Although many functions of activating transcription factor 4 (ATF4) are identified, a role of ATF4 in the hypothalamus in regulating energy homeostasis is unknown. Here, we generated adult-onset agouti-related peptide neuron–specific ATF4 knockout (AgRP-ATF4 KO) mice and found that these mice were lean, with improved insulin and leptin sensitivity and decreased hepatic lipid accumulation. Furthermore, AgRP-ATF4 KO mice showed reduced food intake and increased energy expenditure, mainly because of enhanced thermogenesis in brown adipose tissue. Moreover, AgRP-ATF4 KO mice were resistant to high-fat diet–induced obesity, insulin resistance, and liver steatosis and maintained at a higher body temperature under cold stress. Interestingly, the expression of FOXO1 was directly regulated by ATF4 via binding to the cAMP-responsive element site on its promoter in hypothalamic GT1-7 cells. Finally, Foxo1 expression was reduced in the arcuate nucleus (ARC) of the hypothalamus of AgRP-ATF4 KO mice, and adenovirus-mediated overexpression of FOXO1 in ARC increased the fat mass in AgRP-ATF4 KO mice. Collectively, our data demonstrate a novel function of ATF4 in AgRP neurons of the hypothalamus in energy balance and lipid metabolism and suggest hypothalamic ATF4 as a potential drug target for treating obesity and its related metabolic disorders.

Obesity is strongly associated with metabolic syndrome and predisposes to diseases including type 2 diabetes and liver steatosis (1,2). Changes in body weight normally result from an imbalance between energy intake and energy expenditure (2), controlled by the central nervous system, especially the hypothalamus (3). The center of this regulatory network is the arcuate nucleus (ARC) of the hypothalamus, which contains sets of important neurons devoted to metabolic regulation including orexigenic neurons that coproduce agouti-related peptide (AgRP) and neuropeptide Y, as well as anorexigenic neurons that contain cocaine- and amphetamine-regulated transcript and proopiomelanocortin (POMC)–derived peptides (3,4). AgRP neurons increase feeding by opposing the anorexigenic actions of POMC neurons, in part through the release of AgRP, a competitive inhibitor of melanocortin receptors (4). It also had an effect on energy expenditure via affecting sympathetic nervous system (SNS) activity or leptin sensitivity (5,6).

Activating transcription factor 4 (ATF4), also known as CREBP2, belongs to the CREBP families, characterized by the presence of a leucine zipper dimerization domain and a basic amino acid–rich DNA binding domain (7,8). ATF4 is ubiquitously expressed in many tissues and some parts of the brain, including the hypothalamus (8). It is involved in the regulation of various processes, including memory formation, osteoblast differentiation, amino acid deprivation, and redox homoeostasis (9). Recent studies (912) have demonstrated a role of ATF4 in the control of glucose and lipid metabolism. A role of ATF4 in specific neurons of the hypothalamus, however, has not been previously described. The aim of our current study was to investigate the role of ATF4 expressed in AgRP neurons in energy homeostasis regulation.

Generation of Mice With ATF4 Deletion in AgRP Neurons and Animal Treatment

All animals were on C57BL/6J background. ATF4-floxed mice (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 inducing Cre expression and avoiding the possible toxic effect of tamoxifen (15,16), both control and AgRP-ATF4 mice were intraperitoneally injected with 150 mg/kg body weight tamoxifen (Sigma-Aldrich, St. Louis, MO) for 5 days, between the ages of 5 and 7 weeks (14). The basal metabolic phenotypes in AgRP-ATF4 mice were analyzed by treating them and control mice with corn oil (Standard Food, Shanghai, People’s Republic of China) for 5 days. For high-fat diet (HFD) study, 4-week-old AgRP-ATF4 KO or control mice were 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 control mice a normal chow diet in the same amounts of food eaten by AgRP-ATF4 KO mice during the previous day. The efficiency for ATF4 deletion was evaluated by mating AI9 (tdTomato) reporter mice (18) with transgenic mice expressing Cre under control of the AgRP promoter after tamoxifen treatment. Body weight was monitored weekly throughout the experiments, and mice were kept as previously described (19). All the experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences.

Cell Culture and Treatments

Pshuttle vector–constructed plasmids expressing ATF4 or a dominant-negative form of ATF4 (DN-ATF4) was made based on plasmids described previously (19). The recombinant adenoviruses (Ads) expressing mouse ATF4 (Ad-ATF4) or control green fluorescent protein (Ad-GFP) were generated as previously described (19). Hypothalamic GT1-7 cells were maintained as described previously (20). Plasmids and Ads indicated were transfected into GT1-7 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA).

