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ATF7 ablation prevents diet-induced obesity and insulin resistance
Biochemical and Biophysical Research Communications xxx (2016) 1e7
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ATF7 ablation prevents diet-induced obesity and insulin resistance Yang Liu a, b, Toshio Maekawa a, Keisuke Yoshida a, Tamio Furuse c, Hideki Kaneda c, Shigeharu Wakana c, Shunsuke Ishii a, b, * a
Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan Department of Molecular Genetics and Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan c Technology and Development Team for Mouse Phenotype Analysis, RIKEN BRC, Tsukuba, Ibaraki, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2016 Accepted 2 August 2016 Available online xxx
The activating transcription factor (ATF)2 family of transcription factors regulates a variety of metabolic processes, including adipogenesis and adaptive thermogenesis. ATF7 is a member of the ATF2 family, and mediates epigenetic changes induced by environmental stresses, such as social isolation and pathogen infection. However, the metabolic role of ATF7 remains unknown. The aim of the present study is to examine the role of ATF7 in metabolism using ATF7-dificeint mice. Atf7/ mice exhibited lower body weight and resisted diet-induced obesity. Serum triglycerides, resistin, and adipose tissue mass were all significantly lower in ATF7-deficient mice. Fasting glucose levels and glucose tolerance were unaltered, but systemic insulin sensitivity was increased, by ablation of ATF7. Indirect calorimetry revealed that oxygen consumption by Atf7/ mice was comparable to that of wild-type littermates on a standard chow diet, but increased energy expenditure was observed in Atf7/ mice on a high-fat diet. Hence, ATF7 ablation may impair the development and function of adipose tissue and result in elevated energy expenditure in response to high-fat-feeding obesity and insulin resistance, indicating that ATF7 is a potential therapeutic target for diet-induced obesity and insulin resistance. © 2016 Published by Elsevier Inc.
Keywords: ATF7 Metabolism Obesity Insulin resistance
1. Introduction The growing epidemic of obesity poses serious health risks due to the development of obesity-associated diseases including type 2 diabetes, hypertension, heart diseases, and cancer [1]. Overconsumption of calorie-dense foods and a sedentary lifestyle are thought to be the two most important factors responsible for the steep increase in the prevalence of obesity worldwide [2,3]. When energy intake consistently exceeds energy expenditure, excess calories are stored as triglycerides in white adipose tissue (WAT). By contrast, brown adipose tissue (BAT) and so-called beige/brite cells are responsible for the dissipation of energy as heat by adaptive thermogenesis [4,5]. The activating transcription factor (ATF)2 family of transcription factors is part of the ATF/cAMP response element-binding protein (CRE-BP) superfamily and consists of three members: ATF2 (originally named CRE-BP1) [6,7], CRE-BPa [8], and ATF7 (originally
Abbreviation: ATF, activating transcription factor. * Corresponding author. 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. E-mail address: [email protected] (S. Ishii).
named ATF-a) [9]. Each of these three proteins contains stressactivated protein kinase (SAPK) phosphorylation sites, which are targets of SAPKs, such as c-jun n-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) [10]. We previously showed that Atf2þ/Cre-bpaþ/ double heterozygous mice exhibited reduced WAT mass and improved insulin sensitivity. ATF2 is required for white adipocyte differentiation via p38-dependent induction of peroxisome proliferator-activated receptor (PPAR)g2 [11], which is a key transcription factor mediating adipocyte differentiation [12]. The p38-dependent phosphorylation of ATF2 is also involved in increasing energy expenditure. In response to cold exposure, ATF2 directly activates transcription of the UCP1 gene. This gene encodes uncoupling protein 1 [13], which mediates non-shivering thermogenesis in BAT [14]. In addition, ATF2 stimulates transcription of PPARg coactivator-1a (PGC-1a) and zinc finger protein 516 (Zfp516), both of which also activate UCP1 transcription [13,15]. When ATF2 is phosphorylated by SAPKs in response to various stimuli and stresses, it binds to its coactivator CREB-binding protein (CBP)and activates transcription [16]. Although ATF7 has a similar structure to ATF2, it represses rather than activates gene
http://dx.doi.org/10.1016/j.bbrc.2016.08.009 0006-291X/© 2016 Published by Elsevier Inc.
