👤 Kiyofumi Yamada

In addition to overeating, starvation also reduces fecundity in mammals. However, little is known about the molecular mechanisms linking food intake to fertility, especially in males. Gastric inhibitory polypeptide (GIP), which is released from intestinal K-cells after meal ingestion, stimulates insulin secretion from pancreatic β-cells through the action of incretin and has several extrapancreatic effects. Here, we identified GIP receptor (Gipr) expression in mouse spermatids. Microarray analysis revealed that pregnancy-specific glycoprotein 17 (Psg17), a potential CD9-binding partner, was significantly decreased in GIP receptor-knockout (Gipr-/-) testes. Glycosylphosphatidylinositol-anchored PSG17 was expressed on the surface of acrosome-reacted sperm, and Gipr-/- sperm led to a lower fertilization rate in vitro, compared with that of Gipr+/+ sperm, both in the absence and presence of the zona pellucida. Plasma GIP concentrations and Psg17 messenger RNA (mRNA) were immediately increased in the testis after a single meal, whereas ingestion of a chronic high-fat diet markedly decreased Gipr and Psg17 mRNA. These results suggest that reduced GIP signaling, by decreased GIP levels or the downregulation of Gipr, is associated with the reduction of fecundity due to starvation or overeating. Thus, proper regulation of GIP signaling in the testis could be a potential unique therapeutic target for male infertility in obese and diabetic individuals. Show less

The bone marrow (BM) contains controlled specialized microenvironments, or niches, that regulate the quiescence, proliferation, and differentiation of hematopoietic stem and progenitor cells (HSPC). The glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone that mediates postprandial insulin secretion and has anabolic effects on adipose tissue. Previous studies demonstrated altered bone microarchitecture in mice deficient for GIP receptor ( Show less

The incidence of liver cancer has increased in recent years. Worldwide, liver cancer is common: more than 600000 related deaths are estimated each year. In the USA, about 27170 deaths due to liver cancer are estimated for 2016. Liver cancer is highly resistant to conventional chemotherapy and radiotherapy. For all stages combined, the 5-year survival rate is 15-17%, leaving much to be desired for liver cancer prevention and therapy. Heterogeneity, which can originate from genomic instability, is one reason for poor outcome. About 80-90% of liver cancers are hepatocellular carcinoma (HCC), and recent cancer genome sequencing studies have revealed frequently mutated genes in HCC. In this review, we discuss the cause of the tumor heterogeneity based on the functions of genes that are frequently mutated in HCC. We overview the functions of the genes that are most frequently mutated (e.g. TP53, CTNNB1, AXIN1, ARID1A and WWP1) that portray major pathways leading to HCC and identify the roles of these genes in preventing genomic instability. Notably, the pathway analysis suggested that oxidative stress management may be critical to prevent accumulation of DNA damage and further mutations. We propose that both chromosome instability (CIN) and microsatellite instability (MIN) are integral to the hepatic carcinogenesis process leading to heterogeneity in HCC and that the pathways leading to heterogeneity may be targeted for prognosis, prevention and treatment. Show less

Herein we describe the discovery and characterization of a novel, piperidine-based inhibitor of cholesteryl ester transfer protein (CETP) with a core structure distinct from other reported CETP inhibitors. A versatile synthesis starting from 4-methoxypyridine enabled an efficient exploration of the SAR, giving a lead molecule with potent CETP inhibition in human plasma. The subsequent optimization focused on improvement of pharmacokinetics and mitigation of off-target liabilities, such as CYP inhibition, whose improvement correlated with increased lipophilic efficiency. The effort led to the identification of an achiral, carboxylic acid-bearing compound 16 (TAP311) with excellent pharmacokinetics in rats and robust efficacy in hamsters. Compared to anacetrapib, the compound showed substantially reduced lipophilicity, had only modest distribution into adipose tissue, and retained potency in hypertriglyceridemic plasma in vitro and in vivo. Furthermore, in contrast to torcetrapib, the compound did not increase aldosterone secretion in human adrenocortical carcinoma cells nor in chronically cannulated rats. On the basis of its preclinical efficacy and safety profile, the compound was advanced into clinical trials. Show less

