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Deletion of ATF4 in Agrp Neurons Promotes Fat Loss Mainly Via Increasing Energy Expenditure

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Deletion of ATF4 in Agrp Neurons Promotes Fat Loss Mainly Via Increasing Energy Expenditure 640 Diabetes Volume 66, March 2017 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 Neurons Promotes Fat Loss Mainly via Increasing Energy Expenditure Diabetes 2017;66:640–650 | DOI: 10.2337/db16-0954 Although many functions of activating transcription liver steatosis (1,2). Changes in body weight normally re- factor 4 (ATF4) are identified, a role of ATF4 in the sult from an imbalance between energy intake and energy hypothalamus in regulating energy homeostasis is un- expenditure (2), controlled by the central nervous system, known. Here, we generated adult-onset agouti-related especially the hypothalamus (3). The center of this reg- peptide neuron–specific ATF4 knockout (AgRP-ATF4 KO) ulatory network is the arcuate nucleus (ARC) of the hy- mice and found that these mice were lean, with im- pothalamus, which contains sets of important neurons proved insulin and leptin sensitivity and decreased devoted to metabolic regulation including orexigenic neu- hepatic lipid accumulation. Furthermore, AgRP-ATF4 rons that coproduce agouti-related peptide (AgRP) and KO mice showed reduced food intake and increased neuropeptide Y, as well as anorexigenic neurons that con- energy expenditure, mainly because of enhanced ther- tain cocaine- and amphetamine-regulated transcript and mogenesis in brown adipose tissue. Moreover, AgRP- proopiomelanocortin (POMC)–derived peptides (3,4). AgRP ATF4 KO mice were resistant to high-fat diet–induced neurons increase feeding by opposing the anorexigenic obesity, insulin resistance, and liver steatosis and main- actions of POMC neurons, in part through the release of METABOLISM tained at a higher body temperature under cold stress. Interestingly, the expression of FOXO1 was directly AgRP, a competitive inhibitor of melanocortin receptors regulated by ATF4 via binding to the cAMP-responsive (4). It also had an effect on energy expenditure via affecting element site on its promoter in hypothalamic GT1-7 sympathetic nervous system (SNS) activity or leptin sensi- cells. Finally, Foxo1 expression was reduced in the tivity (5,6). arcuate nucleus (ARC) of the hypothalamus of AgRP- Activating transcription factor 4 (ATF4), also known as ATF4 KO mice, and adenovirus-mediated overexpres- CREBP2, belongs to the CREBP families, characterized by sion of FOXO1 in ARC increased the fat mass in the presence of a leucine zipper dimerization domain and a AgRP-ATF4 KO mice. Collectively, our data demon- basic amino acid–rich DNA binding domain (7,8). ATF4 is strate a novel function of ATF4 in AgRP neurons of ubiquitously expressed in many tissues and some parts of the hypothalamus in energy balance and lipid metab- the brain, including the hypothalamus (8). It is involved in olism and suggest hypothalamic ATF4 as a potential the regulation of various processes, including memory for- drug target for treating obesity and its related meta- mation, osteoblast differentiation, amino acid deprivation, bolic disorders. and redox homoeostasis (9). Recent studies (9–12) have demonstrated a role of ATF4 in the control of glucose and lipid metabolism. A role of ATF4 in specificneuronsofthe Obesity is strongly associated with metabolic syndrome hypothalamus, however, has not been previously described. and predisposes to diseases including type 2 diabetes and The aim of our current study was to investigate the role of 1Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Received 5 August 2016 and accepted 12 December 2016. Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Grad- This article contains Supplementary Data online at http://diabetes ’ uate School of the Chinese Academy of Sciences, Shanghai, People s Republic of .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0954/-/DC1. China J.D. and F.Y. contributed equally to this study. 2Key Laboratory of Human Functional Genomics of Jiangsu Province, Jiangsu Diabetes Center, Nanjing Medical University, Nanjing, People’s Republic of China © 2017 by the American Diabetes Association. Readers may use this article as 3Advanced Institute for Medical Sciences, Dalian Medical University, Dalian, long as the work is properly cited, the use is educational and not for profit, and the People’s Republic of China work is not altered. More information is available at http://www.diabetesjournals .org/content/license. Corresponding author: Feifan Guo, [email protected]. diabetes.diabetesjournals.org Deng and Associates 641 ATF4 expressed in AgRP neurons in energy homeostasis Cold Exposure Treatment regulation. The 2- to 3-month-old mice were housed in individual precooled 4°C cages for 