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

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

Data on Lipocalin 2 and phosphatidylinositol 3-kinase signaling in a methionine- and choline-deficient model of non-alcoholic steatohepatitis

The data presented in this brief report support the research article “Altered mitochondrial and peroxisomal integrity in lipocalin-2-deficient mice with hepatic steatosis” [1, doi: 10.1016/j.bbadis.2017.04.006]. We tested whether the absence of Lipocalin-2 (LCN2) could dysregulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K-PKB) pathway and hepatic homeostasis in Non-Alcoholic-Steatohepatitis (NASH). The article highlights the role of LCN2 in hepatic homeostasis. Keywords: Lipocalin 2, Fat, MCD, NASH, PIP3, Oxidative stress, Mitochondri

Lipocalin 2 (LCN2) Expression in Hepatic Malfunction and Therapy

Lipocalin 2 (LCN2) is a secreted protein that belongs to the Lipocalins, a group of transporters of small lipophilic molecules such as steroids, lipopolysaccharides, iron and fatty acids in circulation. Two decades after its discovery and after a high variety of published findings, LCN2’s altered expression has been assigned to critical roles in several pathological organ conditions, including liver injury and steatosis, renal damage, brain injury, cardiomyopathies, muscle-skeletal disorders, lung infection and cancer in several organs. The significance of this 25-kDa lipocalin molecule has been impressively increased during the last years. Data from several studies indicate the role of LCN2 in physiological conditions as well as in response to cellular stress and injury. LCN2 in the liver shows a protective role in acute and chronic injury models where its expression is highly elevated. Moreover, LCN2 expression is being considered as a potential strong biomarker for pathological conditions, including rheumatic diseases, cancer in human organs, hepatic steatosis, hepatic damage and inflammation. In this review, we summarize experimental and clinical findings linking LCN2 to the pathogenesis of liver disease

Perilipin 5 and Lipocalin 2 Expression in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly cancers worldwide. Therefore, current global research focuses on molecular tools for early diagnosis of HCC, which can lead to effective treatment at an early stage. Perilipin 5 (PLIN5) has been studied as one of the main proteins of the perilipin family, whose role is to maintain lipid homeostasis by inhibiting lipolysis. In this study, we show for the first time that PLIN5 is strongly expressed in tumors of human patients with HCC as well as in mouse livers, in which HCC was genetically or experimentally induced by treatment with the genotoxic agent diethylnitrosamine. Moreover, the secreted acute phase glycoprotein Lipocalin 2 (LCN2) established as a biomarker of acute kidney injury, is also proven to indicate liver injury with upregulated expression in numerous cases of hepatic damage, including steatohepatitis. LCN2 has been studied in various cancers, and it has been assigned roles in multiple cellular processes such as the suppression of the invasion of HCC cells and their metastatic abilities. The presence of this protein in blood and urine, in combination with the presence of α -Fetoprotein (AFP), is hypothesized to serve as a biomarker of early stages of HCC. In the current study, we show in humans and mice that LCN2 is secreted into the serum from liver cancer tissue. We also show that AFP-positive hepatocytes represent the main source for the massive expression of LCN2 in tumoral tissue. Thus, the strong presence of PLIN5 and LCN2 in HCC and understanding their roles could establish them as markers for diagnosis or as treatment targets against HCC

Targeting Activated Hepatic Stellate Cells Using Collagen-Binding Chitosan Nanoparticles for siRNA Delivery to Fibrotic Livers

Activated hepatic stellate cells (aHSCs) are the main orchestrators of the fibrotic cascade in inflamed livers, with transforming growth factor-beta (TGF-β) being the most potent pro-fibrotic cytokine. Hence, aHSCs serve as interesting therapeutic targets. However, drug delivery to aHSCs is hindered by excessive collagen deposition in the extracellular matrix (ECM) and capillarization of liver sinusoids. Chitosan-nanoparticles (CS-NPs) show intrinsic affinity for collagen, holding potential for drug delivery to fibrotic livers. Here, we employed CS-NPs for anti-TGF-β siRNA delivery, promoting delivery into aHSCs via modification with platelet-derived growth factor receptor-beta binding peptides. In-vitro experiments using aHSCs demonstrated the association of unmodified CS-NPs to the collagen-rich ECM, with reduced intracellular accumulation. Peptide-modified CS-NPs showed a higher propensity to localize intracellularly; however, this was only the case upon ECM-collagen reduction via collagenase treatment. Peptide-modified CS-NPs were more potent than unmodified CS-NPs in reducing TGF-β expression, implying that while collagen binding promotes liver accumulation, it hinders cell-specific siRNA delivery. In-vivo, CS-NPs successfully accumulated in fibrotic livers via collagen binding. Similar to in-vitro findings, when mice were pretreated with collagenase-loaded CS-NPs, the accumulation of peptide-modified NPs increased. Our findings demonstrate the usefulness of NPs modification with targeting ligands and collagenase treatment for aHSCs targeting and highlight the importance of chitosan–collagen binding in drug delivery to fibrotic diseases