Autophagy of mitochondria in rat liver assessed by immunogold procedures

Glutamate dehydrogenase and carbamoyl phosphate synthase-I were localized in rat liver by immunogold procedures, using monoclonal and polyclonal antibodies. As expected, there was extensive labeling in mitochondria. Label was also found in lysosomal autophagic vacuoles. When autophagy was stimulated by in vivo administration of the anti-microtubular agent vinblastine we found that: (a) carbamoyl phosphate synthase-I and glutamate dehydrogenase could be found in mitochondria within autophagic vacuoles; (b) the carbamoyl phosphate synthase-I and glutamate dehydrogenase content of the mitochondria sequestered into autophagic vacuoles is the same as that of the nearby "free" mitochondria; and (c) in the whole liver, autophagic vacuoles contain c. 1.5 times more glutamate dehydrogenase than carbamoyl phosphate synthase-I, in contrast to mitochondria which have c. three times more carbamoyl phosphate synthase-I than glutamate dehydrogenase. The latter finding could explain, at least partially, the difference in half-lives of these enzymes.     

Turnover of rat liver ornithine transcarbamylase

The relative half-life of ornithine transcarbamylase from rat liver has been determined using the double isotope technique and affinity chromatography. The calculated half-life (6-9 days) is similar to that of mitochondria and of the other mitochondrial enzyme of the urea cycle, carbamoyl-phosphate synthase. Therefore, both mitochondrial urea cycle enzymes are most probably degraded mainly via the lysosomal (autophagic) pathway of mitochondrial protein degradation.     

Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media

Ammonium ion accumulation in mammalian cell culture media causes toxicity which inhibits cell growth and productivity. To reduce the level of the accumulated ammonium ion, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC) were used, which catalyze the first and second steps of the urea cycle in the liver. To examine the effects of overexpressed CPS I and OTC genes on the concentration of the ammonium ion in culture media, the two genes were introduced into Chinese hamster ovary (CHO) dhfr-cells. The CPS I expressing cell lines (CPS I-CHO) and both CPS I and OTC expressing cell lines (CPS I/OTC-CHO) were confirmed at the mRNA level and analyzed in terms of the cell growth and the accumulation of ammonium ion in culture media. The accumulation of ammonium ion was approximately 25-33% less in CPS I/OTC-CHO than in either CPS I-CHO or the vector-control cell lines. Interestingly however, the cell growth was approximately 15-30% faster in both CPS I-CHO and CPS I/OTC-CHO than in the control cell lines. Forced expression of urea cycle enzymes in the CHO cells revealed that both the expression of CPS I and OTC can reduce the accumulation of ammonium ion in the culture media.     

Role of LAMP-2 in lysosome biogenesis and autophagy

In LAMP-2-deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2-deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2-deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.     

氨甲酰磷酸不同合成途径在大肠杆菌中的比较

【背景】氨甲酰磷酸是生物合成代谢中精氨酸与嘧啶的重要前体物质,在工业微生物生产精氨酸与嘧啶及其衍生物中发挥关键作用。【目的】在大肠杆菌Escherichia coli BW25113中比较氨甲酰磷酸不同合成途径的催化效率。【方法】在大肠杆菌Escherichia coli BW25113中过表达鸟氨酸氨甲酰基转移酶(OTC)的基础上,分别过表达大肠杆菌自身的氨基甲酸激酶(CK)和氨甲酰磷酸合酶(CPSⅡ)并表征其反应效果。通过优化底物供应(调整底物浓度与引入L-谷氨酰胺合成酶)对CK与CPSⅡ的催化反应进行优化。【结果】在大肠杆菌中过表达OTC,建立细胞水平氨甲酰磷酸检测体系。在此基础上比较不同来源的CK,发现大肠杆菌来源的CK效果最好,50mmol/LNH4HCO3条件下全细胞催化9h得到2.95±0.15mmol/LL-瓜氨酸;过表达CPSⅡ时,50mmol/LL-谷氨酰胺催化9h得到3.16±0.29 mmol/L L-瓜氨酸。通过改变底物NH4HCO3浓度和引入外源L-谷氨酰胺合成酶(GS)等方式对CK与CPSⅡ的催化反应分别进行优化后,100 mmol/L NH4HCO3条件下,L-瓜氨酸浓度分别提高至4.67±0.55mmol/L和6.12±0.38mmol/L,且过表达GS后CPSⅡ途径可以利用NH3,不需要额外添加L-谷氨酰胺。【结论】引入L-谷氨酰胺合成酶后的CPSⅡ途径合成氨甲酰磷酸的能力优于CK途径,为精氨酸、嘧啶及其衍生物的合成提供了一种更加高效的策略。     

Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase

Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.     

Inhibition of pyruvate carboxylase degradation and total protein breakdown by lysosomotropic agents in 3T3-L1 cells.