3 h, and rectal temperatures of RESEARCH DESIGN AND METHODS mice were measured every 30 min during this period, as Generation of Mice With ATF4 Deletion in AgRP described previously (23). Body weight was measured im- Neurons and Animal Treatment mediately before the cold exposure. fl All animals were on C57BL/6J background. ATF4- oxed Blood Glucose, Serum Insulin, Glucose Tolerance mice (13) and AgRP Cre-ER mice (14) (provided by Joel K. Tests, Insulin Tolerance Tests, and HOMA-Insulin Elmquist and Tiemin Liu, UT Southwestern Medical Center, Resistance Index fl fl Dallas, TX) were bred to generate AgRP-Cre ATF4 ox/ ox The measurements of blood glucose and serum insulin, fl fl and ATF4 ox/ ox littermates, which were named AgRP- results of glucose tolerance tests (GTTs) and insulin ATF4 knockout (KO) and control mice, respectively. For in- tolerance tests (ITTs), and the calculation of the HOMA- ducing Cre expression and avoiding the possible toxic effect insulin resistance (IR) index were conducted as described of tamoxifen (15,16), both control and AgRP-ATF4 mice previously (19). were intraperitoneally injected with 150 mg/kg body weight Leptin Sensitivity Assay In Vivo tamoxifen (Sigma-Aldrich, St. Louis, MO) for 5 days, be- Mice were intraperitoneally injected with either PBS or tween the ages of 5 and 7 weeks (14). The basal metabolic 3 mg/kg leptin (R&D Systems, Minneapolis, MN) at 9:00 A.M. phenotypes in AgRP-ATF4 mice were analyzed by treating after fasting for 24 h, as described previously (24). Food them and control mice with corn oil (Standard Food, Shang- intakeandbodyweightweremeasuredat1and4hafter hai, People’sRepublicofChina)for5days.Forhigh-fatdiet the injection of leptin. (HFD) study, 4-week-old AgRP-ATF4 KO or control mice were maintained on a normal chow diet or HFD with 60% Metabolic Parameters Measurements kcal fat (Research Diets, New Brunswick, NJ) for 16 weeks. The body fat composition of mice was determined using the Pair-fed experiments (17) were conducted by feeding control Bruker Minispec mq10 NMR Analyzer (Bruker, Billerica, mice a normal chow diet in the same amounts of food eaten MA). Indirect calorimetry was measured in a Comprehensive by AgRP-ATF4 KO mice during the previous day. The effi- Lab Animal Monitoring System (Columbus Instruments, ciency for ATF4 deletion was evaluated by mating AI9 Columbus, OH), as described previously (11). Rectal tem- (tdTomato) reporter mice (18) with transgenic mice express- peratures were measured using a rectal probe attached to ing Cre under control of the AgRP promoter after tamoxifen a digital thermometer (Physitemp Instruments, Clifton, treatment. Body weight was monitored weekly throughout NJ). the experiments, and mice were kept as previously described (19). All the experiments were conducted in accordance with Serum and Liver Measurements the guidelines of the Institutional Animal Care and Use Serum and liver total glycerol, total cholesterol, and free Committee of the Institute for Nutritional Sciences. fatty acid levels were determined using Glycerol Assay Kit Reagent (SSUF-C, Shanghai, People’s Republic of China), Cell Culture and Treatments cholesterol reagent (SSUF-C), and NEFA C reagent (Wako, – Pshuttle vector constructed plasmids expressing ATF4 or Osaka, Japan), respectively. Serum norepinephrine (NE) a dominant-negative form of ATF4 (DN-ATF4) was made level was determined using ELISA kits (R&D Systems). All based on plasmids described previously (19). The recom- of these assays were performed according to manufac- binant adenoviruses (Ads) expressing mouse ATF4 (Ad- turer instructions. ATF4) or control green fluorescent protein (Ad-GFP) were generated as previously described (19). Hypotha- Protein and mRNA Analysis lamic GT1-7 cells were maintained as described previously Western blot analysis was performed with primary anti- (20). Plasmids and Ads indicated were transfected into bodies against actin (Sigma-Aldrich); ATF4, tribbles homo- GT1-7 cells with Lipofectamine 2000 (Invitrogen, Carls- log 3 (TRB3), and uncoupling protein 1 (UCP1) (Santa Cruz bad, CA). Biotechnology, Santa Cruz, CA); and t–hormone-sensitive lipase (HSL), phosphorylated (p)-HSL, p-cAMP-dependent ARC Administration Experiments protein kinase (PKA), and FOXO1 (Cell Signaling Technol- ARC administration experiments were conducted as pre- ogy, Danvers, MA); and visualized by ECL Plus (GE Health- viously described (21). Ad-FOXO1 (22) was provided by care, Chicago, IL), as described previously (11). RT-PCR, Professor Youfei Guan (Dalian Medical University, Dalian, with GAPDH as an internal control gene, was carried out People’s Republic of China). Mice were anesthetized and as described previously (11). The sequences of primers used received bilateral stereotaxic injections of Ad-GFP, Ad-Null, in the current
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