Senescence Marker Protein-30 Knockout Research Articles

Determination of tissue-specific interaction between vitamin C and vitamin E in vivo using senescence marker protein-30 knockout mice as a vitamin C synthesis deficiency model.

Vitamin E (α-tocopherol; VE) is known to be regenerated from VE radicals by vitamin C (L-ascorbic acid; VC) in vitro. However, their in vivo interaction in various tissues is still unclear. Therefore, we alternatively examined the in vivo interaction of VC and VE by measurement of their concentrations in various tissues of senescence marker protein-30 (SMP30) knockout (KO) mice as a VC synthesis deficiency model. Male SMP30-KO mice were divided into four groups (VC+/VE+, VC+/VE-, VC-/VE+ and VC-/VE-), fed diets with or without 500 mg/kg VE and given water with or without 1·5 g/l VC ad libitum. Then, VC and VE concentrations in the plasma and various tissues were determined. Further, gene expression levels of transporters associated with VC and VE, such as α-tocopherol transfer protein (α-TTP) and sodium-dependent vitamin C transporters (SVCTs), were examined. These results showed that the VE levels in the VC-depleted (VC-/VE+) group were significantly lower than those in the VC+/VE+ group in the liver and heart; the VC levels in the VE-depleted (VC+/VE-) group were significantly lower than those in the VC+/VE+ group in the kidneys. The α-TTP gene expression in the liver and kidneys was decreased by VC and/or VE depletion. Moreover, SVCT1 gene expression in the liver was decreased by both VC and VE depletion. In conclusion, these results indicate that VC spares VE mainly in the liver and heart and that VE spares VC in the kidneys of SMP30-KO mice. Thus, interaction between VC and VE is likely to be tissue specific.

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Vitamin C Deficiency Inhibits Nonalcoholic Fatty Liver Disease Progression through Impaired de Novo Lipogenesis

Despite the increasing clinical importance of nonalcoholic fatty liver disease (NAFLD), little is known about its underlying pathogenesis or specific treatment. The senescence marker protein 30 (SMP30), which regulates the biosynthesis of vitamin C (VC) in many mammals, except primates and humans, was recently recognized as a gluconolactonase. However, the precise relation between VC and lipid metabolism in NAFLD is not completely understood. Therefore, this study aimed to clearly reveal the role of VC in NAFLD progression. SMP30 knockout (KO) mice were used as a VC-deficient mouse model. To investigate the precise role of VC on lipid metabolism, 13- to 15-week-old SMP30 KO mice and wild-type mice fed a 60% high-fat diet were exposed to tap water or VC-containing water (1.5 g/L) ad libitum for 11 weeks. Primary mouse hepatocytes isolated from the SMP30 KO and wild-type mice were used to demonstrate the relation between VC and lipid metabolism in hepatocytes. Long-term VC deficiency significantly suppressed the progression of simple steatosis. The high-fat diet-fed VC-deficient SMP30 KO mice exhibited impaired sterol regulatory element-binding protein-1c activation because of excessive cholesterol accumulation in hepatocytes. Long-term VC deficiency inhibits de novo lipogenesis through impaired sterol regulatory element-binding protein-1c activation.