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transcription in the absence of stress [17]. ATF7 silences gene transcription by recruiting the histone H3K9 trimethyltransferase ESET and the histone H3K9 dimethyltransferase G9a to promote the formation of heterochromatin-like structures [18,19]. Social isolation stress stimulates p38-mediated phosphorylation of ATF7 and therefore the release of ATF7 and ESET from the promoter of the serotonin receptor 5b gene (Htr5b), leading to the up-regulation of Htr5b in the dorsal raphe nucleus of mouse brain [18]. In macrophages, ATF7 silences a group of innate immunity genes by recruiting G9a. Pathogen infection-induced phosphorylation of ATF7 leads to a decrease in repressive histone H3K9me2 levels and elevated gene expression [19]. Thus, ATF7 acts as a silencer of transcription in the absence of stress or other stimuli, an effect that is cancelled when such stimuli are present. However, the metabolic role of ATF7 has not been characterized. In this study, we show that ablation of ATF7 protects mice against diet-induced obesity and improves systemic insulin sensitivity. Atf7/ mice have lower adipose tissue mass and serum resistin levels and, when challenged with a high-fat diet (HFD), they display increased energy expenditure. Our data suggest that ATF7 contributes to diet-induced obesity and insulin resistance and therefore constitutes a possible therapeutic target for obesity and type 2 diabetes.
previously [18]. The Atf7/ and wild-type (WT) littermates used in this study were derived from Atf7 heterozygotes on a C57BL/6 background by in vitro fertilization. From 6 weeks old, male mice were fed a standard chow diet (CD) or a HFD (60% of kcal from fat). At 5 months old, body fat percentage was measured by dual-energy X-ray absorptiometry (DEXA) analysis using a Lunar PIXImus densitometer. Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of the RIKEN Institute. 2.2. Plasma parameters Serum glucose, triglycerides, and cholesterol concentrations were measured using an automatic clinical biochemistry analyzer (JCA-BM6070, JEOL). Serum insulin and resistin levels were measured using the Bio-plex system (Bio-Rad Laboratories). 2.3. Histological analysis Epididymal (e)WAT was isolated from 3-month-old mice, and specimens were fixed in 4% paraformaldehyde. Paraffin sections were stained with hematoxylin and eosin (H&E). 2.4. Glucose and insulin tolerance tests
2. Materials and methods 2.1. Mice ATF7-deficient (Atf7/) mice were generated as described
At 14 weeks of age, glucose tolerance testing was performed after fasting for 16 h. Glucose solution (2 g/kg body weight) was injected intraperitoneally into mice. Blood glucose levels were measured before (0 min) and 15, 30, 60, and 120 min after glucose
Fig. 1. ATF7-deficient mice exhibited resistance to obesity. (A) A representative photograph of 5-month-old wild-type (WT, left) and Atf7/ (right) mice. (B) Body weight of Atf7/ and WT littermates fed on a standard chow diet (CD) or a high-fat diet (HFD) (n ¼ 8e9 for each group). (C) Serum cholesterol levels in WT and Atf7/ mice (n ¼ 4e7 for each group). (D) Serum triglycerides levels in WT and Atf7/ mice (n ¼ 4e7 for each group). *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.
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injection, using a Glucocard hand-held analyzer (Arkray, Inc). Insulin tolerance testing was performed after a 4 h fast; mice were injected intraperitoneally with recombinant human insulin (Eli Lilly, 0.75 IU/kg of body mass), and then blood glucose was measured before (0 min) and 30, 60, and 90 min after insulin injection.
ameliorates diet-induced obesity. We measured a variety of metabolic parameters in the blood of 11- and 18-week-old mice. There was no difference in serum cholesterol levels between Atf7/ and WT control mice (Fig. 1C), whereas Atf7/ mice displayed reduced serum triglycerides, particularly in 18-week-old mice (~50% lower than WT) (Fig. 1D).
2.5. Energy expenditure
3.2. Effects of ATF7 deficiency on adipose tissue
Energy expenditure was evaluated using indirect calorimetry, by measuring oxygen consumption as described previously [11]. Briefly, 12-week-old mice were placed in calorimetric chambers for 48 h. After acclimation of mice to the chambers for 24 h, the volumes of O2 consumed and CO2 produced were recorded for ~21 h.
