Zonation of hepatic lipogenic enzymes identified by dual-digitonin-pulse perfusion.
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ABSTRACT:
SUBMITTER: Evans JL
PROVIDER: S-EPMC1138591 | biostudies-other | 1989 May
REPOSITORIES: biostudies-other
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Project description:Heterogeneous zonation of hepatic protein expression over the liver lobule has been recognized by using several analytical techniques, including microdissection, selective cell isolation, immunohistochemistry and hybridization of mRNA in situ. We previously employed the technique of dual-digitonin-pulse perfusion for the highly selective collection and analysis of periportal and perivenous soluble protein. In the present work we have now documented the feasibility of the application of this technique to the study of zonal distribution of mRNA. By using a split-stream design, both protein and RNA fractions can be simultaneously collected from hepatic zones. High-quality RNA (average yield approximately 9-33 micrograms of total RNA per mg of eluted protein) is obtained for analysis. As analysed by immunoblotting and Northern-blot analysis, the zonal distribution of several important cytosolic metabolic enzymes and their mRNAs can be documented. This technique is also applicable to the study of mRNAs for organelle- and membrane-associated proteins that are not recoverable with this digitonin-lysis technique. The application of this experimental technique should allow further molecular insight into the mechanisms underlying zonation of hepatic function.
| S-EPMC1134533 | biostudies-other
Project description:A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed 'dual-digitonin-pulse perfusion'. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.
| S-EPMC1147818 | biostudies-other
Project description:The hepatic lobule is divided into three zones along the portal-central vein axis. Hepatocytes within each zone exhibit a distinctive gene expression profile that coordinates their metabolic compartmentalization. The zone-dependent heterogeneity of hepatocytes has been hypothesized to result from the differential degree of exposure to oxygen, nutrition and gut-derived toxins. In addition, the gradient of Wnt signaling that increases towards the central vein seen in rodent models is believed to play a critical role in shaping zonation. Furthermore, hepatic zonation is coupled to the site of the homeostatic renewal of hepatocytes. Despite its critical role, the regulatory mechanisms that determine the distinctive features of zonation and its relevance to humans are not well understood. The present study first conducted a comprehensive zone-dependent transcriptome analysis of normal human liver using laser capture microdissection. Upstream pathway analysis revealed the signatures of host responses to gut-derived toxins in the periportal zone, while both the canonical Wnt pathway and the xenobiotic response pathway govern the perivenular zone. Furthermore, we found that the hypoxic environment of the perivenular zone promotes Wnt11 expression in hepatocytes, which then regulates unique gene expression via activation of the non-canonical Wnt pathway. In summary, our study reports the comprehensive zonation-dependent transcriptome of the normal human liver. Our analysis revealed that the LPS response pathway shapes the characteristics of periportal hepatocytes. By contrast, the perivenular zone is regulated by a combination of three distinct pathways: the xenobiotic response pathway, canonical Wnt signaling, and hypoxia-induced noncanonical Wnt signaling.
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Project description:The lenticular fiber cells are comprised of extremely long-lived proteins while still maintaining an active biochemical state. Dysregulation of these activities has been implicated in diseases such as age-related cataracts. However, the lenticular protein dynamics underlying health and disease is unclear. We sought to measure the global protein turnover rates in the eye using nitrogen-15 labeling of mice and mass spectrometry. We measured the 14N/15N-peptide ratios of 248 lens proteins, including Crystallin, Aquaporin, Collagen and enzymes that catalyze glycolysis and oxidation/reduction reactions. Direct comparison of lens cortex versus nucleus revealed little or no 15N-protein contents in most nuclear proteins, while there were a broad range of 14N/15N ratios in cortex proteins. Unexpectedly, like Crystallins, many enzymes with relatively high abundance in nucleus were also exceedingly long-lived. The slow replacement of these enzymes in spite of young age of mice suggests their potential roles in age-related metabolic changes in the lens.
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Project description:To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.
| S-EPMC4251831 | biostudies-literature
Project description:Inflammation in response to excess low-density lipoproteins in the blood is an important driver of atherosclerosis development. Due to its ability to enhance ATP-binding cassette A1-dependent (ABCA1-dependent) reverse cholesterol transport (RCT), liver X receptor (LXR) is an attractive target for the treatment of atherosclerosis. However, LXR also upregulates the expression of sterol regulatory element-binding protein 1c (SREBP-1c), leading to increased hepatic triglyceride synthesis, an independent risk factor for atherosclerosis. Here, we developed a strategy to separate the favorable and unfavorable effects of LXR by exploiting the specificity of the coactivator thyroid hormone receptor-associated protein 80 (TRAP80). Using human hepatic cell lines, we determined that TRAP80 selectively promotes the transcription of SREBP-1c but not ABCA1. Adenovirus-mediated expression of shTRAP80 inhibited LXR-dependent SREBP-1c expression and RNA polymerase II recruitment to the LXR responsive element (LXRE) of SREBP-1c, but not to the LXRE of ABCA1. In murine models, liver-specific knockdown of TRAP80 ameliorated liver steatosis and hypertriglyceridemia induced by LXR activation and maintained RCT stimulation by the LXR ligand. Together, these data indicate that TRAP80 is a selective regulator of hepatic lipogenesis and is required for LXR-dependent SREBP-1c activation. Moreover, targeting the interaction between TRAP80 and LXR should facilitate the development of potential LXR agonists that effectively prevent atherosclerosis.
| S-EPMC4382268 | biostudies-literature
Project description:The activities of several hepatic enzymes are preferentially zonated to the periportal or perivenous cells of the liver acinus. Employing dual-digitonin-pulse perfusion of rat liver in the study of acetyl-CoA carboxylase (ACC), we have identified a heretofore unrecognized feature of hepatic zonation, namely an intrahepatic gradient in enzyme specific activity. ACC activity shows a relative periportal localization in normally feeding rats, even when corrected for ACC protein mass. In contrast with results previously reported by us [Evans, Quistorff & Witters (1989) Biochem. J. 259, 821-829], the total mass of both hepatic ACC isoenzymes was not found to differ between the two hepatic zones in the present study. In perfusion eluates from fed animals, periportal ACC displays enhanced citrate reactivity and two kinetic components of acetyl-CoA reactivity; the largest periportal/perivenous gradient (5-fold) is accounted for by a species with a lower Km for acetyl-CoA. The zonal gradient in ACC maximal velocity, measured in eluates from fed rats, does not persist after ACC purification, although the isolated periportal enzyme, like dephosphorylated ACC, has a lower activation constant for citrate. Total ACC protein phosphatase activity is higher in periportal eluates, but no differences in the activities of either a 5'-AMP-activated ACC kinase or the cyclic-AMP-dependent protein kinase are noted between the hepatic zones. The induction of total hepatic ACC mass and specific activity, on fasting/refeeding with a high-carbohydrate diet, abolishes the periportal/perivenous activity gradient, largely owing to a selective activation of perivenous enzyme. Nutritional induction is also accompanied by a marked alteration in ACC acetyl-CoA kinetics and abolition of the gradient in total ACC phosphatase. These studies indicate that hepatic enzyme zonation, which is often attributed to differential expression of enzyme protein, may result from zonal variations in enzyme specific activity, owing to differences in allosteric regulation and/or covalent modification.