Role of Ascorbic Acid in Skeletal Muscle Regeneration

Total amount of L‐ascorbic acid (AsA) in human skeletal muscle is approximately 40%. As satellite cells, muscle specific stem cells, are responsible for providing new myoblast cells during the muscle regeneration process. Myoblast cells construct myofibers in the regenerative process including proliferation and differentiation. AsA is known to prevent scurvy helping dioxygenases for the prolines hydroxylation in collagen. It has been reported that AsA accelerated Ten‐Eleven Translocation (Tet) methylcytosine dioxygenase‐mediated generation of 5‐hydroxymethylcytosine (5hmc) from 5‐methylcytosine. We have previously reported that AsA deficiency causes skeletal muscle atrophy and deterioration in physical performance in senescence marker protein 30 knockout (SMP30‐KO) mice. In this study, we showed that AsA deficiency causes a delay of skeletal muscle regeneration in SMP30‐KO mouse which is unable to biosynthesis of AsA. Moreover, AsA administration after skeletal muscle injury recovered it regeneration. In vitro experiment, AsA treated C2C12 mouse myoblast cells showed higher cell proliferation rate, mRNA expression levels of myogenic differentiation markers such as myogenin and myomaker, and fusion index compared to untreated cells. Finally, we examine the 5hmc levels in AsA‐treated and untreated C2C12 cells. The 5hmc levels was higher in AsA‐treated cells than untreated cells. These results suggest that AsA induced skeletal muscle regeneration by enhancing the myoblast proliferation, differentiation, and DNA demethylation.Support or Funding InformationGrant‐in‐Aid for Early‐Career Scientists. Funding Agency: Japan Society for the Promotion of Science. Project number: 19K19822

Hydrogen-rich pure water prevents cigarette smoke-induced pulmonary emphysema in SMP30 knockout mice

Chronic obstructive pulmonary disease (COPD) is predominantly a cigarette smoke (CS)-triggered disease with features of chronic systemic inflammation. Oxidants derived from CS can induce DNA damage and stress-induced premature cellular senescence in the respiratory system, which play significant roles in COPD. Therefore, antioxidants should provide benefits for the treatment of COPD; however, their therapeutic potential remains limited owing to the complexity of this disease. Recently, molecular hydrogen (H2) has been reported as a preventive and therapeutic antioxidant. Molecular H2 can selectively reduce hydroxyl radical accumulation with no known side effects, showing potential applications in managing oxidative stress, inflammation, apoptosis, and lipid metabolism. However, there have been no reports on the efficacy of molecular H2 in COPD patients. In the present study, we used a mouse model of COPD to investigate whether CS-induced histological damage in the lungs could be attenuated by administration of molecular H2. We administered H2-rich pure water to senescence marker protein 30 knockout (SMP30-KO) mice exposed to CS for 8 weeks. Administration of H2-rich water attenuated the CS-induced lung damage in the SMP30-KO mice and reduced the mean linear intercept and destructive index of the lungs. Moreover, H2-rich water significantly restored the static lung compliance in the CS-exposed mice compared with that in the CS-exposed H2-untreated mice. Moreover, treatment with H2-rich water decreased the levels of oxidative DNA damage markers such as phosphorylated histone H2AX and 8-hydroxy-2′-deoxyguanosine, and senescence markers such as cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 1, and β-galactosidase in the CS-exposed mice. These results demonstrated that H2-rich pure water attenuated CS-induced emphysema in SMP30-KO mice by reducing CS-induced oxidative DNA damage and premature cell senescence in the lungs. Our study suggests that administration of molecular H2 may be a novel preventive and therapeutic strategy for COPD.

Ascorbic Acid Attenuates Acute Ethanol-Induced Liver Injury in SMP30 Knockout Mice

Ascorbic acid (AA) is recognized as a free radical scavenger that protects cells from oxidative stress-induced damage. However, no studies have investigated the role of AA in acute alcoholic liver disease using senescence marker protein-30 (SMP30) knockout (KO) mice. SMP30 is a novel 34-kDa protein involved in AA biosynthesis. The present study aimed to elucidate the physiological functions of AA in acute ethanol-induced liver injury using SMP30 KO mice, which cannot synthesize AA in vivo. After a 4-week experimental period, mice were divided into six groups. The following three groups comprised the ethanol treatment groups: WT-E group (wild-type), KV-E group (AA-supplemented), and KT-E group (AA-deficient). Mice were exposed to an acute dose of ethanol (6 g ethanol/kg) administered by gavage once a day for three days. The other three control groups, namely, WT-C, KV-C, and KT-C control groups, received an equal volume of water via oral administration. Analysis of changes in body weight showed that mice in the KT-E group had significant loss of body weight compared to the control, KV-E, and WT-E groups. Behavioral analysis revealed that alcohol exposure significantly increased alcohol sensitivity in the KT-E group, whereas the WT-E, KV-E, and control groups developed ethanol tolerance. Aspartate transaminase (AST) levels in the KT-E group were significantly higher than those in the control, KV-E, and WT-E groups. The number of large and binucleated hepatocytes was significantly higher in the KT-E group than in the KV-E and WT-E groups. In addition, cytochrome P450 2E1 (CYP2E1) was over expressed in the central vein in the KT-E group when compared to the KV-E and WT-E groups. Our current findings indicate that AA supplementation in SMP30 KO mice can alleviate alcohol-induced liver damage by down regulating CYP2E1 expression. These results suggest that reduced CYP2E1 expression is a novel mechanism responsible for AA-induced reduction of ethanol-mediated oxidative stress.

