CIDE and metabolic disorders Jingyi Gong, Zhiqi Sun and Peng Li

Protein Science Laboratory of Ministry of Education, Purpose of review Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China The cell death-inducing DFF45-like effector (CIDE) family proteins, comprising three members, Cidea, Cideb, and Fsp27 (Cidec), have emerged as important regulators for Correspondence to Dr Peng Li, Department of Biological Sciences and Biotechnology, Tsinghua various aspects of metabolism. This review summarizes our current understanding University, Beijing 100084, China about the physiological roles of CIDE proteins, their transcriptional regulations, and their Tel/fax: +86 10 62797121; e-mail: [email protected] underlying mechanism in controlling the development of metabolic disorders. Recent findings Current Opinion in Lipidology 2009, 20:121–126 Animals with deficiency in Cidea, Cideb, and Fsp27 all display lean phenotypes with higher energy expenditure and are resistant to diet-induced and insulin resistance. CIDE proteins, localized to lipid droplets and endoplasmic reticulum, control lipid metabolism in adipocytes and hepatocytes through regulating AMP-activated kinase stability and influencing lipogenesis or lipid droplet formation. The expression of CIDE proteins is controlled at both transcriptional and posttranslational levels and positively correlates with the development of obesity, liver steatosis, and insulin sensitivity in both rodents and humans. Summary CIDE proteins are important regulators of energy homeostasis and are closely linked to the development of metabolic disorders including obesity, diabetes, and liver steatosis. They may serve as potential molecular targets for the screening of therapeutic drugs for these diseases.

Keywords Cidea, Cideb, Fsp27, lipid droplets, metabolic disorders

Curr Opin Lipidol 20:121–126 ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins 0957-9672

proteins that are ubiquitously expressed, CIDE family Introduction proteins have distinct tissue expression patterns with Metabolic disorders including obesity, hyperlipidemia, Cidea in BAT in rodents [3], Cideb [4] in liver, and insulin resistance, liver steatosis, and hypertension are a Fsp27 in WAT and BAT [3,5,6]. Cidea mRNA is group of diseases associated with metabolic malfunction detected in human WAT tissues, which might reflect a and unbalanced energy homeostasis. The cell death- heterogeneous nature of human WAT [7]. Interestingly, inducing DFF45-like effector (CIDE) proteins, Cidea, mRNAs of Fsp27 and Cidea were also detected in fatty Cideb and Fsp27, are predominantly expressed in brown livers in which excess amount of lipid is accumulated and adipose tissue (BAT), liver and white adipose tissues large lipid droplets are formed [8–10]. (WATs). Studies using animals deficient in CIDE proteins have demonstrated that this class of proteins plays import- CIDE proteins are subjected to tight regulation on both ant roles in lipid storage, lipid droplet formation, lipolysis transcriptional and posttranslational levels. Fsp27 is and the development of obesity, diabetes, and liver stea- expressed in differentiated white adipocytes [5,11,12] tosis. Here we summarize recent findings on the CIDE and controlled by transcription factors CCAAT/enhancer proteins, with particular emphasis on their molecular binding protein (C/EBP) and peroxisome proliferator- mechanisms in controlling energy homeostasis. activated receptor (PPARg) [8,11]. TNF-a, a negative regulator of C/EBP, reduced the Fsp27 expression [13]. Cidec, a human homolog of rodent Fsp27, is induced Tissue distribution and transcriptional by PPARg2 [14]. Retinoid X receptor (RXR) also binds to regulation of CIDE proteins the Fsp27 promoter region in a PPARgg-dependent CIDE proteins contain an evolutionary conserved CIDE- manner [15]. In liver, Fsp27 is a direct target of PPARg N domain that shares to the DNA fragment [9]. Cidea promoter region contains both PPARa and factor 40/45 (DFF40/45) [1] and a CIDE-C domain that is PPARg response elements and can be activated by PPAR unique among CIDE proteins [2]. Unlike DFF40/45 agonist [10]. In addition, PGC-1a can activate Cidea

