Lipocalin 2, an essential multi-regulator of hepatic homeostasis during a Methionine Choline deficient diet-induced non-alcoholic steatohepatitis in mice

Lipocalin-2 (LCN2) is a 25-kDa secretory protein with great importance as a sensitive and specific screening and diagnostic biomarker for hepatic inflammation, acute hepatic injury, hepatic cancer, radiation-induced early phase hepatic damage and lipid abnormalities. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis as well as its actual role in the event of inflammation. The aim of this study was to prospectively evaluate the LCN2 role in the ‘two-hit’ development of Non-alcoholic steatohepatitis (NASH) meaning the steatosis and the injury, observed by the appearance of inflammation. NASH is a condition where fatty liver is accompanied with inflammation. The objective of this study was to determine in wild type (WT) and Lcn2-deficient (Lcn2−/−) mice the possible differences in their biological reactions after a Methionine and Choline deficient diet (MCD), a nutritional model used to induce the pathological condition of NASH. The current study compares initially the intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins known to regulate cellular lipid metabolism. Lcn2−/− mice fed with MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. In in vitro experimentation the restoration of LCN2 in Lcn2−/− primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. The other section of this study targeted the role of LCN2 specifically in the inflammation process accompanying hepatic injury after administration of the MCD diet. Therefore, the hepatic LCN2 and inflammatory cytokines’ interleukin- 6 and interleukin-1β (IL-6, IL-1β), tumor necrosis factor α (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), and chemokine (C-C motif) receptor 2 (CCR2) expression was compared between the two genotypes. The expression of the activated forms of the Signal Transducers and Activators of Transcription (STAT1 and STAT3) were tested as well. Additionally, histology inflammation score and immunohistochemistry stainings with leukocytes and neutrophil granulocyte markers were performed. Both genotypes had inflammation sites as a result of the MCD diet, however the WT mice recruited more inflammatory cells than the Lcn2−/− accompanied with a strong upregulation of LCN2. Moreover, the WT mice fed with MCD diet, presented a remarkably stronger expression of phosphorylated forms of STAT1 and STAT3. In conclusion this study shows that LCN2 is a protein of high significance with multiple functions in MCD-induced NASH in mice. The presented findings indicate that first of all, LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression while a second significant role in inflammation is that its upregulation serves as a ‘HELP-ME’ signal while inflammation appears. The upregulation of the LCN2-‘HELP-ME’-factor, enhances the recruitment of neutrophil granulocytes and leukocytes towards resolution of the inflammation protecting the liver from progression to further hepatic damage agreeing to previous reports of LCN2’s hepato-protective role in liver injury models. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with inflammation and elevated fat accumulation.