Akiko Amano

Hydrogen-rich pure water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/GNL knockout mice.

Hydrogen is an established anti-oxidant that prevents acute oxidative stress. To clarify the mechanism of hydrogens effect in the brain, we administered hydrogen-rich pure water (H(2)) to senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), also a well-known anti-oxidant. These KO mice were divided into three groups; recipients of H(2), VC, or pure water (H(2)O), administered for 33 days. VC levels in H(2) and H(2)O groups were <6% of those in the VC group. Subsequently, superoxide formation during hypoxia-reoxygenation treatment of brain slices from these groups was estimated by a real-time biography imaging system, which models living brain tissues, with Lucigenin used as chemiluminescence probe for superoxide. A significant 27.2% less superoxide formed in the H(2) group subjected to ischemia-reperfusion than in the H(2)O group. Thus hydrogen-rich pure water acts as an anti-oxidant in the brain slices and prevents superoxide formation.

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice.

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(-)]. At 4 and 8 weeks, VC levels in brains from VC(-) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(-) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.

Effect of vitamin C depletion on age-related hearing loss in SMP30/GNL knockout mice

Using senescence marker protein 30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), we examined whether modulating VC level affects age-related hearing loss (AHL). KO and wild-type (WT) C57BL/6 mice were given water containing 1.5 g/L VC [VC(+)] or 37.5mg/L VC [VC(-)]. At 10 months of age, KO VC(-) mice showed significant reduction in VC level in the inner ear, plasma, and liver, increase in auditory brainstem response (ABR) thresholds, and decrease in the number of spiral ganglion cells compared to WT VC(-), WT VC(+), and KO VC(+) mice. There were no differences in VC level in the inner ear, ABR thresholds, or the number of spiral ganglion cells among WT VC(-), WT VC(+), and KO VC(+) mice. These findings suggest that VC depletion can accelerate AHL but that supplementing VC may not increase VC level in the inner ear or slow AHL in mice.

Age-related changes of dopamine, noradrenaline and adrenaline in adrenal glands of mice

Aim:  Catecholamines, which are physiologically important neurotransmitters and hormones, apparently decrease in the brain and plasma as some species age. Because this observation has engendered controversy, we used mice to investigate whether age‐related changes occur in adrenal catecholamine levels and in the expression of catecholamine synthetic enzymes.

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.

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.