The orphan nuclear receptor Nur77 regulates LKB1 ... - Nature

article published online: 16 september 2012 | doi: 10.1038/NChemBio.1069

The orphan nuclear receptor Nur77 regulates LKB1 localization and activates AMPK

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© 2012 Nature America, Inc. All rights reserved.

Yan-yan Zhan1,4, Yan Chen1,4, Qian Zhang1,4, Jia-jia Zhuang2, Min Tian1, Hang-zi Chen1, Lian-ru Zhang1, Hong-kui Zhang2, Jian-ping He1, Wei-jia Wang1, Rong Wu1, Yuan Wang1, Chunfang Shi1, Kai Yang1, An-zhong Li1, Yong-zhen Xin1, Terytty Yang Li1, James Y Yang1, Zhong-hui Zheng1, Chun-dong Yu1, Sheng-Cai Lin1, Chawnshang Chang3, Pei-qiang Huang2, Tianwei Lin1* & Qiao Wu1* Liver kinase B1 (LKB1) has important roles in governing energy homeostasis by regulating the activity of the energy sensor kinase AMP-activated protein kinase (AMPK). The regulation of LKB1 function, however, is still poorly understood. Here we demonstrate that the orphan nuclear receptor Nur77 binds and sequesters LKB1 in the nucleus, thereby attenuating AMPK activation. This Nur77 function is antagonized by the chemical compound ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA), which interacts with Nur77 with high affinity and at specific sites. TMPA binding of Nur77 results in the release and shuttling of LKB1 to the cytoplasm to phosphorylate AMPKa. Moreover, TMPA effectively reduces blood glucose and alleviates insulin resistance in type II db/db and high-fat diet– and streptozotocin-induced diabetic mice but not in diabetic littermates with the Nur77 gene knocked out. This study attains a mechanistic understanding of the regulation of LKB1-AMPK axis and implicates Nur77 as a new and amenable target for the design and development of therapeutics to treat metabolic diseases.

A

MPK is an evolutionarily conserved metabolic sensor crucial for the cellular energy homoeostasis1. In response to reducing energy supply, AMPK switches off ATP-consuming pathways, including those for fatty acid synthesis2 and gluconeogenesis3, and activates ATP-generating pathways, such as fatty acid oxidation and glycolysis4,5. AMPK activity and its mediated metabolic effects are regulated by several hormones, including leptin6, adiponectin7 and α-adrenergic hormones8, as well as antidiabetic drugs such as metformin and thiazolidinediones9. It is generally accepted that the pharmacological activation of AMPK is a useful approach to treat metabolic disorders associated with type II diabetes and obesity. AMPK normally forms a ternary complex, made up of the catalytic α subunit and the regulatory β and γ subunits10. Its signaling usually starts with the binding of AMP and ADP to the γ subunit, which triggers the conformational change in the ternary complex and promotes the full activation of AMPK via three independent mechanisms: (i) promoting Thr172 phosphorylation of AMPKα by various kinases11; (ii) inhibiting Thr172 dephosphorylation of AMPKα11 and (iii) allosterically activating AMPK with phosphorylation at Thr172 by binding AMP12. The first mechanism involves kinases, such as LKB1, calcium/calmodulin– dependent protein kinase β (CaMKKβ) or TGF-β–activated kinase 1 (TAK1). LKB1 (also known as STK11) is required for the activation of AMPK in response to cellular energy depletion and increased AMP/ATP ratio in peripheral tissues, such as the liver, skeletal muscle and adipose tissues13,14. CaMKKβ is particularly important for AMPK activation triggered by the increase of Ca2+, which can induce the depolarization of neurons in the rat brain15, whereas TAK1 (also known as MAP3K7 or MEKK7), a kinase downstream of cytokine receptors, has been implicated in phosphorylation and activation of AMPK at Thr172 by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which leads to

autophagy in epithelial cells and retinal pigment epithelial cells16. Therefore, phosphorylation of AMPK at the same site by different kinases may result in different physiological responses. More recently, the regulation of LKB1 by various nuclear receptors was reported. Retinoic acid receptor-related orphan receptor α (RORα) and its activator cholesterol sulfate induced the phosphorylation of LKB1 and the subsequent activation of AMPK by decreasing cellular ATP and increasing the NAD+/NADH ratio17. Farnesoid X receptor increased the expression of LKB1 by repressing miR-199a-3p, which targeted the LKB1 mRNA18. LKB1 expression was also regulated by estrogen receptor α (ERα) through direct binding to the putative estrogen response element in the LKB1 promoter region in human breast cancer cells19. Notably, LKB1 acted as a coactivator to activate estrogen receptor activity in response to the recruitment of ERα to the promoter of the ERα-responsive gene20. These studies have established the transcriptional and post-transcriptional links between LKB1 and nuclear receptors. Orphan nuclear receptor Nur77 (also known as TR3) belongs to the subfamily 4, group A (NR4A) of nuclear receptors21 and has important roles in metabolism and energy balance in a context-dependent manner. In skeletal muscle, Nur77 promotes glucose utilization by enhancing the activity of insulin to stimulate glucose transport22 and upregulating expression of multiple genes to activate the glucolysis pathway23. Expression of Nur77 is reduced in skeletal muscle of animal models, such as streptozotocin (STZ)-induced mice and mice with obesity and insulin resistance (ob/ob db/db)22. In contrast, expression of Nur77 in hepatocytes promotes gluconeogenesis. Higher Nur77 expression was demonstrated in the livers of mice with type I and type II diabetes, in which Nur77 promoted gluconeogenesis through binding Nur77 response elements in the promoter regions of a variety of genes and modulating their expression24. Mice with genetic deletion of Nur77 were more susceptible to diet-induced obesity and showed

