IUBMB
Life, 64(3): 226–230, March 2012
Critical Review Effects of Activating Transcription Factor 4 Deficiency on Carbohydrate and Lipid Metabolism in Mammals Chunxia Wang and Feifan Guo Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, The Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai, China 200031
Summary It has been shown that the mammalian activating transcription factor 4 (ATF4) is involved in many different physiological events, such as eye development, stress response, learning, and memory. However, several recent studies have demonstrated that ATF4 also plays an important role in the regulation of lipid and glucose metabolism, energy homeostasis, insulin secretion, and sensitivity, suggesting that ATF4 is a master regulator of metabolism. This review summarizes the most recent progress in the understanding of the novel roles of ATF4 in the regulation of metabolism. Ó 2012 IUBMB IUBMB Life, 64(3): 226–230, 2012 Keywords
ATF4; lipid metabolism; energy homeostasis; insulin secretion and sensitivity; glucose metabolism.
INTRODUCTION Mammalian activating transcription factor 4 (ATF4), also known as cyclic AMP (cAMP) response element-binding 2 (CREB2), tax-responsive enhancer element B67, and C/elementbinding protein (EBP)-related ATF (C/ATF), belongs to the family of basic zipper-containing proteins (1–3). This family was originally defined in the late 1980s and its members are distinguished by their ability to bind to the consensus cAMP response element (CRE) site (consensus sequence 50 -GTGACGTACAG30 ) (4). It has been shown that ATF4 is constitutively expressed in a wide variety of tissues, including those of the brain, heart, Received 30 October 2011; accepted 23 November 2011 Address correspondence to: Feifan Guo, Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China. Tel: 186-21-54920250; Fax: 186-2154920291. E-mail:
[email protected] ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.605
white adipose tissue (WAT), brown adipose tissue (BAT), liver, kidney, and muscle (5). ATF4 expression is regulated transcriptionally, translationally, and posttranslationally by phosphorylation, as described in previous reviews (6, 7). The activity of ATF4 is determined not only by its expression and phosphorylation levels but also by its stability and by interactive proteins. ATF4 has a very short half-life of about 30 6 60 min (7). One protein that is very important for maintaining ATF4 stability is histone acetyltransferase p300, which inhibits the ubiquitination of ATF4 (8). ATF4 activity can also be affected by its cofactors. For example, prolyl-4-hydroxylase domain (PHD)1/PHD3 and twist1 can both interact with ATF4 to repress its transcriptional activity (9, 10). Other heterodimers can enhance ATF4 DNA binding function; these include subunit 3 of RNA polymerase II, Zhangfang, and C/EBP homology protein (11–13). As a transcription factor, three conserved binding sites in the promoter regions of ATF4 target genes have been identified. These include the CRE site (sequence: 50 -GTGACGTACAG-30 ), amino acid response element (AARE) site (sequence: 50 -TGATGCAAT-30 ), and nutrient-sensing response element-1 (NSRE-1) site (sequence: 50 -TGATGAAAC-30 ) site. For example, ATF4 regulates the expression of glucose-regulated protein 78 kDa (Grp78), embryonic stem cell phosphatase (Esp), and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) by binding at the CRE site in the promoters of these genes; ATF4 regulates the expression of Chop and pseudokinase tribbles homolog 3 (Trb3) by binding at the AARE site located in their promoters; ATF4 regulates asparagine synthetase expression by binding at the NSRE-1 site in its promoter (14). It has been shown that ATF4 is crucial to many physiological activities. Studies have demonstrated that ATF4 is involved in hematopoiesis (15), lens and skeletal development (16, 17), learning and memory formation (18, 19), hypoxia resistance (20), tumor growth (21), endoplasmic reticulum (ER) stress (22), autophagy (23), amino acid deprivation (14), etc. These physiological functions have been summarized in previous
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reviews (6, 24–26). Over the past several years, the role of ATF4 in various metabolic processes has attracted a great deal of attention. Below, we review some of the most common observations of the involvement of ATF4 in the regulation of lipid and glucose metabolism, energy homeostasis, and insulin secretion and sensitivity.
