Jiali Deng,1 Feixiang Yuan,1 Yajie Guo,1 Yuzhong Xiao,1 Yuguo Niu,1 Yalan Deng,1

Xiao Han,2 Youfei Guan,3 Shanghai Chen,1 and Feifan Guo1

Deletion of ATF4 in AgRP NeuronsPromotes Fat Loss Mainly viaIncreasing Energy ExpenditureDiabetes 2017;66:640–650 | DOI: 10.2337/db16-0954

Although many functions of activating transcriptionfactor 4 (ATF4) are identified, a role of ATF4 in thehypothalamus in regulating energy homeostasis is un-known. Here, we generated adult-onset agouti-relatedpeptide neuron–specific ATF4 knockout (AgRP-ATF4 KO)mice and found that these mice were lean, with im-proved insulin and leptin sensitivity and decreasedhepatic lipid accumulation. Furthermore, AgRP-ATF4KO mice showed reduced food intake and increasedenergy expenditure, mainly because of enhanced ther-mogenesis in brown adipose tissue. Moreover, AgRP-ATF4 KO mice were resistant to high-fat diet–inducedobesity, insulin resistance, and liver steatosis and main-tained at a higher body temperature under cold stress.Interestingly, the expression of FOXO1 was directlyregulated by ATF4 via binding to the cAMP-responsiveelement site on its promoter in hypothalamic GT1-7cells. Finally, Foxo1 expression was reduced in thearcuate nucleus (ARC) of the hypothalamus of AgRP-ATF4 KO mice, and adenovirus-mediated overexpres-sion of FOXO1 in ARC increased the fat mass inAgRP-ATF4 KO mice. Collectively, our data demon-strate a novel function of ATF4 in AgRP neurons ofthe hypothalamus in energy balance and lipid metab-olism and suggest hypothalamic ATF4 as a potentialdrug target for treating obesity and its related meta-bolic disorders.

Obesity is strongly associated with metabolic syndromeand predisposes to diseases including type 2 diabetes and

liver steatosis (1,2). Changes in body weight normally re-sult from an imbalance between energy intake and energyexpenditure (2), controlled by the central nervous system,especially the hypothalamus (3). The center of this reg-ulatory network is the arcuate nucleus (ARC) of the hy-pothalamus, which contains sets of important neuronsdevoted to metabolic regulation including orexigenic neu-rons that coproduce agouti-related peptide (AgRP) andneuropeptide Y, as well as anorexigenic neurons that con-tain cocaine- and amphetamine-regulated transcript andproopiomelanocortin (POMC)–derived peptides (3,4). AgRPneurons increase feeding by opposing the anorexigenicactions of POMC neurons, in part through the release ofAgRP, a competitive inhibitor of melanocortin receptors(4). It also had an effect on energy expenditure via affectingsympathetic nervous system (SNS) activity or leptin sensi-tivity (5,6).

Activating transcription factor 4 (ATF4), also known asCREBP2, belongs to the CREBP families, characterized bythe presence of a leucine zipper dimerization domain and abasic amino acid–rich DNA binding domain (7,8). ATF4 isubiquitously expressed in many tissues and some parts ofthe brain, including the hypothalamus (8). It is involved inthe regulation of various processes, including memory for-mation, osteoblast differentiation, amino acid deprivation,and redox homoeostasis (9). Recent studies (9–12) havedemonstrated a role of ATF4 in the control of glucose andlipid metabolism. A role of ATF4 in specific neurons of thehypothalamus, however, has not been previously described.The aim of our current study was to investigate the role of

1Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences,Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Grad-uate School of the Chinese Academy of Sciences, Shanghai, People’s Republic ofChina2Key Laboratory of Human Functional Genomics of Jiangsu Province, JiangsuDiabetes Center, Nanjing Medical University, Nanjing, People’s Republic of China3Advanced Institute for Medical Sciences, Dalian Medical University, Dalian,People’s Republic of China

Corresponding author: Feifan Guo, ffguo@sibs.ac.cn.

Received 5 August 2016 and accepted 12 December 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0954/-/DC1.

J.D. and F.Y. contributed equally to this study.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

640 Diabetes Volume 66, March 2017

METABOLISM

ATF4 expressed in AgRP neurons in energy homeostasisregulation.

RESEARCH DESIGN AND METHODS

Generation of Mice With ATF4 Deletion in AgRPNeurons and Animal TreatmentAll animals were on C57BL/6J background. ATF4-floxedmice (13) and AgRP Cre-ER mice (14) (provided by Joel K.Elmquist and Tiemin Liu, UT Southwestern Medical Center,Dallas, TX) were bred to generate AgRP-Cre ATF4 flox/flox

and ATF4flox/flox littermates, which were named AgRP-ATF4 knockout (KO) and control mice, respectively. For in-ducing Cre expression and avoiding the possible toxic effectof tamoxifen (15,16), both control and AgRP-ATF4 micewere intraperitoneally injected with 150 mg/kg body weighttamoxifen (Sigma-Aldrich, St. Louis, MO) for 5 days, be-tween the ages of 5 and 7 weeks (14). The basal metabolicphenotypes in AgRP-ATF4 mice were analyzed by treatingthem and control mice with corn oil (Standard Food, Shang-hai, People’s Republic of China) for 5 days. For high-fat diet(HFD) study, 4-week-old AgRP-ATF4 KO or control micewere maintained on a normal chow diet or HFD with 60%kcal fat (Research Diets, New Brunswick, NJ) for 16 weeks.Pair-fed experiments (17) were conducted by feeding controlmice a normal chow diet in the same amounts of food eatenby AgRP-ATF4 KO mice during the previous day. The effi-ciency for ATF4 deletion was evaluated by mating AI9(tdTomato) reporter mice (18) with transgenic mice express-ing Cre under control of the AgRP promoter after tamoxifentreatment. Body weight was monitored weekly throughoutthe experiments, and mice were kept as previously described(19). All the experiments were conducted in accordance withthe guidelines of the Institutional Animal Care and UseCommittee of the Institute for Nutritional Sciences.

