A number of regulatory factors including dietary iron levels can dramatically alter the expression of the intestinal iron transporter DMT1. Here we show that Caco-2 cells exposed to iron for 4 h exhibited a significant decrease in plasma membrane DMT1 protein, though total cellular DMT1 levels were unaltered. Following biotinylation of cell surface proteins, there was a significant increase in intracellular biotin-labelled DMT1 in iron-exposed cells. Furthermore, irontreatment increased levels of DMT1 co-localised with LAMP1, suggesting that the initial response of intestinal epithelial cells to iron involves internalisation and targeting of DMT1 transporter protein towards a late endosomal/lysosomal compartment.
TNFa has dramatic effects on iron metabolism contributing to the generation of hypoferraemia in the anaemia of chronic disease. Interestingly, TNFa is also synthesised and released within the intestinal mucosa, suggesting that this proinflammatory cytokine may play a role in regulating dietary iron absorption. To investigate this possibility, we stimulated intestinal Caco-2 cells with TNFa (10 ng/ml). In TNFa-treated cells, apical iron uptake was significantly decreased and this was accompanied by a reduction in divalent metal transporter protein and mRNA expression. Our data suggest that TNFa could regulate dietary iron absorption and that the apical transport machinery is the target for these actions.
The development of hepatocyte cell models that represent fatty acid partitioning within the human liver would be beneficial for the study of the development and progression of nonalcoholic fatty liver disease (NAFLD). We sought to develop and characterize a novel human liver cell line (LIV0APOLY) to establish a model of lipid accumulation using a physiological mixture of fatty acids under low- and high-glucose conditions. LIV0APOLY cells were compared with a well-established cell line (HepG2) and, where possible, primary human hepatocytes. LIV0APOLY cells were found to proliferate and express some mature liver markers and were wild type for the PNPLA3 (rs738409) gene, whereas HepG2 cells carried the Ile148Met variant that is positively associated with liver fat content. Intracellular triglyceride content was higher in HepG2 than in LIV0APOLY cells; exposure to high glucose and/or exogenous fatty acids increased intracellular triglyceride in both cell lines. Triglyceride concentrations in media were higher from LIV0APOLY compared with HepG2 cells. Culturing LIV0APOLY cells in high glucose increased a marker of endoplasmic reticulum stress and attenuated insulin-stimulated Akt phosphorylation whereas low glucose and exogenous fatty acids increased AMPK phosphorylation. Although LIV0APOLY cells and primary hepatocytes stored similar amounts of exogenous fatty acids as triglyceride, more exogenous fatty acids were partitioned toward oxidation in the LIV0APOLY cells than in primary hepatocytes. LIV0APOLY cells offer the potential to be a renewable cellular model for studying the effects of exogenous metabolic substrates on fatty acid partitioning; however, their usefulness as a model of lipoprotein metabolism needs to be further explored.
The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat 'group-specific protease' [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.
Paclitaxel, a relatively new antineoplastic agent, is associated with numerous side effects, including two reported cases of pancreatitis. Our patient also developed paclitaxel-associated pancreatitis. Several companion drugs, including steroids, diphenhydramine, histamine2 blockers, serotonin type 3 antagonists, and other chemotherapeutic agents administered with paclitaxel, must be considered as possible causes of pancreatitis. In addition, paclitaxel is a hydrophobic agent that requires a vehicle, cremophor (CrEL), for solubility. Intravenous cyclosporine also requires CrEL and has been associated with pancreatitis. In the cerulein-induced pancreatitis rat model, paclitaxel with dimethyl sulfoxide as a vehicle prevents pancreatitis, suggesting that another causal agent is responsible. Animal studies of CrEL as a single agent may be required to settle this question, but for now, awareness that paclitaxel may be associated with pancreatitis may lead to earlier treatment of this potentially fatal complication.
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