Retinyl ester hydrolase(s) of hepatic stellate cells

In mammals, vitamin A (retinol and metabolites) is an essential, fat soluble micronutrient. It exerts its biological activity through two major metabolites: 11-cis-retinaldehyde and all-trans- /9-cis-retinoic acid. Retinaldehyde functions as hn acceptor in the visual cycle. Retinoic acids are ligands for specific nuclear receptors which regulate gene expression and are inevitable for growth, development, and the maintenance of life. Mammals take up vitamin A from the diet and store it in large quantities as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). The mobilization of RE stores requires lipases which hydrolyze REs to liberate retinol which is then secreted into the circulation. Yet, so far no enzyme has been identified which could be responsible for the mobilization of these stores. HSC RE stores have also been recognized in a pathological process, the onset of liver fibrosis. After liver injury HSCs undergo "activation" and transform into myofibroblast-like cells. These cells then form excessive extracellular matrix proteins and, interestingly, loose entirely their RE stores. To date it is unknown why in early phase of fibrosis RE stores of HSCs are depleted and which protein(s) are involved in this process. The objective of this study is to identify RE hydrolase(s) in HSCs. Since REs are stored in lipid droplets of HSCs we aim for a straight forward approach. First, we will isolate lipid droplets from the stellate cell line HSC-T6. Then the lipid droplet proteome will be determined by mass spectrometry, bioinformatically analyzed, and screened for potential RE hydrolases. This will allow us to refine the isolation and analyses procedure with samples which are easily available. Then we will isolate primary HSCs from mouse liver, purify lipid droplets, and determine the proteome. Furthermore, since activated HSCs may overexpress RE hydrolases we will also isolate HSC from mice which had been treated with carbon tetrachloride to induce HSC activation. Alternatively, we will activate HSCs in vitro by cultivation. Then from activated HSCs lipid droplets will be purified, the proteome determined, and bioinformatically analyzed. All identified potential RE hydrolases will be cloned and biochemically characterized in in vitro and cell experiments. It is expected that RE hydrolases are capable to cleave lipid droplet associated RE and to mobilizing RE stores of living cells. In summary, the anticipated work seeks to identify enzymes involved in the mobilization of HSC RE stores.

In mammals, vitamin A is stored in large quantities in specialized liver cells, the hepatic stellate cells. These hepatic vitamin A stores consist of retinyl esters, which are present in cytosolic lipid droplets. To date it is unknown which enzymes (so-called retinyl ester hydrolases) are responsible for the mobilization of these vitamin A stores. The aim of this project was to identify retinyl ester hydrolases from hepatic stellate cells. For this purpose, a direct approach was chosen, namely the proteomic analysis of lipid droplet proteins from hepatic stellate cells. The investigation of lipid droplet proteome resulted in the identification of the known triglyceride hydrolase of the adipose tissue, the adipocyte triglyceride lipase (ATGL), as a lipid droplet protein. Within the framework of this project we were able to show that ATGL is expressed in primary, hepatic stellate cells, capable of hydrolyzing retinyl esters. Primary hepatic stellate cells from mice lacking the enzyme ATGL (from so-called ATGL-ko mice) exhibited increased retinyl ester content. Furthermore, the mobilization of retinyl esters of primary hepatic stellate cells derived from ATGL-ko mice were delayed. Interestingly, however, ATGL-ko mice exhibited normal hepatic retinyl ester content. Together, these observations suggest that ATGL is involved in the mobilization of retinyl esters from hepatic stellate cells, but additional enzymes are involved in this process. Within the scope of this project, we identified lysosomal acid lipase as retinyl ester hydrolase. This enzyme is known to be responsible for the hydrolysis of neutral lipids, in particular cholesterol esters, of the lysosomes. Our investigations showed that lysosomal acid lipase is essential for the hydrolysis of retinyl esters in the lysosome. Mice lacking this enzyme accumulated retinyl esters mainly in the intestine. Furthermore, these mice exhibited markedly impaired nutritional availability of vitamin A. Together, results indicate that lysosomal acid lipase plays an important role in retinoid homeostasis. At the end of the project we initiated a second proteomic analysis of lipid droplets of the human hepatic stellate cell-line LX2. We, once again, identified ATGL as lipid droplet protein of human stellate cells. It can be assumed that the human ATGL, similar as its murine homologue, hydrolyzes retinyl esters and plays a role in the mobilization of vitamin A stores of hepatic stellate cells. Within the scope of this project, a number of collaborations with national and international research groups in the field of lipid metabolism have been carried out. In total 13 scientific papers, one methodological article and two review articles were published.