Combined transcriptomics and proteomics unveil the impact of vitamin C in modulating specific protein abundance in the mouse liver.

BACKGROUND: Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to the key enzyme gulonolactone oxidase (Gulo) unlike humans. In the current investigation, we used Gulo-/- mice, which cannot synthesize their own ascorbate to determine the impact of this vitamin on both the transcriptomics and proteomics profiles in the whole liver. The study included Gulo-/- mouse groups treated with either sub-optimal or optimal ascorbate concentrations in drinking water. Liver tissues of females and males were collected at the age of four months and divided for transcriptomics and proteomics analysis. Immunoblotting, quantitative RT-PCR, and polysome profiling experiments were also conducted to complement our combined omics studies. RESULTS: Principal component analyses revealed distinctive differences in the mRNA and protein profiles as a function of sex between all the mouse cohorts. Despite such sexual dimorphism, Spearman analyses of transcriptomics data from females and males revealed correlations of hepatic ascorbate levels with transcripts encoding a wide array of biological processes involved in glucose and lipid metabolisms as well as in the acute-phase immune response. Moreover, integration of the proteomics data showed that ascorbate modulates the abundance of various enzymes involved in lipid, xenobiotic, organic acid, acetyl-CoA, and steroid metabolism mainly at the transcriptional level, especially in females. However, several proteins of the mitochondrial complex III significantly correlated with ascorbate concentrations in both males and females unlike their corresponding transcripts. Finally, poly(ribo)some profiling did not reveal significant enrichment difference for these mitochondrial complex III mRNAs between Gulo-/- mice treated with sub-optimal and optimal ascorbate levels. CONCLUSIONS: Thus, the abundance of several subunits of the mitochondrial complex III are regulated by ascorbate at the post-transcriptional levels. Our extensive omics analyses provide a novel resource of altered gene expression patterns at the transcriptional and post-transcriptional levels under ascorbate deficiency.

Conservation of a Chromosome 8 Inversion and Exon Mutations Confirm Common Gulonolactone Oxidase Gene Evolution Among Primates, Including H. Neanderthalensis.

Ascorbic acid functions as an antioxidant and facilitates other biochemical processes such as collagen triple helix formation, and iron uptake by cells. Animals which endogenously produce ascorbic acid have a functional gulonolactone oxidase gene (GULO); however, humans have a GULO pseudogene (GULOP) and depend on dietary ascorbic acid. In this study, the conservation of GULOP sequences in the primate haplorhini suborder were investigated and compared to the GULO sequences belonging to the primates strepsirrhini suborder. Phylogenetic analysis suggested that the conserved GULOP exons in the haplorhini primates experienced a high rate of mutations following the haplorhini/strepsirrhini divergence. This high mutation rate has decreased during the evolution of the haplorhini primates. Additionally, indels of the haplorhini GULOP sequences were conserved across the suborder. A separate analysis for GULO sequences and well-conserved GULOP sequences focusing on placental mammals identified an in-frame GULO sequence in the Brazilian guinea pig, and a potential GULOP sequence in the pika. Similar to haplorhini primates, the guinea pig and lagomorph species have experienced a high substitution rate when compared to the mammals used in this study. A shared synteny to examine the conservation of local genes near GULO/GULOP identified a conserved inversion around the GULO/GULOP locus between the haplorhini and strepsirrhini primates. Fischer's exact test did not support an association between GULOP and the chromosomal inversion. Mauve alignment showed that the inversion of the length of the syntenic block that the GULO/GULOP genes belonged to was variable. However, there were frequent rearrangements around ~ 2 million base pairs adjacent to GULOP involving the KIF13B and MSRA genes. These data may suggest that genes acquiring deleterious mutations in the coding sequence may respond to these deleterious mutations with rapid substitution rates.

Ascorbic acid biosynthesis in Pacific abalone Haliotis discus hannai Ino and L-gulonolactone oxidase gene loss as an independent event.