Activation of glucose-dependent insulinotropic polypeptide receptor (GIPR) has been shown to be protective against atherosclerosis. However, effects of GIP on the heart have remained unclear. To address this question, in vitro and in vivo experiments were conducted. In isolated mouse cardiomyocytes, GIPR mRNA was detected by reverse transcription-polymerase chain reaction, and GIP stimulation increased adenosine 3',5'-cyclic monophosphate production. In apolipoprotein E-knockout mice, infusion of angiotensin II (AngII; 2,000 ng·kg(-1)·min(-1)) significantly increased the heart weights, and co-administration of GIP (25 nmol·kg(-1)·day(-1)) reversed this increase (both P<0.01). In the left ventricular walls, GIP suppressed AngII-induced cardiomyocyte hypertrophy by 34%, apoptosis by 77%, and interstitial fibrosis by 79% (all P<0.01). Furthermore, GIP reduced AngII-induced expression of transforming growth factor-β1 (TGF-β1) and hypoxia inducible factor-1α. In wild-type mice, cardiac hypertrophy was induced by AngII to a lesser extent, and prevented by GIP. In contrast, GIP did not show any cardioprotective effect against AngII-induced cardiac hypertrophy in GIPR-knockout mice. In an in vitro experiment using mouse cardiomyocytes, GIP suppressed AngII-induced mRNA expression of B-type natriuretic peptide and TGF-β1. It was demonstrated that cardiomyocytes represent a direct target of GIP action in vitro, and that GIP ameliorated AngII-induced cardiac hypertrophy via suppression of cardiomyocyte enlargement, apoptosis, and fibrosis in vivo. (Circ J 2016; 80: 1988-1997). Show less

The transcription factor cyclic AMP-responsive element-binding protein H (CREBH, encoded by To investigate the influence of intestinal CREBH on cholesterol metabolism, we compared plasma, bile, fecal, and tissue cholesterol levels between wild-type (WT) mice and mice overexpressing active human CREBH mainly in the small intestine (CREBH Tg mice) under different dietary conditions. Plasma cholesterol, hepatic lipid, and cholesterol crystal formation in the gallbladder were lower in CREBH Tg mice fed a lithogenic diet (LD) than in LD-fed WTs, while fecal cholesterol output was higher in the former. These results suggest that intestinal CREBH overexpression suppresses cholesterol absorption, leading to reduced plasma cholesterol, limited hepatic supply, and greater excretion. The expression of Niemann-Pick C1-like 1 ( Intestinal CREBH regulates dietary cholesterol flow from the small intestine by controlling the expression of multiple intestinal transporters. We propose that intestinal CREBH could be a therapeutic target for hypercholesterolemia. Show less

Herein, we report the characterization of Limb expression 1-like, (LIX1L), a putative RNA-binding protein (RBP) containing a double-stranded RNA binding motif, which is highly expressed in various cancer tissues. Analysis of MALDI-TOF/TOF mass spectrometry and RNA immunoprecipitation-sequencing of interacting proteins and the microRNAs (miRNAs) bound to LIX1L revealed that LIX1L interacts with proteins (RIOK1, nucleolin and PABPC4) and miRNAs (has-miRNA-520a-5p, -300, -216b, -326, -190a, -548b-3p, -7-5p and -1296) in HEK-293 cells. Moreover, the reduction of phosphorylated Tyr(136) (pTyr(136)) in LIX1L through the homeodomain peptide, PY136, inhibited LIX1L-induced cell proliferation in vitro, and PY136 inhibited MKN45 cell proliferation in vivo. We also determined the miRNA-targeted genes and showed that was apoptosis induced through the reduction of pTyr(136). Moreover, ROS1, HCK, ABL1, ABL2, JAK3, LCK and TYR03 were identified as candidate kinases responsible for the phosphorylation of Tyr(136) of LIX1L. These data provide novel insights into the biological significance of LIX1L, suggesting that this protein might be an RBP, with implications for therapeutic approaches for targeting LIX1L in LIX1L-expressing cancer cells. Show less

Although genome-wide association studies (GWASs) have identified various genes and loci in predisposition to metabolic syndrome (MetS) or each component of this condition, the genetic basis of MetS in individuals remains to be identified definitively. The aim of the present study was to examine the possible association of MetS in individuals with 29 polymorphisms that were previously identified as susceptibility loci for coronary artery disease or myocardial infarction by meta-analyses of GWASs. The study population comprised 1,822 subjects with MetS and 1,096 controls. Subjects with MetS had ≥3 of the 5 components of the diagnostic criteria for MetS, whereas control individuals had 0-1 of the 5 components. The genotypes for the 29 polymorphisms were determined by the multiplex bead-based Luminex assay. Comparisons of allele frequencies by the χ Show less

