Zones of the liver accumulate fat at different rates

A fatty liver maintains the ability to self-regenerate for a long time

LiSyM-Pillar I focuses its research effort on the early stages of metabolic liver disease. The primary research aim is to understand how non-alcoholic fatty liver disease (NAFLD) is initiated and leads to inflammation in the organ. The onset of inflammation marks the transition to pathological non-alcoholic steatohepatitis (NASH).

Non-alcoholic Fatty Liver Disease, NAFLD

Experts diagnose NAFLD when more than five percent of hepatocytes consist have stored fat and alcohol does not play a role. Estimates indicate between 20 to 30 percent of the population in European is affected. The major causes are an unfavorable genetic disposition and an unhealthy lifestyle with poor diet and lack of exercise. Weight loss can reverse NAFLD in the early stage. Failing that, it will progress gradually. In up to 20 percent of those affected, the non-inflammatory form (simple or bland fatty liver) will graduate to an inflammatory form (NASH: non-alcoholic steatohepatitis). By this stage the liver cell function is in part already damaged. Between 10 to 20 percent of these patients develop severe fibrosis, some a pronounced cirrhosis.

Pillar I has generated many new insights into the metabolism and signaling pathways of the liver, which have led to new approaches for diagnosis and treatment. Researchers have already patented several tests and therapeutic options. Furthermore, Pillar I has developed a new generation of computer models which can predict how well each individual patient’s metabolism can process certain drugs and other substances. Moreover, researchers were able to reconstruct three-dimensional anatomical microstructures of liver lobules and also map the spatial differences for a complete set of active genes and epigenetic changes in the liver. This means Pillar I has successfully provided the foundation for diagnostic and therapeutic innovations.

Epigenetics – molecular factors that influence gene activity without affecting the genetic code itself. Known examples include DNA methylation processes, which lead to poorer binding of transcription factors and make it more difficult for them to read their target genes.
Epigenome – the entire record of a cell’s epigenetic methylation processes.
Acinus, liver acinus – the smallest functional unit of the liver.
Hepatocytes – epithelial liver cells which make up approx. 80 % of the liver volume. They carry out most of the organ’s metabolic tasks.
Messenger RNA – messenger RNAs are copies of base sequence of genes, which ultimately make up specific proteins. The presence of a specific messenger RNA reveals the activity of the corresponding gene.
Transcriptome – set of all messenger RNAs produced by a cell at any one time.
Lipidome – entire lipid structure within a cell.

Networks cause liver zonation on multiple levels

At the onset of NAFLD, some hepatocytes accumulate fat quicker than others. These cells fulfil a number of functions in the healthy liver. “Their primary metabolic tasks vary according to the hepatocyte’s position in the acinus,” Dr Mario Brosch explains. He is Head of the Laboratory in the research group led by Prof. Jochen Hampe, Pillar I Coordinator, at the Technical University and University Hospital, Dresden. An acinus (plural: acini) is the smallest functional unit of the liver. Experts differentiate several zones in the acinus: periportal zones are situated close to the portal vein, whereas the pericentral zones are close to a central vein.

What actually distinguishes hepatocytes from the different zones? This question was investigated in an extensive collaborative effort among Pillar I researchers led by Prof. Hampe. Liver tissue samples from people of average weight as well as obese people without NAFLD, with NAFLD and with early NASH formed the basis of the study. Prof. Clemens Schafmayer’s team at the Kiel Clinic for General Surgery and Thoracic Surgery (UKSH) froze the tissue samples immediately after surgical removal and collected them. From these samples, scientists then isolated 100 to 200 hepatocytes with different aciniar positions. “Today, with laser capture micro-dissection, this procedure is much more accurate than in the past,” Brosch says. Scientists thoroughly analyzed the epigenome – that is all epigenetic markers. They determined the transcriptome – that is all messenger RNAs as copies of the active genes - and the frequency of transcription factors known to be involved in the accumulation of fat in liver cells.

In the transcriptome, there are hundreds of messenger RNA frequency gradients: their abundance increases or decreases continuously depending on their position. Since the main task of the hepatocytes changes zonally, experts had expected these gradients. This is well known in mice and rats. However, they were surprised to discover that portal-central methylation gradients also exist in the epigenome. They affect more than 17 000 hepatocyte DNA sites. Furthermore, methylations occurring at the transcription factor binding sites are reflected in the expression of the corresponding gene: those with a high degree of methylation have only few messenger RNAs whereas those with no methylation have many. In contrast, the frequency of corresponding transcription factors is almost the same everywhere. The overall pattern of parameters investigated proved almost identical throughout all stages of NAFLD covered.

A model scientifically more valuable than gold dust

The cooperation partners published the resulting spatial epigenomic map of the human liver in the scientific journal Nature Communications. Brosch highlights a key finding: “Zonation is largely retained in the epigenome and the transcriptome when fat accumulates in the liver up until the onset of non-alcoholic steatohepatitis.” Healthy cells and newly diseased cells have the same capabilities. As a result, the liver can recover fully from early-stage NAFLD if the correct measures are taken. However, the study points to subtle differences in the regeneration of a healthy and a diseased liver. Furthermore, it revealed clear epigenetic zonation of the liver, as shown by the gradients in the transcriptome. Evidently, a complex network with multiple levels supports the liver zones and maintains them. The data show that a few specific genes play a key role in this. The map also indicates when pharmaceutical substances preferentially take effect in the pericentral hepatocytes, i.e. where NAFLD begins.

Prof. Edda Klipp, Head of Theoretical Biophysics at the Humboldt University, Berlin, will incorporate these new data on epigenetic zonation into her model of the metabolism of the human liver. Dr Lutz Brusch, research group leader at the Centre for Information and High Performance Computing (ZIH) in Dresden, contributes his own model of zonal bile flows. Prof. Lars Kaderli, Head of Bioinformatics and Systems Biology at the University of Greifswald, supports the group with his expertise in the analysis and modeling of complex genomic networks. Dr. Andrej Shevchenko, research group leader at the Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, undertakes lipidome zonation analysis. Prof. Marino Zerial and his colleagues, at the same Max Planck Institute, have been analyzing hepatocytes from the same samples using 3D microscopy. Their results will also be incorporated in the large collective liver model, says Mario Brosch. “Our data will be as valuable as gold dust because a network of highly competent scientists are contributing their expertise on the same set of patient samples.”

“This model will comprise more data and cover more research disciplines than any before it,” Pillar I Coordinator Prof. Hampe agrees. The spatial epigenome map alone has led to many new impulses. The overall model will act as a catalyst in diagnostics, therapy and research. The question that remains: When does NASH become irreversible – how long do hepatocytes retain the potential for complete regeneration? Hampe indicates one of the next steps: “Therefore we will also analyze samples from the advanced stages.” He expressly praises how well the institutes and scientific disciplines in Pillar I are connected. “From the outset, the joint organization of our research has been excellent,” Hampe explains. “The collaboration among the participants could not be better or more successful!”

Back to the 4 pillars