Up to now, it has been believed that invertebrates are unable to synthesize ascorbic acid (AA) in vivo. However, in the present study, the full-length CDs (Coding sequence) of L-gulonolactone oxidase (GLO) from Pacific abalone (Haliotis discus hannai Ino) were obtained through molecular cloning. The Pacific abalone GLO contained a FAD-binding domain in the N-termination, and ALO domain and conserved HWAK motif in the C-termination. The GLO gene possesses 12 exons and 11 introns. The Pacific abalone GLO was expressed in various tissues, including the kidney, digestive gland, gill, intestine, muscle and mantle. The GLO activity assay revealed that GLO activity was only detected in the kidney of Pacific abalone. After a 100-day feeding trial, dietary AA levels did not significantly affect the survival, weight gain, daily increment in shell length, and feed conversion ratio of Pacific abalone. The expression of GLO in the kidney was downregulated by dietary AA. These results implied that the ability to synthesize AA in abalone had not been lost. From the evolutionary perspective, the loss of GLO occurred independently as an independent event by matching with the genomes of various species. The positive selection analysis revealed that the GLO gene underwent purifying selective pressure during its evolution. In conclusion, the present study provided direct evidence to prove that the GLO activity and the ability to synthesize AA exist in abalone. The AA synthesis ability in vertebrates might have originated from invertebrates dating back 930.31 million years.

Vitamin C deficiency reveals developmental differences between neonatal and adult hematopoiesis.

Hematopoiesis, a process that results in the differentiation of all blood lineages, is essential throughout life. The production of 1x1012 blood cells per day, including 200x109 erythrocytes, is highly dependent on nutrient consumption. Notably though, the relative requirements for micronutrients during the perinatal period, a critical developmental window for immune cell and erythrocyte differentiation, have not been extensively studied. More specifically, the impact of the vitamin C/ascorbate micronutrient on perinatal as compared to adult hematopoiesis has been difficult to assess in animal models. Even though humans cannot synthesize ascorbate, due to a pseudogenization of the L-gulono-γ-lactone oxidase (GULO) gene, its generation from glucose is an ancestral mammalian trait. Taking advantage of a Gulo-/- mouse model, we show that ascorbic acid deficiency profoundly impacts perinatal hematopoiesis, resulting in a hypocellular bone marrow (BM) with a significant reduction in hematopoietic stem cells, multipotent progenitors, and hematopoietic progenitors. Furthermore, myeloid progenitors exhibited differential sensitivity to vitamin C levels; common myeloid progenitors and megakaryocyte-erythrocyte progenitors were markedly reduced in Gulo-/- pups following vitamin C depletion in the dams, whereas granulocyte-myeloid progenitors were spared, and their frequency was even augmented. Notably, hematopoietic cell subsets were rescued by vitamin C repletion. Consistent with these data, peripheral myeloid cells were maintained in ascorbate-deficient Gulo-/- pups while other lineage-committed hematopoietic cells were decreased. A reduction in B cell numbers was associated with a significantly reduced humoral immune response in ascorbate-depleted Gulo-/- pups but not adult mice. Erythropoiesis was particularly sensitive to vitamin C deprivation during both the perinatal and adult periods, with ascorbate-deficient Gulo-/- pups as well as adult mice exhibiting compensatory splenic differentiation. Furthermore, in the pathological context of hemolytic anemia, vitamin C-deficient adult Gulo-/- mice were not able to sufficiently increase their erythropoietic activity, resulting in a sustained anemia. Thus, vitamin C plays a pivotal role in the maintenance and differentiation of hematopoietic progenitors during the neonatal period and is required throughout life to sustain erythroid differentiation under stress conditions.

Elucidating the genetic architecture controlling antioxidant status and ionic balance in barley under salt stress.