Although genetic variants, which regulate lipid metabolism, have been extensively investigated in Caucasian populations, the genes, which confer susceptibility to dyslipidemia in Japanese individuals, remain to be elucidated. The aim of the present study was to examine a possible association among hypertriglyceridemia, hypo‑high density lipoprotein (HDL)‑cholesterolemia or hyper‑low density lipoprotein (LDL)‑cholesterolemia in Japanese individuals with 29 polymorphisms observed to confer susceptibility for coronary heart disease. This was performed through meta‑analyses of genome‑wide association studies in Caucasian populations. The study population comprised 2,354 individuals with dyslipidemia (hypertriglyceridemia, hypo‑HDL‑cholesterolemia or hyper‑LDL‑cholesterolemia) and 3,106 control individuals. To compensate for multiple comparisons of genotypes, a false discovery rate (FDR) of <0.05 was adopted to determine the statistical significance of the associations. Comparisons of allele frequencies using the χ2 test revealed that rs964184 of zinc finger gene (ZPR1; FDR=2.1x10‑7), rs4845625 of interleukin 6 receptor (IL6R; FDR=0.032), rs46522 of ubiquitin‑conjugating enzyme E2Z gene (UBE2Z; FDR=0.032) and rs17514846 of furin (FDR=0.041) were significantly associated with hypertriglyceridemia. The χ2 test revealed that rs599839 of proline/serine‑rich coiled‑coil 1 (PSRC1; FDR=0.004) and rs2075650 of translocase of outer mitochondrial membrane 40 homolog (TOMM40; FDR=0.004) were significantly associated with hyper‑LDL‑cholesterolemia. Multivariate logistic regression analysis with adjustment for age, gender and body mass index revealed that rs964184 of ZPR1 (P=5.1x10‑7; odds ratio, 1.37; dominant model), rs4845625 of IL6R (P=0.0019, odds ratio, 1.25; dominant model) and rs46522 of UBE2Z (P=0.0039, odds ratio, 1.19; dominant model) were significantly associated with hypertriglyceridemia, and that rs599839 of PSRC1 (P=0.0004, odds ratio, 0.70; dominant model) and rs2075650 of TOMM40 (P=0.0004, odds ratio, 1.43; dominant model) were significantly associated with hyper‑LDL‑cholesterolemia. Therefore, ZPR1, IL6R, and UBE2Z may be susceptibility loci for hypertriglyceridemia, whereas PSRC1 and TOMM40 may be such loci for hyper-LDL-cholesterolemia in Japanese individuals. Show less

Various loci and genes that confer susceptibility to coronary heart disease (CHD) have been identified in Caucasian populations by genome-wide association studies (GWASs). As type 2 diabetes mellitus (DM) is an important risk factor for CHD, we hypothesized that certain polymorphisms may contribute to the genetic susceptibility to CHD through affecting the susceptibility to type 2 DM. The purpose of the present study was to examine a possible association of type 2 DM in Japanese individuals with 29 polymorphisms identified as susceptibility loci for CHD by meta-analyses of the GWASs. The study subjects comprised of 3,757 individuals (1,444 subjects with type 2 DM and 2,313 controls). The polymorphism genotypes were determined by the multiplex bead-based Luminex assay, which combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. To compensate for multiple comparisons of genotypes, the criterion of a false discovery rate (FDR) ≤0.05 was adopted for testing the statistical significance of the association. The comparisons of allele frequencies by the χ Show less

Gastric inhibitory polypeptide (GIP) is an incretin secreted from the gastrointestinal tract after an ingestion of nutrients, and stimulates an insulin secretion from the pancreatic islets. Additionally, GIP has important roles in extrapancreatic tissues: fat accumulation in adipose tissue, neuroprotective effects in the central nervous system and an inhibition of bone resorption. In the current study, we investigated the effects of GIP signaling on the peripheral nervous system (PNS). First, the presence of the GIP receptor (GIPR) in mouse dorsal root ganglion (DRG) was evaluated utilizing immunohistochemical analysis, western blotting and reverse transcription polymerase chain reaction. DRG neurons of male wild-type mice (WT) were cultured with or without GIP, and their neurite lengths were quantified. Functions of the PNS were evaluated in GIPR-deficient mice (gipr-/-) and WT by using current perception thresholds (CPTs), Thermal Plantar Test (TPT), and motor (MNCV) and sensory nerve conduction velocity (SNCV, respectively). Sciatic nerve blood flow (SNBF) and plantar skin blood flow (PSBF) were also evaluated. We confirmed the expression of GIPR in DRG neurons. The neurite outgrowths of DRG neurons were promoted by the GIP administrations. The gipr-/- showed impaired perception functions in the examination of CPTs and TPT. Both MNCV and SNCV were delayed in gipr-/- compared with these in WT. There was no difference in SNBF and PSBF between WT and gipr-/-. Our findings show that the GIP signal could exert direct physiological roles in the PNS, which might be directly exerted on the PNS. Show less

Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV)-related hepatocellular carcinomas (HCCs) and their matched controls. Comparison of whole genome sequence (WGS) and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3), and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome. Show less

The predominant risk factor of metabolic syndrome is intra-abdominal fat accumulation, which is determined by waist circumference, waist-hip ratio measurements and visceral fat area (VFA); the latter can be accurately measured by performing computed tomography (CT). In addition to environmental factors, genetic factors play an important role in obesity and fat distribution. New genetic loci associated with body mass index (BMI) and adiposity have been identified by genome-wide association studies (GWASs). This study utilized CT to investigate whether single nucleotide polymorphisms (SNPs) that confer susceptibility to higher BMI are associated with VFA, subcutaneous fat area (SFA), and the ratio of VFA to SFA (V/S ratio). We measured the VFA and SFA of 1424 obese Japanese subjects (BMI ≥ 25 kg/m(2), 635 men and 789 women) who were genotyped for 13 single nucleotide polymorphisms (SNPs) reported by recent GWASs, namely, TNNI3K rs1514175, PTBP2 rs1555543, ADCY3 rs713586, IRS1 rs2943650, POC5 rs2112347, NUDT3 rs206936, LINGO2 rs10968576, STK33 rs4929949, MTIF3 rs4771122, SPRY2 rs534870, MAP2K5 rs2241423, QPCTL rs2287019, and ZC3H4 rs3810291. The G-allele of NUDT3 rs206936 was significantly associated with increased BMI (P = 5.3 × 10(-5)) and SFA (P = 0.00039) in the obese Japanese women. After adjustment with BMI, the association between rs206936 and SFA was not observed. This significant association was not observed in the men. The other SNPs analyzed were not significantly associated with BMI, VFA, SFA, or V/S ratio. Our results suggest that NUDT3 rs206936 is associated with BMI in Japanese women. Show less

The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (PRKAG2); transforming growth factor-beta receptor II (TGFBR2); and exostosin 1 (EXT1) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of TGFBR2 and EXT1 enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of TGFBR2 and EXT1 include activation of the TGF-β signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the PRKAG2 mRNA level in HCC tissues was positively correlated with survival period, suggesting that PRKAG2 enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that PRKAG2 mRNA can serve as a prognostic marker for IFN-α/5-FU therapy. Show less

We here report a 2-year-old female with relapsed acute myeloid leukemia (AML) with MLL gene rearrangement in the bone marrow and central nervous system. The 3'-RACE (Rapid Amplification of cDNA Ends) method identified the MLLT10 gene as a fusion partner of the MLL gene. The patient was complicated with hemophagocytic lymphohistiocytosis (HLH) and invasive aspergillosis (IPA) after re-induction treatment with FLAG-IDA following etoposide, cytarabine, and mitoxantrone. Although treatment with systemic anti-fungal drugs was effective for IPA, HLH did not improve. We considered tumor-associated HLH to be initiated from leukemic stem cells (LSCs) in the bone marrow niche because reverse transcription-polymerase chain reaction (RT-PCR) analysis of a bone marrow biopsy sample was positive for MLL-MLLT10. Gemtuzumab ozogamicin and sorafenib had no major effect on acquiring complete remission, and the patient died of progressive AML with an exacerbation of HLH and aspergillosis. LSCs are known to be resistant to conventional chemotherapy due to their quiescence in the cell cycle. Novel therapeutic concepts are important to eradicate LSCs in order to cure AML patients. Show less

Selective Alzheimer's disease (AD) indicator-1 (Seladin-1) gene has been identified as a gene, whose expression is down-regulated in the vulnerable region in the brain of AD patients. Thyroid hormone (TH) is important to maintain the function of central nervous system and TH receptor (TR) is known to crosstalk with liver X receptor (LXR) on the lipid metabolism-related gene promoter. Recently, we have demonstrated that TR-β up-regulates the mouse Seladin-1 gene promoter at the transcriptional levels and LXR-α compensates the promoter activation only when the thyroid function is insufficient. In the current study, we have identified that TH and an LXR artificial agonist, TO901317 (TO) activated the human Seladin-1 promoter (-1024/+57 base pair (bp)) including consensus TH response element (TRE) half site (site A: -381 to -375 bp), and the site A mutation deteriorated the activation by TH and TO. Both TR-β and LXR-α heterodimerize with retinoid X receptor (RXR)-α on the site A, and chromatin immunoprecipitation (ChIP) assay revealed that TR-β, LXR-α and RXR-α are recruited to the site A. Moreover, TR-β and LXR-α functionally compete for the promoter activation in CV1 cells. Taken together, we concluded that TR-β and LXR-α competitively up-regulate the human Seladin-1 promoter, sharing the same response element, site A. Show less

