c-Jun Amino-Terminal Kinase-1 Mediates Glucose-Responsive Upregulation of the RNA Editing Enzyme ADAR2 in Pancreatic Beta-Cells

A-to-I RNA editing catalyzed by the two main members of the adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, represents a RNA-based recoding mechanism implicated in a variety of cellular processes. Previously we have demonstrated that the expression of ADAR2 in pancreatic islet β-cells is responsive to the metabolic cues and ADAR2 deficiency affects regulated cellular exocytosis. To investigate the molecular mechanism by which ADAR2 is metabolically regulated, we found that in cultured β-cells and primary islets, the stress-activated protein kinase JNK1 mediates the upregulation of ADAR2 in response to changes of the nutritional state. In parallel with glucose induction of ADAR2 expression, JNK phosphorylation was concurrently increased in insulin-secreting INS-1 β-cells. Pharmacological inhibition of JNKs or siRNA knockdown of the expression of JNK1 prominently suppressed glucose-augmented ADAR2 expression, resulting in decreased efficiency of ADAR2 auto-editing. Consistently, the mRNA expression of Adar2 was selectively reduced in the islets from JNK1 null mice in comparison with that of wild-type littermates or JNK2 null mice, and ablation of JNK1 diminished high-fat diet-induced Adar2 expression in the islets from JNK1 null mice. Furthermore, promoter analysis of the mouse Adar2 gene identified a glucose-responsive region and revealed the transcription factor c-Jun as a driver of Adar2 transcription. Taken together, these results demonstrate that JNK1 serves as a crucial component in mediating glucose-responsive upregulation of ADAR2 expression in pancreatic β-cells. Thus, the JNK1 pathway may be functionally linked to the nutrient-sensing actions of ADAR2-mediated RNA editing in professional secretory cells.


Introduction
RNA editing through the hydrolytic C6 deamination of adenosine (A) to yield inosine (I) represents a pivotal posttranscriptional mechanism that further diversifies the cellular transcriptome and proteome [1,2,3]. Based upon the RNA substrates that have been found to undergo A to I editing within regions with double-stranded (ds) structural character, this genetic recoding process has been implicated in the functional modifications of proteins [1,2,3,4,5], alternative splicing [6], and micro-RNA biogenesis [7]. A growing body of evidence has established that A to I RNA editing plays essential roles in the function and development of the central nervous system, largely through editing of transcripts encoding the neurotransmitter receptors and ion channels, including the ionotropic glutamate receptors (GluRs), Gprotein-coupled serotonin-2C subtype receptor, and Kv1.1 potassium channel [4,8,9,10,11].
In mammals, two members of the adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, are enzymatically active for catalyzing the A to I deamination reaction [12]. Both ADAR1 and ADAR2 are ubiquitously expressed in many tissues [13,14,15]. Multiple promoters have been identified to control the expression of ADAR1, generating transcripts with alternative exon 1 structures that encode two ADAR1 forms, an interferon (IFN)inducible protein of ,150 kDa and a constitutively expressed Nterminally truncated protein of ,110 kDa [16,17,18]. In addition to the regulatory elements found within the IFN-inducible ADAR1 promoter [19,20], recent studies revealed distinct tissuespecific expression features for different ADAR1 transcripts [21]. In contrast, the promoter that directs the ADAR2 expression has not been functionally characterized, despite that a putative promoter region upstream of a newly identified exon was described for both human and mouse ADAR2 genes [22]. While it is yet to be established whether ADAR2 possesses multiple promoters like ADAR1 to produce multiple transcripts, it also remains unclear if regulatory mechanism(s) exists for the transcriptional control of ADAR2 in a tissue-or cell type-specific fashion.
Many intracellular signaling mechanisms act to modulate the function of pancreatic b-cells, which play a central role in glucose homeostasis through fuel-regulated secretion of insulin [23]. Glucose, the primary physiological stimulator of insulin synthesis and secretion, has been shown to trigger the activation of c-Jun amino-terminal kinase (JNK) [24], the stress-activated protein kinase that belongs to the large mitogen-activated protein kinase (MAPK) family [25]. The JNK pathway is known to integrate signals from a diversity of extracellular stimuli and regulate various cellular processes such as survival, proliferation and apoptosis [25]. Among the three JNK isoforms, JNK1 and JNK2 are found to be ubiquitously expressed, while JNK3 is mainly expressed in brain, pancreatic islets, testis and heart [26]. For JNK1 and JNK2, alternative splicing yields multiple protein forms of ,54 kDa and ,46 kDa [27]. Distinct intracellular mechanisms are operational in mounting cell-or stimulus-specific responses that result in JNK activation, which phosphorylates and activates transcription factors including the c-Jun component of the activating protein-1 (AP-1) [28]. Documented studies have implicated the JNK pathway in metabolic dysregulation associated with obesity, insulin resistance, and type 2 diabetes [29,30]. In pancreatic bcells, JNK is thought to be involved in suppression of insulin gene expression under oxidative stress [31] and cytokine-induced apoptosis [32]. While the JNK isoforms may possess functional redundancy in mediating cellular adaptation responses to various stress stimuli, it remains poorly understood whether JNK1 or JNK2 is specifically linked to regulation of different cellular aspects of the nutrient-sensing actions in b-cells.
Previously we demonstrated that RNA editing by ADAR2 is upregulated selectively in the pancreatic islets of obese mice when chronically fed a high-fat diet, and ADAR2 expression can be stimulated by glucose in the rat insulinoma INS-1 b-cells [33]; moreover, knockdown of the expression of ADAR2 in pancreatic islets and INS-1 cells leads to attenuation of glucose-stimulated insulin secretion, suggesting that ADAR2 is coupled to cellular exocytosis in professional secretory cells [34]. However, the molecular mechanism for the nutritional regulation of ADAR2 expression in b-cells is largely unclear. Here we examined the mechanistic link between glucose-stimulated JNK activation and ADAR2 expression. Our findings indicate that JNK1 serves as a crucial component in mediating glucose-responsive upregulation of ADAR2 in pancreatic b-cells.

