MTORC1 is required for the lipidation and ER‐to‐Golgi transport of prechylomicrons in Caco‐2 cells

Abstract

Dietary fats are efficiently digested and absorbed by enterocytes, assembled into triacylglycerol‐rich chylomicrons and finally secreted in a multistep process. The role of mTORC1 (mechanistic target of rapamycin complex 1) in the fate of absorbed lipids, their intracellular trafficking, triacylglycerol packaging in chylomicrons by enterocytes has not been previously studied. On the basis of our previous work in hepatocytes showing that mTORC1 positively regulate VLDL production, we hypothesized that intestinal mTORC1 supports the production and secretion of chylomicrons. Lentivirus mediated shRNA technology was used to knockdown (>90% down) mTORC1 signaling‐associated proteins, Raptor and TSC2, to create three Caco‐2 cell lines that exhibit basal (shScramble cells), low (shRaptor cells) or high (shTSC2 cells) mTORC1 activity. Chylomicron production was primed with the addition of 1.2 mM oleate/1.5% BSA complex to the apical side of ThinCert™ cell culture inserts. We found that knocking down Raptor disrupts triacylglycerol synthesis and chylomicron assembly. Knockdown of Raptor decreased the mRNA levels of APOB (−87%), MTTP (−94%), FASN (−76%), DGAT1 (−77%) and DGAT2 (−81%) compared with control shScramble cells. The cellular protein abundance of apoB (−89%), MTTP (−63%) and FASN (−80%), and the secretion of apoB100 (−51%), apoB48 (−66%) and triacylglycerols (−69%) were decreased compared with control shScramble cells. Rapamycin (a mTORC1 inhibitor) lowered the secretion of apoB100 and apoB48 in shScramble cells by 47% and 75%, respectively. Knockdown of Raptor decreased the gene expression of prechylomicron transport vesicle‐associated proteins VAMP7 (vesicle‐associated membrane protein 7) and SAR1B (secretion associated Ras related GTPase 1B) by 85% and 70%, respectively, compared with shScramble cells, suggesting that prechylomicron trafficking from the ER to the Golgi was repressed in the absence of Raptor. In conclusion, our studies revealed the critical role of mTORC1 in chylomicron metabolism. These findings have broad implications for the absorption of essential fatty acids and lipid‐soluble vitamins, for chylomicron retention disease, chylomicron overproduction and associated hypertriglyceridemia.Support or Funding InformationUSDA Hatch Act, USDA‐NIFA