1. Exposure to [3H]biotin during the differentiation of 3T3-L1 cells to adipocytes selectively labelled pyruvate carboxylase (EC 6.4.1.1). A subsequent incubation of labelled cells permitted the measurement of the degradation rate constant of this mitochondrial enzyme. 2. In medium without serum, pyruvate carboxylase was degraded with a half-life of 64 h, considerably longer than that found for average cell protein. The long half-life is commensurate with the enzyme being catabolized when whole mitochondria are destroyed. 3. The breakdown of pyruvate carboxylase was inhibited to a greater extent than the breakdown of total cell protein by insulin, NH4Cl and inhibitors of lysosomal proteinases, suggesting that the enzyme is degraded by the autophagic lysosomal system of the cell. 4. The above evidence implies that whole mitochondria are degraded in lysosomes, a conclusion that agrees with earlier electron-microscopic evidence showing mitochondria within autophagic vacuoles. 5. A second degradative pathway must be invoked to account for the breakdown of mitochondrial proteins of short half-life.     

Elevated autophagic sequestration of mitochondria and lipid droplets in steatotic hepatocytes of chronic ethanol-treated rats: an immunohistochemical and electron microscopic study

Ethanol-induced hepatic steatosis may induce the progression of alcoholic liver disease. The involvement of autophagic clearance of damaged mitochondria (mitophagy) and lipid droplets (LDs) (lipophagy) in chronic ethanol-induced hepatic steatosis is not clearly understood. Adult Wistar rats were fed either 5 % ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 10 weeks. Light microscopy showed marked steatosis in hepatocytes of ethanol-treated rats (ETRs), which was further revealed by transmission electron microscopy (TEM), where significant numbers of large LDs and damaged mitochondria were detected in steatotic hepatocytes. Moreover, TEM demonstrated that hepatocyte steatosis was associated with greatly enhanced autophagic vacuole (AV) formation compared to control hepatocytes. Mitochondria and LDs were the predominant contents of AVs in steatotic hepatocytes. Immunohistochemistry of LC3, a specific marker of early AVs (autophagosomes), demonstrated an extensive punctate pattern in hepatocytes of ETRs, while LC3 puncta were much less frequent in control hepatocytes. This was confirmed by immunoelectron microscopy (IEM), which showed localization of LC3 to autophagosomes sequestering damaged mitochondria and LDs. In addition, IEM revealed that PINK1 (a sensor of mitochondrial damage and marker of mitophagy) was overexpressed in mitochondria of ETRs. Enhanced autophagic lysosomal activity was evidenced by increased immunolabeling of LAMP-2, a marker of late AVs (autolysosomes) in hepatocytes of ETRs and colocalization of LC3 and lysosomal cathepsins using double immunofluorescence labeling. Increased AVs in hepatocytes of ETRs reflect ethanol toxicity and could represent a possible protective mechanism via stimulation of mitophagy and lipophagy.     

ATP6v0d2 deficiency increases bone mass, but does not influence ovariectomy-induced bone loss

SIR2 protein, an NAD-dependent deacetylase, is localized to nucleus and is involved in life span extension by calorie restriction in yeast. In mammals, among the seven SIR2 homologues (SIRT1-7), SIRT3, 4, and 5 are localized to mitochondria. As SIRT5 mRNA levels in liver are increased by fasting, the physiological role of SIRT5 was investigated in liver of SIRT5-overexpressing transgenic (SIRT5 Tg) mice. We identified carbamoyl phosphate synthetase 1 (CPS1), a key enzyme of the urea cycle that catalyzes condensation of ammonia with bicarbonate to form carbamoyl phosphate, as a target of SIRT5 by two-dimensional electrophoresis comparing mitochondrial proteins in livers of SIRT5 Tg and wild-type mice. CPS1 protein was more deacetylated and activated in liver of SIRT5 Tg mice than in wild-type. In addition, urea production was upregulated in hepatocytes of SIRT5 Tg mice. These results agree with those of a previous study using SIRT5 knockout (KO) mice. Because ammonia generated during fasting is toxic, SIRT5 protein might play a protective role by converting ammonia to non-toxic urea through deacetylation and activation of CPS1.     

Autophagic-lysosomal and mitochondrial sequestration of [14C]sucrose. Density gradient distribution of sequestered radioactivity

[14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37 degrees C. The sedimentation characteristics of particle-associated [14C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome. Isopycnic banding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08-1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large-size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3-methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy.     

Association of ATP citrate lyase with mitochondria

(1) The association of ATP citrate lyase with mitochondria was studied with isolated rat hepatocytes and mitochondria. (2) When hepatocytes were treated with digitonin, about 25% of the lyase activity was released like a mitochondrial enzyme. (3) The effect of temperature on release of lyase from hepatocytes was different from that on the release of other cytosolic or mitochondrial enzymes. (4) The fraction of total hepatic lyase in mitochondrial preparations made with exogenous MgCl₂ was 30 times greater than that for a cytosolic marker enzyme, phosphoglycerate kinase. (5) Lyase substrates enhanced the release of the enzyme both from hepatocytes and from isolated mitochondria. (6) The metabolic significance of association of ATP citrate lyase with mitochondria is discussed. (7) Data obtained in the course of these experiments indicate that less than 3% of adenylate kinase is cytosolic.     