Selective Alzheimer's disease (AD) indicator 1 (Seladin-1) has been identified as a gene down-regulated in the degenerated lesions of AD brain. Up-regulation of Seladin-1 reduces the accumulation of β-amyloid and neuronal death. Thyroid hormone (TH) exerts an important effect on the development and maintenance of central nervous systems. In the current study, we demonstrated that Seladin-1 gene and protein expression in the forebrain was increased in thyrotoxic mice compared with that of euthyroid mice. However, unexpectedly, no significant decrease in the gene and protein expression was observed in hypothyroid mice. Interestingly, an agonist of liver X receptor (LXR), TO901317 (TO) administration in vivo increased Seladin-1 gene and protein expression in the mouse forebrain only in a hypothyroid state and in the presence of mutant TR-β, suggesting that LXR-α would compensate for TR-β function to maintain Seladin-1 gene expression in hypothyroidism and resistance to TH. TH activated the mouse Seladin-1 gene promoter (-1936/+21 bp) and site 2 including canonical TH response element (TRE) half-site in the region between -159 and -154 bp is responsible for the positive regulation. RXR-α/TR-β heterodimerization was identified on site 2 by gel-shift assay, and chromatin immunoprecipitation assay revealed the recruitment of TR-β to site 2 and the recruitment was increased upon TH administration. On the other hand, LXR-α utilizes a distinct region from site 2 (-120 to -102 bp) to activate the mouse Seladin-1 gene promoter. Taking these findings together, we concluded that TH up-regulates Seladin-1 gene expression at the transcriptional level and LXR-α maintains the gene expression. Show less

Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial-like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin-based cell-cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)-based cell-cell adhesion, and cadherin and claudin were recruited to the nectin-3- and JAM-based cell-cell adhesion sites to form AJ-like and TJ-like domains, respectively. This inversed alignment of the AJ-like and TJ-like domains was reversed by complementary expression of CPMs Par-3, atypical protein kinase C, Par-6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico-basal alignment of TJs and AJs in epithelial cells. Show less

We previously showed that the -1131T→C polymorphism (rs662799) of the apolipoprotein A-V gene (APOA5) and the C→T polymorphism (rs6929846) of the butyrophilin, subfamily 2, member A1 gene (BTN2A1) were significantly associated with an increased serum concentration of triglycerides, a decreased serum concentration of high density lipoprotein (HDL)-cholesterol, and the prevalence of metabolic syndrome (MetS) in Japanese individuals. The purpose of the present study was to examine whether these polymorphisms synergistically affect the prevalence of dyslipidemia and MetS in East Asian populations. The study populations comprised 7471 Japanese and 3529 Korean individuals in the dyslipidemia study, and 3474 Japanese and 1671 Korean individuals in the MetS study. Multivariable logistic regression analysis of combined genotypes with adjustment for age, gender and diabetes mellitus revealed that rs662799 and rs6929846 significantly and synergistically affected dyslipidemia. Japanese or Korean individuals with the C allele of APOA5 and the T allele of BTN2A1 had a 2.05- or 1.92-fold increased risk for hypertriglyceridemia and a 1.82- or 1.56-fold increased risk for hypo-HDL-cholesterolemia, respectively, compared to those with the TT genotype of APOA5 and the CC genotype of BTN2A1. Similar analysis with adjustment for age and gender revealed that Japanese individuals, but not Korean individuals, with the C allele of APOA5 and the T allele of BTN2A1 had a 2.87-fold increased risk for MetS compared to those with the TT genotype of APOA5 and the CC genotype of BTN2A1. Genetic variants of APOA5 and BTN2A1 may synergistically affect the prevalence of dyslipidemia in East Asian populations and of MetS in Japanese individuals. Show less

