👤 S Iwamoto

This study aimed to investigate the anti-atherosclerotic properties of tirzepatide, a dual glucose-dependent insulinotropic polypeptide (GIP)/glucagon-like peptide-1 (GLP-1) receptor agonist, in comparison with semaglutide, a selective GLP-1 receptor agonist. ApoE knockout mice were divided into early diabetes (dosed from 10 to 22 weeks of age), late diabetes (dosed from 18 to 30 weeks of age), and non-diabetic groups after streptozotocin treatment, and each group received semaglutide, tirzepatide, or saline for 12 weeks. In the early diabetes group, both agents significantly suppressed aortic plaque formation compared with control, while modestly improving glycemia and lipid levels. No significant vascular effects were observed in late diabetes or non-diabetic groups. Tirzepatide markedly reduced inflammatory mediators, including Mcp-1, Il-6, I-cam, and Cd68, whereas semaglutide showed partial overlap. Notably, these anti-inflammatory effects were also detected in non-diabetic mice, suggesting vascular protection may involve arterial actions independently of metabolic control. Taken together, our findings demonstrate that tirzepatide exerts anti-atherosclerotic effects comparable to semaglutide, supporting the concept that GIP and GLP-1 signaling can confer vascular benefits. These results highlight the potential clinical relevance of dual incretin receptor agonism for cardiovascular risk reduction, although further studies are required to clarify the specific role of GIP signaling. Show less

A 50-year-old man with a triglyceride (TG) level of 11,397 mg/dL was admitted to our hospital. He consumed a high-fat and high-carbohydrate diet as well as more than 100 g of alcohol per day. He had type 2 diabetes and obesity and had previously suffered from severe acute pancreatitis twice. A genetic analysis revealed compound heterozygous mutations in APOA5 (c.56C>G and c.553G>T). In addition to low-fat meals and alcohol cessation, administration of pemafibrate lowered his triglyceride levels to <150 mg/dL. Show less

N-3 long-chain polyunsaturated fatty acids (n-3LCPUFAs) are crucial for child growth and development particularly for fetal growth in utero and brain development and function. This study examined the relationship between birth outcomes and FADS1 rs174547 genotypes in Japanese mothers and infants. The study included 406 mothers and 373 infants, i.e., 373 infant-mother pairs, from a supplementary survey of the Japan Environment and Children's Study. Multiple regression analysis revealed that infants with the CC genotype had significantly smaller head circumference at birth compared to those with the TT genotype. Moreover, an interaction between infant genotype and cord blood docosahexaenoic acid (DHA; 22:6n-3) composition affected head circumference at birth. The findings suggest that maternal and infant FADS1 genotypes may influence fetal growth. Furthermore, in FADS1 genotype-stratified multiple regression analysis, infants with maternal and infant CC genotypes exhibited a significant positive association between head circumference at birth and maternal erythrocyte DHA/α-linolenic acid (ALA; 18:3n-3) ratio or fish intake. We highlighted lower metabolic efficiency for endogenous long-chain polyunsaturated fatty acids synthesis in infant-mother pairs homozygous for the minor C allele of FADS1 rs174547. In conclusion, for mothers and infants with this genetic background, maternal fish intake during pregnancy may be potentially important for fetal growth and development. Show less