ARC Administration Experiments

ARC administration experiments were conducted as previously described (21). Ad-FOXO1 (22) was provided by Professor Youfei Guan (Dalian Medical University, Dalian, People’s Republic of China). Mice were anesthetized and received bilateral stereotaxic injections of Ad-GFP, Ad-Null, or Ad-FOXO1 (1 μL/5 × 109 plaque-forming units/side/mice) into ARC (−1.4 mm from bregma; ±0.3 mm from midline; −5.90 mm from dorsal surface), and metabolic phenotypes were examined 1–2 weeks after Ad-FOXO1 injection.

Cold Exposure Treatment

The 2- to 3-month-old mice were housed in individual precooled 4°C cages for 3 h, and rectal temperatures of mice were measured every 30 min during this period, as described previously (23). Body weight was measured immediately before the cold exposure.

Blood Glucose, Serum Insulin, Glucose Tolerance Tests, Insulin Tolerance Tests, and HOMA-Insulin Resistance Index

The measurements of blood glucose and serum insulin, results of glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs), and the calculation of the HOMA-insulin resistance (IR) index were conducted as described previously (19).

Leptin Sensitivity Assay In Vivo

Mice were intraperitoneally injected with either PBS or 3 mg/kg leptin (R&D Systems, Minneapolis, MN) at 9:00 a.m. after fasting for 24 h, as described previously (24). Food intake and body weight were measured at 1 and 4 h after the injection of leptin.

Metabolic Parameters Measurements

The body fat composition of mice was determined using the Bruker Minispec mq10 NMR Analyzer (Bruker, Billerica, MA). Indirect calorimetry was measured in a Comprehensive Lab Animal Monitoring System (Columbus Instruments, Columbus, OH), as described previously (11). Rectal temperatures were measured using a rectal probe attached to a digital thermometer (Physitemp Instruments, Clifton, NJ).

Serum and Liver Measurements

Serum and liver total glycerol, total cholesterol, and free fatty acid levels were determined using Glycerol Assay Kit Reagent (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). All of these assays were performed according to manufacturer instructions.

Protein and mRNA Analysis

Western blot analysis was performed with primary antibodies against actin (Sigma-Aldrich); ATF4, tribbles homolog 3 (TRB3), and uncoupling protein 1 (UCP1) (Santa Cruz Biotechnology, Santa Cruz, CA); and t–hormone-sensitive lipase (HSL), phosphorylated (p)-HSL, p-cAMP-dependent protein kinase (PKA), and FOXO1 (Cell Signaling Technology, Danvers, MA); and visualized by ECL Plus (GE Healthcare, Chicago, IL), as described previously (11). RT-PCR, with GAPDH as an internal control gene, was carried out as described previously (11). The sequences of primers used in the current study are available upon request.

Histological Analysis of White Adipose Tissue, Brown Adipose Tissue, and Liver

White adipose tissue (WAT), brown adipose tissue (BAT), and liver were fixed in 4% paraformaldehyde overnight and stained with hematoxylin-eosin (H-E), as well as Oil Red O staining with optimal cutting temperature embedding (25).

Immunofluorescence Staining

Immunofluorescence (IF) staining was performed with antibodies against p-STAT3 (Cell Signaling Technology), ATF4 (Santa Cruz Biotechnology), FOXO1 (Cell Signaling Technology), and c-fos (Santa Cruz Biotechnology), as described previously (24).

Luciferase Assay

pGL3-Foxo1 promoter (−1,137 to 5) (26) were provided by Professor Xiao Han (Nanjing Medical University, Nanjing, People’s Republic of China). GT1-7 cells were cotransfected with the internal control vector pRL Renilla (Promega, Madison, WI) and plasmids expressing ATF4 using Lipofectamine 2000. The Firefly and Renilla luciferase activities were assayed using Dual-Glo Luciferase Assay System (Promega).