Cell Culture and TreatmentsPshuttle vector–constructed plasmids expressing ATF4 ora dominant-negative form of ATF4 (DN-ATF4) was madebased on plasmids described previously (19). The recom-binant adenoviruses (Ads) expressing mouse ATF4 (Ad-ATF4) or control green fluorescent protein (Ad-GFP)were generated as previously described (19). Hypotha-lamic GT1-7 cells were maintained as described previously(20). Plasmids and Ads indicated were transfected intoGT1-7 cells with Lipofectamine 2000 (Invitrogen, Carls-bad, CA).

ARC Administration ExperimentsARC administration experiments were conducted as pre-viously described (21). Ad-FOXO1 (22) was provided byProfessor Youfei Guan (Dalian Medical University, Dalian,People’s Republic of China). Mice were anesthetized andreceived bilateral stereotaxic injections of Ad-GFP, Ad-Null,or Ad-FOXO1 (1 mL/5 3 109 plaque-forming units/side/mice) into ARC (21.4 mm from bregma; 60.3 mm frommidline; 25.90 mm from dorsal surface), and metabolicphenotypes were examined 1–2 weeks after Ad-FOXO1injection.

Cold Exposure TreatmentThe 2- to 3-month-old mice were housed in individualprecooled 4°C cages for 3 h, and rectal temperatures ofmice were measured every 30 min during this period, asdescribed previously (23). Body weight was measured im-mediately before the cold exposure.

Blood Glucose, Serum Insulin, Glucose ToleranceTests, Insulin Tolerance Tests, and HOMA-InsulinResistance IndexThe measurements of blood glucose and serum insulin,results of glucose tolerance tests (GTTs) and insulintolerance tests (ITTs), and the calculation of the HOMA-insulin resistance (IR) index were conducted as describedpreviously (19).

Leptin Sensitivity Assay In VivoMice were intraperitoneally injected with either PBS or3 mg/kg leptin (R&D Systems, Minneapolis, MN) at 9:00 A.M.

after fasting for 24 h, as described previously (24). Foodintake and body weight were measured at 1 and 4 h afterthe injection of leptin.

Metabolic Parameters MeasurementsThe body fat composition of mice was determined using theBruker Minispec mq10 NMR Analyzer (Bruker, Billerica,MA). Indirect calorimetry was measured in a ComprehensiveLab Animal Monitoring System (Columbus Instruments,Columbus, OH), as described previously (11). Rectal tem-peratures were measured using a rectal probe attached toa digital thermometer (Physitemp Instruments, Clifton,NJ).

Serum and Liver MeasurementsSerum and liver total glycerol, total cholesterol, and freefatty acid levels were determined using Glycerol Assay KitReagent (SSUF-C, Shanghai, People’s Republic of China),cholesterol reagent (SSUF-C), and NEFA C reagent (Wako,Osaka, Japan), respectively. Serum norepinephrine (NE)level was determined using ELISA kits (R&D Systems). Allof these assays were performed according to manufac-turer instructions.

Protein and mRNA AnalysisWestern blot analysis was performed with primary anti-bodies against actin (Sigma-Aldrich); ATF4, tribbles homo-log 3 (TRB3), and uncoupling protein 1 (UCP1) (Santa CruzBiotechnology, Santa Cruz, CA); and t–hormone-sensitivelipase (HSL), phosphorylated (p)-HSL, p-cAMP-dependentprotein kinase (PKA), and FOXO1 (Cell Signaling Technol-ogy, Danvers, MA); and visualized by ECL Plus (GE Health-care, Chicago, IL), as described previously (11). RT-PCR,with GAPDH as an internal control gene, was carried outas described previously (11). The sequences of primers usedin the current study are available upon request.

Histological Analysis of White Adipose Tissue, BrownAdipose Tissue, and LiverWhite adipose tissue (WAT), brown adipose tissue (BAT),and liver were fixed in 4% paraformaldehyde overnight and

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stained with hematoxylin-eosin (H-E), as well as Oil Red Ostaining with optimal cutting temperature embedding (25).

Immunofluorescence StainingImmunofluorescence (IF) staining was performed withantibodies against p-STAT3 (Cell Signaling Technology),ATF4 (Santa Cruz Biotechnology), FOXO1 (Cell SignalingTechnology), and c-fos (Santa Cruz Biotechnology), as des-cribed previously (24).

Luciferase AssaypGL3-Foxo1 promoter (21,137 to 5) (26) were providedby Professor Xiao Han (Nanjing Medical University, Nan-jing, People’s Republic of China). GT1-7 cells were cotrans-fected with the internal control vector pRL Renilla (Promega,Madison, WI) and plasmids expressing ATF4 using Lipo-fectamine 2000. The Firefly and Renilla luciferase activi-ties were assayed using Dual-Glo Luciferase Assay System(Promega).

Chromatin Immunoprecipitation AssaysChromatin immunoprecipitation (ChIP) assays were per-formed according to the manufacturer protocol (EMDMillipore, Billerica, MA) with anti-ATF4 antibodies (1:50;Santa Cruz Biotechnology) or normal rabbit IgG (1:50;Santa Cruz Biotechnology) for negative control. Immuno-precipitated FOXO1 promoter was quantified using PCRwith primers designed to amplify the 220 base-pair regionencompassing the cAMP-responsive element (CRE) site(forward, 59-TCAATTCTAAAGCATCCTAGCC-39; reverse,59-TGGGGCACAGCTCGTCTC-39) or a 220 base-pair up-stream region not involved in ATF4 response (forward,59-AACCTTTGTATTGGGGGCAT TGATTG-39; reverse,59-CTGTTGCGATGAGAGCATTTGGTTA-39).

