scholarly journals Distribution of hepatic glutaminase activity and mRNA in perivenous and periportal rat hepatocytes

1990 ◽  
Vol 267 (1) ◽  
pp. 265-267 ◽  
Author(s):  
M Watford ◽  
E M Smith
Keyword(s):  
Glutamine Synthetase ◽  
Relative Abundance ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Rat Hepatocytes ◽  
Perfusion Technique ◽  
Collagenase Perfusion ◽  
Phosphate Activated Glutaminase ◽  
Glutaminase Activity ◽  
Periportal Zone

Perivenous and periportal hepatocytes were isolated by the digitonin/collagenase perfusion technique. The specific activity of phosphate-activated glutaminase was 2.33-fold higher in periportal cells than in perivenous cells. Similarly, the relative abundance of glutaminase mRNA was 2.6-fold higher in samples from periportal cells. The distribution of glutaminase activity and mRNA was compared with those for glutamine synthetase (predominantly perivenous) and phosphoenolpyruvate carboxykinase (predominantly periportal). The results suggest that phosphate-activated glutaminase is predominantly expressed in the periportal zone of the liver acinus.

scholarly journals Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities

1987 ◽  
Vol 243 (1) ◽  
pp. 87-95 ◽  
Author(s):  
B Quistorff ◽  
N Grunnet
Keyword(s):  
Lactate Dehydrogenase ◽  
Glutamine Synthetase ◽  
Rat Liver ◽  
High Purity ◽  
Alanine Aminotransferase ◽  
Specific Activity ◽  
Perfusion Technique ◽  
Short Intervals ◽  
Specific Activities

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.

scholarly journals Gluconeogenesis in periportal and perivenous hepatocytes of rat liver, isolated by a new high-yield digitonin/collagenase perfusion technique

1985 ◽  
Vol 229 (1) ◽  
pp. 221-226 ◽  
Author(s):  
B Quistorff
Keyword(s):  
Glucose Metabolism ◽  
Pyruvate Kinase ◽  
Rat Liver ◽  
Alanine Aminotransferase ◽  
Control Cell ◽  
High Yield ◽  
Perfusion Technique ◽  
Data Support ◽  
Collagenase Perfusion ◽  
Periportal Zone

A technique is described which allows preparations of hepatocytes, enriched in either periportal or perivenous hepatocytes (‘PP-cells’ and ‘PV-cells’ respectively), in a yield of about 30-50% compared with control cell preparations. The liver is first perfused for 40-60s with digitonin (4 mg/ml) to destroy selectively either the periportal or the perivenous part of the microcirculatory unit, and then the remaining hepatocytes are isolated by the ordinary collagenase perfusion technique. In periportal cells the activities of alanine aminotransferase and pyruvate kinase were 29.4 and 18.7 mumol/min per mg of DNA respectively. The rate of gluconeogenesis was 0.402 mumol/min per mg of DNA. In perivenous cells the corresponding values were 9.55, 22.1 and 0.244 mumol/min per mg of DNA respectively. These data support the concept of a zonation of glucose metabolism within the microcirculatory unit of the liver, with the afferent part (periportal zone) having a 2-fold, more active gluconeogenesis than the efferent part (perivenous zone).

Adaptive changes in zonation for gluconeogenic capacity in liver lobules of cold-exposed rats

1993 ◽  
Vol 265 (4) ◽  
pp. E559-E564 ◽  
Author(s):  
M. Shiota ◽  
Y. Fujimoto ◽  
M. Inagami ◽  
M. Hiramatsu ◽  
M. Moriyama ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Mitochondrial Respiration ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Cold Treatment ◽  
Control Group ◽  
Perfusion Technique ◽  
Zonal Distribution ◽  
Collagenase Perfusion ◽  
The Relationship ◽  
Malate Aspartate Shuttle

