Biology subjects

Takeshima, H.

Publications and source records attributed to Takeshima, H..

11 recordsLinked to original sources

bioRxiv2026

A vagal sensory pathway for satiety suppression by intestinal fructose

Mammals primarily use glucose as an energy source, but they can also utilize other isocaloric monosaccharides, such as galactose and fructose. Since the gustatory system cannot distinguish between different sugars in ingested foods, it remains unclear whether and how mammals control the intake of different monosaccharides. Here we perform in vivo calcium imaging of the nodose ganglion in mice and find that intestinal glucose and fructose activate distinct populations of vagal sensory neurons. While glucose suppresses food intake in fed mice, fructose does not, resulting in increased calorie intake. However, after vagotomy, both glucose and fructose suppressed food intake, suggesting that vagal circuits activated by fructose facilitate feeding. This specialized vagal sensory pathway for fructose may partly explain the excess calorie intake induced by a fructose-rich diet.

neuroscience↗

bioRxiv2024

Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but not for live tissues due to its toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is minimally invasive to live cells, allowing for structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices, and the mouse brain in vivo. SeeDB-Live minimally affects the electrophysiological properties and sensory responses of neurons. We demonstrate its utility for widefield imaging of subcellular voltage dynamics, such as backpropagating action potentials, in acute brain slices. We also utilize SeeDB-Live for widefield voltage imaging of dozens of dendrites in vivo, demonstrating population dynamics. Thus, SeeDB-Live expands the scale and modalities of fluorescence imaging of live mammalian tissues.

neuroscience↗

bioRxiv2024

Phosphodiesterase 3 inhibitors boost bone outgrowth

C-type natriuretic peptide (CNP) stimulates skeletal growth by acting on the growth plates of long bones, and a CNP variant is clinically used for achondroplasia treatment. We previously reported that CNP stimulates the autonomic Ca2+ influx mediated by TRPM7 channels in growth plate chondrocytes to facilitate extracellular matrix synthesis for bone growth. Here, we report that phosphodiesterase 3 (PDE3) inhibitors facilitate the Ca2+ influx and promote bone outgrowth. The representative PDE3 inhibitor cilostazol elevated cGMP levels and activated cell-surface K+ channels probably due to protein kinase G-mediated phosphorylation in growth-plate chondrocytes. The resulting hyperpolarization likely facilitated TRPM7-mediated Ca2+ influx by increasing the Ca2+-driving force. Moreover, cilostazol simulated the elongation of cultured bones and enlarged the body size of juvenile mice. Several PDE3 inhibitors have been used for clinical treatment of thrombosis, heart failure and asthma, while our observations suggest that the repositioning of PDE3 inhibitors would provide novel medications for syndromes involving short stature.

pharmacology and toxicology↗

bioRxiv2024

Knock out of the intracellular calcium conducting ion channel Mitsugumin 23 (MG23) protects against pressure overload induced left ventricular hypertrophy and cardiac dysfunction.

BackgroundIn cardiac dysfunction, intracellular Ca2+-dynamics are disrupted leading to leakage of Ca2+ from the sarcoplasmic reticulum (SR). This results in diminished cardiac contractility and impaired cardiac function. In cardiac tissue, the underlying molecular mechanisms responsible for RyR2-independent Ca2+ leak are poorly understood. Mitsugumin 23 (MG23) is an intracellular Ca2+-conducting ion channel located on ER/SR and nuclear membranes. We propose that MG23 contributes to regulation of intracellular Ca2+-homeostasis, and that altered MG23 function may drive progression of cardiac dysfunction. The aim of this research was to investigate the role of MG23 in SR Ca2+ leak, and whether knock out of Mg23 protects the heart against pressure-overload induced left ventricular hypertrophy. MethodsCardiac pressure-overload was induced in wild type (WT) and Mg23-knock out (KO) mice through subcutaneous Angiotensin II (AngII, 1.1 mg/kg/day) infusion via osmotic pump. After 10-days infusion, in vivo pressure-volume dynamics were measured by insertion of a pressure-volume catheter into the left ventricle. MG23 protein expression was assessed through Western blot analysis. Ventricular fibrosis and cardiomyocyte size were measured using histological and immunofluorescence approaches. Cardiomyocytes were isolated from WT and Mg23-KO hearts and intracellular Ca2+ dynamics assessed through live cell imaging using the Ca2+ indicator Fluo-4. ResultsAngII-induced cardiac pressure-overload increased expression of MG23 in WT mouse hearts. Knock out of Mg23 protected hearts against AngII-induced cardiac hypertrophy. Compared to WT animals, AngII treated Mg23-KO mice displayed a significant reduction in left ventricular fibrosis and displayed normal cardiac functioning. In Mg23-KO hearts, no alteration in expression of key Ca2+ handling proteins was identified, but cardiomyocytes displayed altered Ca2+ spark profiles consistent with a role for MG23 in SR Ca2+ leak. ConclusionMG23 plays a key role in driving Ca2+ dysregulation observed in the early pathological stages of pressure-overload induced heart failure.

