4. LCN2 in NAFLD Pathophysiology
LCN2 has proven to be a good target to study as a possible biomarker for NAFLD and subsequent liver pathology (). It has been proven by independent research teams that its high levels can indicate liver damage 
. Since it can be detected in bodily fluids such as blood and urine, it can be routinely used as a part of laboratory tests.
Figure 2. Lipocalin-2 (LCN2) as a component in non-alcoholic steatohepatitis (NASH) pathology and diagnostics. NASH patients show elevated levels of LCN2 in urine, serum, and liver tissue, which makes it a potential biomarker for NASH. It is able to discriminate between simple steatosis (SS) and NASH, while the level of LCN2 correlates with inflammation (e.g., increase of CRP), insulin resistance, and fibrosis. It is induced by proinflammatory cytokines, high fat diet, glucose, and fructose overconsumption. Overexpression experiments have implicated LCN2 in the enhancement of lipolysis, fatty acid oxidation, mitochondrial activity, and immune cells crosstalk. In addition, LCN2 upregulation is associated with reduced de novo lipogenesis. All evidence points out that its role is hepatoprotective. However, the exact mechanisms are ought to be found.
To prove it, one study examined the levels of circulating LCN2 as well as its gene expression in obese women with NAFLD (with either NASH or simple steatosis) and normal liver 
. The research concluded that both gene expression and protein levels were upregulated in obese women with NAFLD. However, gene expression correlated with simple steatosis while protein levels correlated with NASH. The same study showed that treatment with proinflammatory TNF-α, IL6, and resistin causes the upregulation of LCN2 in HepG2 cells 
. Therefore, LCN2 is considered to be a liver’s protective response to inflammation. Another study, done on Chinese subjects, proved that LCN2 serum levels are elevated in patients with NAFLD as opposed to the control and that they highly correlate with both inflammation (C-reactive protein) and insulin resistance 
. The study of Milner and colleagues showed that LCN2 levels correlated with the degree of liver inflammation and the stage of hepatic fibrosis as well as insulin resistance 
. The diagnostic value of serum LCN2 was estimated by Xu and colleagues. Around 500 patients with either NAFLD (steatosis: n
= 83, NASH: n
= 277) or alcoholic fatty liver disease, together with a healthy control, were recruited for the study. The study showed that three variables, including serum LCN2 level, body mass index (BMI), and low-density lipoprotein (LDL) cholesterol, are positively correlated with NAFLD. The predicted value of LCN2 was calculated by multiple regression analysis, and the predicted value was applied to calculate the cut-off value using receiver operating characteristic (ROC) analysis. The area under the ROC curve of serum LCN2 was 0.987 with a specificity of 100% and a sensitivity of 93.5% for NASH diagnosis, and 0.977 with almost the same specificity and sensitivity for steatosis. Both cases showed a low rate of a false positive and a false negative. These data confirmed high diagnostic value of serum LCN2 in NASH and steatosis. However, although the serum LCN2 levels were compared between NAFL and NASH groups, ROC curve analysis was unable to establish an optimal cut-off value of serum LCN2 levels for distinguishing NASH from NAFL subgroups 
. While all these studies portray LCN2 as a potential biomarker, they have all failed to prove that LCN2 can serve as a tool to differentiate the particular stages of NAFLD. Nonetheless, it would be of high importance to pinpoint its role in the pathogenesis. While, as mentioned above, human studies focus on LCN2 as a biomarker, there had been excessive studies to address LCN2’s role in NAFLD pathogenesis performed both in in vitro and in vivo models.
For instance, classical approach of LCN2
overexpression in hepatocytes done by Xu and colleagues resulted in lipolysis and fatty acid oxidation. Through this study, it was demonstrated that LCN2
overexpression prevents de novo
lipogenesis, lipid peroxidation, and apoptosis, thus, preventing steatohepatitis 
. This is one of the many studies that imply that LCN2
regulates liver lipid homeostasis.