Angiopoietin-like protein 3 (ANGPTL3) is expressed predominantly in the liver and plays a major role in regulating the circulating triglyceride and lipoprotein fraction concentrations by inhibiting lipoprotein lipase (LPL) activity. Given these physiological roles, ANGPTL3 may play an important role in metabolic changes related to fat accumulation during the fattening period in Japanese Black. This study aimed to reveal the physiological roles of hepatic ANGPTL3 in Japanese Black steers (Bos taurus) during the fattening period and investigate the regulatory effects of hepatic ANGPTL3. To investigate the gene expression and protein localization of ANGPTL3, 18 tissue samples were collected from tree male Holstein bull calves aged 7 wk. Biopsied liver tissues and blood samples were collected from 21 Japanese Black steers during the early (T1; 13 mo of age), middle (T2; 20 mo), and late fattening phases (T3; 28 mo). Relative mRNA expression, blood metabolite concentrations, hormone concentrations, growth, and carcass traits were analyzed. To identify the regulatory factors of hepatic ANGPTL3, primary bovine hepatocytes collected by two Holstein calves aged 7 wk were incubated with insulin, palmitate, oleate, propionate, acetate, or beta-hydroxybutyric acid (BHBA). The ANGPTL3 gene was most highly expressed in the liver, with minor expression in the renal cortex, lungs, reticulum, and jejunum in Holstein bull calves. In Japanese Black steers, relative ANGPTL3 mRNA expressions were less as fattening progressed, and blood triglyceride, total cholesterol, and nonesterified fatty acid (NEFA) concentrations increased. Relative ANGPTL8 and Liver X receptor alpha (LXRα) mRNA expressions decreased in late and middle fattening phases, respectively. Furthermore, relative ANGTPL3 mRNA expression was positively correlated with ANGPTL8 (r = 0.650; P < 0.01) and ANGPTL4 (r = 0.540; P < 0.05) in T3 and T1, respectively, and LXRα showed no correlation with ANGPTL3. Relative ANGTPL3 mRNA expression was negatively correlated with total cholesterol (r = -0.434; P < 0.05) and triglyceride (r = -0.645; P < 0.01) concentrations in T3 and T1, respectively; There was no significant correlation between ANGTPL3 and carcass traits. Relative ANGTPL3 mRNA expression in cultured bovine hepatocytes was downregulated in oleate treatment. Together, these findings suggest that ANGPTL3 downregulation in late fattening phases is associated with the changes in lipid metabolism. Show less

We aimed to determine the similarities and differences in the roles of genic and regulatory copy number variations (CNVs) in bipolar disorder (BD), schizophrenia (SCZ), and autism spectrum disorder (ASD). Based on high-resolution CNV data from 8708 Japanese samples, we performed to our knowledge the largest cross-disorder analysis of genic and regulatory CNVs in BD, SCZ, and ASD. In genic CNVs, we found an increased burden of smaller (<100 kb) exonic deletions in BD, which contrasted with the highest burden of larger (>500 kb) exonic CNVs in SCZ/ASD. Pathogenic CNVs linked to neurodevelopmental disorders were significantly associated with the risk for each disorder, but BD and SCZ/ASD differed in terms of the effect size (smaller in BD) and subtype distribution of CNVs linked to neurodevelopmental disorders. We identified 3 synaptic genes (DLG2, PCDH15, and ASTN2) as risk factors for BD. Whereas gene set analysis showed that BD-associated pathways were restricted to chromatin biology, SCZ and ASD involved more extensive and similar pathways. Nevertheless, a correlation analysis of gene set results indicated weak but significant pathway similarities between BD and SCZ or ASD (r = 0.25-0.31). In SCZ and ASD, but not BD, CNVs were significantly enriched in enhancers and promoters in brain tissue. BD and SCZ/ASD differ in terms of CNV burden, characteristics of CNVs linked to neurodevelopmental disorders, and regulatory CNVs. On the other hand, they have shared molecular mechanisms, including chromatin biology. The BD risk genes identified here could provide insight into the pathogenesis of BD. Show less