We examined the body composition of the mice using DEXA. Consistent with the reduced body mass of Atf7/ mice, the percentage body fat of Atf7/ mice was lower than that of WT littermates when fed either a CD or a HFD (Fig. 2A). There was no difference in liver mass relative to total body mass between Atf7/ and WT littermates (Fig. 2B). However, the masses of adipose tissue depots, including eWAT, inguinal (i)WAT, and BAT, were significantly decreased in Atf7/ mice (Fig. 2B). Histological analysis revealed that Atf7/ mice possessed smaller adipocytes than WT mice (Fig. 2C), suggesting that the reduction in adipose tissue mass may have been caused by impaired adipocyte differentiation. Because adipose tissue is an important endocrine organ [20], we measured serum adipocytokine levels and found that there was on significant difference in serum leptin and adiponectin between Atf7/ and WT littermates (data not shown). By contrast, serum resistin was reduced by ~50% in Atf7/ mice (Fig. 2D).
2.6. Statistical analysis Data are presented as mean ± SEM, and the numbers of mice used for each procedure were as indicated below. Statistical significance was evaluated using Student's t-test (two-tailed) or ANOVA. P < 0.05 was considered statistically significant. 3. Results 3.1. Atf7/ mice show lower body mass Atf7/ mice appeared leaner than their WT littermates (Fig. 1A); therefore, the body masses of Atf7/ and WT mice fed with a CD and HFD were compared. Atf7/ mice on a CD had a significantly lower body mass compared to WT littermates (Fig. 1B). When challenged with a HFD, Atf7/ mice gained less mass relative to WT littermates (Fig. 1B), suggesting that deficiency in ATF7
3.3. Atf7/ mice demonstrate increased insulin sensitivity Atf7/ mice exhibited a tendency for fasting serum insulin levels to be reduced (P ¼ 0.08), although similar levels of fasting blood glucose were observed in WT and Atf7/ mice on both diets (Fig. 3A, B). Blood glucose increased to comparable levels during
Fig. 2. Reduction in adipose tissue mass in Atf7/ mice. (A) Body fat percentage in 5-month-old WT and Atf7/ mice fed on a CD or a HFD (n ¼ 8e9 for each group). (B) Masses of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), and liver relative to total body mass (n ¼ 6 for each group). (C) Histological analysis of eWAT using H&E staining. Bar ¼ 50 mm. (D) Serum resistin levels in 4-month-old WT and Atf7/ mice (n ¼ 4e7 for each group). *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.
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Fig. 3. ATF7 deficiency improved insulin sensitivity. (A) Serum insulin levels in 4-month-old WT and Atf7/ mice (n ¼ 4e7 for each group). (B) Serum fasting glucose levels in 4month-old WT and Atf7/ mice (n ¼ 4e7 for each group). (C) Glucose tolerance test in WT and Atf7/ mice on a CD and (D) HFD (n ¼ 6 for each group). (E) Insulin tolerance test in WT and Atf7/ mice on a CD and (F) HFD (n ¼ 6 for each group). *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.
glucose tolerance testing of WT and Atf7/ mice on a CD (Fig. 3C) and after mice had been fed a HFD for 5 months (Fig. 3D). However, blood glucose was significantly more suppressed during insulin tolerance testing in Atf7/ mice than in WT mice on a CD (Fig. 3E). Since long-term HFD feeding can induce the development of insulin resistance [21], we also assessed the systemic insulin sensitivity of mice on a HFD. Consistent with the effect of genotype on mice fed a CD, ablation of ATF7 ameliorated HFD-induced resistance, indicated by improved suppression of blood glucose levels during an insulin tolerance test (Fig. 3F). 3.4. Atf7/ mice on a HFD demonstrate increased energy expenditure
adipocyte size were reduced in Atf7/ mice as described above. Therefore, we postulated that ATF7 might also participate in the regulation of energy metabolism. We measured food intake and found that it was not different between Atf7/ mice and their WT littermates (Fig. 4A). Next, we determined energy expenditure by measuring oxygen consumption at room temperature and found that Atf7/ and WT mice on a CD displayed similar levels of energy expenditure (Fig. 4B, C). However, higher oxygen consumption was observed in Atf7/ mice on a HFD during both phases of the circadian cycle (Fig. 4D, E). This implies that the resistance of Atf7/ mice to diet-induced obesity may be due partially to increased energy expenditure. 4. Discussion
ATF2 mediates adaptive thermogenesis by modulating transcription of thermogenic genes [13,15]. Furthermore, fat mass and
In the present study, we showed that the absence of the
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Fig. 4. Higher energy expenditure in Atf7/ mice on a HFD. (A) Food intake of 12-week-old WT and Atf7/ mice on a CD or a HFD (n ¼ 5 for each group). (B, C) Oxygen consumption in 12-week-old WT and Atf7/ mice on a CD (n ¼ 7 for each group). (D, E) Oxygen consumption in 12-week-old WT and Atf7/ mice on a HFD (n ¼ 7 for each group). **, P < 0.01, ***, P < 0.001, NS, not significant.
