Dieter Sasse

Heterogeneous reciprocal localization of fructose-1,6-bis-phosphatase and of glucokinase in microdissected periportal and perivenous rat liver tissue

Liver and kidney catalyze glycolysis as well as gluconeogenesis [l-3] . In isolated liver cell suspensions the antagonistic processes were shown to occur simultaneously, the net process being dependent on substrate concentrations [4]. This mode of action of liver cells and the histochemicaily determined heterogeneous distribution of glucose-6-phosphatase (G6Pase) [5] and of glycogen metabolism [6,7] between periportal+ and perivenous+ zones of liver parenchyma led to the proposal of a metabolic zonation of the organ [4,5 ] . It was suggested that in the peripcrtal zone glucose formation by gluconeogenesis and glycogenolysis might be the predominant process, while in the perivenous zone glycolysis linked to liponeogenesis should prevail. Such a zonation would be analogous to kidney cortex heterogeneity, gluconeogenesis being located in the proximal and glycolysis in the distal tubules [9-l I]. The concept of a metabolic zonation of liver parenchyma was then further strengthened by demonstrating with the microdissection technique [ 121 that the glucogenic enzymes phosphoenolpyruvate carboxykinase [ 131 and glucose-6-phosphatase [ 141 were predominantly located in the periportal and the glycolytic pyruvate kinase [ 131 in the perivenous zone.

Functional heterogeneity of rat liver parenchyma and of isolated hepatocytes

It has been observed recently that rat hepatocyte suspensions catalyze glycolysis and gluconeogenesis simultaneously [ 11. The simultaneous catalysis of the two antagonistic processes may occur in one and the same hepatocyte or, as has been proposed [ 11, in two different types of hepatocytes, one catalyzing glycolysis the other gluconeogenesis. The key enzyme for the differentiation of such two hypothetical types of hepatocytes is glucose-6-phosphatase; only the cells possessing this enzyme will be able to release glucose and hence be “gluconeogenic”. In the present study it is shown that in the rat Liver parenchyma zones with high and low activities of glucose-6-phosphatase, glycogen synthase and.glycogen phosphorylase can be differentiated and that this differentiation is preserved in isolated single hepatocytes. The glucose-6-phosphatase-rich zone is always located around the portal branches; its relative size is subject to dietary changes. The observed doubling of the glucose-6-phosphatase level in the liver upon starvation appears to be due to both an increase in the number of glucose-6-phosphatase-rich liver cells and a rise in enzyme level within the already enzyme-rich hepatocytes.

Heterogeneous distribution of glucose‐6‐phosphatase in microdissected periportal and perivenous rat liver tissue

Liver and kidney catalyze glycolysis as well as gluconeogenesis [l-3] . In the rat nephron these two antagonistic processes are spatially separated between proximal and distal tubules, as could be demonstrated by studies of enzyme activities in microdissected kidney tissue [4-61. In rat liver parenchyma the two pathways might also be catalyzed in different cells, which would best explain the simultaneous catalysis of glycolysis and gluconeogenesis observed [7,8] . Histochemical studies of liver parenchyma showed a heterogeneous distribution of glucose-6-phosphatase (G6Pase). It was proposed that in the G6Pase-rich periportal* zone glucose formation by gluconeogenesis and glycogenolysis should be catalyzed, while in the G6Pase-poor perivenous* zone glycolysis may be the predominant process [9]. Histochemical enzyme determinations mostly provide only a qualitative or at best a semiquantitative information. Therefore, a quantification of histochemi cal findings appeared desirable. In the present investigation G6Pase was quantitatively determined by direct enzyme measurement in periportal and perivenous liver tissue separated by microdissection. It was found that the G6Pase activity of fed animals was about 2.3-fold higher and of fasted animals about 1.7-fold higher,in zone 1 than in zone 3. This finding supports the theory of a ‘metabolic zonation’ of liver parenchyma into functionally different hepatocytes [7-91.

Perinatal development of the metabolic zonation of hamster liver parenchyma

Adult liver catalyzes the antagonistic processes gluconeogenesis and glycolysis [l] . The heterogeneous distribution of glucose-6-phosphatase (G6Pase) over the liver parenchyma of the rat [2,3] and mouse [4] was interpreted to indicate a metabolic zonation of the liver lobule with respect to carbohydrate metabolism [5,6]. In the G6Pase-rich periportal zone glucose should be formed from gluconeogenesis and glycogenolysis. In the G6Pase-poor pericentral zone glycolysis may be the predominant process [7].. This metabolic zonation is influenced by changes of the nutritional state [S] . Prenatal liver parenchyma appears to catalyze only glycolysis [8,9], it should thus not be metabolically zonated. It was, therefore, of interest to study the perinatal development of the metabolic zonation and to try to correlate it with the drastic alteration of the nutritional state occurring with birth and during the following neonatal period. In the present study the following results have been obtained: (1) G6Pase is absent from prenatal liver. It appears rapidly in a homogeneous distribution with birth. From the 3rd or 4th postnatal day the zonation of G6Pase begins to develop; it is completed around the 12th day. (2) Glycogen storage begins 3 days before birth; it reaches a maximum with a homogeneous distribution at birth. Glycogen breakdown occurs rapidly during the first postnatal days predominantly in the G6Pase-rich zones. (3) Both the development of the zonation of G6Pase and of glycagen can be correlated to the perinatal switch from carbohydrateto proteinplus fatand back to carbohydrate-rich nutrition.