KEY MESSAGE: Association genetic analysis empowered us to identify candidate genes underlying natural variation of morpho-physiological, antioxidants, and grain yield-related traits in barley. Novel intriguing genomic regions were identified and dissected. Salinity stress is one of the abiotic stresses that influence the morpho-physiological, antioxidants, and yield-related traits in crop plants. The plants of a core set of 138 diverse barley accessions were analyzed after exposure to salt stress under field conditions during the reproductive phase. A genome-wide association scan (GWAS) was then conducted using 19,276 single nucleotide polymorphisms (SNPs) to uncover the genetic basis of morpho-physiological and grain-related traits. A wide range of responses to salt stress by the accessions was explored in the current study. GWAS detected 263 significantly associated SNPs with the antioxidants, K+/Na+ content ratio, and agronomic traits. Five genomic regions harbored interesting putative candidate genes within LD ± 1.2 Mbp. Choromosome 2H harbored many candidate genes associated with the antioxidants ascorbic acid (AsA) and glutathione (GSH), such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), under salt stress. Markedly, an A:C SNP at 153,773,211 bp on chromosome 7H is located inside the gene HORVU.MOREX.r3.7HG0676830 (153,772,300-153,774,057 bp) that was annotated as L-gulonolactone oxidase, regulating the natural variation of SOD_S and APX_S. The allelic variation at this SNP reveals a negative selection of accessions carrying the C allele, predominantly found in six-rowed spring landraces originating from Far-, Near-East, and central Asia carrying photoperiod sensitive alleles having lower activity of enzymatic antioxidants. The SNP-trait associations detected in the current study constitute a benchmark for developing molecular selection tools for antioxidant compound selection in barley.

The evolution of vitamin C biosynthesis and transport in animals.

BACKGROUND: Vitamin C (VC) is an indispensable antioxidant and co-factor for optimal function and development of eukaryotic cells. In animals, VC can be synthesized by the organism, acquired through the diet, or both. In the single VC synthesis pathway described in animals, the penultimate step is catalysed by Regucalcin, and the last step by L-gulonolactone oxidase (GULO). The GULO gene has been implicated in VC synthesis only, while Regucalcin has been shown to have multiple functions in mammals. RESULTS: Both GULO and Regucalcin can be found in non-bilaterian, protostome and deuterostome species. Regucalcin, as here shown, is involved in multiple functions such as VC synthesis, calcium homeostasis, and the oxidative stress response in both Deuterostomes and Protostomes, and in insects in receptor-mediated uptake of hexamerin storage proteins from haemolymph. In Insecta and Nematoda, however, there is no GULO gene, and in the latter no Regucalcin gene, but species from these lineages are still able to synthesize VC, implying at least one novel synthesis pathway. In vertebrates, SVCT1, a gene that belongs to a family with up to five members, as here shown, is the only gene involved in the uptake of VC in the gut. This specificity is likely the result of a subfunctionalization event that happened at the base of the Craniata subphylum. SVCT-like genes present in non-Vertebrate animals are likely involved in both VC and nucleobase transport. It is also shown that in lineages where GULO has been lost, SVCT1 is now an essential gene, while in lineages where SVCT1 gene has been lost, GULO is now an essential gene. CONCLUSIONS: The simultaneous study, for the first time, of GULO, Regucalcin and SVCTs evolution provides a clear picture of VC synthesis/acquisition and reveals very different selective pressures in different animal taxonomic groups.

Vitamin C Enhances Antiviral Functions of Lung Epithelial Cells.

Vitamin C is well documented to have antiviral functions; however, there is limited information about its effect on airway epithelial cells-the first cells to encounter infections. Here, we examined the effect of vitamin C on human bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B) cells, and observed that sodium-dependent vitamin C transporter 2 (SVCT2) was the primary vitamin C transporter. Transcriptomic analysis revealed that treating BEAS-2B cells with vitamin C led to a significant upregulation of several metabolic pathways and interferon-stimulated genes (ISGs) along with a downregulation of pathways involved in lung injury and inflammation. Remarkably, vitamin C also enhanced the expression of the viral-sensing receptors retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA-5), which was confirmed at the protein and functional levels. In addition, the lungs of l-gulono-γ-lactone oxidase knockout (GULO-KO) mice also displayed a marked decrease in these genes compared to wild-type controls. Collectively, our findings indicate that vitamin C acts at multiple levels to exert its antiviral and protective functions in the lungs.

Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression.

Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo-/-) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo-/- mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species.