Increasing the amount of long-chain polyunsaturated fatty acids (LCPUFA) in human milk is an important strategy for infant growth and development. We investigated the associations of LCPUFA compositions in human milk with maternal diet (especially fish and shellfish intake), with fatty acid Δ5 desaturase gene (FADS1) polymorphisms, and with gene-diet interactions. The present study was performed as part of an adjunct study of the Japan Environment and Children’s Study. The participants were 304 lactating females, who provided human milk 6−7 months after delivery. Fatty acids in human milk were analyzed by gas chromatography, and dietary surveys were conducted using a brief self-administered diet history questionnaire. We also analyzed a single nucleotide polymorphism of FADS1 (rs174547, T/C). There was a significant difference in arachidonic acid (ARA) composition in human milk among the genotype groups, and the values were decreasing in the order of TT > TC > CC. The concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also different between TT and CC genotype, indicating a tendency for decreasing values in the same order. The composition of ARA showed significant gene−dietary interactions in multiple regression analysis, and the positive correlation between fish and shellfish intake and ARA composition in human milk was significant only in the CC genotype. Moreover, the factor most strongly associated with EPA and DHA composition in human milk was fish and shellfish intake. Therefore, it was suggested that increasing fish and shellfish intake in mothers may increase EPA and DHA composition in human milk, while increasing fish and shellfish intake in CC genotype mothers may lead to increased ARA composition in human milk. Show less

We investigated the physiological changes during the fattening period and production characteristics in Japanese Black steers bred and raised using the typical feeding system in Japan. Here, 21 Japanese Black steers aged 12 months were used, with experimental period divided into early (12-14 months of age), middle (15-22 months), and late fattening phases (23-30 months). The liver transcriptome, blood metabolites, hormones, and rumen fermentation characteristics were analyzed. Blood triglyceride and non-esterified fatty acid concentrations increased, whereas blood ketone levels decreased, with fattening phases. Blood insulin increased with fattening phases and was positively correlated with carcass weight and marbling in late fattening phases. Rumen fermentation characteristics showed high propionate levels and low butyrate levels in late fattening phases, likely due to increased energy intake. Genes related to glucose metabolism, such as SESN3, INSR, LEPR, and FOXO3, were down-regulated in late fattening phases. Genes related to lipid metabolism, such as FABP4, were up-regulated, whereas FADS1 and FADS2 were down-regulated. These findings suggest that the physiological changes resulted from changes in the energy content and composition of diets. Liver metabolism changed with changes in fat metabolism. Insulin was strongly associated with physiological changes and productivity in Japanese Black cattle. Show less

Anacetrapib is a novel, powerful cholesteryl ester transfer protein (CETP) inhibitor with bidirectional lipid regulation, which was developed for dyslipidemia. The aim of this study is to evaluate the single- and multiple-dose pharmacokinetics (PK), safety and tolerability of anacetrapib in healthy Chinese subjects and assess the PK difference between Chinese and other populations. Forty subjects were enrolled in an open-label study consisting of three panels (50 mg single dose; 100 mg single dose followed by 100 mg once-daily multiple doses for 10 days; a 200 mg single dose). Safety and tolerability were evaluated by monitoring adverse events, laboratory safety tests, ECGs, vital signs and physical examination. PK were evaluated and compared with historical data in black and white subjects. Anacetrapib was absorbed after administration of a single oral dose, with a median T The PK properties of anacetrapib in Chinese subjects are comparable to those observed in the black population and in white subjects. Single and once-daily administration of anacetrapib was generally well tolerated in healthy Chinese subjects observed in this study. chinadrugtrials.org.cn identifier number CTR20130983. Show less

Long-chain polyunsaturated fatty acids (LC-PUFAs) are involved in the fetal growth in utero, and are essential for the development of visual and cognitive functions during infancy. The purpose of this study was to examine the associations of erythrocyte fatty acid compositions with FADS1 gene polymorphism in Japanese mothers and infants. The subjects were 383 mothers who participated in an adjunct birth cohort study of the Japan Environment and Children's Study (JECS). In maternal FADS1 SNP genotypes, the precursor fatty acids composition of the Δ5 desaturase in the maternal blood showed significant differences in levels among the groups, and showed increasing values in the order of TT < TC < CC genotype groups. On the other hand, many product fatty acids levels were significantly reduced in the order of TT > TC > CC genotype groups, and DHA levels were significantly lower in the CC genotype group relative to the other groups. Likewise, the relationship between fetal genotype group and fatty acid composition in cord blood was very similar to the maternal relationship. These results indicate the maternal and fetal blood fatty acid compositions are strongly influenced by the FADS1 genotypes. With respect to the cord blood DHA composition, the levels in the fetal CC genotype group showed a trend toward lower values in the maternal CC genotype group pair (p = 0.066) compared to the maternal TC genotype group pair. However, in the fetal TT and TC genotype groups (p = 0.131, p = 0.729, respectively), the maternal genotype did not have a significant effect. The DHA composition was more influenced by the maternal genotype in the fetal CC genotype group than in the fetal TT and TC genotype groups. It was shown that DHA transport via the placenta from the mother might be promoted in the fetal CC genotype compared to the other fetal genotype groups. In conclusion, differences in the FADS1 SNP genotypes of pregnant women and their children may greatly affect the supply of LC-PUFAs. Further studies on the involvement of the FADS1 polymorphisms and the fetal LC-PUFA levels in the fetal growth and development are warranted. Show less

