Research Outputs
Sustained blockade of ascorbic acid transport associated with marked SVCT1 loss in rat hepatocytes containing increased ascorbic acid levels after partial hepatectomy
The liver has an extraordinary regenerative capacity in response to partial hepatectomy (PHx), which develops with neither tissue inflammation response nor alterations in the whole organism. This process is highly coordinated and it has been associated with changes in glutathione (GSH) metabolism. However, there are no reports indicating ascorbic acid (AA) levels after partial hepatectomy. AA and GSH act integrally as an antioxidant system that protects cells and tissues from oxidative damage and imbalance observed in a variety of diseases that affect the liver. Although rat hepatocytes are able to synthesize AA and GSH, which are the providers of AA for the whole organism, they also acquire AA from extracellular sources through the sodiumcoupled ascorbic acid transporter-1 (SVCT1). Here, we show that hepatocytes from rat livers subjected to PHx increase their GSH and AA levels from 1 to 7 days post hepatectomy, whose peaks precede the peak in cellproliferation observed at 3 days post-hepatectomy. The increase in both antioxidants was associated with higher expression of the enzymes involved in their synthesis, such as the modifier subunit of enzyme glutamine cysteine ligase (GCLM), glutathione synthetase (GS), gulonolactonase (GLN) and gulonolactone oxidase (GULO). Importantly, rat hepatocytes, that normally exhibit kinetic evidence indicating only SVCT1-mediated transport of AA, lost more than 90% of their capacity to transport it at day 1 after PHx without evidence of recovery at day 7. This observation was in agreement with loss of SVCT1 protein expression, which was undetectable in hepatocytes as early as 2 h after PHx, with partial recovery at day 7, when the regenerated liver weight returns to normal. We conclude that after PHx, rat hepatocytes enhance their antioxidant capacity by increasing GSH and AA levels prior to the proliferative peak. GSH and AA are increased by de novo synthesis, however paradoxically hepatocytes from rat subjected to PHx also suppress their capacity to acquire AA from extracellular sources through SVCT1.
Catalase is more sensible to the inhibitory effect of soluble component of tobacco smoke than Glutathione Peroxidase and Superoxide Dismutase
Tobacco smoke causes oxidative damage directly by the effect of their oxidants or indirectly through the induction of endogenously produced oxidants and/or inactivation of antioxidants. The oxidative effect of tobacco smoke depends on many variables: dose, time of exposure, tissue or cell type and endogenous antioxidant status. In an attempt to simplify this complex scenario, we examined the effect of a soluble extract of tobacco smoke on the activity of purified antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase) and in human plasma. Our results revealed that catalase and glutathione peroxidase were inhibited with an IC50 of 18 and 80 smoker equivalents (arbitrary units), respectively; meanwhile superoxide dismutase was not affected. A similar effect of soluble extract of tobacco smoke was obtained for antioxidant enzymes in human plasma, where catalase was inhibited, while superoxide dismutase was little affected, and glutathione peroxidase increased 20% its activity. Benzo[a]pyrene, a well-known component of tobacco smoke, was partly responsible for catalase inactivation. Although soluble extract of tobacco smoke and benzo[a]pyrene both induced carbonylation of plasma proteins, we ruled out that catalase inhibition would be caused by carbonylation, since the inhibition was reversed by dialysis. Considering the higher sensitivity of catalase to inhibition induced by soluble extract of tobacco smoke and its important role in peroxide elimination, we conceived that benzo[a]pyrene and other compounds of tobacco smoke extract promote a transient peroxide accumulation which could be one of the factors responsible for the oxidative damage in respiratory tract and other tissues in smokers.
GLUT1 and GLUT8 support lactose synthesis in Golgi of murine mammary epithelial cells
The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (p < 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (p < 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (p < 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane–associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in cis-Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.