Mitotic Spindle Positioning (MISP) Facilitates Colorectal Cancer Progression by Forming a Complex with Opa Interacting Protein 5 (OIP5) and Activating the JAK2-STAT3 Signaling Pathway.

Patients with inflammatory bowel disease (IBD) who experience long-term chronic inflammation of the colon are at an increased risk of developing colorectal cancer (CRC). Mitotic spindle positioning (MISP), an actin-binding protein, plays a role in mitosis and spindle positioning. MISP is found on the apical membrane of the intestinal mucosa and helps stabilize and elongate microvilli, offering protection against colitis. This study explored the role of MISP in colorectal tumorigenesis using a database, human CRC cells, and a mouse model for colitis-induced colorectal tumors triggered by azoxymethane (AOM)/dextran sodium sulfate (DSS) treatment. We found that MISP was highly expressed in colon cancer patient tissues and that reduced MISP expression inhibited cell proliferation. Notably, MISP-deficient mice showed reduced colon tumor formation in the AOM/DSS-induced colitis model. Furthermore, MISP was found to form a complex with Opa interacting protein 5 (OIP5) in the cytoplasm, influencing the expression of OIP5 in a unidirectional manner. We also observed that MISP increased the levels of phosphorylated STAT3 in the JAK2-STAT3 signaling pathway, which is linked to tumorigenesis. These findings indicate that MISP could be a risk factor for CRC, and targeting MISP might provide insights into the mechanisms of colitis-induced colorectal tumorigenesis.

The neutrophil-osteogenic cell axis promotes bone destruction in periodontitis.

The immune-stromal cell interactions play a key role in health and diseases. In periodontitis, the most prevalent infectious disease in humans, immune cells accumulate in the oral mucosa and promote bone destruction by inducing receptor activator of nuclear factor-κB ligand (RANKL) expression in osteogenic cells such as osteoblasts and periodontal ligament cells. However, the detailed mechanism underlying immune-bone cell interactions in periodontitis is not fully understood. Here, we performed single-cell RNA-sequencing analysis on mouse periodontal lesions and showed that neutrophil-osteogenic cell crosstalk is involved in periodontitis-induced bone loss. The periodontal lesions displayed marked infiltration of neutrophils, and in silico analyses suggested that the neutrophils interacted with osteogenic cells through cytokine production. Among the cytokines expressed in the periodontal neutrophils, oncostatin M (OSM) potently induced RANKL expression in the primary osteoblasts, and deletion of the OSM receptor in osteogenic cells significantly ameliorated periodontitis-induced bone loss. Epigenomic data analyses identified the OSM-regulated RANKL enhancer region in osteogenic cells, and mice lacking this enhancer showed decreased periodontal bone loss while maintaining physiological bone metabolism. These findings shed light on the role of neutrophils in bone regulation during bacterial infection, highlighting the novel mechanism underlying osteoimmune crosstalk.

Ameliorated Renal Pathological Feature in MRL/MpJ-Faslpr/lpr Background Interleukin-36 Receptor-Deficient Mice.

Systemic autoimmune diseases frequently induce lupus nephritis, causing altered balance and expression of interleukin 36 receptor (IL-36R) ligands, including agonists (IL-36α, ß, γ) and antagonists (IL-36Ra, IL-38), in kidneys. Here, we established and analyzed a mouse model of lupus nephritis, MRL/MpJ-Faslpr/lpr with IL-36R-knockout (KO), compared to wild-type (WT) mice. In both genotypes, indices for immune abnormalities and renal functions were comparable, although female WT mice showed higher serum autoantibody levels than males. IL-36R ligand expression did not differ significantly between genotypes at the mRNA level or in IL-36α and IL-38 scores. However, glomerular lesions, especially mesangial matrix expansion, were significantly ameliorated in both sexes of IL-36R-KO mice compared to WT mice. Cell infiltration into the tubulointerstitium with the development of tertiary lymphoid structures was comparable between genotypes. However, the positive correlation with the IL-36α score in WT mice was not evident in IL-36R-KO mice. Fibrosis was less in female IL-36R-KO mice than in WT mice. Importantly, some IL-36α+ nuclei co-localized with acetylated lysine and GCN5 histone acetyltransferase, in both genotypes. Therefore, IL-36R ligands, especially IL-36α, contribute to the progression of renal pathology in lupus nephritis via IL-36R-dependent and IL-36R-independent pathways.

