Journal: Hepatology communications

Article Title: Lipocalin-2 Protects Against Diet-Induced Nonalcoholic Fatty Liver Disease by Targeting Hepatocytes.

doi: 10.1002/hep4.1341

Figure Lengend Snippet: FIG. 1. AAV-mediated overexpression of human LCN2 in hepatocytes protects against HFCF diet–induced steatohepatitis. C57BL/6 mice were intravenously injected with AAV8-ALB-null or AAV8-ALB-hLCN2 (n = 8). The mice were then fed an HFCF diet for 20 weeks. (A) Plasma ALT and AST levels. (B) Hepatic TG levels. (C) Hepatic FFA levels. (D) Hepatic MDA levels. (E) Liver sections were used for H&E staining (left top panel), ORO (left bottom panel), sirius red staining (right top panel) or immunostaining with an antibody against F4/80 (right bottom panel) (arrows point to fibrosis [sirius red staining] or macrophages [F4/80 staining]). (F) Hepatic hydroxyproline levels (top panel) or F4/80 staining–positive area (%) (bottom panel) were quantified. In (E). *P < 0.05, **P < 0.01.

Article Snippet: Some wildtype primary hepatocytes were infected with either Ad-empty or Ad-LCN2, or treated with phosphate-buffered saline or recombinant LCN2 protein (500 ng/mL; R&D Systems).

Techniques: Over Expression, Injection, Clinical Proteomics, Staining, Immunostaining

Journal: Hepatology communications

Article Title: Lipocalin-2 Protects Against Diet-Induced Nonalcoholic Fatty Liver Disease by Targeting Hepatocytes.

doi: 10.1002/hep4.1341

Figure Lengend Snippet: FIG. 2. Overexpression of human LCN2 in hepatocytes regulates the hepatic genes implicated in lipogenesis, lipolysis, FAO, inflammation, and fibrogenesis. Mice have been described in the legend of Fig. 1 (n = 8). Real-time quantitative polymerase chain reaction (PCR) was used to quantify hepatic genes involved in lipogenesis, lipolysis, FAO (A), inflammation, or fibrogenesis (B). (C) Hepatic proteins were detected by western blotting (left panel) and then quantified (right panel). (D) Plasma proteins were analyzed by western blotting (left panel) and then quantified (right panel). (E) Plasma β-HB levels. *P < 0.05, **P < 0.01. Abbreviations: BP1c, Srebp-1c.

Article Snippet: Some wildtype primary hepatocytes were infected with either Ad-empty or Ad-LCN2, or treated with phosphate-buffered saline or recombinant LCN2 protein (500 ng/mL; R&D Systems).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Clinical Proteomics

Journal: Hepatology communications

Article Title: Lipocalin-2 Protects Against Diet-Induced Nonalcoholic Fatty Liver Disease by Targeting Hepatocytes.

doi: 10.1002/hep4.1341

Figure Lengend Snippet: FIG. 3. Human LCN2 induces lipolysis and FAO and inhibits DNL in an LXR-SREBP-1-dependent manner. (A) C57BL/6 mice were infected with AAV8-ALB-null or AAV8-ALB-hLCN2 and then fed an HFCF diet for 20 weeks (n = 8). TGH activity was determined using [3H]Triolein as substrate. (B) HepG2 cells were co-transfected with pTK-3 × PPRE-luc, pCMV-β-gal, pCMV- hLCN2, or pCMV-empty. Relative luciferase activity units (RLUs) were determined after 36 hours (n = 7). (C) Primary hepatocytes were infected with Ad-Empty or Ad-hLCN2 for 36 hours. FAO was determined using [3H]palmitic acid as substrate (n = 6). (D) HepG2 cells were co-transfected with pGL3-SREBP-1-luc, CMV-β-gal, CMV-hLCN2, or CMV-empty in the presence or absence of the LXRα ligand T0901317 (10 µM). After 36 hours, RLUs were determined (n = 6). (E) Primary hepatocytes were isolated from Srebp1+/+ or Srebp1−/− mice, and then infected with Ad-Empty or Ad-hLCN2 for 48 hours. TG levels were quantified. (F) C57BL/6 mice were intravenously injected with AAV8-ALB-null (Null) or AAV8-ALB-hLCN2 (hLCN2) and then fed an HFCF diet for 20 weeks (n = 8). Four hours before euthanization, mice were intraperitoneally injected with 2H2O. Newly synthesized palmitate (PA) (left panel) or TG (right panel) levels were quantified by GC-MS. *P < 0.05, **P < 0.01.

Article Snippet: Some wildtype primary hepatocytes were infected with either Ad-empty or Ad-LCN2, or treated with phosphate-buffered saline or recombinant LCN2 protein (500 ng/mL; R&D Systems).