State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China. 2Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, China. 3George H. Whipple Lab for Cancer Research, Departments of Pathology and Urology and the Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York, USA. 4These authors contributed equally to this work. *e-mail: [email protected] or [email protected] 1

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RESULTS Nur77 inhibits AMPKa activation via interaction with LKB1

To investigate the functional interaction between Nur77 and AMPK, we first knocked down Nur77 in cells under glucose starvation or metformin treatment, which showed that the depletion of Nur77 increased AMPK activity as shown by its phosphorylation of Thr172 in human LO2 hepatocytes (Fig. 1a). Conversely, overexpression of Nur77 repressed AMPKα phosphorylation (Fig. 1a). The results in the mouse model were consistent with the observations in vitro, in which AMPKα phosphorylation was elevated in the liver of Nur77 knockdown mice (Fig. 1b). Furthermore, in nondiabetic (m/m) mice and diabetic (db/db) mice, the inverse relationship between AMPKα phosphorylation and Nur77 expression was clear (Fig. 1b). These results led to the conclusion that Nur77 has a role in the suppression of AMPKα phosphorylation. However, there is no indication that Nur77 binds the AMPKαβγ trimer in total cell lysates in coexpression experiments (Supplementary Results, Supplementary Fig. 1a), suggesting that Nur77 modulation of AMPK activity might not occur through direct interaction but by modification of the activity of other proteins that control AMPK. LKB1 is the major kinase for AMPK phosphorylation in the liver14. In LKB1-deficient HeLa cells14, overexpression of Nur77 alone did not suppress the phosphorylation of AMPKα, but introduction of LKB1 led to the increase of AMPKα phosphorylation, and this LKB1-induced AMPKα phosphorylation was suppressed in cells cotransfected with Nur77 (Fig. 1c). Like LKB1, CaMKKβ is also a kinase for phosphorylation of AMPKα at Thr172 (ref. 15). However, overexpression of CaMKKβ could not rescue Nur77’s repressive function on AMPKα phosphorylation in HeLa cells (Fig. 1c). Therefore, the Nur77-induced suppression of AMPKα phosphorylation may be LKB1 dependent. Several lines of evidence implicate LKB1 in Nur77’s control of AMPKα phosphorylation. First, Nur77 bound LKB1 both endogenously and exogenously in different cell lines (Supplementary Fig. 1b), which perturbed the interaction between LKB1 and AMPKα in total cell lysates when they were coexpressed (Fig. 1d). As a negative control, RXRα did not show any interaction with LKB1 (Supplementary Fig. 1c). Second, the Nur77 interaction with LKB1 could be defined to a specific domain. It was the ligandbinding domain of Nur77 (LBD) that interacted with LKB1. Neither the transactivation domain nor the DNA-binding domain of Nur77 had such a role (Supplementary Fig. 1d). Because AMPKα was autoinhibitory and LKB1 could not interact with and phosphorylate 898

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reduced insulin resistance in both skeletal muscle and liver. The protective effect of Nur77 deletion on hepatic insulin sensitivity in normal diet–fed mice vanished when challenged with a high-fat diet, and these mice even developed hepatic steatosis and exacerbated insulin resistance in the liver25. Therefore, it is likely that the target genes and physiological functions of Nur77 are tissue dependent. We previously reported that the administration of cytosporone B, an agonist to Nur77, resulted in the elevation of blood glucose in C57BL/6J mice by the induction of a group of genes involved in gluconeogenesis26. Although some of the downstream effectors in gluconeogenetic activation have been determined, the underlying mechanism of Nur77 regulation of glucose metabolism is still to be investigated. In this study, we demonstrate that the phosphorylation of AMPKα in the cytoplasm by LKB1 is strongly suppressed by Nur77 binding and sequestering LKB1 in the nucleus. We have also identified TMPA as a new antidiabetic compound with high affinity to Nur77 and antagonism to Nur77’s interaction with LKB1, which leads to the subsequent shuttle of free LKB1 to the cytoplasm to phosphorylate AMPK. These findings not only result in a better understanding of the regulation of the LKB1-AMPK axis in controlling the glucose metabolism but also identify a new drug target of Nur77 for the treatment of metabolic disorders.

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Figure 1 | Nur77 interaction with LKB1 suppresses AMPKa phosphorylation. (a) Negative modulation of Nur77 in AMPKα phosphorylation. Nur77 in LO2 cells was knocked down by using lentivirusbased RNA interference or was overexpressed by transfection with FlagNur77 plasmids. The scrambled shRNA (shCtrl) or pCMV-Flag vector (Flag) was used as a control. After treatment with metformin (2 mM, 8 h) or glucose starvation (2 h), the amount of AMPKα phosphorylation was determined by western blotting with an antibody specific to the phosphorylated Thr172 of AMPKα. (b) Left, comparison of the amount of AMPKα phosphorylation in the liver of wild-type (WT; C57BL/6J background) and Nur77 knockout (KO) mice (C57BL/6J background) (n = 6). Right, the relationship between Nur77 expression and AMPKα phosphorylation in the liver of m/m mice (nondiabetic mice; C57BL/KsJ background) and db/db mice (C57BL/KsJ background) (n = 6). (c) Nur77 downregulation of AMPKα phosphorylation is LKB1 dependent and CaMKKβ independent. LKB1-null HeLa cells were transfected with hemagglutinin (HA)-Nur77 and Flag-LKB1 or Flag-CaMKKβ as indicated. The amount of AMPKα phosphorylation was determined by western blotting. (d) Transfection of Nur77 reduces the interaction of AMPKα and LKB1. 293T cells were cotransfected with HA-Nur77, HA-AMPKα and Flag-LKB1. Coimmunoprecipitation (IP) assay was performed with antibody against Myc. (e) Nur77 and LBD suppress AMPKα phosphorylation. Different expression vectors were introduced into LO2 cells as indicated. After transfection, total cell lysates were prepared and subjected to western blotting with antibody against phospho-AMPKα (p-AMPKα).