Cell Lines and Pancreatic Islet Isolation
The rat insulinoma INS-1 cell line [35], a generous gift from Dr. Christopher B. Newgard (Duke University Medical Center), were maintained in RPMI 1640 containing 11.1 mM D-glucose, 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 mg/ ml), 10 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.05 mM 2-b-mercaptoethanol. Cells with passage numbers of 19 to 30 were used. Pancreatic islets were isolated from mice at 14-24 weeks of age using the Liberase (Roche) digestion method as previously described in detail [33].

RT-PCR Analysis
Total RNA was isolated from INS-1 cells with TRIzol (Invitrogen) or from primary islets with RNeasy Plus Mini Kit (Qiagen). After treatment with RNase-free DNase I (Roche Applied Science), first-strand cDNA was synthesized with M-MLV Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was conducted using an ABI 7500 Fast Real-Time PCR System and Power SYBR Green PCR Master Mix (Applied Biosystems) following the manufacturer's recommendations. Cyclohphilin or Gapdh was used as an internal control for normalization. Primers used for each target gene were listed in Table S1. For quantitative analysis of ADAR2 auto-editing, regular PCR was performed using the TaKaRa Taq Kit (Takara). After analysis by 3.0% agarose gel electrophoresis, editing efficiency was quantified using the Quantity One software (Bio-Rad).

Generation of Recombinant Adenoviruses and Viral Infection
Recombinant adenoviruses Ad-shJNK1 and Ad-shLacZ, which express shRNAs directed against rat JNK1 and LacZ genes, respectively, were generated using the BLOCK-iT TM Adenoviral RNAi Expression System (Invitrogen, Carlsbad, CA) as previously described [34]. The adenovirus used for JNK1 knockdown contains the following target sequence: 59 -GCAGAAG-TAAACGTGACAACA-39. The viral titers were determined by the tissue culture infectious dose 50 (TCID50) method. INS-1 cells were infected by adenoviruses at an MOI of 20 for 72 hours.