The rate of gluconeogenesis from lactate increased in perfused livers after exposure of rats to cold for 5 days, and it returned to the control rate after 20 days [M. Shiota, T. Tanaka, and T. Sugano. Am. J. Physiol. 249 (Endocrinol. Metab. 12): E281-E286, 1985.]. The relationship between the increased gluconeogenic activity and its zonal distribution in liver lobules was studied in cold-exposed rats that had been starved for 24 h by examination of preparations enriched for periportal hepatocytes (PP-H) and for perivenous hepatocytes (PV-H), which had been isolated by the digitonin-collagenase perfusion technique. In the control group, the rate of gluconeogenesis from lactate or alanine was three times higher in PP-H than in PV-H. The rate of gluconeogenesis from these substrates in PP-H was not changed by exposure of rats to cold. The rates of PV-H increased to the level in PP-H after 5 days of exposure of rats to cold and then returned to the control rates after 20 days. The rate of gluconeogenesis from fructose was not altered in either preparation of cells by cold treatment of rats. The change in gluconeogenic capacity in PV-H caused by exposure of rats to cold was unrelated to changes in the activity of the malate-aspartate shuttle and of pyruvate kinase. The increased capacity in mitochondrial respiration was observed in both preparations of cells by cold treatment of rats for 5 days. The activity of phosphoenolpyruvate carboxykinase was higher in PP-H than in PV-H in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The regulation of phosphate-activated glutaminase activity and glutamine metabolism in the streptozotocin-diabetic rat

1984 ◽  
Vol 224 (1) ◽  
pp. 207-214 ◽  
Author(s):  
M Watford ◽  
E M Smith ◽  
E J Erbelding
Keyword(s):  
Drinking Water ◽  
Small Intestine ◽  
Glutamine Metabolism ◽  
Diabetic Rat ◽  
Complete Suppression ◽  
Chemically Induced ◽  
Phosphate Activated Glutaminase ◽  
Kidney Liver ◽  
Nh4cl Solution ◽  
Glutaminase Activity

The activity of phosphate-activated glutaminase was increased in the kidney, liver and small intestine of rats made diabetic for 6 days with injection of streptozotocin (75 mg/kg body wt.). Insulin prevented this increase in all three tissues. Treatment with NaHCO3, to correct the acidosis that accompanies diabetes, prevented the increase in renal glutaminase activity, but not that in liver or small intestine. Chemically induced acidosis (NH4Cl solution as drinking water) or alkalosis (NaHCO3 solution as drinking water) increased and decreased, respectively, glutaminase activity in the kidney, but were without significant effect on the activity in liver and small intestine. The increase in glutaminase activity in the small intestine during diabetes was due to an overall increase in the size of this organ, and was only detectable when activity was expressed in terms of whole organ, not mucosal scrapings or isolated enterocytes. Prolonged diabetes (40 days) resulted in an even greater increase in the size and glutaminase activity of the small intestine. Despite this marked increase in capacity for glutamine catabolism, arteriovenous-difference measurements showed a complete suppression of plasma glutamine utilization by the small intestine during diabetes, confirming the report by Brosnan, Man, Hall, Colbourne & Brosnan [(1983) Am. J. Physiol. 235, E261-E265].

scholarly journals Involvement of a local Fenton reaction in the reciprocal modulation by O2 of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene and the insulin-dependent activation of the glucokinase gene in rat hepatocytes

1998 ◽  
Vol 335 (2) ◽  
pp. 425-432 ◽  
Author(s):  
Thomas KIETZMANN ◽  
Torsten PORWOL ◽  
Karl ZIEROLD ◽  
Kurt JUNGERMANN ◽  
Helmut ACKER
Keyword(s):  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Fenton Reaction ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Gene Activation ◽  
Three Dimensional ◽  
Rat Hepatocytes ◽  
Confocal Laser ◽  
Radical Scavenger ◽  
Insulin Dependent

H2O2 mimicked the action of periportal pO2 in the modulation by O2 of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene and the insulin-dependent activation of the glucokinase (GK) gene. H2O2 can be converted in the presence of Fe2+ in a Fenton reaction into hydroxyl anions and hydroxyl radicals (•OH). The hydroxyl radicals are highly reactive and might interfere locally with transcription factors. It was the aim of the present study to investigate the role of and to localize such a Fenton reaction. Hepatocytes cultured for 24 h were treated under conditions mimicking periportal or perivenous pO2 with glucagon or insulin plus the iron chelator desferrioxamine (DSF) or the hydroxyl radical scavenger dimethylthiourea (DMTU) to inhibit the Fenton reaction. PCK mRNA was induced by glucagon maximally under conditions of periportal pO2 and half-maximally under venous pO2. GK mRNA was induced by insulin with reciprocal modulation by O2. DSF and DMTU reduced the induction of PCK mRNA to about half-maximal and increased the induction of GK mRNA to maximal under both O2 tensions. Hydroxyl radical formation was maximal under arterial pO2. Perivenous pO2, DSF and DMTU each decreased the formation of •OH to about 70% of control. The Fenton reaction could be localized in a perinuclear space by confocal laser microscopy and three-dimensional reconstruction techniques. In the same compartment, iron could be detected by electron-probe X-ray microanalysis. Thus a local Fenton reaction is involved in the O2 signalling, which modulated the glucagon- and insulin-dependent PCK gene and GK gene activation.