In mouse embryos, some primordial germ cells (PGCs) are eliminated by apoptosis, but the molecular pathways that lead to PGC survival versus apoptosis have not been fully characterized. Here, we found that REST (repressor element 1-silencing transcription factor), a transcription factor that binds a conserved regulatory element, NRSE/RE1, played a role in PGC survival. REST expression was higher in PGCs than in surrounding somatic cells. Moreover, in mouse embryos with a PGC-specific conditional REST mutation, the PGC population experienced more apoptosis and was significantly smaller than that in control embryos; these findings indicated that REST functioned in a cell-autonomous fashion that was critical for PGC survival. Several anti-apoptotic genes were among the previously identified REST-target gene candidates; moreover, some of these genes were downregulated in the REST-deficient PGCs. Mek5, which encodes a component in the a MAP kinase cascade, was one of these downregulated REST-target gene candidates, and a Mek5 mutation, like the REST mutation, caused an increase in PGC apoptosis; these finding suggested that REST promoted PGC survival via regulation of the Mek5 expression. Importantly, there were a normal number of PGCs in the REST mutants at birth, and both the male and female REST-mutant adults were fertile; these final observations revealed that the PGC population was very robust and could recover from a genetically induced reduction in cell number. Show less

The liver X receptor α (LXRα) is a nuclear receptor that is involved in regulation of lipid metabolism, cellular proliferation and apoptosis, and immunity. In this report, we characterize three human LXRα isoforms with variation in the ligand-binding domain (LBD). While examining the expression of LXRα3, which lacks 60 amino acids within the LBD, we identified two novel transcripts that encode LXRα-LBD variants (LXRα4 and LXRα5). LXRα4 has an insertion of 64 amino acids in helix 4/5, and LXRα5 lacks the C-terminal helices 7 to 12 due to a termination codon in an additional exon that encodes an intron in the LXRα1 mRNA. LXRα3, LXRα4, and LXRα5 were expressed at lower levels compared with LXRα1 in many human tissues and cell lines. We also observed weak expression of LXRα3 and LXRα4 in several tissues of mice. LXR ligand treatment induced differential regulation of LXRα isoform mRNA expression in a cell type-dependent manner. Whereas LXRα3 had no effect, LXRα4 has weak transactivation, retinoid X receptor (RXR) heterodimerization, and coactivator recruitment activities. LXRα5 interacted with a corepressor in a ligand-independent manner and inhibited LXRα1 transactivation and target gene expression when overexpressed. Combination of LXRα5 cotransfection and LXRα antagonist treatment produced additive effects on the inhibition of ligand-dependent LXRα1 activation. We constructed structural models of the LXRα4-LBD and its complexes with ligand, RXR-LBD, and coactivator peptide. The models showed that the insertion in the LBD can be predicted to disrupt RXR heterodimerization. Regulation of LXRα pre-mRNA splicing may be involved in the pathogenesis of LXRα-related diseases. Show less

Metabolic syndrome is defined as a cluster of multiple risk factors, including central obesity, dyslipidemia, hypertension and impaired glucose tolerance, that increase cardiovascular disease morbidity and mortality. Genetic factors are important in the development of metabolic syndrome, as are environmental factors. However, the genetic background of metabolic syndrome is not yet fully clarified. There is evidence that obesity and obesity-related phenotypes are associated with variations in several genes, including NEGR1, SEC16B, TMEM18, ETV5, GNPDA2, BDNF, MTCH2, SH2B1, FTO, MAF, MC4R, KCTD15, SCG3, MTMR9, TFAP2B, MSRA, LYPLAL1, GCKR and FADS1. To investigate the relationship between metabolic syndrome and variations in these genes in the Japanese population, we genotyped 33 single-nucleotide polymorphisms (SNPs) in 19 genes from 1096 patients with metabolic syndrome and 581 control individuals who had no risk factors for metabolic syndrome. Four SNPs in the FTO gene were significantly related to metabolic syndrome: rs9939609 (P=0.00013), rs8050136 (P=0.00011), rs1558902 (P=6.6 × 10(-5)) and rs1421085 (P=7.4 × 10(-5)). rs3764220 in the SCG3 gene (P=0.0010) and rs2293855 in the MTMR9 gene (P=0.0015) were also significantly associated with metabolic syndrome. SNPs in the FTO, SCG3 and MTMR9 genes had no SNP × SNP epistatic effects on metabolic syndrome. Our data suggest that genetic variations in the FTO, SCG3 and MTMR9 genes independently influence the risk of metabolic syndrome. Show less