Animal Studies
All experimental protocols were approved by the Institutional Animal Care and Use Committee at the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of sciences. Jnk1 2/2 and Jnk2 2/2 mice were housed in laboratory cages at a temperature of 2363uC and a humidity of 3565% under a 12 h dark/light cycle (lights on at 6:30 a.m.) in accredited animal facilities at the Shanghai Institutes for Biological Sciences, CAS. Heterozygote mice were subsequently intercrossed to yield homozygotes and wild-type littermates. For diet-induced obesity, female mice maintained on a normal chow diet were fed ad libitum a low-fat diet (LFD, 10 kcal% fat) or high-fat diet (HFD, 60 kcal% fat) (Research Diets, Inc., New Brunswick, NJ). The body weight was monitored monthly. Total body fat content was measured by nuclear magnetic resonance (NMR) using the Minispec Mq7.5 (Bruker, Germany). Fasting glucose was determined in blood collected from tail vein of female mice at 24 weeks after a 6 hours fasting (from 8:30-14:30) using a glucometer (FreeStyle, Alameda, California, USA).

Mouse Adar2 Promoter Constructs and Luciferase Reporter Assays
The promoter of mouse Adar2 gene, which corresponds to the region of -4921 to +186 with respect to the putative transcription start site (denoted nucleotide +1), was amplified by PCR using mouse genomic DNA as the template. The DNA fragment was subsequently subcloned into plasmid pGL3-basic (Promega, Madison, WI) via Mlu I and Xho I restriction sites to generate the P Adar2 -4921-Luc construct. Three constructs with 59-terminal deletions of the promoter, P Adar2 -1256-Luc, P Adar2 -219-Luc, and P Adar2 -63-Luc were derived from P Adar2 -4921-Luc. A PCR-based mutagenesis strategy was used to generate the mutant P Adar2 -219-Luc with a four-nucleotide substitution within the AP1 site, using the Muta-direct site-directed mutagenesis kit (SBS Genetech) and the following primers: 59-CAGGAGGCGCGCATT-GAAGCGTGGAGCGCT-39 (forward) and 59 -AGCGCTC-CACGCTTCAATGCGCGCCTCCTG-39 (reverse). Rat Insulin I promoter reporter construct (410RIP1-Luc) was kindly provided by Dr. M. German (University of California at San Francisco, Medical School). INS-1 cells with 40-60% confluence were maintained at 2.8 mM glucose for 6 hours before transfection using Lipofectamine 2000 (Invitrogen, Grand Island, NY). Renilla luciferase reporter plasmid pRL-TK (Promega, Madison, WI) was used as an internal control. Cells were treated at 24 hours after transfection with 16.7 mM glucose for 16-24 hours, and the luciferase activities were measured using Dual-luciferase Assay Kit (Promega, Madison, WI) according to the manufacturer's instructions.

Statistical Analysis
Statistical analysis was performed with unpaired two-tailed t-test or one-way or two-way analysis of variance (ANOVA). p,0.05 was considered to be statistically significant.

Glucose-stimulated JNK Phosphorylation Accompanies Upregulation of ADAR2 in INS-1 b-cells
To investigate the signaling mechanism by which ADAR2 is regulated in b-cells, we first tested the association of JNK activation with glucose-dependent upregulation of ADAR2. Consistent with our previous finding that Adar2 is a glucoseinducible gene [33], glucose stimulation dose-responsively increased the mRNA abundance of Adar2 but not Adar1 in INS-1 bcells, as shown by quantitative RT-PCR analysis (Fig. 1A). Western immunoblotting also revealed significant elevations of ADAR2 protein but not the p110 form of ADAR1 protein in response to increasing glucose concentrations (Fig. 1B). This corresponded with increased efficiency of auto-editing of Adar2 transcripts (Fig. 1B), as determined by RT-PCR that detected the 47-nucleotide insertion resulting from ADAR2 editing-initiated alternative splicing [6]. Interestingly, glucose stimulation of ADAR2 expression and RNA editing was concomitantly accompanied by elevated phosphorylation of JNK1/2 at Thr 183 /Tyr 185 (Fig. 1B). Furthermore, the time-course analysis showed that stimulation by glucose of ADAR2 protein expression for different time intervals was also paralleled with induced JNK1/2 phos-phorylation as well as increased phosphorylation of c-Jun (Fig. 1C), the major target of JNK kinases. Notably, the protein level of c-Jun was also augmented upon glucose stimulation (Fig. 1C), which could be largely ascribable to JNK phosphorylation-dependent enhancement of c-Jun protein stability [36]. These results raised the possibility that the JNK pathway is coupled to glucose regulation of ADAR2-mediated RNA editing in b-cells.

