Akinori Ishihara

Time of day and nutrients in feeding govern daily expression rhythms of the gene for sterol regulatory element-binding protein (SREBP)-1 in the mouse liver.

Sterol regulatory element-binding protein-1 (SREBP-1) plays a central role in transcriptional regulation of genes for hepatic lipid synthesis that utilizes diet-derived nutrients such as carbohydrates and amino acids, and expression of SREBP-1 exhibits daily rhythms with a peak in the nocturnal feeding period under standard housing conditions of mice. Here, we report that the Srebp-1 expression rhythm shows time cue-independent and Clock mutation-sensitive circadian nature, and is synchronized with varied photoperiods apparently through entrainment of locomotor activity and food intake. Fasting caused diminution of Srebp-1 expression, while diabetic db/db and ob/ob mice showed constantly high expression with loss of rhythmicity. Time-restricted feedings during mid-light and mid-dark periods exhibited differential effects, the latter causing more severe damping of the oscillation. Therefore, “when to eat in a day (the light/dark cycle),” rather than “whenever to eat in a day,” is a critical determinant to shape the daily rhythm of Srebp-1 expression. We further found that a high-carbohydrate diet and a high-protein diet, as well as a high-fat diet, cause phase shifts of the oscillation peak into the light period, underlining the importance of “what to eat.” Daily rhythms of SREBP-1 protein levels and Akt phosphorylation levels also exhibited nutrient-responsive changes. Taken together, these findings provide a model for mechanisms by which time of day and nutrients in feeding shape daily rhythms of the Srebp-1 expression and possibly a number of other physiological functions with interindividual and interdaily differences in human beings and wild animals subjected to day-by-day changes in dietary timing and nutrients.

The Vacuolar-ATPase of Paramecium multimicronucleatum: Gene Structure of the B Subunit and the Dynamics of the V-ATPase-rich Osmoregulatory Membranes

Abstract Previous studies have shown that the vacuolar-ATPase (V-ATPase) of the contractile vacuole complexes (CVCs) in Paramecium multimicronucleatum is necessary for fluid segregation and osmoregulation. In the current study, immunofluorescence showed that the development of a new CVC begins with the formation of a new pore around which the collecting canals form. The decorated membranes are then deposited around the newly formed collecting canals. Quick-freeze deep-etch techniques reveal that six 10-nm-wide V-ATPase V1 sectors, tightly packed into a 20 × 30-nm rectangle, form two rows of these compacted sectors that helically wrap around the cytosolic side of decorated membrane tubules. During new CVC formation, packing of decorated tubules around mature CVCs was temporarily disrupted so that some of these decorated tubules became transformed into decorated vesicles. Freeze-fracturing of these decorated vesicles revealed a highly pitted E-face and a particulate P-face. The V-ATPase was purified for the first time in any ciliated protozoan and shown to contain, as in other cells, the V1 subunits A to E, and four 14–20 kDa polypeptides. The B subunit was cloned and found to be encoded by one gene containing four short introns. This subunit has 510 amino acid residues with a predicted molecular weight of 56.8 kDa, a value similar to B subunits of other organisms. Except for the N- and C-termini, it has a 75% sequence identity with other B subunits, suggesting that the B subunits in Paramecium, like other species, have been conserved and that the entire surface of this subunit may be important in interacting with other subunits.

Evolutionary changes to transthyretin: developmentally regulated and tissue‐specific gene expression

A survey of the expression of the transthyretin and thyroxine‐binding globulin genes in various species during development provides clues as to how the present thyroid hormone distribution network in extracellular compartments developed during vertebrate evolution. Albumin may be the ‘oldest’ component of the thyroid hormone distribution network as it is found in the plasma of all vertebrates investigated. Subsequent to albumin, transthyretin appeared as the second component in this network during the evolution of vertebrates. The strong expression of transthyretin genes in the liver coincides with the presence of recognition site(s) for liver‐enriched transcription factors, such as HNF‐3β (Foxa2), in the transthyretin promoter regions of vertebrates. Finally, the addition of thyroxine‐binding globulin to this network occurred at postnatal stages in some marsupials and rodents and in perinatal to adult stages in most eutherians. All vertebrates have defined developmental stages when thyroid hormone‐dependent transition from larval to juvenile forms occurs. The inclusion of transthyretin and thyroxine‐binding globulin in the thyroid hormone distribution network may be correlated with the increased requirement of thyroid hormones for thyroid hormone‐dependent tissue remodeling during these stages and/or increased metabolism in thyroid hormone‐target tissues with the acquisition of homeothermy.

The effects of endocrine-disrupting chemicals on thyroid hormone binding to Xenopus laevis transthyretin and thyroid hormone receptor.