Obstructive cholestasis induces TNF-α- and IL-1β-mediated periportal downregulation of Bsep and zonal regulation of Ntcp, Oatp1a4, and Oatp1b2

2007 ◽  
Vol 293 (6) ◽  
pp. G1134-G1146 ◽  
Author(s):  
Markus G. Donner ◽  
Stephanie Schumacher ◽  
Ulrich Warskulat ◽  
Jane Heinemann ◽  
Dieter Häussinger
Keyword(s):  
Glutamine Synthetase ◽  
Bile Duct Ligation ◽  
Normal Liver ◽  
Polymorphonuclear Cells ◽  
Adaptive Mechanisms ◽  
Duct Ligation ◽  
Tnf Α ◽  
Obstructive Cholestasis ◽  
Periportal Zone ◽  
Lps Treatment

Inverse acinar regulation of Mrp2 and 3 represents an adaptive response to hepatocellular cholestatic injury. We studied whether obstructive cholestasis (bile duct ligation) and LPS treatment affect the zonal expression of Bsep (Abcb11), Mrp4 (Abcc4), Ntcp (Slc10a1), and Oatp isoforms (Slco1a1, Slco1a4, and slco1b2) in rat liver, as analyzed by semiquantitative immunofluorescence. Contribution of TNF-α and IL-1β to transporter zonation in obstructive cholestasis was studied by cytokine inactivation. In normal liver Bsep, Mrp4, Ntcp, and Oatp1a1 were homogeneously distributed in the acinus, whereas Oatp1a4 and Oatp1b2 expression increased from zone 1 to 3. Glutamine synthetase-positive pericentral hepatocytes exhibited markedly lower Oatp1a4 expression than the remaining zone 3 hepatocytes. In cholestatic liver Bsep and Ntcp immunofluorescence in periportal hepatocytes significantly decreased to 66 ± 4% ( P < 0.01) and 67 ± 7% ( P < 0.05), whereas it was not altered in pericentral hepatocytes. Oatp1a4 was significantly induced in hepatocytes with a primarily low expression, i.e., in periportal hepatocytes and in glutamine synthetase-positive pericentral hepatocytes. Likewise, Oatp1b2 was upregulated in periportal hepatocytes. Mrp4 zonal induction was homogeneous. Inactivation of TNF-α and IL-1β prevented periportal downregulation of Bsep. Recruitment of neutrophils and polymorphonuclear cells mainly occurred in the periportal zone. Likewise, IL-1β induction was largely found periportally. No significant transporter zonation was seen following LPS treatment. In conclusion, zonal downregulation of Bsep in obstructive cholestasis is associated with portal inflammation and is mediated by TNF-α and IL-1β. Periportal downregulation of Ntcp and induction of Oatp1a4 and Oatp1b2 may represent adaptive mechanisms to reduce cholestatic injury in hepatocytes with profound downregulation of Bsep and Mrp2.

Stimulation of albumin gene transcription by insulin in primary cultures of rat hepatocytes

1987 ◽  
Vol 252 (2) ◽  
pp. C205-C214 ◽  
Author(s):  
C. E. Lloyd ◽  
J. E. Kalinyak ◽  
S. M. Hutson ◽  
L. S. Jefferson
Keyword(s):  
Gene Transcription ◽  
Relative Abundance ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Complementary Strand ◽  
Albumin Secretion ◽  
Single Stranded Dna ◽  
Albumin Gene ◽  
Albumin Mrna ◽  
Stimulation Of

