Endotoxemia by Porphyromonas gingivalis Injection Aggravates Non-alcoholic Fatty Liver Disease, Disrupts Glucose/Lipid Metabolism, and Alters Gut Microbiota in Mice

Abstract

Many risk factors related to the development of non-alcoholic fatty liver disease (NAFLD) have been proposed, including the most well-known of diabetes and obesity as well as periodontitis. As periodontal pathogenic bacteria produce endotoxins, periodontal treatment can result in endotoxemia. The aim of this study was to investigate the effects of intravenous, sonicated Porphyromonas gingivalis (Pg) injection on glucose/lipid metabolism, liver steatosis, and gut microbiota in mice. Endotoxemia was induced in C57BL/6J mice (8 weeks old) by intravenous injection of sonicated Pg; Pg was deactivated but its endotoxin remained. The mice were fed a high-fat diet and administered sonicated Pg (HFPg) or saline (HFco) injections for 12 weeks. Liver steatosis, glucose metabolism, and gene expression in the liver were evaluated. 16S rRNA gene sequencing with metagenome prediction was performed on the gut microbiota. Compared to HFco mice, HFPg mice exhibited impaired glucose tolerance and insulin resistance along with increased liver steatosis. Liver microarray analysis demonstrated that 1278 genes were differentially expressed between HFco and HFPg mice. Gene set enrichment analysis showed that fatty acid metabolism, hypoxia, and TNFα signaling via NFκB gene sets were enriched in HFPg mice. Although sonicated Pg did not directly reach the gut, it changed the gut microbiota and decreased bacterial diversity in HFPg mice. Metagenome prediction in the gut microbiota showed enriched citrate cycle and carbon fixation pathways in prokaryotes. Overall, intravenous injection of sonicated Pg caused impaired glucose tolerance, insulin resistance, and liver steatosis in mice fed high-fat diets. Thus, blood infusion of Pg contributes to NAFLD and alters the gut microbiota.

Keywords: Porphyromonas gingivalis; endotoxemia; gut microbiota; non-alcoholic fatty liver disease; periodontitis.

FIGURE 1

Comparison of body weight, fasting glucose/insulin concentration and body fat between HFco and HFPg mice. (A) Body weight (B) fasting blood glucose at 12 weeks (C) fasting plasma insulin at 12 weeks (D) Photographs of Micro-CT imaging. Yellow region represents visceral fat area and orange region represents subcutaneous fat area; (E) The volume of total fat area, subcutaneous fat area, and visceral fat area evaluated by Micro-CT imaging at 12 weeks (n = 5). ^∗P < 0.05, ^∗∗P < 0.01 HFco vs. HFPg (unpaired t-test).

FIGURE 2

Comparison of glucose tolerance and insulin resistance between HFco and HFPg mice. (A) OGTT (1 g/kg) and (B) ITT (1.5 U/kg) performed 6 h fasting at 12 weeks (n = 9).^∗P < 0.05 HFco vs. HFPg (unpaired t-test).

FIGURE 3

evaluation of liver steatosis. Oil red o staining of liver tissue from (A) HFco, (B) HFg mice (row magnification × 200, yellow bar = 100 μm), and (C) lipid area (%). (D) Triglyceride and (E) glycogen in the liver of HFco and HFPg mice at 12 weeks (n = 5). (F) Glut2, G6p, Glck, and Pepck; (G) Srebp1c and Acc1; (H) Tnfa and Ilb; (I) Tgfb expressions in HFco and HFPg mice at 12 weeks (n = 5).^∗P < 0.05, ^∗∗P < 0.01 (unpaired t-test).

FIGURE 4

Microarray analysis in the liver between HFco and HFPg mice (n = 4). (A) Volcano plots, (B) PCA analysis.

FIGURE 5

Microarray analysis in the liver between HFco and HFPg mice (n = 4). Gene Ontology in DEGs.

FIGURE 6

Microarray analysis in the liver between HFco and HFPg mice. (A) KEGG pathways in the upregulated DEGs (n = 4), (B) Acot1, Acot2, Acot3, Acot4, Aldh3a2, Cpt1b, Cyp4a10, Cyp4a14, Cyp4a31, and Ehhadh expressions in the HFco and HFPg mice at 12 weeks (n = 5) ^∗P < 0.05, ^∗∗P < 0.01 (unpaired t-test).

FIGURE 7

GSEA with hallmark gene sets enriched in HFPg mice compared to HFco mice (n = 4). (A) Gene sets showing FDR q < 0.25. NES: normalized enrichment score. (B) Gene set related to fatty acid metabolism. A heatmap provided illustrating gene expression levels for each gene in the core enrichment subset (blue: low, red: high).

FIGURE 8

Evaluation of gut microbiome compositions based on 16S rRNA gene sequences between HFco and HFPg mice (n = 4). (A) PCoA analysis, (B) rarefaction curve, (C) number of OTUs, (D) Shannon index, (E) Chao1 index between HFco and HFPg mice.

FIGURE 9

Evaluation of gut microbiome compositions based on 16S rRNA gene sequences between HFco and HFPg mice at a Phylum level (n = 4) (unpaired t-test with FDR).

FIGURE 10

Evaluation of gut microbiome compositions based on 16S rRNA gene sequences between HFco and HFPg mice at a Family level (n = 4). Dendrogram and heatmap was constructed based on read abundance. ^∗adjusting P < 0.05 between HFco and HFPg mice (unpaired t-test with FDR).

FIGURE 11

Evaluation of gut microbiome compositions based on 16S rRNA gene sequences between HFco and HFPg mice at a Genus level (n = 4). Dendrogram and heatmap constructed based on read abundance. ^∗adjusting P < 0.05 between HFco and HFPg mice (unpaired t-test with FDR).

FIGURE 12

Evaluation of gut microbiome compositions based on 16S rRNA gene sequences between HFco and HFPg mice (n = 4). Distributions of the species between HFco and HFPg mice (>1.0% relative abundance). The species name or 16S ribosomal RNA database ID in DDBJ is shown. ^∗adjusting P < 0.05 between HFco and HFPg mice (unpaired t-test with FDR).

FIGURE 13

Metagenome prediction of level-2 subsystem between HFco and HFPg mice (n = 4). ^∗adjusting P < 0.05 between HFco and HFPg mice (unpaired t-test with FDR).

FIGURE 14

Metagenome prediction between HFco and HFPg mice. (n = 4) (A) Dendrogram and heatmap constructed based on metagenome prediction. (B) Predicted KEGG pathways present in any of samples for HFco (upper figure) and HFPg (lower figure). Middle figure shows significantly enriched pathway. Blue, HFco. Red, HFPg.

FIGURE 15

A mechanistic model summarizing the findings of the study.