Dietary omega-3 fatty acid does not improve male infertility caused by lysophospholipid acyltransferase 3 (LPLAT3/AGPAT3) deficiency.

Docosahexaenoic acid (DHA), an omega-3 fatty acid, usually presents as a constituent of phospholipids in the cellular membrane. Lysophospholipid acyltransferase 3 (LPLAT3; AGPAT3) is the primary enzyme that incorporates DHA into phospholipids. LPLAT3-KO mice show male infertility and visual dysfunction accompanied by decreased phospholipids (PLs) containing DHA (PL-DHA) in the testis and retina, respectively. In this study, we evaluated the effect of diets consisting mainly of triacylglycerol-bound DHA (fish oil) and PL-bound DHA (salmon roe oil) on the amount of PL-DHA in a broad range of tissues and on reproductive functions. Both diets elevated phosphatidylcholines (PCs)-containing DHA in most tissues of wild type (WT) mice. Although LPLAT3-KO mice acquired a minimal amount of PC-DHA in the testes and sperm by eating either of the diets, reproductive function did not improve. The present study suggests that DHA-rich diets do not restore sufficient PL-DHA to improve male infertility in LPLAT3-KO mice. Alternatively, PL-DHA can be biosynthesized by LPLAT3 but not by external supplementation, which may be necessary for normal reproductive function.

Molecular and Pathological Analyses of IARS1-Deficient Mice: An IARS Disorder Model.

Most mitochondrial diseases are hereditary and highly heterogeneous. Cattle born with the V79L mutation in the isoleucyl-tRNA synthetase 1 (IARS1) protein exhibit weak calf syndrome. Recent human genomic studies about pediatric mitochondrial diseases also identified mutations in the IARS1 gene. Although severe prenatal-onset growth retardation and infantile hepatopathy have been reported in such patients, the relationship between IARS mutations and the symptoms is unknown. In this study, we generated hypomorphic IARS1V79L mutant mice to develop an animal model of IARS mutation-related disorders. We found that compared to wild-type mice, IARSV79L mutant mice showed a significant increase in hepatic triglyceride and serum ornithine carbamoyltransferase levels, indicating that IARS1V79L mice suffer from mitochondrial hepatopathy. In addition, siRNA knockdown of the IARS1 gene decreased mitochondrial membrane potential and increased reactive oxygen species in the hepatocarcinoma-derived cell line HepG2. Furthermore, proteomic analysis revealed decreased levels of the mitochondrial function-associated protein NME4 (mitochondrial nucleoside diphosphate kinase). Concisely, our mutant mice model can be used to study IARS mutation-related disorders.

Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis.

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3-/-mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3-/-embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3-/-mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3-/-mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3-/-mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3-/- mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases.

Dystrophin Short Product, Dp71, Interacts with AQP4 and Kir4.1 Channels in the Mouse Cerebellar Glial Cells in Contrast to Dp427 at Inhibitory Postsynapses in the Purkinje Neurons.

