Dual role of Rab18 in liver cells

The protein Rab18 fulfils several tasks in the metabolism of lipid droplets

Systems biologist, Dr. Nachiket Vartak, LiSyM Junior Group Leader at the Leibniz Research Centre for Working Environment and Human Factors (IfADo), in Dortmund, has discovered that in hepatocytes, the protein Rab18 regulates lipid metabolism by controlling lipid droplets. These proteins play an important role in non-alcoholic fatty liver disease (NAFLD), a widespread condition that can lead to serious health problems. Vartak’s research results, primarily drawn from modern microscopy techniques, should improve early detection, prognosis and therapeutic treatment options in the future.

Rab18 – a protein from the large family of Rab GTPases.

Rab proteins are bound to intracellular membranes where they aid the formation, identification, transportation and fusion of vesicles.
Hepatocytes – epithelial cells of the liver comprise about 80% of the liver volume. They are responsible for most of the organ’s metabolic functions.
Lipid droplets (LD) – intracellular fat deposits enclosed by a membrane. Almost all eukaryotic cells produce them to store fat. The organelles play a role in lipometabolism.
Endoplasmic reticulum – intracellular membrane system in which and on which many metabolic processes occur. Lipid droplets (LD) are also formed here.

“According to studies, sixty per cent of Europeans have fat deposits in the liver, and NAFLD is already prevalent in more than twenty percent,” Vartak says. These deposits can lead to NAFLD. The condition often reveals itself very late since years can pass without any apparent symptoms. “By the time it becomes evident to us, it is already irreversible,” he explains. NAFLD can lead to serious liver damage: at worst, it can cause cirrhosis.

Non-alcoholic fatty liver disease, NAFLD

Experts speak of NAFLD when more than five per cent of hepatocytes have fat deposits, and when alcohol consumption does not play a role. Estimates of frequency of incidence in Europe lie between 20 and 30 per cent. The most relevant factors are an unfavourable genetic predisposition and a lifestyle characterised by poor diet and physical inactivity. Weight reduction can cure NAFLD in its early stages. If unnoticed, the disease will progress gradually. In up to 20% of those affected, the non-inflammatory form (i.e. simple or bland fatty liver) progresses to an inflammatory form (i.e. non-alcoholic steatohepatitis, or NASH). At this point, the function of hepatocytes is partially impaired. Between 10 and 20 per cent of those affected develop a more severe fibrosis, some a pronounced cirrhosis.

“We do not know what triggers the disease,” Vartak explains. This is another reason why early detection is difficult. The first visible indication of NAFLD is a high lipid droplet count. Large, balloon-like lipid droplets form in the diseased liver and impact upon the various hepatocyte functions. “They squash everything inside the liver cells!” The 35-year-old adds: “We have evidence suggesting that Rab18 controls the formation and size of the lipid droplets.”

The GTPase Rab18 promotes the formation of lipid droplets in hepatocytes. A high lipid droplet count is an early indicator of non-alcoholic fatty liver disease. Yet it seems Rab18 simultaneously hinders the formation of large lipid bodies, which are more dangerous than small ones.

Free fatty acids are toxic to hepatocytes, and so the hepatocytes restructure and store them safely in lipid droplets. This takes place on the membrane of the endoplasmic reticulum. Here a fat bubble forms, which expands as long as Rab18 is bound to its membrane. “Then Rab 18 cuts the bubble off and a lipid droplet is formed,” Vartak explains. “With Rab18, hepatocytes form more lipid droplets.” However, something else also happens: While Rab18 is active on a new lipid droplet, it seems to also cut the lipid droplet membrane. Consequently, instead of dangerous large lipid droplets being formed, many little ones are produced. As long as Rab18 performs this dual function correctly, there is neither a positive nor a negative impact on the liver.

“We believe the activity of Rab18 and its localisation in hepatocytes to be precisely balanced,” says Vartak. He hopes to discover details of how and when this balance becomes disrupted in NAFLD, leading to aberrant lipid droplet dynamics. To achieve this, Vartak correlates data from conventional molecular biology and biochemistry with data from modern microscopy e.g. intravital correlative microscopy. This approach combines the advantages of several processes. First, the intravital method depicts the dynamics of biological processes in a living organism. Then computers calibrate and process the position of the area of interest, allowing the results to be amalgamated with those from a second process, such as coherent anti-Stokes Raman spectroscopy (CARS) imaging. In the case of the CARS microscopy, two lasers render the molecules visible by their contrasting molecular vibrations. The results reveal detailed chemical information on structures in cells – a fingerprint of chemical composition – for example, on fat distribution.

The next step for Vartak and his colleagues is to analyse liver biopsy samples from adolescent patients with fatty liver disease. Does the cellular localisation of Rab18 correlate with the size and number of lipid droplets? Which distribution is good, which is bad? The Junior Group Leader hopes to gain more information about the onset and severity of NAFLD from the biopsy samples. “We can also control the localisation of Rab18.” In liver cells, specific fats determine whether Rab18 attaches to the endoplasmic reticulum or to lipid droplets. Furthermore, the systems biologist wants to test the extent to which other pharmacological substances are able to control the lipid accumulation and subsequent disruption of hepatocyte function, and hence prevent NAFLD. “We have already identified a few promising candidates.”

How do bile acids exit the liver?

Vartak’s second LiSyM project concerns bile acids. Choleostatic liver diseases such as gallstones cause a build-up of bile fluid in the liver. Furthermore, bile acids are toxic – though no one knows exactly why. Vartak wants to learn more about this as well as about how the acids produced in the liver find their way out. He has already discovered that “in most areas of the liver, bile is cleared through diffusion.” Now he is searching for ways to enhance the clearance of bile acids.

Vartak, a microscopy expert who studied life sciences, biochemistry and biotechnology in Mumbai, knows what he wants to achieve with research in the long-term. “Some equipment for certain liver tests fills entire rooms,” he says. After obtaining his PhD. at the Max Planck Institute for Molecular Physiology in Dortmund and continuing his research there for some time as a postdoctoral researcher, Vartak joined IfADo in 2014. Nachiket Vartak wants to contribute to significantly reducing the size of electronic liver testing equipment in future: “Patients should be able to monitor their liver at home with a small device – such as those for testing blood sugar levels today.”

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