ATF4 DEFICIENCY AND LIPID METABOLISM Maintenance of optimal lipid homeostasis is required for most of the body’s normal functions. Excessive accumulation or abnormal distribution of lipids can cause significant health issues, such as obesity, fatty liver, and type 2 diabetes. Understanding the regulation of lipid metabolism may help define novel targets for future therapeutic intervention. Lipid metabolism includes the hydrolysis of lipids into glycerol and fatty acids and the synthesis of lipids, which is called lipogenesis. Over the past 2 years, studies have indicated a novel role for ATF4 in the regulation of many aspects of lipid metabolism. Seo et al. (27) have recently shown that ATF4-deficient mice exhibit reduced body fat content when fed a normal diet. These mice were also resistant to age-associated and high-fatdiet (HFD)-induced obesity and to HFD-induced hepatic steatosis. Furthermore, they found that mammalian target of rapamycin (mTOR) activity is decreased in the livers and adipose tissues of these mice, suggesting that the effects of ATF4 deficiency might be mediated by mTOR signaling (27). Consistent with this hypothesis, several aspects of the ATF4 deficiency phenotype were found to resemble that of mice with mutations in the components of the mTOR pathway. The mTOR signals were conveyed mainly through ribosomal S6 protein kinase (S6K) and 4E-BP1, which closely communicated with metabolic regulation (28). For example, 4E-BP1-deficient mice showed unusually small white fat pads and increased metabolic rates (29). In addition to this, S6K1-deficient mice were protected against obesity via enhanced b-oxidation (30). Molecular mechanisms underlying ATF4 regulation of the mTOR pathway, however, are yet to be described in detail and merit further investigation. Consistent with these observations (27), the results of the experiments performed in our laboratory also show ATF4 to be a key regulator of lipid metabolism (31). Compared with wildtype mice, ATF4-deficient mice exhibited increased expression of genes related to lipolysis and b-oxidation, including hormone-sensitive lipase, carnitine palmitoyltransferase 1, and medium-chain acyl-CoA dehydrogenase. They also showed decreased expression of lipogenic genes, including fatty acid synthase, stearoyl CoA desaturase 1 (SCD1), and sterol regulatory element-binding protein 1c (SREBP1c) in WAT (31). The molecular mechanisms underlying ATF4 regulation of the expression of these genes remain unknown. One possibility is that peroxisome proliferator-activated receptor gamma coactivator (PGC)1a, the master regulator of lipid metabolism, may be involved. There are several reasons why this is likely: (1)
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PGC1a expression is largely induced in WAT and BAT in ATF4-deficient mice. (2) It has been shown that PGC1 a expression can be stimulated by CREB via binding to CRE site in its promoter (32). (3) ATF4 belongs to the same family as CREB, and it can also regulate target gene expression via the CRE site. Therefore, it is likely that ATF4 regulates lipid metabolism through regulation of PGC1 a expression as a suppressor. Another possibility is that the ER stress response may be involved in ATF4 regulation. ATF4 is a key factor in this process and it has recently been shown to induce intracellular lipid accumulation through the activation of SREBPs (33, 34). ATF4 deficiency may cause the ER stress response to fail, contributing to the decreased expression of SREBP1c in WAT and BAT in experimental mice. In addition to the roles that ATF4 plays in lipid metabolism in mice fed normal chow diets or HFD, another study conducted in our laboratory has suggested that ATF4 deficiency can also protect mice from high-carbohydrate-diet (HCD)-induced hepatic steatosis (35). Further study has indicated that these protective effects are caused by suppressed expression of SCD1. This hypothesis is supported by the fact that oral supplementation of the main product of SCD1 oleate (18:1) increases TAG accumulation in livers of mice deficient in ATF4 (35). It is unclear that how ATF4 regulates the expression of SCD1.
