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ATF4/ATG5 Signaling in Hypothalamic Proopiomelanocortin Neurons Regulates Fat Mass Via Affecting Energy Expenditure

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ATF4/ATG5 Signaling in Hypothalamic Proopiomelanocortin Neurons Regulates Fat Mass Via Affecting Energy Expenditure 1146 Diabetes Volume 66, May 2017 ATF4/ATG5 Signaling in Hypothalamic Proopiomelanocortin Neurons Regulates Fat Mass via Affecting Energy Expenditure Yuzhong Xiao, Yalan Deng, Feixiang Yuan, Tingting Xia, Hao Liu, Zhigang Li, Zhixue Liu, Hao Ying, Yi Liu, Qiwei Zhai, Shanghai Chen, and Feifan Guo Diabetes 2017;66:1146–1158 | DOI: 10.2337/db16-1546 Although many biological functions of activating tran- between food intake and energy expenditure (2–4). It has scription factor 4 (ATF4) have been identified, a role of been shown that certain populations of neurons in the arcu- hypothalamic ATF4 in the regulation of energy homeo- ate nucleus (ARC) of the hypothalamus play key roles in the stasis is poorly understood. In this study, we showed regulation of energy homeostasis (5,6). These neurons include that hypothalamic proopiomelanocortin (POMC) neuron– those expressing orexigenic neuropeptides neuropeptide Y specific ATF4 knockout (PAKO) mice are lean and have and agouti-related protein, along with neurons expressing higher energy expenditure. Furthermore, PAKO mice were anorexigenic neuropeptides cocaine and amphetamine-related – resistant to high-fat diet induced obesity, glucose intoler- transcript and proopiomelanocortin (POMC) (5,6). ance, and leptin resistance. Moreover, the expression of The activating transcription factor 4 (ATF4) belongs to autophagy protein 5 (ATG5) was increased or decreased the family of basic zipper-containing proteins (bZIP) by ATF4 knockdown or overexpression, respectively, and (7,8), with broad expression in various tissues, including ATF4 inhibited the transcription of ATG5 by binding to the brain (9). Previous studies have shown that ATF4 global METABOLISM basic zipper-containing protein sites on its promoter. Im- knockout mice are lean (8,10) and resistant to high-fat portantly, mice with double knockout of ATF4 and ATG5 in – – POMC neurons gained more fat mass and reduced energy diet (HFD) or high-carbohydrate diet induced obesity expenditure compared with PAKO mice under a high-fat and hyperglycemia (10,11). Recently, studies using tis- fi fi diet. Finally, the effect of ATF4 deletion in POMC neurons sue-speci c knockout mice have identi ed that ATF4 reg- was possibly mediated via enhanced ATG5-dependent ulates glucose metabolism through its expression in liver autophagy and a-melanocyte–stimulating hormone pro- and osteoblasts (12,13). In contrast, the role of ATF4 in duction in the hypothalamus. Taken together, these results POMC neurons in metabolic control is largely unknown. identify the beneficial role of hypothalamic ATF4/ATG5 axis Autophagy is a cellular process through which cells in the regulation of energy expenditure, obesity, and engulf and degrade damaged cytoplasmic components obesity-related metabolic disorders, which suggests (14). Recent studies have implicated the involvement of that ATF4/ATG5 axis in the hypothalamus may be a new autophagy in the regulation of obesity and energy expen- potential therapeutic target for treating obesity and diture (15–21). For example, autophagy is reduced in the obesity-related metabolic diseases. hypothalamus of aged mice or mice fed an HFD (17,22). Consistently, mice with POMC neuron knockout of autophagy- related gene 7 (ATG7), one of the key regulators of autophagy Decreased energy expenditure and/or increased food (23), are obese and have lower energy expenditure (16,17,21), intake contributes to the development of obesity (1,2). The and autophagy protein 5 (ATG5)-transgenic mice have the central nervous system (CNS), especially the hypothalamus, opposite phenotype (19). Previous studies have shown that plays an important role in the regulation of the balance ATF4 can regulate autophagy and the expression of some of Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, This article contains Supplementary Data online at http://diabetes Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Uni- .