Enterobacter cloacae administration induces hepatic damage and subcutaneous fat accumulation in high-fat diet fed mice

Abstract

Accumulating evidence indicates that gut microbiota plays a significant role in obesity, insulin resistance and associated liver disorders. Family Enterobacteriaceae and especially Enterobacter cloacae strain B29 have been previously linked to obesity and hepatic damage. The underlying mechanisms, however, remain unclear. Therefore, we comprehensively examined the effects of E. cloacae subsp. cloacae (ATCC® 13047™) administration on host metabolism of mice fed with high-fat diet (HFD). C57BL/6N mice were randomly divided into HFD control, chow control, and E. cloacae treatment groups. The E. cloacae treatment group received live bacterial cells in PBS intragastrically twice a week, every other week for 13 weeks. Both control groups received PBS intragastrically. After the 13-week treatment period, the mice were sacrificed for gene and protein expression and functional analyses. Our results show that E. cloacae administration increased subcutaneous fat mass and the relative proportion of hypertrophic adipocytes. Both subcutaneous and visceral fat had signs of decreased insulin signaling and elevated lipolysis that was reflected in higher serum glycerol levels. In addition, E. cloacae -treated mice had significantly higher hepatic AST and AST/ALT ratio, and their liver histology indicated fibrosis, demonstrating that E. cloacae subsp. cloacae administration promotes hepatic damage in HFD fed mice.

Fig 1. E. cloacae administration increases subcutaneous adipose tissue mass and reduces insulin receptor β expression in SAT.

(A) Weight gain during the treatment period. (B) Food consumption during the treatment period. (C) Average daily energy intake during the treatment period. At necropsy: (D) SAT mass; (E) Insr expression; (F) Xbp1 expression; (G) Ccl expression; (H) Tlr4 expression. All data are presented as mean ± SD. n was 4-6/group when statistical outliers were excluded. The statistical significance was set to p<0.05 and the significant differences are presented with lines and * between the groups.

Fig 2. E. cloacae administration increases lipolysis in SAT.

At necropsy: (A) ACC and HSL phosphorylation; (B) Mgll expression; (C) Serum glycerol levels. All data are presented as mean ± SD. n was 4-6/group when statistical outliers were excluded. The statistical significance was set to p<0.05 and the significant differences are presented with lines and * between the groups.

Fig 3. E.cloacae adminstration decreases the number of leukocytes and increases the size of adipocytes.

(A) and (B) The histological images present the adipose tissues with CD45-staining indicated with arrows. (C) The relative numbers of CD45-positive cells as determined with manual cell counting from the stained tissues, represented as average cell numbers per field of view. (D) The percentages of adipocytes with diameters of 10–20 μm, 20–30 μm and 30–40 μm in each group. 500 cells were randomly selected from each sample. All data are presented as mean ± SD. n was 5-6/group. The statistical significance was set to p<0.05 and the significant differences are presented with lines and * between the groups.

Fig 4. E. cloacae administration increases insulin resistance and lipolysis, but also increases adiponectin expression in VAT.

At necropsy (A) visceral fat mass; (B) Adipoq expression; (C) Rela expression; (D) Insr expression; (E) AKT phosphorylation; (F) HSL phosphorylation; (G) Mgll expression; (H) Ccl2 expression; (I) Tlr4 expression; (J) The relative numbers of CD45-positive cells as determined with manual cell counting from the stained tissues, represented as average cell numbers per field of view. All data are presented as mean ± SD. n was 4-6/group when statistical outliers were excluded. The statistical significance was set to p<0.05 and the significant differences are presented with lines and * between the groups.

Fig 5. E. cloacae administration increases hepatic AST activity and triglyceride synthesizing Dgat2 expression without increasing triglyceride content.

At necropsy: (A) hepatic AST and ALT activity; (B) Dgat2 expression; (C) Acc2 expression; (D) triglyceride content; (E) Adipor expression; (F) AS160 and HSL phosphorylation; (G) Mmp9 expression; (H) ERK phosphorylation. All data are presented as mean ± SD. n was 4-6/group when statistical outliers were excluded. The statistical significance was set to p<0.05 and the significant differences are presented with lines and * between the groups.

Fig 6. Fibrosis is increased in the liver of E. cloacae -treated mice.

(A), (B), and (C) H&E staining; (D), (E) and (F) overexposed anti -smooth muscle actin stainings of the frozen liver tissues. H&E staining shows more ballooning and an increase in lipid droplets in HFD controls and E. cloacae -treated mice compared to chow controls. In SMA staining, an increase in E. cloacae -treated mice compared to chow controls and HFD controls can be seen both in blood vessels (discontinuous arrows) and sites outside the vessels (continuous arrows).

Fig 7. E. cloacae administration increases TLR5 and IL-22 gene expression.

At necropsy: (A) Tjp1 expsession; (B) Tlr5 expression; (C) Il1b expression; (D) Rela expression; (E) Ccl2 expression; (F) Il22 expression. All data are presented as mean ± SD. n was 4-6/group when statistical outliers were excluded. The statistical significance was set to p<0.05 and the significant differences are presented with lines and * between the groups.

Fig 8. Summary of the main findings.

E. cloacae administration increased adipose tissue hypertrophy and hepatic damage in the HFD fed mice. The subcutaneous adipose tissue of the E. cloacae -treated mice appeared to be partly insulin resistant, and the increased lipolysis and adipocyte hypertrophy led to increased glycerol release. Liver fat accumulation did not increase in response to the E. cloacae treatment, but AST activity measurements and histology revealed hepatic damage in the E. cloacae -treated mice.