ATF4 DIFICIENCY AND ENERGY HOMEOSTASIS Energy homeostasis is the balance between energy intake and energy expenditure, which is correlated with several organ systems that coordinate food intake, energy stores, metabolic demands, and energy expenditure (36, 37). ATF4-deficient mice are small and lean (27, 31). This suggests that ATF4 may be involved in the regulation of food intake. However, the effects of ATF4 deficiency on food intake have been found to vary depending on the genetic background of the mice. For example, among ATF4-deficient and wild-type on 129SV background, ATF4-deficient mice consumed 35% more food when normalized against body weight, whereas, on C57 background, food intake was equivalent in the ATF4-deficient and wild-type mice (27). The molecular mechanisms underlying ATF4 regulation of food intake have not been investigated. ATF4 has been found to directly affect levels of neuropeptide, which have been shown to play important roles in the regulation of appetite (38). Alternatively, the effects of ATF4 on food intake may be mediated by emotional control, which is supported by a recent study. Overexpression of ATF4 in the nucleus accumbens is found to decrease emotional reactivity and increase depression-like behavior. This process has also been shown to affect eating and control food choice (39). These possibilities need to be clarified in the future. Basic metabolism, physical activity, and thermogenesis are the major components of energy expenditure. ATF4-deficient mice display higher oxygen consumption than other mice, suggesting higher energy expenditure (27, 31). However, no
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difference in physical activity was observed between ATF4-deficient mice and wild-type mice of either the 129SV or C57 genetic backgrounds (27, 31). This suggests a possible increase in thermogenesis in these mice. Thermogenesis is regulated by increased expression of uncoupling protein 1 (UCP1) expression and its upstream regulator, PGC1a, in BAT (31). Consistent with the increase in thermogenesis, body temperature tends to be much higher and expression of PGC1a and UCP1 more elevated in the BAT of ATF4-deficient mice (27, 31). In addition, UCP1 protein levels are also significantly increased in the WAT of ATF4-deficient mice (27). Consistent with these changes, ATF4-deficient mice have been found to have multilocular adipocytes in WAT depots, which are characteristic of BAT (27). These results suggest that ATF4 deficiency not only induces UCP1 expression in BAT but also induces WAT transdifferentiation into BAT, both of which can contribute to the higher energy expenditure as observed in ATF4-deficient mice. Taken together, ATF4 deficiency has profound effects on both food intake and energy expenditure. However, It is not known whether this is a direct effect of ATF4 on either of these processes or whether one of the changes is a compensatory response to the other. Furthermore, because these results are obtained using global ATF4 knockout (KO) mice, energy homeostasis is regulated mainly in the central nervous system (CNS). The effects of ATF4 on the CNS merit further investigation.
ATF4 DIFICIENCY AND INSULIN SECRETION AND SENSITIVITY AND GLUCOSE METABOLISM Glucose is one of the most important energy sources to the maintenance of normal body function. Glucose metabolism is controlled by central metabolic hormone, insulin. Several studies have suggested that ATF4 is involved in the regulation of insulin levels. For example, Yoshizawa et al. (40) showed that insulin levels were higher in ATF4-deficient mice fed with normal chow than in wild-type mice fed with the same diet. Studies indicate that increased insulin levels are caused by increased insulin secretion, which is controlled by ATF4 expression in osteoblasts. Researchers have found that ATF4 deficiency inhibits Esp expression, which increases the osteocalcin activity that promotes insulin secretion (40). In addition, ATF4 deficiency was associated with increased pancreatic b-cell area and proliferation, which may also contribute to increased insulin levels (40). Consistent with these results, ATF4 was found to negatively regulate insulin exocytosis by interacting with TRB3 to competitively inhibit CREB activation of insulin exocytosis genes in pancreatic b-cells (41). Taken together, these results suggest that ATF4 regulates insulin levels, possibly as a suppressor of insulin secretion. However, the effects of ATF4 on insulin levels, are not always the same. ATF4 deficiency has also been shown to have no effect on insulin levels when ATF4-deficient mice are compared with wild-type mice fed the same normal chow diet (27, 31). Seo et al. (27) showed that insulin levels are even