AMPKα directly without the regulatory subunits in vitro, we used a truncated version of AMPKα containing only residues 1–312 (AMPKα(1–312)) that could be directly phosphorylated by LKB1 without AMPKβ and AMPKγ27. As expected, the LBD alone was sufficient to affect the interaction between LKB1 and AMPKα(1–312) (Supplementary Fig. 1e), and the amount of LKB1-dependent AMPKα phosphorylation was greatly reduced by the addition of either the full-length Nur77 or LBD (Supplementary Fig. 1e). Third, introduction of Nur77 or the LBD into LO2 cells modulated the phosphorylation of AMPKα (Fig. 1e). Collectively, these data implicate Nur77’s interference in the LKB1 phosphorylation of AMPKα and support a model that Nur77 suppresses the activation of AMPKα by impairing LKB1 binding to AMPKα.

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Figure 2 | TMPA antagonizes the Nur77-LKB1 interaction. (a) The chemical structure of TMPA. (b) TMPA increases the amount of phosphorylation of AMPKα in a dose- and time-dependent manner. LO2 cells were treated with different doses of TMPA for 6 h (left) or at 10 μM for different durations (right). The amount of AMPKα phosphorylation was determined by western blotting. (c) TMPA increases phosphorylation of AMPKα via Nur77 mediation. Endogenous Nur77 in LO2 cells was knocked down by lentivirus-based RNA interference, and the cells were treated with TMPA at 10 μM for 6 h. AMPKα phosphorylation was detected (Supplementary Fig. 2a) and quantified by densitometry. Basal AMPKα phosphorylation (shCtrl) was normalized to one. Data are presented as the means ± s.e.m. from three independent experiments. Differences between groups were assessed by a twotailed unpaired Student’s t-test (*) or by a two-way ANOVA (#) using SPSS software. #P < 0.05; **P < 0.01; NS, nonsignificant versus control. (d) TMPA induces LKB1-dependent phosphorylation of AMPKα. HeLa cells were transfected with Flag-LKB1 and then treated with 10 μM TMPA for 6 h. AMPKα phosphorylation was detected. (e) TMPA treatment (10 μM, 6 h) rescues the LKB1-AMPKα interaction by reducing the Nur77-LKB1 interaction in LO2 cells, as detected by coimmunopreciptation assays. HC, heavy chain.

TMPA antagonizes the Nur77-LKB1 interaction

It is conceivable that small-molecule binding to Nur77 can modulate the interaction between Nur77 and LKB1 to alter the pattern of the subsequent LKB1 phosphorylation of AMPKα. A screen of an in-house chemical library of a series of compounds derived from cytosporone B unexpectedly identified a compound, TMPA (1) (Fig. 2a), which enhanced rather than downregulated the phosphorylation of AMPKα in hepatic LO2 cells in both dose- and time-dependent manners (Fig. 2b). When endogenous Nur77 was knocked down by shRNA, the amount of AMPKα phosphorylation rose, whereas TMPA lost its ability to elevate AMPKα phosphorylation (Fig. 2c and Supplementary Fig. 2a). Similarly, TMPA failed to elevate AMPKα phosphorylation in HeLa cells that are deficient in LKB1. In contrast, ectopic expression of LKB1 could lead to the HeLa cell response to TMPA, which is activation of AMPKα phosphorylation (Fig. 2d). Moreover, LKB1 was associated with both Nur77 and AMPKα in the total LO2 cell lysates, and TMPA enhanced the LKB1-AMPKα interaction but decreased the LKB1Nur77 interaction under physiological conditions (Fig. 2e). Because the absorption spectrum of TMPA overlaps with the emission spectrum of the LBD fluorescence (Supplementary Fig. 2b), TMPA can function as a quencher to the LBD fluorescence. It was shown that TMPA has a strong affinity for both Nur77 and LBD in the quenching experiments but not for monomeric or heterotrimeric AMPKα, AMPKβ, AMPKγ or LKB1 (Supplementary Fig. 2c). In addition, TMPA did not show any effect on Nur77 transactivation activity (Supplementary Fig. 2d). Together, these results strongly suggest that TMPA binding to Nur77 would impair the Nur77-LKB1 interaction.

with TMPA (Fig. 3 and Supplementary Fig. 3b). There are two sites in molecule I identified with comparable density for TMPA (Supplementary Fig. 3c). Site A is a shallow cleft formed among helices 4, 11 and 12, and site B is similar in size to site A and is formed among helices 1, 5, 7 and 8. The two sites are on the different faces of the LBD molecule and are close to the surface rather than in the ligand-binding pockets deep in the protein interior, as in the other nuclear receptors28–31. TMPA adopts different poses at the two different sites. At site A, the side chains of Arg515 and Glu445 are involved in an interaction with the carboxyl moiety of TMPA that is mediated by water molecules. The oxygen atoms of the three trimethoxy groups are involved in an interaction with the

