SKD1 AAA ATPase-dependent endosomal transport is involved in autolysosome formation

Mouse SKD1 AAA ATPase is involved in the sorting and transport from endosomes; cells overexpressing a dominant-negative mutant, SKD1(E235Q) were defective in endosomal transport to both the plasma membranes and lysosomes (Yoshimori et al., 2000). In the present study, we demonstrated that overexpression of SKD1(E235Q) using an adenovirus delivery system caused a defect in autophagy-dependent bulk protein degradation. Morphological observations suggested that this inhibition of autophagy results from an impairment of autolysosome formation. SKD1(E235Q) overexpression also inhibited transport from endosomes to autophagosomes, an event normally occurring prior to fusion with lysosomes. These results indicate that SKD1-dependent endosomal membrane trafficking is required for formation of autolysosomes.     

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: mechanism of suppression

We have shown that nitric oxide production is dramatically decreased in rat primary hepatocyte cultures exposed to galactosamine. Cotreatment of the cells with uridine, which is known to prevent cytotoxicity, was found to also attenuate NO loss. In the present study, two possible mechanisms for the decreased nitric oxide production were examined. First, we examined the possibility that galactosamine could interfere with the uptake of extracellular arginine by the cultured hepatocytes. Cellular uptake of arginine was determined after addition of 14C-arginine at the time of hepatocyte attachment. Uptake of arginine was rapid in control cultures, and both the rate and level of uptake were unchanged by the addition of a cytotoxic concentration of galactosamine (4 mM). In addition, increased concentrations of arginine in the cell culture medium did not ameliorate the galactosamine-induced decrease in production of nitric oxide. Second, we determined whether the synthesis of inducible nitric oxide synthase in the hepatocyte cultures was inhibited by addition of galactosamine. Hepatocyte levels of inducible nitric oxide synthase were determined immunochemically at various times after the addition of galactosamine (4 mM). In control cultures, inducible nitric oxide synthase was detectable at 7 and 24 hours after attachment. In contrast, no nitric oxide synthase protein was detectable at any time in the galactosamine-treated cultures. Furthermore, addition of galactosamine after inducible nitric oxide synthase had already been synthesized (6.5 h after attachment) did not result in suppression of nitric oxide production in the hepatocyte cultures. The present studies suggest that galactosamine suppresses nitric oxide production in hepatocyte cultures by inhibiting synthesis of inducible nitric oxide synthase, rather than by interference in cellular uptake of arginine.     

PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation

Autophagosome formation is promoted by the PI3 kinase complex and negatively regulated by myotubularin phosphatases, indicating that regulation of local phosphatidylinositol 3‐phosphate (PtdIns3P) levels is important for this early phase of autophagy. Here, we show that the Caenorhabditis elegans myotubularin phosphatase MTM‐3 catalyzes PtdIns3P turnover late in autophagy. MTM‐3 acts downstream of the ATG‐2/EPG‐6 complex and upstream of EPG‐5 to promote autophagosome maturation into autolysosomes. MTM‐3 is recruited to autophagosomes by PtdIns3P, and loss of MTM‐3 causes increased autophagic association of ATG‐18 in a PtdIns3P‐dependent manner. Our data reveal critical roles of PtdIns3P turnover in autophagosome maturation and/or autolysosome formation.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation

How myosin II localizes to the cleavage furrow in Dictyostelium and metazoan cells remains largely unknown despite significant advances in understanding its regulation. We designed a genetic selection using cDNA library suppression of 3xAsp myosin II to identify factors involved in myosin cleavage furrow accumulation. The 3xAsp mutant is deficient in bipolar thick filament assembly, fails to accumulate at the cleavage furrow, cannot rescue myoII-null cytokinesis, and has impaired mechanosensitive accumulation. Eleven genes suppressed this dominant cytokinesis deficiency when 3xAsp was expressed in wild-type cells. 3xAsp myosin II''s localization to the cleavage furrow was rescued by constructs encoding rcdBB, mmsdh, RMD1, actin, one novel protein, and a 14-3-3 hairpin. Further characterization showed that RMD1 is required for myosin II cleavage furrow accumulation, acting in parallel with mechanical stress. Analysis of several mutant strains revealed that different thresholds of myosin II activity are required for daughter cell symmetry than for furrow ingression dynamics. Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp. Overall, myosin II accumulation is the result of multiple parallel and partially redundant pathways that comprise a cellular contractility control system.     

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization

Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ⁹-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.     

Ferritin is increased in diethylnitrosamine-altered rat hepatocytes

Ferritin was localized by immunoperoxidase in rat liver during the early stages of experimental carcinogenesis induced by diethylnitrosamine. Carcinogen - altered hepatocytes, indentified by their reduction in membrane-bound ATPase activity, showed a highly elevated content in ferritin (or ferritin subunits), compared to normal hepatocytes. This could correspond to an accumulation of free subunits in the cytoplasm or to a “non-specific” increase in ferritin synthesis related to the carcinogenic process. Our results suggest that some cytoplasmic proteins other than enzymes can be modified during the early stages of carcinogenesis and that ferritin accumulation detected by immunolocalization can be used as a valuable marker to identify foci of cellular alterations.     

