The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes Blommaart, E.F.C.; Krause, P.; Schellens, J.P.M.; Vreeling-Sindelárová, H.; Meijer, A.J. Published in:European Journal of Biochemistry DOI:10.1111/j. 1432-1033.1997.0240a.x Link to publication Citation for published version (APA): Blommaart, E. F. C., Krause, P., Schellens, J. P. M., Vreeling-Sindelárová, H., & Meijer, A. J. (1997). The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. European Journal of Biochemistry, 243, 240-246. https://doi.org/10.1111/j.1432-1033.1997.0240a.x General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date: 22 Mar 2019Eur. J. Biochem. 243, 240-246 (1997) Recent studies indicate that phosphatidylinositol 3-kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3-kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenylchromone (LY294002).The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC=, o 30 nM) and LY294002 (IC5,, 10 pM) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids.3-Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3-kinase, thus providing a target for its action.It is proposed that phosphatidylinositol 3-kinase activity is required for autophagy. 3-Methyladenine inhibits autophagy by inhibition of this enzyme.Keywords: phosphatidylinositol 3-kinase; lysosome; proteolysis; 3-methyladenine; liver.Phosphatidylinositol 3-kinase ...

Colon cancer preferentially metastasizes to the liver. To determine cellular backgrounds of this preference, we generated an enhanced green fluorescent protein (eGFP)-expressing rat adenocarcinoma cell line (CC531s) that forms metastases in rat liver after administration to the portal vein. Intravital videomicroscopy (IVVM) was used to visualize early events in the development of tumors in livers of live animals from the time of injection of the cancer cells up to 4 days afterward. Based on information obtained with IVVM, tissue areas were selected for further analysis using confocal laser scanning microscopy (CLSM), electron microscopy (EM), and electron tomography. M any types of cancer of the gastrointestinal tract preferentially form metastases in the liver. 1 Metastasis of colon cancer in liver is found in 25% of patients at the time of colorectal resection, whereas 50% of patients eventually develop liver metastases. Approximately 90% of patients who die from colorectal cancer have liver metastases. 2 Metastasis is a multistep and dynamic process. Several steps are considered essential for the formation of metastases. These steps include detachment of cancer cells from the primary tumor, entry into the circulation and exit from the circulation at a secondary site by interactions with endothelial cells, and subsequent extravasation. 3 Completion of all these steps is considered necessary for metastasis.Two processes have been proposed to explain the high incidence of colon cancer metastasis in liver. One explanation is based on the seed-and-soil theory of Paget (1875) in which preferential adhesion of colon cancer cells (homing) forms the basis. 4,5 Alternatively, experimental data have shown that cancer cells cannot pass the liver because they are arrested physically in the too-narrow sinusoids of the liver. [6][7][8] The next steps after cancer cell arrest are also a matter of debate. Extravasation is considered the next step, but experimental data have shown that cancer cells can then either be dormant 8 or form micrometastases in the circulation. 9 Experimental end-point animal models of metastasis allow for quantification of numbers and sizes of metastases 10 but provide little information on processes that are involved in the development of metastases. Early events in metastasis were thus far not easy to study because identification of single cancer cells or micrometastases in tissues was difficult. To enable the analysis of early events in the formation of metastases, cancer cells have been transfected with the Escherichia coli ␤-galactosidase (lacZ) gene, which enables detection of micrometastases in tissue sections [11][12][13] and quantification of cancer cells in tumor tissue homogenates. 14 However, lacZ does not allow direct visualization of cancer cells in live animals, which would help us understand dynamic processes related to invasion and metastasis of cancer. 15 Green fluorescent protein is a suitable heritable marker and has been proven Abbreviations: CLSM, confocal laser scanning microsc...

Unfiued cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisdmes, whereas the crystalline core remained unstained. Control incubatiom were performed in the absence of substrate; the lack of final reac-

Ultrastructural morphometric analysis was used to study time-dependent variations in macro- and microautophagy in rat hepatocytes. Except during periods of short-term starvation for up to 24 h, animals were kept under standardized conditions of food intake. In hepatocytes of meal-fed rats the volume fraction of macroautophagic vacuoles is significantly higher at 23:00 h, i.e., immediately before food intake, compared to 11:00 h, i.e., 12 h following feeding. During fasting, macroautophagy drops to a low level. Microautophagic vacuoles in hepatocytes of meal-fed rats, sacrificed at 11:00 or 23:00 h respectively, do not show any significant quantitative differences. However, during 12 h of starvation, the volume fraction of microautophagic vacuoles rises significantly, whereas the numerical density remains constant. Subsequently, during the second 12-h period of fasting, the volume fraction of microautophagic vacuoles remains unchanged, but the numerical density increases. Over a period of 24 h of starvation the volume fraction of the total lysosomal system does not change significantly, whereas the numerical density rises. The time-dependent changes of the macroautophagic vacuolar system correlate with the circadian, food-related variations in the protein content of individual hepatocytes from meal-fed animals. The increase in volume fraction and thereafter in number of microautophagic vacuoles, as observed during starvation, coincides with a large decrease in protein content of individual hepatocytes.

Summary(Pre)neoplastic lesions in livers of rats induced by diethylnitrosamine are characterized by elevated activity of the first irreversible enzyme of the oxidative branch of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PD), for production of NADPH. In the present study, the activity of G6PD, and the other NADPH-producing enzymes, phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (ICD) and malate dehydrogenase (MD) was investigated in (pre)neoplastic lesions by metabolic mapping. Transketolase (TKT), the reversible rate-limiting enzyme of the non-oxidative branch of the PPP, mainly responsible for ribose production, was studied as well. Activity of G6PD in (pre)neoplastic lesions was highest, whereas activity of PGD and ICD was only 10% and of MD 5% of G6PD activity, respectively. Glucose-6-phosphate dehydrogenase activity in (pre)neoplastic lesions was increased 25 times compared with extralesional parenchyma, which was also the highest activity increase of the four NADPH-producing dehydrogenases. Transketolase activity was 0.1% of G6PD activity in lesions and was increased 2.5-fold as compared with normal parenchyma. Transketolase activity was localized by electron microscopy exclusively at membranes of granular endoplasmic reticulum in rat hepatoma cells where G6PD activity is localized as well. It is concluded that NADPH in (pre)neoplastic lesions is mainly produced by G6PD, whereas elevated TKT activity in (pre)neoplastic lesions is responsible for ribose formation with concomitant energy supply by glycolysis. The similar localization of G6PD and TKT activity suggests the channelling of substrates at this site to optimize the efficiency of NADPH and ribose synthesis.