Adaptative changes of metabolic zonation in liver cirrhosis

Le métabolisme des hépatocytes diffère en fonction de leur localisation proximale (zone périportale) ou distale (périvenulaire) le long des sinusoïdes hépatiques. Certaines voies métaboliques ont une localisation préférentielle, voire exclusive, dans une zone acinaire, soit périportale, soit périvenulaire. Cette hétérogénéité fonctionnelle des hépatocytes est appelée « zonation métabolique ». Grâce à ce système, les voies métaboliques agissent de manière séquentielle et complémentaire le long du sinusoïdes. La direction du flux sanguins dans le sinusoïde semble jouer un rôle prépondérant dans l’organisation de cette zonation métabolique.
La technique de cytochimie quantitative et de microdensitométrie développée au laboratoire permet de mesurer de façon précise l’activité de différentes enzymes au niveau de la cellule hépatique individuelle, tout en corrélant directement cette activité enzymatique avec l’histologie et les lésions pathologiques éventuelles. Le principe de la technique consiste en une mesure in situ, par microspectrophotométrie, du produit final de réactions cytochimiques sur sections cryocongelées de tissus hépatiques.
Nous avons démontré le reproductibilité et la spécificité de la technique, également appliquée à l’examen de biopsies humaines telles qu’obtenues par ponction à l’aiguille. La zonation métabolique s’établit pogressivement en période postnatale, et ce développement ontogénique explique les différences d’activité observées tant chez l’homme que chez l’animal en fonction de l’âge.
Afin de déterminer si la zonation métabolique décrite dans le parenchyme hépatique normal persistait également dans le nodule cirrhotique, nous avons développé au laboratoire différents modèles de cirrhoses expérimentales, et nous avons étudié également du foie humain cirrhotique en provenance de patients transplantées pour cirrhose biliaire.
la zonation métabolique persiste dans le module cirrhotique malgré les bouleversements architecturaux qui caractérisent la cirrhose. La zonation métabolisque s’établit entre le centre et la périphérie des nodules cirrhotiques, plaidant pour une organisation fonctionnelle du nodule comparable à celle de l’acinus. Néanmoins, la zonation diffère dans la cirrhose toxique (CC14) par rapport à une cirrhose biliaire (ligature des voies biliaires) : dans le premier modèle, le centre du nodule exprime une activité de type périportale, plaidant pour une direction centrifuge du flux sanguin dans les sinusoïdes du nodule. Dans la cirrhose biliaire expérimentale et humaine, l’activité de type périportale se retrouve en périphérie du nodule, plaidant pour une direction centripète du flux sanguin sinusoïdal. La perte de la zonation métabolique et de la sous-spécialisation hépatocytaire pourrait être un facteur déterminant de la décompensation fonctionnelle hépatique dans la cirrhose terminale

Hepatocytes differ in their composition and metabolism according to their own localization along the liver cell plate, close or distal to the portal tract. This heterigenous distribution of liver metabolism is called “liver metabolic zonation”, and seems to play a key role in achieving physiological concentrations of solutes and optimal composition of the blood leaving the organ. The zonal distribution of enzymes is a very efficient means to combine complementary metabolic pathways, acting sequentially along the liver cell plate. Antagonist metabolic processes occur in different hepatocytes. This liver cell heterogeneity may be partially pre-determined, according to the anatomy of the organ, and to the distribution of cellular receptors.
Beside this predetermination, the hepatocyte metabolism is also influenced by its microenvironment, which varies along the sinusoid due to uptake and release of substances by the liver cells themselves.
We have developed in our laboratory a technique of quantitative histochemistry, using microdensitometry to measure the activities of several enzymes in individual liver cells on frozen liver sections. This technique allows direct correlation of enzyme activities with adjacent histology. We choose to measure the activities of 5 different enzymes, representative of major metabolic pathways carbohydrate metabolism (glucose-6-phosphatase dehydrogenase, mitochondrial, more active in the perivenual zone), drug metabolism (NADPH dehydrogenase and alcohol dehydrogenase, microsome and cytosol, more active in the perivenular zone) and oxidative metabolism (succinate dehydrogenase, mitochondria, more active in the periportal zone). The technique of quantitative histochemisty was first validated in animals, and we thereafter demonstrated its applicability to human clinical biopsy samples.