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor. Show less

The liver X receptors (LXR-α and -β) are nuclear oxysterol receptors that play pivotal roles in regulating the expression of genes involved in cholesterol transport and metabolism. Recently, several groups have reported that the LXRs also regulate adrenal steroidogenesis. In the previous report, we demonstrated that LXR-α is dominantly expressed in the pituitary and that LXR-α positively regulates the proopiomelanocortin (POMC) gene promoter at the transcriptional level. In this report, we evaluated the expression levels of LXR-α and -β gene in the human pituitary tumor. Even though LXR-α mRNA levels are not significantly increased in ACTH-secreting adenomas, LXR-α/β expression ratio is significantly higher than other pituitary tumors including normal pituitaries. Furthermore, in At-T20 cells, which express POMC gene, overexpression of LXR-β decreased POMC gene promoter activities. Thus, we concluded that LXR-α/β gene expression ratio is a critical factor to activate POMC gene expression in ACTH-secreting pituitary adenomas. Show less

Visceral fat accumulation has an important role in increasing morbidity and mortality rate by increasing the risk of developing several metabolic disorders, such as type 2 diabetes, dyslipidemia and hypertension. New genetic loci that contribute to the development of obesity have been identified by genome-wide association studies in Caucasian populations. We genotyped 1279 Japanese subjects (556 men and 723 women), who underwent computed tomography (CT) for measuring visceral fat area (VFA) and subcutaneous fat area (SFA), for the following single-nucleotide polymorphisms (SNPs): NEGR1 rs2815752, SEC16B rs10913469, TMEM18 rs6548238, ETV5 rs7647305, GNPDA2 rs10938397, BDNF rs6265 and rs925946, MTCH2 rs10838738, SH2B1 rs7498665, MAF rs1424233, and KCTD15 rs29941 and rs11084753. In the additive model, none of the SNPs were significantly associated with body mass index (BMI). The SH2B1 rs7498665 risk allele was found to be significantly associated with VFA (P=0.00047) but not with BMI or SFA. When the analysis was performed in men and women separately, no significant associations with VFA were observed (P=0.0099 in men and P=0.022 in women). None of the other SNPs were significantly associated with SFA. Our results suggest that there is a VFA-specific genetic factor and that a polymorphism in the SH2B1 gene influences the risk of visceral fat accumulation. Show less

Allergic rhinitis is a global health problem that causes major illnesses and disability worldwide. Allergen-specific immunotherapy (SIT) is the only available treatment that can alter the natural course of allergic disease. However, the precise mechanism underlying allergen-SIT is not well understood. The aim of the current study was to identify protein expression signatures reflective of allergen-SIT-more specifically, sublingual immunotherapy (SLIT). Serum was taken twice from patients with seasonal allergic rhinitis caused by Japanese cedar: once before the pollen season and once during the season. A total of 25 patients was randomly categorized into a placebo-treated group and an active-treatment group. Their serum protein profiles were analyzed by 2-dimensional electrophoresis. Sixteen proteins were found to be differentially expressed during the pollen season. Among the differentially expressed proteins, the serum levels of complement C4A, apolipoprotein A-IV (apoA-IV), and transthyretin were significantly increased in SLIT-treated patients but not in placebo-treated patients. Among these proteins, the serum levels of apoA-IV correlated with the clinical symptom-medication scores (r = -0.635; P < .05) and with quality of life scores (r = -0.516; P < .05) in the case of SLIT-treated patients. The amount of histamine released from the basophils in vitro was greatly reduced after the addition of recombinant apoA-IV in the medium (P < .01). Our data will increase the understanding of the mechanism of SLIT and may provide novel insights into the treatment of allergic rhinitis. Show less

The predominant risk factor of metabolic syndrome is intra-abdominal fat accumulation, which is determined by waist circumference and waist-hip ratio measurements and visceral fat area (VFA) that is measured by computed tomography (CT). There is evidence that waist circumference and waist-hip ratio in the Caucasian population are associated with variations in several genes, including neurexin 3 (NRXN3), transcription factor AP-2β (TFAP2B), methionine sulfoxide reductase A (MSRA), lysophospholipase-like-1 (LYPLAL1), fat mass and obesity associated (FTO) and melanocortin 4 receptor (MC4R) genes. To investigate the relationship between VFA and subcutaneous fat area (SFA) and these genes in the recruited Japanese population, we genotyped 8 single-nucleotide polymorphisms (SNPs) in these 6 genes from 1228 subjects. Multiple regression analysis revealed that gender, age, and rs1558902 and rs1421085 genotypes (additive model) in FTO were significantly associated with body mass index (BMI; P=0.0039 and 0.0039, respectively), SFA (P=0.0027 and 0.0023, respectively) and VFA (P=0.045 and 0.040, respectively). However, SNPs in other genes, namely, NRXN3, TFAP2B, MSRA, LYPLAL1 and MC4R were not significantly associated with BMI, SFA or VFA. Our data suggest that some SNPs, which were identified in genome-wide studies in the Caucasians, also confer susceptibility to fat distribution in the Japanese subjects. Show less

