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

Abstract

Aim & backgroundAs 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 on mTOR signalling and inflammation/metabolic markers in three different cell types -based two-dimensional and three-dimensional cell culture models. MethodsFirst, mono-cultivation of 3D Hepatocytes and 3D human aortic endothelial cells was established in 3D scaffolds. Then, the 2D enterocyte monolayers and 3D Hepatocytes were co-cultured. Thereafter, the 2D enterocyte - 3D macrophages - 3D Hepatocytes triple co-culture models were developed. Monolayer barrier integrity was assessed by measuring TEER and leakage of LPS. Cell-based ELISA and Western blot 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. ResultsLAPB inhibited oleate/LPS-induced IL-1β, IL-6, TNF-α, and cellular TG, 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. ConclusionLAPB 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 LAPB enhance intrinsic cellular tolerance against excess calorie consumption and participate in homeostatic regulation of metabolic processes in vivo.