Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-a-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.

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The facilitative glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in adipocytes and muscles, and the participation of GLUT4 in the pathogenesis of various clinical conditions associated with obesity, visceral fat accumulation and insulin resistance has been proposed. Glucose uptake by some members of the GLUT family, mainly GLUT1, is inhibited by flavonoids, the natural polyphenols present in fruits, vegetables and wine. Therefore it is of interest to establish if these polyphenolic compounds present in the diet, known to be effective antioxidants but also endowed with several other biological activities such as protein-tyrosine kinase inhibition, interfere with GLUT4 function. In the present study, we show that three flavonoids, quercetin, myricetin and catechin-gallate, inhibit the uptake of methylglucose by adipocytes over the concentration range of 10-100 microM. These three flavonoids show a competitive pattern of inhibition, with K(i)=16, 33.5 and 90 microM respectively. In contrast, neither catechin nor gallic acid inhibit methylglucose uptake. To obtain a better understanding of the interaction among GLUT4 and flavonoids, we have derived a GLUT4 three-dimensional molecular comparative model, using structural co-ordinates from a GLUT3 comparative model and a mechanosensitive ion channel [PDB (Protein Data Bank) code 1MSL] solved by X-ray diffraction. On the whole, the experimental evidence and computer simulation data favour a transport inhibition mechanism in which flavonoids and GLUT4 interact directly, rather than by a mechanism related to protein-tyrosine kinase and insulin signalling inhibition. Furthermore, the results suggest that GLUT transporters are involved in flavonoid incorporation into cells.

Despite the physiologic importance of vitamin E, in particular its alpha-tocopherol (alpha-T) isoform, the molecular mechanisms involved in the cellular uptake of this antioxidant from plasma lipoproteins have not been well-defined. Recent studies have suggested that selective lipid uptake, rather than endocytosis, is important for alpha-T delivery to cells. Here we show that the scavenger receptor class B type I (SR-BI), which mediates cellular selective cholesteryl ester uptake from lipoproteins, facilitates efficient transfer of alpha-T from HDL to cultured cells. In SR-BI-deficient mutant mice, relative to wild-type control animals, there was a significant increase in plasma alpha-T levels (1.1- to 1.4-fold higher) that was mostly due to the elevated alpha-T content of their abnormally large plasma HDL-like particles. This increase in plasma alpha-T in SR-BI knockout mice was accompanied by a significant decrease (65-80%) in the alpha-T concentrations in bile and several tissues including ovary, testis, lung and brain. SR-BI deficiency did not alter the alpha-T concentrations of the liver, spleen, kidney or white fat. These data show that SR-BI plays an important role in transferring alpha-T from plasma lipoproteins to specific tissues. Also, in the case of the liver as was previously shown for SR-BI-dependent hepatic cholesterol transport, SR-BI-mediated uptake of alpha-T was primarily coupled to biliary excretion rather than to tissue accumulation. Defective tissue uptake of lipoprotein alpha-T in SR-BI-deficient mice may contribute to the reproductive and cardiovascular pathologies exhibited by these animals.