A study done by Semba and colleagues aimed to detect if LCN2 can be a factor in the differentiation of simple steatosis and NASH 
. For that purpose, DNA microarray analysis of the liver transcriptomes, RT-qPCR, and immunohistochemistry were done on murine models of simple steatosis (dd Shionogi mice) and NASH (fatty liver Shionogi mice). This study showed that LCN2 is overexpressed in mice with NASH, together with chemokines CXCL1 and CXCL9, while their overexpression was missed in mice with steatosis. What seems to be most interesting, all three proteins have specific localization, likely correlating with their role. LCN2 seems to be localized in hepatocytes and correlates to inflammatory cell clusters due to its implication in neutrophil signaling.
Another study from our group showed a hepatoprotective role of LCN2
both in vivo
and in vitro
. We fed wild type (WT) and Lcn2
) mice with a methionine and choline deficient (MCD) diet as a nutritional model of NASH. We found that LCN2 maintains lipid homeostasis through, among others, the induction of proteins important for lipid droplet formation named Perilipin 5 (PLIN5). It became clear that depletion of LCN2 or PLIN5 prevented normal intracellular lipid droplet formation in murine models as well as cell lines. The homeostasis was restored after transfection or adenoviral vector infection, which only confirmed the importance of LCN2 in liver protection 
. In another previous investigation, we also performed a comparative analysis of Lcn2−/−
and WT, high fat diet-fed mice. We managed to detect the proteins BRIT1/MCPH1, HMGB1, FABP5, and PLIN5 as important factors in LCN2-mediated lipid homeostasis. Besides that, it was shown that LCN2 increased mitochondrial activity, intra-mitochondrial chelatable iron pool, and a peroxisome number, suggesting that it is possible for LCN2 to act as a sensor that measures fat content and adjusts homeostasis by modifying peroxisome numbers and/or mitochondrial activity 
Another role has been assigned to LCN2
by Ye and colleagues. In their study, they induced NASH by either a high fat, high cholesterol (HFHC) diet or an MCD diet in mice. They could show how LCN2
mediates NASH by promoting neutrophil-macrophage crosstalk via the induction of CXCR2 
. It was noticed that the infiltration of neutrophils and macrophages was substantially attenuated by genetic depletion of Lcn2
, but was augmented by chronic infusion of recombinant LCN2, thus, promoting inflammation. Mice lacking CXCR2 are resistant to LCN2-evoked liver inflammation. Even though the mechanism by which LCN2 may upregulate CXCR2 is still not known, the authors believe that it could be through an NF-κB-dependent mechanism.
As mentioned above, major sources of NAFLD pathogenesis seem to be glucose and fructose lipogenesis-derived FFA. However, a series of recent studies have indicated that sugars can induce NAFLD by means independent of de novo
lipogenesis. A study conducted in our group showed how excess fructose leads to hepatic steatosis. However, in this context, fructose appears to directly affect liver homeostasis, thereby, manipulating fat metabolism 
. Fructose might disturb liver homeostasis by promoting lipid uptake into the liver, while LCN2 counteracts lipid uptake. The same study showed there are potential differences between the sexes in LCN2-mediated lipid metabolism. This finding is of the utmost importance because it also shows a potential influence of oestrogens on LCN2-mediated lipid homeostasis. This goes in agreement with another study made by Alwash and colleagues who fed rats a fructose high diet that provoked a gradual increase of the LCN2 level over the course of eight weeks 
. LCN2 levels seemed to correlate both with increased indicators of oxidative stress and mitochondrial dysfunction. Their model suggests a hepatoprotective role of LCN2 since its expression in a rat model seems to be provoked by endotoxins and inflammatory cytokines, as mentioned by many more 
. Although these studies have managed to portray LCN2 as a hepatoprotective element in the liver, they have not managed to provide an exact mechanism, or mechanisms of its action regarding NAFLD pathology. As a matter of fact, they have not even explained whether LCN2 is a mere consequence of the pathology or its driver.