transcription factor ATF7 in mice results in resistance to dietinduced obesity and reduced serum triglycerides. Atf7/ mice exhibited lower fat mass and smaller adipocytes, suggesting that ATF7 may be involved in the regulation of adipose tissue differentiation. Lower level of plasma resistin can reverse HFD-induced hepatic insulin resistance [22]. Serum levels of resistin were 50% lower in Atf7/ mice than in their WT littermates, indicating that ATF7 ablation may also alter the endocrine function of adipose tissue, which may be associated with the improved systemic insulin sensitivity in Atf7/ mice. ATF2 and ATF7 are thought to bind to the same target DNA sequences. Furthermore, both ATF2 and ATF7 were recently identified as proteins that interact with CCAAT/enhancer-binding protein beta or zinc finger E-box-binding homeobox 1, both of which are essential transcriptional factors during the early phase of
adipogenesis [23,24]. ATF2 is required for WAT differentiation [11], while ATF2 and ATF7 have opposite effects on transcription of target genes, activating and silencing transcription, respectively [16e19]. Based on this, we speculated that loss of ATF7 might enhance the differentiation of adipose tissue, but in fact Atf7/ mice exhibited lower adipose tissue mass. Thus, the molecular mechanisms whereby ATF7 regulates adipocyte differentiation may be more complex than predicted. The presence of obesity is closely associated with the development of insulin resistance [21,25]. Thus, we wondered whether the reduced body weight and fat mass of Atf7/ mice would be associated with improved systemic insulin sensitivity in mice fed either a RD or a HFD. In fact, ATF deficiency did not alter glucose tolerance and there was no difference in serum glucose concentration between Atf7/ and WT mice, although this may be due to the
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decreased serum insulin observed in Atf7/ mice. Of relevance, ATF2 cooperates with other transcription factors, including MafA, to increase transcription of the insulin gene in b-cells [26]. If, conversely, ATF7 reduces insulin gene expression, Atf7/ mice should exhibit a higher serum insulin. Thus, the molecular mechanism underlying the observed decrease in serum insulin in Atf7/ mice may not be simple. The development of obesity is tightly linked to the dysregulation of energy balance [27], whereby an imbalance between food intake and energy expenditure leads to the development of obesity and insulin resistance. There were no differences in food intake and energy expenditure between WT and Atf7/ mice on a RD, indicating that the lean phenotype of the Atf7/ mice is not due to an imbalance between food intake and energy expenditure. However, Atf7/ mice fed a HFD showed higher energy expenditure during both light and dark phases, implying that ATF7 may contribute to the regulation of adaptive thermogenesis. Since ATF2 is involved in increasing energy expenditure by both directly and indirectly activating transcription of the UCP1 gene [13,15], loss of the repressor ATF7 may also enhance UCP1 transcription and thus energy expenditure. One reason why increased energy expenditure was observed when Atf7/ mice were fed a HFD, but not a RD, could be altered production of reactive oxygen species (ROS), which are generated as a byproduct of oxidative phosphorylation in mitochondria [28]. Increased production of ROS in mice fed a HFD results in p38 activation [29], and thus may induce higher levels of ATF7 phosphorylation and the release of ATF7 from the UCP1 promoter, resulting in increased UCP1 transcription. Alternatively, loss of ATF7 could contribute to enhanced energy expenditure via altered macrophage activity. Upon cold exposure, expression of thermogenic genes, including UCP1, is induced in iWAT, leading to “browning” of the adipocytes [30]. However, cold exposure can also stimulate muscle and adipose tissue to release factors that recruit macrophages to the iWAT, also resulting in browning [31,32]. We previously reported that ATF7 could regulate macrophage function via the p38 signaling pathway [19]; thus ATF7 may regulate energy expenditure by promoting macrophagedependent browning. In conclusion, our data show that ATF7 deficiency protects mice against diet-induced obesity by inhibiting adipose tissue differentiation and increasing energy expenditure. This suggests that ATF7 may play an important role in the regulation of energy balance and provides a potential target for novel treatments of obesity.
Conflict of interest The authors declare no conflict of interest.
Acknowledgements We thank the members of the Experimental Animal Division of RIKEN Tsukuba Institute for maintenance of the mice. This work was supported in part by AMED (Japan Agency for Medical Research and Development) (16gm0510015h0004), and by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (Grant No. 16H01413).
Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.08.009.
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ATF7 ablation prevents diet-induced obesity and insulin resistance Yang Liu a, b, Toshio Maekawa a, Keisuke Yoshida a, Tamio Furuse c, Hideki Kaneda c, Shigeharu Wakana c, Shunsuke Ishii a, b, * a
Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan Department of Molecular Genetics and Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan c Technology and Development Team for Mouse Phenotype Analysis, RIKEN BRC, Tsukuba, Ibaraki, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2016 Accepted 2 August 2016 Available online xxx
The activating transcription factor (ATF)2 family of transcription factors regulates a variety of metabolic processes, including adipogenesis and adaptive thermogenesis. ATF7 is a member of the ATF2 family, and mediates epigenetic changes induced by environmental stresses, such as social isolation and pathogen infection. However, the metabolic role of ATF7 remains unknown. The aim of the present study is to examine the role of ATF7 in metabolism using ATF7-dificeint mice. Atf7/ mice exhibited lower body weight and resisted diet-induced obesity. Serum triglycerides, resistin, and adipose tissue mass were all significantly lower in ATF7-deficient mice. Fasting glucose levels and glucose tolerance were unaltered, but systemic insulin sensitivity was increased, by ablation of ATF7. Indirect calorimetry revealed that oxygen consumption by Atf7/ mice was comparable to that of wild-type littermates on a standard chow diet, but increased energy expenditure was observed in Atf7/ mice on a high-fat diet. Hence, ATF7 ablation may impair the development and function of adipose tissue and result in elevated energy expenditure in response to high-fat-feeding obesity and insulin resistance, indicating that ATF7 is a potential therapeutic target for diet-induced obesity and insulin resistance. © 2016 Published by Elsevier Inc.
Keywords: ATF7 Metabolism Obesity Insulin resistance
1. Introduction The growing epidemic of obesity poses serious health risks due to the development of obesity-associated diseases including type 2 diabetes, hypertension, heart diseases, and cancer [1]. Overconsumption of calorie-dense foods and a sedentary lifestyle are thought to be the two most important factors responsible for the steep increase in the prevalence of obesity worldwide [2,3]. When energy intake consistently exceeds energy expenditure, excess calories are stored as triglycerides in white adipose tissue (WAT). By contrast, brown adipose tissue (BAT) and so-called beige/brite cells are responsible for the dissipation of energy as heat by adaptive thermogenesis [4,5]. The activating transcription factor (ATF)2 family of transcription factors is part of the ATF/cAMP response element-binding protein (CRE-BP) superfamily and consists of three members: ATF2 (originally named CRE-BP1) [6,7], CRE-BPa [8], and ATF7 (originally
Abbreviation: ATF, activating transcription factor. * Corresponding author. 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. E-mail address: [email protected] (S. Ishii).
named ATF-a) [9]. Each of these three proteins contains stressactivated protein kinase (SAPK) phosphorylation sites, which are targets of SAPKs, such as c-jun n-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) [10]. We previously showed that Atf2þ/Cre-bpaþ/ double heterozygous mice exhibited reduced WAT mass and improved insulin sensitivity. ATF2 is required for white adipocyte differentiation via p38-dependent induction of peroxisome proliferator-activated receptor (PPAR)g2 [11], which is a key transcription factor mediating adipocyte differentiation [12]. The p38-dependent phosphorylation of ATF2 is also involved in increasing energy expenditure. In response to cold exposure, ATF2 directly activates transcription of the UCP1 gene. This gene encodes uncoupling protein 1 [13], which mediates non-shivering thermogenesis in BAT [14]. In addition, ATF2 stimulates transcription of PPARg coactivator-1a (PGC-1a) and zinc finger protein 516 (Zfp516), both of which also activate UCP1 transcription [13,15]. When ATF2 is phosphorylated by SAPKs in response to various stimuli and stresses, it binds to its coactivator CREB-binding protein (CBP)and activates transcription [16]. Although ATF7 has a similar structure to ATF2, it represses rather than activates gene
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transcription in the absence of stress [17]. ATF7 silences gene transcription by recruiting the histone H3K9 trimethyltransferase ESET and the histone H3K9 dimethyltransferase G9a to promote the formation of heterochromatin-like structures [18,19]. Social isolation stress stimulates p38-mediated phosphorylation of ATF7 and therefore the release of ATF7 and ESET from the promoter of the serotonin receptor 5b gene (Htr5b), leading to the up-regulation of Htr5b in the dorsal raphe nucleus of mouse brain [18]. In macrophages, ATF7 silences a group of innate immunity genes by recruiting G9a. Pathogen infection-induced phosphorylation of ATF7 leads to a decrease in repressive histone H3K9me2 levels and elevated gene expression [19]. Thus, ATF7 acts as a silencer of transcription in the absence of stress or other stimuli, an effect that is cancelled when such stimuli are present. However, the metabolic role of ATF7 has not been characterized. In this study, we show that ablation of ATF7 protects mice against diet-induced obesity and improves systemic insulin sensitivity. Atf7/ mice have lower adipose tissue mass and serum resistin levels and, when challenged with a high-fat diet (HFD), they display increased energy expenditure. Our data suggest that ATF7 contributes to diet-induced obesity and insulin resistance and therefore constitutes a possible therapeutic target for obesity and type 2 diabetes.