StatisticsAll results are expressed as the mean 6 SEM. Significantdifferences were assessed by two-tailed Student t test,one-way ANOVA followed by the Student-Newman-Keulstest, or ANCOVA. For energy expenditure, respiratoryexchange ratio (RER), and locomotor activity presentedwith lines, GTTs, and ITTs, a t test was used to comparethe differences between different groups of mice at eachtime point examined. P , 0.05 was considered to bestatistically significant.

RESULTS

Deletion of ATF4 in AgRP Neurons Promotes Fat LossIn this study, we generated AgRP-ATF4 KO mice bytamoxifen treatment that allows temporal control of Crerecombinase activity and can be combined with flox mice toenable adult-onset deletion (14). IF staining of tdTomato(reflecting AgRP neurons) and ATF4 showed that ATF4was colocalized with AgRP neurons in control mice butwas absent in AgRP neurons of AgRP-ATF4 KO mice(Supplementary Fig. 1A and B). In addition, Atf4mRNA levels were significantly decreased in ARC, butnot other brain areas or tissues, of AgRP-ATF4 KO micecompared with control mice (Supplementary Fig. 1C).

The body weight of male AgRP-ATF4 KO mice was lowerthan that of control mice starting from 8 weeks of age(tamoxifen was given as treatment at 6 weeks of age)and was associated with decreased fat and lean mass (Fig.1A and B). Consistently, decreased weights of subcutane-ous WAT (sWAT), epididymal WAT (eWAT), BAT, andliver were observed in AgRP-ATF4 KO mice (Fig. 1C).

Histological analysis of eWAT showed that the lossof ATF4 expression in AgRP neurons resulted in a 40%reduction in adipocyte volume, but had no effect oneWAT cell numbers as determined by DNA content anal-ysis (Fig. 1D–F). The diminished adipocyte volume observedin AgRP-ATF4 KO mice suggested a possible enhanced li-polysis. Consistently, levels of p-HSL, the rate-limiting en-zyme for triglyceride (TG) lipolysis, and substrate for PKA,the kinase that phosphorylates HSL (27), were signifi-cantly increased in eWAT of AgRP-ATF4 KO mice com-pared with control mice (Fig. 1G). Genes related to otherlipid metabolism including lipogenesis, fatty acid oxidationand TG secretion (28,29), however, were not significantlyaffected in the WAT of AgRP-ATF4 KO mice (Supplemen-tary Fig. 1D).

AgRP-ATF4 KO Mice Have Decreased Food Intake andIncreased Energy ExpenditureTo assess the possible reasons for the reduced fat massin AgRP-ATF4 KO mice, we measured food intake andenergy expenditure, the two aspects determining body fatmass (27). Daily food intake and feeding efficiency (30)were decreased in AgRP-ATF4 KO mice compared withcontrol mice (Fig. 2A). Total energy expenditure (24-h O2

consumption) adjusted to lean mass (31,32) or calculatedby ANCOVA (33) and heat generation were signifi-cantly increased and RER (VCO2/VO2) was decreased inAgRP-ATF4 KO mice during both the dark and lightphases (Fig. 2B–D and Supplementary Fig. 1E). No differ-ence in total physical activity was observed betweenAgRP-ATF4 KO mice and control mice (Fig. 2E). In con-trast, rectal temperature was higher in AgRP-ATF4 KOmice (Fig. 2F). The higher body temperature observed inAgRP-ATF4 KO mice was most likely caused by increasedthermogenesis, which is regulated by UCP1 in BAT (34).Consistently, BAT UCP1 expression was significantly el-evated and the BAT cells were denser with fewer lipids inAgRP-ATF4 KO mice compared with control mice (Fig.2G–I). BAT thermogenesis is activated by SNS with therelease of NE (34). Not surprisingly, AgRP-ATF4 KO micehad higher serum NE levels (Fig. 2J). A pair-fed experiment(17) showed that AgRP-ATF4 KO mice had lower bodyweight, fat mass, lean mass, and tissue weight (liver andadipose tissue), and higher body temperature (Supplemen-tary Fig. 2).

AgRP-ATF4 KO Mice Exhibit Improved InsulinSensitivity, Decreased Hepatic Lipid Accumulation,and Increased Leptin SensitivityWe then investigated whether ablation of ATF4 in AgRPneurons had any impact on insulin sensitivity and liver

642 ATF4 Deletion in AgRP Neurons Promotes Fat Loss Diabetes Volume 66, March 2017

steatosis, which is associated with a change in fat mass(1). Though levels of fed blood glucose and fed and fastingserum insulin were comparable between two genotypes,fasting blood glucose levels were decreased in AgRP-ATF4KO mice (Supplementary Fig. 3A and B). Consistently,HOMA-IR was significantly reduced in AgRP-ATF4 KOmice (Supplementary Fig. 3C). GTTs and ITTs furtherrevealed that AgRP-ATF4 KO mice had significantly in-creased glucose clearance and improved insulin sensitivity(Supplementary Fig. 3D). Possibly because of the reducedfat mass in AgRP-ATF4 KO mice, hepatic lipid accumula-tion was also slightly reduced in these mice as examinedby Oil Red O and H-E staining (Supplementary Fig. 3E).Furthermore, hepatic and serum TG levels were also re-duced, although total cholesterol and free fatty acids wereunchanged, in AgRP-ATF4 KO mice (Supplementary Fig.3F and G).

