👤 Takahiro Sakai

Ghrelin is abundantly produced in the stomach. Here, we found that glutamate decreased ghrelin expression and release in ghrelin-producing cells, and decreased levels of food intake and plasma acyl-ghrelin in mice. Treatment with siRNA of G protein-coupled receptor, family C, group 5, member B (GPRC5B) in ghrelin-producing cell lines completely blocked the effect of glutamate-induced ghrelin suppression. In addition, glutamate inhibited ghrelin release via the extracellular signal-regulated kinase (ERK) activity pathway, and stimulated CREB2 mRNA expression in ghrelin-producing cell lines. These results suggest that glutamate inhibits ghrelin release via ERK-CREB2 pathway. These results suggest that the GPRC5B-ERK-CREB2 pathway is involved in the inhibition of ghrelin expression and secretion in ghrelin cells. Show less

Epigenetic regulation is required to ensure the precise spatial and temporal pattern of gene expression that is necessary for embryonic development. Although the roles of some epigenetic modifications in embryonic development have been investigated in depth, the role of methylation at lysine 79 (H3K79me) is poorly understood. Dot1L, a unique methyltransferase for H3K79, forms complexes with distinct sets of co-factors. To further understand the role of H3K79me in embryogenesis, we generated a mouse knockout of Mllt10, the gene encoding Af10, one Dot1L complex co-factor. We find homozygous Mllt10 knockout mutants (Mllt10-KO) exhibit midline facial cleft. The midfacial defects of Mllt10-KO embryos correspond to hyperterolism and are associated with reduced proliferation of mesenchyme in developing nasal processes and adjacent tissue. We demonstrate that H3K79me level is significantly decreased in nasal processes of Mllt10-KO embryos. Importantly, we find that expression of AP2α, a gene critical for midfacial development, is directly regulated by Af10-dependent H3K79me, and expression AP2α is reduced specifically in nasal processes of Mllt10-KO embryos. Suppression of H3K79me completely mimicked the Mllt10-KO phenotype. Together these data are the first to demonstrate that Af10-dependent H3K79me is essential for development of nasal processes and adjacent tissues, and consequent midfacial formation. Show less

We performed a replication study in a Japanese population to evaluate the association between type 2 diabetes and 7 susceptibility loci originally identified by European genome-wide association study (GWAS) in 2012: ZMIZ1, KLHDC5, TLE1, ANKRD55, CILP2, MC4R, and BCAR1. We also examined the association of 3 additional loci: CCND2 and GIPR, identified in sex-differentiated analyses, and LAMA1, which was shown to be associated with non-obese European type 2 diabetes. We genotyped 6,972 Japanese participants (4,280 type 2 diabetes patients and 2,692 controls) for each of the 10 single nucleotide polymorphisms (SNPs): rs12571751 in ZMIZ1, rs10842994 near KLHDC5, rs2796441 near TLE1, rs459193 near ANKRD55, rs10401969 in CILP2, rs12970134 near MC4R, rs7202877 near BCAR1, rs11063069 near CCND2, rs8108269 near GIPR, and rs8090011 in LAMA1 using a multiplex polymerase chain reaction invader assay. The association of each SNP locus with the disease was evaluated using a logistic regression analysis. All SNPs examined in this study had the same direction of effect (odds ratio > 1.0, p = 9.77 × 10(-4), binomial test), as in the original reports. Among them, rs12571751 in ZMIZ1 was significantly associated with type 2 diabetes [p = 0.0041, odds ratio = 1.123, 95% confidence interval 1.037-1.215, adjusted for sex, age and body mass index (BMI)], but we did not observe significant association of the remaining 9 SNP loci with type 2 diabetes in the present Japanese population (p ≥ 0.005). A genetic risk score, constructed from the sum of risk alleles for the 7 SNP loci identified by un-stratified analyses in the European GWAS meta-analysis were associated with type 2 diabetes in the present Japanese population (p = 2.3 × 10(-4), adjusted for sex, age and BMI). ZMIZ1 locus has a significant effect on conferring susceptibility to type 2 diabetes also in the Japanese population. Show less

The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1(ko) /H2(ko) ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1(ko) /H2(eko) ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1(ko) /H2(ko) phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1(ko) /H2(eko) embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency. Show less

