Degradation of peroxisomal catalase and urate oxidase of rat liver

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5–9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation.After [³H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37°C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process.From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.     

Activation of stem cells in hepatic diseases

The liver has enormous regenerative capacity. Following acute liver injury, hepatocyte division regenerates the parenchyma but, if this capacity is overwhelmed during massive or chronic liver injury, the intrinsic hepatic progenitor cells (HPCs) termed oval cells are activated. These HPCs are bipotential and can regenerate both biliary epithelia and hepatocytes. Multiple signalling pathways contribute to the complex mechanism controlling the behaviour of the HPCs. These signals are delivered primarily by the surrounding microenvironment. During liver disease, stem cells extrinsic to the liver are activated and bone-marrow-derived cells play a role in the generation of fibrosis during liver injury and its resolution. Here, we review our current understanding of the role of stem cells during liver disease and their mechanisms of activation. This work was supported by a Wellcome Trust Clinical Training Fellowship to T.G.B.; S.L. is supported by an EASL Sheila Sherlock Fellowship Post-Doctoral Fellowship.     

Quantitative proteomic comparison of mouse peroxisomes from liver and kidney

The peroxisome plays a central role in the catabolic and anabolic pathways that contribute to the lipid homeostasis. Besides this main function, this organelle has gained functional diversity. Although several approaches have been used for peroxisomal proteome analysis, a quantitative protein expression analysis of peroxisomes from different tissues has not been elucidated yet. Here, we applied a 2-DE-based method on mouse liver and kidney peroxisomal enriched fractions to study the tissue-dependent protein expression. Ninety-one spots were identified from the 2-DE maps from pH 3.0-10.0 and 51 spots from the basic range corresponding to 31 peroxisomal proteins, 10 putative peroxisomal, 6 cytosolic, 17 mitochondrial and 1 protein from endoplasmic reticulum. Based on the identification and on the equivalent quality of both tissue preparations, the differences emerging from the comparison could be quantified. In liver, proteins involved in pathways such as alpha- and beta-oxidation, isoprenoid biosynthesis, amino acid metabolism and purine and pyrimidine metabolism were more abundant whereas in kidney, proteins from the straight-chain fatty acid beta-oxidation were highly expressed. These results indicate that tissue-specific functional classes of peroxisomal proteins could be relevant to study peroxisomal cellular responses or pathologies. Finally, a web-based peroxisomal proteomic database was built.     

Microbodies in fungi: a review

Summary Microbodies are ubiquitous organelles in fungal cells, occurring in both vegetative hyphae and spores. They are bounded by a single membrane and may contain a crystalloid inclusion with subunits spaced at regular intervals. Typically, they contain catalase which reacts with the cytochemical stain 3,3-diaminobenzidine to yield an electron-opaque product, urate oxidase,l--hydroxy acid oxidase andd-amino acid oxidase. Their fragility and the necessity to disrupt the tough fungal cell wall before isolating them make them difficult to isolate. Analysis of enzymes in purified or partially purified microbodies from fungi indicates that they participate in fatty acid degradation, the glyoxylate cycle, purine metabolism, methanol oxidation, assimilation of nitrogenous compounds, amine metabolism and oxalate synthesis. In organisms where microbodies are known to contain enzymes of the glyoxylate cycle, they are known as glyoxysomes; where they are known to contain peroxidatic activity, they are known as peroxisomes. In some cases microbodies contain enzymes for only a portion of a pathway or cycle. Thus, they must be involved in metabolic cooperation with other organelles, particularly mitochondria. The number, size and shape of microbodies in cells, their buoyant density and their enzyme contents may vary with the composition of the medium; their proliferation in cells is regulated by the growth environment. The isolation from the same organism of microbodies with different buoyant densities and different enzymes suggests strongly that more than one type of microbody can be formed by fungi.     

Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor alpha

Peroxisome proliferators are potent rodent liver carcinogens that act via a non-genotoxic mechanism. The mode of action of these agents in rodent liver includes increased cell proliferation, decreased apoptosis, secondary oxidative stress and other events; however, it is not well understood how peroxisome proliferators are triggering the plethora of the molecular signals leading to cancer. Epigenetic changes have been implicated in the mechanism of liver carcinogenesis by a number of environmental agents. Short-term treatment with peroxisome proliferators and other non-genotoxic carcinogens leads to global and locus-specific DNA hypomethylation in mouse liver, events that were suggested to correlate with a burst of cell proliferation. In the current study, we investigated the effects of long-term exposure to a model peroxisome proliferator WY-14,643 on DNA and histone methylation. Male SV129mice were fed a control or WY-14,643-containing (1000ppm) diet for one week, five weeks or five months. Treatment with WY-14,643 led to progressive global hypomethylation of liver DNA as determined by an HpaII-based cytosine extension assay with the maximum effect reaching over 200% at five months. Likewise, trimethylation of histone H4 lysine 20 and H3 lysine 9 was significantly decreased at all time points. The majority of cytosine methylation in mammals resides in repetitive DNA sequences. In view of this, we measured the effect of WY-14,643 on the methylation status of major and minor satellites, as well as in IAP, LINE1 and LINE2 elements in liver DNA. Exposure to WY-14,643 resulted in a gradual loss of cytosine methylation in major and minor satellites, IAP, LINE1 and LINE2 elements. The epigenetic changes correlated with the temporal effects of WY-14,643 on cell proliferation rates in liver, but no sustained effect on c-Myc promoter methylation was observed. Finally, WY-14,643 had no effect on DNA and histone methylation status in Pparalpha-null mice at any of the time points considered in this study. These data indicate the importance of epigenetic alterations in the mechanism of action of peroxisome proliferators and the key role of Pparalpha.     

Mitochondrial dysfunction in liver disease and organ transplantation

The mitochondria play a crucial role in maintaining hepatocyte integrity and functions. Mitochondrial defects are either inherited or acquired. Mitochondria dysfunction occurs when the hepatocyte experience excessive physiologic stress. Its clinical presentation depends on the severity of the stress. It varies from mild abnormalities in liver biochemical tests to manifestations of acute or chronic liver failure. Mitochondria dysfunction is implicated in most liver disease and in early graft dysfunction after liver transplantation. This review will address the role of mitochondria in liver disease.     

Deficiency of manganese superoxide dismutase in hepatocytes disrupts zonated gene expression in mouse liver

The liver acinus displays a physiological periportal to perivenous oxygen gradient. This gradient was implicated to use reactive oxygen species (ROS) as mediators for the zonal gene expression. Mitochondria use oxygen and produce ROS, therefore they may contribute to the zonation of gene expression. To further elucidate this, we used the Cre-loxP system to generate a hepatocyte-specific null mutation of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) in mice. We found that ROS levels were enhanced in livers of MnSOD(-/-) mice which were reduced in size and displayed signs of liver failure such as intracellular protein droplets, increased apoptotic bodies and Bax levels as well as multinuclear hepatocytes. Further, the zonation of glutamine synthetase, glucokinase and phosphoenolpyruvate carboxykinase was no longer preserved. We conclude that deficiency of mitochondrial MnSOD initiates a dysregulation of zonated gene expression in liver.     

Mitochondria attached to desmosomes in the ciliary epithelia of human,monkey, and rabbit eyes

In the normal ciliary epithelia of the rhesus monkey, owl monkey, albino rabbit, and human eye, a previously unreported relationship exists between mitochondria and certain desmosomes. At these sites, two mitochondria appear like "sentinels" attached to the cytoplasmic surfaces of their respective sides of a desmosome. In other instances, only one side of the junction may be afforded an associated mitochondrion. In each case the cytoplasmic filaments of the desmosome are seen to blend with the outer membrane of the mitochondrion. The relationship between desmosomes and mitochondria in the ciliary epithelium is unique among ocular tissues. A survey of ocular epithelia in the various species examined, failed to give any evidence of similar junctional/organelle complexes. Various functional roles for this relationship are discussed including the possibility that the mitochondria could control the cytoplasmic calcium ion concentration in the microenvironment of their associated desmosomal junctions.     

Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells

In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the β-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.     

Unaltered hepatic oxidative phosphorylation and mitochondrial permeability transition in wistar rats treated with nimesulide: Relevance for nimesulide toxicity characterization

Nonsteroidal anti-inflammatory drugs have been associated with hepatotoxicity in susceptible patients. One such example is nimesulide, a preferential cyclooxygenase 2-inhibitor, widely used for the treatment of inflammation and pain. It was suggested that nimesulide could exert its hepatotoxicity by altering hepatic mitochondrial function, which was demonstrated in vitro. The objective of this study was to verify whether liver mitochondria isolated from rats treated with doses of nimesulide well above therapeutic levels possessed decreased calcium tolerance and oxidative phosphorylation, which indicates in vivo nimesulide mitochondrial toxicity. Male and female rats received nimesulide or its vehicle twice daily, for 5 days, and were killed on the seventh day for the isolation of liver mitochondria. Mitochondrial respiration, transmembrane electric potential, and calcium tolerance were characterized in all experimental groups. Nimesulide had no effect on liver mitochondrial function. Indexes of mitochondrial integrity, calcium loading capacity, and oxidative phosphorylation efficiency were unchanged between liver mitochondria from treated and control animals. In the animals tested, no evidence of degraded mitochondrial function due to nimesulide administration could be found. The results corroborate the notion that despite recognized in vitro mitochondrial toxicity, nimesulide does not cause detectable mitochondrial dysfunction in Wistar rats, even when administered in much higher concentrations than those known to have anti-inflammatory effects.     

