Lipocalin-2 activates hepatic stellate cells and promotes nonalcoholic steatohepatitis in high-fat diet-fed Ob/Ob mice

Abstract

Background and aims: In obesity and type 2 diabetes mellitus, leptin promotes insulin resistance and contributes to the progression of NASH via activation of hepatic stellate cells (HSCs). However, the pathogenic mechanisms that trigger HSC activation in leptin-deficient obesity are still unknown. This study aimed to determine how HSC-targeting lipocalin-2 (LCN2) mediates the transition from simple steatosis to NASH.

Approach and results: Male wild-type (WT) and ob/ob mice were fed a high-fat diet (HFD) for 20 weeks to establish an animal model of NASH with fibrosis. Ob/ob mice were subject to caloric restriction or recombinant leptin treatment. Double knockout (DKO) mice lacking both leptin and lcn2 were also fed an HFD for 20 weeks. In addition, HFD-fed ob/ob mice were treated with gadolinium trichloride to deplete Kupffer cells. The LX-2 human HSCs and primary HSCs from ob/ob mice were used to investigate the effects of LCN2 on HSC activation. Serum and hepatic LCN2 expression levels were prominently increased in HFD-fed ob/ob mice compared with normal diet-fed ob/ob mice or HFD-fed WT mice, and these changes were closely linked to liver fibrosis and increased hepatic α-SMA/matrix metalloproteinase 9 (MMP9)/signal transducer and activator of transcription 3 (STAT3) protein levels. HFD-fed DKO mice showed a marked reduction of α-SMA protein compared with HFD-fed ob/ob mice. In particular, the colocalization of LCN2 and α-SMA was increased in HSCs from HFD-fed ob/ob mice. In primary HSCs from ob/ob mice, exogenous LCN2 treatment induced HSC activation and MMP9 secretion. By contrast, LCN2 receptor 24p3R deficiency or a STAT3 inhibitor reduced the activation and migration of primary HSCs.

Conclusions: LCN2 acts as a key mediator of HSC activation in leptin-deficient obesity via α-SMA/MMP9/STAT3 signaling, thereby exacerbating NASH.

Graphical abstract

FIGURE 1

Lipocalin‐2 (LCN2) expression is upregulated in liver injury. (A) Representative hematoxylin and eosin (H&E) and Sirius Red staining of liver sections. Scale bars, 100 μm. (B) Hepatic triglyceride (TG; n = 4–5). (C) Sirius Red–positive area. (D,E) Serum leptin and LCN2 levels (n = 5–6). (F) Western blot analysis and quantification of LCN2 protein (n = 5–6). (G) Representative LCN2 and F4/80 staining of liver sections. Scale bars, 100 μm. (H) Quantification of LCN2‐ and F4/80‐positive areas. Significance was determined by one‐way analysis of variance. HFD, high‐fat diet; ND, normal diet. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Increased hepatic fibrosis is linked to increased lipocalin‐2 (LCN2) levels in high‐fat diet (HFD)‐fed Ob/Ob mice. Wild‐type (WT) and Ob/Ob mice were fed a normal diet (ND) or HFD for 20 weeks to induce NASH with fibrosis. (A) Representative images of livers from mice after 20 weeks of ND or HFD feeding. Scale bars, 1 cm. (B–D) Liver weight (n = 10–14), serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (n = 9–10), and hepatic triglycerides (TG; n = 4–7). (E) Representative hematoxylin and eosin (H&E), Nile Red, and Sirius Red staining of liver tissue. Scale bars, 50 μm. (F–H) NAFLD activity score (NAS), Nile Red–positive area, and Sirius Red–positive area. (I) Western blot analysis and quantification of α‐smooth muscle actin (α‐SMA) protein in liver lysates (n = 5–6). (J–L) Serum LCN2 (n = 10–14), hepatic LCN2 messenger RNA, and protein levels (n = 5–7). Significance was determined by two‐way analysis of variance. *p < 0.05, **p < 0.01, ***p < 0.001. ob/obHFD, HFD‐fed ob/ob; ob/obND, ND‐fed ob/ob; WTHFD, HFD‐fed WT; WTND, ND‐fed WT.

