👤 Eurim C Hwang

G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power. Show less

Peripheral blood mononuclear cell (PBMC)-derived gene signatures were investigated for their potential use in the early detection of non-small cell lung cancer (NSCLC). In our study, 187 patients with NSCLC and 310 age- and gender-matched controls, and an independent set containing 29 patients for validation were included. Eight significant NSCLC-associated genes were identified, including DUSP6, EIF2S3, GRB2, MDM2, NF1, POLDIP2, RNF4, and WEE1. The logistic model containing these significant markers was able to distinguish subjects with NSCLC from controls with an excellent performance, 80.7% sensitivity, 90.6% specificity, and an area under the receiver operating characteristic curve (AUC) of 0.924. Repeated random sub-sampling for 100 times was used to validate the performance of classification training models with an average AUC of 0.92. Additional cross-validation using the independent set resulted in the sensitivity 75.86%. Furthermore, six age/gender-dependent genes: CPEB4, EIF2S3, GRB2, MCM4, RNF4, and STAT2 were identified using age and gender stratification approach. STAT2 and WEE1 were explored as stage-dependent using stage-stratified subpopulation. We conclude that these logistic models using different signatures for total and stratified samples are potential complementary tools for assessing the risk of NSCLC. Show less

Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions. Show less

Genome-wide association studies have been used extensively to identify genetic variants linked to metabolic syndrome (MetS), but most of them have been conducted in non-Asian populations. This study aimed to evaluate the association between MetS and previously studied single nucleotide polymorphisms (SNPs), and their interaction with health-related behavior in Korean men. Seventeen SNPs were genotyped and their association with MetS and its components was tested in 1193 men who enrolled in the study at Seoul National University Hospital. We found that rs662799 near APOA5 and rs769450 in APOE had significant association with MetS and its components. The SNP rs662799 was associated with increased risk of MetS, elevated triglyceride (TG) and low levels of high-density lipoprotein, while rs769450 was associated with a decreased risk of TG. The SNPs showed interactions between alcohol drinking and physical activity, and TG levels in Korean men. We have identified the genetic association and environmental interaction for MetS in Korean men. These results suggest that a strategy of prevention and treatment should be tailored to personal genotype and the population. Show less

Kaempferol is a dietary flavonol previously shown to regulate cellular lipid and glucose metabolism. However, its molecular mechanisms of action and target proteins have remained elusive, probably due to the involvement of multiple proteins. This study investigated the molecular targets of kaempferol. Ligand binding of kaempferol to liver X receptors (LXRs) was quantified by time-resolved fluorescence resonance energy transfer and surface plasmon resonance analyses. Kaempferol directly binds to and induces the transactivation of LXRs, with stronger specificity for the β-subtype (EC50 = 0.33 μM). The oral administration of kaempferol in apolipoprotein-E-deficient mice (150 mg/day/kg body weight) significantly reduced plasma glucose and increased high-density lipoprotein cholesterol levels and insulin sensitivity compared with the vehicle-fed control. Kaempferol also reduced plasma triglyceride concentrations and did not cause liver steatosis, a common side effect of potent LXR activation. In immunoblotting analysis, kaempferol reduced the nuclear accumulation of sterol regulatory element-binding protein-1 (SREBP-1). Our results show that the suppression of SREBP-1 activity and the selectivity for LXR-β over LXR-α by kaempferol contribute to the reductions of plasma and hepatic triglyceride concentrations in mice fed kaempferol. They also suggest that kaempferol activates LXR-β and suppresses SREBP-1 to enhance symptoms in metabolic syndrome. Show less

Hepatic resection is the most curative treatment option for early-stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early-stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high-copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer-specific and recurrence-free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti-FGF19 treatment in these patients. Show less

The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium. Show less

The perturbation of the steady state of reactive oxygen species (ROS) due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 (responsive to ABA protein 21) promoter with/without a selection marker, the bar gene. A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol (DTT). An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice. Show less