Ruthenium methylimidazole complexes induced apoptosis in lung cancer A549 cells through intrinsic mitochondrial pathway

Ruthenium(II) methylimidazole complexes, with the general formula [Ru(MeIm)₄(N?N)]²⁺ (N?N = tip (RMC1), iip (RMC2), dppz (RMC3), dpq (RMC4); MeIm = 1-methylimidazole, tip = 2-(thiophene-2-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, iip = 2-(1H-imidazol-4-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, dppz = dipyrido[3,2-a:2′,3′-c]phenazine, dpq = pyrazino [2,3-f] [1,10]phenanthroline), were synthesized and characterized. As determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, these complexes displayed potent anti-proliferation activity against various cancer cells. RMC1 inhibited the growth of A549 (human lung adenocarcinoma) lung cells through induction of apoptotic cell death, as evidenced by the accumulation of cell population in sub-G1 phase. RMC1 also induced the depletion of mitochondrial membrane potential in A549 cells by regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Another experiment showed that Bid protein was also activated by RMC1, which implied that RMC1 could existed two pathways crosstalk, namely, have exogenous death receptor signaling pathway. These results demonstrated that RMC1 induced cancer cell death by acting on both mitochondrial and death receptor apoptotic pathways, suggesting that RMC1 could be a candidate for further evaluation as a chemotherapeutic agent against human cancers.     

Isolated mouse liver mitochondria are devoid of glucokinase

Glucokinase is a hexokinase isoform with low affinity for glucose that has previously been identified as a cytosolic enzyme. A recent report claims that glucokinase physically associates with liver mitochondria to form a multi-protein complex that may be physiologically important in apoptotic signaling [N.N. Danial, C.F. Gramm, L. Scorrano, C.Y. Zhang, S. Krauss, A.M. Ranger, S.R. Datta, M.E. Greenberg, L.J. Licklider, B.B. Lowell, S.P. Gygi, S.J. Korsmeyer, Nature 424 (2003) 952-956]. Here, we re-examined the association of glucokinase with isolated mouse liver mitochondria. When glucokinase activity was measured by coupled enzyme assay, robust activity was present in whole liver homogenates and their 9500 g supernatants (cytosol), but activity in the purified mitochondrial fraction was below detection (<0.2% of homogenate). Furthermore, addition of 45 mM glucose in the presence of ATP did not increase mitochondrial respiration, indicating the absence of ADP formation by glucokinase or any other hexokinase isoform. Immunoblots of liver homogenates and cytosol revealed strong glucokinase bands, but no immunoreactivity was detected in mitochondria. In conclusion, mouse liver mitochondria lack measurable glucokinase. Thus, functional linkage of glucokinase to mitochondrial metabolism and apoptotic signaling is unlikely to be mediated by the physical association of glucokinase with mitochondria.     

Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells

The fetal liver serves as the predominant hematopoietic organ until birth. However, the mechanisms underlying this link between hematopoiesis and hepatogenesis are unclear. Previously, we reported the isolation of a monoclonal antibody (anti-Liv8) that specifically recognizes an antigen (Liv8) present in murine fetal livers at embryonic day 11.5 (E11.5). Liv8 is a cell surface molecule expressed by hematopoietic cells in both fetal liver and adult mouse bone marrow. Here, we report that Liv8 is also transiently expressed by hepatoblasts at E11.5. Using protein purification and mass spectrometry, we have identified Liv8 as the CD44 protein. Interestingly, the expression of Liv8/CD44 in fetal liver was completely lost in AML1^−/− murine embryos, which lack definitive hematopoiesis. These results show that hepatoblasts change from Liv8/CD44-negative to Liv8/CD44-positive status in a hematopoiesis-dependent manner by E11.5, and indicate that Liv8/CD44 expression is an important link between hematopoiesis and hepatogenesis during fetal liver development.     

Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition

Background

Chronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-differentiate into activated myofibroblasts, which produce extracellular matrix.Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study showed that succinate and its specific receptor, GPR91, are involved in the activation of HSCs and the overexpression of α-smooth muscle actin (α-SMA).Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model. However, the role of metformin in HSC activation and succinate-GPR91 signaling has not been clarified.