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TMPA binding to LBD was investigated by X-ray crystallography. The structure of LBD was determined to 2.06-Å resolution by molecular replacement (Supplementary Table 1). There were two LBD molecules in the asymmetric unit. One of the conformations (molecule II) could be superimposed with a previously determined apo LBD structure (Protein Data Bank code (PDB) code 2QW4) that was used as the phasing model to solve the current structure. The other conformation (molecule I) included movements in several helices, notably helices 9 and 10 (Supplementary Fig. 3a). The complex structure of TMPA and LBD was determined to 2.20-Å resolution. Although the conformations of the apo- and TMPA-bound protein were essentially the same, only molecule I, whose conformation was different from that of the previous structure, was associated

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Figure 3 | TMPA binds LBD with a specific conformation. Ribbon diagram of LBD (molecule I in blue) in complex with two TMPA molecules at site A (yellow) and site B (pink). Residues involved in TMPA binding are shown in sticks, with oxygen atoms in red and nitrogen atoms in blue. Water molecules are indicated as red spheres. Hydrogen bonds are shown as gray dotted lines. H, helix.


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In LO2 cells and primary hepatic cells from wild-type mice, AMPKα is primarily in the cytoplasm, yet Nur77 and LKB1 generally localize in the nucleus (Supplementary Fig. 5a), which raises the question of how nuclear LKB1 phosphorylates cytoplasmic AMPKα. It was reported that phosphorylation of LKB1 at distinct sites had different roles in controlling its cellular localization, enzymatic activity and 900

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The critical Nur77 residues for LKB1 binding were further investigated by mutational and functional studies. As shown by the results from X-ray crystallography, three mutants in LBD were generated. LBD(A2) had a double mutation at site A, LBD(B3) involved triple point mutations at site B, and LBD(A2/B3) combined the changes in both LBD(A2) and LBD(B3) (Supplementary Table 1). The mutations did not perturb the overall structure, as shown by CD (Supplementary Fig. 4a). In addition, the mutants show similar unfolding responses to the native protein, as demonstrated by fluorescence quenching and the associated red shift (Supplementary Fig. 4b). Although the binding of TMPA was weakened to various degrees in LBD(A2) and LBD(B3), only LBD(A2/B3) completely lost the ability to interact with TMPA (Supplementary Fig. 4c), which is consistent with the crystallographic observations. To further corroborate the data shown above, two additional point mutations were made on the scaffold of LBD(B3), producing two mutants that could not bind TMPA at site B, which we named LBD(B3)C566R and LBD(B3)T595E (Supplementary Table 1). LBD(B3)C566R completely lost the ability to bind TMPA, as shown by fluorescence quenching experiments (Fig. 4a), indicating that the single mutation is sufficient to interfere with the TMPA binding at site A, but its binding affinity for LKB1 was not affected, as shown by Biolayer interferometry (Fig. 4b and Supplementary Fig. 4d). In contrast, although LBD(B3)T595E retained the ability to bind TMPA (Fig. 4a), its interaction with LKB1 was greatly weakened (Fig. 4b and Supplementary Fig. 4d). As the mutations in LBD(B3) only impair the binding of LKB1 marginally (Fig. 4b and Supplementary Fig. 4d) and Thr595 is at the upper edge of site A but apparently not critical for TMPA binding, it can be concluded that LKB1 interacts with LBD with direct contacts around site A, which can be disrupted by either TMPA binding or mutations at the region. Rescue assays in LO2 cells were also carried out with the full-length Nur77 as well as with its variants with mutations at the relevant residues (Nur77(B3)C566R and Nur77(B3)T595E in Supplementary Table 1). After more than 90% of endogenous Nur77 was knocked down, reintroduction of Nur77 into cells still inhibited AMPKα phosphorylation, which could be recovered by TMPA. However, Nur77(B3)T595E failed to inhibit AMPKα phosphorylation, and Nur77(B3)C566R lost its response to TMPA-induced activation of AMPKα phosphorylation (Fig. 4c), consistent with the findings that the interaction was minimal between Nur77(B3)T595E and LKB1 and between TMPA and Nur77(B3)C566R (Fig. 4d and Supplementary Fig. 4e). Molecular docking experiments also showed preferential LKB1 interaction at the face of the LBD molecule where site A was located (Supplementary Fig. 4f). Taken together, Thr595 is important for Nur77 interaction with LKB1, whereas a single mutation at Cys566 of Nur77 abolishes the interaction with TMPA and its antagonism.

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main chain atoms of Nur77 as well as the side chain of Thr595 that is mediated with a water molecule. In site B, the carbonyl oxygen of TMPA forms hydrogen bonds with the side chain of Lys456. The oxygen atom of the 3-trimethoxy group interacts with the side chain of His372. The carboxyl group of TMPA is involved in an inter action with the carbonyl oxygen of Arg450 that is mediated by a water molecule. Tyr453, Leu492 and Val498 provide a hydrophobic environment for TMPA (Fig. 3 and Supplementary Fig. 3c).