The glucagon (GCG) peptide family consists of GCG, glucagon-like peptide 1 (GLP1), and GLP2, which are derived from a common GCG precursor, and the glucose-dependent insulinotropic polypeptide (GIP). These peptides interact with cognate receptors, GCGR, GLP1R, GLP2R, and GIPR, which belong to the secretin-like G protein-coupled receptor (GPCR) family. We used bioinformatics to identify genes encoding a novel GCG-related peptide (GCRP) and its cognate receptor, GCRPR. The GCRP and GCRPR genes were found in representative tetrapod taxa such as anole lizard, chicken, and Xenopus, and in teleosts including medaka, fugu, tetraodon, and stickleback. However, they were not present in mammals and zebrafish. Phylogenetic and genome synteny analyses showed that GCRP emerged through two rounds of whole genome duplication (2R) during early vertebrate evolution. GCRPR appears to have arisen by local tandem gene duplications from a common ancestor of GCRPR, GCGR, and GLP2R after 2R. Biochemical ligand-receptor interaction analyses revealed that GCRP had the highest affinity for GCRPR in comparison to other GCGR family members. Stimulation of chicken, Xenopus, and medaka GCRPRs activated Gαs-mediated signaling. In contrast to chicken and Xenopus GCRPRs, medaka GCRPR also induced Gαq/11-mediated signaling. Chimeric peptides and receptors showed that the K(16)M(17)K(18) and G(16)Q(17)A(18) motifs in GCRP and GLP1, respectively, may at least in part contribute to specific recognition of their cognate receptors through interaction with the receptor core domain. In conclusion, we present novel data demonstrating that GCRP and GCRPR evolved through gene/genome duplications followed by specific modifications that conferred selective recognition to this ligand-receptor pair. Show less

Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, which catalyzes the formation of cyclic adenosine-3',5'-monophosphate (cAMP). However, our transcriptome analysis of gastric cancer tissue samples (NCBI GEO GSE30727) revealed that ADCY3 expression was specifically altered in cancer samples. Here we investigated the tumor-promoting effects of ADCY3 overexpression and confirmed a significant correlation between the upregulation of ADCY3 and Lauren's intestinal-type gastric cancers. ADCY3 overexpression increased cell migration, invasion, proliferation, and clonogenicity in HEK293 cells; conversely, silencing ADCY3 expression in SNU-216 cells reduced these phenotypes. Interestingly, ADCY3 overexpression increased both the mRNA level and activity of matrix metalloproteinase 2 (MMP2) and MMP9 by increasing the levels of cAMP and phosphorylated cAMP-responsive element-binding protein (CREB). Consistent with these findings, treatment with a protein kinase A (PKA) inhibitor decreased MMP2 and MMP9 expression levels in ADCY3-overexpressing cells. Knockdown of ADCY3 expression by stable shRNA in human gastric cancer cells suppressed tumor growth in a tumor xenograft model. Thus, ADCY3 overexpression may exert its tumor-promoting effects via the cAMP/PKA/CREB pathway. Additionally, bisulfite sequencing of the ADCY3 promoter region revealed that gene expression was reduced by hypermethylation of CpG sites, and increased by 5-Aza-2'-deoxycytidine (5-Aza-dC)-induced demethylation. Our study is the first to report an association of ADCY3 with gastric cancer as well as its tumorigenic potentials. In addition, we demonstrate that the expression of ADCY3 is regulated through an epigenetic mechanism. Further study on the mechanism of ADCY3 in tumorigenesis will provide the basis as a new molecular target of gastric cancer. Show less