previously [18]. The Atf7/ and wild-type (WT) littermates used in this study were derived from Atf7 heterozygotes on a C57BL/6 background by in vitro fertilization. From 6 weeks old, male mice were fed a standard chow diet (CD) or a HFD (60% of kcal from fat). At 5 months old, body fat percentage was measured by dual-energy X-ray absorptiometry (DEXA) analysis using a Lunar PIXImus densitometer. Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of the RIKEN Institute. 2.2. Plasma parameters Serum glucose, triglycerides, and cholesterol concentrations were measured using an automatic clinical biochemistry analyzer (JCA-BM6070, JEOL). Serum insulin and resistin levels were measured using the Bio-plex system (Bio-Rad Laboratories). 2.3. Histological analysis Epididymal (e)WAT was isolated from 3-month-old mice, and specimens were fixed in 4% paraformaldehyde. Paraffin sections were stained with hematoxylin and eosin (H&E). 2.4. Glucose and insulin tolerance tests
2. Materials and methods 2.1. Mice ATF7-deficient (Atf7/) mice were generated as described
At 14 weeks of age, glucose tolerance testing was performed after fasting for 16 h. Glucose solution (2 g/kg body weight) was injected intraperitoneally into mice. Blood glucose levels were measured before (0 min) and 15, 30, 60, and 120 min after glucose
Fig. 1. ATF7-deficient mice exhibited resistance to obesity. (A) A representative photograph of 5-month-old wild-type (WT, left) and Atf7/ (right) mice. (B) Body weight of Atf7/ and WT littermates fed on a standard chow diet (CD) or a high-fat diet (HFD) (n ¼ 8e9 for each group). (C) Serum cholesterol levels in WT and Atf7/ mice (n ¼ 4e7 for each group). (D) Serum triglycerides levels in WT and Atf7/ mice (n ¼ 4e7 for each group). *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.
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injection, using a Glucocard hand-held analyzer (Arkray, Inc). Insulin tolerance testing was performed after a 4 h fast; mice were injected intraperitoneally with recombinant human insulin (Eli Lilly, 0.75 IU/kg of body mass), and then blood glucose was measured before (0 min) and 30, 60, and 90 min after insulin injection.
ameliorates diet-induced obesity. We measured a variety of metabolic parameters in the blood of 11- and 18-week-old mice. There was no difference in serum cholesterol levels between Atf7/ and WT control mice (Fig. 1C), whereas Atf7/ mice displayed reduced serum triglycerides, particularly in 18-week-old mice (~50% lower than WT) (Fig. 1D).
2.5. Energy expenditure
3.2. Effects of ATF7 deficiency on adipose tissue
Energy expenditure was evaluated using indirect calorimetry, by measuring oxygen consumption as described previously [11]. Briefly, 12-week-old mice were placed in calorimetric chambers for 48 h. After acclimation of mice to the chambers for 24 h, the volumes of O2 consumed and CO2 produced were recorded for ~21 h.
We examined the body composition of the mice using DEXA. Consistent with the reduced body mass of Atf7/ mice, the percentage body fat of Atf7/ mice was lower than that of WT littermates when fed either a CD or a HFD (Fig. 2A). There was no difference in liver mass relative to total body mass between Atf7/ and WT littermates (Fig. 2B). However, the masses of adipose tissue depots, including eWAT, inguinal (i)WAT, and BAT, were significantly decreased in Atf7/ mice (Fig. 2B). Histological analysis revealed that Atf7/ mice possessed smaller adipocytes than WT mice (Fig. 2C), suggesting that the reduction in adipose tissue mass may have been caused by impaired adipocyte differentiation. Because adipose tissue is an important endocrine organ [20], we measured serum adipocytokine levels and found that there was on significant difference in serum leptin and adiponectin between Atf7/ and WT littermates (data not shown). By contrast, serum resistin was reduced by ~50% in Atf7/ mice (Fig. 2D).
