivered short-hairpin RNA (shRNA) (39, 45). Fgf21 shRNA re-versed the beneficial effects of CNOT6L loss on liver weight, hepaticlipid droplet content, and the resistance to HFD-induced tri-glyceride accumulation (Fig. 6 J–L). In addition to partial res-toration of Ucp1 expression in BAT and insulin levels in serum(SI Appendix, Fig. S6 D and E), Fgf21 knockdown in Cnot6l−/−
mice resulted in increased body weight (Fig. 6M and SI Appendix,Fig. S6F). These findings further support the tenet that FGF21 isa major mediator of the systemic metabolic effects of CCR4–NOT and that the hepatic CCR4–NOT/TTP/FGF21 axis plays anessential role in whole-body energy homeostasis.FGF21-based therapies have shown promise for the treatment
of metabolic disorders in humans; however, concerns have beenraised due to side effects reported in mice, including changes inbone development and homeostasis (29, 46). Strikingly, Cnot6l−/−
mice did not display any defects in bone homeostasis (SI Appendix,Fig. S6 G–J), suggesting that induction of FGF21 via inhibition ofCNOT6L could circumvent bone homeostasis issues associatedwith administration of FGF21. In summary, our data establish astrong link between CNOT6L deadenylase-mediated regulationof the hormone-like protein FGF21 and downstream systemic
metabolic control (SI Appendix, Fig. S6K). Thus, targeting CNOT6Lcould potentially provide better options for the treatment of meta-bolic disorders with fewer side effects than FGF21-based therapies.
DiscussionSelective deadenylation by the CCR4–NOT complex contributessignificantly to the wide range of mRNA half-lives and is medi-ated by specific RBPs that recruit the complex to target mRNAs,as has been described for TTP (9), Roquin (10), and miRISC (14–16). RIP-CHIP analysis of CNOT6L-associated mRNAs in theliver revealed a TTP-dependent posttranscriptional programthat systemically alters mammalian metabolism. CNOT6L tar-gets a subset of metabolism-related mRNAs, such as Fgf21mRNA, whose expression is rapidly altered in response to changesin feeding conditions (37, 39, 47). In accordance with previousreports (9, 11, 12), the association between the CCR4–NOT com-plex and ARE mRNAs depends on TTP expression (Figs. 2 and 3).Together, these data demonstrate that CCR4–NOT selectivelycontrols TTP-specific ARE mRNAs encoding metabolic factors inhepatocytes. Although the impact of TTP on immune regulation inmammals is well documented (36), these results ascribe a functionfor TTP in organismal metabolism.
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Hormone-like proteins, whose expression is tightly controlledin response to nutrients, systemically control whole-body me-tabolism (25, 26). Most studies have focused on transcriptionalregulators of hormone-like proteins. For example, Fgf21 tran-scription is stimulated by the transcription factor PPAR-α underfasting conditions, leading to hepatic lipid oxidation, triglycerideclearance, and ketogenesis (37–39, 47). In contrast, Fgf21mRNAis rapidly suppressed 2 h after refeeding by an unknown mech-anism (37, 39). We show that this suppression is less pronouncedin Cnot6l−/− liver (Fig. 3I), which demonstrates that feeding-induced suppression of Fgf21 mRNA is at least in part con-trolled by the CCR4–NOT deadenylase. Accordingly, the CCR4–NOT complex is activated following feeding (Fig. 3J). Thus,
posttranscriptional regulation of Fgf21 mRNA by the CCR4–NOT deadenylase is critical for the repression of triglycerideclearance and fatty acid oxidation following feeding. Consistentwith the increased FGF21 serum level in Cnot6l−/− mice,CNOT6L ablation leads to an increase in serum ketone bodies,oxygen consumption, and expression of genes involved in energyexpenditure and fatty acid oxidation (Figs. 4 and 5). Thus, ourdata show that Fgf21 mRNA stability, in addition to transcrip-tion, plays an important role in balancing serum FGF21 levels tomaintain metabolic homeostasis in response to nutrients.The obesity-resistant phenotype of Cnot6l−/− mice is largely,
but not completely, reversed by reexpression of CNOT6L in theliver (Fig. 6), demonstrating a major role for hepatic CNOT6L in
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mice fed on an HFD. HFD feeding started at 8 wk of age. n = 14–17 per group. (B–E) Levels of blood glucose (B), serum triglycerides (C), serum insulin (D), andserum β-hydroxybutyrates (E) in wild-type or Cnot6l−/− mice fed on an HFD during ad libitum feeding or fasting. n = 7–10 per group. (F and G) Intraperitonealglucose tolerance tests (GTTs) (F) and insulin tolerance tests (ITTs) (G) in wild-type and Cnot6l−/− mice fed on an HFD or standard diet (SD). Blood glucose levelswere measured at the indicated time points following intraperitoneal injection of glucose or insulin. n = 7–10 per group. *P < 0.05, **P < 0.01 for Cnot6l−/− onHFD versus wild-type on HFD. (H and I) Oxygen consumption (VO2) over 24 h (H) and average VO2 (I) in wild-type and Cnot6l−/− mice fed on HFD. VO2 werenormalized to body weight. n = 5 per group. (J–M) Daily food intake per body weight (J), locomotor activity (K), calculated heat production (L), and re-spiratory exchange ratio (M) in wild-type and Cnot6l−/− mice fed on an HFD. n = 5 per group. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001;two-way ANOVA with Tukey’s post hoc test for A–G and I and Student’s t test for J–M.