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

Multiple independent L-gulonolactone oxidase (GULO) gene losses and vitamin C synthesis reacquisition events in non-Deuterostomian animal species.

BACKGROUND: L-ascorbate (Vitamin C) is an important antioxidant and co-factor in eukaryotic cells, and in mammals it is indispensable for brain development and cognitive function. Vertebrates usually become L-ascorbate auxothrophs when the last enzyme of the synthetic pathway, an L-gulonolactone oxidase (GULO), is lost. Since Protostomes were until recently thought not to have a GULO gene, they were considered to be auxothrophs for Vitamin C. RESULTS: By performing phylogenetic analyses with tens of non-Bilateria and Protostomian genomes, it is shown, that a GULO gene is present in the non-Bilateria Placozoa, Myxozoa (here reported for the first time) and Anthozoa groups, and in Protostomians, in the Araneae family, the Gastropoda class, the Acari subclass (here reported for the first time), and the Priapulida, Annelida (here reported for the first time) and Brachiopoda phyla lineages. GULO is an old gene that predates the separation of Animals and Fungi, although it could be much older. We also show that within Protostomes, GULO has been lost multiple times in large taxonomic groups, namely the Pancrustacea, Nematoda, Platyhelminthes and Bivalvia groups, a pattern similar to that reported for Vertebrate species. Nevertheless, we show that Drosophila melanogaster seems to be capable of synthesizing L-ascorbate, likely through an alternative pathway, as recently reported for Caenorhabditis elegans. CONCLUSIONS: Non-Bilaterian and Protostomians seem to be able to synthesize Vitamin C either through the conventional animal pathway or an alternative pathway, but in this animal group, not being able to synthesize L-ascorbate seems to be the exception rather than the rule.

Gulo regulates the proliferation, apoptosis and mesenchymal-to-epithelial transformation of metanephric mesenchyme cells via inhibiting Six2.

During kidney development, the balance between self-renewal and differentiation of metanephric mesenchyme (MM) cells, mainly regulated by Sine oculis-related homeobox 2 (Six2), is critical for forming mature kidney. L-gulono-γ-lactone oxidase (Gulo), a crucial enzyme for vitamin C synthesis, reveals a different expression at various stages during kidney development, but its function in the early renal development remains unknown. In this work, we aim to study the role of Gulo in MM cells at two differentiation stages. We found that Gulo expression in undifferentiated MM (mK3) cells was lower than in differentiated MM (mK4) cells. Over-expression of Gulo can promote mesenchymal-to-epithelial transformation (MET) and apoptosis and inhibit the proliferation in mK3 cells. Knock-down of Gulo in mK4 cells made its epithelial character cells unstabilized, facilitated the proliferation and restrained the apoptosis. Furthermore, we found that Six2 was negatively regulated by Gulo, and over-expression or knock-down of Six2 was able to rescue partially the MET, proliferation and apoptosis of MM cells caused by Gulo. In conclusion, these findings reveal that Gulo promotes the MET and apoptosis, and inhibits proliferation in MM cells by down-regulating Six2.

Oxidative Stress Levels in the Brain Are Determined by Post-Mortem Interval and Ante-Mortem Vitamin C State but Not Alzheimer's Disease Status.

The current study highlighted several changes in measures of oxidative stress and antioxidant status that take place in the mouse brain over the course of 24 h post-mortem. Ascorbic acid (vitamin C) and glutathione both decreased significantly in cortex in as little as 2 h and malondialdehyde levels increased. Further change from baseline was observed up to 24 h, including carbonyl and sulfhydryl formation. The greatest changes were observed in brains that began with low ascorbic acid levels (gulo−/− mice) compared to wild-type or 5XFAD mice. Cortical samples from nine Alzheimer’s Disease cases and five controls were also assayed under the same conditions. Post mortem intervals ranged from 6 to 47 h and all samples had low ascorbic acid levels at time of measurement. Malondialdehyde levels were lower in Alzheimer’s Disease cases. Despite a strong positive correlation between ascorbic acid and glutathione levels, no other correlations among oxidative stress measures or post mortem interval were observed. Together the data suggest that molecular changes occurring within the first hours of death may mask differences between patient groups. Care must be taken interpreting studies in human brain tissue where ante-mortem nutrient status is not known to avoid bias or confounding of results.