FIGURE 3

A high‐fat diet (HFD) upregulates lipocalin‐2 (Lcn2) gene expression in mouse livers as revealed by transcriptomic profiling. Groups of wild‐type (WT) or Ob/Ob mice fed a normal diet (ND) or high‐fat diet (HFD; n = 3 per group) were discriminated clearly based on significant changes in the expression of 6280 genes in the liver (p < 0.05) as indicated by two‐way analysis of variance using unsupervised analysis. (A) Hierarchical clustering and (B) principal components analysis. Dendrograms were generated based on hierarchical clustering of correlation distance between individual RNA‐sequencing expression profiles. (C) Venn diagram showing differentially expressed genes (DEGs) in the liver affected by an HFD (log₂FC ≥ 1, adjusted p < 0.05). (D) Eight HFD‐induced DEGs upregulated in both WT and Ob/Ob mice were considered key regulatory genes, among which Lcn2 was the most highly expressed gene on average. (E) Protein–protein interactions of eight DEG products were constructed with no more than 10 interactors using the STRING database. Three clusters were found based on a Markov clustering algorithm, and both Lcn2 and matrix metalloproteinase 9 (Mmp9) were implicated as hub genes in the regulation of each subnetwork. (F) Functional enrichment plot of both Lcn2 and Mmp9 showing seven gene sets (false discovery rate <0.05). Cbl, Casitas B‐lineage lymphoma; Ccl5, Chemokine (C‐C motif) ligand 5; Ccr, Chemokine (C‐C motif) receptor; Ciart, Circadian associated repressor of transcription; Cxcl, Chemokine (C‐X‐C motif) ligand; Cxcr, CXC chemokine receptor; Egfr, Epidermal growth factor receptor; FDR, False Discovery Rate; Frs, FAR1 related sequences; Gab, GRB2 associated binding protein; Gm43912, predicted gene 43912; Got1l1, glutamic‐oxaloacetic transaminase 1‐like 1; Grb, Growth factor receptor bound protein; Irs, Insulin receptor substrate; Lrp, Leucine‐responsive transcriptional regulator; PCs, principal components; Shc, SHC‐adaptor protein; Slc, Solute carrier; Sos, SOS Ras/Rac guanine nucleotide exchange factor; Spry, Sprouty; Themis, Thymocyte selection associated.

FIGURE 4

Lipocalin‐2 (LCN2) regulates hepatic fibrosis in high‐fat diet (HFD)‐fed Ob/Ob mice by interacting with matrix metalloproteinase 9 (MMP9). Wild‐type (WT) and Ob/Ob mice were fed a normal diet (ND) or HFD for 20 weeks to induce NASH. (A) Western blot analysis and quantification of MMP9 protein in liver lysates (n = 6). (B) Photographs of zymography. Bands corresponding to MMP9 and MMP2 are indicated by arrows. (C) Immunoprecipitation of LCN2 and MMP9 in liver lysates. (D) Representative Duolink PLA staining of LCN2 and MMP9 in liver sections. Nuclei were stained with DAPI. Scale bar, 50 μm. Quantification of the LCN2+MMP9–positive area. (E) Western blot analysis and quantification of phosphorylated signal transducer and activator of transcription 3 (pSTAT3)/signal transducer and activator of transcription 3 (STAT3) protein in liver lysates (n = 4–5). Significance was determined by two‐way analysis of variance. *p < 0.05, **p < 0.01, ***p < 0.001. DAPI, 4′,6‐diamidino‐2‐phenylindole; IP, immunoprecipitation.

FIGURE 5

Lipocalin‐2 (LCN2) deficiency protects against high‐fat diet (HFD)‐induced liver fibrosis in Ob/Ob mice. Ob/Ob and double knockout (DKO) mice were fed an HFD for 20 weeks to induce NASH with fibrosis. (A) Western blot analysis and quantification of LCN2, matrix metalloproteinase 9 (MMP9), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) protein in liver lysates (n = 5–6). (B) Serum MMP9 was assessed by enzyme‐linked immunosorbent assay (n = 5–6). (C) Representative images and quantification of Sirius Red staining in liver sections. Scale bars, 100 μm. (D) Hepatic α‐smooth muscle actin (α‐SMA) messenger RNA was measured by real‐time polymerase chain reaction (n = 5–6). (E) Western blot analysis and quantification of α‐SMA protein in liver lysates from HFD‐fed Ob/Ob and DKO mice (n = 6). (F) Western blot analysis of cytosolic and nuclear NF‐κBp65 protein and the nuclear/cytosolic (N/C) ratio of NF‐κBp65 in liver lysates (n = 6). β‐Actin and p84 were used as loading controls for total and nuclear proteins, respectively. Significance was determined by a two‐tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001. STAT3, signal transducer and activator of transcription 3.