2.6. Statistical analysis Data are presented as mean ± SEM, and the numbers of mice used for each procedure were as indicated below. Statistical significance was evaluated using Student's t-test (two-tailed) or ANOVA. P < 0.05 was considered statistically significant. 3. Results 3.1. Atf7/ mice show lower body mass Atf7/ mice appeared leaner than their WT littermates (Fig. 1A); therefore, the body masses of Atf7/ and WT mice fed with a CD and HFD were compared. Atf7/ mice on a CD had a significantly lower body mass compared to WT littermates (Fig. 1B). When challenged with a HFD, Atf7/ mice gained less mass relative to WT littermates (Fig. 1B), suggesting that deficiency in ATF7
3.3. Atf7/ mice demonstrate increased insulin sensitivity Atf7/ mice exhibited a tendency for fasting serum insulin levels to be reduced (P ¼ 0.08), although similar levels of fasting blood glucose were observed in WT and Atf7/ mice on both diets (Fig. 3A, B). Blood glucose increased to comparable levels during
Fig. 2. Reduction in adipose tissue mass in Atf7/ mice. (A) Body fat percentage in 5-month-old WT and Atf7/ mice fed on a CD or a HFD (n ¼ 8e9 for each group). (B) Masses of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), and liver relative to total body mass (n ¼ 6 for each group). (C) Histological analysis of eWAT using H&E staining. Bar ¼ 50 mm. (D) Serum resistin levels in 4-month-old WT and Atf7/ mice (n ¼ 4e7 for each group). *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.
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Fig. 3. ATF7 deficiency improved insulin sensitivity. (A) Serum insulin levels in 4-month-old WT and Atf7/ mice (n ¼ 4e7 for each group). (B) Serum fasting glucose levels in 4month-old WT and Atf7/ mice (n ¼ 4e7 for each group). (C) Glucose tolerance test in WT and Atf7/ mice on a CD and (D) HFD (n ¼ 6 for each group). (E) Insulin tolerance test in WT and Atf7/ mice on a CD and (F) HFD (n ¼ 6 for each group). *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.
glucose tolerance testing of WT and Atf7/ mice on a CD (Fig. 3C) and after mice had been fed a HFD for 5 months (Fig. 3D). However, blood glucose was significantly more suppressed during insulin tolerance testing in Atf7/ mice than in WT mice on a CD (Fig. 3E). Since long-term HFD feeding can induce the development of insulin resistance [21], we also assessed the systemic insulin sensitivity of mice on a HFD. Consistent with the effect of genotype on mice fed a CD, ablation of ATF7 ameliorated HFD-induced resistance, indicated by improved suppression of blood glucose levels during an insulin tolerance test (Fig. 3F). 3.4. Atf7/ mice on a HFD demonstrate increased energy expenditure
adipocyte size were reduced in Atf7/ mice as described above. Therefore, we postulated that ATF7 might also participate in the regulation of energy metabolism. We measured food intake and found that it was not different between Atf7/ mice and their WT littermates (Fig. 4A). Next, we determined energy expenditure by measuring oxygen consumption at room temperature and found that Atf7/ and WT mice on a CD displayed similar levels of energy expenditure (Fig. 4B, C). However, higher oxygen consumption was observed in Atf7/ mice on a HFD during both phases of the circadian cycle (Fig. 4D, E). This implies that the resistance of Atf7/ mice to diet-induced obesity may be due partially to increased energy expenditure. 4. Discussion
ATF2 mediates adaptive thermogenesis by modulating transcription of thermogenic genes [13,15]. Furthermore, fat mass and
In the present study, we showed that the absence of the
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Fig. 4. Higher energy expenditure in Atf7/ mice on a HFD. (A) Food intake of 12-week-old WT and Atf7/ mice on a CD or a HFD (n ¼ 5 for each group). (B, C) Oxygen consumption in 12-week-old WT and Atf7/ mice on a CD (n ¼ 7 for each group). (D, E) Oxygen consumption in 12-week-old WT and Atf7/ mice on a HFD (n ¼ 7 for each group). **, P < 0.01, ***, P < 0.001, NS, not significant.