Intraacinar profiles of alcohol dehydrogenase and aldehyde dehydrogenase activities in human liver

The intraacinar activity profiles of alcohol dehydrogenase and the aldehyde dehydrogenases (I, I plus II, and total) were determined, using liver biopsy samples from eight male and eight female patients. Microchemical assays were performed in microdissected tissue samples from the whole length of the sinusoid. Alcohol dehydrogenase activity in men less than 53 years of age showed a maximum in the intermediate zone, whereas in women less than 50 years of age an increase in the gradient toward the perivenous zone was observed. Furthermore, alcohol dehydrogenase activity in the livers of women was significantly higher than in men. After the age of 53 in men and 50 in women, the sex specificity of the distribution profiles was no longer apparent. The intraacinar profiles of aldehyde dehydrogenase isoenzymes showed only minor variations in the different groups; they were not statistically significant. This is also true for low-Michaelis constant (Km) aldehyde dehydrogenase, which is most important for acetaldehyde oxidation in vivo. Thus, of the variations in zonal heterogeneity of ethanol-degrading enzymes, it is mainly the activity of alcohol dehydrogenase that may contribute to the sex- and age-related susceptibility of liver parenchyma.

Proteolytic enzymes of rat liver mitochondria. Evidence for a mast cell origin.

When rat liver mitochondria were subjected to step gradient centrifugation, a sediment of high density was obtained [ 11. This sediment contained an insoluble proteinase and a carboxypeptidase. Both enzymes have been purified and their physicochemical properties were studied [2-41. After subcellular fractionation, digitonin treatment and step gradient centrifugation both enzymes were localized in mitochondria [ 1,3]. After submitochondrial fractionation both proteolytic enzymes were found in the inner mitochondrial membrane [ 1,3]. From detailed studies on the properties of the proteinase [2,4] it became likely that the histone degrading enzyme isolated in our laboratory is identical with the ‘group-specific’ proteinase described [S]. The proteinase in the inner membrane of mitochondria has also been localized [6,7]. Recent experiments [8,9] have shown that the group specific proteinases isolated from small intestine [8] and from skeletal muscle [9] are of mast cell origin. In the light of these findings it became important to examine this possibility, although mast cells have not been described as an integral part of the liver [lo]. It will be shown here that indeed the proteinase as well as the carboxypeptidase isolated from the mitochondrial fraction originate from mast cells.

POSSIBLE METABOLIC ZONATION OF LIVER PARENCHYMA INTO GLUCOGENIC AND GLYCOLYTIC HEPATOCYTES

With hepatocyte suspensions it was found that (a) in the C3 part of carbohydrate metabolism glycolysis can be shifted to gluconeogenesis, dependent only on substrate concentrations rather than on hormones, and that (b) in both the C6 and C3 part glycolysis and gluconeogenesis are catalyzed simultaneously. This mode of action of the liver led to the hypothesis that there are at least two types of metabolically different hepatocytes forming a gluconeogenic and a glycolytic organ zone. Histochemical studies and biochemical analysis after microdissection of liver tissue indicated that the key glucogenic enzyme glucose-6-phosphatase (G6Pase) is highly active in periportal, and hardly active in perivenous hepatocytes, and that in the post-absorptive period glycogen is degraded first in the G6Pase rich cells. These results together with findings reported by other investigators support the model of “metabolic zonation” of liver parenchyma.

The Development of Functional Heterogeneity in the Liver Parenchyma of the Golden Hamster

SummaryPrenatal and postnatal stages of the development of golden hamsters were studied histochemically and biochemically. It was shown that, beginning with the 12th gestational day, the fetal liver starts to store glycogen, and that this process reaches its maximum a birth. Glycogen phosphorylase and glucose-6-phosphatase (G6Pase)-activity increased drastically in the last two days before birth, glycogen phosphorylase preceding G6Pase. As a histochemical characteristic, an even distribution of glycogen, glycogen phosphorylase and G6Pase activity is found in the liver parenchyma at birth. During the first two postnatal weeks typical heterogeneous patterns of distribution developed: glycogen depletion could be demonstrated predominantly in zone 1 of the liver acinus, this being at the same time the area of highest glycogen phosphorylase and G6Pase-activity. The periportal zone 1 thus became characterized as the primary site of glycogenolysis (glycogen phosphorylase) and gluco(neo)genesis (G6Pase). “Metabolic Zonation” is interpreted as the chemomorphological equivalent of the regulatory function of the liver as a glucostat.

Localization and Some Properties of a Proteinase and a Carboxypeptidase from Rat Liver

When chromatin was sedimented through a 1.7 M sucrose layer the cosedimentation of an insoluble proteinase from the mitochondrial fraction was observed (1). Although the enzyme was purified from rat liver mitochondria (2), it was not clear whether it is a mitochondrial enzyme or a contamination from lysosomes, peroxisomes, or endoplasmic reticulum. The existence of a mitochondrial proteinase has been discussed by several authors (3–9). Until now, however, no such enzyme has been unambiguously localized in mitochondria.

Microquantitative determination of lactate dehydrogenase isoenzymes in the myocardium and the conducting system

SummaryA newly developed technique was used for the electrophoretic separation of lactate dehydrogenase (LDH) isoenzymes from lyophilized tissue samples in the nanogram range. In this study portions of 10–200 ng from the myocardium and the conducting system of cattle, sheep, pig and man were microdissected and analysed.In the heart tissues of cattle, sheep and pig, the isoforms LDH1, LDH2 and LDH3 were detected in species-specific varying amounts. In all these animals, the conducting system is marked by high LDH1 activity, which is present at a ratio of about 2:1 compared with the myocardium. The values in man, however, differ from these values, but this might be due to post-mortem changes. The findings are discussed with respect to possible aerobic-anaerobic functions.