JNK Activation-associated ADAR2 Upregulation in b-cells is a Glucose-sensing Event
As glucose-induced upregulation of ADAR2 and the concurrent JNK activation in INS-1 cells may represent a glucose-sensing event, we asked if glucose can affect the stability of Adar2 mRNA or if glucose metabolism is required for the stimulation of ADAR2 expression and JNK phosphorylation. Upon treatment with actinomycin D for up to 6 hours, comparable decreases in the Adar2 mRNA abundance were detected by quantitative RT-PCR in INS-1 cells when maintained at 2.8 or 16.7 mM glucose ( Fig. 2A), suggesting that glucose had no effect upon the Adar2 mRNA decay and glucose augmentation of the Adar2 mRNA level may reflect an induction of its transcription. Moreover, inhibition of glucose glycolysis in INS-1 cells with 2-deoxyglucose (2-DG), the non-metabolizable glucose analogue [37], not only blunted glucose induction of the ADAR2 mRNA and protein expression ( Fig. 2B-C), but also attenuated glucose stimulation of JNK and c-Jun phosphorylation (Fig. 2C). Next, we examined whether mobilization of intracellular calcium, a critical cellular signaling event that mediates glucose-regulated insulin secretion in b cells [38], is also involved. Indeed, blocking calcium influx with EGTA or calcium release from the endoplasmic reticulum with 2-aminoethoxydiphenyl borate (2-APB) significantly diminished glucose-dependent upregulation of Adar2 mRNA expression (Fig. 2D). However, it is unclear how the calcium-triggered downstream signaling events are linked to regulation of ADAR2. To test whether an indirect autocrine action from glucose-stimulated insulin secretion is involved, we examined the effects of insulin or inhibitors of insulin signaling or secretion in INS-1 cells. Whereas insulin treatment of cells cultured at 2.8 mM glucose did not show a stimulatory effect upon the expression of ADAR2 protein, inhibition of insulin signaling with HNMPA-(AM) 3 or its secretion with somatostatin-14 in cells cultured at 16.7 mM glucose had no detectable effects on glucose-stimulated expression of ADAR2 protein (Fig. 2E). Together, these data indicate that the upregulation of ADAR2 is selective to glucose stimulation and directly relies on glucose metabolism in b-cells, and JNK activation may be an integral part of this glucose-sensing event.

Inhibition of JNK Activity Abrogates Glucose Enhancement of ADAR2 Expression and Auto-editing
To determine if the JNK pathway is critical for glucosestimulated ADAR2 expression and ADAR2-mediated RNA editing, we evaluated the impact of SP600125, a specific inhibitor of JNK activity (JNKi) [39]. Glucose-stimulated expression of Adar2 mRNA was reduced by ,30% and ,55%, respectively, in INS-1 cells that were cultured at 16.7 mM glucose for 3 hours in the presence of 10 mM and 20 mM JNKi, while it was totally blocked in cells when cultured for 24 hours (Fig. 3A). Treatment of INS-1 cells with 10 mM or 20 mM JNKi for 24 hours did not affect the expression of ADAR1-p110 protein but resulted in partial (by ,65%) or complete abrogation of glucose-induced increases in the level of ADAR2 protein; these were accompanied by partial (,50%) or total inhibition of glucose-stimulated c-Jun phosphorylation (Fig. 3B). Consistent with the reported findings [39], we also observed a marked decrease in JNK phosphorylation when cells were treated with 20 mM JNKi (Fig. 3B). In parallel, analysis of ADAR2 auto-editing showed that treatment with JNKi for 24 hours markedly reduced the stimulation by glucose of ADAR2catalyzed RNA editing (Fig. 3C). These results thus suggest that the JNK pathway may play a crucial role in mediating the glucoseresponsive upregulation of ADAR2 expression and ADAR2associated RNA editing.