Abstract We investigated the effects of medical, industrial and agricultural chemicals on 3,3′,5-L-[125I]triiodothyronine ([125I]T3) binding to purified recombinant Xenopus laevis (X. laevis) transthyretin (xTTR), a plasma thyroid hormone-binding protein, and to the ligand-binding domain of thyroid hormone receptor-β (xTR LBD). xTTR derived from X. laevis serum had about 80 times higher affinity for T3 than for L-thyroxine. The xTTRs relative affinities for diethylstilbestrol, pentachlorophenol and ioxynil were 10−1- to 10−2-fold less than that for T3. However, all chemicals investigated had either a weak or no influence on [125I]T3 binding to xTR LBD. The concentration of diethylstilbestrol, the most potent chemical, required for 50% inhibition of [125I]T3 binding to xTR LBD was 104 times greater than that of unlabeled T3. These results indicate the existence of several chemicals that interact with xTTR but not with xTR LBD.

Thyroid system-disrupting chemicals: interference with thyroid hormone binding to plasma proteins and the cellular thyroid hormone signaling pathway.

In vertebrates, thyroid hormones are essential for post-embryonic development, such as establishing the central nervous system in mammals and metamorphosis in amphibians. The present paper summarizes the possible extra-thyroidal processes that environmental chemicals are known to or suspected to target in the thyroid hormone-signaling pathway. We describe how such chemicals interfere with thyroid-hormone-binding protein functions in plasma, thyroid-hormone-uptake system, thyroid-hormone-metabolizing enzymes, and activation or suppression of thyroid-hormone-responsive genes through thyroid-hormone receptors in mammals and amphibian tadpoles. Several organohalogens affect different aspects of the extra-thyroidal thyroid-hormone-signaling pathway but hardly affect thyroid hormone binding to receptors. Rodents and amphibian tadpoles are most sensitive to the effects of environmental chemicals during specific thyroid-hormone-related developmental windows. Possible mechanisms by which environmental chemicals exert multipotent activities beyond one hormone-signaling pathway are discussed.

Multifactorial Regulation of Daily Rhythms in Expression of the Metabolically Responsive Gene Spot14 in the Mouse Liver

Spot14 is a putative transcriptional regulator for genes involved in fatty acid synthesis. The Spot14 gene is activated in response to lipogenic stimuli such as dietary carbohydrate and is also under circadian regulation. The authors investigated factors responsible for daily oscillation of Spot14 expression. If mice were kept under a 12-h light/12-h dark cycle with ad libitum feeding, Spot14 mRNA levels in the liver reached a peak at an early dark period when mice, as nocturnal animals, start feeding. Under fasting, while Spot14 mRNA levels were generally decreased, the rhythmicity was still maintained, suggesting contribution of both nutritional elements and circadian clock factors on robust rhythmicity of Spot14 expression. Effects of circadian clock factors were confirmed by the observations that the circadian rhythm of Spot14 expression was seen also under the constant darkness and that the rhythmicity was lost in Clock mutant mice. When mice were housed in short-photoperiod (6-h light/18-h dark) and long-photoperiod (18-h light/6-h dark) cycles, rhythms of Spot14 mRNA levels were phase advanced and phase delayed, respectively, being concordant with the notion that Spot14 expression is under the control of the light-entrainable oscillator. As for nutritional mediators, in the liver of db/db mice exhibiting hyperinsulinemia-accompanied hyperglycemia, Spot14 mRNA levels were constantly high without apparent rhythmicity, consistent with previous observations for strong activation of the Spot14 gene by glucose and insulin. Restricted feeding during the 4-h mid-light period caused a phase advance of the Spot14 expression rhythm. On the other hand, restricted feeding during the 4-h mid-dark period led to damping of the rhythmicity, apparently resulting from the separation of phases between effects of the light/dark cycle and feeding on Spot14 expression. Thus, the daily rhythm of Spot14 expression in the liver is under the control of the light-entrainable oscillator, food-entrainable oscillator, and food-derived nutrients, in a separate or cooperative manner.