FIGURE 6

Effects of a high‐fat diet (HFD) on perilipin 2 expression in the liver of Ob/Ob mice. (A) Representative images of immunofluorescence staining of perilipin 2 and α‐smooth muscle actin (α‐SMA) in liver sections. Nuclei were counterstained with 4',6‐diamidino‐2‐phenylindole (DAPI). Scale bar, 50 μm. (B) Western blot analysis and quantification of hepatic perilipin 2 protein in wild‐type (WT) and Ob/Ob mice fed a normal diet (ND) or HFD (n = 5–8). (C) Western blot analysis of perilipin 2, α‐SMA, and lipocalin‐2 (LCN2) protein in hepatocytes, Kupffer cells, and hepatic stellate cells (HSCs) purified from livers of Ob/Ob mice fed an ND or HFD. (D) Western blot analysis and quantification of hepatic perilipin 2 protein in HFD‐fed Ob/Ob or double knockout (DKO) mice (n = 6). (E) Western blot analysis of perilipin 2, α‐SMA, and LCN2 protein in HSCs purified from livers of HFD‐fed Ob/Ob or DKO mice (n = 3). (F) Western blot analysis and quantification of acetyl‐CoA carboxylase 1 (ACC1), stearoyl‐CoA desaturase 1 (SCD1), and fatty acid synthase (FAS) protein in liver lysates (n = 5–8). For (B) and (F), significance was determined by two‐way analysis of variance. For (D), significance was determined by a two‐tailed t test. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Lipocalin‐2 (LCN2) activates and induces secretion of matrix metalloproteinase 9 (MMP9) from isolated primary mouse hepatic stellate cells (mHSCs) from Ob/Ob mice. (A) Expression of messenger RNA Lcn2 and collagen 1α in LPS‐treated medium (LTM)‐treated mHSCs was measured by real‐time polymerase chain reaction (n = 3). (B) Western blot analysis of LCN2, 24p3R, α‐smooth muscle actin (α‐SMA), phosphorylated signal transducer and activator of transcription 3 (pSTAT3), and signal transducer and activator of transcription 3 (STAT3) in mHSCs after LTM treatment. Western blot analysis of MMP9 and LCN2 in the supernatant medium of mHSCs after LTM treatment. (C) Western blot analysis of LCN2, 24p3R, α‐SMA, pSTAT3, and STAT3 in mHSCs after recombinant LCN2 (rLCN2) treatment. Western blot analysis of MMP9 and LCN2 in the supernatant medium of mHSCs after rLCN2 treatment. (D) Western blot analysis of LCN2, 24p3R, α‐SMA, pSTAT3, and STAT3 in mHSCs after S31‐201 treatment. Western blot analysis of MMP9 in the supernatant medium of mHSCs after S31‐201 treatment (n = 3). For (A–C), significance was determined by one‐way analysis of variance (ANOVA). For (D), significance was determined by two‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. CTL, control; DMSO, Dimethyl sulfoxide; Gapdh, glyceraldehyde 3‐phosphate dehydrogenase.

FIGURE 8

Effects of 24p3R inhibition on activation and migration of primary mouse hepatic stellate cells (mHSCs). (A) Western blot analysis of 24p3R, lipocalin‐2 (LCN2), α‐smooth muscle actin (α‐SMA), phosphorylated signal transducer and activator of transcription 3 (pSTAT3), and signal transducer and activator of transcription 3 (STAT3) in mHSCs from Ob/Ob mice after LPS‐treated medium (LTM) and si24p3R treatment. Western blot analysis of matrix metalloproteinase 9 (MMP9) and LCN2 in the supernatant medium of mHSCs after LTM and si24p3R treatment. (B) Representative images of Nile Red staining of LTM‐treated mHSCs with or without si24p3R and quantification of Nile Red–stained lipid droplets in mHSCs. Nuclei were counterstained with DAPI. Scale bar, 10 μm. (C,D) Wound healing migration assay was used to investigate the effect of 24p3R on the migration of (C) LX‐2 cells and (D) mHSCs following 24 h of cotreatment with LTM and si24p3R (n = 3). Scale bar, (C) 500 μm, (D) 50 μm. Significance was determined by two‐way analysis of variance. *p < 0.05, **p < 0.01, ***p < 0.001. CTL, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; Scr, Scramble.