Negative regulation of thyroid adenoma-associated protein (THADA) in the cardiac glycoside-induced anti-cancer effect.

Cardiac glycosides, known as inhibitors of Na+,K+-ATPase, have anti-cancer effects such as suppression of cancer cell proliferation and induction of cancer cell death. Here, we examined the signaling pathway elicited by cardiac glycosides in the human hepatocellular carcinoma HepG2 cells and human epidermoid carcinoma KB cells. Three kinds of cardiac glycosides (ouabain, oleandrin, and digoxin) inhibited the cancer cell proliferation and decreased the expression level of thyroid adenoma-associated protein (THADA). Interestingly, the knockdown of THADA inhibited cancer cell proliferation, and the proliferation was significantly rescued by re-expression of THADA in the THADA-knockdown cells. In addition, the THADA-knockdown markedly decreased the expression level of L-type amino acid transporter LAT1. Cardiac glycosides also reduced the LAT1 expression. The LAT1 inhibitor, JPH203, significantly weakened the cancer cell proliferation. These results suggest that the binding of cardiac glycosides to Na+,K+-ATPase negatively regulates the THADA-LAT1 pathway, exerting the anti-proliferative effect in cancer cells.

No-observed-adverse-effect-level (NOAEL) clothianidin, a neonicotinoid pesticide, impairs hippocampal memory and motor learning associated with alteration of gene expression in cerebellum.

Neonicotinoid pesticides (NNs) have been associated with numerous neurobehavioral effects in rodents, raising concerns about their impact on cognitive function. Clothianidin (CLO), a type of NN, was orally administered to male mice (10 weeks old, C57BL/6N) at the no-observed-adverse-effect level (NOAEL) of 50 mg/kg/day as indicated in the pesticide risk assessment report. Behavioral tests (novel location recognition and rotarod tests) evaluated hippocampal memory and cerebellar motor learning. After each test, plasma monoamines (3-methoxytyramine, histamine, serotonin, tryptamine) were measured by LC-ESI/MS/MS (Liquid chromatography-electrospray ionization/tandem mass spectrometry), and cerebellar mRNA expression was quantified by microarray and qRT-PCR analyses. The NOAEL of CLO was found to impair hippocampal memory, leading to decreased spontaneous locomotor activity and motor function. We reported, for the first time, multiple alterations of gene expression in the cerebellum associated with motor dysfunction.

An adverse outcome pathway-based approach to assess the neurotoxicity by combined exposure to current-use pesticides.

Exposure to multiple pesticides in daily life has become an important public health concern. However, the combined effects of pesticide mixtures have not been fully elucidated by the conventional toxicological testing used for individual chemicals. Grouping of chemicals by mode of action using common key events (KEs) in the adverse outcome pathway (AOP) as endpoints could be applied for efficient risk assessment of combined exposure to multiple chemicals. The purpose of this study was to investigate whether exposure to multiple pesticides has synergistic neurotoxic effects on mammalian nervous systems. According to the AOP-based approach, we evaluated the effects of 10 current-use pesticides (4 neonicotinoids, 4 pyrethroids and 2 phenylpyrazoles) on the common KEs in AOPs for neurotoxicity, such as KEs involving mitochondrial and proteolytic functions, in a mammalian neuronal cell model. Our data showed that several pyrethroids and phenylpyrazoles partly shared the effects on several common KEs, including decreases in mitochondrial membrane potential and proteasome activity and increases in autophagy activity. Furthermore, we also found that combined exposure to a type-I pyrethroid permethrin or a type-II pyrethroid deltamethrin and the phenylpyrazole fipronil decreased the cell viability and the benchmark doses much more than either single exposure, indicating that the pair exhibited synergistic effects, since the combination indexes were less than 1. These findings revealed that novel pairs of different classes of pesticides with similar effects on common KEs exhibited synergistic neurotoxicity and provide new insights into the risk assessment of combined exposure to multiple chemicals.