Abstract 11394: Decrease of Senescence Marker Protein-30 in Aging Impairs Collateral Growth Under Ischemia

Aging decreases collateral-dependent flow recovery following ischemic arterial obstruction. However, the critical management has been lacked in clinical setting. Senescence marker protein-30 (SMP30) is a novel aging marker, which is assumed to act as an anti-aging factor in various organs. Therefore, we studied the effect of SMP30 on ischemia-induced collateral growth in C57BL/6 mice with young [8 weeks old (W)] and old (92 W), and SMP30 knockout (KO) mice (8 W). The SMP30 expression in gastrocnemius tissue was decreased in old mice compared to that of young mice. The recoveries of cutaneous blood flow in hindlimb after femoral artery ligation and tissue capillary density were suppressed in old and SMP30 KO mice compared to those in young mice. Nitric oxide generation induced by L-arginine was lower in old and SMP30 KO mice than that in young mice. The levels of NADPH oxidase activity and superoxide production in the ischemic tissue were higher in old and SMP30 KO mice than in young mice. The phosphorylated eNOS and Akt levels and VEGF levels in ischemic muscle were lower in old and SMP30 KO mice than in young mice (VEGF: Old mice 266±32, SMP30 KO 238±34, Young 414±48 pg/mg, P<0.01 ). Decrease of SMP30 exacerbates oxidative stress related to NADPH oxidase activity enhancement and impairs Akt-eNOS pathway, which leads to rarefaction of vasculogenesis induced by ischemia. These results suggest that SMP30 plays a key role in disrupting collateral growth under ischemia in aging.

Deficiency of Senescence Marker Protein 30 Exacerbates Cardiac Injury after Ischemia/Reperfusion.

Early myocardial reperfusion is an effective therapy but ischemia/reperfusion (I/R) causes lethal myocardial injury. The aging heart was reported to show greater cardiac damage after I/R injury than that observed in young hearts. Senescence marker protein 30 (SMP30), whose expression decreases with age, plays a role in reducing oxidative stress and apoptosis. However, the impact of SMP30 on myocardial I/R injury remains to be determined. In this study, the left anterior descending coronary artery was occluded for 30 min, followed by reperfusion in wild-type (WT) and SMP30 knockout (KO) mice. After I/R, cardiomyocyte apoptosis and the ratio of infarct area/area at risk were higher, left ventricular fractional shortening was lower, and reactive oxygen species (ROS) generation was enhanced in SMP30 KO mice. Moreover, the previously increased phosphorylation of GSK-3β and Akt was lower in SMP30 KO mice than in WT mice. In cardiomyocytes, silencing of SMP30 expression attenuated Akt and GSK-3β phosphorylation, and increased Bax to Bcl-2 ratio and cardiomyocyte apoptosis induced by hydrogen peroxide. These results suggested that SMP30 deficiency augments myocardial I/R injury through ROS generation and attenuation of Akt activation.

Involvement of senescence marker protein-30 in glucose metabolism disorder and non-alcoholic fatty liver disease.

