Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis

Abstract

Genetically obese fatty/fatty rats and obese/obese mice exhibit increased sensitivity to endotoxin hepatotoxicity, quickly developing steatohepatitis after exposure to low doses of lipopolysaccharide (LPS). Among obese animals, females are more sensitive to endotoxin liver injury than males. LPS induction of tumor necrosis factor alpha (TNF alpha), the proven affecter of endotoxin liver injury, is no greater in the livers, white adipose tissues, or sera of obese animals than in those of lean controls. Indeed, the lowest serum concentrations of TNF occur in female obese rodents, which exhibit the most endotoxin-induced liver injury. Several cytokines that modulate the biological activity of TNF are regulated abnormally in the livers of obese animals. After exposure to LPS, mRNA of interferon gamma, which sensitizes hepatocytes to TNF toxicity, is overexpressed, and mRNA levels of interleukin 10, a TNF inhibitor, are decreased. The phagocytic activity of liver macrophages and the hepatic expression of a gene encoding a macrophage-specific receptor are also decreased in obesity. This new animal model of obesity-associated liver disease demonstrates that hepatic macrophage dysfunction occurs in obesity and suggests that this might promote steatohepatitis by sensitizing hepatocytes to endotoxin.

Figure 1

Liver histology before and after treatment with LPS. (A) Micro- and macrovesicular steatosis in a representative fa/fa rat before LPS treatment. Area of (B) hepatocyte apoptosis and (C) steatohepatitis in two distinct, but representative, fa/fa rats 24 h after treatment with LPS (0.5 μg/g). (D) Normal liver histology in a representative ?/fa rat before LPS. (E and F) Normal liver histology in two distinct, but representative, ?/fa rats 24 h after treatment with LPS (0.5 μg/g). (Final magnification ×400.)

Figure 2

Effect of LPS on serum activities of liver-associated enzymes. (A) AST and ALT in fa/fa rats and ?/fa controls 24 h after exposure to 0.5 μg/g LPS. (B) ALT in fa/fa and ?/fa rats 24 h after treatment with 100 or 200 μg of LPS. (C) AST and ALT in ob/ob and ?/ob mice 24 h after treatment with LPS (0.5 μg/g). (D) AST and ALT before and 24 h after treatment with LPS (0.5 μg/g) in female (F) and male (M) fa/fa rats. IU/L, units per liter for A–D.

Figure 3

TNFα expression in fa/fa and ?/fa rats pre- and 1 h posttreatment with LPS (0.5 μg/g). (A) Total liver RNA was isolated from three distinct rats/group at each time point, reverse transcribed, and amplified with TNF-specific primers. PCR products were separated on nondenaturing agarose gels, transferred to nylon membranes, and visualized by hybridization with TNF-specific probes. (B) Serum TNF concentrations were evaluated by ELISA using rat recombinant TNFα as standard. In both males and females, TNF expression was greater in serum and liver at 1 h after LPS than at any other time point (0, 0.5, 6, or 24 h) evaluated.

Figure 4

Hepatic expression of TNF regulatory cytokines pre- and posttreatment with LPS (0.5 μg/g). Liver RNA was isolated from three distinct rats/group either before (pre) or 1 and 24 h after LPS treatment and subjected to reverse transcriptase PCR analysis with sets of primers specific for IL-12, IFNγ, IL-10, or TGFβ-1 under semiquantitative conditions.

Figure 5

Comparison of Kupffer cell function in fa/fa and ?/fa rats. (A) Total liver RNA was isolated from two distinct rats/group either before (pre) or 1 and 24 h after LPS (0.5 μg/g), separated by electrophoresis on denaturing polyacrylamide gels (20 μg RNA/lane), transferred to nylon membranes, and cohybridized with [³²P]-labeled cDNAs for KCR and a constitutively expressed monocyte transcription factor (Pu-1). (B and C) Rats were injected intraportally with fluorescent beads, and intravital microscopy was used to evaluate (B) the distribution of beads in acinar zones 1 (periportal), 2 (midacinus), and 3 (pericentral) 30 minutes after injection and (C) the cumulative hepatic uptake of the beads over time. Results shown are representative of replicate experiments.