Dystrophin is the causative gene for Duchenne and Becker muscular dystrophy (DMD/BMD), and it produces full-length and short dystrophin, Dp427 and Dp71, respectively, in the brain. The existence of the different dystrophin molecular complexes has been known for a quarter century, so it is necessary to derive precise expression profiles of the molecular complexes in the brain to elucidate the mechanism of cognitive symptoms in DMD/BMD patients. In order to investigate the Dp71 expression profile in cerebellum, we employed Dp71-specific tag-insertion mice, which allowed for the specific detection of endogenous Dp71 in the immunohistochemical analysis and found its expressions in the glial cells, Bergmann glial (BG) cells, and astrocytes, whereas Dp427 was exclusively expressed in the inhibitory postsynapses within cerebellar Purkinje cells (PCs). Interestingly, we found different cell-type dependent dystrophin molecular complexes; i.e., glia-associated Dp71 was co-expressed with dystroglycan (DG) and dystrobrevinα, whereas synapse-associated Dp427 was co-expressed with DG and dystrobrevinß. Furthermore, we investigated the molecular relationship of Dp71 to the AQP4 water channel and the Kir4.1 potassium channel, and found biochemical associations of Dp71 with AQP4 and Kir4.1 in both the cerebellum and cerebrum. Immunohistochemical and cytochemical investigations revealed partial co-localizations of Dp71 with AQP4 and Kir4.1 in the glial cells, indicating Dp71 interactions with the channels in the BG cells and astrocytes. Taken together, different cell-types, glial cells and Purkinje neurons, in the cerebellum express different dystrophin molecular complexes, which may contribute to pathological and physiological processes through the regulation of the water/ion channel and inhibitory postsynapses.

Identification of SARS-CoV-2 Mpro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

Mitotic spindle positioning protein (MISP) deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis in mice.

Inflammatory bowel disease (IBD) is classified into two types: Crohn's disease and ulcerative colitis. In IBD, the imbalance between the pro-inflammatory and anti-inflammatory cytokines prevents recovery from the inflammatory state, resulting in chronic inflammation in the colon. The mitotic spindle positioning protein (MISP) is localized to the apical membrane in the colon. In this study, we observed increased expression of MISP in the intestinal epithelial cells in dextran sulfate sodium (DSS)-induced colitis in mice. MISP-deficient mice receiving DSS showed significant exacerbation of colitis (e.g., weight loss, loss of the crypts). The intestinal epithelial cells of the MISP-deficient mice showed a trend towards decreased cell proliferation after DSS treatment. Reverse transcription followed by quantitative polymerase chain reaction revealed that the expression levels of Tgfb1, an anti-inflammatory cytokine, were significantly reduced in the colon of MISP-deficient mice compared with the wild-type mice regardless of DSS treatment. These findings indicate that MISP may play a role in the recovery of the colon after inflammation through its anti-inflammatory and proliferative activities, suggesting that MISP may be a new therapeutic target for IBD.

Potent and biostable inhibitors of the main protease of SARS-CoV-2.

Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection in vitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.

Generation of dystrophin short product-specific tag-insertion mouse: distinct Dp71 glycoprotein complexes at inhibitory postsynapse and glia limitans.

Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA-tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71, although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinß (Dtnb), whereas glia-associated Dp71 did with Dg and dystrobrevinα (Dtna). Thus, our data indicate that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.

The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator.

Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genome-editing efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.

A single amino acid substitution in PRKDC is a determinant of sensitivity to Adriamycin-induced renal injury in mouse.

Adriamycin (ADR)-induced nephropathy is frequently utilized in rodent models of podocytopathy. However, the application of this model in mice is limited to a few strains, such as BALB/c mice. The most commonly used mouse strain, C57BL/6 (B6), is resistant to ADR-induced nephropathy, as are all mouse strains with a B6 genetic background. Reportedly, the R2140C variant of the Prkdc gene is the cause of susceptibility to ADR-induced nephropathy in mice. To verify this hypothesis, we produced Prkdc mutant B6 mice, termed B6-PrkdcR2140C, that possess the R2140C mutation. After administration of ADR, B6-PrkdcR2140C mice exhibited massive proteinuria and glomerular and renal tubular injuries. In addition, there was no significant difference in the severity between B6-PrkdcR2140C and BALB/c. These findings demonstrated that B6-PrkdcR2140C show ADR-induced nephropathy susceptibility at a similar level to BALB/c, and that the PRKDC R2140C variant causes susceptibility to ADR-induced nephropathy. In future studies, ADR-induced nephropathy may become applicable to various kinds of genetically modified mice with a B6 background by mating with B6-PrkdcR2140C.