Synovial sarcoma (SS) is a rare high-grade malignant mesenchymal tumour with a relatively poor prognosis despite intensive multimodal therapy. Although pazopanib, a multi-kinase inhibitor, is often used for advanced SS, most cases eventually become resistant to pazopanib. In the present study, we investigated the mechanisms of acquired pazopanib resistance in SS. To examine acquired pazopanib resistance, two SS cell lines, SYO-1 and HS-SY-II, were isolated after multiple selection steps with increasing concentrations of pazopanib. SYO-1 was also used in vivo. Then, pazopanib-resistant clones were investigated to assess potential mechanisms of acquired pazopanib resistance. Stable pazopanib-resistant clones were established and exhibited enhanced cell cycle progression, cell growth with increased ERK1/2 phosphorylation, and higher sensitivity than parental cells to a MEK-inhibitor, trametinib, both in vitro and in vivo. Furthermore, addition of low-dose trametinib partially reversed the pazopanib resistance. In the pazopanib-resistant clones, dual specificity phosphatase 6 (DUSP6) was downregulated. Inhibition of DUSP6 expression in parental HS-SY-II cells partially recapitulated acquired pazopanib resistance. Acquired pazopanib resistance in SS was associated with activation of ERK1/2 through downregulation of DUSP6 expression. Simultaneous treatment with pazopanib and a MEK inhibitor could be a promising strategy to overcome pazopanib resistance in SS. Show less

The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of ∼30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Show less

The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of the MAPK kinase MEK and its targets extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we elucidated network structures for the reversible threonine and tyrosine phosphorylation of ERK in both cell types. In addition to differences in the phosphorylation and dephosphorylation reactions, the HaCaT network model required two feedback mechanisms, which, as the experimental data suggested, involved the induction of the dual-specificity phosphatase DUSP6 and the scaffold paxillin. We assayed and modeled the accumulation of the double-phosphorylated and active form of ERK1/2, as well as the dynamics of the changes in the monophosphorylated forms of ERK1/2. Modeling the differences in the dynamics of the changes in the distributions of the phosphorylated forms of ERK1/2 suggested that different amounts of MEK activity triggered context-specific responses, with primary hepatocytes favoring the formation of double-phosphorylated ERK1/2 and HaCaT A5 cells that produce both the threonine-phosphorylated and the double-phosphorylated form. These differences in phosphorylation distributions explained the threshold, sensitivity, and saturation of the ERK response. We extended the findings of differential ERK phosphorylation profiles to five additional cultured cell systems and matched liver tumor and normal tissue, which revealed context-specific patterns of the various forms of phosphorylated ERK. Show less