Protein activities controlled by receptor protein tyrosine phosphatases (RPTPs) play comparably important roles in transducing cell surface signals into the cytoplasm by protein tyrosine kinases. Previous studies showed that several RPTPs are involved in neuronal generation, migration, and axon guidance in Drosophila, and the vertebrate hippocampus, retina, and developing limbs. However, whether the protein tyrosine phosphatase type O (ptpro), one kind of RPTP, participates in regulating vertebrate brain development is largely unknown. We isolated the zebrafish ptpro gene and found that its transcripts are primarily expressed in the embryonic and adult central nervous system. Depletion of zebrafish embryonic Ptpro by antisense morpholino oligonucleotide knockdown resulted in prominent defects in the forebrain and cerebellum, and the injected larvae died on the 4th day post-fertilization (dpf). We further investigated the function of ptpro in cerebellar development and found that the expression of ephrin-A5b (efnA5b), a Fgf signaling induced cerebellum patterning factor, was decreased while the expression of dusp6, a negative-feedback gene of Fgf signaling in the midbrain-hindbrain boundary region, was notably induced in ptpro morphants. Further analyses demonstrated that cerebellar defects of ptpro morphants were partially rescued by inhibiting Fgf signaling. Moreover, Ptpro physically interacted with the Fgf receptor 1a (Fgfr1a) and dephosphorylated Fgfr1a in a dose-dependant manner. Therefore, our findings demonstrate that Ptpro activity is required for patterning the zebrafish embryonic brain. Specifically, Ptpro regulates cerebellar formation during zebrafish development through modulating Fgf signaling. Show less

Sudden cardiac death due to malignant ventricular arrhythmia is a devastating manifestation of cardiac hypertrophy. Sarcomere protein myosin binding protein C is functionally related to cardiac diastolic function and hypertrophy. Zebrafish is a better model to study human electrophysiology and arrhythmia than rodents because of the electrophysiological characteristics similar to those of humans. We established a zebrafish model of cardiac hypertrophy and diastolic dysfunction by genetic knockdown of myosin binding protein C gene (mybpc3) and investigated the electrophysiological phenotypes in this model. We found expression of zebrafish mybpc3 restrictively in the heart and slow muscle, and mybpc3 gene was evolutionally conservative with sequence homology between zebrafish and human mybpc3 genes. Zebrafish with genetic knockdown of mybpc3 by morpholino showed ventricular hypertrophy with increased myocardial wall thickness and diastolic heart failure, manifesting as decreased ventricular diastolic relaxation velocity, pericardial effusion, and dilatation of the atrium. In terms of electrophysiological phenotypes, mybpc3 knockdown fish had a longer ventricular action potential duration and slower ventricular diastolic calcium reuptake, both of which are typical electrophysiological features in human cardiac hypertrophy and heart failure. Impaired calcium reuptake resulted in increased susceptibility to calcium transient alternans and action potential duration alternans, which have been proved to be central to the genesis of malignant ventricular fibrillation and a sensitive marker of sudden cardiac death. mybpc3 knockdown in zebrafish recapitulated the morphological, mechanical, and electrophysiological phenotypes of human cardiac hypertrophy and diastolic heart failure. Our study also first demonstrated arrhythmogenic cardiac alternans in cardiac hypertrophy. Show less

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important roles in insulin secretion through their receptors, GLP1R and GIPR. Although GLP-1 and GIP are attractive candidates for treatment of type 2 diabetes and obesity, little is known regarding the molecular interaction of these peptides with the heptahelical core domain of their receptors. These core domains are important not only for specific ligand binding but also for ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R/GIPR, we determined that evolutionarily conserved amino acid residues such as Ile(196) at transmembrane helix 2, Leu(232) and Met(233) at extracellular loop 1, and Asn(302) at extracellular loop 2 of GLP1R are responsible for interaction with ligand and receptor activation. Application of chimeric GLP-1/GIP peptides together with molecular modeling suggests that His(1) of GLP-1 interacts with Asn(302) of GLP1R and that Thr(7) of GLP-1 has close contact with a binding pocket formed by Ile(196), Leu(232), and Met(233) of GLP1R. This study may provide critical clues for the development of peptide and/or nonpeptide agonists acting at GLP1R. Show less