Senescence marker protein-30 (SMP30) was found to decrease in the liver, kidneys and lungs of mice during aging. SMP30 is a pleiotropic protein that acts to protect cells from apoptosis by enhancing plasma membrane Ca(2+) -pump activity and is bona fide gluconolactonase (EC 3.1.1.17) that participates in the penultimate step of the vitamin C biosynthetic pathway. For the past several years, we have obtained strong evidence showing the close relationship between SMP30, glucose metabolism disorder and non-alchoholic fatty liver disease in experiments with SMP30 knockout mice. Emerging proof links the following abnormalities: (i) the reduction of SMP30 by aging and/or excessive dietary fat or genetic deficiency causes a loss of Ca(2+) pumping activity, which impairs acute insulin release in pancreatic β-cells, initiates inflammatory responses with oxidative stress and endoplasmic reticulum stress in non-alchoholic steatohepatitis, exacerbates renal tubule damage, and introduces tubulointerstitial inflammation and fibrosis in diabetic nephropathy; (ii) vitamin C insufficiency also impairs acute insulin secretion in pancreatic β-cells by a mechanism distinct from that of the SMP30 deficiency; and (iii) the increased oxidative stress by concomitant deficiencies of SMP30, superoxide dismutase 1 and vitamin C similarly causes hepatic steatosis. Here, we review recent advances in our understanding of SMP30 in glucose metabolism disorder and non-alchoholic fatty liver disease.

Senescence marker protein-30 deficiency impairs angiogenesis under ischemia

Aging decreases collateral-dependent flow recovery following acute arterial obstruction. However, the mechanisms are partially understood, therefore critical management has been lacked in clinical setting. Senescence marker protein-30 (SMP30) is a novel aging marker, which is assumed to act as an anti-aging factor in various organs. Therefore, we studied the effect of SMP30 on ischemia-induced collateral growth in SMP30 knockout (KO) mice, young and old C57BL/6 mice. The SMP30 expression in gastrocnemius tissue was decreased in old mice compared to that of young mice. The recovery of cutaneous blood flow in hind limb after femoral artery ligation and tissue capillary density recoveries were suppressed in SMP30 KO and old mice compared to those in young mice. Nitric oxide generation induced by l-arginine and GSH/GSSG in aorta of SMP30 KO and old mice were lower than those in young mice. The levels of NADPH oxidase activity and superoxide production in the ischemic tissue were higher in SMP30 KO and old mice than in young mice. The phosphorylated eNOS and Akt levels and VEGF levels in ischemic muscle were lower in SMP30 KO and old mice than in young mice. Deficiency of SMP30 exacerbates oxidative stress related to NADPH oxidase activity enhancement and impairs eNOS activity, which leads to rarefaction of angiogenesis induced by ischemia. These results suggest that SMP30 plays a key role in disrupting collateral growth under ischemia in aging.

Deficiency of Senescence Marker Protein‐30 Impairs Collateral Growth by Supressing Nitric Oxide Bioactivity under Ischemia

Aging impairs collateral growth under ischemia and reduces the expression of VEGF, however the exact mechanism is unclear. Senescence marker protein‐30 (SMP30) decreases with aging. Mice with SMP30 deficiency, a model of aging, have a short lifespan with increased oxidant stress. To elucidate the effect of SMP30 on ischemia‐induced collateral growth, we examined the recovery of cutaneous blood flow (CBF) after femoral artery ligation by laser Doppler imaging in mice. Recoveries of CBF and tissue capillary density were suppressed in SMP30 knockout (KO) mice compared to those of wild type (WT) mice. Acetylcholine (ACh, 1 µM)‐induced NO generation in aorta of SMP30 KO mice was lower than those of WT mice. Superoxide generation and NADPH oxidase activity were greater in aorta of SMP30 KO mice compared to that of WT mice (superoxide level: 3.3±0.5 vs. 1.6±0.2 nmol/mg protein; NADPH oxidase activity: 3240±254 vs. 982±106 RLU; P<0.01, respectively). VEGF content in the biceps femoris muscle in SMP30 KO mice did not increase in 14 days post‐ischemia compared to that in pre‐ischemia (SMP30 KO mice 1.1±0.2 fold vs. WT mice 2.2±0.3 fold, P<0.01). Akt phosphorylation in the biceps femoris muscle was attenuated in SMP30 KO mice compared to that of WT mice (p‐Akt/Akt: SMP30 KO mice 0.4±0.3 vs. WT mice 1.2±0.4, P<0.01). Thus, SMP30 deficiency exacerbates oxidant stress due to NADPH oxidase activation and impairs eNOS activity, which leads to rarefaction of collateral growth induced by ischemia related to Akt‐VEGF pathway. These results suggest that the decrease of SMP30 in aging impairs VEGF/NO‐dependent angiogenesis associated with overproduction of superoxide.