0957-9672 ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI:10.1097/MOL.0b013e328328d0bb 122 Genetics and molecular biology expression through binding to estrogen-related receptor and ectopic expression of Cideb induces caspase-inde- alpha (ERRa) and nuclear respiratory factor-1 (NRF-1), pendent cell death in a dimer-dependent manner [23]. while RIP140, a corepressor of nuclear receptors, inter- Cideb expression was also correlated with neuronal apop- acts with PGC-1a and represses its transcriptional tosis after nerve injury [32]. Furthermore, Cideb was activity on Cidea promoter [16,17]. Investigation in tumor shown to interact with NS2, a nonstructural protein cells indicates that CpG methylation on the Cidea pro- encoded by HCV, which inhibits Cideb-induced cell moter region may play a crucial role in establishing death [33]. Furthermore, Cidea could regulate and maintaining tissue-specific and cell-specific Cidea induced by TGF-b pathway in human breast epithelial expression [18]. TNF-a negatively regulates transcrip- cells [22]. In addition, Fsp27 and Cidec were reported to tion of Cidea through NF-kB activation [19]. The play a role in cell death and tumorigenesis [34,35]. expression of human Cideb is regulated by upstream and internal promoters. While CpG methylation in the We generated Cidea-deficient mice and observed no upstream promoter region controls the production of obvious difference in cell death between wild-type and longer transcript, hepatocyte nuclear factor-4a (HNF4a) CideaÀ/À brown adipocytes. Surprisingly, CideaÀ/À mice binds to the internal promoter and controls the pro- showed a drastic lean phenotype with lower adiposity, duction of shorter transcripts of Cideb [20]. decreased leptin and plasma lipid levels and are resistant to diet-induced obesity. In particular, lipid content in On the posttranslational level, Cidea is degraded through CideaÀ/À mice is dramatically reduced upon cold treat- ubiquitin-mediated proteasome pathway in adipocytes ment, suggesting that Cidea is important in controlling [21]. Furthermore, Cidea was shown to be modified by energy expenditure in adipose tissues. In addition, O-linked glycosylation that may control its subcellular CideaÀ/À mice have higher glucose disposition rate and localization and cell death-inducing activity [22]. How- improved insulin sensitivity. The lean phenotype of ever, it remains to be determined if Cideb and Fsp27 are Cidea-null mice may be due to increased whole body controlled by similar posttranslational modifications. metabolism, higher basal lipolysis, and fatty acid oxi- dation in their brown adipocytes [36] (Table 1, Fig. 1).

Subcellular localization of CIDE proteins The role of CIDE proteins in regulating metabolic dis- Much effort has been made toward subcellular localiz- orders was further confirmed in CidebÀ/À mice. Deficiency ation of the CIDE proteins. When overexpressed in of Cideb resulted in lower plasma triacylglycerol (TAG) and heterologous cells, Cidea and Cideb proteins showed a fatty acid levels, decreased white fat mass and increased distribution pattern similar to that of mitochondria whole body metabolism, and a lean phenotype [4,36]. specific marker – mitotracker [3,23]. Later, it was dis- In addition, CidebÀ/À mice are resistant to diet-induced covered that this particular pattern was associated specifi- obesity and liver steatosis. More interestingly, CidebÀ/À cally with the later stage of cell death. Recently, Cidea mice showed decreased plasma insulin concentration, was found to be localized to endoplasmic reticulum (ER) increased hepatic insulin sensitivity manifested by [24] and lipid droplet [25]. Similarly, Fsp27 targets increased IRS-1 and AKT2 phosphorylation in response to lipid droplets in adipocytes [5,26,27,28]. Lipid to insulin stimulation. The lean phenotype of CidebÀ/À droplets are highly dynamic organelles that are in close mice is likely due to a combination of increased fatty acid contact with multiple subcellular structures including ER, oxidation and decreased lipogenesis in CidebÀ/À hepato- endosomes, mitochondria, and plasma membrane [29]. cytes. Consistently, SREBP-1c, a crucial factor in regulat- Therefore, the discrepancy of these observations might ing fatty acid synthesis, was significantly downregulated, be due to the overexpression of proteins in heterologous resulting in the lower expression levels of its downstream cells that undergo specific morphological changes during target such as ACC1/2, fatty acid synthase (FAS), apoptosis or the diverse localization of lipid droplets. As and stearoyl-CoA desaturase (SCD) [4,36] (Table 1, nascent lipid droplet was proposed to be derived from the Fig. 2). The molecular mechanism by which Cideb con- cytosolic leaflet of ER [30], the ER and lipid droplet trols the expression levels of SREBP1c remains to be an localization of CIDE family proteins indicate their import- intensive area of future research. ant functions in lipid droplet formation. Fsp27À/À mice also have dramatically lower levels of TAG and much smaller lipid droplets in their white adipocytes CIDE proteins and the development of and are protected from diet-induced obesity. Fsp27À/À metabolic disorders mice also have higher glucose uptake rates and improved Due to their to DFF40/45, early insulin sensitivity [5,6]. Furthermore, Fsp27 deficiency studies on CIDE family proteins mainly focused on their leads to a reduction in fat accumulation and improved cell death-inducing activity. Cideb proteins could form insulin sensitivity in leptin-deficient ob/ob mice. The dimers through the N-terminal [31] or C-terminal region, increased insulin sensitivity in Fsp27À/À mice is likely CIDE proteins and metabolic disorders Gong et al. 123