Increase in Paracellular Leakage of Amino Acids Mediated by Aging-Induced Reduction of Claudin-4 Expression.

BACKGROUND: Claudins (CLDNs), major components of tight junctions, control paracellular permeabilities of mineral ions and wastes. The absorption of nutrients including glucose and amino acids (AAs) is regulated by intestinal epithelial cells. However, the role of CLDNs is not fully understood. OBJECTIVES: The purpose of this study was to clarify the effect of AA deprivation on the expression of AA transporters and CLDNs, as well as the role of CLDNs in the regulation of paracellular AA fluxes. METHODS: The messenger RNA and protein expression of various CLDNs were examined by real-time quantitative polymerase chain reaction and Western blot analyses, respectively. The AA selectivity of CLDNs was estimated using liquid chromatography-tandem mass spectrometry (LC-MS) analysis. RESULTS: The expression levels of some AA transporters, CLDN4, and CLDN15 were increased by AA deprivation in normal mouse colon-derived MCE301 cells. The expression of AA transporters and CLDN15 in the mouse colon was positively correlated with aging but the expression of CLDN4 was not. The AA deprivation-induced elevation of CLDN4 expression was inhibited by MHY1485, a mammalian target of rapamycin (mTOR) activator. Furthermore, CLDN4 expression was increased by rapamycin, an mTOR inhibitor. mTOR may be involved in the transcriptional activation of CLDN4. The fluxes of AAs from the basal to apical compartments were decreased and increased by CLDN4 overexpression and silencing, respectively. LC-MS analysis showed that the fluxes of all AAs, especially Lys, His, and Arg, were enhanced by CLDN4 silencing. CONCLUSIONS: CLDN4 is suggested to form a paracellular barrier to AAs, especially alkaline AAs, which is attenuated with aging.

Effect of Oral Administration of Polyethylene Glycol 400 on Gut Microbiota Composition and Diet-Induced Obesity in Mice.

Polyethylene glycol (PEG) is a commonly used dispersant for oral administration of hydrophobic agents. PEG is partly absorbed in the small intestine, and the unabsorbed fraction reaches the large intestine; thus, oral administration of PEG may impact the gut microbial community. However, to the best of our knowledge, no study evaluated the effects of PEG on gut commensal bacteria. Herein, we aimed to determine whether oral administration of PEG modifies the gut microbiota. Administration of PEG400 and PEG4000 altered gut microbial diversity in a concentration-dependent manner. Taxonomic analysis revealed that Akkermansia muciniphila and particularly Parabacteroides goldsteinii were overrepresented in mice administered with 40% PEG. PEG400 administration ameliorated the high-fat diet (HFD)-induced obesity and adipose tissue inflammation. Fecal microbiome transplantation from PEG400-administered donors counteracted the HFD-induced body and epididymal adipose tissue weight gain, indicating that PEG400-associated bacteria are responsible for the anti-obesity effect. Conversely, carboxymethyl cellulose, also used as a dispersant, did not affect the abundance of these two bacterial species or HFD-induced obesity. In conclusion, we demonstrated that oral administration of a high concentration of PEG400 (40%) alters the gut microbiota composition and ameliorates HFD-induced obesity.

Data on plasma cortisol levels in nibbler fish Girella punctata reared under high-density conditions in either surface seawater or deep ocean water.

