Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations

Osypiw, Jacqueline C.; Allen, Roger L.; Billington, David C.

doi:10.1042/bj3040617

Cited by 20 publications

(13 citation statements)

References 45 publications

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“…To determine whether this heterogeneity was associated with a portal-central gradient of cytosolic ␤-glucosidase expression along the hepatic acinus, we employed density gradient centrifugation to separate periportal from pericentral hepatocytes. 25 The specific activity in the lowest-density (pericentral) hepatocytes (362 U/mg) was only slightly greater than the specific activity in the highest density (periportal) hepatocytes (270 U/mg). However, incubation of fresh liver slices in tissue culture medium containing RESGlc, followed by preparation of frozen sections, resulted in diffuse labeling of hepatocytes, with no obvious portal-central gradient (Fig.…”

Section: Resultsmentioning

confidence: 89%

“…Freshly isolated hepatocytes were fractionated by density gradient centrifugation using methods similar to those described for rat hepatocytes. 25 Viable hepatocytes (1.8 ϫ 107) were suspended in 10 mL of 25% (wt/vol) isotonic Percoll and layered over 10 mL each of 50%, 55%, 60%, and 65% isotonic Percoll in a 50-mL centrifuge tube. The tube was centrifuged at 530g for 5 minutes, and six fractions were collected.…”

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Expression of cytosolic ?-glucosidase in guinea pig liver cells

et al. 1998

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The cytosolic ␤-glucosidase of mammalian liver has been implicated in the metabolic transformation of plant glycosides, such as vicine and amygdalin, which are associated with the development of toxic syndromes. We investigated which cell types express cytosolic ␤-glucosidase in guinea pig liver, and characterized the contribution of this enzyme to the hydrolysis of aromatic glucosides in cultured cells and in tissue slices. Cytosolic ␤-glucosidase was expressed in hepatocytes and not in Kupffer or endothelial cells as determined by enzyme-specific activity and Western blots of liver cell extracts. Intracellular ␤-glucosidase activity was visualized using the fluorescent ␤-glucosidase substrate, resorufin ␤-D-glucoside, and shown to be caused by the cytosolic ␤-glucosidase using the inhibitors, conduritol ␤-epoxide and dinitrophenol-2-deoxy-2-fluoro-␤-D-glucopyranoside (DNP2FGlc). Staining of fresh liver slices with resorufin ␤-glucoside revealed that cytosolic ␤-glucosidase is expressed in all hepatocytes, with no significant portalcentral gradient. These data indicate that cytosolic ␤-glucosidase is a hepatocyte-specific enzyme, and support the hypothesis that cytosolic ␤-glucosidase in the liver functions to hydrolyze small glucosides absorbed by the intestine. Furthermore, toxic injury to cultured hepatocytes by CCl 4 resulted in release of cytosolic ␤-glucosidase in parallel with the hepatocyte marker enzymes alanine transaminase and lactate dehydrogenase. This suggests that acute increases in serum levels of cytosolic ␤-glucosidase in animal models of liver injury may reflect direct injury of hepatocytes. (HEPATOLOGY 1998;28:156-163.)A principal physiological function of the liver is to dispose of xenobiotic compounds absorbed from the intestinal tract. The detoxification and excretion pathways for numerous compounds are classified into two stages. Phase I reactions involve either oxidation or reduction reactions, and are typically catalyzed by cytochrome P450 enzymes. 1 Phase II reactions then conjugate the modified compound, typically with glucuronic acid, sulfate, or glutathione, before excretion of the conjugate into the bile. 2 In plants, however, a variety of physiologically relevant compounds are produced and stored as O-linked ␤-D-glucosides. 3-5 Although the metabolic disposal of such glucosides by mammals is largely uncharacterized, it is likely that removal of the glucose moiety occurs at some point before ultimate excretion. Furthermore, the aglycone moieties of ␤-D-glucosides found in dietary plants have been implicated as the etiologic agents in acute cyanide intoxication resulting from ingestion of cassava, 6 apricot seeds, 7 and administration of amygdalin (laetrile), 8 as well as neurodegenerative disorders such as western pacific amyotrophic lateral sclerosis/parkinsonism-dementia. 6 The cytosolic ␤-glucosidase of mammalian liver has been characterized and studied by several groups. 9 The enzyme has been described in the livers of humans, 10 cattle, 11 rabbits, 12 and guinea pigs. 13 Cyto...

