Yuki Kishimoto

Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts.

Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.

Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes

BackgroundSenescence-accelerated mice (SAM) are a series of mouse strains originally derived from unexpected crosses between AKR/J and unknown mice, from which phenotypically distinct senescence-prone (SAMP) and -resistant (SAMR) inbred strains were subsequently established. Although SAMP strains have been widely used for aging research focusing on their short life spans and various age-related phenotypes, such as immune dysfunction, osteoporosis, and brain atrophy, the responsible gene mutations have not yet been fully elucidated.ResultsTo identify mutations specific to SAMP strains, we performed whole exome sequencing of 6 SAMP and 3 SAMR strains. This analysis revealed 32,019 to 38,925 single-nucleotide variants in the coding region of each SAM strain. We detected Ogg1 p.R304W and Mbd4 p.D129N deleterious mutations in all 6 of the SAMP strains but not in the SAMR or AKR/J strains. Moreover, we extracted 31 SAMP-specific novel deleterious mutations. In all SAMP strains except SAMP8, we detected a p.R473W missense mutation in the Ldb3 gene, which has been associated with myofibrillar myopathy. In 3 SAMP strains (SAMP3, SAMP10, and SAMP11), we identified a p.R167C missense mutation in the Prx gene, in which mutations causing hereditary motor and sensory neuropathy (Dejerine-Sottas syndrome) have been identified. In SAMP6 we detected a p.S540fs frame-shift mutation in the Il4ra gene, a mutation potentially causative of ulcerative colitis and osteoporosis.ConclusionsOur data indicate that different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains.

Antioxidant nutrients in plasma of Japanese patients with chronic obstructive pulmonary disease (COPD), asthma-COPD overlap syndrome, and bronchial asthma.

Few studies to date have investigated the antioxidant nutrients such as vitamin C (ascorbic acid), vitamin E (α‐tocopherol), retinol and carotenoids in plasma from patients with pulmonary disease in Japan. To clarify the role of antioxidant nutrients such as vitamin C, vitamin E, retinol and various carotenoids in plasma of Japanese patients with chronic obstructive lung diseases (COPD), asthma‐COPD overlap syndrome (ACOS) and/or bronchial asthma (BA), we compared to healthy elderly controls.

Ascorbic acid deficiency affects genes for oxidation-reduction and lipid metabolism in livers from SMP30/GNL knockout mice.

BACKGROUND We sought to elucidate the effect of an ascorbic acid (AA) deficiency on gene expression, because the water soluble antioxidant AA is an important bioactive substance in vivo. METHODS We performed microarray analyses of the transcriptome in the liver from senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which are unable to synthesize AA in vivo. RESULTS Our microarray analysis revealed that the AA deficiency increased gene expression related to the oxidation-reduction process, i.e., the nuclear factor, erythroid derived 2, like 2 (Nrf2) gene, which is a reactive oxygen species-sensitive transcriptional factor. Moreover, this AA deficiency increased the expression of genes for lipid metabolism including the cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1), which is a late-limiting enzyme of the primary bile acid biosynthesis pathway. Although an AA deficiency increased the Cyp7a1 protein level, bile acid levels in the liver and gallbladder decreased. Since Cyp7a1 has a heme iron at the active site, AA must function as a reductant of the iron required for the continuous activation of Cyp7a1. CONCLUSIONS This experimental evidence strongly supports a role for AA in the physiologic oxidation-reduction process and lipid metabolism including bile acid biosynthesis. GENERAL SIGNIFICANCE Although many effects of AA supplementation have been reported, no microarray analysis of AA deficiency in vivo is available. Results from using this unique model of AA deficiency, the SMP30/GNL-KO mouse, now provide new information about formerly unknown AA functions that will implement further study of AA in vivo.

Leprdb/db Mice with Senescence Marker Protein-30 Knockout (Leprdb/dbSmp30Y/−) Exhibit Increases in Small Dense-LDL and Severe Fatty Liver Despite Being Fed a Standard Diet

Background/Aims The senescence marker protein-30 (SMP30) is a 34 kDa protein originally identified in rat liver that shows decreased levels with age. Several functional studies using SMP30 knockout (Smp30Y/−) mice established that SMP30 functions as an antioxidant and protects against apoptosis. To address the potential role of SMP30 in nonalcoholic fatty liver disease (NAFLD) pathogenesis, we established Smp30Y/− mice on a Leprdb/db background (Leprdb/dbSmp30Y/− mice). Research Design/Principal Findings Male Leprdb/dbSmp30Y/− mice were fed a standard diet (340 kcal/100 g, fat 5.6%) for 16 weeks whereupon the lipid/lipoprotein profiles, hepatic expression of genes related to lipid metabolism and endoplasmic reticulum stress markers were analyzed by HPLC, quantitative RT-PCR and western blotting, respectively. Changes in the liver at a histological level were also investigated. The amount of SMP30 mRNA and protein in livers was decreased in Leprdb/dbSmp30Y/+ mice compared with Leprdb/+Smp30Y/+ mice. Compared with Leprdb/dbSmp30Y/+ mice, 24 week old Leprdb/dbSmp30Y/− mice showed: i) increased small dense LDL-cho and decreased HDL-cho levels; ii) fatty liver accompanied by numerous inflammatory cells and increased oxidative stress; iii) decreased mRNA expression of genes involved in fatty acid oxidation (PPARα) and lipoprotein uptake (LDLR and VLDLR) but increased CD36 levels; and iv) increased endoplasmic reticulum stress. Conclusion Our data strongly suggest that SMP30 is closely associated with NAFLD pathogenesis, and might be a possible therapeutic target for NAFLD.