High dietary intake of vitamin C suppresses age-related thymic atrophy and contributes to the maintenance of immune cells in vitamin C-deficient senescence marker protein-30 knockout mice.

Vitamin C (VC) is an essential nutrient for humans and certain other animals. It has antioxidant properties and has been reported to ameliorate oxidative damage to lipids, DNA and proteins. However, the effects of VC on immune function are poorly understood, especially the influence of long-term high-dose VC intake on the number and function of immune cells. In the present study, to evaluate the immune effects of VC, VC-deficient senescence marker protein-30 knockout (SMP30KO) mice were fed a diet containing the recommended level of VC (20 mg/kg per d; 0·02 % VC) or a high level of VC (200 mg/kg per d; 0·2 % VC) for 1 year. The plasma VC concentration of the 0·02 % group was the same as that of age-matched C57BL/6 mice after 1 year of feeding; however, plasma VC concentration and thymus weight were significantly higher in the 0·2 % VC group than in the 0·02 % VC group. The total counts of leucocytes, lymphocytes, granulocytes and monocytes in the peripheral blood, as well as the number of splenocytes and thymocytes, were all significantly higher in the 0·2 % VC group than in the 0·02 % VC group. In addition, the number of naive T cells in peripheral blood lymphocytes, the number of memory T-cell populations in splenocytes, and the number of cluster of differentiation (CD)4⁺CD8⁺ or CD4⁺CD8⁻ or CD4⁻CD8⁺ T cells in thymocytes were all markedly higher in the 0·2 % VC group than in the 0·02 % VC group after 1 year of dietary treatment. These results suggest that a long-term high-dose intake of VC is effective in the maintenance of immune cells, partly through the suppression of age-related thymic involution in VC-deficient SMP30KO mice.

Abstract 11474: Deficiency of Senescence Marker Protein-30 Impairs Collateral Growth by Supressing Nitric Oxide Bioactivity Under Ischemia

Aging impairs collateral growth under ischemia and reduces the expression of VEGF, however the exact mechanism is unclear. Senescence marker protein-30 (SMP30) decreases with aging. Mice with SMP30 deficiency, a model of aging, have a short lifespan with increased oxidant stress. To elucidate the effect of SMP30 on ischemia-induced collateral growth, we examined the recovery of cutaneous blood flow (CBF) after femoral artery ligation by laser Doppler imaging in mice. Recoveries of CBF and tissue capillary density were suppressed in SMP30 knockout (KO) mice compared to those of wild type (WT) mice (Figure). Acetylcholine (ACh, 1 μM)-induced NO generation, total thiol and reduced glutathione (GSH) levels in aorta of SMP30 KO mice were lower than those of WT mice (NO: 2.6±4.5 vs. 86.2±9.2 nM; total thiol: 6.3±1.6 vs. 33.2±4.6 nmol/mg/protein; GSH: 5.1±0.8 vs. 18.6±2.1 nmol/mg/protein; P<0.01, respectively). Superoxide generation and NADPH oxidase activity were greater in aorta of SMP30 KO mice compared to that of WT mice (superoxide level: 3.3±0.5 vs. 1.6±0.2 nmol/mg protein; NADPH oxidase activity: 3240±254 vs. 982±106 RLU; P<0.01, respectively). Asymmetric dimethylarginine (ADMA) level was higher in aorta of SMP30 KO mice than that of WT mice (6.4±0.8 vs. 2.4±0.4 nmol/mg protein, P<0.01). VEGF content in the crural muscle in SMP30 KO mice did not increase in 14 days post-ischemia compared to that in pre-ischemia (SMP30 KO mice 1.1±0.2 fold vs. WT mice 2.2±0.3 fold, P<0.01). Akt phosphorylation in the crural muscle was attenuated in SMP30 KO mice compared to that of WT mice (p-Akt/Akt: SMP30 KO mice 0.4±0.3 vs. WT mice 1.2±0.4, P<0.01). Thus, SMP30 deficiency exacerbates oxidant stress due to NADPH oxidase activation, which leads to the rarefaction of collateral growth induced by ischemia related to Akt-VEGF pathway. These results suggest that the decrease of SMP30 in aging impairs VEGF/NO-dependent angiogenesis associated with overproduction of superoxide.