Screening of matrix metalloproteinase (MMP)-14 substrates in human plasma using a proteomics approach previously identified apolipoprotein A-IV (apoA-IV) as a novel substrate for MMP-14. Here, we show that among the tested MMPs, purified apoA-IV is most susceptible to cleavage by MMP-7, and that apoA-IV in plasma can be cleaved more efficiently by MMP-7 than MMP-14. Purified recombinant apoA-IV (44-kDa) was cleaved by MMP-7 into several fragments of 41, 32, 29, 27, 24, 22 and 19 kDa. N-terminal sequencing of the fragments identified two internal cleavage sites for MMP-7 in the apoA-IV sequence, between Glu(185) and Leu(186), and between Glu(262) and Leu(263). The cleavage of lipid-bound apoA-IV by MMP-7 was less efficient than that of lipid-free apoA-IV. Further, MMP-7-mediated cleavage of apoA-IV resulted in a rapid loss of its intrinsic anti-oxidant activity. Based on the fact that apoA-IV plays important roles in lipid metabolism and possesses anti-oxidant activity, we suggest that cleavage of lipid-free apoA-IV by MMP-7 has pathological implications in the development of hyperlipidemia and atherosclerosis. Show less

Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD. Show less

A novel liver X receptor (LXR) modulator, iristectorigenin B isolated from Belamcanda chinensis, stimulated the transcriptional activity of both LXR-α and LXR-β. In macrophages, iristectorigenin B suppressed cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes, ATP-binding cassette transporters A1 and G1. It did not induce hepatic lipid accumulation nor the expression of the lipogenesis genes sterol regulatory element-binding protein-1c, fatty acid synthase, and stearoyl-CoA desaturase-1. Iristectorigenin B thus is a dual-LXR agonist that regulates the expression of key genes in cholesterol homeostasis in macrophage cells without inducing hepatic lipid accumulation. Show less

Taurine, which is abundant in seafood, has antiatherogenic activities in both animals and humans; however, its molecular target has been elusive. We examined whether taurine could activate liver X receptor-α (LXR-α), a critical transcription factor in the regulation of reverse cholesterol transport in macrophages. Taurine bound directly to LXR-α in a reporter gene assay, time-resolved fluorescence resonance energy transfer analysis, and limited protease digestion experiment. Macrophage cells incubated with taurine showed reduced cellular cholesterol and induced medium cholesterol in a dose-dependent manner with the induction of ATP-binding cassette transporter A1 and G gene and protein expression. In hepatocytes, taurine significantly induced Insig-2a levels and delayed nuclear translocation of the sterol regulatory element-binding protein 1 (SREBP-1) protein, resulting in a dose-dependent reduction in the cellular lipid levels without inducing the expression of fatty acid synthesis genes. Taurine is a direct LXR-α ligand, represses cholesterol accumulation, and modulates the expression of genes involved in reverse cholesterol transport in macrophages, without inducing hepatic lipogenesis. The induction of Insig-2a suppressed the nuclear translocation of SREBP-1c. Show less

The present study reports a novel liver X receptor (LXR) activator, ethyl 2,4,6-trihydroxybenzoate (ETB), isolated from Celtis biondii. Using a reporter gene assay, time-resolved fluorescence resonance energy transfer (TR-FRET), and surface plasmon resonance (SPR) analysis, we showed that ETB directly bound to and stimulated the transcriptional activity of LXR-α and LXR-β. In macrophages, hepatocytes, and intestinal cells, ETB suppressed cellular cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes. Notably, ETB did not induce lipogenic gene expression or cellular triglyceride accumulation in hepatocytes. These results suggest that ETB is a dual-LXR modulator that regulates the expression of key genes in cholesterol homeostasis in multiple cells without inducing lipid accumulation in HepG2 cells. Show less