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Figure 4 | Critical residues of Nur77 for TMPA binding and LKB1 interaction. (a) TMPA binds LBD(B3)T595E but not LBD(B3)C566R. The fluorescence spectra were obtained for various mutation proteins (5 μM) with increasing amounts of TMPA. Binding affinity (Kd) is represented as means ± s.e.m. of three independent reactions. R2 is the coefficient of determination. (b) Binding kinetic analysis of GST-LKB1 to His-LBD or mutants by BioLayer interferometry. A comparison for the binding abilities between LBD and its mutants with the same concentration of GST-LKB1 (1.778 μM) from Supplementary Figure 4d is shown. Biotinylated His-LBD and its mutants were immobilized to streptavidin-coated biosensors to analyze binding to GST-LKB1, and the binding affinities are calculated using a 1:1 model. The average Kd is extracted from binding results for five different concentrations of the analytes using OctetRed system (Supplementary Fig. 4d). (c) Differential induction of AMPKα phosphorylation by Nur77 and its mutants in the presence of TMPA. Endogenous Nur77 in LO2 cells was first knocked down by lentivirus-based RNA interference, and Nur77 or its mutants were retransfected into cells as indicated. After treatment of cells with TMPA (10 μM, 6 h), the amount of AMPKα phosphorylation was determined. (d) Nur77(B3)T595E does not bind LKB1. Flag-Nur77 or its mutants, together with Myc-LKB1 and Flag-AMPKα, were cotransfected into 293T cells and subjected to coimmunoprecipitation (co-IP) assays.

other biological functions32–35. Transfection and expression of Nur77 inhibited LKB1 phosphorylation at Ser428 but not at Thr189, Ser307 or Ser334 in LO2 cells. In contrast, TMPA treatment led to an increase of LKB1 phosphorylation at Ser428 (Fig. 5a), implicating that the Nur77- and TMPA-associated AMPK activity is dependent on Ser428 phosphorylation of LKB1. To verify that this TMPA role is Nur77 dependent in vivo, diabetic Nur77-null mice (db/db Nr4a1−/−) were generated by crossing db/db mice and Nur77 knockout (Nr4a1−/−) mice. As shown in Figure 5b, TMPA could no longer induce LKB1 phosphorylation in the liver of db/db Nr4a1−/− mice, unlike in the liver of db/db Nr4a1+/+ mice. As a result, Nur77 inhibited and TMPA activated AMPKα phosphorylation in LO2 cells and db/db mice, respectively (Fig. 5a,b). Our data also exclude the possibility that Nur77 is a substrate for LKB1 phosphorylation (Supplementary Fig. 5b).


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Figure 5 | TMPA affects subcellular localization of LKB1 through phosphorylation. (a) Nur77 and TMPA influence LKB1 phosphorylation at Ser428 in LO2 cells. Left, transfection of Nur77 inhibited the phosphorylation of LKB1 at Ser428. Right, TMPA treatment (6 h) elevated the LKB1 phosphorylation at Ser428. Phosphorylations at different LKB1 sites were indicated by using corresponding antibodies specific for phosphorylation. (b) Phosphorylation of LKB1 induced by TMPA is dependent on Nur77. Liver samples were taken from fasting db/db Nr4a1+/+ and their littermate db/db Nr4a1−/− mice (n = 6). The amount of phosphorylation of LKB1 was determined by western blotting. (c) Subcellular localization of LKB1 in the livers of mice. The amount of LKB1 was determined by western blotting using an LKB1-specific antibody. The similar liver samples as above were used. C, cytosol; N, nucleus. (d) Mutation of Ser428 site of LKB1 (S428A) or deletion of nuclear localization sequence (ΔNLS) attenuated the effect of Nur77 (top) or TMPA (bottom) on AMPKα phosphorylation as compared to the wild-type LKB1. Different plasmids indicated were transfected into HeLa cells, then the cells were treated with TMPA (10 μM, 6 h). Cell lysates were subjected to western blotting.

The potency of metformin, a drug that lowers glucose in blood, mainly originates from the phosphorylation of LKB1 at Ser428, which results in trafficking LKB1 from the nucleus to the cytoplasm for the subsequent phosphorylation of AMPK33. As Nur77 also affects LKB1 phosphorylation primarily at Ser428, it is possible that the function of TMPA is to release LKB1 by disrupting its interactions with Nur77 for the subsequent LKB1 translocation to the cytoplasm to phosphorylate AMPKα. Indeed, higher cytoplasmic LKB1 was detected in the liver of db/db Nr4a1−/− mice than in the liver of db/db mice, and an increase of cytoplasmic LKB1 in the liver of db/db mice was observed in response to TMPA administration (Fig. 5c). The nuclear location of LKB1 in intact HeLa or LO2 cells can be observed by confocal microscopy (Supplementary Fig. 5c). Moreover, an obvious overlap between Nur77 and LKB1 in the nucleus of intact HeLa or LO2 cells was detected. When the cells were treated by TMPA, LKB1 but not Nur77 was trafficked to the cytoplasm (Supplementary Fig. 5c). However, when Ser428 was mutated to alanine (S428A), the mutant located mainly in the nucleus of HeLa cells, as expected, and the TMPA-induced nucleo cytoplasmic translocation was not observed (Supplementary Fig. 5c). Consequently, Nur77-inhibited or TMPA-induced AMPKα phosphorylation could not be detected (Fig. 5d). It was also demonstrated that Nur77 and LKB1 formed a complex primarily in the nucleus, and TMPA antagonized the formation of the LKB1–Nur77 complex in the nucleus while enhancing the formation of the cytoplasmic complex of LKB1–AMPKα (Supplementary Fig. 5d). These results support the notion that

