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).

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 Digitonin-collagenase perfusion for efficient separation of periportal or perivenous hepatocytes

1985 ◽  
Vol 228 (3) ◽  
pp. 757-760 ◽  
Author(s):  
K O Lindros ◽  
K E Penttilä
Keyword(s):  
Lactate Dehydrogenase ◽  
Alanine Aminotransferase ◽  
High Yield ◽  
Cell Populations ◽  
Gamma Glutamyltransferase ◽  
Efficient Separation ◽  
Rat Liver Cells ◽  
Collagenase Perfusion ◽  
Portal Infusion

Intact rat liver cells from the perivenous region were isolated by collagenase perfusion after first destroying the periportal region by a brief portal infusion of digitonin. Periportal cells were isolated after retrograde digitonin infusion. Significantly higher alanine aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase activities and lower glutamate dehydrogenase and pyruvate kinase activities in periportal than in perivenous cells demonstrate marked separation. The high yield allows further characterization in vitro of the cell populations.

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.

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)

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