DDR1 (discoidin domain receptor tyrosine kinase 1) kinase s highly expressed in a variety of human cancers and occasionally mutated in lung cancer and leukemia. It is now clear that aberrant signaling through the DDR1 receptor is closely associated with various steps of tumorigenesis, although little is known about the molecular mechanism(s) underlying the role of DDR1 in cancer. Besides the role of DDR1 in tumorigenesis, we previously identified DDR1 kinase as a transcriptional target of tumor suppressor p53. DDR1 is functionally activated as determined by its tyrosine phosphorylation, in response to p53-dependent DNA damage. In this study, we report the characterization of the Notch1 protein as an interacting partner of DDR1 receptor, as determined by tandem affinity protein purification. Upon ligand-mediated DDR1 kinase activation, Notch1 was activated, bound to DDR1, and activated canonical Notch1 targets, including Hes1 and Hey2. Moreover, DDR1 ligand (collagen I) treatment significantly increased the active form of Notch1 receptor in the nuclear fraction, whereas DDR1 knockdown cells show little or no increase of the active form of Notch1 in the nuclear fraction, suggesting a novel intracellular mechanism underlying autocrine activation of wild-type Notch signaling through DDR1. DDR1 activation suppressed genotoxic-mediated cell death, whereas Notch1 inhibition by a γ-secretase inhibitor, DAPT, enhanced cell death in response to stress. Moreover, the DDR1 knockdown cancer cells showed the reduced transformed phenotypes in vitro and in vivo xenograft studies. The results suggest that DDR1 exerts prosurvival effect, at least in part, through the functional interaction with Notch1. Show less

The nuclear receptor liver X receptor-α (LXRα) stimulates lipogenesis, leading to steatosis. Nuclear factor erythroid-2-related factor-2 (Nrf2) contributes to cellular defense mechanism by upregulating antioxidant genes, and may protect the liver from injury inflicted by fat accumulation. However, whether Nrf2 affects LXRα activity is unknown. This study investigated the inhibitory role of Nrf2 in hepatic LXRα activity and the molecular basis. A deficiency of Nrf2 enhanced the ability of LXRα agonist to promote hepatic steatosis, as mediated by lipogenic gene induction. In hepatocytes, Nrf2 overexpression repressed gene transactivation by LXR-binding site activation. Consistently, treatment of mice with sulforaphane (an Nrf2 activator) suppressed T0901317-induced lipogenesis, as confirmed by the experiments using hepatocytes. Nrf2 activation promoted deacetylation of farnesoid X receptor (FXR) by competing for p300, leading to FXR-dependent induction of small heterodimer partner (SHP), which was responsible for the repression of LXRα-dependent gene transcription. In human steatotic samples, the transcript levels of LXRα and SREBP-1 inversely correlated with those of Nrf2, FXR, and SHP. Our findings offer the mechanism to explain how decrease in Nrf2 activity in hepatic steatosis could contribute to the progression of NAFLD, providing the use of Nrf2 as a molecular biomarker to diagnose NAFLD. As certain antioxidants have the abilities to activate Nrf2, clinicians might utilize the activators of Nrf2 as a new therapeutic approach to prevent and/or treat NAFLD. Nrf2 activation inhibits LXRα activity and LXRα-dependent liver steatosis by competing with FXR for p300, causing FXR activation and FXR-mediated SHP induction. Our findings provide important information on a strategy to prevent and/or treat steatosis. Show less

