Identification of gut microbiota and microbial metabolites regulated by an antimicrobial peptide lipocalin 2 in high fat diet-induced obesity - PubMed (original) (raw)
Identification of gut microbiota and microbial metabolites regulated by an antimicrobial peptide lipocalin 2 in high fat diet-induced obesity
Xiaoxue Qiu et al. Int J Obes (Lond). 2021 Jan.
Abstract
Lipocalin 2 (Lcn2), as an antimicrobial peptide is expressed in intestine, and the upregulation of intestinal Lcn2 has been linked to inflammatory bowel disease. However, the role of Lcn2 in shaping gut microbiota during diet-induced obesity (DIO) remains unknown. We found that short-term high fat diet (HFD) feeding strongly stimulates intestinal Lcn2 expression and secretion into the gut lumen. As the HFD feeding prolongs, fecal Lcn2 levels turn to decrease. Lcn2 deficiency accelerates the development of HFD-induced intestinal inflammation and microbiota dysbiosis. Moreover, Lcn2 deficiency leads to the remodeling of microbiota-derived metabolome, including decreased production of short-chain fatty acids (SCFAs) and SCFA-producing microbes. Most importantly, we have identified Lcn2-targeted bacteria and microbiota-derived metabolites that potentially play roles in DIO and metabolic dysregulation. Correlation analyses suggest that Lcn2-targeted Dubosiella and Angelakisella have a novel role in regulating SCFAs production and obesity. Our results provide a novel mechanism involving Lcn2 as an antimicrobial host factor in the control of gut microbiota symbiosis during DIO.
Conflict of interest statement
Conflict of Interests
There were no potential conflicts of interest relevant to this article.
Figures
Figure 1:. Regulation of Lcn2 expression and secretion in the gut by HFD feeding.
(A-E) Male mice were fed either a RCD or a HFD for 12 weeks and euthanized at the age of 20 weeks. Small intestines (A-B) and large intestines (C-D) were collected and homogenized in RIPA buffer. Lcn2 protein levels were determined by western blotting (A and C) and quantified by ImageJ (B and D). (E-F) Fecal Lcn2 levels were assessed using an ELISA kit. Data are presented as mean ± SEM. (B, D and E). Data was analyzed using a two-tailed unpaired student t-test (B, D, and E) or one-way ANOVA with the Tukey HSD post hoc test (F). n = 4 (B); n = 3 (D); n = 5 (E); n =8 (F). * P < 0.05, ** P < 0.01.
Figure 2:. HFD-induced reshaping of gut microbiota in LKO mice.
Male WT mice (n = 9, 3 cages) and LKO mice (n = 7, 3 cages) were fed a HFD starting at the age of 8 weeks. Fecal samples were collected at 0 (RCD), 2, 4, 8 and 16 weeks of HFD feeding. (A) Alpha diversity metrics: Chao1 and Simpson were shown to demonstrate the richness and evenness of gut microbiota communities. (B-C) Beta diversity (unweighted unifrac) indicates the dissimilarity for all-time points (B), the genotypic difference at each time point (C). Data Analysis: (A) Chao1: Repeated measures ANOVA for WT and LKO, followed by the post hoc test Tukey’s; Simpson: linear quantile mixed model, followed by a FDR-based multiple testing correction procedure. At all time points, n = 9 for WT; n = 7 for LKO. # P < 0.05 versus RCD-fed WT.
Figure 3:. HFD-induced reshaping of gut microbiota in LKO mice.
(A and C) Distribution of bacteria at the phylum level, (B) The ratio of Firmicutes to Bacteroidetes (F/B), and (D) Distribution of bacteria at the major families between WT and LKO mice fed a HFD at all different time points. Data are presented as mean (A, C and D) or as mean ± SEM (B). Data was analyzed using a linear quantile mixed model, followed by a FDR-based multiple testing correction procedure. At all time points, n = 9 for WT; n = 7 for LKO. * q < 0.05 versus WT mice at indicated time point. # q < 0.05, ## q < 0.01 versus RCD-fed WT. + q < 0.05, ++ q < 0.01, +++ q < 0.001, versus RCD-fed LKO mice.