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1546/-/DC1. versity of Chinese Academy of Sciences, Shanghai, China © 2017 by the American Diabetes Association. Readers may use this article as Corresponding author: Feifan Guo, [email protected]. long as the work is properly cited, the use is educational and not for profit, and the Received 15 December 2016 and accepted 13 February 2017. work is not altered. More information is available at http://www.diabetesjournals .org/content/license. diabetes.diabetesjournals.org Xiao and Associates 1147 the autophagy regulators in vitro under different stimula- mice after overnight fasting or 0.75 units/kg insulin to mice tions (24–27); however, the relationship has not been after 4-h fasting, respectively, at 21 weeks old. HOMA of tested in vivo yet. insulin resistance index and areas under the curve were Given the knowledge mentioned above, the aim of our calculated as described previously (33). current study was to explore the role of ATF4 in POMC Serum Norepinephrine, Leptin, Growth Hormone, neurons in the regulation of obesity and energy expenditure and Corticosterone Measurements and the possible involvement of autophagy in this regulation. Serum norepinephrine (NE), leptin, growth hormone, and RESEARCH DESIGN AND MATERIALS corticosterone levels were measured by an NE ELISA kit (Novus Biologicals, Littleton, CO), leptin ELISA kit (Merck Animals and Treatment Millipore, Frankfurter, Germany), growth hormone ELISA C57BL/6J wild-type (WT) mice were purchased from kit (Merck Millipore), and corticosterone ELISA kit (Novus Shanghai Laboratory Animals Co. Ltd (Shanghai, China). To Biologicals), respectively, according to the manufacturer’s generate hypothalamic POMC neuron–specificATF4knock- instructions. out mice (PAKO) mice, ATF4-floxed mice (13) were inter- crossed with POMC-Cre mice (28), provided by Joel K. Histological Analysis of Tissues Elmquist and Tiemin Liu from Southwestern Medical Paraformaldehyde-fixed, paraffin-embedded sections of Center (Dallas, TX). The mice lacking ATF4 and ATG5 in the white adipose tissue (WAT) and brown adipose tissue POMC neurons (double knockout [DKO]) were generated (BAT) were stained with hematoxylin and eosin for histology. fl fl by intercrossing ATF4- oxed and ATG5- oxed mice (29) fl – Immuno uorescence Staining with POMC-Cre mice. To visualize POMC protein expressing Immunofluorescence (IF) staining with anti-ATF4 anti- neurons under fluorescence microscope, POMC-Cre, PAKO, body (Santa Cruz Biotechnology, Santa Cruz, CA), anti–a- and DKO mice were intercrossed with tdTomato reporter/Ai9 melanocyte–stimulating hormone (a-MSH) antibody mice (30). All animals were under the C57BL/6J background (Merck Millipore), and anti-p62 antibody (Progen, Heidel- and housed in animal cages with a 12-h dark/light cycle at 25°C, berg, Germany) were performed as described previously with free access to water and normal chow diet (NCD). For HFD (31). Phosphorylated (p-)STAT3 staining was performed studies, 8-week-old male mice were fed an HFD with 60% kcal as described previously (32,34). from fat (Research Diets, New Brunswick, NJ) for 3 months. At Construction of Plasmids the age of 22 weeks, mice were sacrificed by CO2 inhalation. All of the experiments were conducted in accordance with the The coding sequence region of ATF4 was amplified from guidelines of the Institutional Animal Care and Use Committee mouse hepatic cDNA and inserted into the eukaryotic of the Institute for Nutritional Sciences, Shanghai Institutes for expression plasmid pCMV-myc. The ATG5 promoter Biological Sciences, Chinese Academy of Sciences. (22085 to +1) was amplified from mouse genomic DNA and inserted into the pGL3-basic report plasmid. The Metabolic Parameter Measurements ATG5 promoter with the bZIP sites (A: 21962/21958 The mice body composition was measured by a nuclear and B: 21321/21317) deleted was generated by site- magnetic resonance system (Bruker, Rheinstetten, Ger- directed mutagenesis (35). many). Indirect calorimetry was measured in a compre- hensive laboratory animal-monitoring system (Columbus Cell Culture and Treatments Instruments, Columbus, OH), as previously described The primary culture of hypothalamic neurons was per- (31). Rectal temperature of mice was measured at 14:00 formed as described previously (31). Recombinant adeno- by a rectal probe attached to a digital thermometer viruses expressing green fluorescent protein (GFP), ATF4, (Physitemp, Clifton, NJ). The measurement of food intake or dominant-negative (DN) ATF4 were purified and admin- 7 was conducted as reported previously (6). istrated at the dose of 1 3 10 plaque-forming units/well in 12-well plates for 48 h (36). The autophagic flux assays Leptin Sensitivity Assay were performed as reported previously (37). 293T cells Leptin sensitivity assay was performed as reported pre- were cultured in DMEM with 10% FBS. Plasmids were viously (32). Mice were individually housed and intraper- transfected by Lipofectamine 2000 (Invitrogen, Carlsbad, itoneally (i.p.) injected with PBS for 5 days prior to i.p. CA) according to the manufacturer’s instructions. injection of leptin (R&D Systems, Minneapolis, MN) twice a day (at 8:00 and 19:00) for 3 days. RNA Isolation and Relative Quantitative RT-PCR The RNA isolation and RT-PCR were performed as Blood Glucose, Serum Insulin, Glucose Tolerance described previously (31). The sequence of primers used Tests, Insulin Tolerance Tests, and HOMA of Insulin Resistance Index is available upon request. Levels of blood glucose and serum insulin were measured by
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