Leptin signaling in hypothalamus is a key regulatorfor energy homeostasis (35). To examine the effect of ATF4deletion in AgRP neurons on leptin sensitivity, we in-traperitoneally administered leptin (24) to AgRP-ATF4KO and control mice and monitored the effects of lep-tin injection on changes in food intake and body weight.As shown previously (24), food intake and body weightwere reduced after 1 or 4 h after leptin injection in con-trol mice (Supplementary Fig. 4A and B). Notably, the

effects of leptin were much more significant in AgRP-ATF4 KO mice (Supplementary Fig. 4A and B). Consistentwith the stronger effect of leptin in AgRP-ATF4 KO mice,leptin produced more signals of p-STAT3, the marker ofcellular leptin action (35), in ARC of hypothalamus ofthese mice (Supplementary Fig. 4C).

AgRP-ATF4 KO Mice Are Resistant to HFD-InducedMetabolic DisordersTo test whether AgRP-ATF4 mice may play a roleunder an HFD, we fed 4-week-old male AgRP-ATF4 KOmice and control mice an HFD for 16 weeks. Similarphenotypic changes were observed as those in micemaintained on a normal chow diet. AgRP-ATF4 KOmice showed thin appearance, decreased body weight,lean and fat mass, and weight of tissues, includingliver, sWAT, eWAT, and BAT (Fig. 3A–C). Consis-tently, the size of adipocytes was much smaller inAgRP-ATF4 KO mice, associated with increased levelsof p-PKA substrates and p-HSL in eWAT (Fig. 3D andE). Food intake was comparable between AgRP-ATF4KO mice and control mice under HFD, whereas HFD-fed AgRP-ATF4 KO mice exhibited markedly increasedoxygen consumption, adjusted to lean mass or calcu-lated by ANCOVA (33), and rectal temperature, withdecreased RER and no change in locomotor activity

Figure 1—AgRP-ATF4 KO mice are lean. A: Body weight at the age of 6, 8, and 16 weeks. Fat and lean mass (B) and tissue weights(liver, sWAT, eWAT, and BAT) (C ). D: H-E staining of representative abdominal eWAT sections (scale bars, 250 mm). E: Analysis ofabdominal eWAT cell volume. F: DNA content of total abdominal eWAT. G: p-PKA substrate proteins, p-HSL, and HSL Western blotand densitometric quantification in eWAT. All studies were conducted in 12-week-old (or as indicated) male control or AgRP-ATF4 KOmice maintained on a normal chow diet. The data are expressed as the mean 6 SEM (n = 5–7/group) and analyzed by two-tailedStudent t test. *P < 0.05.

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(Fig. 3F–K). Consistently, BAT UCP1 expression was sig-nificantly higher, cells were denser with fewer lipids, andserum NE levels were elevated in AgRP-ATF4 KO mice(Fig. 3L–O). In addition, the deletion of ATF4 in AgRPneurons protected mice from HFD-induced IR and liversteatosis (Supplementary Fig. 5).

AgRP-ATF4 KO Mice Maintains at a Higher BodyTemperature Under Cold ExposureBased on the above results, we investigated whether AgRP-ATF4 KO mice have enhanced thermogenic responseunder cold exposure, a process that has been shown toinduce thermogenesis (34). After exposure to an ambienttemperature of 4°C, AgRP-ATF4 KO mice constantlymaintained at a relatively higher core body temperaturefor the period examined, with significantly reducedweight and cell volume of sWAT and eWAT, comparedwith control mice (Fig. 4A–C). Although BAT weightremained unchanged, BAT was much denser with fewlipid droplets in AgRP-ATF4 KO mice compared with

control mice (Fig. 4B and D). UCP1 expression and serumNE levels were induced by cold exposure in BAT of controlmice; however, the extent was much higher in AgRP-ATF4KO mice (Fig. 4E–G).

ATF4 Regulates Expression of FOXO1 via DirectBinding to the CRE Site on Its PromoterIt is shown that FOXO1 in AgRP neurons is critical for themaintenance of energy homeostasis (36), leading us toinvestigate the possible involvement of FOXO1 in medi-ating the effects of ATF4 deletion in AgRP neurons. In-hibition of ATF4 by a plasmid expressing DN-ATF4 (37),as evaluated by the increased ATF4 expression and de-creased expression of its downstream target gene TRB3(19), significantly decreased FOXO1 expression in AgRP-expressing hypothalamic GT1-7 cells (20) (Fig. 5A and B).Opposite effects were observed when ATF4 was over-expressed (Fig. 5C and D). ATF4 regulates the expres-sion of downstream target genes via direct binding toCRE sites in their promoters (7). Similarly, ATF4 can

Figure 2—AgRP-ATF4 KOmice have decreased food intake and enhanced energy expenditure. Daily food intake and feeding efficiency (A),24-h oxygen consumption normalized by lean mass (B) or analyzed by ANCOVA (C), RER (VCO2/VO2) (D), locomotor activity (E), and rectaltemperature (F ). Gene expression (G) and Western blot and densitometric quantification (H) of UCP1 in BAT. I: H-E staining of represen-tative BAT sections (scale bars, 250 mm). J: Serum NE levels. All studies were conducted in 10- to 12-week-old male control or AgRP-ATF4KO mice maintained on a normal chow diet. The data are expressed as the mean 6 SEM (n = 4–6/group) and analyzed by two-tailedStudent t test in all panels except for C, or ANCOVA in C. *P < 0.05.

644 ATF4 Deletion in AgRP Neurons Promotes Fat Loss Diabetes Volume 66, March 2017

Figure 3—AgRP-ATF4 KO mice are resistant to HFD-induced obesity. A: Body weight at the age of 12, 16, and 20 weeks. Fat and leanmass (B) and tissue weights (liver, sWAT, eWAT, and BAT) (C). D: H-E staining of representative eWAT sections (scale bars, 250 mm). E:p-PKA substrate proteins, p-HSL, and HSL Western blot and densitometric quantification in eWAT. Daily food intake (F ), 24-h oxygenconsumption normalized by lean mass (G) or analyzed by ANCOVA (H), RER (VCO2/VO2) (I), locomotor activity (J), and rectal temperature (K).Gene expression (L) and Western blot and densitometric quantification (M) of UCP1 in BAT. N: H-E staining of representative BAT sections(scale bars, 250 mm). O: Serum NE levels. All studies were conducted in 16- to 20-week-old (or as indicated) male control or AgRP-ATF4KO mice under an HFD (HFD feeding starting from age of 4 weeks). The data are expressed as the mean 6 SEM (n = 8–10/group) andanalyzed by two-tailed Student t test for all panels except for H (analyzed by ANCOVA in H). *P < 0.05.