Vitamin C modulates the metabolic and cytokine profiles, alleviates hepatic endoplasmic reticulum stress, and increases the life span of Gulo-/- mice.

Suboptimal intake of dietary vitamin C (ascorbate) increases the risk of several chronic diseases but the exact metabolic pathways affected are still unknown. In this study, we examined the metabolic profile of mice lacking the enzyme gulonolactone oxidase (Gulo) required for the biosynthesis of ascorbate. Gulo-/- mice were supplemented with 0%, 0.01%, and 0.4% ascorbate (w/v) in drinking water and serum was collected for metabolite measurements by targeted mass spectrometry. We also quantified 42 serum cytokines and examined the levels of different stress markers in liver. The metabolic profiles of Gulo-/- mice treated with ascorbate were different from untreated Gulo-/- and normal wild type mice. The cytokine profiles of Gulo-/-mice, in return, overlapped the profile of wild type animals upon 0.01% or 0.4% vitamin C supplementation. The life span of Gulo-/- mice increased with the amount of ascorbate in drinking water. It also correlated significantly with the ratios of serum arginine/lysine, tyrosine/phenylalanine, and the ratio of specific species of saturated/unsaturated phosphatidylcholines. Finally, levels of hepatic phosphorylated endoplasmic reticulum associated stress markers IRE1α and eIF2α correlated inversely with serum ascorbate and life span suggesting that vitamin C modulates endoplasmic reticulum stress response and longevity in Gulo-/- mice.

Ascorbic acid regulation in stress responses during acute cold exposure and following recovery in juvenile Chinese soft-shelled turtle (Pelodiscus sinensis).

Intense temperature change often leads to increased oxidative stress in many animals with a few exceptions, including the turtle. To date, little is known about the mechanism of protective antioxidative defenses in turtles during acute temperature change, specifically the role that the antioxidant ascorbic acid (AA) plays. In this study, Chinese soft-shelled turtles (Pelodiscus sinensis) were initially acclimated at 28°C (3 wks), exposed to acute cold condition (8°C, 8 h) and finally placed in recovery (28°C, 24 h). L-Gulonolactone oxidase (GLO) mRNA exhibited a stable transcription pattern during the intense thermal fluctuation. GLO activity also remained stable, which validated the mRNA expression pattern. The similar Q10 values for GLO activity in the different treatment groups at incubation temperatures of 28°C and 8°C indicated that the GLO activity response to thermal change exhibited a temperature-dependent enzymatic kinetic characteristic. The AA storage was tissue-specific as well as the AA re-supply in the recovery period, with brain as the priority. Despite the insufficient transport during cold exposure, the plasma AA reservoir greatly contributed to the redistribution of AA during recovery. Depending on the prominent GLO activity, the high level of tissue-specific AA storage and the extraordinary plasma AA transport potential, the Chinese soft-shelled turtle endured severe thermal fluctuations with no apparent oxidative stress. However, the significant decrease in AA concentration in the brain tissue during acute cold exposure suggested that such a strategy may not be sufficient for prolonged cold exposure.

L-gulono-γ-lactone oxidase expression and vitamin C synthesis in the brain and kidney of the African lungfish, Protopterus annectens.

This study aimed to test the hypothesis that the brain of Protopterus annectens expressed L-gulono-γ-lactone oxidase (gulo/Gulo), the enzyme catalyzing the last step of ascorbate biosynthesis, and could maintain high concentrations of ascorbate during estivation. We cloned and sequenced gulo from the kidney of P. annectens and performed quantitative PCR to determine its mRNA expression in kidney and brain. Gulo activity was assayed and its protein abundance was determined by Western blot using custom-made anti-Gulo antibody. Effects of estivation on concentrations of ascorbate and dehydroascorbate in the kidney and brain were also determined. Both brain and kidney, but not liver, of P. annectens expressed gulo/Gulo. Desiccation induced P. annectens to estivate, and 6 mo of estivation led to drastic decreases in gulo/Gulo expression and ascorbate concentration in the kidney. However, high concentrations of ascorbate and ascorbate + dehydroascorbate were maintained in the brain during estivation, probably resulting from in situ ascorbate synthesis. Control fish were placed in freshwater, where they were fully active in a favorable environment unlike estivation on land. The ability to synthesize ascorbate to ameliorate oxidative stress directly in the brain might contribute to the ability of P. annectens to undergo prolonged estivation on land.

