Age-related sensitivity to endotoxin-induced liver inflammation: Implication of inflammasome/IL-1β for steatohepatitis

Abstract

Aging is associated with increased vulnerability to inflammatory challenge. However, the effects of altered inflammatory response on the metabolic status of tissues or organs are not well documented. In this study, we present evidence demonstrating that lipopolysaccharide (LPS)-induced upregulation of the inflammasome/IL-1β pathway is accompanied with an increased inflammatory response and abnormal lipid accumulation in livers of aged rats. To monitor the effects of aging on LPS-induced inflammation, we administered LPS (2 mg kg(-1) ) to young (6-month old) and aged (24-month old) rats and found abnormal lipid metabolism in only aged rats with increased lipid accumulation in the liver. This lipid accumulation in the liver was due to the dysregulation of PPARα and SREBP1c. We also observed severe liver inflammation in aged rats as indicated by increased ALT levels in serum and increased Kupffer cells in the liver. Importantly, among many inflammation-associated factors, the aged rat liver showed chronically increased IL-1β production. Increased levels of IL-1β were caused by the upregulation of caspase-1 activity and inflammasome activation. In vitro studies with HepG2 cells demonstrated that treatment with IL-1β significantly induced lipid accumulation in hepatocytes through the regulation of PPARα and SREBP1c. In summary, we demonstrated that LPS-induced liver inflammation and lipid accumulation were associated with a chronically overactive inflammasome/IL-1β pathway in aged rat livers. Based on the present findings, we propose a mechanism of aging-associated progression of steatohepatitis induced by endotoxin, delineating a pathogenic role of the inflammasome/IL-1β pathway involved in lipid accumulation in the liver.

Keywords: IL-1β; LPS; aging; inflammasome; inflammation; lipid accumulation.

Fig 1

Aging increases sensitivity to lipopolysaccharide (LPS)-induced changes to lipid metabolism. Lipopolysaccharide was injected intraperitoneally (2 mg kg⁻¹body weight) in young and aged rats. Rats were euthanized 12 h, 72 h, and 7 days after injection. (A) Measurement of triglycerides (TG) in serum following LPS injection in young and aged rats. (B) Measurement of free fatty acids (FFAs) in serum following LPS injection in young and aged rats. (C) Measurement of TG in the liver following LPS injection in young and aged rats. (D) Measurement of FFAs in the liver following LPS injection in young and aged rats. (E) Lipid accumulation in the livers was visualized by hematoxylin–eosin (H&E) staining. (F) Lipid accumulation in the livers was visualized by Oil red O staining. Oil red O positive areas were quantified. Data are expressed as the mean ± SEM. *P < 0.05 in comparison with the corresponding controls.

Fig 2

Effects of aging on lipid metabolism-associated transcription factors changes induced by lipopolysaccharide (LPS) in liver. (A) The nuclear fraction of liver homogenates was used to detect transcription factors associated with lipid metabolism. Western blots were performed to detect nuclear protein levels of FXR, LXR, PPARα, PPARβ, PPARγ, SREBP1c, and SREBP2. TF-IIB was used as control. The blots of PPARα and SREBP1c were quantified by densitometry (n = 5). (B) qRT–PCR of PPARα target genes (Acox1, Cpt1, and Cyp4a1) in LPS-treated young and aged rat livers. mRNA levels were normalized to GAPDH level. (C) qRT–PCR of SREBP1c target genes (ACCa and FASN) in LPS-treated young and aged rat livers. mRNA levels were normalized to GAPDH level. Data are expressed as the mean ± SEM. *P < 0.05 vs. corresponding control group.

Fig 3

Effects of aging on lipopolysaccharide (LPS)-induced inflammasome activation and IL-1β production. (A) Measurement of serum levels of IL-1β and MCP1 following LPS injection in young and aged rats. (B) Measurement of liver levels of IL-1β and MCP1 following LPS injection in young and aged rats. (C) Pro-form (upper panel, short exposure) and cleaved form (lower panel, long exposure) of IL-1β were analyzed by Western blot. The blots of pro- and cleaved IL-1β were quantified by densitometry (n = 5). (D) Expression of NALP3, ASC, and Casp-1 in the liver was measured using qPCR. (E) Caspase-1 activity was measured using a colorimetric assay. Data are expressed as the mean ± SEM. *P < 0.05 vs. corresponding control group.

Fig 4

Effects of aging on lipopolysaccharide (LPS)-induced liver inflammation. (A) Liver injury was quantified by measuring serum ALT/GPT levels. (B) Oxidative stress was measured using reactive species(RS)-specific fluorescent dye. (C) Activation of liver Kupffer cells was used as a marker of liver inflammation. Immunohistochemistry was used to evaluate recruitment of CD68 positive Kupffer cells. DAB-based brown-colored region indicates the CD68⁺ Kupffer cell. Percentage of Kupffer cells was calculated. Data are expressed as the mean ± SEM. *P < 0.05 vs. control group.

Fig 5

Observation of lipid accumulation in hepatic cells after treatment with IL-1β. (A) HepG2 cells were incubated with or without 20 ng mL⁻¹ of IL-1β as indicated. Accumulation of lipids was visualized by Nile red staining. Cells were counterstained with Hoechst. (B) The concentration of triglycerides (TG) in HepG2 cells was measured as described in Experimental procedures. Data are expressed as the mean ± SEM. *P < 0.05 vs. control group.

Fig 6

IL-1β exerts lipogenic effects on hepatocytes through the regulation of PPARα and SREBP1c transcription factors. (A) Western blot for SREBP1c from HepG2 cells treated with IL-1β. Analysis of total lysates (upper panel) reveals upregulation of total SREBP1c (short exposure) as well as cleaved form of SREBP1c (long exposure). Actin was used as control. Analysis of nuclear fractions (lower panel) reveals increased translocation of SREBP1c after IL-1β treatment in HepG2 cells. TF-IIB was used as control. (B) Western blot for PPARα from HepG2 cells treated with IL-1β. TF-IIB was used as control. (C) Expression of SREBP1c target genes (ACCa, FASN) was analyzed by qPCR. Results were normalized to GAPDH level. (D) Expression of PPARα target genes (Cpt1, Acox1) was analyzed by qPCR. Results were normalized to GAPDH level.*P < 0.05 vs. control group.