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).

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.

Novel murine model of congenital diabetes: The insulin hyposecretion mouse.

AIMS/INTRODUCTION: Diabetic animal models have made an enormous contribution to our understanding of the etiology of diabetes and the development of new medications. The aim of the present study was to develop and characterize a novel, non-obese murine strain with spontaneous diabetes - the insulin hyposecretion (ihs) mouse. MATERIALS AND METHODS: During the development of the ICGN.B6-Tns2WT strain as the control for the ICGN-Tns2nph congenital nephrotic strain, diabetic mice were discovered and named ihs mice. Intraperitoneal insulin tolerance test, oral glucose tolerance test and an insulin secretion experiment by the pancreas perfusion system were carried out on ihs mice. The pancreatic islets were examined histologically, and the mRNA expression of pancreatic ß-cell-specific genes or genes associated with monogenic diabetes was examined by RT-qPCR. RESULTS: The ihs mice showed several distinctive diabetes-related characteristics: (i) the onset of diabetes was observed only in the male mice; (ii) there were no differences in insulin content between the ihs and control mice; (iii) impaired insulin secretion was elicited by glucose, potassium chloride and sulfonylureas; (iv) there was a significant reduction of relative ß-cell volume with no signs of inflammation or fibrosis; (v) they showed a normal glycemic response to exogenous insulin; and (vi) the mice were not obese. CONCLUSIONS: The ihs mouse provides a novel murine model of congenital diabetes that shows insulin secretion failure. This model allows not only an analysis of the progression of diabetes, but also the identification of unknown genes involved in insulin secretion.

A deletion in the Ctns gene causes renal tubular dysfunction and cystine accumulation in LEA/Tohm rats.

The Long-Evans Agouti (LEA/Tohm) rat has recently been established as a new rat model of type 2 diabetes. The onset of diabetes mellitus was observed only in male LEA/Tohm rats; however, urinary glucose appeared before the onset of diabetes. To clarify the genetic basis of urinary glucose, we performed genetic linkage analysis using (BN × LEA) F2 intercross progeny. A recessively acting locus responsible for urinary glucose excretion (ugl) was mapped to a 7.9 Mb region of chromosome 10, which contains the cystinosin (Ctns) gene. The Ctns gene encodes the cystine transporter, which transports cystine out of the lysosome and is responsible for nephropathic cystinosis in humans. Sequence analysis identified a 13-bp deletion in the Ctns gene, leading to a truncated and loss-of-function protein, which cause cystine accumulation in various tissues. We also developed a novel congenic rat strain harboring the Ctnsugl mutation on the F344 genetic background. Phenotypic analysis of F344-Ctnsugl rats indicated that the incidence of urinary glucose was 100% in both males and females at around 40 weeks of age, and marked cystine accumulation was observed in the tissues, as well as remarkable renal lesions and cystine crystals in the lysosomes of the renal cortex. Furthermore, treatment with cysteamine depleted the cystine contents in F344-Ctnsugl rat embryonic fibroblasts. These results indicated that the F344-Ctnsugl rat provides a novel rat model of cystinosis, which allows not only a better understanding of the pathogenesis and pathophysiology of cystinosis but will also contribute to the development of new therapies.

Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in ß-arrestin 2-Akt signaling and dopaminergic behaviors.

The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-ß-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and ß-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with ß-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a ß-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-ß-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in ß-arrestin-dependent, G protein-independent signaling.

IL-10 production in murine IgM+ CD138hi cells is driven by Blimp-1 and downregulated in class-switched cells.

In contrast to antibody-induced inflammatory responses, some B-cell subpopulations suppress inflammation through the production of interleukin (IL)-10. However, the mechanisms underlying Il10 gene expression during B-cell development is elusive. Here, we identify IgM+ B220lo CD138hi cells responsible for marked IL-10 production in the bone marrow and spleen of mice. These murine IL-10-producing cells predominantly secrete IgM and have unique characteristics of long-lived plasma cells in spite of high expression of surface IgM. We found that IL-10 production is strongly correlated with the expression level of Prdm1 (encoding the Blimp-1 protein), an essential regulator of plasma cell development. Furthermore, overexpression of Prdm1 induces Il10 expression in naïve B cells. Immunoglobulin class-switching recombination events resulted in the downregulation of both Il10 and Prdm1 expression in differentiating B cells. Thus, the prolonged elevation of Blimp-1 expression during the formation of IgM+ CD138hi cells without class-switching elicits IL-10 production. Adoptive transfer of Il10-deficient B cells into B-cell-deficient mice demonstrated that IgM+ CD138hi cell-derived IL-10 supports the survival of class-switched plasma cells and their antibody production in response to antigen challenge. These findings reveal an important role for IL-10 secretion by IgM+ CD138hi cells in the complete and efficient humoral response.