We investigated whether the single nucleotide polymorphism rs174547 (T/C) of the fatty acid desaturase-1 gene, FADS1, is associated with changes in erythrocyte membrane and plasma phospholipid (PL) long-chain polyunsaturated fatty acid (LCPUFA) composition in elderly Japanese participants (n=124; 65 years or older; self-feeding and oral intake). The rs174547 C-allele carriers had significantly lower arachidonic acid (ARA; n-6 PUFA) and higher linoleic acid (LA, n-6 PUFA precursor) levels in erythrocyte membrane and plasma PL (15% and 6% ARA reduction, respectively, per C-allele), suggesting a low LA to ARA conversion rate in erythrocyte membrane and plasma PL of C-allele carriers. α-linolenic acid (n-3 PUFA precursor) levels were higher in the plasma PL of C-allele carriers, whereas levels of the n-3 LCPUFAs eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) were unchanged in erythrocyte membrane and plasma PL. Thus, rs174547 genotypes were significantly associated with different ARA compositions of the blood of elderly Japanese. Show less

The mammalian Class III phosphoinositide 3-kinase (PIK3C3, also known as mammalian vacuolar protein sorting 34 homologue, Vps34) is a regulator of vesicular trafficking, autophagy, and nutrient sensing. In this study, we generated a specific antibody against PIK3C3, and carried out expression and morphological analyses of PIK3C3 during mouse brain development. In Western blotting, PIK3C3 was detected throughout the developmental process with higher expression in the early embryonic stage. In immunohistochemical analyses with embryonic day 16 mouse brain, PIK3C3 was detected strongly in the axon of cortical neurons. While PIK3C3 was distributed at the soma, nucleus, axon, and dendrites in primary cultured mouse hippocampal neurons at 3 days in vitro (div), it was also found in a punctate distribution with partial colocalization with synaptic marker, synaptophysin, at 21 div. The obtained results indicate that PIK3C3 is expressed and may have a physiological role in central nervous system during corticogenesis. Show less

Apolipoprotein A5 gene promoter region T-1131C polymorphism (APOA5 T-1131C) is known to be associated with elevated plasma TG levels, although little is known of the influence of the interaction between APOA5 T-1131C and lifestyle modification on TG levels. To investigate this matter, we studied APOA5 T-1131C and plasma TG levels of subjects participating in a three-month lifestyle modification program. A three-month lifestyle modification program was conducted with 297 participants (Age: 57 ± 8 years) in Izumo City, Japan, from 2001-2007. Changes in energy balance (the difference between energy intake and energy expenditure) and BMI were used to evaluate the participants' responses to the lifestyle modification. Even after adjusting for confounding factors, plasma TG levels were significantly different at baseline among three genotype subgroups: TT, 126 ± 68 mg/dl; TC, 134 ± 74 mg/dl; and CC, 172 ± 101 mg/dl. Lifestyle modification resulted in significant reductions in plasma TG levels in the TT, TC, and CC genotype subgroups: -21.9 ± 61.0 mg/dl, -20.9 ± 51.0 mg/dl, and -42.6 ± 78.5 mg/dl, respectively, with no significant differences between them. In a stepwise regression analysis, age, APOA5 T-1131C, body mass index (BMI), homeostasis model assessment-insulin resistance (HOMA-IR), and the 18:1/18:0 ratio showed independent association with plasma TG levels at baseline. In a general linear model analysis, APOA5 T-1131C C-allele carriers showed significantly greater TG reduction with decreased energy balance than wild type carriers after adjustment for age, gender, and baseline plasma TG levels. The genetic effects of APOA5 T-1131C independently affected plasma TG levels. However, lifestyle modification was effective in significantly reducing plasma TG levels despite the APOA5 T-1131C genotype background. Show less

Animal studies have demonstrated that glucose-dependent insulinotropic polypeptide (GIP) and GIP receptor (GIPR) contribute to the etiology of obesity. In humans, genomewide association studies have identified single nucleotide polymorphisms (SNPs) in the GIPR gene that are strongly associated with body mass index (BMI); however, it is not clear whether genetic variations in the GIP gene are involved in the development of obesity. In the current study, we assessed the impact of GIP SNPs on obesity-related traits in Japanese adults. Six tag SNPs were tested for associations with obesity-related traits in 3,013 individuals. Multiple linear regression analyses showed that rs9904288, located at the 3'-end of GIP, was significantly associated with visceral fat area (VFA). Moreover, rs1390154 and rs4794008 showed significant associations with plasma triglyceride levels and hemoglobin A1c levels, respectively. Among the significant SNPs, rs9904288 and rs1390154 were independently linked with SNPs in active enhancers of the duodenum mucosa, the main GIP-secreting tissue. The haplotypes of these two SNPs exhibited stronger associations with VFA. Numbers of VFA-increasing alleles of rs9904288 and BMI-increasing alleles of previously identified GIPR SNPs showed a strong additive effect on VFA, waist circumference, and BMI in the subject population. These novel results support the notion that the GIP-GIPR axis plays a role in the etiology of central obesity in humans, which is characterized by the accumulation of visceral fat. Show less