Liver X receptor-α (LXRα) functions as a major regulator of lipid homeostasis through activation of sterol regulatory element binding protein-1c (SREBP-1c), which promotes hepatic steatosis and steatohepatitis. NF-E2-related factor 2 (Nrf2) is the crucial transcription factor that is necessary for the induction of antioxidant enzymes. This study investigated the potential of liquiritigenin (LQ), a hepatoprotective flavonoid in licorice, to inhibit LXRα-induced hepatic steatosis, and the underlying mechanism of the action. LQ treatment attenuated fat accumulation and lipogenic gene induction in the liver of mice fed a high fat diet. Also, LQ had the ability to inhibit oxidative liver injury, as shown by decreases in thiobarbituric acid reactive substances formation and nitrotyrosinylation. Moreover, LQ treatment antagonized LXRα agonist (T0901317)-mediated SREBP-1c activation, and transactivation of the lipogenic target genes. LQ was found to activate Nrf2, and the ability of LQ to inhibit LXRα-mediated SREBP-1c activation was reversed by Nrf2 deficiency, which supports the inhibitory role of Nrf2 in LXRα-dependent lipogenesis. Consistently, treatment with other Nrf2 activators or forced expression of Nrf2 also inhibited LXRα-mediated SREBP-1c activation. Our results demonstrate that LQ has an efficacy to activate Nrf2, which contributes to inhibiting the activity of LXRα that leads to SREBP-1c induction and hepatic steatosis. Show less

GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P₁) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics. Show less

Glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released in response to food intake and potentiate insulin secretion from pancreatic beta cells through their distinct yet related G protein-coupled receptors, GLP1R and GIPR. While GLP-1 and GIP exhibit similarity in their N-terminal sequence and overall alpha-helical structure, GLP-1 does not bind to GIPR and vice versa. To determine which amino acid residues of these peptide ligands are responsible for specific interaction with their respective receptors, we generated mutant GIP in which several GLP-1-specific amino acid residues were substituted for the original amino acids. The potency of the mutant ligands was examined using HEK293 cells transfected with GLP1R or GIPR expression plasmids together with a cAMP-responsive element-driven luciferase (CRE-luc) reporter plasmid. A mutated GIP peptide in which Tyr(1), Ile(7), Asp(15), and His(18) were replaced by His, Thr, Glu, and Ala, respectively, was able to activate both GLP1R and GIPR with moderate potency. Replacing the original Tyr(1) and/or Ile(7) in the N-terminal moiety of this mutant peptide allowed full activation of GIPR but not of GLP1R. However, reintroducing Asp(15) and/or His(18) in the central alpha-helical region did not significantly alter the ligand potency. These results suggest that Tyr/His(1) and Ile/Thr(7) of GIP/GLP-1 peptides confer differential ligand selectivity toward GIPR and GLP1R. Show less

Apolipoprotein A-V (apo A-V) exerts a potent triglyceride (TG)-lowering effect through enhanced intravascular TG-hydrolysis with increased uptake of TG-derived free fatty acids into muscle and adipose tissue. Genetic variants in the APOA5 gene were strongly associated with plasma TG concentrations. The aim of this study was to examine whether APOA5 genetic variation was associated with obesity. We genotyped the missense c.553 G>T polymorphism (p.G185C) in the APOA5 gene in 1,085 Chinese (333 obese subjects and 752 nonobese controls). We analyzed the association between the c.553 G>T polymorphism and obesity and related metabolic phenotypes. The T allele at the c.553 G>T polymorphism was associated with higher plasma TG concentrations. Each additional T allele was associated with an increased TG concentration of 53.5 mg/dl (95% confidence interval (CI) 29.6-76.0, P < 0.0001). However, the T allele was associated lower risk of obesity (odds ratio (OR), 0.48; 95% CI 0.32-0.73, P = 0.0004). Each additional copy of the T allele was associated with a BMI decrease of 0.73 kg/m(2) (95% CI 0.26-1.16, P = 0.002), equivalent to 2.11 kg in a person 1.7 m tall. We may then conclude that the TG-raising APOA5 genetic variant was associated with a decrease in BMI and reduced risk of obesity in the Chinese population. Show less