High-dose of vitamin C supplementation reduces amyloid plaque burden and ameliorates pathological changes in the brain of 5XFAD mice.

Blood-brain barrier (BBB) breakdown and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Besides vitamin C being as one of the important antioxidants, recently, it has also been reported as a modulator of BBB integrity and mitochondria morphology. Plasma levels of vitamin C are decreased in AD patients, which can affect disease progression. However, investigation using animal models on the role of vitamin C in the AD pathogenesis has been hampered because rodents produce with no dependence on external supply. Therefore, to identify the pathogenic importance of vitamin C in an AD mouse model, we cross-bred 5 familial Alzheimer's disease mutation (5XFAD) mice (AD mouse model) with ι-gulono-γ-lactone oxidase (Gulo) knockout (KO) mice, which are unable to synthesize their own vitamin C, and produced Gulo KO mice with 5XFAD mice background (KO-Tg). These mice were maintained on either low (0.66 g/l) or high (3.3 g/l) supplementation of vitamin C. We found that the higher supplementation of vitamin C had reduced amyloid plaque burden in the cortex and hippocampus in KO-Tg mice, resulting in amelioration of BBB disruption and mitochondrial alteration. These results suggest that intake of a larger amount of vitamin C could be protective against AD-like pathologies.

Chronic vitamin C insufficiency aggravated thioacetamide-induced liver fibrosis in gulo-knockout mice.

Given the involvement of oxidative stress in liver-disease- or hepato-toxicant-induced hepatic damage and fibrosis, antioxidants are an effective preventive and therapeutic tool. The beneficial results of vitamin C, one of the physiological antioxidants, have been observed both in experimental animals and in humans. However, most of these studies have been concerned with supplementary vitamin C; the effects of under vitamin C insufficiency, which humans sometimes confront, have not been substantially investigated. In the present study, we established a vitamin C-insufficient animal model (half-to-normal serum vitamin C concentration) with gulo(-/-) mice that cannot synthesize vitamin C, and induced hepatotoxicity by means of thioacetamide (TAA) injections twice a week for 18 weeks. Additionally, we explored the direct effects of vitamin C both on immortalized human hepatic stellate LX-2 cells and on rat primary hepatic stellate cells. Vitamin C insufficiency resulted in a decreased survival rate and increased serum markers for hepatocyte damage, such as alanine aminotransferase and aspartate aminotransferase. Concomitantly, the levels of reactive oxygen species (ROS) and lipid peroxides in the liver were increased. Histological examinations of the vitamin C-insufficient liver revealed increases in collagen fiber deposition and activated-hepatic-stellate-cell number. Vitamin C, when directly applied to the LX-2 cells as well as the rat primary hepatic stellate cells, suppressed not only proliferation but hydrogen peroxide-induced collagen expression as well. In conclusion, vitamin C insufficiency exacerbated TAA-induced hepatotoxicity. These effects seem to be mainly from insufficient scavenging of ROS in the liver, and possibly in part, by directly affecting hepatic stellate cells.

Senescence marker protein-30/gluconolactonase expression in the mouse ovary during gestation.

