|dc.description.abstracteng||The liver is the vital organ for fat and carbohydrate metabolism as well as detoxification and excretion of metabolites. Any functional impairment will thus affect the whole organism and is absolutely undesirable. Nonalcoholic fatty liver disease (NAFLD) is a multifactorial worldwide disease which coincides with metabolic syndrome. The spectrum of NAFLD ranges from simple steatosis to nonalcoholic steatohepatitis (NASH), with consecutive fibrosis, cirrhosis, and might ultimately lead to hepatocellular carcinoma. The prevalence of NAFLD is estimated to be 20-30% of the general population in Europe, and it is paralleling with the increase in fructose consumption. Fructose, a monosaccharide, is naturally present in fruit, and it can be metabolized into glucose and lipid within the liver. Industrially, fructose is used in soft drinks as a sweetener, and it is integrated in pre-packed food as an additive. Fructose is considered a risk factor for NAFLD. Previous reports found an upregulation of liver lipocalin-2 (LCN2) expression in inflammation and metabolic syndrome. LCN2 is a 25-kDa secretory glycoprotein, found abundantly in tissues that are exposed to microorganisms. It can bind a variety of lipophilic substances.
Study motivations. Although ultrasonography, CT scan, and MRI are reliable tools for detecting a fatty liver, they are inadequate to classify the disease. Instead, a liver biopsy is needed to distinguish between simple steatosis and NASH. However, this is an invasive procedure which carries the risk of bleeding and injury to gallbladder, lungs and kidney. Thus, serum biomarkers would be most welcome. In addition, the pathogenesis of NAFLD is still poorly defined. The available animal models, such as Lcn2-/-, db/db, ob/ob, and ZF fa/fa do not mimic the physiological and/or histological features of NAFLD seen in humans. Since fructose is included in commercial/fast food as well as juice beverages and as it has been proposed to induce the most NAFLD manifestations noticed in humans; this study selected fructose as an inducer for metabolic syndrome and NAFLD.
This study aimed to establish diet-induced fatty liver models in non-genetically-modified rats, to compare the expressional changes of inflammatory and metabolic parameters and LCN2 in these models, to explore whether serum LCN2 is a diagnostic indicator for NASH, and to investigate the potential mechanism(s) that induce(s) hepatic LCN2 expression.
Male Sprague-Dawley rats were randomly assigned (n= 4 per group/ time point) as follows: chow pellets (control (Co)), liquid Lieber-DeCarli (LDC, high fat), and LDC + high (70% of the total calorie) fructose (L-HFr) diet. After feeding for 4 or 8 wks; the animals were deprived of any food 10 h before sacrifice.
Blood from the vena cava and liver samples were harvested. Blood samples were examined for fasting glucose, lipid profile, serum transaminases activities, and leptin concentration. Histochemical studies for lipid deposition, inflammation, and fibrosis were performed on the liver sections. In addition, the hepatic transcript of Lcn2, the inflammatory mediators Il-8, Mcp-1 (Ccl2), alpha-2 macroglobulin (α2m), Tnf-α, Inos, Tlr4, and fructose transporter (Glut5) and leptin receptor (Lep-r) were evaluated by qRT-PCR. Furthermore, ELISA and Western blots were performed to assess LCN2 levels in serum, while the localization of LCN2 in liver tissue was detected by double-immunofluorescence staining. Western immunoblots for hepatic LCN2, CD14, IκB1α, pMAPK, casp 9, Cyt c, 4-hydroxynonenal (4-HNE adducts): by-products of lipid peroxidation, GRP78: an ER chaperone, and PGC-1α: a mitochondrial-biogenic protein, were carried out. In vitro experiments were done as well to study Lcn2 transcription. Primary hepatocytes (HCs) were isolated from control rats and cultured in M199 media supplemented with either 20 mM glucose or fructose for 0, 3, 6, 12, and 24 h.
Hallmarks of the metabolic syndrome, including a significant elevation of fasting blood glucose and triglycerides as well as a decrease in HDL-cholesterol levels, were observed in the LHFr group. The activities of AST and ALT were augmented in L-HFr fed rats. Concurrently, feeding with L-HFr augmented fasting leptin levels by about 3-fold vs. Co (P<0.001). Histologically, hepatic simple steatosis was featured in the LDC group. Parallel to the metabolic syndrome, the L-HFr group evidenced NASH criteria, such as inflammation (grade 2) and mild fibrosis (stage 2) in the portal triads, and hepatocellular ballooning in the centrilobular areas. Hepatic Glut5 and Lep-r expressions were substantially upregulated in the L-HFr group. While most tested biochemical parameters under the LDC regimen were slightly higher than in control rats, a significant increase in the plasma triglyceride level was seen in this group at wk 4.
In the liver, specific transcripts of Mcp-1, α2-m, and Il-8 were increased in the L-HFr group at both time points (P<0.001), whereas the transcription of Tlr4, Inos, and Tnf-α was significantly enhanced at wk 4 and then dropped to the control level at wk 8. In contrast, no significant change was observed in the LDC group. Interestingly, of the studied parameters, hepatic transcription of Lcn2 mRNA was the most pronounced (90- and 507-fold higher in the L-HFr rats vs. control at wk 4 and 8, respectively (P<0.001)). These data were further validated by Western blots. Of note, while hepatic LCN2 expression reached its maximum at wk 8, the expression of inflammatory mediators in the liver decreased to control levels pointing to a possible hepatoprotective effect of LCN2. Likewise, a significant elevation of systemic LCN2 levels was observed in the L-HFr regimen. In contrast to the mild changes seen in the LDC group, the hepatic expression of CD14, pMAPK, casp 9, Cyt c and 4-HNE proteins was increased in the L-HFr group. Conversely, the expression of PGC-1α in the liver was reduced in the L-HFr group at wk 8. In the liver, the localization of LCN2 was restricted to MPO+ recruited granulocytes. LCN2 showed a high consecutive expression in fructose-treated primary HCs compared to glucose treatment.
Both LDC and L-HFr diets led NAFLD. While the LDC diet resulted in a simple steatosis, the L-HFr diet led to NASH. Fructose supplementation appears to worsen liver pathology. The features of the metabolic syndrome and NASH with progressive fibrosis obtained with fructose-enriched diet represent an animal model broadly similar to the conditions in human liver disease. These findings highlight the impact of dietary composition in the development of NAFLD. The fructose diet upregulates hepatic Lcn2 gene expression in fructose-induced NASH. This correlates with the increased indicators of inflammation, oxidative stress and mitochondrial dysfunction. This novel study could introduce serum LCN2 as a useful new diagnostic to discriminate between simple steatosis and NASH. Evidently, an interaction exists between the metabolic impetus and inflammatory processes in the animals. The current model is valuable for investigation of the role(s) of LCN2 and other markers for inflammation and metabolism in greater depth and the development of new insights for preventing NASH.||de