Mammalian tribbles homolog 1 (TRIB1) regulates hepatic lipogenesis and is genetically associated with plasma triglyceride (TG) levels and cholesterol, but the molecular mechanisms remain obscure. We explored these mechanisms in mouse livers transfected with a TRIB1 overexpression, a shRNA template or a control (LacZ) adenovirus vector. The overexpression of TRIB1 reduced, whereas induction of the shRNA template increased, plasma glucose, TG, and cholesterol and simultaneously hepatic TG and glycogen levels. The involvement of TRIB1 in hepatic lipid accumulation was supported by the findings of a human SNP association study. A TRIB1 SNP, rs6982502, was identified in an enhancer sequence, modulated enhancer activity in reporter gene assays, and was significantly (P=9.39 × 10(-7)) associated with ultrasonographically diagnosed non-alcoholic fatty liver disease in a population of 5570 individuals. Transcriptome analyses of mouse livers revealed significant modulation of the gene sets involved in glycogenolysis and lipogenesis. Enforced TRIB1 expression abolished CCAAT/enhancer binding protein A (CEBPA), CEBPB, and MLXIPL proteins, whereas knockdown increased the protein level. Levels of TRIB1 expression simultaneously affected MKK4 (MAP2K4), MEK1 (MAP2K1), and ERK1/2 (MAPK1/3) protein levels and the phosphorylation of JNK, but not of ERK1/2. Pull-down and mammalian two-hybrid analyses revealed novel molecular interaction between TRIB1 and a hepatic lipogenic master regulator, MLXIPL. Co-expression of TRIB1 and CEBPA or MLXIPL reduced their protein levels and proteasome inhibitors attenuated the reduction. These data suggested that the modulation of TRIB1 expression affects hepatic lipogenesis and glycogenesis through multiple molecular interactions. Show less

The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8-47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC. Show less

Heparan sulfate proteoglycans (HSPGs) regulate a number of major developmental processes, but their roles in synovial joint formation remain unknown. Here we created conditional mouse embryo mutants lacking Ext1 in developing joints by mating Ext1(f/f) and Gdf5-Cre mice. Ext1 encodes a subunit of the Ext1/Ext2 Golgi-associated protein complex responsible for heparan sulfate (HS) synthesis. The proximal limb joints did form in the Gdf5-Cre;Ext1(f/f) mutants, but contained an uneven articulating superficial zone that expressed very low lubricin levels. The underlying cartilaginous epiphysis was deranged as well and displayed random patterns of cell proliferation and matrillin-1 and collagen IIA expression, indicative of an aberrant phenotypic definition of the epiphysis itself. Digit joints were even more affected, lacked a distinct mesenchymal interzone and were often fused likely as a result of local abnormal BMP and hedgehog activity and signaling. Interestingly, overall growth and lengthening of long bones were also delayed in the mutants. To test whether Ext1 function is needed for joint formation at other sites, we examined the spine. Indeed, entire intervertebral discs, normally composed by nucleus pulposus surrounded by the annulus fibrosus, were often missing in Gdf5-Cre;Ext1(f/f) mice. When disc remnants were present, they displayed aberrant organization and defective joint marker expression. Similar intervertebral joint defects and fusions occurred in Col2-Cre;β-catenin(f/f) mutants. The study provides novel evidence that local Ext1 expression and HS production are needed to maintain the phenotype and function of joint-forming cells and coordinate local signaling by BMP, hedgehog and Wnt/β-catenin pathways. The data indicate also that defects in joint formation reverberate on, and delay, overall long bone growth. Show less