Chromatin Immunoprecipitation Assays

Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer protocol (EMD Millipore, Billerica, MA) with anti-ATF4 antibodies (1:50; Santa Cruz Biotechnology) or normal rabbit IgG (1:50; Santa Cruz Biotechnology) for negative control. Immunoprecipitated FOXO1 promoter was quantified using PCR with primers designed to amplify the 220 base-pair region encompassing the cAMP-responsive element (CRE) site (forward, 5′-TCAATTCTAAAGCATCCTAGCC-3′; reverse, 5′-TGGGGCACAGCTCGTCTC-3′) or a 220 base-pair upstream region not involved in ATF4 response (forward, 5′-AACCTTTGTATTGGGGGCAT TGATTG-3′; reverse, 5′-CTGTTGCGATGAGAGCATTTGGTTA-3′).

Statistics

All results are expressed as the mean ± SEM. Significant differences were assessed by two-tailed Student t test, one-way ANOVA followed by the Student-Newman-Keuls test, or ANCOVA. For energy expenditure, respiratory exchange ratio (RER), and locomotor activity presented with lines, GTTs, and ITTs, a t test was used to compare the differences between different groups of mice at each time point examined. P < 0.05 was considered to be statistically significant.

Deletion of ATF4 in AgRP Neurons Promotes Fat Loss

In this study, we generated AgRP-ATF4 KO mice by tamoxifen treatment that allows temporal control of Cre recombinase activity and can be combined with flox mice to enable adult-onset deletion (14). IF staining of tdTomato (reflecting AgRP neurons) and ATF4 showed that ATF4 was colocalized with AgRP neurons in control mice but was absent in AgRP neurons of AgRP-ATF4 KO mice (Supplementary Fig. 1A and B). In addition, Atf4 mRNA levels were significantly decreased in ARC, but not other brain areas or tissues, of AgRP-ATF4 KO mice compared with control mice (Supplementary Fig. 1C). The body weight of male AgRP-ATF4 KO mice was lower than 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 subcutaneous WAT (sWAT), epididymal WAT (eWAT), BAT, and liver were observed in AgRP-ATF4 KO mice (Fig. 1C).

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 µm). E: Analysis of abdominal eWAT cell volume. F: DNA content of total abdominal eWAT. G: p-PKA substrate proteins, p-HSL, and HSL Western blot and densitometric quantification in eWAT. All studies were conducted in 12-week-old (or as indicated) male control or AgRP-ATF4 KO mice maintained on a normal chow diet. The data are expressed as the mean ± SEM (n = 5–7/group) and analyzed by two-tailed Student t test. *P < 0.05.

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 µm). E: Analysis of abdominal eWAT cell volume. F: DNA content of total abdominal eWAT. G: p-PKA substrate proteins, p-HSL, and HSL Western blot and densitometric quantification in eWAT. All studies were conducted in 12-week-old (or as indicated) male control or AgRP-ATF4 KO mice maintained on a normal chow diet. The data are expressed as the mean ± SEM (n = 5–7/group) and analyzed by two-tailed Student t test. *P < 0.05.

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Histological analysis of eWAT showed that the loss of ATF4 expression in AgRP neurons resulted in a 40% reduction in adipocyte volume, but had no effect on eWAT cell numbers as determined by DNA content analysis (Fig. 1D–F). The diminished adipocyte volume observed in AgRP-ATF4 KO mice suggested a possible enhanced lipolysis. Consistently, levels of p-HSL, the rate-limiting enzyme for triglyceride (TG) lipolysis, and substrate for PKA, the kinase that phosphorylates HSL (27), were significantly increased in eWAT of AgRP-ATF4 KO mice compared with control mice (Fig. 1G). Genes related to other lipid metabolism including lipogenesis, fatty acid oxidation and TG secretion (28,29), however, were not significantly affected in the WAT of AgRP-ATF4 KO mice (Supplementary Fig. 1D).