Chronic kidney disease (CKD) is a significant public health problem, and recent genetic studies have identified common CKD susceptibility variants. The CKDGen consortium performed a meta-analysis of genome-wide association data in 67,093 individuals of European ancestry from 20 predominantly population-based studies in order to identify new susceptibility loci for reduced renal function as estimated by serum creatinine (eGFRcrea), serum cystatin c (eGFRcys) and CKD (eGFRcrea < 60 ml/min/1.73 m(2); n = 5,807 individuals with CKD (cases)). Follow-up of the 23 new genome-wide-significant loci (P < 5 x 10(-8)) in 22,982 replication samples identified 13 new loci affecting renal function and CKD (in or near LASS2, GCKR, ALMS1, TFDP2, DAB2, SLC34A1, VEGFA, PRKAG2, PIP5K1B, ATXN2, DACH1, UBE2Q2 and SLC7A9) and 7 loci suspected to affect creatinine production and secretion (CPS1, SLC22A2, TMEM60, WDR37, SLC6A13, WDR72 and BCAS3). These results further our understanding of the biologic mechanisms of kidney function by identifying loci that potentially influence nephrogenesis, podocyte function, angiogenesis, solute transport and metabolic functions of the kidney. Show less

Insulin resistance results in dysregulated hepatic gluconeogenesis that contributes to obesity-related hyperglycemia and progression of type 2 diabetes mellitus (T2DM). Recent studies show that MAPK phosphatase-3 (MKP-3) promotes gluconeogenic gene transcription in hepatoma cells, but little is known about the physiological role of MKP-3 in vivo. Here, we have shown that expression of MKP-3 is markedly increased in the liver of diet-induced obese mice. Consistent with this, adenovirus-mediated MKP-3 overexpression in lean mice promoted gluconeogenesis and increased fasting blood glucose levels. Conversely, shRNA knockdown of MKP-3 in both lean and obese mice resulted in decreased fasting blood glucose levels. In vitro experiments identified forkhead box O1 (FOXO1) as a substrate for MKP-3. MKP-3-mediated dephosphorylation of FOXO1 at Ser256 promoted its nuclear translocation and subsequent recruitment to the promoters of key gluconeogenic genes. In addition, we showed that PPARγ coactivator-1α (PGC-1α) acted downstream of FOXO1 to mediate MKP-3-induced gluconeogenesis. These data indicate that MKP-3 is an important regulator of hepatic gluconeogenesis in vivo and suggest that inhibition of MKP-3 activity may provide new therapies for T2DM. Show less

Higher resting heart rate is associated with increased cardiovascular disease and mortality risk. Though heritable factors play a substantial role in population variation, little is known about specific genetic determinants. This knowledge can impact clinical care by identifying novel factors that influence pathologic heart rate states, modulate heart rate through cardiac structure and function or by improving our understanding of the physiology of heart rate regulation. To identify common genetic variants associated with heart rate, we performed a meta-analysis of 15 genome-wide association studies (GWAS), including 38,991 subjects of European ancestry, estimating the association between age-, sex- and body mass-adjusted RR interval (inverse heart rate) and approximately 2.5 million markers. Results with P < 5 × 10(-8) were considered genome-wide significant. We constructed regression models with multiple markers to assess whether results at less stringent thresholds were likely to be truly associated with RR interval. We identified six novel associations with resting heart rate at six loci: 6q22 near GJA1; 14q12 near MYH7; 12p12 near SOX5, c12orf67, BCAT1, LRMP and CASC1; 6q22 near SLC35F1, PLN and c6orf204; 7q22 near SLC12A9 and UfSp1; and 11q12 near FADS1. Associations at 6q22 400 kb away from GJA1, at 14q12 MYH6 and at 1q32 near CD34 identified in previously published GWAS were confirmed. In aggregate, these variants explain approximately 0.7% of RR interval variance. A multivariant regression model including 20 variants with P < 10(-5) increased the explained variance to 1.6%, suggesting that some loci falling short of genome-wide significance are likely truly associated. Future research is warranted to elucidate underlying mechanisms that may impact clinical care. Show less