Disappearance of visible bile canaliculi caused by vinblastine in primary cultures of rat hepatocytes

Treatment of cultured hepatocytes with vinblastine resulted in the inhibition of the reformation of biliary spaces (at times earlier than 16 h) or in the disappearance of preformed bile canaliculi (at times later than 24 h) detectable on both the light and electron microscopical level. Concomitantly the preferential localization of leucine aminopeptidase around the lucid biliary spaces was lost without any change in the specific activity of this enzyme. Despite these alterations the performance of the cultured cells (e.g., urea synthesis) was not impaired by the exposure to the drug. The effect of vinblastine was mimicked by colchicine, but not by lumicolchicine, indicating that microtubules might play a role in the structural organization of the biliary pole.     

Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation

Effects of various preservation solutions were compared in an experimental hypothermic preservation model using cultured rat hepatocytes. Hepatocytes prepared by the collagenase perfusion method were cultured for 48 hr, then the medium in each culture dish was exchanged for various preservation solutions, and the dishes were hypothermically (0-2 degrees C) stored in a refrigerator for 12-72 hr. After the preservation period, the hepatocytes were cultured again at 37 degrees C for 2 hr. Hepatocytes' viability after 18-hr preservation and reculture was greater when they were preserved in "intracellular" rather than "extracellular" solutions. Even with Euro-Collins solution (intracellular solution), hepatocyte viability decreased to approximately 20% after 24-hr preservation, and an increase in the cellular lipid peroxide content was observed. However, when this solution contained a submillimolar concentration of calcium, lipid peroxidation was significantly suppressed and hepatocyte viability was dramatically improved. Vitamin E was almost equally effective and a marked synergistic effect was observed with calcium. Calcium was found to be capable of maintaining the cellular glutathione level during cold storage, which seems to suppress lipid peroxidation and consequently improve hepatocyte survival.     

Rapid stimulation of free glucuronate formation by non-glucuronidable xenobiotics in isolated rat hepatocytes

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle.     

Evidence that cyclic AMP-induced inhibition of phosphatidylcholine biosynthesis is caused by a decrease in cellular diacylglycerol levels in cultured rat hepatocytes.

The mechanism by which glucagon and cAMP analogues inhibit phosphatidylcholine biosynthesis was investigated in rat hepatocytes. The studies were facilitated by preparation of an antibody to a synthetic peptide (D-F-V-A-H-D-D-I-P-Y-S-S-A) corresponding to residues 164-176 of CTP:phosphocholine cytidylyl-transferase. The antibody, which was purified by affinity chromatography, quantitatively immunoprecipitated cytidylyltransferase from rat liver cytosol. Various analogues of cAMP had no effect on the labeling of cytidylyltransferase with 32Pi in rat hepatocytes. Nor did the cAMP analogues have any effect on the distribution of cytidylyltransferase between cytosol and membranes. These results indicate that the supply of CDP-choline does not limit phosphatidylcholine biosynthesis in hepatocytes treated with cAMP analogues. A decreased supply of diacylglycerol was considered as an alternative mechanism for inhibition of phosphatidylcholine biosynthesis. An approximately 30% decrease in diacylglycerol concentration was observed in hepatocytes treated with the cAMP analogues or glucagon, compared with controls. A similar decrease of phosphatidylcholine biosynthesis was observed. The cAMP-mediated decrease in diacylglycerol levels and inhibition of phosphatidylcholine biosynthesis were reversed by addition of 0.5-1.5 mM oleic acid to the treated hepatocytes. A correlation coefficient of 0.93 was calculated between the levels of diacylglycerol and the rate of phosphatidylcholine biosynthesis. In another approach, the diacylglycerol levels were increased by an inhibitor of diacylglycerol lipase (U-57908) which also reversed the cAMP effects on diacylglycerol levels and phosphatidylcholine biosynthesis. We conclude that the cAMP-mediated inhibition of phosphatidylcholine biosynthesis was not due to an effect on the phosphorylation of cytidylyltransferase. Instead, phosphatidylcholine biosynthesis appears to be inhibited due to a decreased level of diacylglycerol, a substrate for CDP-choline: 1,2-diacylglycerol cholinephosphotransferase.     

Leucine inhibition of autophagic vacuole formation in isolated rat hepatocytes

An electron microscopic, morphometric analysis of isolated rat hepatocytes revealed a 70% decrease in the early forms of autophagic vacuoles after administration of leucine. The lysosomal degradation of protein was reduced by only about 30% under the same conditions. These observations suggest that leucine is a major regulator of the bulk autophagy observable in the electron microscope, but that this type of autophagy contributes only about one-half of the total amount of protein degraded in lysosomes. Asparagine inhibited lysosomal protein degradation more strongly than did leucine, but had no significant effect on the amount of autophagic vacuoles. Leucine and asparagine would therefore seem to exert their effects on lysosomal protein degradation through different mechanisms.     

