Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients.

Ascorbate requiring Fe2+/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR-γ gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease.

Inhibition of hepatic glucose 6-phosphatase system by the green tea flavanol epigallocatechin gallate.

Effect of 5-100 microM epigallocatechin gallate (EGCG) on hepatic glucose 6-phosphatase (G6Pase) system was investigated. EGCG inhibited G6Pase in intact but not in permeabilized rat liver microsomes, suggesting the interference with the transport. However, EGCG did not hinder microsomal glucose 6-phosphate (G6P) uptake. Instead, it increased the accumulation of radioactivity after the addition of [(14)C]G6P, presumably due to a slower release of [(14)C]glucose, the product of luminal hydrolysis. Indeed, EGCG was found to inhibit microsomal glucose efflux. Since G6Pase activity is depressed by glucose in a concentration-dependent manner, we concluded that EGCG inhibits G6Pase through an elevated luminal glucose level.

Glucuronide transport across the endoplasmic reticulum membrane is inhibited by epigallocatechin gallate and other green tea polyphenols.

Toxic endogenous or exogenous compounds can be inactivated by various conjugation reactions. Glucuronidation (i.e. conjugation with glucuronate) is especially important due to the large number of drugs and chemical carcinogens that are detoxified through this pathway. Stable and harmless glucuronides can be reactivated by enzymatic hydrolysis thus inhibitors of glucuronidase activity reduce the risk of chemical carcinogenesis. The aim of this study was to reveal whether this mechanism contributes to the anti-cancer effect of green tea flavanols, which has been shown in various animal models. Therefore, we investigated the effect of these polyphenols on deglucuronidation in rat liver microsomes and in Hepa 1c1c7 mouse hepatoma cells, using 4-methylumbelliferyl glucuronide as model substrate. Tea flavanols inhibited beta-glucuronidase in intact vesicles, where glucuronide transport across the microsomal membrane is rate-limiting, but were almost ineffective in permeabilized vesicles. Epigallocatechin gallate, the major green tea flavanol was shown to have a concentration-dependent inhibitory effect on both beta-glucuronidase activity and glucuronide transport in native vesicles. Epigallocatechin gallate also inhibited beta-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Our findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage. This phenomenon might potentially contribute to the cancer-preventing dietary or pharmacological effect attributed to these catechins.

Scurvy leads to endoplasmic reticulum stress and apoptosis in the liver of Guinea pigs.

Insufficient ascorbate intake causes scurvy in certain species. Beyond its known functions, it has been suggested that ascorbate participates in oxidative protein folding in the endoplasmic reticulum (ER). Because redox imbalance in this organelle might cause ER stress and apoptosis, we hypothesized that this might contribute to the pathology of scurvy. Guinea pigs were divided into 7 groups: the control group was fed a commercial guinea pig food containing 0.1 g/100 g ascorbate for 4 wk, 5 groups consumed an ascorbate-free food for 0, 1, 2, 3, or 4 wk and 1 group was fed this scorbutic diet for 2 wk and then the commercial food plus 1 g/L ascorbate in drinking water for 2 wk. TBARS generation and the expression of some ER chaperones and foldases were determined in hepatic microsomes. The apoptotic index was assessed in histological sections. Although ascorbate, measured by HPLC, was undetectable in the livers of the guinea pigs after they had consumed the scorbutic diet for 2 wk, the microsomal TBARS level was elevated relative to the initial value only at wk 4. Western blot revealed the induction of GRP78, GRP94, and protein disulfide isomerase at wk 3 and 4. Apoptosis was greater than in the control, beginning at wk 3. None of the alterations occurred in the groups fed the commercial guinea pig food or ascorbate-free food followed by ascorbate supplementation. Therefore, persistent ascorbate deficiency leads to ER stress, unfolded protein response, and apoptosis in the liver, suggesting that insufficient protein processing participates in the pathology of scurvy.