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the binding of TMPA to Nur77 results in the LKB1 release and exit from the nucleus to phosphorylate cytoplasmic AMPKα. LKB1 has a nuclear location signal (NLS)36. To further demonstrate the role of LKB1 nucleocytoplasmic shuttling in mediating the effect of Nur77 and TMPA, an NLS-deletion mutant of LKB1 (ΔNLS) was generated. This ΔNLS localized in the cytoplasm of HeLa cells (Supplementary Fig. 5c) and elevated the amount of AMPKα phosphorylation with or without either Nur77 or TMPA (Fig. 5d). Moreover, LKB1 could interact with either STRAD or Mo25, two accessory proteins essential in activating LKB1 activity and facilitating LKB1 nucleocytoplasmic translocation37, which was enhanced by TMPA but impaired by Nur77 (Supplementary Fig. 5e). The regulatory mechanism of Nur77 for STRAD and the Mo25 interaction with LKB1 are beyond the scope of this study and awaits further investigation. LKB1 has the ability to phosphorylate several other AMPKrelated kinases besides AMPK, some of which have overlapping functions with AMPK in metabolism, such as MARK38. We investigated the possibility that TMPA modulates the activity of MARK. Although TMPA increased the phosphorylation levels of MARK, its effect was marginal compared to that of AMPKα (Supplementary Fig. 5f), which, although it did not discount the possibility of TMPA’s influence on other LKB1-regulated kinases, demonstrated the importance of activating AMPKα by TMPA on modulating the metabolic processes in liver.

TMPA effectively lowers blood glucose in diabetic mice

Activation of AMPK in liver can efficiently inhibit gluconeogenesis to control the blood glucose1,39. The effect of TMPA on the amount of blood glucose in fasting type II diabetic mice was assessed. As shown in Figure 6a, a significant (P > 0.05, P < 0.01, P < 0.01, P < 0.01 and P < 0.05 for day 0, 7, 11, 15 and 19, respectively) reduction in blood glucose was observed in the TMPA-treated group starting at day 7. The hypoglycemic effects of TMPA were comparable to those of metformin and persisted during the remainder of the test. After 19 d, both TMPA-treated and metformin-treated mice were able to metabolize glucose more efficiently at all time points than the vehicle-treated mice (Fig. 6b), and TMPA was of the same potency as metformin. Moreover, in comparison with vehicle-treated mice, TMPA-treated mice had lower insulin (Fig. 6c), suggesting an improvement in insulin resistance. TMPA administration increased the amount of phosphorylated AMPKα in the liver of mice from each treatment group (Supplementary Fig. 6a), and the mRNA levels from the genes of glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (Pepck), two critical gluconeogenic genes, were substantially downregulated by TMPA in the same samples, probably as a consequence of AMPKα activation (Supplementary Fig. 6b). Clearly, TMPA is capable of lowering blood glucose and improving glucose tolerance in type II diabetic mice. To better understand the role of Nur77 in the TMPA-regulated glucose metabolism, we examined the amount of blood glucose in db/db mice and db/db Nr4a1−/− mice. From week 6, the amount of blood glucose in the mice were tracked, and both db/db Nr4a1+/+ and db/db Nr4a1−/− mice (C57BL/6J background) showed relatively lower glucose than age-matched db/db mice (C57BL/KsJ background), which was consistent with the data from a report using C57BL/6J-Leprdb mice40. The db/db Nr4a1−/− mice had much lower glucose compared to the db/db Nr4a1+/+ mice owing to the knockout of Nur77-encoding gene (Nr4a1) and were very sensitive to fasting. It is clear that Nr4a1 knockout directly reduces the glucose of type II diabetic mice. After mice were treated with TMPA by intraperitoneal injection once daily for 19 d, a significant (P = 0.011) hypoglycemic effect of TMPA was observed in the db/db mice but not in the db/db Nr4a1−/− mice (Fig. 6d). Similarly, TMPA was able to increase AMPKα phosphorylation (Supplementary Fig. 6c) and repress mRNA expression of G6pc and Pepck (Supplementary Fig. 6d) in the livers of db/db mice but not in db/db Nr4a1−/− mice.


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The roles of TMPA on glucose amounts and insulin responses were further investigated by using nongenetic obese mice as another model for type II diabetes, which were generated by a combination

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Figure 6 | TMPA is capable of lowering blood glucose in diabetic mice. (a) Effect of TMPA on blood glucose of db/db mice. The male db/db mice were separated into three groups and injected intraperitoneally with vehicle (negative control), TMPA (treatment group) or metformin (positive control) daily for 19 consecutive days. Blood glucose was measured after 18 h of fasting. (b) Glucose-tolerance test in db/db mice. At day 19, the mice described above were injected intraperitoneally with D-glucose, and blood glucose was then measured at indicated times. (c) The plasma insulin of db/db mice was measured in each group as in a. Plasma samples were prepared as described in the Methods section. (d) TMPA lowers blood glucose in db/db mice (Nr4a1+/+) but not db/db Nr4a1−/− mice. The amount of blood glucose after 8 h of fasting was measured. (e) TMPA lowers blood glucose in wild-type (left, Nr4a1+/+) but not Nr4a1−/− (right) mice with type II diabetes induced by HFD and STZ. The amount of blood glucose after 12 h of fasting was measured. Representatives of three independent experiments with similar results are shown in the above experiments. Data are presented as the means ± s.e.m. *P < 0.05; **P < 0.01; NS, not significant versus control. (f) A proposed model to illustrate the regulation of AMPKα phosphorylation by Nur77-LKB1 interaction and the possible functions of TMPA in the regulation of gluconeogenesis and diabetes. 9 02

