Fig 2: Antibiotics treatment decreases hepatic Lbp expression and supernatant from RAW 264.7 macrophages pretreated with LPS induces Lbp expression in primary hepatocytes. (A) Bacterial count in the cecum. (B) Hepatic Lbp expression. (C) Expression of Lbp in primary hepatocytes treated with LPS or supernatant from RAW 264.7 macrophages treated with LPS. n = 6–8 (A–B); n = 4 (C). ∗P < 0.05∗∗, P < 0.01, and ∗∗∗P < 0.001 determined by Student's t-test (A–B) or one-way analysis of variance and using Tukey's multiple comparisons test (C).

Fig 3: Interaction between LPS and LBP increases JNK phosphorylation and decreases IRS1 phosphorylation in response to insulin in primary hepatocytes. (A) Immunoblot showing the effect of LPS exposure on phosphorylation of IRS1 (Y612) and JNK in response to insulin. (B) Immunoblot showing the effect of LBP exposure on phosphorylation of IRS1 (Y612) and JNK in response to insulin in the presence of LPS. (C) Semi-quantitative analysis of the phosphorylation levels in panel B. (D) Immunoblot showing the effect of the LBP-blocking peptide LBPK95A on phosphorylation of IRS1 (Y612) and JNK in response to insulin in the presence of LPS and LBP. (E) Semi-quantitative analysis of the phosphorylation levels in panel D. n = 3. ∗∗P < 0.01 and ∗∗∗P < 0.001 determined by one-way analysis of variance and using Tukey's multiple comparisons test between all of the groups.

Fig 4: Analysis of hepatic gene expression quantified by microarray in the CONV-R and GF wild-type and Myd88 KO mice. (A) Principle component analysis of transcriptional data. PC1, principal component 1; PC2, principal component 2. (B) Venn diagram showing the numbers of genes significantly regulated by the gut microbiota (at a 5% false discovery rate) in the wild-type mice (beige), Myd88 KO mice (red), and both strains (intersection between circles). Genes more than 2-fold upregulated (C) or downregulated (D) in the CONV-R wild-type mice compared to the CONVR Myd88 KO mice, GF wild-type mice, and GF Myd88 KO mice. Wild-type CONV-R, n = 5; wild-type GF, n = 3; Myd88 CONV-R, n = 5; Myd88 GF, n = 6. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 determined by one-way analysis of variance and using Tukey's multiple comparisons test. Hierarchical multivariate statistical analysis of the genes presented in C-D resulted in the following P values: Lbp: Pmicro = 0.00015, Pgeno = 0.30, Pinteraction = 0.0092; Saa2: Pmicro = 0.00020, Pgeno = 0.45, Pinteraction = 0.0075; Atp11a: Pmicro = 6.8e-06, Pgeno = 0.044, Pinteraction = 0.0045; Adgrf1: Pmicro = 7.9e-05, Pgeno = 0.047, Pinteraction = 0.026; Hsd3b2: Pmicro = 2.4e-06, Pgeno = 0.10, Pinteraction = 0.0063.

Fig 5: Blocking of interaction between LPS and LBP with LBPK95A in vivo improves systemic glucose metabolism. (A) Fasting glucose levels, (B) fasting insulin levels, and (C–D) glucose tolerance in the mice treated with LBPK95A or saline control. n = 5. ∗P < 0.05 and ∗∗P < 0.01 determined by Student's t-test.