Ascorbic acid prevents protein oxidation in livers of senescence marker protein‐30/gluconolactonase knockout mice

Senescence marker protein‐30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice are incapable of synthesizing L‐ascorbic acid (AA) in vivo. As AA is known to be a water‐soluble anti‐oxidant, we assessed protein oxidation levels in livers from SMP30/GNL KO mice maintained in an AA‐insufficient condition.

Insufficient ascorbic acid intake during gestation induces abnormal cardiac dilation in fetal and neonatal SMP30/GNL knockout mice.

Background:Despite the acknowledged importance of ascorbic acid (AA) in maintaining pregnancy and normal fetal development, its precise actions remain obscure. Therefore, we investigated the impact of maternal AA content on the growth of fetal mice during the gestation period using senescence marker protein-30/gluconolactonase (SMP30/GNL) knockout (KO) mice, which cannot synthesize AA in vivo.Methods:SMP30/GNL KO mice gave birth after a gestation period under conditions of absent, low, or normal AA intake. AA was measured using high-performance liquid chromatography and electrochemical detection. Whole-body sections were stained with hematoxylin and eosin, Elastica van Gieson, and Azan.Results:The mothers in the group absent AA intake failed to bear young because of incomplete fetal development. Offspring born under the low-AA condition generally died within a few days after birth. Morphological analysis revealed that the latter neonates of SMP30/GNL KO mothers whose intake of AA was low during gestation manifested abnormal cardiac dilation, congestion of the liver and lungs, incompletely expanded pulmonary alveoli, and impaired vertebral bodies. In contrast, a normal AA diet produced healthy progeny.Conclusion:A diet sufficiently replete with AA is essential during the gestational period for normal tissue development in the fetus and neonate.

Bone degeneration and its recovery in SMP30/GNL-knockout mice

Senescence marker protein-30 (SMP30) decreases androgen-independently with aging and is a lactone-hydrolyzing enzyme gluconolactonase (GNL) that is involved in vitamin C biosynthesis. In the present study, bone properties of SMP30/GNL knockout (KO) mice with deficiency in vitamin C synthesis were investigated to reveal the effects of SMP30/GNL and exogenous vitamin C supplementation on bone formation. Mineral content (BMC) and mineral density (BMD) of the mandible and femur of SMP30/GNL KO and wild-type mice at 2 and 3 months of age with or without vitamin C supplementation were measured by dual-energy X-ray absorptiometry. Body and bone weight of both age groups decreased and became significantly lower than those of wild-type mice. The bones of SMP30/GNL KO mice were rough and porous, with BMC and BMD significantly below wild-type. Oral supplementation with vitamin C eliminated differences in body weight, bone weight, BMC, and BMD between SMP30/GNL KO and wild-type mice at each age. These results indicate that bone degeneration in SMP30/GNL KO mice was caused by lack of vitamin C, and that this mouse strain is an appropriate model for bone metabolism in humans, which have no ability to synthesize vitamin C.

Time-Dependent Alterations of Vancomycin-Induced Nephrotoxicity in Mice

Vancomycin hydrochloride (VCM) is a glycopeptide antibiotic that is commonly used against methicillin-resistant, Gram-positive cocci despite the nephrotoxic side effects. VCM-induced nephrotoxicity has been reported in 5-28% of recipient patients. Therefore, renal failure induced by VCM has become an important clinical problem. However, the exceedingly complex mechanism of VCM-induced nephrotoxicity is not fully understood. Therefore, this study was designed to clarify time-dependent alterations of VCM-induced nephrotoxicity in mice as a step toward decreasing the risks of kidney injury associated with VCM therapy. VCM was injected intraperitoneally into mice at a dose of 400 mg/kg body weight at 24-h intervals for 3, 5, 7, and 14 d. At 24 h after the last injection, we examined histopathological alterations of the kidney as well as blood biochemistry. VCM administration resulted in a decrease of body weight and increase of kidney weight. Histological examination revealed renal damage such as dilated proximal tubules with occasional casts and interstitial fibrosis in VCM-treated mice. Furthermore, immunohistochemical staining with anti-CD10 and anti-single-stranded DNA antibodies highlighted damaged renal proximal tubules with marked dilatation as well as numerous apoptotic cells as early as day 4 of VCM-treatment. The severity of symptoms progressed until day 15. These results suggest that VCM-induced renal damage and incipient renal failure begin soon after the start of treatment and progressively worsen. This is the first report describing the time-dependence of VCM-induced nephrotoxicity in mice and depicting a model that clarifies the mechanisms of this tissue damage.