Techniques: Infection, Activity Assay, Transfection, Luciferase, Isolation, Injection, Synthesized, Gas Chromatography-Mass Spectrometry

Journal: Hepatology communications

Article Title: Lipocalin-2 Protects Against Diet-Induced Nonalcoholic Fatty Liver Disease by Targeting Hepatocytes.

doi: 10.1002/hep4.1341

Figure Lengend Snippet: FIG. 4. Loss of LCN2 exacerbates diet-induced NAFLD. Lcn2+/+ or Lcn2−/− mice were fed an HFCF diet for 16 weeks (n = 6-9). (A) Plasma ALT or AST levels. (B) Body or liver weight. (C) Hepatic total cholesterol or TG levels. (D) Hepatic MDA levels. (E) Representative images of H&E, ORO, or sirius red staining or immunostaining using an antibody against F4/80. (F) Hepatic hydroxyproline levels (top panel) or F4/80 staining–positive area (%) (bottom panel). Arrows point to fibrosis (sirius red staining) or macrophages (immunostaining with F4/80 antibody). *P < 0.05, **P < 0.01. Abbreviations: TC, total cholesterol.

Article Snippet: Some wildtype primary hepatocytes were infected with either Ad-empty or Ad-LCN2, or treated with phosphate-buffered saline or recombinant LCN2 protein (500 ng/mL; R&D Systems).

Techniques: Clinical Proteomics, Staining, Immunostaining

Journal: Hepatology communications

Article Title: Lipocalin-2 Protects Against Diet-Induced Nonalcoholic Fatty Liver Disease by Targeting Hepatocytes.

doi: 10.1002/hep4.1341

Figure Lengend Snippet: FIG. 5. Loss of LCN2 induces genes involved in lipogenesis, inflammation, and fibrogenesis. Lcn2+/+ or Lcn2−/− mice were fed an HFCF diet for 16 weeks (n = 6-9). (A-D) Hepatic mRNA levels involved in lipogenesis (A), lipolysis or FAO (B), inflammation (C), or fibrogenesis (D) were quantified by real-time quantitative polymerase PCR. (E) Hepatic proteins were analyzed by western blotting (left panel) and quantified (right panel). (F) Plasma β-HB levels (left panel) and hepatic FFA levels (right panel). *P < 0.05, **P < 0.01.

Article Snippet: Some wildtype primary hepatocytes were infected with either Ad-empty or Ad-LCN2, or treated with phosphate-buffered saline or recombinant LCN2 protein (500 ng/mL; R&D Systems).

Techniques: Western Blot, Clinical Proteomics

Journal: Hepatology communications

Article Title: Lipocalin-2 Protects Against Diet-Induced Nonalcoholic Fatty Liver Disease by Targeting Hepatocytes.

doi: 10.1002/hep4.1341

Figure Lengend Snippet: FIG. 6. Loss of LCN2 or treatment with recombinant LCN2 protein regulates lipolysis, FAO, and lipogenesis in hepatocytes. (A) Lcn2+/+ or Lcn2−/− mice were fed an HFCF diet for 16 weeks. Hepatic TGH activity was determined (n = 7-8). (B) FAO was determined using [3H]palmitic acid as substrate in primary hepatocytes isolated from Lcn2+/+ mice or Lcn2−/− mice (n = 6). (C) mRNA levels were quantified in primary hepatocytes isolated from Lcn2+/+ mice or Lcn2−/− mice (n = 4). (D-F) Primary hepatocytes were isolated from Lcn2+/+ or Lcn2−/− mice and then treated with phosphate-buffered saline (control) or 500 ng/mL LCN2 for 30 hours (n = 4-5 per group). TGH (D) and FAO (E) were determined. The mRNA levels of genes involved in lipogenesis, FAO, or lipolysis were determined. *P < 0.05, **P < 0.01. Abbreviations: PBS, phosphate-buffered saline.

Article Snippet: Some wildtype primary hepatocytes were infected with either Ad-empty or Ad-LCN2, or treated with phosphate-buffered saline or recombinant LCN2 protein (500 ng/mL; R&D Systems).

Techniques: Recombinant, Activity Assay, Isolation, Saline, Control

Journal: Hepatology communications

Article Title: Lipocalin-2 Protects Against Diet-Induced Nonalcoholic Fatty Liver Disease by Targeting Hepatocytes.

doi: 10.1002/hep4.1341

Figure Lengend Snippet: FIG. 7. LCN2 inhibits apoptosis. (A-E) C57BL/6 mice were injected intravenously with AAV8-ALB-null or AAV8-ALB-hLCN2 (n = 8), and then fed an HFCF diet for 20 weeks. Lcn2+/+ or Lcn2−/− mice were fed an HFCF diet for 16 weeks (n = 6-9). Hepatic DCF (ROS) levels were quantified (A). Hepatic protein levels were determined by western blotting (B) and then quantified (C) (top and bottom panels). Apoptosis was analyzed by TUNEL assays (D) and then quantified (E) (top and bottom panels). (F) A model for hepatocyte LCN2 to regulate the development of NAFLD. LCN2 stimulates lipolysis to release FFAs from TGH. Freshly released FFAs act as ligands for PPARα to stimulate FAO, and antagonize LXR activity to inhibit DNL. As a result, hepatic levels of FFAs, ROS, and apoptosis are reduced, leading to decreased steatosis, inflammation, and fibrosis in the liver. *P < 0.05, **P < 0.01. Abbreviations: DCF, 2′,7′-dichlorofluorescein.

Article Snippet: Some wildtype primary hepatocytes were infected with either Ad-empty or Ad-LCN2, or treated with phosphate-buffered saline or recombinant LCN2 protein (500 ng/mL; R&D Systems).

Techniques: Injection, Western Blot, TUNEL Assay, Activity Assay