Table 1 A comparison of the phenotype of Cidea, Cideb, and Fsp27 knockout mice Gene name Cidea Cideb Fsp27

Tissue distribution BAT Liver, kidney, small intestine, stomach WAT, BAT Phenotype after gene knockout Blood chemistry Plasma TAG Decreased Decreased Decreased Plasma NEFA Decreased Decreased Decreased Plasma ketone body N/D Decreased Decreased Plasma insulin No change Decreased No change Plasma glucose Decreased Decreased No change Plasma adiponectin N/D Increased N/D Plasma leptin Decreased Decreased Decreased Metabolic parameters Glucose disposal Increased Increased Increased Insulin sensitivity No change Increased Increased Adiposity index Decreased Decreased Decreased Food intake No change Increased Increased Body temperature Increased N/D Decreased Whole body metabolic rate Increased Increased Increased WAT Lipid droplet morphology Smaller Smaller Smaller Lipid content Decreased Decreased Decreased Fatty acid b-oxidation N/D N/D Increased Fatty acid synthesis No difference N/D Increased Induced lipolysis rate No difference N/D Increased BAT Lipid droplet morphology Smaller No change Larger Lipid content Decreased No change Increased Fatty acid b-oxidation Increased N/D Decreased Basal lipolysis rate Increased N/D N/D Induced lipolysis rate Increased N/D N/D Liver Lipid droplet morphology No change Smaller No change Lipid content No change Decreased Decreased Fatty acid b-oxidation N/D Increased N/D Fatty acid synthesis N/D Decreased N/D BAT, brown adipose tissue; N/D, not determined; NEFA, non-esterified fatty acids; TAG, triacylglycerol; WAT; white adipose tissues. due to increased expression and phosphorylation of crucial have higher lipolysis rates, especially the basal lipolysis factors such as GLUT4, IRS-1, and AKT2 in insulin rate. Significantly increased mitochondrial volume and signaling pathway in the WAT. The Fsp27À/À mice also activity was observed in the WAT of Fsp27À/À mice as well. Interestingly, Fsp27À/À WAT tends to acquire prop- Figure 1 Role of Cidea in regulating energy metabolism in brown adipocytes erties of BAT, such as smaller lipid droplets, increased mitochondrial activity, and enhanced expression of

Figure 2 Cideb controls plasma triacylglycerol levels and insulin sensitivity in hepatocytes

Insulin sensitivity Hepatocyte Cideb p-AKT p-IRS-1

ACC2 SREBP−1c

ACC1 FAS β-Oxidation SCD1

Decreased TAG secretion Lipogenesis Cidea, localized on ER and lipid droplets, induces large lipid droplet resistant to diet-induced obesity formation and inhibit lipolysis, resulting in the promotion of lipid storage and fatty liver and inhibition of fatty acid b-oxidation. Cidea can be regulated by ubiquitin-dependent proteasomal degradation pathway. Cidea also con- trols fatty acid oxidation by interacting with AMPK b-subunit and pro- CidebÀ/À mice have increased insulin sensitivity due to higher levels of motes AMPK degradation through ubiquitin-dependent pathway. Cidea phospho-AKT and phospho-IRS-1. Deficiency in Cideb leads to reduced can be positively regulated by PPAR and PGC-1 and lipogenesis and increased fatty acid b-oxidation due to lower levels of negatively regulated by RIP140. AMPK, AMP-activated protein kinase; SREBP-1c and its downstream target genes ACC1/2, FAS and SCD1. ER, endoplasmic reticulum; LD, lipid droplets; PPAR, peroxisome pro- FAS, fatty acid synthase; p-AKT, phospho-AKT; SCD, stearoyl-CoA liferator-activated receptor. desaturase; TAG, triacylglycerol. 124 Genetics and molecular biology