MLXIPL is a transcription factor integral to the regulation of glycolysis and lipogenesis in the liver. Common variants of the MLXIPL gene (MLXIPL) are known to influence plasma triglyceride levels in people of European descent. As MLXIPL has a key role in energy storage, genetic variations of the MLXIPL may be relevant to physiological adaptations to nutritional stresses that have occurred during the evolution of modern humans. In the present study, we assessed the phenotypic consequences of the Q241H variant of MLXIPL in populations of Asian and Oceanian origin and also surveyed the prevalence of Q241H variant in populations worldwide. Multiple linear regression models based on 2373 individuals of Asian origin showed that the H allele was significantly associated with decreased concentrations of plasma triglycerides (P=0.0003). Direct genotyping of 1455 individuals from Africa, Asia and Oceania showed that the triglyceride-lowering H allele was found at quite low frequencies (0.00-0.16) in most of the populations examined. The exceptions were some Central Asian populations, including Mongolians, Tibetans and Uyghurs, which exhibited much higher frequencies of the H allele (0.21-0.26). The high prevalence of the H allele in Central Asia implies that the Q241H variant of MLXIPL might have been significant for utilization of carbohydrates and fats in the common ancestors of these populations, who successfully adapted to the environment of Central Asia by relying on nomadic livestock herding. Show less

Recent genome-wide association studies (GWASs) showed that single nucleotide polymorphisms (SNPs) in FADS1/FADS2 were associated with plasma lipid concentrations in populations with European ancestry. We investigated the associations between the SNPs in FADS1/FADS2 and plasma concentrations of triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in two Asian groups, i.e., Japanese and Mongolians. The genotype of rs174547 (T/C), found to be associated with triglyceride and HDL-C concentrations in the GWAS, was determined in 21,004 Japanese and 1,203 Mongolian individuals. Genotype-phenotype association was assessed by using multiple linear regression models, assuming an additive model of inheritance. The copy number of the rs174547 C allele was significantly associated with increased triglyceride levels (P = 1.5 x 10(-6)) and decreased HDL-C levels (P = 0.03) in the Japanese population. On the other hand, in the Mongolian population, the rs174547 C allele copy number was strongly associated with decreased LDL-C levels (P = 2.6 x 10(-6)), but was not associated with triglyceride and HDL-C levels. The linkage disequilibrium pattern and haplotype structures of SNPs around the FADS1/FADS2 locus showed no marked dissimilarity between Japanese and Mongolian individuals. The present data indicate that the FADS1/FADS2 locus can be added to the growing list of loci involved in polygenic dyslipidemia in Asians. Furthermore, the variable effects of FADS1/FADS2 on plasma lipid profiles in Asians may result from differences in the dietary intake of polyunsaturated fatty acids, which serve as substrates for enzymes encoded by FADS1/FADS2. Show less

Recent genome wide association studies discovered seven novel loci that influence plasma concentrations of triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol in Europeans. To date, large scale replication studies using populations with known differences in genome-wide linkage disequilibrium (LD) pattern have not been undertaken. To address this issue, we tested associations between single nucleotide polymorphisms (SNPs) within the seven novel loci and plasma lipid profiles in 21 010 Japanese individuals. Multiple linear regression analyses showed that the rs3812316 in MLXIPL was strongly associated with triglyceride concentrations (p approximately 3.0x10(-11), 7.1 mg/dl decrease per minor C allele) and that rs599839 in CELSR2/PSRC1/SORT1 was strongly associated with LDL cholesterol concentrations (p approximately 3.1x10(-11), 4.7 mg/dl decrease per minor G allele) in the Japanese population. SNPs near ANGPTL3, TRIB1 and GALNT2 showed evidence for associations with triglyceride concentrations (3.6x10(-6)Show less

