Chronic oral LPS administration does not increase inflammation or induce metabolic dysregulation in mice fed a western-style diet

Abstract

Introduction: Lipopolysaccharides (LPS) present in the intestine are suggested to enter the bloodstream after consumption of high-fat diets and cause systemic inflammation and metabolic dysregulation through a process named "metabolic endotoxemia." This study aimed to determine the role of orally administered LPS to mice in the early stage of chronic low-grade inflammation induced by diet.

Methods: We supplemented the drinking water with E. coli derived LPS to mice fed either high-fat Western-style diet (WSD) or standard chow (SC) for 7 weeks (n = 16-17). Body weight was recorded weekly. Systemic inflammatory status was assessed by in vivo imaging of NF-κB activity at different time points, and glucose dysregulation was assessed by insulin sensitivity test and glucose tolerance test near the end of the study. Systemic LPS exposure was estimated indirectly via quantification of LPS-binding protein (LBP) and antibodies against LPS in plasma, and directly using an LPS-sensitive cell reporter assay.

Results and discussion: Our results demonstrate that weight development and glucose regulation are not affected by LPS. We observed a transient LPS dependent upregulation of NF-κB activity in the liver region in both diet groups, a response that disappeared within the first week of LPS administration and remained low during the rest of the study. However, WSD fed mice had overall a higher NF-κB activity compared to SC fed mice at all time points independent of LPS administration. Our findings indicate that orally administered LPS has limited to no impact on systemic inflammation and metabolic dysregulation in mice fed a high-fat western diet and we question the capability of intestinally derived LPS to initiate systemic inflammation through a healthy and uncompromised intestine, even when exposed to a high-fat diet.

Keywords: LPS; NF-κB activation; intestinal permeability; low-grade inflammation; obesity.

Figure 1

LPS content measured by the HEK-Blue^™ TLR4 assay, and gene expression in the small intestine. (A) Duodenal and ileal LPS levels in luminal content from SC fed mice given LPS (0.33 mg/mL, estimated daily intake of 2 mg LPS) through drinking water for 1, 4 or 8 days. N = 4–7 per group. One-way ANOVA compared to the mean of control group (non-treated mice). (B) Duodenal and ileal LPS levels in mice fed WSD or SC diet for 7 weeks, and 35 days of oral LPS administration (0.33 mg/mL, estimated daily intake of 2 mg LPS), n = 11–17. Relative mRNA expression of (C) interleukin 1 beta (Il1b) and (D) Tumor necrosis factor alpha (Tnfa) in mucosa from the small intestine. n = 15–17. Two-way ANOVA with assessment of simple main effects (treatment, no-LPS versus LPS; diet, WSD versus SC) and interaction effect (treatment × diet) with Tukey’s correction for multiple comparisons (B–D). Data are presented as mean ± SEM. LPS, lipopolysaccharide; SC, standard chow; WSD, western-style diet.

Figure 2

Weight and food intake assessed in mice fed WSD or SC diet for 7 weeks, and 35 days of oral LPS administration (0.33 mg/mL, estimated 2 mg LPS/mouse/day). (A) Body weight gain (G) of WSD or SC fed mice for 7 weeks. Week 0 (yellow triangle) indicates the start of LPS administration. (B) AUC body weight gain (g). (C) Weekly energy intake per diet group expressed as kcal/mouse/day. Week 0 (yellow triangle) indicates the start of LPS administration. (D) Total energy intake per diet group expressed as kcal/mouse. Two-way ANOVA with assessment of simple main effects (treatment, no-LPS versus LPS; diet, WSD versus SC) and interaction effect (treatment × diet) with Tukey’s correction for multiple comparisons (B–D). Data are presented as mean ± SEM. N = 16–17 per group. AUC, area under the curve; LPS, lipopolysaccharide; ns, non-significant; SD, standard chow; WSD, western-style diet.

Figure 3

Oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IpITT) in mice fed WSD or SC diet for 7 weeks, and 35 days of oral LPS administration (0.33 mg/mL, estimated 2 mg LPS/mouse/day). (A) OGTT. (B) Total glucose excursion after OGTT expressed as area under the curve. (C) IpITT. (D) AUC IpITT. (E) IAP activity from duodenal lumen content. Two-way ANOVA with assessment of simple main effects (treatment, no-LPS versus LPS; diet, WSD versus SC) and interaction effect (treatment × diet) with Tukey’s correction for multiple comparisons (B,D,E). Data are presented as mean ± SEM. AUC, area under the curve; IAP, intestinal alkaline phosphatase; LPS, lipopolysaccharide; ns, non-significant, WSD, western-style diet.

Figure 4

In vivo imaging of NF-κB activation during 35 days of LPS administration in drinking water (0.33 mg/mL, estimated 2 mg LPS/mouse/day). Day 0 indicates start of oral LPS administration Graphs display the photon emission in region of interest (ROIs) representing NF-κB activity in the liver region (A) and abdominal region (B) N = 8 per group. (C) Panel displays representative images of one mouse per group at different time points. Color bar indicates intensity of light emission from the mice. Two-way ANOVA with assessment of simple main effects (treatment, no-LPS versus LPS; diet, WSD versus SC) and interaction effect (treatment × diet) with Tukey’s correction for multiple comparisons (B,C). Data are presented as mean ± SEM. LPS, lipopolysaccharide; NF-κB, nuclear factor-kappaB; SC-W, standard chow + water; SC-LPS, standard chow + LPS; WSD, western-style diet; WSD-W, western-style diet + water; WSD-LPS, western-style diet + LPS.

Figure 5

Plasma measurements of LBP, IL-6, immunoglobulins and LPS. (A) Measurement of plasma LBP, (B) IL-6, (C) Anti-LPS (O55:B5) IgG, (D) Anti-LPS IgG (O128:B12) and (E) Anti-flagellin IgG in mice fed WSD or chow diet for 7 weeks, and 35 days of oral LPS administration (0.33 mg/mL, estimated 2 mg LPS/mouse/day). Two-way ANOVA with assessment of simple main effects (treatment, no-LPS versus LPS; diet, WSD versus SC) and interaction effect (treatment × diet) with Tukey’s correction for multiple comparisons (A–E). (F) Relative plasma LPS levels with normalized values from mice administered with oral LPS for 8 days compared to control group (water). One-way ANOVA compared to the mean of control group (non-treated mice). Data are presented as mean ± SEM. N = 16–17 (LBP), 7 (IL-6, anti-flagellin IgG and anti-LPS IgG), and 5 (LPS) per group. IgG, immunoglobulin G; LBP, LPS-binding protein; LPS, lipopolysaccharide; ns, non-significant; WSD, western-style diet.