AgRP-ATF4 KO Mice Have Decreased Food Intake and Increased Energy Expenditure

To assess the possible reasons for the reduced fat mass in AgRP-ATF4 KO mice, we measured food intake and energy expenditure, the two aspects determining body fat mass (27). Daily food intake and feeding efficiency (30) were decreased in AgRP-ATF4 KO mice compared with control mice (Fig. 2A). Total energy expenditure (24-h O2 consumption) adjusted to lean mass (31,32) or calculated by ANCOVA (33) and heat generation were significantly increased and RER (VCO2/VO2) was decreased in AgRP-ATF4 KO mice during both the dark and light phases (Fig. 2B–D and Supplementary Fig. 1E). No difference in total physical activity was observed between AgRP-ATF4 KO mice and control mice (Fig. 2E). In contrast, rectal temperature was higher in AgRP-ATF4 KO mice (Fig. 2F). The higher body temperature observed in AgRP-ATF4 KO mice was most likely caused by increased thermogenesis, which is regulated by UCP1 in BAT (34). Consistently, BAT UCP1 expression was significantly elevated and the BAT cells were denser with fewer lipids in AgRP-ATF4 KO mice compared with control mice (Fig. 2G–I). BAT thermogenesis is activated by SNS with the release of NE (34). Not surprisingly, AgRP-ATF4 KO mice had higher serum NE levels (Fig. 2J). A pair-fed experiment (17) showed that AgRP-ATF4 KO mice had lower body weight, fat mass, lean mass, and tissue weight (liver and adipose tissue), and higher body temperature (Supplementary Fig. 2).

Figure 2

AgRP-ATF4 KO mice 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 rectal temperature (F). Gene expression (G) and Western blot and densitometric quantification (H) of UCP1 in BAT. I: H-E staining of representative BAT sections (scale bars, 250 µm). J: Serum NE levels. All studies were conducted in 10- to 12-week-old male control or AgRP-ATF4 KO mice maintained on a normal chow diet. The data are expressed as the mean ± SEM (n = 4–6/group) and analyzed by two-tailed Student t test in all panels except for C, or ANCOVA in C. *P < 0.05.

Figure 2

AgRP-ATF4 KO mice 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 rectal temperature (F). Gene expression (G) and Western blot and densitometric quantification (H) of UCP1 in BAT. I: H-E staining of representative BAT sections (scale bars, 250 µm). J: Serum NE levels. All studies were conducted in 10- to 12-week-old male control or AgRP-ATF4 KO mice maintained on a normal chow diet. The data are expressed as the mean ± SEM (n = 4–6/group) and analyzed by two-tailed Student t test in all panels except for C, or ANCOVA in C. *P < 0.05.

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AgRP-ATF4 KO Mice Exhibit Improved Insulin Sensitivity, Decreased Hepatic Lipid Accumulation, and Increased Leptin Sensitivity

We then investigated whether ablation of ATF4 in AgRP neurons had any impact on insulin sensitivity and liver steatosis, which is associated with a change in fat mass (1). Though levels of fed blood glucose and fed and fasting serum insulin were comparable between two genotypes, fasting blood glucose levels were decreased in AgRP-ATF4 KO mice (Supplementary Fig. 3A and B). Consistently, HOMA-IR was significantly reduced in AgRP-ATF4 KO mice (Supplementary Fig. 3C). GTTs and ITTs further revealed that AgRP-ATF4 KO mice had significantly increased glucose clearance and improved insulin sensitivity (Supplementary Fig. 3D). Possibly because of the reduced fat mass in AgRP-ATF4 KO mice, hepatic lipid accumulation was also slightly reduced in these mice as examined by Oil Red O and H-E staining (Supplementary Fig. 3E). Furthermore, hepatic and serum TG levels were also reduced, although total cholesterol and free fatty acids were unchanged, in AgRP-ATF4 KO mice (Supplementary Fig. 3F and G).

Leptin signaling in hypothalamus is a key regulator for energy homeostasis (35). To examine the effect of ATF4 deletion in AgRP neurons on leptin sensitivity, we intraperitoneally administered leptin (24) to AgRP-ATF4 KO and control mice and monitored the effects of leptin injection on changes in food intake and body weight. As shown previously (24), food intake and body weight were reduced after 1 or 4 h after leptin injection in control 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). Consistent with the stronger effect of leptin in AgRP-ATF4 KO mice, leptin produced more signals of p-STAT3, the marker of cellular leptin action (35), in ARC of hypothalamus of these mice (Supplementary Fig. 4C).