We aim to investigate the effect of transforming growth factor (TGF)-beta1 on the expression of enhancer of split- and hairy-related protein-2 (SHARP-2) messenger RNA (mRNA) and its signaling pathway. In this study, several cell lines including LLC-PK1 (a porcine kidney tubular epithelial cell line), MDCK (Madin-Darby canine kidney) and CTLL-2 (cytotoxic T-lymphocyte line) were treated with recombinant human TGF-beta1, and a series of experiments were carried out, involving Northern blot analysis of total RNA from these cells. Further, several specific chemical inhibitors were applied before TGF-beta1 treatment to probe the signaling pathway. The results showed that TGF-beta1 can significantly up-regulate SHARP-2 mRNA expression in the LLC-PK1 cell line. The peak level of induction was found 2 h after TGF-beta1 stimulation. While one phosphoinositide 3-kinases (PI-3) kinase inhibitor, LY294002, completely blocked the effect of TGF-beta1 on SHARP-2 mRNA expression in LLC-PK1 cells at a low concentration, other inhibitors, including PD98059, staurosporine, AG490, wortmannin, okadaic acid and rapamycin, had no effect. The effect of LY294002 was dose-dependent. We conclude that, in LLC-PK1 cells at least, TGF-beta1 can effectively induce the SHARP-2 mRNA expression and that the PI-3 kinase pathway can mediate this effect. Show less

The molecular mechanism of thyroid hormone (TH) effects to fatty acid metabolism in liver is yet to be clear. The carbohydrate response element-binding protein (ChREBP) as well as sterol response element-binding protein (SREBP)-1c plays a pivotal role in hepatic lipogenesis. Both SREBP-1c and ChREBP are target genes of liver X receptors (LXRs). Because LXRs and TH receptors (TRs) cross talk mutually in many aspects of transcription, we examined whether TRs regulate the mouse ChREBP gene expression. In the current study, we demonstrated that TH up-regulated mouse ChREBP mRNA and protein expression in liver. Run-on and luciferase assays showed that TH and TR-beta1 positively regulated the ChREBP gene transcription. The mouse ChREBP gene promoter contains two direct repeat-4 sites (LXRE1 and LXRE2) and EMSAs demonstrated that LXR-alpha and TR-beta1 prefer to bind LXRE1 and LXRE2, respectively. The direct repeat-4 deletion and LXRE2 mutants of the promoter deteriorate the positive regulation by TR-beta1, indicating that LXRE2 is functionally important for the regulation. We also showed that human ChREBP gene expression and promoter activities were up-regulated by TH. These data suggest that ChREBP mRNA expression is positively regulated by TR-beta1 and TH at the transcriptional level in mammals. This novel observation indicates that TH fine-tunes hepatic lipogenesis via regulating SREBP-1c and ChREBP gene expression reciprocally. Show less

The liver X receptors (LXR-alpha and -beta) are nuclear oxysterol receptors that play pivotal roles in regulating the expression of genes involved in cholesterol transport and metabolism. Recently, several groups have reported that the LXRs also regulate adrenal steroidogenesis. However, the roles of LXRs in the hypothalami-pituitary-adrenal axis, especially whether they regulate proopiomelanocortin (POMC) gene expression in the pituitary, remain to be elucidated. In this report, we demonstrate that LXR mRNA is expressed in the pituitary and that at the protein level, LXR-alpha is dominantly expressed. Next, we show that the LXR agonist TO901317 (TO) increased POMC mRNA levels and the number of cells immunostained with anti-ACTH antibody in the mouse pituitary. We also confirmed that TO elevated plasma ACTH and serum corticosterone levels in vivo and increased the total tissue content of immunoreactive ACTH in the pituitary. TO activated the rat POMC gene promoter (-706/+64 bp) in GH3 and AtT-20 cells. Silencing of LXR-alpha mRNA expression in GH3 cells with small interfering RNA specific to LXR-alpha caused a loss of promoter activity induced by the LXR ligand, suggesting that LXR-alpha directly regulates the POMC gene promoter. EMSAs also demonstrated that the retinoid X receptor-alpha/LXR-alpha heterodimer bound to the region between -73 and -52 bp in the rat POMC gene promoter, and this site was responsible for the induction by TO, as confirmed by chromatin immunoprecipitation assays using AtT-20 cells. Our findings provide the first evidence that LXR-alpha positively regulates the POMC gene promoter at the transcriptional level and suggest LXR-alpha to be a coordinator for cross talk between lipid metabolism and neuroendocrinology. Show less