Functional validation of tensin2 SH2-PTB domain by CRISPR/Cas9-mediated genome editing.

Podocytes are terminally differentiated and highly specialized cells in the glomerulus, and they form a crucial component of the glomerular filtration barrier. The ICGN mouse is a model of glomerular dysfunction that shows gross morphological changes in the podocyte foot process, accompanied by proteinuria. Previously, we demonstrated that proteinuria in ICR-derived glomerulonephritis mouse ICGN mice might be caused by a deletion mutation in the tensin2 (Tns2) gene (designated Tns2nph). To test whether this mutation causes the mutant phenotype, we created knockout (KO) mice carrying a Tns2 protein deletion in the C-terminal Src homology and phosphotyrosine binding (SH2-PTB) domains (designated Tns2ΔC) via CRISPR/Cas9-mediated genome editing. Tns2nph/Tns2ΔC compound heterozygotes and Tns2ΔC/Tns2ΔC homozygous KO mice displayed podocyte abnormalities and massive proteinuria similar to ICGN mice, indicating that these two mutations are allelic. Further, this result suggests that the SH2-PTB domain of Tns2 is required for podocyte integrity. Tns2 knockdown in a mouse podocyte cell line significantly enhanced actin stress fiber formation and cell migration. Thus, this study provides evidence that alteration of actin remodeling resulting from Tns2 deficiency causes morphological changes in podocytes and subsequent proteinuria.

Fatty acid remodeling by LPCAT3 enriches arachidonate in phospholipid membranes and regulates triglyceride transport.

Polyunsaturated fatty acids (PUFAs) in phospholipids affect the physical properties of membranes, but it is unclear which biological processes are influenced by their regulation. For example, the functions of membrane arachidonate that are independent of a precursor role for eicosanoid synthesis remain largely unknown. Here, we show that the lack of lysophosphatidylcholine acyltransferase 3 (LPCAT3) leads to drastic reductions in membrane arachidonate levels, and that LPCAT3-deficient mice are neonatally lethal due to an extensive triacylglycerol (TG) accumulation and dysfunction in enterocytes. We found that high levels of PUFAs in membranes enable TGs to locally cluster in high density, and that this clustering promotes efficient TG transfer. We propose a model of local arachidonate enrichment by LPCAT3 to generate a distinct pool of TG in membranes, which is required for normal directionality of TG transfer and lipoprotein assembly in the liver and enterocytes.

The thymic cortical epithelium determines the TCR repertoire of IL-17-producing γδT cells.

The thymus provides a specialized microenvironment in which distinct subsets of thymic epithelial cells (TECs) support T-cell development. Here, we describe the significance of cortical TECs (cTECs) in T-cell development, using a newly established mouse model of cTEC deficiency. The deficiency of mature cTECs caused a massive loss of thymic cellularity and impaired the development of αßT cells and invariant natural killer T cells. Unexpectedly, the differentiation of certain γδT-cell subpopulations-interleukin-17-producing Vγ4 and Vγ6 cells-was strongly dysregulated, resulting in the perturbation of γδT-mediated inflammatory responses in peripheral tissues. These findings show that cTECs contribute to the shaping of the TCR repertoire, not only of "conventional" αßT cells but also of inflammatory "innate" γδT cells.

Deletion of CDKAL1 affects high-fat diet-induced fat accumulation and glucose-stimulated insulin secretion in mice, indicating relevance to diabetes.