AgRP-ATF4 KO Mice Are Resistant to HFD-Induced Metabolic Disorders

To test whether AgRP-ATF4 mice may play a role under an HFD, we fed 4-week-old male AgRP-ATF4 KO mice and control mice an HFD for 16 weeks. Similar phenotypic changes were observed as those in mice maintained on a normal chow diet. AgRP-ATF4 KO mice showed thin appearance, decreased body weight, lean and fat mass, and weight of tissues, including liver, sWAT, eWAT, and BAT (Fig. 3A–C). Consistently, the size of adipocytes was much smaller in AgRP-ATF4 KO mice, associated with increased levels of p-PKA substrates and p-HSL in eWAT (Fig. 3D and E). Food intake was comparable between AgRP-ATF4 KO mice and control mice under HFD, whereas HFD-fed AgRP-ATF4 KO mice exhibited markedly increased oxygen consumption, adjusted to lean mass or calculated by ANCOVA (33), and rectal temperature, with decreased RER and no change in locomotor activity (Fig. 3F–K). Consistently, BAT UCP1 expression was significantly higher, cells were denser with fewer lipids, and serum NE levels were elevated in AgRP-ATF4 KO mice (Fig. 3L–O). In addition, the deletion of ATF4 in AgRP neurons protected mice from HFD-induced IR and liver steatosis (Supplementary Fig. 5).

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 lean mass (B) and tissue weights (liver, sWAT, eWAT, and BAT) (C). D: H-E staining of representative eWAT sections (scale bars, 250 µm). E: p-PKA substrate proteins, p-HSL, and HSL Western blot and densitometric quantification in eWAT. Daily food intake (F), 24-h oxygen consumption 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 µm). O: Serum NE levels. All studies were conducted in 16- to 20-week-old (or as indicated) male control or AgRP-ATF4 KO mice under an HFD (HFD feeding starting from age of 4 weeks). The data are expressed as the mean ± SEM (n = 8–10/group) and analyzed by two-tailed Student t test for all panels except for H (analyzed by ANCOVA in H). *P < 0.05.

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 lean mass (B) and tissue weights (liver, sWAT, eWAT, and BAT) (C). D: H-E staining of representative eWAT sections (scale bars, 250 µm). E: p-PKA substrate proteins, p-HSL, and HSL Western blot and densitometric quantification in eWAT. Daily food intake (F), 24-h oxygen consumption 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 µm). O: Serum NE levels. All studies were conducted in 16- to 20-week-old (or as indicated) male control or AgRP-ATF4 KO mice under an HFD (HFD feeding starting from age of 4 weeks). The data are expressed as the mean ± SEM (n = 8–10/group) and analyzed by two-tailed Student t test for all panels except for H (analyzed by ANCOVA in H). *P < 0.05.

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AgRP-ATF4 KO Mice Maintains at a Higher Body Temperature Under Cold Exposure

Based on the above results, we investigated whether AgRP-ATF4 KO mice have enhanced thermogenic response under cold exposure, a process that has been shown to induce thermogenesis (34). After exposure to an ambient temperature of 4°C, AgRP-ATF4 KO mice constantly maintained at a relatively higher core body temperature for the period examined, with significantly reduced weight and cell volume of sWAT and eWAT, compared with control mice (Fig. 4A–C). Although BAT weight remained unchanged, BAT was much denser with few lipid droplets in AgRP-ATF4 KO mice compared with control mice (Fig. 4B and D). UCP1 expression and serum NE levels were induced by cold exposure in BAT of control mice; however, the extent was much higher in AgRP-ATF4 KO mice (Fig. 4E–G).

Figure 4

AgRP-ATF4 KO mice maintain at a higher body temperature under cold exposure. A: Rectal temperature of mice. B: Tissue weights (sWAT, eWAT, BAT). C: H-E staining of representative eWAT and sWAT sections (scale bars, 250 µm). D: H-E staining of representative BAT sections (scale bars, 250 µm). Gene expression (E) and Western blot and densitometric quantification (F) of UCP1 in BAT. G: Serum NE levels. All studies were conducted in 10-week-old male control or AgRP-ATF4 KO mice maintained on a normal chow diet and exposed to a 4°C environment for 3 h. The data were expressed as the mean ± SEM (n = 4–6/group) and analyzed by two-tailed Student 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 group of 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 cold exposure in E–G.

Figure 4

AgRP-ATF4 KO mice maintain at a higher body temperature under cold exposure. A: Rectal temperature of mice. B: Tissue weights (sWAT, eWAT, BAT). C: H-E staining of representative eWAT and sWAT sections (scale bars, 250 µm). D: H-E staining of representative BAT sections (scale bars, 250 µm). Gene expression (E) and Western blot and densitometric quantification (F) of UCP1 in BAT. G: Serum NE levels. All studies were conducted in 10-week-old male control or AgRP-ATF4 KO mice maintained on a normal chow diet and exposed to a 4°C environment for 3 h. The data were expressed as the mean ± SEM (n = 4–6/group) and analyzed by two-tailed Student 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 group of 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 cold exposure in E–G.