Figure 3 Role of Fsp27 in white adipocytes

Fsp27, a lipid droplet-associated protein, inhibits lipolysis and promotes large lipid droplet formation in white adipocytes. Although the exact identity needs to be further determined, certain lipid metabolites released from lipid droplets after lipolysis and secondary metabolites from mitochondriain Fsp27À/À white adipocytes might function as potent ligands to activate the expression of PPAR, PGC-1, NRF-1, FoxC2 and inhibit the expression of Rb, p107, and RIP140. The altered expression of these regulatory factors leads to the upregulation of mitochondrial proteins for oxidative phosphorylation, b-oxidation, and increased levels of brown fat-specific genes (Ucp1, Cidea, and Dio2). The levels of lipases (HSL, ATGL), lipid droplet-associated proteins (perilipin and adipophilin), and proteins in insulin signaling pathway (GLUT4, AKT2, and IRS-1) are increased in Fsp27À/À white adipocytes. AGTL, adipose triglyceride lipase; BAT, brown adipose tissue; ER, endoplasmic reticulum; HSL, hormone-sensitive lipase; LD, lipid droplets.

BAT-specific genes such as Ucp1, Cidea, PPARa, and novel regulators of the development of metabolic dis- Dio2. The attainment of BAT-like property in the WAT eases, such as obesity, type 2 diabetes, and liver steatosis. of Fsp27À/À mice is likely due to reduced expression levels of factors such as Rb, p107, and RIP140 that help to maintain WAT identity and increased levels of key meta- Molecular mechanism for CIDE proteins in bolic regulators such as FoxC2, PPAR, and PGC-1a [37] regulating metabolic disorders (Table 1, Fig. 3). Although the role of CIDE proteins in the development of metabolic disorders is well established, the molecular Importantly, function of Cidea seems to be conserved basis by which CIDE proteins control metabolism in between mouse and human. It has been reported that a adipose tissues and hepatocytes has just begun to be V115F polymorphism in human Cidea is associated with elucidated. It has been shown that Cidea controls the obesity in both Swedish and Japanese populations protein levels and activity of AMP-activated protein [38,39]. Its expression levels are inversely correlated with kinase (AMPK), a pivotal enzyme regulating energy basal metabolic rates [40]. Consistent with the obser- homoeostasis in BAT [3]. Cidea can specifically interact vation that Cidea regulates lipolysis in BAT, Cidea was with AMPK-b subunit and promote the ubiquitination- shown to control lipolysis in human adipocyte [7]. In mediated proteosome degradation of AMPK complex. addition, elevated Cidea was observed in the liver of Consistent with this, AMPK protein levels and enzymatic diabetic mice [41]. In WAT of BMI-matched obese activity were significantly increased in the BAT of humans, levels of Cidea and Cidec are positively corre- CideaÀ/À mice and in differentiated mouse embryonic lated with insulin sensitivity indicating their role in fibroblasts (MEFs)-derived CideaÀ/À mice. Therefore, controlling adipose lipolysis and thus circulating fatty increased AMPK levels and its enzymatic activity lead acids [25]. Furthermore, levels of Cidec were reduced to significantly increased fatty acid b-oxidation in in response to a reduced caloric intake in obese patients CideaÀ/À adipocytes. This provides a molecular expla- [42]. Overall, these data suggest that CIDE proteins are nation for the increased energy expenditure and lean CIDE proteins and metabolic disorders Gong et al. 125 phenotype in CideaÀ/À mice (Fig. 1). The physiological and lipid storage in adipocytes and hepatocytes. Much role of Cidea in controlling AMPK stability and activity effort will be needed to elucidate the mechanism by might be extended to tissues other than BAT. It has been which CIDE proteins modulate metabolic pathways and reported that resveratrol improves high-calorie diet- gene expression. Furthermore, CIDE proteins may well induced fatty liver and extends the life span in mice at represent new drug targets for developing therapeutical least in part through activating liver AMPK. Notably, this drugs for the treatment of metabolic disorders. drug dramatically downregulates Cidea mRNA levels in liver [43]. Further experiments are needed to determine Acknowledgements whether these two events are directly related. We thank members in Peng Li’s laboratory in Tsinghua University for helpful discussion and Dr S.C. Lin for the critical editing of the Recently, it is shown that when overexpressed, Fsp27 can manuscript. This work was supported by grants (30530350 to P.L.) from National Natural Science Foundation of China; (704002 to P.L.) increase lipid droplet size and enhance the accumulation from Ministry of Education of China; National Basic Research program of neutral lipids [6,12,27,44]. 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