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bind to FOXO1 promoter as demonstrated by lucifer-ase assays and ChIP assays in hypothalamic GT1-7cells transfected with plasmid or adenovirus expressingATF4 (Fig. 5E and F).

Overexpression of FOXO1 Increases Fat Mass inAgRP-ATF4 KO MiceAs observed above in vitro, expression of Foxo1 wasdecreased in ARC of AgRP-ATF4 KO mice (Fig. 6A).To confirm a role for FOXO1 in mediating the effectsof ATF4 deletion in AgRP neurons, we injectedAd-FOXO1 or control Ad-GFP to ARC of AgRP-ATF4KO and control mice. As predicted, IF staining showedthat FOXO1 expression was increased in both controland AgRP-ATF4 KO mice, most of which were localizedat ARC (Supplementary Fig. 6A and B). Consistently,RT-PCR results showed that Foxo1 was overexpressedin ARC but not in other brain areas (SupplementaryFig. 6C). Although there was no significant difference inbody weight and lean mass, Ad-FOXO1 increased fat massand tissue weights, including sWAT and eWAT, and foodintake; and decreased WAT p-HSL, rectal temperature,BAT UCP1 expression, and serum NE levels in AgRP-ATF4 KO mice (Fig. 6B–M). As shown previously (36,38),Ad-FOXO1 also reversed the suppressed neuronal ac-tivity, as measured by IF staining of c-fos, a markerused to reflect neuronal activity (39), and the expression

of neuronal peptides, including Agrp, Npy, and Pomc,except for Cart, in ARC of AgRP-ATF4 KO mice (Supple-mentary Fig. 7).

DISCUSSION

In this study, we used an AgRP-Cre-ER transgenic mouseline that allowed for spatiotemporal gene manipulationspecifically in AgRP neurons after tamoxifen inductionof Cre recombinase expression to avoid developmentalissues or compensating actions (14,40), which may hap-pen in AgRP-Cre mice (14). As expected, no differencein metabolic parameters examined was observed betweenthe two phenotypes before tamoxifen treatment whenadministered with corn oil as the control vehicle (Supple-mentary Fig. 8). In contrast, after tamoxifen treatment,AgRP-ATF4 KO mice became lean and resistant to HFD-induced obesity, with improved insulin sensitivity anddecreased lipid accumulation in liver. A pair-fed experi-ment showed that the decreased fat mass was mainlycaused by increased energy expenditure in AgRP-ATF4KO mice. Our current study for the first time demon-strated a novel function of ATF4 in hypothalamic AgRPneurons for systematic metabolic control. Our results alsoprovided novel insights into understanding the signals inspecific neurons that are critical for the regulation ofenergy homeostasis.

Figure 4—AgRP-ATF4 KO mice maintain at a higher body temperature under cold exposure. A: Rectal temperature of mice. B: Tissueweights (sWAT, eWAT, BAT). C: H-E staining of representative eWAT and sWAT sections (scale bars, 250 mm). D: H-E staining ofrepresentative BAT sections (scale bars, 250 mm). Gene expression (E) and Western blot and densitometric quantification (F ) of UCP1in BAT. G: Serum NE levels. All studies were conducted in 10-week-old male control or AgRP-ATF4 KO mice maintained on a normal chowdiet and exposed to a 4°C environment for 3 h. The data were expressed as the mean 6 SEM (n = 4–6/group) and analyzed by two-tailedStudent t test. *P < 0.05 for the effects of AgRP-ATF4 KO mice vs. control mice after cold exposure in A and B, for the effects of any groupof mice vs. control mice prior to cold exposure in E–G; #P < 0.05 for the effects of AgRP-ATF4 KO mice vs. control mice after coldexposure in E–G.

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In this study, we found that the deletion of ATF4 inAgRP neurons also improves insulin sensitivity and reduceshepatic lipid accumulation in normal chow diet–fed miceand protects mice from HFD-induced IR and liver stea-tosis. We speculated that the above effects in AgRP-ATF4KO mice may result from the decreased fat mass in thesemice, as many studies (1,35,41) have demonstrated thata reduction in body weight helps to improve insulin sen-sitivity and decrease lipid accumulation in liver. On theother hand, it might be directly regulated by signals fromhypothalamus (42), possibly via vagus nerve (19,42,43).Therefore, we could not exclude the possibility that ATF4in AgRP neurons may have a direct effect on hepatic in-sulin sensitivity and lipid accumulation, particularly giventhe fact that hypothalamic ATF4 has a significant impacton regulating hepatic insulin sensitivity via the vagusnerve (19).

Leptin binds to its receptors expressed in the hypo-thalamus and regulates neural circuits that suppress foodintake and increase energy expenditure (35,44). Here, weshowed that the deletion of ATF4 in AgRP neurons im-proves leptin sensitivity, as demonstrated by the muchmore significant inhibitory effects of leptin on food in-take and body weight, and p-STAT3 signaling in ARC of

AgRP-ATF4 KO mice. Given the importance for leptinsensitivity in body weight control, we speculated thatthe increased leptin sensitivity may contribute to thedecreased food intake and enhanced energy expenditurein AgRP-ATF4 KO mice. This possibility, however, mustbe studied in the future.