of high-fat diet (HFD) and low-dose STZ treatment, as described previously41,42. This model was shown to be of closer resemblance to humans with the same type of disease41,42. TMPA lowered the blood glucose in HFD- and STZ-induced diabetic mice (Fig. 6e), with an increase of phosphorylated AMPKα and decrease of G6pc and Pepck expression (Supplementary Fig. 6e,f), which were not observed in the corresponding Nr4a1−/− diabetic mice. These results are consistent with those from the db/db mice and support the notion that the TMPA-induced hypoglycemic effect involves Nur77. It also indicates that TMPA has a greater influence on glucose levels and insulin responses in diet-induced obesity.

DISCUSSION

Nur77 has multiple effects on glucose metabolism. It was reported to stimulate gluconeogenesis in liver as a transcriptional modulator24, promote glucose utilization through the upregulation of gene expression23 and enhance insulin sensitivity in skeletal muscle22. In addition, Nr4a1−/− mice showed increased susceptibility to dietinduced obesity and insulin resistance25. In the current study, the db/db Nr4a1−/− mice are shown to have substantially lower blood glucose compared to the corresponding db/db mice, and the former mice are also very sensitive to fasting. These results indicate that Nur77 has a variety of regulatory roles, some of which can even have diametrically opposing effects in the glucose metabolism of different tissues or under different stimuli. We describe here a mechanism involving Nur77 in the negative regulation of AMPK activity mediated by the major upstream kinase LKB1. Although Nur77 does not interact directly with AMPKα, Nur77 downregulates AMPKα phosphorylation by interacting with and sequestering LKB1 in the nucleus to reduce cytoplasmic AMPKα phosphorylation (Fig. 6f). This mechanism of regulating glucose homeostasis suggests a new strategy to battle insulin resistance by controlling the Nur77-LKB1 interaction. In addition, it is advantageous to use antagonists to Nur77, such as TMPA, to treat type II diabetes. TMPA is target-specific and not only lowers blood glucose but also improves glucose tolerance and insulin resistance as demonstrated in db/db mice. By directly binding to Nur77, TMPA is able to disrupt the interaction between Nur77 and LKB1, resulting in increased trafficking of nuclear LKB1 to phosphorylate the cytoplasmic AMPKα (Fig. 6f). LKB1 is phosphorylated at Thr189, Ser307, Ser334 and Ser428 by upstream kinases. Phosphorylation of LKB1 at these different sites has different roles in controlling LKB1 activity, cellular localization and other biological functions32–35. Although phosphorylation of LKB1 at Ser307 is required for metformin-induced LKB1 cytosolic localization in endothelial cells35, it is evident that LKB1 phosphorylation at Ser307 has a negative role in adiponectinstimulated AMPK activation by sequestering LKB1 in the nucleus of C2C12 mouse myoblast cells43. In the current case, TMPA shows no effect on Ser307 phosphorylation of LKB1 in liver and glucose metabolism, but phosphorylation at Ser428 was effectively influenced by TMPA binding to Nur77. The phosphorylation of LKB1 at Ser428 has been reported to be required for AMPK activation by metformin in bovine aortic endothelial cells33 and by glucagon in perfused rat liver44. It is the metformin-induced phosphorylation at Ser428 that enhances LKB1 translocation from the nucleus to the cytoplasm, with the subsequent LKB1 phosphorylation of its downstream targets such as AMPK. The TMPA-associated increase of LKB1 translocation from the nucleus to the cytoplasm also depends on Ser428 phosphorylation, but via a different mechanism. TMPA interacts with Nur77 to release LKB1 sequestered in the nucleus to exercise its kinase activity in the cytoplasm. In summary, we have uncovered a critical mechanism for regulating the activity of the LKB1-AMPK axis in response to stimuli. Orphan nuclear receptor Nur77 is implicated as an important regulatory factor for interfering in type II diabetes, and TMPA is


Nature chemical biology doi: 10.1038/NChemBio.1069 identified as a promising drug candidate to lower blood glucose without increasing insulin secretion.