Isoliquiritigenin (ILQ), a flavonoid obtained from Glycyrrhizae species, has an antioxidant effect. This study investigated the potential of ILQ for inhibiting liver X receptor-α (LXRα)-mediated lipogenesis and steatosis in hepatocytes and its underlying molecular basis. Treatment with ILQ antagonized the ability of an LXRα agonist (T0901317) to activate sterol regulatory element binding protein-1c (SREBP-1c), thereby repressing transcription of fatty acid synthase, acetyl-CoA carboxylase, ATP-binding cassette transporter-A1, and stearoyl-CoA desaturase-1. ILQ treatment inhibited activating phosphorylation of JNK1 elicited by palmitate or TNFα. JNK1, but not JNK2, increased LXRα phosphorylation at serine residues, promoting LXRα activation. The ability of ILQ to inhibit JNK1 downstream of ASK1-MKK7 led to the repression of T0901317-inducible LXRα and SREBP-1c activation. In mice fed a high-fat diet, ILQ treatment inhibited hepatic steatosis, as shown by a decrease in fat accumulation and repression of lipogenic genes. The results of blood biochemistry and histopathology confirmed attenuation of high-fat diet-induced liver injury by ILQ. Moreover, ILQ inhibited oxidative stress, as indicated by decreases in thiobarbituric acid-reactive substance formation, iNOS and COX2 induction, and nitrotyrosinylation. Our results demonstrate that ILQ has the ability to repress LXRα-dependent hepatic steatosis through JNK1 inhibition and protect hepatocytes from oxidative injury inflicted by fat accumulation. Show less

Sauchinone, as an AMP-activated kinase (AMPK)-activating lignan in Saururus chinensis, has been shown to prevent iron-induced oxidative stress and liver injury. Sterol regulatory element binding protein-1c (SREBP-1c) plays a key role in hepatic steatosis, which promotes oxidative stress in obese subjects. Previously, we identified the role of AMPK in liver X receptor-alpha (LXRalpha)-mediated SREBP-1c-dependent lipogenesis. Because sauchinone as an antioxidant has the ability to activate AMPK, this study investigated its effects on SREBP-1c-dependent lipogenesis in hepatocytes and in high-fat diet (HFD)-induced hepatic steatosis and oxidative injury. Sauchinone prevented the ability of an LXRalpha agonist (T0901317) to activate SREBP-1c, repressing transcription of the fatty acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase-1, ATP-binding cassette transporter A1, and LXRalpha genes. Consistent with this, an HFD in mice caused fat accumulation in the liver with SREBP-1c induction, which was attenuated by sauchinone treatment. Also, sauchinone had the ability to inhibit oxidative stress as shown by decreases in thiobarbituric acid-reactive substance formation, nitrotyrosinylation, and 4-hydroxynonenal production. Moreover, it prevented not only the liver injury, but also the AMPK inhibition elicited by HFD feeding. These results demonstrate that sauchinone has the capability to inhibit LXRalpha-mediated SREBP-1c induction and SREBP-1c-dependent hepatic steatosis, thereby protecting hepatocytes from oxidative stress induced by fat accumulation. Show less

Human infection by Campylobacter jejuni is mainly through the consumption of contaminated poultry products, which results in gastroenteritis and, rarely, bacteremia and polyneuropathies. In this study, six C. jejuni-specific bacteriophages (CPS1-6) were isolated by the spot-on-the-lawn technique from chicken samples in Korea and characterized for potential use as biocontrol agents. All isolated bacteriophages exhibited a high specificity, being able to lyse only C. jejuni, but not other Gram-negative bacteria, including C. coli, Escherichia coli, Salmonella spp., and Gram-positive bacteria. Bacteriophages contain an icosahedral head and a contractile tail sheath in transmission electron microscopy, and possess ds-DNA with an average genome size of approximately 145 kb; therefore, all bacteriophages are categorized into the Myoviridae family. Bacterial lysis studies in liquid media revealed that CPS2 could be used to control the growth of C. jejuni. Show less