Figure 4:. Identification of differentially abundant bacteria between WT and LKO mice.
Shown are hierarchal clustering analysis (HCA)-based heatmaps on differentially abundant microbes at the genus level between WT (n = 9) and LKO (n = 7) mice based on a negative binomial generalized linear model. The bacteria with significantly increased (A) and decreased (B) abundances in LKO mice. q value < 0.05 and log2 scale fold change > 1 are the criteria to determine the significance. n = 9 for WT; n = 7 for LKO.
Figure 5:. Remodeling of fecal metabolites in LKO mice during HFD consumption.
Stool samples from WT and LKO mice fed a HFD for 0, 4, and 16 weeks were homogenized in 50% ACN. (A) Data from LC-MS analyses of fecal extracts were processed by principal component analysis (PCA) modeling. Shown is the scores plot of a PCA model on the metabolites in fecal extracts. (B-C) Supernatants were collected either for HQ reaction to detect SCFAs (B) or for DC reaction to detect amino acids (C). Data are presented as mean ± SEM and analyzed using a linear quantile mixed model, followed by a FDR-based multiple testing correction procedure. At all time points, n = 9 for WT; n = 7 for LKO. * q < 0.05 versus WT mice at indicated time point. # q < 0.05, ## q < 0.01, ### q < 0.001 versus RCD-fed WT. + q < 0.05, ++ q < 0.01, +++ q < 0.001 versus RCD-fed LKO mice.
Figure 6:. Correlation of body weight with microbes and metabolites.
The correlations between body weight, bacteria and metabolites were evaluated by Spearman’s correlation analyses. _P_-values were corrected by the Benjamini-Hochberg method. P < 0.05 is considered to be significantly associated. Body weight was significantly associated with 14 fecal metabolites, including 3 short-chain fatty acids (negatively in dark green bar), 5 medium/long-chain fatty acids and 6 amino acids (positively in dark red bar), as well as significantly associated with 36 bacteria at the genus level (18 negatively in dark green bar, 18 positively in dark red bar). HCA-based heatmap showed the correlation between the selected fecal metabolites and microbes. Based on the values of Spearman’s rho, Red tiles indicate positive associations and blue tiles negative associations; gray tiles indicate non-significant associations (_P_ > 0.05). Bacteria and metabolites labeled by red rectangle were significantly increased in LKO mice, while those labeled by green were decreased in LKO.
Figure 7:. Time-dependent effect of HFD on intestinal inflammation in LKO mice.
(A-B) Colon tissues were collected from male WT and LKO mice fed a HFD for 2 weeks for the examination of phosphor (S536)-NFκB and total NFκB levels by western blotting (A) and quantified by ImageJ (B). (C) H&E staining of colon from RCD-fed or HFD-fed male WT and LKO mice. Scale bars = 200 μm, x 200. (D-E) Immunohistochemistry of CD11c on paraffin-embedded colon sections from RCD-fed, 2-week HFD-fed, 4-week HFD-fed WT and LKO male mice. (D) Representative images of CD11c stained colon sections, scale bars = 50 μm, x200. (E) Results were quantified by counting the number of CD11c+ cells and measuring the mucosal length. Data are presented as mean ± SEM. Data was analyzed by a two-tailed unpaired student t-test (B) or 2-way ANOVA with the post hoc test Tukey’s; the genotype and diet interaction _P_-value < 0.001 (E). n = 5 for both WT and LKO on RCD and 2-week HFD; n = 6 for both WT and LKO on 4-week HFD; n = 9 for WT and n = 7 for LKO on 16-week HFD. * P < 0.05, ** P < 0.01 versus WT mice. # P < 0.05, ### P < 0.001 versus RCD-fed WT. + P < 0.05, ++ P < 0.01, +++ P < 0.001 versus RCD-fed LKO mice.
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