Selective Knockdown of JNK1 Attenuates Glucosestimulated ADAR2 Expression
To rule out the possible nonspecific effect of the JNK inhibitor and investigate which JNK isoform is specifically involved, we employed the recombinant adenovirus Ad-shJNK1 expressing a small hairpin (sh) RNA directing against the coding region of Jnk1 to knock down its expression. In comparison with the control virus Ad-shLacZ, Ad-shJNK1 infection of INS-1 cells significantly decreased the mRNA expression level of Jnk1, but did not affect that of Jnk2 (Fig. 4A). Furthermore, Ad-shJNK1-mediated knockdown of JNK1 markedly reduced (by ,70%) the induction of Adar2 mRNA expression in response to stimulation by glucose at 16.7 mM (Fig. 4A; from 6.460.44-fold to 1.860.03-fold). Consistently, Western immunoblot analysis showed that knockdown of JNK1 expression in INS-1 cells infected with Ad-shJNK1 blunted the phosphorylation of c-Jun and significantly decreased (by ,45%) the protein expression level of ADAR2 upon stimulation by 16.7 mM glucose (Fig. 4B). These data indicate that JNK1 may be an important player that contributes to the glucose-dependent upregulation of ADAR2 in b-cells. glucose for 6 hours and then stimulated with 16.7 mM glucose for the indicated time intervals. Cell lysates were analyzed by immunoblotting using the indicated antibodies. Shown are representative immunoblots from three independent experiments. Phosphorylated c-Jun (p-c-Jun) was detected by anti-c-Jun (pSer63) antibody. doi:10.1371/journal.pone.0048611.g001

JNK1 is a Critical Driver of ADAR2 Expression in Pancreatic Islets
To further determine if JNK1 is specifically essential for regulating the expression of ADAR2 in b-cells, we measured the mRNA expression of Adar2 in the primary islets isolated from mice with whole-body deletion of the Jnk1 or Jnk2 gene [29]. As shown by quantitative RT-PCR assessment, the mRNA expression of Adar2 displayed a significant reduction (by ,50%) in the islets of JNK1 2/2 mice (Fig. 5A) but not JNK2 2/2 mice (Fig. 5B). By contrast, the expression levels of Adar1 mRNAs, including those encoding the IFN-inducible p150 form or both the p150 and p110 forms, did not exhibit significant alterations (Fig. 5). Moreover, Western immunoblot assessment showed that the protein expression level of ADAR2 was apparently decreased in the islets of JNK1 2/2 mice (Fig. 5C), which was accompanied by reduced protein level of c-Jun expression. Therefore, JNK1 is likely to serve as a critical driver of ADAR2 expression in pancreatic islets.

High-fat Diet-induced Expression of ADAR2 is Dependent on JNK1 in Pancreatic Islets
We have previously reported that Adar2 transcripts were selectively upregulated in the pancreatic islets isolated from the insulin-resistant obese mice which were chronically fed a high-fat diet (HFD) [33]. We then asked if deficiency in JNK1 signaling could affect the upregulation of islet Adar2 in response to HFD feeding. When challenged with HFD for up to 24 weeks, the wildtype (WT) littermates exhibited substantial increases in their body weight gain and whole-body fat content as compared with the lowfat diet (LFD)-fed control mice (Fig. 6A), which was associated with elevated blood glucose levels in the fasted state (Fig. S1). In contrast, JNK1 2/2 mice displayed marked resistance to the HFDinduced obesity (Fig. 6A) as well as normalized fasting glucose levels ( Figure S1). This is in accordance with the documented studies demonstrating the crucial role of JNK1 in protecting mice against diet-induced obesity and insulin resistance [29]. Importantly, quantitative RT-PCR analysis showed that the expression of Adar2 mRNA was augmented by ,2-fold in the islets from HFD-fed WT mice, which was abolished in JNK1-deficient islets (Fig. 6B). On the other hand, JNK1 ablation led to no significant changes of islet Adar1 mRNA expression, which did not respond to HFD feeding (Fig. 6B). Hence, JNK1 is a key component that mediates the metabolic regulation of Adar2 in the islets in response to overnutrition. Moreover, as ADAR2 knockdown could lead to impaired insulin exocytosis [34], the failure of ADAR2 upregulation in the islets of HFD-fed JNK1 2/2 mice may contribute to the lowered circulating insulin level that was reported for the JNK1 2/ 2 mice when fed HFD [29].