Deep ocean water (DOW) is the water obtained from depth of >200 m below the surface of Earth's oceans and is characterized by rich nutrients and cleanliness [1,2]. We have recently reported that DOW suppresses the high-density-induced increase of plasma cortisol levels (i.e., a stress marker) in Japanese flounder (Paralichthys olivaceus) [1]. The current study aimed to examine whether the cortisol-reducing effect of DOW was observed in other marine organisms as well by comparing the plasma cortisol levels of nibbler fish Girella punctata reared under high-density conditions between surface seawater (SSW) and DOW. The nibbler fish were caught from Tsukumo Bay of Noto Peninsula (Ishikawa Prefecture, Japan). The DOW was obtained from seawater 320 m below the Noto Bay surface at a facility (Aquas Noto, Ishikawa Prefecture, Japan), whereas SSW was obtained from Tsukumo Bay (Noto Peninsula, Ishikawa Prefecture). The dissolved oxygen was maintained at approximately 7 mg/L in DOW as well as in SSW. Before they were transferred to the high-density condition, nibbler fish were acclimated in SSW at 20°C for 1 week at a mean density of 100 g/62.5 L. To expose them to the high-density stress, each of fish was kept at a density of 10 kg/m3 in a single aquarium (60 × 25 × 30 cm) containing either SSW or DOW (n = 8). Subsequently, the fish were reared with SSW or DOW for 10 days at 20°C ± 1°C under a 12:12-h light-dark cycle. A heparin containing syringe was used to obtain the blood samples from the caudal vessels of the fish anesthetized with a 0.04% 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation). The blood sampling was performed on days 0, 5, and 10 after rearing in the small aquaria. The plasma samples were prepared from the collected blood by centrifuging it at 5200 × g for 5 min and the cortisol concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cosmo Bio Co. Ltd., Tokyo, Japan) from those samples. The plasma cortisol concentration of nibbler fish reared in SSW on day 10 was significantly higher than that on day 0, whereas those reared in DOW did not show significant difference on the respective days. The current data contributes to the generalization of the cortisol-reducing effect of DOW on fish, which has been proposed in Japanese flounder [1]. These data could be used for developing and designing experiments to analyze the mechanisms underlying the cortisol-reducing effects by using small fish such as zebrafish, a well-established animal model.

Deep ocean water alters the cholesterol and mineral metabolism of squid Todarodes pacificus and suppresses its weight loss.

This study is the first to demonstrate that deep ocean water (DOW) has physiological significant effects on squid. After 36 h of rearing squids, those reared with DOW had significantly higher total and free cholesterol levels and lower alanine transaminase activity in hemolymph as compared with those reared with surface sea water (SSW). SSW rearing also resulted in 6.95% weight loss, while DOW rearing caused only 2.5% weight loss, which might be due to liver metabolism suppression. Furthermore, both monovalent (sodium, chloride, and potassium ions) and divalent (calcium, inorganic phosphorus, and magnesium ions) ions in hemolymph were elevated when reared with DOW compared to those when reared with SSW. A study of genes expressed in the brain revealed that five genes were specifically remarked in DOW rearing. Most altered genes were neuropeptides, including those from vasopressin superfamily. These neuropeptides are involved in cholesterol and/or mineral metabolisms and physiological significant effects on squid. This study is the first report the effects of DOW on cholesterol and mineral metabolism of squid and will contribute to squid aquaculture using DOW.

Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus.

Deep ocean water (DOW) exerts positive effects on the growth of marine organisms, suggesting the presence of unknown component(s) that facilitate their aquaculture. We observed that DOW suppressed plasma cortisol (i.e., a stress marker) concentration in Japanese flounder (Paralichthys olivaceus) reared under high-density condition. RNA-sequencing analysis of flounder brains showed that when compared to surface seawater (SSW)-reared fish, DOW-reared fish had lower expression of hypothalamic (i.e., corticotropin-releasing hormone) and pituitary (i.e., proopiomelanocortin, including adrenocorticotropic hormone) hormone-encoding genes. Moreover, DOW-mediated regulation of gene expression was linked to decreased blood cortisol concentration in DOW-reared fish. Our results indicate that DOW activated osteoblasts in fish scales and facilitated the production of Calcitonin, a hypocalcemic hormone that acts as an analgesic. We then provide evidence that the Calcitonin produced is involved in the regulatory network of genes controlling cortisol secretion. In addition, the indole component kynurenine was identified as the component responsible for osteoblast activation in DOW. Furthermore, kynurenine increased plasma Calcitonin concentrations in flounders reared under high-density condition, while it decreased plasma cortisol concentration. Taken together, we propose that kynurenine in DOW exerts a cortisol-reducing effect in flounders by facilitating Calcitonin production by osteoblasts in the scales.

