Sensitivity of mice to lipopolysaccharide is increased by a high saturated fat and cholesterol diet

Abstract

Background: It was hypothesized that a pro-atherogenic, high saturated fat and cholesterol diet (HCD) would increase the inflammatory response to E. coli endotoxin (LPS) and increase its concentration in plasma after administration to mice.

Methods: C57Bl/6 mice were fed a HCD or a control diet (CD) for 4 weeks, and then treated with saline, 0.5, 1 or 2 mg LPS/kg, ip. Liver injury (alanine:2-oxoglutarate aminotransferase and aspartate aminotransferase, collagen staining), circulating cytokines (tumor necrosis factor-alpha, interleukin-6 and interferon-gamma), factors that can bind LPS (serum amyloid A, apolipoprotein A1, LPS binding protein, and CD14), and plasma levels of LPS were measured. The hepatic response was assessed by measuring vascular cell adhesion molecule (VCAM)-1, inducible nitric oxide synthase (iNOS) and signal transducer and activator of transcription-1 proteins, and VCAM-1 and iNOS mRNAs. Hepatic mRNA encoding the LPS receptor, Toll like receptor 4, was also determined.

Results: Two mg LPS/kg killed 100% of mice fed HCD within 5 d, while no mice fed CD died. All mice treated with 0 to 1 mg LPS/kg survived 24 h. HCD increased plasma alanine:2-oxoglutarate aminotransferase and aspartate aminotransferase, and the enzymes were increased more by LPS in HCD than CD mice. Induction of plasma tumor necrosis factor-alpha, interleukin-6, and interferon-gamma by LPS was greater with HCD than CD. Hepatic VCAM-1 and iNOS protein and mRNA were induced by LPS more in mice fed HCD than CD. Tyrosine phosphorylation of signal transducer and activator of transcription-1 caused by LPS was prolonged in HCD compared with CD mice. Despite the hepatic effects of HCD, diet had no effect on the LPS plasma concentration-time profile. HCD alone did not affect circulating levels of plasma apolipoprotein A1 or LPS binding protein. However, plasma concentrations of serum amyloid A and CD14, and hepatic toll-like receptor-4 mRNA were increased in mice fed HCD.

Conclusion: HCD increased the sensitivity of mice to LPS without affecting its plasma level. Although increased serum amyloid A and CD14 in the circulation may inhibit LPS actions, their overexpression, along with hepatic toll-like receptor-4 or other factors, may contribute to the heightened sensitivity to LPS.

Figure 1

The effect of LPS on survival of mice. C57Bl/6 mice (8 per group), were fed HCD or CD for 4 wk, and then treated with a single dose of LPS (2 mg/kg, ip). The median survival time for mice fed HCD was 2 d and the curves differed significantly (p < 0.0001).

Figure 2

The effect of diet on plasma ALT and AST. CD or HCD mice received a single ip injection of 0, 0.5, or 1.0 mg LPS/kg and blood was sampled after 12 h. Values are mean + SE of 5 mice per group. *: p < 0.05 for comparison with 0 mg LPS/kg. +:p < 0.05 for comparison with CD mice treated with the same dose of LPS.

Figure 3

The effect of diet and LPS on plasma levels of pro-inflammatory TNFα (A), IL-6 (B), and IFNγ (C). Plasma levels were measured 0.5, 2, 4, 12, or 24 h after treatment. Values are the mean ± SE of 5 mice per group. *: p < 0.05 for comparison with 0 mg LPS/kg. +: p < 0.05 for comparison with CD treated with the same dose of LPS.

Figure 4

The effect of diet and LPS on hepatic VCAM-1 expression. mRNA levels (A) were measured with RT-PCR. Densitometric intensity of images of ethidium bromide-stained agarose gels were normalized to β-actin RT-PCR signal. Induction of VCAM-1 RT-PCR signal ratios (n = 5 in each group) are expressed as fold increase compared to vehicle-treated CD mice, represented by 1 on the y-axis. Values are expressed mean ± SE of 5 mice are the fold increases in VCAM-1/β-actin mRNA signal ratio relative CD mice treated with 0 mg LPS/ml (represented by 1 on the y-axis). The level of VCAM-1 protein (B) was measured by western blotting 4 and 12 h after treatment (representative images and image analysis). Values are the mean + SE of total integrated signal intensity obtained from densitometry for 5 mice in each group. *: p < 0.05 for comparison with 0 mg LPS/kg. +: p < 0.05 for comparison with CD treated with the same dose of LPS.

Figure 5

The effect of diet and LPS on hepatic iNOS expression. iNOS mRNA (A) was assessed as in figure 4. Values are expressed mean ± SE of 5 mice are the fold increases in iNOS/β-actin mRNA signal ratio relative CD mice treated with 0 mg LPS/ml, represented by 1 on the y-axis. The level of iNOS protein (B) was measured by western blotting 4 and 12 h after treatment (representative images and image analysis). Values are the mean + SE of total integrated signal intensity obtained from densitometry for 5 mice in each group. *: p < 0.05 for comparison with 0 mg LPS/kg. +: p < 0.05 for comparison with CD treated with the same dose of LPS.

Figure 6

The effect of diet and LPS on STAT1 activation in liver. Total and tyrosine 701 phosphorylated (pY701) STAT1 protein levels in hepatic tissues from CD and HCD mice were measured by western blotting. A representative image from samples 12 h after LPS treatment is shown in panel A. STAT1α and the shorter β form are visible. Densitometric intensity of total and pY701 STAT1α was measured and means + SE of 5 mice fed each diet 4 and 12 h after treatment were calculated (B and C). *: p < 0.05 for comparison with 0 mg LPS/kg.

Figure 7

The effect of diet on plasma pharmacokinetics of LPS. CD or HCD mice received a single ip injection of 0, 0.5, or 1.0 mg LPS/kg. Values are mean ± SE of 5 mice per group. *: p < 0.05 for comparison with 0 mg LPS/kg for each diet.

Figure 8

The effect of diet on plasma CD14, ApoA1 and LBP. CD14 (A), LBP (B), and ApoA1 (C) were measured by densitometry after western blotting of plasma samples from mice fed CD or HCD for 4 weeks. Representative blots are shown. Bars depict the mean + SE for 5 mice in each group. *: p < 0.05 for comparison with CD mice.

Figure 9

The effect of diet and LPS on hepatic TLR4 mRNA. Representative images of ethidium-stained agarose electrophoresis gels of RT-PCR products of RNA extracted from livers of mice fed CD or HCD for 4 weeks (A). MW is a Lambda HindIII molecular weight marker. Signal intensities were measured as in figure 4A and normalized to the RT-PCR signal for β-actin in each sample (B). Bars depict mean normalized signal ratios + SE for 5 mice fed each diet, relative to the CD group, represented by 1 on the y-axis. *: p < 0.05 for comparison with CD.