A Functional mTORC1 Containing Raptor Controls Luminal Fat Processing and Epithelium Barrier Function in Transwell Caco‐2 Cultures

Abstract

As a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. We created three stable human colorectal adenocarcinoma Caco-2 cell lines exhibiting normal, low (Raptor knockdown) or high (tuberin knockdown) mTORC1 kinase activity, and grew these cells in Transwell to investigate the contribution of mTORC1 in fat absorption, processing, and barrier permeability. Raptor knockdown impaired lipid and apoB-containing lipoprotein metabolism in Caco-2 cultures provided exogenous oleate in the apical chamber. Raptor-depleted Caco-2 cells secreted less apoB100 (-51%), apoB48 (-66%) and triacylglycerol (-69%) compared with control Caco-2 cells. Genes involved in lipogenesis (FASN, fatty acid synthase), fatty acyl-CoA formation (FATP2, very long-chain acyl-CoA ligase; DBI, diazepam binding inhibitor acyl-CoA binding protein), triacylglycerol synthesis (DGAT1, diacylglycerol O-acyltransferase 1; DGAT2, diacylglycerol O-acyltransferase 2), lipoprotein assembly (APOB, apolipoprotein B; MTTP, microsomal triglyceride transfer protein), and pre-chylomicron transport (VAMP7,vesicle-associated membrane protein 7; SAR1B, secretion associated Ras related GTPase 1B) were downregulated by at least 60% in Caco-2 cells depleted of Raptor versus control Caco-2 cells. The cellular protein abundance of apoB (-89%), MTTP (-63%) and FASN (-80%) were decreased in Raptor-depleted versus control Caco-2 cells. When mTORC1 activity was abrogated, Transwell-type Caco-2 cultures were impermeable to ions (high transepithelial electrical resistance, TEER) and had elevated paracellular transport of 4-kDa FITC dextran. Raptor knockdown likewise rapamycin, a mTORC1 inhibitor, decreased the abundance of small-sized solute pore-forming claudins 2 and 15, and the levels of tight junction proteins occludin and tricellulin. In contrast, resistance against ion diffusion decreased (low TEER) and paracellular transport of 4-kDa FITC dextran increased as mTORC1 activity rose. In conclusion, a functional intestinal mTORC1 is critically important for processing dietary lipids into chylomicrons and establishing the permeability characteristics of the epithelium.