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ATF4 Regulates Expression of FOXO1 via Direct Binding to the CRE Site on Its Promoter

It is shown that FOXO1 in AgRP neurons is critical for the maintenance of energy homeostasis (36), leading us to investigate the possible involvement of FOXO1 in mediating the effects of ATF4 deletion in AgRP neurons. Inhibition of ATF4 by a plasmid expressing DN-ATF4 (37), as evaluated by the increased ATF4 expression and decreased 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 overexpressed (Fig. 5C and D). ATF4 regulates the expression of downstream target genes via direct binding to CRE sites in their promoters (7). Similarly, ATF4 can bind to FOXO1 promoter as demonstrated by luciferase assays and ChIP assays in hypothalamic GT1-7 cells transfected with plasmid or adenovirus expressing ATF4 (Fig. 5E and F).

Figure 5

ATF4 regulates the expression of FOXO1 via direct binding to the CRE site in its promoter in vitro. A: Western blot and densitometric 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 and densitometric 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 was assessed in GT1-7 cells expressing the Foxo1 promoter vector with or without a plasmid expressing ATF4. F: ChIP assay was performed in GT1-7 cells infected with Ad-GFP or Ad-ATF4. NC, negative control. All studies were conducted in GT1-7 cells with at least three independent experiments. The data are expressed as the mean ± SEM and analyzed by two-tailed Student t test. *P < 0.05, for any treatment compared with control group.

Figure 5

ATF4 regulates the expression of FOXO1 via direct binding to the CRE site in its promoter in vitro. A: Western blot and densitometric 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 and densitometric 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 was assessed in GT1-7 cells expressing the Foxo1 promoter vector with or without a plasmid expressing ATF4. F: ChIP assay was performed in GT1-7 cells infected with Ad-GFP or Ad-ATF4. NC, negative control. All studies were conducted in GT1-7 cells with at least three independent experiments. The data are expressed as the mean ± SEM and analyzed by two-tailed Student t test. *P < 0.05, for any treatment compared with control group.

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Overexpression of FOXO1 Increases Fat Mass in AgRP-ATF4 KO Mice

As observed above in vitro, expression of Foxo1 was decreased in ARC of AgRP-ATF4 KO mice (Fig. 6A). To confirm a role for FOXO1 in mediating the effects of ATF4 deletion in AgRP neurons, we injected Ad-FOXO1 or control Ad-GFP to ARC of AgRP-ATF4 KO and control mice. As predicted, IF staining showed that FOXO1 expression was increased in both control and AgRP-ATF4 KO mice, most of which were localized at ARC (Supplementary Fig. 6A and B). Consistently, RT-PCR results showed that Foxo1 was overexpressed in ARC but not in other brain areas (Supplementary Fig. 6C). Although there was no significant difference in body weight and lean mass, Ad-FOXO1 increased fat mass and tissue weights, including sWAT and eWAT, and food intake; 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 activity, as measured by IF staining of c-fos, a marker used 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 (Supplementary Fig. 7).

Figure 6

Overexpression of FOXO1 increases fat mass in AgRP-ATF4 KO mice. A: Gene expression of Foxo1 in ARC. B: Body weight at the seventh day after virus injection. Fat and lean mass (C) and tissue weights (liver, sWAT, eWAT, and BAT) (D). E: H-E staining of representative eWAT sections (scale bars, 250 µm). F: Analysis of abdominal eWAT cell volume. G: P-PKA substrate proteins, p-HSL, and HSL Western blot and densitometric quantification in eWAT. Daily food intake (H) and rectal temperature (I). Gene expression (J) and Western blot and densitometric quantification (K) of UCP1 in BAT. L: H-E staining of representative BAT sections (scale bars, 250 µm). 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 male control or AgRP-ATF4 KO mice maintained on a normal chow diet. The data are expressed as the mean ± SEM (n = 6–7/group) and analyzed by one-way ANOVA followed by the Student-Newman-Keuls test. *P < 0.05 for the effects of any group of mice vs. control mice injected with Ad-GFP; #P < 0.05 for the effects of Ad-FOXO1 vs. Ad-GFP in AgRP-ATF4 KO mice.