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Section: Resultsmentioning

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Expression of cytosolic ?-glucosidase in guinea pig liver cells

et al. 1998

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“…47,48 These disparities may be caused by contaminating activated Kupffer cells in the hepatocyte culture or due to the activation of liver-resident Kupffer cells producing TNF during the liver perfusion. We therefore purified the hepatocytes using a multistep Percoll gradient, 49 a method that excludes any macrophage contamination. Again, CPT alone induced apoptosis of hepatocytes (20 h: con, 25.5%; CPT, 54.9%; CPT/TNF, 54.5%).…”

Section: Discussionmentioning

confidence: 99%

Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytesin vitroandin vivoto TNF-mediated apoptosis

et al. 2004

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Topoisomerases are nuclear enzymes that maintain and modulate DNA structure. Inhibitors of topoisomerases like camptothecin (CPT), etoposide, and others are widely used antitumor drugs that interfere with transcription, induce DNA strand breaks, and trigger apoptosis preferentially in dividing cells. Because transcription inhibitors (actinomycin D, galactosamine, ␣-amanitin) sensitize primary hepatocytes to the cytotoxic action of tumor necrosis factor (TNF), we reasoned whether topoisomerase inhibitors would act similarly. CPT alone was not toxic to primary cultured murine hepatocytes. When incubated with CPT, murine hepatocytes displayed an inhibition of protein synthesis and were thereby rendered sensitive to apoptosis induction by TNF. Apoptosis was characterized by morphology (condensed/fragmented nuclei, membrane blebbing), caspase-3-like protease activity, fragmentation of nuclear DNA, and late cytolysis. T opoisomerases are important nuclear enzymes that regulate DNA tertiary structure by mediating transient, protein-shielded DNA breaks. Topoisomerases are involved in DNA replication, repair, recombination, and in transcriptional regulation. Thus, their activity is particularly required during mitosis to facilitate DNA re-organisation. Type I topoisomerases cut and pass single strands of DNA, whereas type II enzymes cut and pass double-stranded DNA.Topoisomerase II inhibitors (etoposide [ETP], doxorubicin) and the topoisomerase I inhibitor camptothecin (CPT) and derivatives are in clinical use or clinical trials as chemotherapeutics. 1 By covalently trapping topoisomerases on DNA, the compounds preferentially target dividing cells that require high topoisomerase activity. Under topoisomerase inhibition, DNA strand breaks occur during mitosis, which triggers premature termination of replication and inhibits

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“…The mass isotopomer distribution was acquired and corrected for natural 13 C enrichment (18), and f was calculated (11). Hepatocyte Fractionation-Hepatocytes were fractionated on a step Percoll gradient with slight modifications of a published method (19). Briefly, 9 ml of stock Percoll (1.13 g/ml) was diluted with 1 ml of 1.5 M NaCl.…”

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Limitations of the Mass Isotopomer Distribution Analysis of Glucose to Study Gluconeogenesis

Previs

Hallowell

Neimanis

et al. 1998

Journal of Biological Chemistry

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We ascribed the labeling heterogeneity to major decreases in glycerol concentration and enrichment across the liver. We concluded that [13 C]glycerol is unsuitable for tracing the contribution of gluconeogenesis to total glucose production. We now report isotopic heterogeneity of gluconeogenesis in hepatocytes, even when all cells are in contact with identical concentrations and enrichments of gluconeogenic substrates. Total rat hepatocytes were incubated with concentrations of glycerol, lactate, and pyruvate that were kept constant by substrate infusions. To modulate competition between substrates, the (glycerol)/(lactate ؉ pyruvate) infusion ratio ranged from 0.23 to 3.60. Metabolic and isotopic steady states were achieved in all cases. The apparent contribution of gluconeogenesis to glucose production (f) was calculated from the mass isotopomer distribution of glucose. When all substrates were 13 Clabeled, f was 97%, as expected in glycogen-deprived hepatocytes. As the infusion ratio ([ 13 C]glycerol)/(lactate ؉ pyruvate) increased, f increased from 73% to 94%. In contrast, as the infusion ratio (glycerol)/([ 13 C]lactate ؉ [ 13 C]pyruvate) increased, f decreased from 93% to 76%. In all cases, f increased with the rate of supply of the substrate that was labeled. Variations in f show that the 13 C labeling of triose phosphates was not equal in all hepatocytes, even when exposed to the same substrate concentrations and enrichments. We also showed that zonation of glycerol kinase activity is minor in rat liver. We conclude that zonation of other processes than glycerol phosphorylation contributes to the heterogeneity of triose phosphate labeling from glycerol in rat liver.

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Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations

Cited by 20 publications

References 45 publications

Expression of cytosolic ?-glucosidase in guinea pig liver cells

Expression of cytosolic ?-glucosidase in guinea pig liver cells

Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytesin vitroandin vivoto TNF-mediated apoptosis

Limitations of the Mass Isotopomer Distribution Analysis of Glucose to Study Gluconeogenesis

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