Modulation of GSK – 3β/β – catenin cascade by commensal bifidobateria plays an important role for the inhibition of metaflammation-related biomarkers in response to LPS or non-physiological concentrations of fructose: An in vitro study

Highlights:

We describe a new regulatory mechanism in which commensal bifidobacteria inhibit the NF-κB-mediated transcriptional activation of metabolic disorder-causing factors (IL-1β, IL-6, TNF-α) and interfere with the Wnt/β – catenin pathway, which positively regulates NF-κB activity, thereby simultaneously reducing cellular triglyceride concentrations and enhancing cellular consumption of energy-yielding substrates.

The abbreviations used are:

Akt,	Protein kinase B
ASK1,	Apoptosis signal-regulating kinase 1
ECL,	Enhanced chemiluminescence
Fetuin-A,	Major carrier protein of free fatty acids in the blood stream
GS,	Glycogen synthase
GSK-3β,	Glycogen synthase kinase-3beta
HMDMs,	Human monocytes-derived macrophages
IkBα,	Inhibitor of NF-kB alpha
IL-1β,	Interleukin-1 beta
IL-6,	Interleukin-6
LAPB,	Lactic acid producing bacteria
LPS,	Lipopolysaccharide
NF-kB,	Nuclear factor-kobo
p-Akt,	phosphorylated Akt
RT	reverse transcription
siRNA,	Small interfering RNA (or short interfering RNA or silencing RNA)
TEER,	Transepithelial Electrical Resistance
TLR,	Toll-like receptor
TNF-α,	Tumor necrosis factor-alpha
WB,	Western blotting
Wnt,	Wingless/integrated
TG,	Triglycerides

Aim & Background:

Increasing evidence indicates that commensal bifidobacteria of the natural intestinal microbiota play a critical role in energy metabolism in vivo. However, the precise molecular basis and biochemical causes are not well understood. It has been shown that there is a reciprocal regulation of the TLR4/NF-kB and the Wnt/β-catenin pathway in vivo. We, therefore, aimed at investigating the possible role of this system of reciprocal regulation in the modulation of cellular energy metabolism by Bifidobacterium breve (B. breve) and Lactobacillus rhamnosus GG (LGG) in three different cell types-based 2D and 3D co-culture models.

Methods & Material:

We first used a traditional triple 2D co-culture model of intestinal epithelial HT-29/B6 cell line with human monocyte-derived macrophage (HMDM) cells and hepatocyte-derived HepG2 cell line. We then assembled and combined a traditional triple 2D co-culture model of 2D intestinal epithelial HT-29/B6 cell line with scaffold-based 3D human monocyte-derived macrophage (HMDM) cells and 3D hepatocyte-derived HepG2 cell line, combined in 2D and 3D conditions. Then, B. breve (DSMZ 20213) and LGG (ATCC 53103) were added to the apical side of the triple cell co-cultures, in the absence or presence of LPS and/or fructose in non-physiological concentration. Monolayer barrier integrity was assessed by measuring TEER. Cell-based ELISA was used to assess protein expression of IL-1β, IL-6, IL-17, IL-37, and TNF-α, as well as cellular TG and Fetuin-A. WB was used to assess protein expression of Akt, GSK3β, GS, ASK1, β-catenin, IkBα, NF-kB p65 subunit, p38 MAPK, and Wnt3a/5a. TaqMan quantitative RT-PCR was used to assess also mRNA transcription of wnt3a and wnt5a. NF-κB-dependent luciferase activity was assessed using a TECAN GENios multimode microplate reader. To elucidate the regulatory role of β-catenin, expression was knocked down using siRNA.

Results:

Bifidobacteria inhibited LPS- and fructose-induced expression of inflammatory cytokines (IL-1β, IL-6, TNF-α) in luminal cell milieu of co-cultured cells, concomitant with decreases in cellular TG, Fetuin-A, and Hepcidin in 3D HepG2 cells. Exposure of co-cultured cells to bifidobacteria inhibited the LPS- and fructose-reduced GSK-3β and Akt phosphorylation, and blocked the NF-kB activity through inhibition of IκBα, ASK1, GS, p38 MAPK and NF-kB 65 subunit phosphorylation. The loss of NF-κB activity was also associated with a significant increase of total β-catenin protein levels, as reflected by a decrease in phosphorylation at the serine 33/37 residues. Quantitative RT- PCR and western blot analysis revealed that Bifidobacteria inhibited LPS- and/or fructose-induced mRNA and protein expression of wnt5a (as known ligand for TLR4). However, the pattern of wnt3a mRNA and protein expression was quite the opposite of wnt5a.

Conclusion:

We describe a regulatory mechanism in which commensal bifidobacteria prevent LPS- and/or fructose-mediated transcriptional activation of metabolic disorder-causing factors (IL-1β, IL-6, TNF-α) and disruption of normal cellular energy metabolism by attenuating NF-kB activity and interfering with the Wnt/β-catenin pathway. The latter positively regulates NF-κB activity, thereby simultaneously reducing cellular triglyceride concentrations and enhancing cellular glucose consumption. This may be a molecular basis by which commensal bifidobacteria enhance intrinsic cellular tolerance against excess consumption of energy – yielding substrates and participate in homeostatic regulation of metabolic processes in vivo.