Figure 6

Overexpression of FOXO1 increases fat mass in AgRP-ATF4 KO mice. A: Gene expression of Foxo1 in ARC. B: Body weight at the seventh day after virus injection. Fat and lean mass (C) and tissue weights (liver, sWAT, eWAT, and BAT) (D). E: H-E staining of representative eWAT sections (scale bars, 250 µm). F: Analysis of abdominal eWAT cell volume. G: P-PKA substrate proteins, p-HSL, and HSL Western blot and densitometric quantification in eWAT. Daily food intake (H) and rectal temperature (I). Gene expression (J) and Western blot and densitometric quantification (K) of UCP1 in BAT. L: H-E staining of representative BAT sections (scale bars, 250 µm). 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 male control or AgRP-ATF4 KO mice maintained on a normal chow diet. The data are expressed as the mean ± SEM (n = 6–7/group) and analyzed by one-way ANOVA followed by the Student-Newman-Keuls test. *P < 0.05 for the effects of any group of mice vs. control mice injected with Ad-GFP; #P < 0.05 for the effects of Ad-FOXO1 vs. Ad-GFP in AgRP-ATF4 KO mice.

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In this study, we used an AgRP-Cre-ER transgenic mouse line that allowed for spatiotemporal gene manipulation specifically in AgRP neurons after tamoxifen induction of Cre recombinase expression to avoid developmental issues or compensating actions (14,40), which may happen in AgRP-Cre mice (14). As expected, no difference in metabolic parameters examined was observed between the two phenotypes before tamoxifen treatment when administered with corn oil as the control vehicle (Supplementary Fig. 8). In contrast, after tamoxifen treatment, AgRP-ATF4 KO mice became lean and resistant to HFD-induced obesity, with improved insulin sensitivity and decreased lipid accumulation in liver. A pair-fed experiment showed that the decreased fat mass was mainly caused by increased energy expenditure in AgRP-ATF4 KO mice. Our current study for the first time demonstrated a novel function of ATF4 in hypothalamic AgRP neurons for systematic metabolic control. Our results also provided novel insights into understanding the signals in specific neurons that are critical for the regulation of energy homeostasis.

In this study, we found that the deletion of ATF4 in AgRP neurons also improves insulin sensitivity and reduces hepatic lipid accumulation in normal chow diet–fed mice and protects mice from HFD-induced IR and liver steatosis. We speculated that the above effects in AgRP-ATF4 KO mice may result from the decreased fat mass in these mice, as many studies (1,35,41) have demonstrated that a reduction in body weight helps to improve insulin sensitivity and decrease lipid accumulation in liver. On the other hand, it might be directly regulated by signals from hypothalamus (42), possibly via vagus nerve (19,42,43). Therefore, we could not exclude the possibility that ATF4 in AgRP neurons may have a direct effect on hepatic insulin sensitivity and lipid accumulation, particularly given the fact that hypothalamic ATF4 has a significant impact on regulating hepatic insulin sensitivity via the vagus nerve (19).

Leptin binds to its receptors expressed in the hypothalamus and regulates neural circuits that suppress food intake and increase energy expenditure (35,44). Here, we showed that the deletion of ATF4 in AgRP neurons improves leptin sensitivity, as demonstrated by the much more significant inhibitory effects of leptin on food intake and body weight, and p-STAT3 signaling in ARC of AgRP-ATF4 KO mice. Given the importance for leptin sensitivity in body weight control, we speculated that the increased leptin sensitivity may contribute to the decreased food intake and enhanced energy expenditure in AgRP-ATF4 KO mice. This possibility, however, must be studied in the future.

Because body fat mass is maintained by a balance between food intake and energy expenditure, we explored the possible reasons responsible for the decreased fat mass in AgRP-ATF4 KO mice from these two aspects. It is previously shown that global deletion of ATF4 had no effect on food intake (9,11). It is also reported that the activation of a certain signal in ARC is sufficient to inhibit food intake, associated with ATF4 overexpression (45). In contrast, food intake was reduced in AgRP-ATF4 KO mice. The difference in the effect of ATF4 deletion on food intake, however, might be due to the difference in the way of deleting ATF4 under each different circumstance.