decreased in ATF4-deficient mice fed with HFD. The reasons for these decreased insulin levels could be related to increased expression of the 4E-BP1 that is required for normal b-cell function and survival (42). Because ATF4 is involved in the ER stress response and in the upstream regulation of 4E-BP1, ATF4 deficiency cannot prevent HFD-induced ER stress or any deleterious effects on b-cell survival. This can cause decreased insulin secretion. In addition, the different effects of ATF4 on insulin levels may be the result of the differences in the genetic backgrounds and ages of the mice used in the experiments. In addition to affecting insulin secretion, ATF4 deficiency enhances insulin sensitivity. Yoshizawa et al. (40) showed that ATF4 deficiency could enhance insulin sensitivity in the liver, muscle, and WAT of ATF4-deficient mice fed with a normal chow diet. Other studies have indicated that ATF4 deficiency also protects mice from HFD- and HCD-induced insulin resistance (27, 35). The effect of ATF4 on insulin sensitivity is mediated by the regulation of the expression of Esp and by bioactivity of osteocalcin in osteoblasts. This hypothesis is supported by the observation that ATF4 deficiency in ostoblasts alone enhances insulin sensitivity to the same extent as global ATF4 deficiency in mice (40). This may also be due to decreased mTOR activity in the livers and WAT observed in ATF4-deficient mice. However, mice deficient for S6K1 exhibited improved insulin sensitivity (30). The third possibility is the involvement of ER stress. HFD has been shown to induce ER stress, which has recently been implicated in insulin resistance (43, 44). ATF4 deficiency may prevent HFD-induced insulin resistance by decreasing ER stress (22). Insulin levels and insulin sensitivity affect blood glucose levels. ATF4 KO mice of the C57 genetic background showed lower random and fasting glucose levels, and glucose tolerance tests showed that these ATF4 KO mice had improved glucose metabolism under normal chow diet conditions (27, 40). This indicated that the decreases in blood glucose levels are caused by decreased gluconeogenesis, as demonstrated by significantly reduced expression of phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase (G6pase) and by increased glycolysis as demonstrated by increased expression of glucokinase (Gck) and decreased expression of pyruvate dehydrogenase kinase 4 (Pdk4) in the livers of ATF4-deficient mice (40). Further study demonstrated that ATF4 deficiency in osteoblasts alone could cause changes similar to those caused by global ATF4 deficiency (40). This suggested the importance of ATF4 expression in osteoblasts in the regulation of glucose metabolism. However, the random glucose levels in ATF4 KO mice and wild-type mice on the 129SV genetic background were comparable. Overnight-fasted blood glucose levels were much lower in ATF4 null mice fed on either normal chow diet or HCD than in wild-type mice (35). These results suggest that the effects of ATF4 on glucose homeostasis may depend on genetic background.
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CONCLUSIONS AND FUTURE DIRECTIONS In conclusion, mounting evidence reveals that ATF4 is a master regulator of various metabolic pathways and physiological functions. The role it plays in lipid and glucose metabolism, energy homeostasis, and in insulin secretion and sensitivity merits more attention. Because ATF4 plays a key role in osteoblast differentiation, and also interacts with C/EBPb which is a key regulator for adipogenesis, it is very likely that ATF4 is involved in adipocyte adipogenesis to regulate lipid metabolism. However, the molecular mechanisms underlying ATF4 regulation of these physiological processes remain largely unknown. ER stress pathway, PGC1a pathway, and mTOR pathways are the most likely mediators of the effect of ATF4 on the regulation of metabolism. The contribution of each should be addressed in future studies. Because metabolism is regulated by multiple organs and most of the conclusions regarding ATF4 that are mentioned in current reviews have been based on global ATF4 KO mice; the role of ATF4 in different tissues requires further investigation in mice with tissue-specific deficiencies. Because of the extensive functions of ATF4 in regulation of metabolic processes, ATF4 might be a potential therapeutic target for treating metabolic diseases.
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