Anti-inflammatory activity of SMP30 modulates NF-κB through protein tyrosine kinase/phosphatase balance.

Recent studies on senescence marker protein-30 (SMP30) have shown that it has an important functional role in the aging process, but its precise participation in cellular works has not been fully determined. We hypothesize that SMP30 plays crucial roles in signaling processes by modulating the balance of protein tyrosine kinase (PTK)/protein tyrosine phosphatase (PTP) and in activating proinflammatory NF-κB. An experimental paradigm of gain and loss of SMP30 function was established using SMP30-overexpressed YPEN-1 cells (herein referred to as "SMP30(+) cells") and SMP30 (Y/-) knockout mouse kidneys. The resulting data show that SMP30 expression suppressed oxidative stress-induced PTK/PTP dysregulation and PP1/2A inactivation in SMP30(+) cells, leading to the suppression of NF-κB activation. In the kidneys of SMP30 (Y/-) mice, SMP30 deficiency was found to induce NF-κB activation via the upstream signaling of NIK/IKK and MAPKs and to upregulate downstream NF-κB-responsive gene expression. In this study, we also demonstrate for the first time that SMP30 deficiency induced PTK activity in SMP30 (Y/-) kidneys, thereby significantly increasing the tyrosine phosphorylation of a catalytic subunit of PP2A (PP2Ac-Tyr307). Based on these findings, we propose that SMP30 involves NF-κB regulation through the PTK/PTP balance and that the age-related decrease of SMP30 causes NF-κB activation, which contributes to an exacerbation of the inflammatory process during aging. SMP30-deficient mice induced a shorter lifespan and redox changes. Overexpression of SMP30 prevented oxidative stress insults. The depletion of SMP30 increased redox-related PTK/PTP imbalance and PP1/PP2A inactivation. The depletion of SMP30 caused an elevation of NF-κB-responsive inflammatory markers. SMP30 may be a potent inhibitory protein against oxidative stress and chronic inflammation.

Senescence marker protein-30/gluconolactonase deficiency exacerbates diabetic nephropathy through tubular injury in a mouse model of type1 diabetes.

Aims/IntroductionSenescence marker protein-30 (SMP30) is abundantly expressed in renal proximal tubule cells, but its expression decreases with age. Previous studies have shown that reduced SMP30 expression could contribute to aging-associated deterioration of cellular function and tissue injury. In the present study, we investigated the effects of SMP30 deficiency on the pathogenesis of diabetic nephropathy.Materials and MethodsDiabetes was induced using streptozotocin in male SMP30 knockout mice (KO) and wild-type mice at 7 weeks-of-age. Vitamin C was added to the drinking water to prevent vitamin C deficiency in KO mice. The mice were killed 12 weeks after the induction of diabetes.ResultsUrinary biomarkers for proximal tubule damage were significantly increased in non-diabetic KO mice compared with wild-type mice. Furthermore, diabetes-induced tubular damage was significantly exacerbated by SMP30 deletion. Morphological analysis showed a link between cortical tubulointerstitial fibrosis area and the degree of tubular damage. However, SMP30 deletion did not affect mesangial expansion. Tubular injury was associated with accumulation of hypoxia-inducible factor-1α and increased hypoxia-inducible factor-1α targeted gene expression. SMP30 deletion initiated oxidative stress; however, it did not exacerbate the oxidative stress seen in diabetic mice. In contrast, tubular inflammation was associated with SMP30 deletion only in diabetic mice.ConclusionsBased on this evidence, we concluded that SMP30 deficiency exacerbates proximal tubule injury in diabetic mice. Decreased SMP30 could contribute to the increased incidence of various chronic kidney diseases, including diabetic nephropathy, with age.