Fibroblast growth factor 1 (FGF-1) enhances apolipoprotein E (apoE) expression and apoE-HDL biogenesis in autocrine fashion in astrocytes (Ito, J., Y. Nagayasu, R. Lu, A. Kheirollah, M. Hayashi, and S. Yokoyama. Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action. J. Lipid Res. 2005. 46: 679-686) associated with healing of brain injury (Tada,T., J-i. Ito, M. Asai, and S. Yokoyama. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 2004. 45: 23-30). FGF-1 stimulates mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) to increase cholesterol biosynthesis and phosphatidylinositol 3-OH kinase (PI3K)/Akt to enhance apoE-HDL secretion (Ito, J., Y. Nagayasu, K. Okumura-Noji, R. Lu, T. Nishida, Y. Miura, K. Asai, A. Kheirollah, S. Nakaya, and S. Yokoyama. Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes. J. Lipid Res. 2007. 48: 2020-2027). We investigated the mechanism for FGF-1 to upregulate apoE transcription. FGF-1 increased apoE and liver X receptor alpha (LXRalpha) mRNAs in rat astrocytes. Increase of LXRalpha mRNA was suppressed by inhibition of the FGF-1 receptor-1 and MEK/ERK but not by inhibition of PI3K/Akt. The increases of apoE mRNA and apoE-HDL secretion were both inhibited by downregulation or inhibition of LXRalpha, while they were partially suppressed by inhibiting cholesterol biosynthesis. We identified the liver X receptor element responsible for activation of the rat apoE promoter by FGF-1 located between -450 and -320 bp, and the direct repeat 4 (DR4) element in this region (-448 to -433 bp) was responsible for the activation. Chromatin immunoprecipitation analysis supported that FGF-1 enhanced association of LXR with the rat apoE promoter. FGF-1 partially activated the apoE promoter even in the presence of an MEK inhibitor that inhibits the FGF-1-mediated enhancement of cholesterol biosynthesis. On the other hand, FGF-1 induced production of 25-hydroxycholesterol by MEK/ERK as an sterol regulatory element-dependent reaction besides cholesterol biosynthesis. We concluded that FGF-1-induced apoE expression in astrocytes depends on LXRalpha being mediated by both LXRalpha expression and an LXRalpha ligand biosynthesis. Show less

In response to environmental stress, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs are released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs) where dynamic changes in protein-RNA interaction determine the subsequent fate of mRNAs. Here we show that the DEAH box RNA helicase RHAU is a novel SG-associated protein. Although RHAU protein was originally identified as an AU-rich element-associated protein involved in urokinase-type plasminogen activator mRNA decay, it was not clear whether RHAU could directly interact with RNA. We have demonstrated that RHAU physically interacts with RNA in vitro and in vivo through a newly identified N-terminal RNA-binding domain, which was found to be both essential and sufficient for RHAU localization in SGs. We have also shown that the ATPase activity of RHAU plays a role in the RNA interaction and in the regulation of protein retention in SGs. Thus, our results show that RHAU is the fourth RNA helicase detected in SGs, after rck/p54, DDX3, and eIF4A, and that its association with SGs is dynamic and mediated by an RHAU-specific RNA-binding domain. Show less

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures. Show less

RHAU (RNA helicase associated with AU-rich element) is a DExH protein originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The discovery that RHAU is predominantly localized in the nucleus, despite mRNA degradation occurring in the cytoplasm, prompted us to consider the nuclear functions of RHAU. In HeLa cells, RHAU was found to be localized throughout the nucleoplasm with some concentrated in nuclear speckles. Transcriptional arrest altered the localization to nucleolar caps, where RHAU is closely localized with RNA helicases p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by actinomycin D chase. There was no change in the half-lives of most transcripts whose steady-state levels were affected by RHAU knockdown, suggesting that these transcripts are subjected to transcriptional regulation. We propose that RHAU has a dual function, being involved in both the synthesis and degradation of mRNA in different subcellular compartments. Show less