Polyploidization of rat hepatocytes due to cell fusion caused by radiations of different LET

The method of flow cytometry was used to study polyploidization of hepatocytes following X-, gamma-, and neutron-irradiation. Ionizing radiation was shown to induce cell polyploidization by two different ways: (1) cells and nuclei fusion, and (2) restriction of mitosis after DNA replication. RBE of 14 MeV neutrons with respect to fusion was about 5.10(3). With neutron irradiation, the sensitivity of cells by fusion was not lower than that by chromosome mutations.     

Inhibition of glucagon-stimulated cAMP accumulation and fatty acid oxidation by E-series prostaglandins in isolated rat hepatocytes

E-series prostaglandins have been shown to inhibit hepatic glucagon-stimulated glycogenolysis without inhibiting glycogenolysis stimulated by cAMP analogs. In the present studies, prostaglandin E2 and 16,16-dimethylprostaglandin E2 inhibited glucagon-stimulated cAMP accumulation in isolated rat hepatocytes by 25% and 46%, respectively, without affecting basal cAMP levels. Half-maximal inhibition of glucagon-stimulated cAMP accumulation occurred at approx. 10(-7) M 16,16-dimethylprostaglandin E2. 16,16-Dimethylprostaglandin E2 inhibited glucagon-stimulated palmitate oxidation in intact hepatocytes without affecting basal rates of palmitate oxidation. 16,16-Dimethylprostaglandin E2 had no effect on palmitate oxidation in a liver homogenate system. These studies demonstrate that prostaglandin E antagonizes the effects of glucagon on hepatic metabolism by inhibiting glucagon-stimulated cAMP accumulation.     

Arachidonic acid suppression of fatty acid synthase gene expression in cultured rat hepatocytes

Rat hepatocytes were maintained in a serum-free, hormonally defined medium supplemented with 50-500 microM albumin-bound 20:1 (n-9) vs 20:4 (n-6). The induction of fatty acid synthase mRNA by a mix of insulin/dexamethasone/T3 was inhibited in a dose dependent fashion by 20:4 (n-6). The abundance of beta-actin mRNA was not suppressed by 20:4 (n-6). The expression of fatty acid synthase was actually stimulated 2-fold by 20:1 (n-9). It would appear that the in vivo inhibition of fatty acid synthase gene expression by dietary polyunsaturated fatty acids is a specific hepatocelluar event.     

Receptor-linked degradation of 125I-insulin is mediated by internalization in isolated rat hepatocytes

When hepatocytes were freshly isolated from rat liver and incubated for various periods of time at 37 degrees C, the media from the incubation, when completely separated from the cells, actively degraded 125I-insulin. THis soluble protease activity was strongly inhibited by bacitracin but was unaffected by the lysosomatropic agent ammonium chloride (NH4Cl). When hepatocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of 8 mM NH4Cl the ligand initially bound to the plasma membrane and was subsequently internalized as a function of time. When hepatocytes were incubated at 37 degrees C for 30 minutes with 125I-insulin in the presence of bacitracin and NH4Cl or bacitracin alone and the cells were washed, diluted, and the cell-bound radioactivity allowed to dissociate, the percent intact 125I-insulin in the cell pellet and in the incubation media was greater in the presence of NH4Cl at each time point of incubation. Under these same conditions a higher proportion of the cell-associated radioactivity was internalized and a higher proportion was associated with lysosomes. The data suggest that receptor-mediated internalization is required for insulin degradation by the cell, and that this process, at least in part, involves lysosomal enzymes. Furthermore, the data demonstrate that internalization is not blocked by the presence of bacitracin or NH4Cl in the incubation media, but that degradation is inhibited.     

Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells

The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC-like structures developed between rising/piled-up cells. We investigated how BC-like structures developed with maturation of SHs and whether the structures were functionally active as BC. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Immunocytochemistry and immunoblotting for BC proteins, such as ectoATPase, 5'-nucleotidase, dipeptidylpeptidase IV, and multidrug-resistance associated protein 2, were examined and time-lapse microscopy was used for the observation of BC contractions. Secretion of bilirubin into the reconstructed BC was also observed. The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins were localized in the intercellular space that coincided with BC-like structures reconstructed between rising/piled-up cells. Tight junction-associated protein ZO-1 was also expressed along the BC-like structures. Bilirubin added to the medium were secreted into BC-like structure and accumulated without leakage. Time-lapse microscopy showed continuous contractions of reconstructed BC. In conclusion, BC-like structures reconstructed by SHs may be functional with membrane polarity, secretory ability, and motility. These results show that this culture system may suitable for investigating the mechanism of the formation of BC and their functions.