Apolipoprotein AIV (apo AIV) and cholecystokinin (CCK) are satiation factors secreted by the small intestine in response to lipid meals. Apo AIV and CCK-8 has an additive effect to suppress food intake relative to apo AIV or CCK-8 alone. In this study, we determined whether CCK-8 (1, 3, or 5 μg/kg ip) reduces food intake in fasted apo AIV knockout (KO) mice as effectively as in fasted wild-type (WT) mice. Food intake was monitored by the DietMax food system. Apo AIV KO mice had significantly reduced 30-min food intake following all doses of CCK-8, whereas WT mice had reduced food intake only at doses of 3 μg/kg and above. Post hoc analysis revealed that the reduction of 10-min and 30-min food intake elicited by each dose of CCK-8 was significantly larger in the apo AIV KO mice than in the WT mice. Peripheral CCK 1 receptor (CCK1R) gene expression (mRNA) in the duodenum and gallbladder of the fasted apo AIV KO mice was comparable to that in WT mice. In contrast, CCK1R mRNA in nodose ganglia of the apo AIV KO mice was upregulated relative to WT animals. Similarly, upregulated CCK1R gene expression was found in the brain stem of apo AIV KO mice by in situ hybridization. Although it is possible that the increased satiating potency of CCK in apo AIV KO mice is mediated by upregulation of CCK 1R in the nodose ganglia and nucleus tractus solitarius, additional experiments are required to confirm such a mechanism. Show less

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome. Show less

In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT. Show less

Liver X receptor (LXR) is a nuclear receptor that acts as a sterol sensor and metabolic regulator of cholesterol and lipid homeostasis. The foam cell transformation of macrophages (Mvarphi) is considered a critical process in atherosclerotic lesions. The relationship, however, of the foam cell transformation of Mvarphi and LXR is not fully understood. The purpose of the present study was to examine the expression of LXRalpha, retinoid X receptor (RXR)alpha, ATP-binding cassette transporter (ABCA1), and macrophage scavenger receptor A (MSR-A), and lipid accumulation in human monocyte-derived Mvarphi. The expression of LXRalpha, ABCA1, MSR-A in 7 day cultured granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced Mvarphi (GM-Mvarphi) was significantly higher than that in 7 day cultured M-CSF-induced Mvarphi (M-Mvarphi). The expression levels of LXRalpha, ABCA1 and MSR-A protein decreased from 48 h to 5 days after the addition of lipopolysaccharide (LPS) in GM-Mvarphi, but only MSR-A protein decreased at 5 days after the addition of LPS in M-Mvarphi. Intracellular lipid accumulation was clearly observed when GM-Mvarphi was pre-stimulated with LPS for 48 h and incubated with oxidized LDL for an additional 5 days. These findings suggest that the inhibitory activity of LXRalpha, ABCA1 and MSR-A by LPS may be related to the transformation of Mvarphis, especially GM-Mvarphi into foam cells. Show less

Apolipoprotein AIV (apo AIV) and cholecystokinin (CCK) are peptides that act both peripherally and centrally to reduce food intake by decreasing meal size. The present study examined the effects of intraperitoneally administered bolus doses of recombinant apo AIV, CCK-8, and a combination of subthreshold doses of apo AIV and CCK on 4-h food intake in rats that were fasted overnight. Apo AIV at 100 microg/kg reduced food intake significantly relative to the saline control for 1 h, as did doses of CCK-8 at or above 0.125 microg/kg. Doses of apo AIV (50 microg/kg) or CCK (0.06 microg/kg) alone had no effect on food intake. However, when these subthreshold doses of apo AIV and CCK were administered together, the combination produced a significant inhibition of food intake relative to saline controls (P < 0.001), and the duration of the effect was longer than that caused by the administration of either apo AIV or CCK alone. The satiation effect produced by CCK-8 + apo AIV was attenuated by lorglumide, a CCK1 receptor antagonist. We conclude that, whereas the intraperitoneal administration of doses of either recombinant apo AIV or CCK at or above threshold levels reduces food intake, the coadministration of subthreshold doses of the two peptides is highly satiating and works via CCK1 receptor. Show less

Apolipoprotein A-IV (apo A-IV) is a satiety factor involved in the control of food intake and body weight. Our previous studies demonstrated that apo A-IV is present in areas of the hypothalamus where leptin acts to influence energy homeostasis. In the present studies, we found that leptin-deficient obese (ob/ob) mice have significantly reduced hypothalamic apo A-IV mRNA levels. Intragastric infusion of a lipid emulsion significantly stimulated hypothalamic apo A-IV gene expression in lean controls but not in ob/ob mice. Daily ip administration of leptin (3 microg/g) for 5 d significantly increased hypothalamic apo A-IV mRNA levels of ob/ob mice relative to pair-fed controls. In addition, centrally administered leptin raised the reduced apo A-IV gene expression induced by fasting. Using immunohistochemistry, we demonstrated that apo A-IV is present in leptin-sensitive phosphorylated signal transducer and activator of transcription 3 (pSTAT3)-positive cells of the arcuate nucleus of the hypothalamus. Knockdown of STAT3 expression by small interfering RNA significantly attenuated the stimulatory effect of leptin on apo A-IV protein expression in cultured primary hypothalamic neurons, implying that the hypothalamic apo A-IV is regulated by leptin, at least partially, via the STAT3 signaling pathway. Third-ventricular (intracerebroventricular) administration of a subthreshold dose of leptin (1 microg) potentiated apo A-IV-induced (subthreshold dose, 0.5 microg) reduction of feeding, indicating the existence of a functional synergistic interaction between leptin and apo A-IV, leading to suppression of food intake. Show less