ENA-A actimineral resource A extends lifespan associated with antioxidant mechanism in SMP30 knockout mice

ENA-actimineral resource A (ENA-A) is an alkaline mineral water and has a few biological activities such as antioxidant activity. The aim of this study was to examine the effects of ENA-A on lifespan in mice using senescence marker protein-30 knockout mice. The present study had groups of 18-week-old mice (n = 24), 26-week-old mice (n = 12), and 46-week-old mice (n = 20). Each differently aged mice group was divided into three subgroups: a control group, a 5 % ENA-A-treated group, and a 10 % ENA-A-treated group. Mice in the 18-week-old group were treated with vitamin C drinking water 1.5 g/L. However, the mice in the 26-week-old and 46-week-old groups were not treated with vitamin C. The experiments were done for 18 weeks. All vitamin C-treated mice were alive at week 18 (100% survival rate). In the non-vitamin C group, the 10% ENA-A-treated mice were alive at week 18. The control and 5% ENA-A-treated mice died by week 15. As expected, vitamin C was not detected in the non-vitamin C-treated group. However, vitamin C levels were increased in an ENA-A dose-dependent manner in the vitamin C-treated group. In the TUNEL assay, a number of positive hepatocytes significantly decreased in an ENA-A dose-dependent manner. Periodic acid Schiff positive hepatocytes were significantly increased in an ENA-A dose-dependent manner. In addition, the expression level of CuZnSOD was increased by the ENA-A treatment. These data suggest that the intake of ENA-A has a critical role in the anti-aging mechanism and could be applied toward the lifespans of humans.

Senescence Marker Protein 30 Has a Cardio-Protective Role in Doxorubicin-Induced Cardiac Dysfunction

BackgroundSenescence marker protein 30 (SMP30), which was originally identified as an aging marker protein, is assumed to act as a novel anti-aging factor in the liver, lungs and brain. We hypothesized that SMP30 has cardio-protective function due to its anti-aging and anti-oxidant effects on doxorubicin (DOX)-induced cardiac dysfunction.Methods and ResultsSMP30 knockout (SMP30 KO) mice, SMP30 transgenic (SMP30 TG) mice with cardiac-specific overexpression of SMP30 gene and wild-type (WT) littermate mice at 12–14 weeks of age were given intra-peritoneal injection of DOX (20 mg/kg) or saline. Five days after DOX injection, echocardiography revealed that left ventricular ejection fraction was more severely reduced in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but was preserved in the DOX-treated SMP30 TG mice. Generation of reactive oxygen species and oxidative DNA damage in the myocardium were greater in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but much less in the SMP30 TG mice. The numbers of deoxynucleotidyltransferase-mediated dUTP nick end-labeling positive nuclei in the myocardium, apoptotic signaling pathways such as caspase-3 activity, Bax/Bcl-2 ratio and phosphorylation activity of c-Jun N-terminal kinase were increased in SMP30 KO mice and decreased in SMP30 TG mice compared with WT mice after DOX injection.ConclusionsSMP30 has a cardio-protective role by anti-oxidative and anti-apoptotic effects in DOX-induced cardiotoxicity, and can be a new therapeutic target to prevent DOX-induced heart failure.