| S-EPMC1131783 | biostudies-other
Project description:ContextIncreased hepatic de novo lipogenesis (DNL) in response to dietary sugar is implicated in dyslipidemia, fatty liver, and insulin resistance.ObjectiveThe aim of the study was to develop a simple outpatient tolerance test for lipogenic sensitivity to dietary sugar.Design and settingIn inpatients given repeated doses of fructose, protocol 1 compared the acute increase in DNL determined from the percentage of palmitate ("new palmitate") and the percentage of isotopically labeled palmitate ("%DNL") in very low-density lipoprotein triglyceride (TG). Protocol 2 compared the increase in new palmitate in outpatients given three different sugar beverages in a randomized crossover design.ParticipantsThere were 15 lean and overweight volunteers in protocol 1 and 15 overweight volunteers in protocol 2.InterventionsIn protocol 1, subjects received 1.4 g/kg fructose in divided oral doses over 6 h; in protocol 2, subjects received 0.5 g/kg fructose, 0.5 g/kg fructose plus 0.5 g/kg glucose, or 1 g/kg fructose plus 1 g/kg glucose each as a single oral bolus.Main outcome measuresWe measured the increase in DNL by two methods.ResultsAfter repeated doses of fructose, new palmitate was significantly correlated with the increase in %DNL (Δ, r = 0.814; P < 0.001) and with fasting insulin levels (area under the curve, r = 0.754; P = 0.001). After a single sugar dose, new palmitate showed a dose effect and was greater after fructose plus glucose. Very low-density lipoprotein TG and total TG significantly increased in both protocols.ConclusionsA single oral bolus of fructose and glucose rapidly increases serum TG and TG palmitate in overweight subjects. A dual sugar challenge test could prove useful to identify individuals at risk for carbohydrate-induced dyslipidemia and other adverse effects of increased DNL.
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Project description:Background/aimsThe importance of epigenetic changes in etiology and pathogenesis of disease has been increasingly recognized. However, the role of epigenetic alterations in the genesis of hepatic steatosis and cause of individual susceptibilities to this pathological state are largely unknown.MethodsMale inbred C57BL/6J and DBA/2J mice were fed a lipogenic methyl-deficient diet (MDD) that causes liver injury similar to human non-alcoholic steatohepatitis (NASH) for 6, 12, or 18 weeks, and the status of global and repetitive elements cytosine methylation, histone modifications, and expression of proteins responsible for those epigenetic modifications in livers was determined.ResultsThe development of hepatic steatosis in inbred C57BL/6J and DBA/2J mice was accompanied by prominent epigenetic abnormalities. This was evidenced by pronounced loss of genomic and repetitive sequences cytosine methylation, especially at major and minor satellites, accompanied by increased levels of repeat-associated transcripts, aberrant histone modifications, and alterations in expression of the maintenance DNA methyltransferase 1 (DNMT1) and de novo DNMT3A proteins in the livers of both mouse strains. However, the DBA/2J mice, which were characterized by an initially lower degree of methylation of repetitive elements and lower extent of histone H3 lysine 9 (H3K9) and H3 lysine 27 (H3K27) trimethylation in the normal livers, as compared to those in the C57BL/6J mice, developed more prominent NASH-specific pathomorphological changes.ConclusionsThese results mechanistically link epigenetic alterations to the pathogenesis of hepatic steatosis and strongly suggest that differences in the cellular epigenetic status may be a predetermining factor to individual susceptibilities to hepatic steatosis.
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Project description:Photoactivated atomic force microscopy (pAFM), which integrates light excitation and mechanical detection of the deflections of a cantilever tip, has become a widely used tool for probing nanoscale structures. Raising the illuminating laser power is an obvious way to boost the signal-to-noise ratio of pAFM, but strong laser power can damage both the sample and cantilever tip. Here, we demonstrate a dual-pulse pAFM (DP-pAFM) that avoids this problem by using two laser pulses with a time delay. The first laser heats the light absorber and alters the local Grüneisen parameter value, and the second laser boosts the mechanical vibration within the thermal relaxation time. Using this technique, we successfully mapped the optical structures of small-molecule semiconductor films. Of particular interest, DP-pAFM clearly visualized nanoscale cracks in organic semiconductor films, which create crucial problems for small-molecule semiconductors. DP-pAFM opens a promising new optical avenue for studying complex nanoscale phenomena in various research fields.
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