transcription factor ATF7 in mice results in resistance to dietinduced obesity and reduced serum triglycerides. Atf7/ mice exhibited lower fat mass and smaller adipocytes, suggesting that ATF7 may be involved in the regulation of adipose tissue differentiation. Lower level of plasma resistin can reverse HFD-induced hepatic insulin resistance [22]. Serum levels of resistin were 50% lower in Atf7/ mice than in their WT littermates, indicating that ATF7 ablation may also alter the endocrine function of adipose tissue, which may be associated with the improved systemic insulin sensitivity in Atf7/ mice. ATF2 and ATF7 are thought to bind to the same target DNA sequences. Furthermore, both ATF2 and ATF7 were recently identified as proteins that interact with CCAAT/enhancer-binding protein beta or zinc finger E-box-binding homeobox 1, both of which are essential transcriptional factors during the early phase of
adipogenesis [23,24]. ATF2 is required for WAT differentiation [11], while ATF2 and ATF7 have opposite effects on transcription of target genes, activating and silencing transcription, respectively [16e19]. Based on this, we speculated that loss of ATF7 might enhance the differentiation of adipose tissue, but in fact Atf7/ mice exhibited lower adipose tissue mass. Thus, the molecular mechanisms whereby ATF7 regulates adipocyte differentiation may be more complex than predicted. The presence of obesity is closely associated with the development of insulin resistance [21,25]. Thus, we wondered whether the reduced body weight and fat mass of Atf7/ mice would be associated with improved systemic insulin sensitivity in mice fed either a RD or a HFD. In fact, ATF deficiency did not alter glucose tolerance and there was no difference in serum glucose concentration between Atf7/ and WT mice, although this may be due to the
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decreased serum insulin observed in Atf7/ mice. Of relevance, ATF2 cooperates with other transcription factors, including MafA, to increase transcription of the insulin gene in b-cells [26]. If, conversely, ATF7 reduces insulin gene expression, Atf7/ mice should exhibit a higher serum insulin. Thus, the molecular mechanism underlying the observed decrease in serum insulin in Atf7/ mice may not be simple. The development of obesity is tightly linked to the dysregulation of energy balance [27], whereby an imbalance between food intake and energy expenditure leads to the development of obesity and insulin resistance. There were no differences in food intake and energy expenditure between WT and Atf7/ mice on a RD, indicating that the lean phenotype of the Atf7/ mice is not due to an imbalance between food intake and energy expenditure. However, Atf7/ mice fed a HFD showed higher energy expenditure during both light and dark phases, implying that ATF7 may contribute to the regulation of adaptive thermogenesis. Since ATF2 is involved in increasing energy expenditure by both directly and indirectly activating transcription of the UCP1 gene [13,15], loss of the repressor ATF7 may also enhance UCP1 transcription and thus energy expenditure. One reason why increased energy expenditure was observed when Atf7/ mice were fed a HFD, but not a RD, could be altered production of reactive oxygen species (ROS), which are generated as a byproduct of oxidative phosphorylation in mitochondria [28]. Increased production of ROS in mice fed a HFD results in p38 activation [29], and thus may induce higher levels of ATF7 phosphorylation and the release of ATF7 from the UCP1 promoter, resulting in increased UCP1 transcription. Alternatively, loss of ATF7 could contribute to enhanced energy expenditure via altered macrophage activity. Upon cold exposure, expression of thermogenic genes, including UCP1, is induced in iWAT, leading to “browning” of the adipocytes [30]. However, cold exposure can also stimulate muscle and adipose tissue to release factors that recruit macrophages to the iWAT, also resulting in browning [31,32]. We previously reported that ATF7 could regulate macrophage function via the p38 signaling pathway [19]; thus ATF7 may regulate energy expenditure by promoting macrophagedependent browning. In conclusion, our data show that ATF7 deficiency protects mice against diet-induced obesity by inhibiting adipose tissue differentiation and increasing energy expenditure. This suggests that ATF7 may play an important role in the regulation of energy balance and provides a potential target for novel treatments of obesity.
Conflict of interest The authors declare no conflict of interest.
Acknowledgements We thank the members of the Experimental Animal Division of RIKEN Tsukuba Institute for maintenance of the mice. This work was supported in part by AMED (Japan Agency for Medical Research and Development) (16gm0510015h0004), and by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (Grant No. 16H01413).
Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.08.009.
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