We have previously shown that expression of SIAH1 is frequently down-regulated in HCCs and associated with their advanced stages. It has been shown that SIAH1 functions in the phosphorylation-independent degradation of beta-catenin and induces apoptosis and growth arrest. To examine if the effects of SIAH1 overexpression depend on the altered beta-catenin signaling pathway, we transferred the SIAH1 gene into three hepatoma cell lines with different genetic backgrounds: HepG2 (mutant beta-catenin), SNU475 (mutant AXIN1), and Huh7 cells (wild type beta-catenin and AXIN1). SIAH1 significantly decreased aberrant beta-catenin signal in HepG2 and SNU475 cells and induced growth arrest and apoptosis. However, SIAH1 also induced apoptosis in Huh7 cells, which retained a normal membranous distribution pattern of beta-catenin. Immunoblotting study demonstrated that SIAH1 also reduces the amount of PEG10 protein, which is known to be frequently overexpressed in HCC and to promote cell proliferation. These data suggest that PEG10 is another target protein of SIAH1 to induce apoptosis in hepatoma cells. Our results should lead to a better understanding of the relationship between deregulation of beta-catenin signals and hepatocarcinogenesis. Further investigations into the mechanisms by which SIAH1 promotes apoptosis and suppresses cell growth should also allow for the discovery of new therapeutic strategies. Show less

To further investigate the role of intestinal aplipoprotein A-IV (apo A-IV) in the management of daily food intake, we examined the diurnal patterns in apo A-IV gene and protein expression in freely feeding (FF) and food-restricted (FR; food provided 4 h daily for 4 wk) rats that were killed at 3-h intervals throughout the 24-h diurnal cycle. In FF rats, the intestinal apo A-IV mRNA and protein levels showed a circadian rhythm concomitant with the feeding pattern. The daily pattern of fluctuation of apo A-IV, however, was altered in FR rats, which had a marked increase in intestinal apo A-IV levels during the 4-h feeding period of light phase. In both FF and FR rats, increased plasma corticosterone (Cort) levels temporally coincided with the increasing phase of intestinal apo A-IV mRNA and protein expression. Depletion of Cort by adrenalectomy abolished the diurnal rhythm by decreasing the apo A-IV expression during the dark period but did not change the feeding rhythm. Exposure of adrenalectomized rats to consistent Cort level (50-mg continuous release Cort pellet) resulted in fixed apo A-IV levels throughout the day. These results indicate that intestinal apo A-IV exhibits a diurnal rhythm, which can be regulated by endogenous Cort independently of the light-dark cue. The fact that intestinal apo A-IV levels were consistent with the food intake during the normal diurnal cycle as well as during the cycle of 4-h feeding each day suggests that intestinal apo A-IV is involved in the regulation of daily food intake. Show less

Apolipoprotein A-IV (apo A-IV) is a satiety protein synthesized in the small intestine and hypothalamus. To further understand the roles of central apo A-IV in the management of daily food intake, we have examined the diurnal patterns of hypothalamic apo A-IV gene and protein expression in freely feeding and food-restricted (food provided 4 h daily between 1000 h and 1400 h) rats. In freely feeding rats, the hypothalamic apo A-IV mRNA and protein levels fluctuated, with high levels during the light phase, peaking at 0900 h (3 h after lights on), and low levels during the dark phase, with a nadir at 2100 h (3 h after lights off). The daily patterns of the fluctuation, however, were altered in food-restricted rats, which had a marked decrease in hypothalamic apo A-IV mRNA and protein levels during the 4 h-feeding period of the light phase. Although corticosterone (CORT) secretion temporally coincided with the decreasing phase of apo A-IV in the hypothalamus, depletion of CORT by adrenalectomy significantly decreased, rather than increased, hypothalamic apo A-IV mRNA and protein levels. These results indicate that the diurnal expression of hypothalamic apo A-IV is regulated by factors other than the circulating CORT, for example, the reduced food intake and body weight in adrenalectomized animals. The fact that hypothalamic apo A-IV level and food intake were inversely related during the normal diurnal cycle as well as in the period of restricted feeding suggests that hypothalamic apo A-IV is involved in the regulation of daily food intake. Show less