BACKGROUND/OBJECTIVE: The CDKAL1 gene is among the best-replicated susceptibility loci for type 2 diabetes, originally identified by genome-wide association studies in humans. To clarify a physiological importance of CDKAL1, we examined effects of a global Cdkal1-null mutation in mice and also evaluated the influence of a CDKAL1 risk allele on body mass index (BMI) in Japanese subjects. METHODS: In Cdkal1-deficient (Cdkal1⁻/⁻) mice, we performed oral glucose tolerance test, insulin tolerance test, and perfusion experiments with and without high-fat feeding. Based on the findings in mice, we tested genetic association of CDKAL1 variants with BMI, as a measure of adiposity, and type 2 diabetes in Japanese. PRINCIPAL FINDINGS: On a standard diet, Cdkal1⁻/⁻ mice were modestly lighter in weight than wild-type littermates without major alterations in glucose metabolism. On a high fat diet, Cdkal1⁻/⁻ mice showed significant reduction in fat accumulation (17% reduction in %intraabdominal fat, P = 0.023 vs. wild-type littermates) with less impaired insulin sensitivity at an early stage. High fat feeding did not potentiate insulin secretion in Cdkal1⁻/⁻ mice (1.0-fold), contrary to the results in wild-type littermates (1.6-fold, P<0.01). Inversely, at a later stage, Cdkal1⁻/⁻ mice showed more prominent impairment of insulin sensitivity and glucose tolerance. mRNA expression analysis indicated that Scd1 might function as a critical mediator of the altered metabolism in Cdkal1⁻/⁻ mice. In accordance with the findings in mice, a nominally significant (P<0.05) association between CDKAL1 rs4712523 and BMI was replicated in 2 Japanese general populations comprising 5,695 and 12,569 samples; the risk allele for type 2 diabetes was also associated with decreased BMI. CONCLUSIONS: Cdkal1 gene deletion is accompanied by modestly impaired insulin secretion and longitudinal fluctuations in insulin sensitivity during high-fat feeding in mice. CDKAL1 may affect such compensatory mechanisms regulating glucose homeostasis through interaction with diet.

Involvement of retrotransposition of long interspersed nucleotide element-1 in skin tumorigenesis induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate.

Tumor development induced by 7,12-dimethylbenz[a]anthracene (DMBA) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) is a well-characterized model of multistep carcinogenesis. DMBA mutates the Ha-ras gene, whereas TPA promotes the growth of transformed cells by activating cellular signaling molecules. It remains to be clarified how repeated TPA treatment endows transformed cells with autonomous cell growth. Long interspersed nucleotide element-1 (L1) is an endogenous retroelement, and 80-100 copies of L1 function as autonomous mobile elements. Although the L1 retrotransposition (RTP) has been found in various human tumors, implying the possible mobility of L1 during carcinogenesis, little is known about how L1-RTP arises in tumor cells, owing to a lack of experimental models. To dissect the mechanism of L1-RTP during carcinogenesis, we established a line of transgenic mice carrying human L1 and enhanced green fluorescent protein (hL1-EGFP mice) and subjected them to DMBA/TPA-induced skin tumorigenesis. Of 15 skin tumors examined, 13 were positive for L1-RTP; L1-RTP was not detected in normal skin tissues adjacent to the tumors. Moreover, nine L1-RTP-positive tumors were positive for activated Ha-ras, and immunohistochemical analysis revealed cells positive for both L1-RTP and phosphorylated Stat3, a marker of tumor cells. Additional in vivo experiments suggested that L1-RTP occurred during tumor promotion by TPA. This is the first report on the involvement of L1-RTP in chemical carcinogenesis. We propose hL1-EGFP mice as a versatile system for investigating the mode of L1-RTP in tumor development and discuss the possible role of L1-RTP in tumorigenesis.

The solute carrier family 15A4 regulates TLR9 and NOD1 functions in the innate immune system and promotes colitis in mice.

BACKGROUND & AIMS: Solute carrier family 15 (SLC15) A4 is a proton-coupled histidine and oligopeptide cotransporter expressed by the immune and nervous systems and associated with disorders such as inflammatory bowel diseases and systemic lupus erythematosus. High levels of SLC15A4 transcripts were observed in human antigen-presenting cells, including dendritic cells, activated macrophages, and B cells. However, the roles of SLC15A4 in the immune regulation are not known. We investigated the function of SLC15A4 in the innate immune system. METHODS: We created SLC15A4-deficient (SLC15A4(-/-)) mice and compared Toll-like receptor 9 and NOD1-dependent innate immune responses between SLC15A4(-/-) and control (SLC15A4(+/+)) mice. RESULTS: SLC15A4 deficiency impaired CpG-induced production of interleukin-12, interleukin-15, and interleukin-18 by dendritic cells. Correspondingly, SLC15A4(-/-) mice developed a less severe form of Th1-dependent colitis than SLC15A4(+/+) mice. Increased lysosomal histidine, in the absence of SLC15A4, appears to negatively regulate Toll-like receptor 9 function by inhibiting the proteolytic activities of cathepsins B and L. SLC15A4(-/-) mice also had a severe defect in NOD1-dependent cytokine production, indicating that SLC15A4 functions as a transporter of the NOD1 ligand. CONCLUSIONS: SLC15A4 promotes colitis through Toll-like receptor 9 and NOD1-dependent innate immune responses. Histidine homeostasis within intracellular compartments is important for eliciting effective innate immune responses.