physiology↗

bioRxiv2024

Mitochondrial DNA analysis in ayu (Plecoglossus altivelis) raises an urgent conservation need for its southernmost population

The central-marginal hypothesis predicts that genetic diversity decreases and genetic differentiation increases toward the species distribution margin. We analyzed the mitochondrial DNA control region of ayu (Plecoglossus altivelis) collected from different localities, including central and marginal populations, to test the hypothesis. The results show that the ayu populations at southern marginal habitats (Tien Yen River in Vietnam and Yakugachi River in Amami-oshima Island) displayed low genetic diversity (Haplotype diversity, H = 0.52 and 0.67; Nucleotide diversity, Pi = 0.01 and 0.006, respectively) and significantly differed from the central populations (p < 0.001), consistent with the central-marginal hypothesis. However, ayu populations at the center of their distribution range (Shinano River in Honsyu and Hamoji River in Sado Island) and the northern marginal habitats (Yoichi River in Hokkaido and Aonae River in Okushiri Island) exhibited high genetic diversity (H = 1 for all rivers; Pi = 0.024-0.029). The genetic differences between the populations at the center and the northern marginal habitats were insignificant (p > 0.05). This study showed that in ayu, the central-marginal hypothesis is valid in the southern direction but not in the northern direction. Urgent conservation is needed for the genetically distinct population in the degraded southern habitat with severely low genetic diversity.

ecology↗

bioRxiv2024

Non-physiologic interaction of MG53 with insulin receptor and lack of evidence for MG53's role in controlling insulin-stimulated Akt phosphorylation in muscle, heart and liver tissues.

RationaleMG53s known function in facilitating tissue repair and anti-inflammation has broad applications to regenerative medicine. There is controversy regarding MG53s role in the development of type 2 diabetes mellitus (T2DM). ObjectiveThis study aims to address this controversy - whether MG53s myokine function contributes to inhibition of insulin signaling in muscle, heart, and liver tissues. Study DesignWe determined the binding affinity of the recombinant human MG53 (rhMG53) to the insulin receptor extracellular domain (IR-ECD) and found low affinity of interaction with Kd (>480 nM). Using cultured C2C12 myotubes and HepG2 cells, we found no effect of rhMG53 on insulin-stimulated Akt phosphorylation (p-Akt). We performed in vivo assay with C57BL/6J mice subjected to insulin stimulation (1 U/kg, intraperitoneal injection) and observed no effect of rhMG53 on insulin-stimulated p-Akt in muscle, heart and liver tissues. ConclusionOverall, our data suggest that rhMG53 can bind to the IR-ECD, however has a low likelihood of a physiologic role, as the Kd for binding is [~]10,000 higher than the physiologic level of MG53 present in the serum of rodents and humans ([~]10 pM). Our findings question the notion proposed by Xiao and colleagues - whether targeting circulating MG53 opens a new therapeutic avenue for T2DM and its complications.

physiology↗

bioRxiv2023

Probe capture enrichment sequencing of amoA genes discloses diverse ammonia-oxidizing archaeal and bacterial populations

The ammonia monooxygenase subunit A (amoA) gene has been used to investigate the phylogenetic diversity, spatial distribution, and activity of ammonia-oxidizing archaeal (AOA) and bacterial (AOB), which contribute significantly to the nitrogen cycle in various ecosystems. Amplicon sequencing of amoA is a widely used method; however, it produces inaccurate results owing to the lack of a universal primer set. Moreover, currently available primer sets suffer from amplification biases, which can lead to severe misinterpretation. Although shotgun metagenomic and metatranscriptomic analyses are alternative approaches without amplification bias, the low concentration of target genes in heterogeneous environmental DNA restricts a comprehensive analysis to a realizable sequencing depth. In this study, we developed a method for amoA enrichment sequencing using a hybridization capture technique. Using metagenomic mock community samples, our approach effectively enriched amoA genes with low compositional changes, outperforming amplification and meta-omics sequencing analyses. Following the analysis of metatranscriptomic marine samples, we predicted 80 operational taxonomic units (OTUs) assigned to either AOA or AOB, of which 30 OTUs were unidentified using simple metatranscriptomic or amoA gene amplicon sequencing. Mapped read ratios to all the detected OTUs were significantly higher for the capture samples (50.4 {+/-} 27.2%) than for non-capture samples (0.05 {+/-} 0.02%), demonstrating the high enrichment efficiency of the method. The analysis also revealed the spatial diversity of AOA ecotypes with high sensitivity and phylogenetic resolution, which are difficult to examine using conventional approaches.