METHODS

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Cell culture, transfection and drug treatment. Human embryonic kidney HEK293T cells were purchased from the Institute of Cell Biology (China). Normal human liver (LO2) cells, human cervical cancer HeLa cells, human colon cancer (HCT116) cells and human non-small-cell lung carcinoma (H1299) cells were purchased from the American Type Culture Collection. Cells were cultured in RPMI1640 medium (for LO2 and HCT116 cell lines) or DMEM (for all other cell lines), supplemented with 10% (v/v) FBS (Hyclone), 100 U penicillin and 100 μg ml−1 streptomycin (all from Invitrogen). Transfection was performed using GeneJuice transfection reagent (Novagen) according to the manufacturer’s instructions, and cells were harvested 36 h after transfection. Synthetic TMPA was dissolved in DMSO, which was used as the vehicle control. Metformin (Sigma) was dissolved in PBS as a positive control. Before drug treatment, the medium was exchanged with low-glucose DMEM without the addition of serum. For glucose starvation, cells were rinsed three times with PBS and then exposed to glucose-free DMEM (Invitrogen). Mouse models. The experiments were carried out in and approved by Xiamen University Laboratory Animal Center, Xiamen University. Male mice were used in all experiments. (i) db/db mice: Ten-week-old male C57BL/KsJ-Leprdb/Leprdb (db/db) mice with diabetic features (provided by the Model Animal Research Center of Nanjing University) were randomly assigned into three groups. TMPA powder was dissolved in DMSO to a final concentration of 1 M and then dissolved in 5.0% (v/v) Tween-80 in 0.9% (w/v) saline. One group of the mice was injected intraperitoneally with TMPA once daily at a dose of 50 mg per kg mouse body weight for 19 d. The other groups were injected with metformin (250 mg per kg mouse body weight dissolved in normal saline) or vehicle (DMSO in 5.0% (v/v) Tween-80 in 0.9% (w/v) saline) as the positive and negative controls, respectively. Before blood glucose measurement, the mice were fasted for 18 h. The blood glucose of the mice was assayed every 4 d using an OneTouch Ultra glucometer (LifeScan). After 19 d of treatment, mice were injected intraperitoneally with D-glucose (1 g per kg mouse body weight) for the glucose tolerance test. Finally, the mice were fasted for 5 h before blood was sampled from the retro-orbital sinus on day 20. Mice were then killed, and the liver samples were used in real-time PCR and western blot analyses. Plasma was obtained after centrifugation at 3,000g and was estimated for plasma insulin using the rat and mouse insulin ELISA kit (Millipore). (ii) Crossed mice: For wild-type and Nr4a1−/− mice with type II diabetes, the heterozygous (db/m; C57BL/6J) mice were crossed with Nr4a1 knockout mice (Nr4a1−/−, C57BL/6J) for five generations to produce db/db Nr4a1+/+ and their littermate db/db Nr4a1−/− mice (genotypes of mice were determined through genotyping PCR). From week 6, the blood glucose of the mice was monitored. At 10 weeks of age, the mice were treated with TMPA by intraperitoneal injection once daily for 19 d, and then the liver samples from the mice were used in real-time PCR, western blotting and the nuclear and cytoplasmic protein fractional analyses. (iii) HFD- and STZ-induced mice with type II diabetes: For diabetes induction, wild-type and Nr4a1−/− C57BL/6J mice (8-week-old mice, provided by the Jackson Laboratory) were fed with a HFD (Research Diets) containing 60% kcal. After 4 weeks of dietary manipulation, mice were treated with a low dose of STZ (Sigma; 35 mg per kg body weight dissolved in 0.1 M sodium citrate buffer (pH 4.5)) once daily for 7 d. To evaluate whether the type II diabetes mice model was successfully established, the blood glucose of the mice was monitored, by which the frank hyperglycemia was observed in HFD- and STZ-induced mice but not in normal diet-fed mice after STZ treatment. After that, the mice were treated with TMPA by intraperitoneal injection once daily for 19 d, and then the liver samples from the mice were used in real-time PCR and western blot analyses. Other methods. Detailed descriptions of the other methods used in this study, including crystallization, data collection and processing, molecular modeling for LKB1 binding to Nur77, plasmid constructions, antibody and drug use, knockdown of Nur77 by lentivirus-based RNA interference, preparation of nuclear and cytoplasmic fractions from tissue, measurement of protein-compound binding affinity by fluorescence quenching assay, measurement of protein-protein binding affinity using BioLayer interferometry, CD spectroscopy, coimmunoprecipitation and western blot analysis, luciferase reporter assay, immunofluorescence staining, the in vitro glutathione S-transferase pull-down assay, the in vitro kinase assay, real-time PCR, statistical analysis, synthesis of TMPA and structure characterization for TMPA are provided in the Supplementary Methods. Accession codes. PDB: the crystal structures of the human Nur77 LBD alone and in complex are deposited under accession codes 3V3E and 3V3Q.

Received 28 February 2012; accepted 21 August 2012; published online 16 September 2012; corrected online 28 September 2012

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Acknowledgments

We are grateful to P. Li (Tsinghua University) for the plasmids encoding AMPKβ, AMPKγ and AMPKα(1–312) and to J. Wu (Tsinghua University) for the tricistronic expression plasmids for all three subunits of AMPK. This work was supported by the grants from ‘973’ Project of the Ministry of Science and Technology (2011CB910802), the National Natural Science Fund of China (30810103905, 30630070, 30921005 and 30870479), the Program of Introducing Talents of Discipline to Universities (B12001 and B06016) and the Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University (SKLCSB2012KF002). We also gratefully acknowledge the use of Beamline BL17U1 at Shanghai Synchrotron Radiation Facility for crystallographic data collection.

Author contributions

Q.W. and T.L designed the experiments and wrote the manuscript. Y.Z., Y.C., M.T., H.C., J.H., W.W., R.W., Y.W., A.L., Y.X. and T.Y.L. carried out the experiments on molecular and cellular biology as well as studies on mouse models. Q.Z., L.Z., C.S. and K.Y. carried out the structural studies. J.Z. and H.Z. synthesized the chemical compounds, and S.-C.L., P.H., C.C., J.Y.Y., Z.Z. and C.Y. were involved in the design of this project as well as in reading and commenting on the manuscript, and S.-C.L. helped on writing the final version.

Competing financial interests

The authors declare no competing financial interests.

Additional information

Supplementary information and chemical compound information is available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Correspondence and requests for materials should be addressed to Q.W. or T.L.

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