Because body fat mass is maintained by a balancebetween food intake and energy expenditure, we exploredthe possible reasons responsible for the decreased fatmass in AgRP-ATF4 KO mice from these two aspects. It ispreviously shown that global deletion of ATF4 had noeffect on food intake (9,11). It is also reported that theactivation of a certain signal in ARC is sufficient to inhibitfood intake, associated with ATF4 overexpression (45). Incontrast, food intake was reduced in AgRP-ATF4 KO mice.The difference in the effect of ATF4 deletion on foodintake, however, might be due to the difference in theway of deleting ATF4 under each different circumstance.

In addition, AgRP-ATF4 KO mice had increased energyexpenditure and body temperature, which reflect an in-crease in thermogenesis. BAT is of major importance inthe regulation of thermogenesis and energy expendi-ture via affecting UCP1 expression (34). It is shown thatUCP1 expression in BAT is induced by the activation of

Figure 5—ATF4 regulates the expression of FOXO1 via direct binding to the CRE site in its promoter in vitro. A: Western blot anddensitometric quantification of FOXO1, ATF4, and TRB3 in GT1-7 cells transfected with plasmids expressing DN-ATF4 or control vector.B: Gene expression of Foxo1 and Trb3 in GT1-7 cells transfected with plasmids expressing DN-ATF4 or control vector. C:Western blot anddensitometric quantification of FOXO1, ATF4, and TRB3 in GT1-7 cells transfected with plasmids expressing ATF4 or control vector. D:Gene expression of Foxo1 and Trb3 in GT1-7 cells transfected with plasmids expressing ATF4 or control vector. E: Luciferase activity wasassessed in GT1-7 cells expressing the Foxo1 promoter vector with or without a plasmid expressing ATF4. F: ChIP assay was performed inGT1-7 cells infected with Ad-GFP or Ad-ATF4. NC, negative control. All studies were conducted in GT1-7 cells with at least threeindependent experiments. The data are expressed as the mean 6 SEM and analyzed by two-tailed Student t test. *P < 0.05, for anytreatment compared with control group.

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Figure 6—Overexpression of FOXO1 increases fat mass in AgRP-ATF4 KO mice. A: Gene expression of Foxo1 in ARC. B: Body weight atthe seventh day after virus injection. Fat and lean mass (C) and tissue weights (liver, sWAT, eWAT, and BAT) (D). E: H-E staining ofrepresentative eWAT sections (scale bars, 250 mm). F: Analysis of abdominal eWAT cell volume. G: P-PKA substrate proteins, p-HSL, andHSL Western blot and densitometric quantification in eWAT. Daily food intake (H) and rectal temperature (I). Gene expression (J) andWestern blot and densitometric quantification (K) of UCP1 in BAT. L: H-E staining of representative BAT sections (scale bars, 250 mm).M: Serum NE levels. All studies were conducted 7 days after receiving Ad-GFP or Ad-FOXO1 bilaterally in ARC in 10- to 12-week-old malecontrol or AgRP-ATF4 KO mice maintained on a normal chow diet. The data are expressed as the mean 6 SEM (n = 6–7/group) andanalyzed by one-way ANOVA followed by the Student-Newman-Keuls test. *P < 0.05 for the effects of any group of mice vs. control miceinjected with Ad-GFP; #P < 0.05 for the effects of Ad-FOXO1 vs. Ad-GFP in AgRP-ATF4 KO mice.

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SNS, which stimulates the release of NE binding tob3-adrenergic receptor on the surface of BAT (2,34). Theupregulation of UCP1 expression results in increased ther-mogenesis and energy expenditure, which helps in protec-tion from fat accumulation (34). In this study, we foundthat AgRP-ATF4 KO mice exhibit increased body tempera-ture, BAT UCP1 expression, and serum NE levels, suggest-ing increased thermogenesis. It is well known that fatmobilization is promoted by SNS activation, which stimu-lates the release of NE binding to b3-adrenergic receptor onthe surface of adipocytes and sequentially phosphorylatesPKA and HSL (34). Our results show that WAT cell volumewas decreased in AgRP-ATF4 KO mice, suggesting increasedlipolysis and altered nutrient partitioning that may contrib-ute to the maintenance of the energy balance. The alterednutrient partitioning in AgRP-ATF4 KO mice could be dueto the insufficient energy intake (27,34), which is needed tomobilize more fat to be used as an energy source. On theother hand, however, it might also be induced by activatedSNS activity or increased insulin sensitivity (34). The in-creased thermogenesis in BAT and possibly the increasedlipolysis in WAT should certainly contribute to the de-creased fat mass in AgRP-ATF4 KO mice.

In this study, we also explored the possible role ofATF4 in AgRP neurons after treatment with an HFD orcold exposure. Thermogenesis is one of the most impor-tant adaptive changes in response to these environmentalstimuli and functions to maintain metabolic homeostasisor to protect the organism from cold exposure (34). Asobserved in mice maintained on a control diet, we foundthat AgRP-ATF4 KO mice also exhibit enhanced thermo-genesis, resulting in the resistance to HFD-induced obe-sity, IR, and liver steatosis, and a higher body temperatureafter cold exposure. Interestingly, food intake was com-parable between HFD-fed control and AgRP-ATF4 KOmice, which is different from the observed decreasedfood intake in these mice maintained on a control diet.Although it is shown AgRP neurons are important forenergy intake control, however, when palatable food isprovided, AgRP neurons are dispensable for an appropri-ate feeding response (46). These results suggest that AgRPneurons are indispensible in the feeding response under acontrol chow diet, but not under HFD, which might ex-plain the difference in food intake between AgRP-ATF4KO and control mice maintained on a normal chow diet orHFD.