Abstract 9362: Senescence Marker Protein 30 has a Cardio-protective Role in Doxorubicin-induced Cardiac Dysfunction

Background: Senescence marker protein 30 (SMP30), which was originally identified as an aging marker protein, is assumed to act as a novel anti-aging factor in the liver, lungs and brain. Anthracyclines are the drugs most closely related to acute and late cardiac toxicity. One of the mechanisms responsible for doxorubicin (DOX) cardiotoxicity is the formation of reactive oxygen species (ROS) which can harm membrane lipids and other cellular components, leading to cardiomyocyte apoptosis and death. We hypothesized that SMP30 has cardio-protective function due to its anti-aging and anti-oxidant effects on doxorubicin (DOX)-induced cardiac dysfunction. Methods and Results: SMP30 knockout (SMP30 KO) mice, SMP30 transgenic (SMP30 TG) mice with cardiac-specific overexpression of SMP30 gene and wild-type (WT) littermate mice were given intra-peritoneal injections of DOX (20 mg/kg) or saline. Seven days after DOX injection, echocardiography revealed that left ventricular ejection fraction was more severely reduced in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but was preserved in the DOX-treated SMP30 TG mice. Generation of ROS and oxidative DNA damage in the myocardium were greater in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but much less in the SMP30 TG mice. The number of deoxynucleotidyl transferase-mediated dUTP nick end-labeling positive cardiomyocytes was higher in the DOX-treated SMP30 KO mice and lower in the DOX-treated SMP30 TG mice compared to that in the DOX-treated WT mice. Caspase-3 activity was higher in the DOX-treated SMP30 KO mice and lower in the DOX-treated SMP30 TG mice compared with the DOX-treated WT mice. The ratio of Bax to Bcl-2 was higher in the DOX-treated SMP30 KO mice and lower in the DOX-treated SMP30 TG mice than in the DOX-treated WT mice. Phosphorylation activity of SAPK/JNK was increased in the DOX-treated SMP30 KO mice and was decreased in the DOX-treated SMP30 TG mice compared with the DOX-treated WT mice. Conclusions: SMP30 has a cardio-protective role by anti-oxidative and anti-apoptotic effects in DOX-induced cardiotoxicity, and can be a new therapeutic target to prevent DOX-induced heart failure.

Vitamin C Prevents Cigarette Smoke–Induced Pulmonary Emphysema in Mice and Provides Pulmonary Restoration

Vitamin C (VC) is a potent antioxidant and is essential for collagen synthesis. We investigated whether VC treatment prevents and cures smoke-induced emphysema in senescence marker protein-30 knockout (SMP30-KO) mice, which cannot synthesize VC. Two smoke-exposure experiments using SMP30-KO mice were conducted. In the first one (a preventive study), 4-month-old mice received minimal VC (0.0375 g/l) [VC(L)] or physiologically sufficient VC (1.5 g/l) [VC(S)] and exposed to cigarette smoke or smoke-free air for 2 months. Pulmonary evaluations followed when the mice were 6 months of age. The second study began after the establishment of smoke-induced emphysema (a treatment study). These mice no longer underwent smoke exposure but received VC(S) or VC(L) treatment for 2 months. Morphometric analysis was performed, and measurements of oxidative stress, collagen synthesis, and vascular endothelial growth factor in the lungs were evaluated. Chronic smoke exposure caused emphysema (29.6% increases of mean linear intercepts [MLI] and 106.5% increases of destructive index compared with the air-only group) in 6-month-old SMP30-KO mice, and this emphysema closely resembled human chronic obstructive pulmonary disease. Smoke-induced emphysema persisted in the VC(L) group after smoking cessation, whereas VC treatment provided pulmonary restoration (18.5% decrease of MLI and 41.3% decrease of destructive index compared with VC(L) group). VC treatment diminished oxidative stress, increased collagen synthesis, and improved vascular endothelial growth factor levels in the lungs. Our results suggest that VC not only prevents smoke-induced emphysema in SMP30-KO mice but also restores emphysematous lungs. Therefore, VC may provide a new therapeutic strategy for treating chronic obstructive pulmonary disease in humans.

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/AimsThe 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 FindingsMale 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.ConclusionOur data strongly suggest that SMP30 is closely associated with NAFLD pathogenesis, and might be a possible therapeutic target for NAFLD.