The first goal of the work reported here was to prepare single-stranded DNA sequences for use in studies on the regulation of albumin gene expression. A double-stranded rat albumin cDNA clone was subcloned into the bacteriophage vector M13mp7. Single-stranded recombinant clones were screened for albumin sequences containing either the mRNA strand or the complementary strand. Two clones were selected that contained the 1,200 nucleotide long 3' end of the albumin sequence. DNA from the clone containing the mRNA strand was used as a template for DNA polymerase I to prepare a radiolabeled, single-stranded cDNA to albumin mRNA. This radiolabeled cDNA probe was used to quantitate the relative abundance of albumin mRNA in samples of total cellular RNA. DNA from the clone containing the complementary strand was used to measure relative rates of albumin gene transcription in isolated nuclei. The second goal was to use the single-stranded DNA probes to investigate the mechanism of the insulin-mediated stimulation of albumin synthesis in primary cultures of rat hepatocytes. Addition of insulin to hepatocytes maintained in hepatocytes. Addition of insulin to hepatocytes maintained in a chemically defined, serum-free medium for 40 h in the absence of any hormones resulted in a specific 1.5- to 2.5-fold stimulation of albumin gene transcription that was maximal at 3 h and was maintained above control values for at least 24 h. The relative abundance of albumin mRNA and albumin secretion increased correspondingly within 24 to 30 h. These parameters remained above control levels for at least 60 h after addition of insulin. Maximal responses were attained at an insulin concentration of 100 nM and there was a close correspondence between albumin gene transcription and albumin secretion at each concentration tested. The rate of albumin gene transcription in nuclei isolated from livers of diabetic rats was reduced to 50% of the value recorded in control nuclei. Taken together, these findings demonstrate that insulin regulates synthesis of albumin at the level of gene transcription.

Nonenzymatic versus Enzymatic Hepatocyte Isolation from Pig Livers for Larger Scale Investigations of Liver Cell Perfusion Systems

1993 ◽  
Vol 16 (9) ◽  
pp. 677-681 ◽  
Author(s):  
J.C. Gerlach ◽  
J. Brombacher ◽  
J.M. Courtney ◽  
P. Neuhaus
Keyword(s):  
Liver Cell ◽  
Perfusion Technique ◽  
Hepatocyte Cultures ◽  
Hepatocyte Isolation ◽  
Collagenase Perfusion

A comparison of nonenzymatic and enzymatic hepatocyte isolation was performed on pig livers. The collagenase perfusion showed superior results: mean viability 72 ± 10% versus a maximum viability of 21% using EDTA-perfusion. A five-step collagenase perfusion technique, developed for pig livers enables larger scale investigations, in order to develop methods for hepatocyte cultures in therapeutical liver cell perfusion systems.

Separation of enzymatically distinct populations of trout hepatocytes

1991 ◽  
Vol 69 (2) ◽  
pp. 420-426 ◽  
Author(s):  
Thomas P. Mommsen ◽  
Eva Danulat ◽  
M. Emilia Gavioli ◽  
Glen D. Foster ◽  
Thomas W. Moon
Keyword(s):  
Hepatic Vein ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Citrate Synthase ◽  
Short Pulse ◽  
Gradient Centrifugation ◽  
Cellular Heterogeneity ◽  
Relative Distribution ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Collagenase Perfusion ◽  
Glucagon Like Peptide

Two different approaches were chosen to investigate the heterogeneity of rainbow trout (Oncorhynchus mykiss) hepatocytes. (i) Hepatocytes were isolated following selective liver infusion with a short pulse of digitonin from either the portal or the hepatic vein. Cell isolates, presumably enriched in cells from either "periportal" or "perivenous" liver regions, were then compared with cells obtained from the nondigitonized part of the same liver (control). No significant differences between hepatocytes were found in the activities of six key metabolic enzymes or in the rate of pyruvate oxidation. All cell isolates were responsive to glucagon, glucagon-like peptide, and insulin (all at 10 nM). If the liver was digitonized through the hepatic vein, resulting cell isolates revealed higher rates of gluconeogenesis from pyruvate, and gluconeogenic flux was more responsive to glucagon (at 10 nM) than controls. The experimental evidence for metabolic zonation in trout liver is weak. (ii) Enzymatically different subpopulations of hepatocytes were successfully separated by Percoll gradient centrifugation after isolation of cells by the conventional collagenase perfusion technique. Two prominent subpopulations of hepatocytes differed in the activities of glycogen phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, glutamate dehydrogenase, phosphoenolpyruvate carboxykinase, and alanine and aspartate aminotransferases, but not in their content of glycogen. However, the relative distribution of enzymes did not follow the mammalian pattern of perivenous or periportal hepatocytes. These data support cellular heterogeneity in the trout liver, with gluconeogenesis and oxidative metabolism dominating in some hepatocytes, whereas others display a relatively larger scope for glycogen breakdown, glycolysis, and amino acid metabolism.

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