Monoclonal antibodies used in immunocytochemical localization by electron microscopy of carbamoyl phosphate synthetase I in liver from rats fed high-protein diets

Carbamoyl phosphate synthetase I (CPS-I) is the most abundant protein of rat liver mitochondria. Biochemical measurements in liver homogenates have shown that the liver from rats fed a high-protein diet contains more CPS-I per gram tissue protein than controls. However, there is no information on changes in the intact tissue at the cellular and mitochondrial level. Therefore, monoclonal antibodies to beef liver CPS-I were produced by the hybridoma technique. Four clones, C-241/1A, B, C, and D secreted immunogammaglobulin (IgG) IgG1. Using C-241/C, we measured by electron microscopy immunogold procedures the labeling of CPS-I in mitochondria from liver of rats fed high protein (casein, 50 and 80% of total food intake) diets. CPS-I (expressed as gold particles/micron2 of mitochondrial cross-sectional area) was greater than in mitochondria from control rats (20% casein diet), whether the rats were fed for 1, 6, or 14 months on the high-protein diets. The immunocytochemical measurements shown here demonstrate that the increase in the level of CPS-I in high-protein diets is a reflection of both the larger number of CPS-I molecules per mitochondrial area and the larger proportion of the total hepatocyte volume occupied by mitochondria. Similar measurements were carried out with glutamate dehydrogenase (GDH) using previously characterized monoclonal antibodies. No differences in GDH labeling were found with high-protein diets. Interestingly, when mitochondria from hepatocytes of rats fed a high-protein diet were divided into two subpopulations on the basis of mitochondrial cross-sectional size (i.e., greater or less than 0.7 micron2), the large mitochondria had 1.2 times more CPS-I and 0.8 times less GDH than the small mitochondria nearby.     

Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial- lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.     

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.     

Proteolysis in mitochondrial preparations and in lysosomal preparations derived from rat liver

A method of preparing rat liver mitochondria with low residual contamination by lysosomal proteases is described. Preparations of mitochondria are divided into two equal portions, one of which is supplemented with a lysosomal fraction. The addition of the lysosomal fraction causes an increase in proteolysis of between 26- and 56-fold at pH 5.0 in four similar experiments. This increase matches the increase in the lysosomal marker beta-glucuronidase and indicates that all proteolysis at pH 5.0 is due to enzymes of the lysosomal fraction. Above pH 7.0, the addition of a lysosomal supplement increases proteolysis by 1.5- to 5-fold only, suggesting that in the absence of a lysosomal supplement very little of the observed proteolysis is due to enzymes of lysosomal origin. A method of calculating the contribution to total proteolysis of enzymes of the lysosomal fraction or of the mitochondrial fraction is described. The calculations show that at pH 7.0 and above, more than 93% of the observed proteolysis is due to enzymes originating in the mitochondrial fraction. The results support the view of other workers that rat liver mitochondria contain an endogenous neutral proteolytic system capable of degrading mitochondrial proteins to acid-soluble products.     

Liver autophagy in the influenza B virus model of Reye's syndrome in mice

Biochemical evidence is presented for the autophagic destruction of liver mitochondria in the influenza B virus model of Reye's syndrome in mice. Separation of lysosomes and autophagic vacuoles from mitochondria was accomplished by prior treatment of the mice with Triton WR-1339, resulting in uptake of detergent by these organelles (tritosomes), reducing their densities. The organelles were banded in a discontinuous sucrose gradient. Total protein in the heavy tritosomal fraction increased from 1-2% in controls to 7-8% in virus-treated animals. Ornithine carbamoyl transferase (OCTase), a mitochondrial marker, increased from 2-3% (controls) to 11-15% (virus-treated), and glucose-6-phosphatase, a marker for endoplasmic reticulum, increased from 1-2% (controls) to 8-10% (virus-treated). beta-Galactosidase, a soluble enzyme in the lysosome, and OCTase also increase in the cell extract fraction following virus treatment, indicating that there was turnover of heavy lysosomal contents.     

Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes.

In isolated rat hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.     

Intracellular Location and Properties of Plant L-Glutamate Dehydrogenases

A study of some properties of L-glutamate dehydrogenase (GDH)in the supernatant and mitochondrial fractions of mung beanhypocotyls failed to reveal any differences between the twoenzymes. GDH in lettuce leaf chloroplasts was solubilized withTriton-X-100 and marked differences in a number of propertieswere found between this enzyme and the one solubilized fromlettuce leaf mitochondria. It was concluded that the mitochondrialand chloroplastic GDH's are distinct enzymes. The small amountof GDH activity detected in isolated lettuce leaf peroxisomescould have been due to adsorption of the enzyme.     

Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion-exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0. 1% Triton X-100-treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K(m) value for NADP(+) than NAD(+), whereas the K(m) value for NAD(+) was higher than that for NADP(+) in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42 degrees C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N-terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH.