Adipocytes as a Link Between Gut Microbiota-Derived Flagellin and Hepatocyte Fat Accumulation

Abstract

While the role of both elevated levels of circulating bacterial cell wall components and adipose tissue in hepatic fat accumulation has been recognized, it has not been considered that the bacterial components-recognizing adipose tissue receptors contribute to the hepatic fat content. In this study we found that the expression of adipose tissue bacterial flagellin (FLG)-recognizing Toll-like receptor (TLR) 5 associated with liver fat content (r = 0.699, p = 0.003) and insulin sensitivity (r = -0.529, p = 0.016) in humans (n = 23). No such associations were found for lipopolysaccharides (LPS)-recognizing TLR4. To study the underlying molecular mechanisms of these associations, human HepG2 hepatoma cells were exposed in vitro to the conditioned culture media derived from FLG or LPS-challenged human adipocytes. The adipocyte-mediated effects were also compared to the effects of direct HepG2 exposure to FLG and LPS. We found that the media derived from FLG-treated adipocytes stimulated fat accumulation in HepG2 cells, whereas either media derived from LPS-treated adipocytes or direct FLG or LPS exposure did not. This is likely due to that FLG-treatment of adipocytes increased lipolysis and secretion of glycerol, which is known to serve a substrate for triglyceride synthesis in hepatocytes. Similarly, only FLG-media significantly decreased insulin signaling-related Akt phosphorylation, IRS1 expression and mitochondrial respiratory chain ATP5A. In conclusion, our results suggest that the FLG-induced TLR5 activation in adipocytes increases glycerol secretion from adipocytes and decreases insulin signaling and mitochondrial functions, and increases fat accumulation in hepatocytes. These mechanisms could, at least partly, explain the adipose tissue TLR5 expression associated with liver fat content in humans.

Fig 1

Correlation of liver fat content and Matsuda index (= insulin sensitivity) with (A) adipose tissue TLR5 mRNA and (B) adipose tissue TLR4 mRNA in humans (n = 23).

Fig 2

The (A) direct and (B) adipocyte-mediated effects of flagellin and LPS on the lipid metabolism of HepG2 cells. The figure presents the direct and adipocyte-mediated effects of FLG and LPS on the DGAT2 mRNA fold changes. The values are means + SD (n = 5). The results were normalized to β-actin and the fold changes were calculated with the ΔΔC_t method. The bars on the right side present graphically the total lipid content of HepG2 cells measured spectrophotometrically using Oil Red O after (A) direct 24 hours exposure to FLG and LPS, and (B) 24 hours exposure to conditioned media derived from FLG and LPS-treated adipocytes. The values are means + SD (n = 12). * indicates a p-value of <0.05.

Fig 3. Glycerol is secreted from adipocyte lipid droplets due to increased lipolysis in response to flagellin treatment.

(A) Adipocytes were treated with FLG for 3 hours and glycerol was measured from the cell culture media. The values are means + SD (n = 4). * indicates a p-value of <0.05. (B) Adipocytes were treated with FLG for 1 and 4 hours and changes in the expression of ATGL, PLIN1, FABP3 and MGL mRNA were determined. The values are means + SD (n = 4). ** indicates a p-value of <0.001. The results were normalized to GAPDH and the fold changes were calculated with the ΔΔC_t method. (C) Adipocytes with and without 3-hours FLG treatment were labeled with TLR5 and perilipin antibodies, and imaged with confocal microscope. Compared to 30% in control cells, in 70% of FLG-treated adipocytes degradation of lipid droplets membranes was observed (perilipin-labeling in red). In mature adipocytes TLR5 is mainly located in intracellular pool near lipid droplets and nuclei without co-localization, and only a minority on cell membranes.

Fig 4

(A) The direct, and (B) adipocyte-mediated effects of FLG and LPS on inflammation in HepG2 cells. The figure presents the mRNA fold changes of NF-κB and MMP-9 in response to 4 and 24 hour treatments. The results were normalized to β-actin and the fold changes were calculated with the ΔΔC_t method. The values are means + SD (n = 5). The bars below present graphically the amount of luminometrically measured ROS produced after 24 hours exposure. The values are means + SD (n = 5). * indicates a p-value of <0.05.

Fig 5

(A) The direct, and (B) adipocyte-mediated effects of FLG and LPS on insulin signaling in HepG2 cells. The figure presents the mRNA fold changes of IRS1 in response to 1, 4 and 24 hour treatments (n = 5) and the phosphorylation levels of Akt in response to 30 min, 1 and 4 hour treatments (n = 4). The mRNA results were normalized to β-actin and the fold changes were calculated with the ΔΔC_t method. Akt phosphorylation levels were normalized to GAPDH and the determined intensities of the Western blot bands are presented as arbitrary units. The values are means + SD. * indicates a p-value of <0.05.

Fig 6

(A) The direct and (B) adipocyte-mediated effects of FLG and LPS on the expression of mitochondrial respiratory chain complex subunits in HepG2 cells. The figure presents the expression levels of mitochondrial respiratory chain complex subunits ATP5A (Compl V), UQCRC2 (Compl III), MTCO1 (Compl IV), SDHB (Compl II) and NDUFS8 (Compl I) in response to 4 and 24 hours exposure to FLG and LPS. All expression levels were normalized to GAPDH and the determined intensities of the Western blot bands are presented as arbitrary units. The values are means + SD (n = 3). * indicates a p-value of <0.05.