microbiology↗

bioRxiv2023

Atypical cell death in the growth plate chondrocytes of Tric-b-knockout mice

TRIC-A and TRIC-B proteins form homotrimeric cation-permeable channels in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes and are thought to contribute to counterionic flux coupled with store Ca2+ release in various cell types. Serious mutations in the TRIC-B locus cause autosomal recessive osteogenesis imperfecta (OI), which is characterized by insufficient bone mineralization. We have reported that Tric-b-knockout mice can be used as an OI model. Here we report irregular cell death in proliferating growth plate chondrocytes in developing Tric-b-knockout bones. In the knockout chondrocytes, excess pro-collagen fibers were occasionally accumulated in severely dilated ER elements. Of the major ER stress pathways, the PERK pathway was preferentially hyperactivated in the knockout chondrocytes, and most likely altered gene expression to induce apoptosis-related proteins including CHOP and caspase 12. In Ca2+ imaging experiments, the knockout chondrocytes exhibited aberrant Ca2+ handling; ER Ca2+ release was impaired, and intracellular Ca2+ concentration was elevated. Our data suggest that Tric-b deficiency directs growth plate chondrocytes to pro-apoptotic stages by compromising cellular Ca2+-handling and exacerbating ER stress, leading to atypical apoptotic cell death.

developmental biology↗

bioRxiv2021

C-type natriuretic peptide facilitates autonomic Ca2+ entry in growth plate chondrocytes for stimulating bone growth

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide experimental evidence indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel-TRPM7 channel-CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.

cell biology↗

bioRxiv2020

Calcium-induced calcium release and type 3 ryanodine receptors modulate the slow afterhyperpolarising current, sIAHP, and its potentiation in hippocampal pyramidal neurons

The slow afterhyperpolarising current, sIAHP, is a Ca2+-dependent current that plays an important role in the late phase of spike frequency adaptation. sIAHP is activated by voltage-gated Ca2+ channels, while the contribution of calcium from ryanodine-sensitive intracellular stores, released by calcium-induced calcium release (CICR), is controversial in hippocampal pyramidal neurons. Three types of ryanodine receptors (RyR1-3) are expressed in the hippocampus, with RyR3 showing a predominant expression in CA1 neurons. We investigated the specific role of CICR, and particularly of its RyR3-mediated component, in the regulation of the sIAHP amplitude and time course, and the activity-dependent potentiation of the sIAHP in rat and mouse CA1 pyramidal neurons. Here we report that enhancement of CICR by caffeine led to an increase in sIAHP amplitude, while inhibition of CICR by ryanodine caused a small, but significant reduction of sIAHP. Inhibition of ryanodine-sensitive Ca2+ stores by ryanodine or depletion by the SERCA pump inhibitor cyclopiazonic acid caused a substantial attenuation in the sIAHP activity-dependent potentiation in both rat and mouse CA1 pyramidal neurons. Neurons from mice lacking RyR3 receptors exhibited a sIAHP with features undistinguishable from wild-type neurons, which was similarly reduced by ryanodine. However, the lack of RyR3 receptors led to a faster and reduced activity-dependent potentiation of sIAHP. We conclude that ryanodine receptor-mediated CICR contributes both to the amplitude of the sIAHP at steady state and its activity-dependent potentiation in rat and mouse hippocampal pyramidal neurons. In particular, we show that RyR3 receptors play an essential and specific role in shaping the activity-dependent potentiation of the sIAHP. The modulation of activity-dependent potentiation of sIAHP by RyR3-mediated CICR contributes to plasticity of intrinsic neuronal excitability and is likely to play a critical role in higher cognitive functions, such as learning and memory.

neuroscience↗

bioRxiv2018

A new targeted capture method using bacterial artificial chromosome (BAC) libraries as baits for sequencing relatively large genes

To analyze a specific genome region using next-generation sequencing technologies, the enrichment of DNA libraries with targeted capture methods has been standardized. For enrichment of mitochondrial genome, a previous study developed an original targeted capture method that use baits constructed from long-range polymerase chain reaction (PCR) amplicons, common laboratory reagents, and equipment. In this study, a new targeted capture method is presented, that of bacterial artificial chromosome (BAC) double capture (BDC), modifying the previous method, but using BAC libraries as baits for sequencing a relatively large gene. We applied the BDC approach for the 214 kb autosomal region, ring finger protein 213, which is the susceptibility gene of moyamoya disease (MMD). To evaluate the reliability of BDC, cost and data quality were compared with those of a commercial kit. While the ratio of duplicate reads was higher, the cost was less than that of the commercial kit. The data quality was sufficiently the same as that of the kit. Thus, BDC can be an easy, low-cost, and useful method for analyzing individual genome region with substantial length.

genomics↗