Commensal probiotic bacteria affect host cellular lipid metabolism through various cellular metabolic pathways: Role of mTOR, FOXO1, and autophagy machinery system

Aim & Background:

Although in vivo studies revealed that lactic acid bacteria (LAB) of the natural intestinal microflora prevent metabolic diseases through the control of host energy metabolism, the precise molecular bases have not been fully elucidated. As mTOR signalling and autophagy machinery system negatively cross-regulate each other's outcome and regulate cellular lipid metabolism, we investigated the influence of Bifidobacterium breve (B.b) and Lactobacillus rhamnosus GG (LGG) on mTOR signalling and inflammation/metabolic markers in different cell types -based two-dimensional (2D) and three-dimensional (3D) cell culture models.

Methods:

To mimic the natural situation of macrophage-controlled mucosal epithelia in the gut- immune system- liver-axis, different cell types -based 2D and 3D cell culture models (mono-, co-, and tri-cultivation) were investigated. First, mono-cultivation of 3D Hepatocytes (Hu7h and HepG2 cells) and 3D human aortic endothelial cells (HAECs) was established in 3D scaffolds to verify whether they, if any, secrete inflammatory markers in response to our desired stimuli. Then, the epithelial monolayers – 3D Hepatocytes co-culture model were investigated to find the most effective model in terms of tight junction resistance of the human gastrointestinal epithelial cells. Thereafter, the 2D enterocyte (HT-29/B6 cells) – 3D macrophages (HMDMs)- 3D Hepatocytes (HepG2 cell) triple co-culture model were developed to assess the transmit of the bacterial effects in the gut- immune system- liver-axis, which are intricately linked. Monolayer barrier integrity was assessed by measuring TEER and leakage of LPS. Cell-based ELISA and Western blot (WB) were used to assess expression of IL-1β, IL-6, IL-8, TNF-α, CB1, FOXO1, PEPCK, G6Pase, mTOR, FOXO1, adhesion molecules (VCAM-1, ICAM-1), autophagy markers (LC3-I, LC3-II, Beclin-1, p62) as well as mTOR kinase activity and cellular triglycerides. Autophagy was also quantitatively assessed by fluorescence microscopy and Tecan GENios Multi-Detection Microplate Reader. DNA-binding activity, nuclear translocation of NF-kB (IkBα degradation, expression of p50/p65 subunits), and NF-κB-dependent luciferase activity were assessed using EMSA, WB and a TECAN GENios microplate reader respectively. ApoC-III gene expression was assessed by qRT-PCR. FOXO1/mTOR siRNA was used to silence FOXO1 and mTOR gene.

Results:

LAB inhibited oleate/LPS-induced IL-1β, IL-6, TNF-α, and cellular triglycerid, accompanied by reduction of mTOR and FOXO1 activity and enhanced occurrence of autophagy, as manifested by increased LC3-II/LC3-I ratio and decreased expression of Beclin-1 and p62.

Conclusion:

We describe a new mechanism whereby LAB prevent high fat diet/endotoxin/inflammation-mediated disruption of normal cellular lipid metabolism by inhibiting mTOR/FOXO1/NF-kB activity and enhancing the occurrence of autophagy. This may be a molecular basis by which LAB enhance intrinsic cellular tolerance against excess calorie consumption and participate in homeostatic regulation of metabolic processes in vivo.