Several signaling pathways in AgRP neurons have beenidentified to be important regulators of energy homeo-stasis (20,47–49). In this study, we found that the ef-fects of ATF4 deletion in AgRP neurons are mediated byinhibited expression of FOXO1, based on the effects ofFOXO1 overexpression in ARC on increasing fat mass inAgRP-ATF4 KO mice. However, body weight was not af-fected by FOXO1 overexpression, possibly because of thesimultaneously decreased tendency of lean mass. A pre-vious study (36) has shown that FOXO1 regulates energyhomeostasis via affecting AgRP neuronal activity and Agrp

expression. Consistently, we found neuronal activity asexamined by IF staining of c-fos, a marker reflecting neu-ronal activity (39), and Agrp expression are changed inthe ARC of AgRP-ATF4 KO mice after overexpression ofFOXO1, indicating that similar mechanisms may mediateFOXO1 regulation of energy homeostasis in AgRP-ATF4KO mice. Thus, our study demonstrates an important rolefor FOXO1 as a downstream target for ATF4. In addition,we identified a direct effect of ATF4 on the regulation ofFOXO1 expression. In contrast to our observation, it isshown that FOXO1 expression is not affected by ATF4 inosteoblast cells (50). The difference might be caused bytissue-specific regulatory mechanisms, which require fur-ther investigation. The possible influence from FOXO1expressed in other neurons by ARC injection and theinvolvement of other pathways in ATF4 regulation ofenergy homeostasis, however, needs to be studied inthe future.

Taken together, these results demonstrate a critical rolefor ATF4 in hypothalamic AgRP neurons in the regulationof energy homeostasis, and lipid and glucose metabolismmainly by the increased energy expenditure via affectingFOXO1 expression. These results also suggest hypotha-lamic ATF4 as a potential novel drug target in treatingobesity and its related metabolic disorders.

Acknowledgments. The authors thank Joel K. Elmquist and Tiemin Liu(UT Southwestern Medical Center, Dallas, TX) for providing AgRP Cre-ER mice.Funding. This work was supported by grants from National Natural ScienceFoundation (81130076, 81325005, 31271269, 81300659, 81400792, 81471076,81570777, 81500622, and 81390350), Basic Research Project of ShanghaiScience and Technology Commission (16JC1404900), and International S&T Co-operation Program of China (Singapore 2014DFG32470) and by the internationalPartnership Program For Creative Research Teams (CAS/SAFEA). F.G. was alsosupported by the One Hundred Talents Program of the Chinese Academy ofSciences.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. J.D. and F.Y. researched the data and wrote,reviewed, and edited the manuscript. Y. Guo and Y.N. researched the data. Y.X. andY.D. contributed to the writing of the manuscript and helpful discussion. X.H.,Y.Gua., and S.C. provided research material. F.G. directed the project, contributed todiscussion, and wrote, reviewed, and edited the manuscript. F.G. is the guarantor ofthis work and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

References1. Grundy SM. Adipose tissue and metabolic syndrome: too much, too little orneither. Eur J Clin Invest 2015;45:1209–12172. Spiegelman BM, Flier JS. Obesity and the regulation of energy balance. Cell2001;104:531–5433. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Centralnervous system control of food intake and body weight. Nature 2006;443:289–2954. Cowley MA, Smart JL, Rubinstein M, et al. Leptin activates anorexigenicPOMC neurons through a neural network in the arcuate nucleus. Nature 2001;411:480–4845. Blouet C, Liu SM, Jo YH, Chua S, Schwartz GJ. TXNIP in Agrp neuronsregulates adiposity, energy expenditure, and central leptin sensitivity. J Neurosci2012;32:9870–9877

diabetes.diabetesjournals.org Deng and Associates 649

6. Yang L, McKnight GS. Hypothalamic PKA regulates leptin sensitivity andadiposity. Nat Commun 2015;6:82377. Karpinski BA, Morle GD, Huggenvik J, Uhler MD, Leiden JM. Molecularcloning of human CREB-2: an ATF/CREB transcription factor that can negativelyregulate transcription from the cAMP response element. Proc Natl Acad Sci USA1992;89:4820–48248. Vallejo M, Ron D, Miller CP, Habener JF. C/ATF, a member of the activatingtranscription factor family of DNA-binding proteins, dimerizes with CAAT/enhancer-binding proteins and directs their binding to cAMP response ele-ments. Proc Natl Acad Sci USA 1993;90:4679–46839. Seo J, Fortuno ES 3rd, Suh JM, et al. Atf4 regulates obesity, glucose ho-meostasis, and energy expenditure. Diabetes 2009;58:2565–257310. Li K, Zhang J, Yu J, et al. MicroRNA-214 suppresses gluconeogenesis bytargeting activating transcriptional factor 4. J Biol Chem 2015;290:8185–819511. Wang C, Huang Z, Du Y, Cheng Y, Chen S, Guo F. ATF4 regulates lipidmetabolism and thermogenesis. Cell Res 2010;20:174–18412. Yoshizawa T, Hinoi E, Jung DY, et al. The transcription factor ATF4 regulatesglucose metabolism in mice through its expression in osteoblasts. J Clin Invest2009;119:2807–281713. Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG. Central nervoussystem control of food intake. Nature 2000;404:661–67114. Wang Q, Liu C, Uchida A, et al. Arcuate AgRP neurons mediate orexigenicand glucoregulatory actions of ghrelin. Mol Metab 2013;3:64–7215. Ye R, Wang QA, Tao C, et al. Impact of tamoxifen on adipocyte lineagetracing: inducer of adipogenesis and prolonged nuclear translocation of Cre re-combinase. Mol Metab 2015;4:771–77816. Mottillo EP, Desjardins EM, Crane JD, et al. Lack of adipocyte AMPK ex-acerbates insulin resistance and hepatic steatosis through brown and beigeadipose tissue function. Cell Metab 2016;24:118–12917. Herman AM, Ortiz-Guzman J, Kochukov M, et al. A cholinergic basal forebrainfeeding circuit modulates appetite suppression. Nature 2016;538:253–25618. Madisen L, Zwingman TA, Sunkin SM, et al. A robust and high-throughputCre reporting and characterization system for the whole mouse brain. NatNeurosci 2010;13:133–14019. Zhang Q, Yu J, Liu B, et al. Central activating transcription factor 4 (ATF4)regulates hepatic insulin resistance in mice via S6K1 signaling and the vagusnerve. Diabetes 2013;62:2230–223920. Kaushik S, Rodriguez-Navarro JA, Arias E, et al. Autophagy in hypothalamicAgRP neurons regulates food intake and energy balance. Cell Metab 2011;14:173–18321. Nakajima K, Cui Z, Li C, et al. Gs-coupled GPCR signalling in AgRP neuronstriggers sustained increase in food intake. Nat Commun 2016;7:1026822. Li J, Chi Y, Wang C, et al. Pancreatic-derived factor promotes lipogenesis inthe mouse liver: role of the Forkhead box 1 signaling pathway. Hepatology 2011;53:1906–191623. Bal NC, Maurya SK, Sopariwala DH, et al. Sarcolipin is a newly identified reg-ulator of muscle-based thermogenesis in mammals. Nat Med 2012;18:1575–157924. Zhang Q, Liu B, Cheng Y, et al. Leptin signaling is required for leucinedeprivation-enhanced energy expenditure. J Biol Chem 2014;289:1779–178725. Carson FL. Histotechnology: A Self-Instructional Text. Chicago, AmericanSociety for Clinical Pathology Press, 199726. Chen F, Zhu Y, Tang X, et al. Dynamic regulation of PDX-1 and FoxO1expression by FoxA2 in dexamethasone-induced pancreatic b-cells dysfunction.Endocrinology 2011;152:1779–178827. Zechner R, Zimmermann R, Eichmann TO, et al. FAT SIGNALS–lipases andlipolysis in lipid metabolism and signaling. Cell Metab 2012;15:279–291