In addition, AgRP-ATF4 KO mice had increased energy expenditure and body temperature, which reflect an increase in thermogenesis. BAT is of major importance in the regulation of thermogenesis and energy expenditure via affecting UCP1 expression (34). It is shown that UCP1 expression in BAT is induced by the activation of SNS, which stimulates the release of NE binding to β3-adrenergic receptor on the surface of BAT (2,34). The upregulation of UCP1 expression results in increased thermogenesis and energy expenditure, which helps in protection from fat accumulation (34). In this study, we found that AgRP-ATF4 KO mice exhibit increased body temperature, BAT UCP1 expression, and serum NE levels, suggesting increased thermogenesis. It is well known that fat mobilization is promoted by SNS activation, which stimulates the release of NE binding to β3-adrenergic receptor on the surface of adipocytes and sequentially phosphorylates PKA and HSL (34). Our results show that WAT cell volume was decreased in AgRP-ATF4 KO mice, suggesting increased lipolysis and altered nutrient partitioning that may contribute to the maintenance of the energy balance. The altered nutrient partitioning in AgRP-ATF4 KO mice could be due to the insufficient energy intake (27,34), which is needed to mobilize more fat to be used as an energy source. On the other hand, however, it might also be induced by activated SNS activity or increased insulin sensitivity (34). The increased thermogenesis in BAT and possibly the increased lipolysis in WAT should certainly contribute to the decreased fat mass in AgRP-ATF4 KO mice.

In this study, we also explored the possible role of ATF4 in AgRP neurons after treatment with an HFD or cold exposure. Thermogenesis is one of the most important adaptive changes in response to these environmental stimuli and functions to maintain metabolic homeostasis or to protect the organism from cold exposure (34). As observed in mice maintained on a control diet, we found that AgRP-ATF4 KO mice also exhibit enhanced thermogenesis, resulting in the resistance to HFD-induced obesity, IR, and liver steatosis, and a higher body temperature after cold exposure. Interestingly, food intake was comparable between HFD-fed control and AgRP-ATF4 KO mice, which is different from the observed decreased food intake in these mice maintained on a control diet. Although it is shown AgRP neurons are important for energy intake control, however, when palatable food is provided, AgRP neurons are dispensable for an appropriate feeding response (46). These results suggest that AgRP neurons are indispensible in the feeding response under a control chow diet, but not under HFD, which might explain the difference in food intake between AgRP-ATF4 KO and control mice maintained on a normal chow diet or HFD.

Several signaling pathways in AgRP neurons have been identified to be important regulators of energy homeostasis (20,4749). In this study, we found that the effects of ATF4 deletion in AgRP neurons are mediated by inhibited expression of FOXO1, based on the effects of FOXO1 overexpression in ARC on increasing fat mass in AgRP-ATF4 KO mice. However, body weight was not affected by FOXO1 overexpression, possibly because of the simultaneously decreased tendency of lean mass. A previous study (36) has shown that FOXO1 regulates energy homeostasis via affecting AgRP neuronal activity and Agrp expression. Consistently, we found neuronal activity as examined by IF staining of c-fos, a marker reflecting neuronal activity (39), and Agrp expression are changed in the ARC of AgRP-ATF4 KO mice after overexpression of FOXO1, indicating that similar mechanisms may mediate FOXO1 regulation of energy homeostasis in AgRP-ATF4 KO mice. Thus, our study demonstrates an important role for FOXO1 as a downstream target for ATF4. In addition, we identified a direct effect of ATF4 on the regulation of FOXO1 expression. In contrast to our observation, it is shown that FOXO1 expression is not affected by ATF4 in osteoblast cells (50). The difference might be caused by tissue-specific regulatory mechanisms, which require further investigation. The possible influence from FOXO1 expressed in other neurons by ARC injection and the involvement of other pathways in ATF4 regulation of energy homeostasis, however, needs to be studied in the future.

Taken together, these results demonstrate a critical role for ATF4 in hypothalamic AgRP neurons in the regulation of energy homeostasis, and lipid and glucose metabolism mainly by the increased energy expenditure via affecting FOXO1 expression. These results also suggest hypothalamic ATF4 as a potential novel drug target in treating obesity 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 Science Foundation (81130076, 81325005, 31271269, 81300659, 81400792, 81471076, 81570777, 81500622, and 81390350), Basic Research Project of Shanghai Science and Technology Commission (16JC1404900), and International S&T Cooperation Program of China (Singapore 2014DFG32470) and by the international Partnership Program For Creative Research Teams (CAS/SAFEA). F.G. was also supported by the One Hundred Talents Program of the Chinese Academy of Sciences.

Duality of Interest. No potential conflicts of interest relevant to this article were 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. and Y.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 to discussion, and wrote, reviewed, and edited the manuscript. F.G. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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