Senescence marker protein-30 (SMP30) was first described as a physiologic entity that decreases in the rat liver and kidney with aging. Previously, we established that SMP30 is the lactone-hydrolyzing enzyme gluconolactonase (GNL), which is involved in ascorbic acid (AA) biosynthesis. In the present study, we found SMP30/GNL mRNA expressed in the mouse ovary. To ascertain the reason for ovarian SMP30/GNL expression, we examined mice during gestation. SMP30/GNL mRNA expression was evident at the start of gestation, increased for the next eight days then decreased rapidly. Moreover, L-gulono-γ-lactone oxidase (Gulo) mRNA, which catalyzes the last step of AA, was found in the ovaries of these mice. The variations of these genes' expression showed an inverse pattern to that of Cyp19a1 (aromatase) mRNA expression. Therefore, the SMP30/GNL and Gulo mRNA expression might be regulated by estrogen levels in the ovary. Since the presence of both SMP30/GNL and Gulo mRNAs could indicate that AA synthesis occurs in the ovary, we quantified AA levels in mouse ovaries during gestation. However, no correlation was found between changes of AA content and SMP30/GNL or Gulo mRNAs expression at this site. Moreover, we compared the changes of AA content during gestation between wild-type and SMP30/GNL knockout mice, which cannot synthesize AA, and found no significant differences between them. These results indicated that, although AA synthesis might occur in the ovaries, the amount of AA which is synthesized in ovaries must be quite low and insufficient to influence the AA content in ovary.

Helicobacter pylori infection combined with DENA revealed altered expression of p53 and 14-3-3 isoforms in Gulo-/- mice.

Unlike most other mammals, human bodies do not have the ability to synthesize vitamin C inside of their own bodies. Therefore, humans must obtain vitamin C through daily diet. Gulo(-/-) mice strain is known with deficiency, in which vitamin C intake can be controlled by diet like human, and would be valuable for investigating the molecular mechanism of various diseases. In the present study, we established Gulo(-/-) mice model and investigated the differentially expressed proteins in stomach tissue of Gulo(-)(/-) mice after Helicobacter pylori-infected, and followed by DENA, using immunohistochemistry and proteomic approach. The results of immunohistochemistry analysis of stomach tissue showed that the tumor suppressor, p53 protein, expression was significantly decreased (p<0.05) but not messenger RNA (mRNA) transcriptional level, and 14-3-3 ε, 14-3-3 δ, Ki-67 and cleaved caspase 3 expressions were significantly increased (p<0.05) by H. Pylori infection, and followed by DENA treatment in Gulo(-/-) mice. Moreover, knockdown of 14-3-3 isoforms (14-3-3 ε, 14-3-3 σ, 14-3-3 ζ and 14-3-3 η) were significantly increased sub-G1 phase (characteristics of apoptosis) in AGS cells and, phenotypic changes like cell shrinkage, density and cleaved nuclei were also observed. Proteome analyses showed that 14-3-3 σ, 14-3-3 η, and tropomyosin alpha-1 chain were down-regulated, and Hspd1 protein and HSC70 were up-regulated after H. Pylori-infection, and followed by DENA. The combined results of immunohistochemistry and proteomic analysis suggest that H. pylori altered the p53 and 14-3-3 isoforms expression and DENA further enhanced the H. pylori effect, which might be involved in carcinogenesis and metastasis of gastric cancer on Gulo(-/-) mice.

Genome-wide gene expression profiles in antioxidant pathways and their potential sex differences and connections to vitamin C in mice.

Vitamin C (VC) is well known as an antioxidant in humans, primates and guinea pigs. Studies have suggested gender differences in VC requirements in humans, and gender differences in oxidant injury vulnerability in early life may represent a biological mechanism contributing to gender disparity in later life. Using spontaneous bone fracture (sfx) mice, which lack the gene for L-Gulonolactone oxidase (Gulo), we studied the potential sex difference in expression profiles of oxidative genes at the whole-genome level. Then, we analyzed data of gene expressions in a mouse population of recombinant inbred (RI) strains originally derived by crossing C57BL/6J (B6) and DBA/2J (D2) mice. Our data indicated that there were sex differences in the regulation of pre- and pro-oxidative genes in sfx mice. The associations of expression levels among Gulo, its partner genes and oxidative genes in the BXD (B6 × D2) RI strains showed a sex difference. Transcriptome mapping suggests that Gulo was regulated differently between female and male mice in BXD RI strains. Our study indicates the importance of investigating sex differences in Gulo and its oxidative function by using available mouse models.