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regulating systemic metabolism. The partial rescue in metabolicsensitivity suggests that CNOT6L activity in other metabolictissues, such as BAT and WAT, contributes to the regulation ofwhole-body metabolism. Furthermore, knockdown of Fgf21 inthe liver of Cnot6l−/− mice restores sensitivity to nutrient excessand reverses the decrease seen in diet-induced weight gain (Fig.6). It is evident that there is a significant rescue of liver-specificCNOT6L function by Fgf21 knockdown by comparing the extentof rescue by CNOT6L reexpression versus Fgf21 knockdown onbody weight. These results provide compelling evidence thatFGF21 is the major effector of CNOT6L function in the liver,but also indicate that CNOT6L controls some Fgf21-unrelatedpathways relevant to systemic metabolism. Our RIP-CHIP datashow that ∼27% of mRNAs associated with CNOT6L encodemetabolism-related factors. Characterization of these CNOT6Ltargets should provide additional insight into metabolic controlby CCR4–NOT.
FGF21 is a bona fide therapeutic target that has been exploredin the clinic (29). Treatment with FGF21 ameliorated severalmetabolic disorders, such as obesity, hyperlipidemia, and hy-perglycemia in a variety of species, including rodents, monkeys,and humans (41, 42, 48–54). However, the development ofFGF21 as a drug is challenging due to its short half-life in blood(T1/2 = 0.5 ∼ 2 h), and the aggregation of its recombinant form(55). An FGF21 analog, LY2405319, whose efficacy was validatedin humans, was developed to address these issues (54). Un-fortunately, its half-life remains relatively short (T1/2 = 1.5 ∼ 3 h),which motivated a search for other strategies to increaseFGF21 levels in vivo (29). Additionally, exogenous administra-tion of FGF21 has been used at a concentration of 5- to 10-foldhigher than endogenous levels (29). We found that CNOT6Lablation alleviated metabolic disorders with only a 2.5-fold in-crease in serum FGF21 levels (Fig. 3G). These findings dem-onstrate that a modest increase in basal FGF21 levels throughmRNA stabilization is sufficient to ameliorate hepatic steatosis,
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Fig. 6. Systemic regulation of whole-body metabolism by the CCR4–NOT/FGF21 axis. (A–I) Liver weight (A), liver triglyceride contents (B), representative H&Estaining of the liver, BAT, and eWAT (C), gain of body weight (D), Ucp1 mRNA level in BAT (E) and eWAT (F), eWAT weight (G), serum insulin (H), and bloodglucose levels (I) of wild-type and Cnot6l−/− mice injected with adenovirus expressing EGFP, CNOT6L-WT, or CNOT6L-E239A. Ten-week-old mice were ad-ministered with adenovirus and fed on HFD for 2 wk. (Scale bars, 50 μm.) Liver triglyceride contents were normalized to liver weight. n = 7–8 per group. (J–M)Liver weight (J), representative liver H&E staining (K), liver triglyceride contents (L), and gain of body weight (M) of wild-type and Cnot6l−/− mice injected withadenovirus expressing control (shGFP) or Fgf21 shRNA (shFGF21). Ten-week-old mice were injected with adenovirus and fed on HFD for 2 wk. (Scale bars,50 μm.) Liver triglyceride contents were normalized to liver weight. n = 10 per group. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Tukey’s post hoc test.
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and result in more sustained effects compared with transientadministration of FGF21. Importantly, the 2.5-fold increase weobserved did not cause deleterious effects on bone density, un-like exogenous FGF21 (SI Appendix, Fig. S6). Coupled with theobservation in humans, which show a common variant in thelocus of CNOT6L correlating with altered blood cholesterollevels (NCBI PheGenI) (56), posttranscriptional control ofFGF21 by CNOT6L underscores the therapeutic potential oftargeting CNOT6L for metabolic disorders.
MethodsAll animal experiments were conducted according to the guidelines foranimal use issued by the Committee of Animal Experiments,McGill University,and Institute of Medical Science, University of Tokyo. Molecular studies wereperformed according to routine protocol previously published by our group(57). Differences among groups were compared using two-way ANOVAfollowed by between-group comparison with Tukey’s post hoc test, one-wayANOVA with Bonferroni’s post hoc test, or Student’s t test (two-tailed,
unpaired) when there were only two groups. All statistical analyses wereperformed using IBM SPSS Statistics v22 software, and the differences wereconsidered significant when P < 0.05. For detailed in vivo and in vitro ex-perimental methods, see SI Appendix.
ACKNOWLEDGMENTS. We thank I. Saito for the pAxCAwtit vector; M. Fisherfor providing the pAd-shFGF21 vector; J. St-Pierre, D. Pearl, C. Chapat, andS. Tahmasebi for text proofreading; A. Sylvestre, K. Kitazawa, and H. Adachifor technical assistance; the Animal Facility and the Histology Facility at theGoodman Cancer Research Centre for mouse work and tissue processing; andC. Lister, I. Harvey, C. Sgherri, and S. Perreault for assistance. This work wassupported by Canadian Institutes of Health Research Grants CIHR MOP-93607(to N. Sonenberg) and MOP-125885 (to V.G.); Terry Fox Research InstituteGrant TFF-116128 (to V.G., I.T., and N. Sonenberg); and the Ministry of Educa-tion, Culture, Sports, Science and Technology, Japan Grant-in-Aid for ScientificResearch 19390070 (to T.Y.) and 18K07237 (to M.M.). M.M. is supported by theUT Rising Stars Award from the University of Texas System. O.L. is supported bythe Wallenberg Academy Fellows Program and the Swedish Research Council.I.T. is a Junior 2 Research Scholar of the Fonds de Recherche du Québec–Santé.
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