Parkinson's disease-associated ATP13A2/PARK9 functions as a lysosomal H+,K+-ATPase.

Mutations in the human ATP13A2 (PARK9), a lysosomal ATPase, cause Kufor-Rakeb Syndrome, an early-onset form of Parkinson's disease (PD). Here, we demonstrate that ATP13A2 functions as a lysosomal H+,K+-ATPase. The K+-dependent ATPase activity and the lysosomal K+-transport activity of ATP13A2 are inhibited by an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase, thapsigargin, and K+-competitive inhibitors of gastric H+,K+-ATPase, such as vonoprazan and SCH28080. Interestingly, these H+,K+-ATPase inhibitors cause lysosomal alkalinization and α-synuclein accumulation, which are pathological hallmarks of PD. Furthermore, PD-associated mutants of ATP13A2 show abnormal expression and function. Our results suggest that the H+/K+-transporting function of ATP13A2 contributes to acidification and α-synuclein degradation in lysosomes.

Adaptation to the shallow sea floor environment of a species of marine worms, Oligobrachia mashikoi, generally inhabiting deep-sea water.

Beard worms from the family Siboglinidae, are peculiar animals and are known for their symbiotic relationships with sulfur bacteria. Most Siboglinids inhabit the deep-sea floor, thus making difficult to make any observations in situ. One species, Oligobrachia mashikoi, occurs in the shallow depths (24.5 m) of the Sea of Japan. Taking advantage of its shallow-water habitat, the first ecological survey of O. mashikoi was performed over a course of 7 years, which revealed that its tentacle-expanding behavior was dependent on the temperature and illuminance of the sea water. Furthermore, there were significantly more O. mashikoi with expanding tentacles during the nighttime than during the daytime, and the prevention of light eliminated these differences in the number of expending tentacles. These results confirmed that the tentacle-expanding behavior is controlled by environmental light signals. Consistent with this, we identified a gene encoding a photoreceptor molecule, neuropsin, in O. mashikoi, and the expression thereof is dependent on the time of day. We assume that the described behavioral response of O. mashikoi to light signals represent an adaptation to a shallow-water environment within the predominantly deep-sea taxon.

Effect of Wheat-Derived Arabinoxylan on the Gut Microbiota Composition and Colonic Regulatory T Cells.

The health benefits of wheat-derived arabinoxylan, a commonly consumed dietary fiber, have been studied for decades. However, its effect on the gut microenvironment and inflammatory bowel disease remains unclear. The objective of this study was to understand the effect of wheat-derived arabinoxylan on gut microbiota, colonic regulatory T cells (Tregs), and experimental colitis. In this study, healthy and chronic colitis model mice were fed chow containing cellulose or wheat-derived arabinoxylan for 2-6 weeks and subjected to subsequent analysis. A 16S-based metagenomic analysis of the fecal DNA revealed that Lachnospiraceae, comprising butyrate-producing and Treg-inducing bacteria, were overrepresented in arabinoxylan-fed mice. In line with the changes in the gut microbiota, both the fecal butyrate concentration and the colonic Treg population were elevated in the arabinoxylan-fed mice. In a T cell transfer model of chronic colitis, wheat-derived arabinoxylan ameliorated body weight loss and colonic tissue inflammation, which may, in part, be mediated by Treg induction. Moreover, wheat-derived arabinoxylan suppressed TNFα production from type 1 helper T cells in this colitis model. In conclusion, wheat-derived arabinoxylans, by altering the gut microenvironment, may be a promising prebiotic for the prevention of colitis.

Regulation of Paracellular Fluxes of Amino Acids by Claudin-8 in Normal Mouse Intestinal MCE301 Cells.