Characterization of the Glucose-inducible Promoter of Mouse Adar2 Gene
Because JNK kinases exert their physiological functions through regulating transcription factors such as c-Jun, we investigated if the JNK pathway mediates glucose stimulation of ADAR2 at the transcription level in b-cells. We characterized the glucoseresponsive region within the mouse Adar2 promoter in INS-1 cells through generating a series of luciferase reporter constructs (Fig. 7A). Transient transfection and luciferase assays identified a 219-nuceotide region (P Adar2 -219) that retained full capacity for glucose induction (Fig. 7B), which exhibited comparable transcriptional responsiveness to glucose stimulation to that of the 410nuceotide rat Insulin 1 promoter (RIP) ( Figure S2). Importantly, inhibition of the JNK activity by 20 mM JNKi in INS-1 cells dramatically blunted the glucose induction of the transcriptional activity of P Adar2 -219 (Fig. 7C). To further examine the potential trans-factors that may be involved in JNK-mediated glucose regulation of ADAR2, we analyzed the 219-nucleotide sequence that conferred the glucose-responsive property using TRANSFAC [40] and TESS programs (http://www.cbil.upenn.edu/cgi-bin/ tess/tess). Among several putative binding sites for transcription factors, there exists a potential CREB/AP-1 (activating protein-1) binding site (ACGTCA) in the -78/273 region of the mouse Adar2  promoter that may account for the stimulatory effect of JNK1. Supporting this idea, co-transfection experiments showed that expression of c-Jun, but not JunB that is not phosphorylated by JNKs [41], significantly enhanced both the basal and glucosestimulated transcriptional activity of P Adar2 -219 (Fig. 7E); moreover, c-Jun was unable to activate the mutant version of P Adar2 -219 (Fig. 7F), in which this AP-1 site was mutated to ATTGAA (Mut). Thus, these results implicated the JNK-c-Jun pathway in the glucose-responsive control of Adar2 transcription in b-cells.