New inducible mast cell-deficient mouse model (Mcpt5/Cma1DTR).

Mast cell-deficient mice are helpful for understanding the roles of mast cells in vivo. To date, a dozen mouse models for mast cell deficiency have been reported. However, mice with a specific depletion of all populations of mast cells have not been reported. We generated knock-in mice, termed Mcpt5/Cma1DTR mice, expressing human diphtheria toxin A (DT) receptor under the endogenous promoter of Mcpt5 (also known as Cma1), which encodes mouse mast cell protease-5. Flow cytometry and histological analysis showed that intraperitoneal injection of DT induced almost complete depletion of mast cells in heterozygote Mcpt5/Cma1DTR/+ mice. The deletion rates of mast cells in peritoneal cavity, mesentery, abdominal skin, ear skin, and glandular stomach were 99.9%, 100%, 98.7%, 97.7%, and 100%, respectively. Passive cutaneous anaphylaxis reaction also revealed mast cell deficiency in ear skin after DT treatment. Other than mast cells, a small percentage of marginal zone B cells in Mcpt5/Cma1DTR/+ mice were killed by DT treatment. In conclusion, the Mcpt5/Cma1DTR/+ mouse model is valuable for achieving conditional depletion of all populations of mast cells without inducing a marked reduction in other cells.

OPG Production Matters Where It Happened.

Osteoprotegerin (OPG) is a circulating decoy receptor for RANKL, a multifunctional cytokine essential for the differentiation of tissue-specific cells in bone and immune systems such as osteoclasts, medullary thymic epithelial cells (mTECs), and intestinal microfold cells (M cells). However, it is unknown whether OPG functions only at the production site or circulates to other tissues acting in an endocrine fashion. Here we explore the cellular source of OPG by generating OPG-floxed mice and show that locally produced OPG, rather than circulating OPG, is crucial for bone and immune homeostasis. Deletion of OPG in osteoblastic cells leads to severe osteopenia without affecting serum OPG. Deletion of locally produced OPG increases mTEC and M cell numbers while retaining the normal serum OPG level. This study shows that OPG limits its functions within the tissue where it was produced, illuminating the importance of local regulation of the RANKL system.

Fibroblasts as a source of self-antigens for central immune tolerance.

Fibroblasts are one of the most common but also neglected types of stromal cells, the heterogeneity of which underlies the specific function of tissue microenvironments in development and regeneration. In the thymus, autoreactive T cells are thought to be negatively selected by reference to the self-antigens expressed in medullary epithelial cells, but the contribution of other stromal cells to tolerance induction has been poorly examined. In the present study, we report a PDGFR+ gp38+ DPP4- thymic fibroblast subset that is required for T cell tolerance induction. The deletion of the lymphotoxin ß-receptor in thymic fibroblasts caused an autoimmune phenotype with decreased expression of tissue-restricted and fibroblast-specific antigens, offering insight into the long-sought target of lymphotoxin signaling in the context of the regulation of autoimmunity. Thus, thymic medullary fibroblasts play an essential role in the establishment of central tolerance by producing a diverse array of self-antigens.

d-Amino acid oxidase deficiency is caused by a large deletion in the Dao gene in LEA rats.

d-Amino acids, enantiomers of l-amino acids, are increasingly recognized as physiologically active molecules as well as potential biomarkers for diseases. d-Amino acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids and is present in a wide variety of organisms from yeasts to humans. Previous studies indicated that LEA rats lacked DAO activity, and levels of d-Ser and d-Ala were markedly increased in their tissues, suggesting a mutated locus responsible for the lack of Dao activity (ldao) existed in the LEA genome. Sequence analysis identified deletion breakpoints located in intron 4-5 of the Dao gene and intron 1-2 of the Svop gene, resulting in a 54.1-kb deletion which encompassed exons 5-12 of the Dao gene and exons 2-16 of the Svop gene. We developed a novel congenic rat strain, F344-Daoldao, harboring the Daoldao mutation from LEA rats delivered onto the F344 genetic background. Compared to the parental F344 strain, in F344-Daoldao rats d-Ala was markedly increased in both cerebrum and cerebellum, while d-Ser content was increased in cerebellum but not cerebrum. d-Ala, d-Ser, d-Pro and d-Leu levels were also elevated in F344-Daoldao plasma. F344-Daoldao rats represent a novel model system that will aid in elucidating the physiological functions of d-amino acids in vivo. (203 words).

Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells.

Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6.

To identify factors involved in the earliest phase of the differentiation of human embryonic stem cells (hESCs) into brown adipocytes (BAs), we performed multi-time point microarray analyses. We found that growth/differentiation factor 15 (GDF15) expressions were specifically upregulated within three days of differentiation, when expressions of immature hESC markers were sustained. Although GDF15 expressions continued to increase in the subsequent differentiation phases, GDF15-deficient hESCs differentiated into mature BAs (Day 10) without apparent abnormalities. In addition, GDF15-deficient mice had normal brown adipose tissue (BAT) and were metabolically healthy. Unexpectedly, we found that interleukin-6 (IL6) expression was significantly lowered in the BAT of GDF15-/- mice. In addition, GDF15-/- hESCs showed abortive IL6 expressions in the later phase (>Day 6) of the differentiation. Interestingly, GDF15 expression was markedly repressed throughout the whole course of the differentiation of IL6-/- hESCs into BAs, indicating IL6 is essential for the induction of GDF15 in the differentiation of hESCs. Finally, intraperitoneally transplanted BAT grafts of GDF15-/- donor mice, but not those of wild-type (WT) mice, failed in the long-term survival (12 weeks) in GDF15-/- recipient mice. Collectively, GDF15 is required for long-term survival of BAT grafts by creating a mutual gene induction loop with IL6.

Genetic locus responsible for diabetic phenotype in the insulin hyposecretion (ihs) mouse.

Diabetic animal models have made significant contributions to understanding the etiology of diabetes and to the development of new medications. Our research group recently developed a novel diabetic mouse strain, the insulin hyposecretion (ihs)mouse. The strain involves neither obesity nor insulitis but exhibits notable pancreatic ß-cell dysfunction, distinguishing it from other well-characterized animal models. In ihs mice, severe impairment of insulin secretion from pancreas has been elicited by glucose or potassium chloride stimulation. To clarify the genetic basis of impaired insulin secretion, beginning with identifying the causative gene, genetic linkage analysis was performed using [(C57BL/6 × ihs) F1 × ihs] backcross progeny. Genetic linkage analysis and quantitative trait loci analysis for blood glucose after oral glucose loading indicated that a recessively acting locus responsible for impaired glucose tolerance was mapped to a 14.9-Mb region of chromosome 18 between D18Mit233 and D18Mit235 (the ihs locus). To confirm the gene responsible for the ihs locus, a congenic strain harboring the ihs locus on the C57BL/6 genetic background was developed. Phenotypic analysis of B6.ihs-(D18Mit233-D18Mit235) mice showed significant glucose tolerance impairment and markedly lower plasma insulin levels during an oral glucose tolerance test. Whole-genome sequencing and Sanger sequencing analyses on the ihs genome detected two ihs-specific variants changing amino acids within the ihs locus; both variants in Slc25a46 and Tcerg1 were predicted to disrupt the protein function. Based on information regarding gene functions involving diabetes mellitus and insulin secretion, reverse-transcription quantitative polymerase chain reaction analysis revealed that the relative abundance of Reep2 and Sil1 transcripts from ihs islets was significantly decreased whereas that of Syt4 transcripts were significantly increased compared with those of control C57BL/6 mice. Thus, Slc25a46, Tcerg1, Syt4, Reep2 and Sil1 are potential candidate genes for the ihs locus. This will be the focus of future studies in both mice and humans.