28. Tang Y, Wallace M, Sanchez-Gurmaches J, et al. Adipose tissue mTORC2regulates ChREBP-driven de novo lipogenesis and hepatic glucose metabolism.Nat Commun 2016;7:1136529. Xiao F, Deng J, Guo Y, et al. BTG1 ameliorates liver steatosis by decreasingstearoyl-CoA desaturase 1 (SCD1) abundance and altering hepatic lipid metab-olism. Sci Signal 2016;9:ra5030. Hinton AO Jr, He Y, Xia Y, et al. Estrogen receptor-a in the medial amygdalaprevents stress-induced elevations in blood pressure in females. Hypertension2016;67:1321–133031. Himms-Hagen J. On raising energy expenditure in ob/ob mice. Science1997;276:1132–113332. Butler AA, Kozak LP. A recurring problem with the analysis of energy ex-penditure in genetic models expressing lean and obese phenotypes. Diabetes2010;59:323–32933. Tschöp MH, Speakman JR, Arch JR, et al. A guide to analysis of mouseenergy metabolism. Nat Methods 2011;9:57–6334. Lowell BB, Spiegelman BM. Towards a molecular understanding of adaptivethermogenesis. Nature 2000;404:652–66035. Myers MG Jr, Olson DP. Central nervous system control of metabolism.Nature 2012;491:357–36336. Ren H, Orozco IJ, Su Y, et al. FoxO1 target Gpr17 activates AgRP neurons toregulate food intake. Cell 2012;149:1314–132637. He CH, Gong P, Hu B, et al. Identification of activating transcription factor4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 generegulation. J Biol Chem 2001;276:20858–2086538. Kim MS, Pak YK, Jang PG, et al. Role of hypothalamic Foxo1 in the regu-lation of food intake and energy homeostasis. Nat Neurosci 2006;9:901–90639. Curran T, Morgan JI. Fos: an immediate-early transcription factor in neu-rons. J Neurobiol 1995;26:403–41240. Joly-Amado A, Denis RG, Castel J, et al. Hypothalamic AgRP-neuronscontrol peripheral substrate utilization and nutrient partitioning. EMBO J 2012;31:4276–428841. Könner AC, Brüning JC. Selective insulin and leptin resistance in metabolicdisorders. Cell Metab 2012;16:144–15242. Pocai A, Obici S, Schwartz GJ, Rossetti L. A brain-liver circuit regulatesglucose homeostasis. Cell Metab 2005;1:53–6143. van den Hoek AM, van Heijningen C, Schröder-van der Elst JP, et al. In-tracerebroventricular administration of neuropeptide Y induces hepatic insulinresistance via sympathetic innervation. Diabetes 2008;57:2304–231044. Friedman JM, Halaas JL. Leptin and the regulation of body weight inmammals. Nature 1998;395:763–77045. Maurin AC, Benani A, Lorsignol A, et al. Hypothalamic eIF2a signalingregulates food intake. Cell Rep 2014;6:438–44446. Denis RG, Joly-Amado A, Webber E, et al. Palatability can drive feedingindependent of AgRP neurons. Cell Metab 2015;22:646–65747. Kitamura T, Feng Y, Kitamura YI, et al. Forkhead protein FoxO1 mediatesAgrp-dependent effects of leptin on food intake. Nat Med 2006;12:534–54048. Liu T, Kong D, Shah BP, et al. Fasting activation of AgRP neurons requiresNMDA receptors and involves spinogenesis and increased excitatory tone.Neuron 2012;73:511–52249. Claret M, Smith MA, Batterham RL, et al. AMPK is essential for energyhomeostasis regulation and glucose sensing by POMC and AgRP neurons. J ClinInvest 2007;117:2325–233650. Kode A, Mosialou I, Silva BC, et al. FoxO1 protein cooperates with ATF4protein in osteoblasts to control glucose homeostasis. J Biol Chem 2012;287:8757–8768

650 ATF4 Deletion in AgRP Neurons Promotes Fat Loss Diabetes Volume 66, March 2017