Discussion
In the current study, we aimed to explore the molecular mechanism by which the RNA editing enzyme ADAR2 in pancreatic b-cells or primary islets is regulated in response to glucose stimulation or changes in the nutritional state. We demonstrated that the stress-activated protein kinase JNK1 is a crucial signaling molecule in mediating the metabolic control of the transcriptional expression of ADAR2 in b-cells. These findings provide new insight with respect to the mechanism for cell typespecific modulation of ADAR2 expression and ADAR2-dependent RNA editing under physiological conditions. RNA editing by ADAR2 is known to play essential roles in the central nervous system [12,42,43]. However, limited knowledge has been obtained regarding the functions and regulation of ADAR2 in the periphery. We previously found that in professional secretory cells such as pancreatic b-cells, ADAR2 expression is regulated in response to nutritional and metabolic cues [33] and its deficiency can influence the cellular exocytotic output [34]. In the state of excess energy intake, ADAR2 may be connected to overnutrition-associated chronic inflammatory pathways. The JNK signaling cascade is an ancient stress pathway that mediates cellular adaptation responses to environmental changes, thereby promoting the survival of an organism [44]. On the other hand, chronic activation of the JNK pathway is known to play an important role in the pathogenesis of obesity-related insulin resistance and metabolic dysfunctions, including impairment of b-cell function in diabetes [29,45,46]. Here our results indicate that activation of the JNK pathway is metabolically coupled to the glucose-dependent upregulation of ADAR2, which occurs in a manner that requires the metabolism of glucose. The results from the islets of HFD-fed mice further suggest that JNK1mediated regulation of ADAR2 expression may represent a unique aspect of the nutrient-sensing events in b-cells, and glucose may be one of the various metabolic signals that contribute to JNK1 activation and ADAR2 upregulation in the state of overnutrition. In this scenario, it remains to be investigated whether JNK signaling exerts its deleterious effect upon b-cell functions through  affecting ADAR2-mediated RNA editing actions, particularly under the condition of obesity-related metabolic stress.
Gene-targeting studies in mice have revealed distinctive as well as redundant functions of the different JNK isoforms [46]. Deficiency of JNK1 but not JNK2 was reported to result in reduced adiposity and steatohepatitis as well as improved insulin sensitivity in mouse models of obesity [29,45]. While studies in pancreatic islets suggested important roles for both JNK1 and JNK2 in the survival and function of b-cells [32,47], the precise metabolic action of each JNK isoform is yet to be delineated. We observed that JNK1, instead of JNK2, mediates the glucoseresponsive upregulation of ADAR2 expression, providing another example for JNK isoform-selective regulatory action. In addition, JNK3 was shown to be expressed in human pancreatic islets, but given that knockdown of JNK3 increased c-Jun levels [48], it is unclear whether JNK3 contributes to the glucose-responsive regulation of ADAR2 in pancreatic b-cells.
Of note, it has been shown that JNK1 phosphorylates the transcription factor c-Jun for its activation, whereas JNK2 lacks this activity [49,50]. Consistently, we found that the mouse Adar2 promoter contains several potential binding sites for trans-factors including AP-1, which is likely to link JNK1 signaling to the upregulation of Adar2 transcription. The AP-1 family of transcription factors comprise homodimers and heterodimers of Jun, Fos or activating transcription factor (e.g. ATF2) protein [28]. It has been established that Jun-Jun and Jun-Fos dimers bind preferentially to a 12-O-tetradecanoylphorbol-13-acetate-responsive element (TGACTCA), whereas the Jun-ATF dimer can bind to the cAMP-responsive element (TGACGTCA) [51]. As ATF2 can also be directly phosphorylated by JNK [52], it remains to be deciphered whether a c-Jun-ATF2 complex is involved in mediating JNK1-dependent regulation of ADAR2 expression. Furthermore, as suggested by the presence of other putative sequence elements within the Adar2 promoter for transcriptional regulators such as glucocorticoid receptor (GR), CCAAT/ enhancer-binding protein a (C/EBPa) or D site of albumin promoter-binding protein (DBP), ADAR2 may be transcriptionally controlled in the context of a wide range of biological processes that remain to be defined.
The glucose-sensing mechanism is employed by many endocrine cells to control the secretion of hormones, which is best exemplified by glucose stimulation of insulin secretion and inhibition of glucagon secretion in pancreatic islets [53]. Moreover, glucose also regulates the activity of glucose-responsive neurons that are present in the brain stem and hypothalamus [54]. The glucose-excited or -inhibited neurons respond to changes in the local glucose concentration and play critical roles in glucose and energy homeostasis [53,55]. Documented studies in the brain have implicated ADAR2 in the control of energy balance [56], and it remains enigmatic whether ADAR2 exerts its actions in glucose-responsive neurons. In addition, many of the ADAR-targeted substrate RNAs encode key components of neurotransmission that are known to play crucial roles in the control of glucose and energy homeostasis, e.g. serotonin receptor-2C [57,58] and GABA [59]. Given the functional importance of JNK1 in the brain with respect to metabolic control [60,61] as well as the widespread A to I RNA editing events as recently revealed by the human editome analysis [62], it remains an open question whether the JNK pathway can respond to nutritional changes and exerts its regulatory action upon ADAR2-dependent RNA editing in specific nutrient-sensing neurons. As the JNK signaling cascade has been implicated in various physiological and pathological processes including inflammation, metabolic disorders and neurodegenerative diseases [46,63,64], the functional connection between the JNK pathway and ADAR2-mediated RNA editing warrants further investigations.

Supporting Information
Figure S1 JNK1 deletion protects mice against hyperglycemia resulting from HFD feeding. After a fast of 6 hours, blood glucose concentration was determined for JNK1 2/2 mice and their WT littermates, which were fed HFD or LFD for 16 weeks (n = 5-7/group). Data are shown as the mean6SEM. *P,0.05, **P,0.01. (TIF) Figure S2 The 219-nucleotide region of the mouse Adar2 promoter has similar glucose-responsive transcription activity as that of the rat insulin promoter. INS-1 cells were transfected for 40 hours with the P Adar2 -219-Luc or the 410nucleotide RIP-Luc reporter constructs. Luciferase activities were analyzed after cells were cultured at 2.8 mM or 16.7 mM glucose for 16 hours. Shown are fold increases in the luciferase activities upon high glucose stimulation after normalization to Renilla luciferase activity. Data are presented as the mean6SEM from three independent experiments. *P,0.05. (TIF)