The ingested proteins are catabolized to di/tri-peptides and amino acids (AAs), which are absorbed through various transporters in the small intestinal and colonic epithelial cells. Tight junctions (TJs) are formed between neighboring cells and restrict paracellular fluxes to mineral ions and aqueous molecules. However, it is unknown whether the TJs are implicated in the control of paracellular fluxes to AAs. The paracellular permeability is controlled by claudins (CLDNs), which comprise a family of over 20 members. Here, we found that CLDN8 expression is decreased by AAs deprivation in normal mouse colon-derived MCE301 cells. The reporter activity of CLDN8 was not significantly changed by AAs deprivation, whereas the stability of CLDN8 protein was decreased. MicroRNA analysis showed that AAs deprivation increases the expression of miR-153-5p which targets CLDN8. The AAs deprivation-induced decline of CLDN8 expression was reversed by a miR-153-5p inhibitor. The CLDN8 silencing enhanced the paracellular fluxes to AAs, especially middle molecular size AAs. The expression levels of colonic CLDN8 and miR-153-5p in aged mice were lower and higher than those in young mice, respectively. We suggest that AAs deprivation downregulates CLDN8-dependent barrier function, mediated by the elevation of miR-153-5p expression in the colon, in order to enhance the AAs absorption.

Developmental stage-specific exposure and neurotoxicity evaluation of low-dose clothianidin during neuronal circuit formation.

Neonicotinoid pesticides (NN) were recently reported to exhibit adverse effects in higher vertebrates. Moreover, NNs are routinely transferred from mother to offspring, raising concerns about their effects on future generations. The fetal and neonatal periods are the most critical to the formation of neural circuits in the brain through neurogenesis and differentiation, neuronal migration, axon guidance, and synaptogenesis. NN exposure throughout the fetal and neonatal periods was found to affect the neurobehavior of the offspring, but the stage-specific neurobehavioral effects are unclear. We exposed fetal and neonatal mice to a no-observed-adverse-effect level (NOAEL) of clothianidin (CLO) for 4 days during each of four developmental stages: neurite proliferation and differentiation (fetal days 9-12, CLO-1), neurite outgrowth (fetal days 15-18, CLO-2), synapse formation and astrocyte differentiation (days 1-4 after birth, CLO-3), and synapse remodeling (days 11-14 after birth, CLO-4). CLO's neurobehavioral effects were evaluated in juveniles and adults, revealing that CLO-1 and CLO-2 caused behavioral abnormalities in adult mice. CLO-3 significantly increased locomotor activity and decreased juvenile neurons in the hippocampal dentate gyrus in adulthood. Comprehensive gene analysis of CLO-3 revealed high expression of genes related to neurite outgrowth and axonal branching in the hippocampus in juveniles and adults. These results revealed developmental stage-specific effects of a NOAEL of CLO in the fetal and neonatal periods, suggesting that the susceptibility of the fetus and neonate to CLO varies by developmental stage.

Effect of soluble oat fiber on intestinal microenvironment and TNBS-induced colitis.

Soluble oat fibers, including ß-glucan, have been shown to alter the gut microbiome composition and ameliorate DSS-induced colitis; however, the beneficial effect of soluble oat fiber on colonic inflammation is not yet fully understood. In this study, we demonstrated that soluble oat fibers ameliorate T cell-dependent colitis through the induction of peripherally induced regulatory T cells (pTregs). Soluble oat fibers elevated colonic butyrate production dose-dependently, which coincided with the overrepresentation of Faecalibaculum rodentium (an analog of butyrate-producing Holdemanella biformis) in the gut microbiome. Soluble oat fibers promoted the growth of F. rodentium and H. biformis even in vitro, and increased the concentration of butyrate in the culture supernatant. These results indicate that soluble oat fibers are an energy source for butyrate-producing bacteria and are a fermentation substrate. Soluble oat fibers increased the percentage of colonic pTregs and ameliorated the weight loss and inflammation in acute 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis; this may in part be mediated by the increase in IL-10-producing T cells. In conclusion, our results suggest that the administration of soluble oat fibers is a promising prebiotic treatment for the prevention of colitis mediated via altered gut microbiota composition and elevated butyrate production.