In preliminary experiments, we demonstrated that the liver metabolic zonation described in animals is also observed in human liver. However, activities and “zonation” pattern was different in infants as compared with older children and adults, and we concluded that age differences could influence the zonation pattern observed. To investigate further this possibility, zonal changes of enzyme activities with age were systematically analyzed from birth in growing rats Periportal and perivenular activities of SDH and NADPH increased with age during the first postnatal weeks. The increase was more important for SDH in the periportal zone and for DN in the perivenular zone, so that the distribution gradient became more prominent with time, giving birth to the “adult” type of zonation pattern. G6P activity decreased after birth in the perivenular zone while increasing concomitantly in the periportal zone, this evolution being responsible for a maximal distribution gradient, with a ratio of periportal to perivenular activity of 2.27, at day 26. G6P activity decreased then slowly in both zones to reach lower adult values.
In these animals, marked changes in zonal enzyme activities were observed at the time of weaning, reflecting changes in nutriment intake from a high fat/low carbohydrate diet (maternal milk) to the laboratory diet rich in carbohydrate, lowering the need for glucose production.
Liver metabolic zonation incirrhosis:
Pathological conditions affecting the liver do not necessarily impair immediately the liver function itself, and cirrhosis may even develop without liver insufficiency. In view of the key role played by the metabolic zonation in maintaining optimal liver function, we hypothesized that a liver metabolic zonation should be preserved in compensated cirrhosis, despite the intense architectural changes observed in this condition. This would suggest adaptive changes of the metabolic zonation from an “acinar” zonation pattern towards a new zonation pattern and enzyme distribution across the cirrhotic nodule.
To answer this question, the zonal metabolism was investigated by quantitative histochemistry in various human and animal models of cirrhosis.
In a first experiment, cirrhosis of the liver was induced in growing rats by chronic administration of CC1 4 and phenobarbitone. Enzyme activities were deeply affected by this treatment, especially G6P and SDH in the perivenular zone. When cirrhosis had developed, treatment was discontinued, and enzyme activities returned to control values. However, a new pattern of metabolic zonation was observed in the cirrhotic nodules: the distribution gradient of activities was now established between the periphery and centre of the cirrhotic nodule. The “nodule periphery” zone displayed activities similar to the control perivenular zone, whilst activities at the nodule centre were similar to the control periportal activities. This zonation pattern may suggest that the blood flows in the nodule from the centre to the periphery.
Because in this model hepatocyte damage are mainly observed in the perivenular zone, we suggested that a different cirrhotic zonation pattern would be observed in a biliary cirrhosis, in which initial liver damages are found in the periportal zone.
To investigate this possibility, we developed and animal model of pediatric biliary cirrhosis: Bile duct ligation and excision was performed in growing rats, and zonal metabolism analyzes after the development of cirrhosis (mean of 35 days). A metabolic zonation persisted for all enzymes analyzed in this model. As hypothesized, the zonation pattern in this model was exactly the reserve as that observed in the CCl 4 model of cirrhosis. Enzyme activities displayed at the nodule periphery were similar to control periportal activities, whilst at the nodule centre, they corresponded to control perivenular activities.
To further confirm this finding in human pathology, we analyzed liver biopsy samples from children with extra-hepatic biliary atresia compensated cirrhosis, and compared the zonation observed to that of normal human liver obtained after graft reduction during transplantation. In these extra-hepatic biliary atresia livers, a similar zonation pattern was observed as in the animal model of bile duct ligation, with a periportal like activity at the nodule periphery and a perivenular like activity at the nodule centre.
We conclude from these studies that quantitative histochemistry is a reproducible means to investigate the metabolism of individual liver cells and to correlate directly enzyme activities with pathological damages. The liver metabolic zonation described in animals is also observed in human livers, and this physiological organisation plays a key role in adjusting the final composition of fluids leaving the liver. Ontogenic changes of metabolic zonation are observed in both animals and human, and these changes may affect differently activities of the same enzyme in different part of the liver acinus.
A metabolic zonation persists in liver cirrhosis, despite the intense architectural changes disrupting the acinus. In toxic cirrhosis, the metabolic zonation is established between the centre and periphery a perivenular like activity. A mirror image of metabolic zonation is observed in animal and human biliary cirrhosis, reflecting a different type of injury which in these models interest mainly the periportal area.
Study of liver metabolic zonation may therefore be a useful tool to determine the site of the initial liver damage in cryptogenic cirrhosis. Loss of metabolic zonation in cirrhosis may e an important step towards the development of functional impairment, by disrupting the sequential complementary metabolic activities along the liver cell plate