The secretogranin II gene is a signal integrator of glutamate and dopamine inputs

Cooperative gene regulation by different neurotransmitters likely underlies the long‐term forms of associative learning and memory, but this mechanism largely remains to be elucidated. Following cDNA microarray analysis for genes regulated by Ca2+ or cAMP, we found that the secretogranin II gene (Scg2) was cooperatively activated by glutamate and dopamine in primary cultured mouse hippocampal neurons. The Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM) and the mitogen‐activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 prevented Scg2 activation by glutamate or dopamine; thus, the Ca2+/MEK pathway is predicted to include a convergence point(s) of glutamatergic and dopaminergic signaling. Unexpectedly, the protein kinase A inhibitor KT5720 enhanced Scg2 activation by dopamine. The protein‐synthesis inhibitor cycloheximide also enhanced Scg2 activation, and the proteasome inhibitor ZLLLH diminished the KT5720‐mediated augmentation of Scg2 activation. These results are concordant with the notion that dopaminergic input leads to accumulation of a KT5720‐sensitive transcriptional repressor, which is short‐lived because of rapid degradation by proteasomes. This repression pathway may effectively limit the time window permissive to Scg2 activation by in‐phase glutamate and dopamine inputs via the Ca2+/MEK pathway. We propose that the regulatory system of Scg2 expression is equipped with machinery that is refined for the signal integration of in‐phase synaptic inputs.

Gene expression profiling to examine the thyroid hormone-disrupting activity of hydroxylated polychlorinated biphenyls in metamorphosing amphibian tadpole

Hydroxylated polychlorinated biphenyls are the metabolites produced from parent compounds by the drug‐metabolizing enzyme cytochrome P450. These compounds are suspected to disrupt postembryonic neural development in the brains of mammals including humans. We studied the effects of these compounds on thyroid hormone function in the brain by using metamorphosing tadpoles of the African clawed toad (Xenopus laevis) as a model for mammalian postembryonic development. The metamorphosis assay revealed that these compounds inhibit thyroid hormone‐induced metamorphosis. Genome‐wide gene expression analysis in the brain following short‐term exposure demonstrated that delayed metamorphosis could partially be caused by disruption of thyroid hormone‐induced gene expression. Furthermore, we associated the terms of functional ontology with the genes, whose expression was disrupted by these compounds. We suggest that the use of a genome‐wide analysis coupled with bioinformatics might provide an overview of the molecular mechanism underlying thyroid‐disrupting activities in vivo.

Interaction of diethylstilbestrol and ioxynil with transthyretin in chicken serum

The association of suspected endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), ioxynil and pentachlorophenol (PCP), with chicken serum proteins was investigated in relation to thyroid system disruption. All of these chemicals strongly inhibited l-[(125)I]thyroxine ([(125)I]T(4)) binding to purified transthyretin (TTR) whereas PCP was less potent inhibitor than DES and ioxynil of [(125)I]T(4) binding to diluted whole chicken serum. This result suggested that PCP interacted with serum proteins other than TTR in whole chicken serum. Following the incubation of chicken serum with each chemical (final concentrations 0.25-1.0 microM), serum proteins were fractionated by gel filtration chromatography (Cellulofine GCL-1000) and affinity chromatography (human retinol-binding protein coupled to Sepharose 4B). Although all chemicals were detected in the gel filtration chromatography 50-100 kDa fractions, DES and ioxynil, but not PCP, were co-eluted with TTR during affinity chromatography. Our results indicated that a significant proportion of DES and ioxynil, but a low proportion of PCP, interacted with TTR in whole chicken serum.

Histochemical Analyses of Biliary Development During Metamorphosis of Xenopus laevis Tadpoles

In mammalian liver development, intrahepatic biliary morphogenesis takes place in periportal, but not in pericentral, regions. Liver progenitor cells transiently form epithelial plate structures and then intrahepatic bile ducts around the portal veins under the influence of the mesenchyme. The present study was undertaken to histochemically examine normal biliary development and its dependence on the action of the thyroid hormone triiodothyronine (T3) in Xenopus laevis tadpoles. In these tadpoles, the development of hepatic ducts and intrahepatic biliary ducts commenced along the portal veins at NF stages 48–50 and stages 50–52, respectively, when the blood concentration of thyroid hormone may be still low. Some periportal hepatocytes expressed carbamoylphosphate synthase I and SOX9, which are hepatocyte and biliary cell markers, respectively, suggesting that periportal hepatocytes give rise to biliary epithelial cells. Periportal biliary cells did not form ductal plates, nor was the periportal mesenchyme well developed as seen in fetal mouse livers. jag1 mRNA was moderately expressed in cells of portal veins and biliary epithelial cells, and notch1 and notch2 mRNAs were weakly detectable in biliary epithelial cells during metamorphosis as seen in developing mammalian livers. These results suggest that Notch signaling plays a decisive role in biliary cell differentiation and morphogenesis of Xenopus tadpoles. Anti-thyroid agent treatment of the tadpoles resulted in delayed biliary morphogenesis, suggesting that biliary development may depend on T3. However, T3 treatment of the tadpoles did not enhance biliary development. Thus, T3 may act positively on biliary development at a very low concentration.