Physiological consequences of space flight, including abnormal bone metabolism, space radiation injury, and circadian clock dysregulation: Implications of melatonin use and regulation as a countermeasure.

Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight.

ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.

Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis.

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C- cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c-/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.

The Impact of Low-Viscosity Soluble Dietary Fibers on Intestinal Microenvironment and Experimental Colitis: A Possible Preventive Application of Alpha-Cyclodextrin in Intestinal Inflammation.

SCOPE: The purpose of this study is to compare the impact of four low-viscosity soluble dietary fibers (DFs) on the intestinal microenvironment, in terms of microbiota composition, short-chain fatty acid (SCFA) production, proportion of colonic peripherally induced regulatory T cells (pTregs), and experimental colitis in mice. METHODS AND RESULTS: Mice are administered 5% w/v low-viscosity soluble DFs in drinking water for 2 weeks. The gut microbiota composition is determined using 16S rRNA sequencing. Luminal SCFAs are quantified by gas chromatography, and colonic pTregs are analyzed using flow cytometry. All low-viscosity soluble DFs promote the growth of beneficial bacteria such as Akkermansia muciniphila and Bacteroides acidifaciens, while eliminating pathogenic bacteria such as Clostridium perfringens. Moreover, two low-viscosity soluble DFs significantly increase the abundance of commensal bacteria and promote the accumulation of propionate and butyrate, leading to marked induction of colonic pTregs. Consistently, these two fibers, in particular α-cyclodextrin, show remarkable anti-inflammatory properties in a colitis mouse model. CONCLUSION: Mice administered any low-viscosity soluble DF show comparable gut microbiota compositions, but differ in terms of bacterial abundance, SCFA concentration, pTreg population, and colitis development. This exploratory study suggests that administration of α-cyclodextrin may be a possible strategy for the prevention of colitis.

Osteoclastic and Osteoblastic Responses to Hypergravity and Microgravity: Analysis Using Goldfish Scales as a Bone Model.

It is known that the bone matrix plays an important role in the response to physical stresses such as hypergravity and microgravity. In order to accurately analyze the response of bone to hypergravity and microgravity, a culture system under the conditions of coexistence of osteoclasts, osteoblasts, and bone matrix was earnestly desired. The teleost scale is a unique calcified organ in which osteoclasts, osteoblasts, and the two layers of bone matrix, i.e., a bony layer and a fibrillary layer, coexist. Therefore, we have developed in vitro organ culture systems of osteoclasts and osteoblasts with the intact bone matrix using goldfish scales. Using the scale culture system, we examined the effects of hypergravity with a centrifuge and simulated ground microgravity (g-µG) with a three-dimensional clinostat on osteoclasts and osteoblasts. Under 3-gravity (3G) loading for 1 day, osteoclastic marker mRNA expression levels decreased, while the mRNA expression of the osteoblastic marker increased. Upon 1 day of exposure, the simulated g-µG induced remarkable enhancement of osteoclastic marker mRNA expression, whereas the osteoblastic marker mRNA expression decreased. In response to these gravitational stimuli, osteoclasts underwent major morphological changes. By simulated g-µG treatments, morphological osteoclastic activation was induced, while osteoclastic deactivation was observed in the 3G-treated scales. In space experiments, the results that had been obtained with simulated g-µG were reproduced. RNA-sequencing analysis showed that osteoclastic activation was induced by the down-regulation of Wnt signaling under flight-microgravity. Thus, goldfish scales can be utilized as a bone model to analyze the responses of osteoclasts and osteoblasts to gravity.

Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na+ ,K+ -ATPase α3-isoform in human cancer cells.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+ ,K+ -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+ ,K+ -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+ ,K+ -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.