Insulin increases cholesterol uptake, lipid droplet content, and apolipoprotein B secretion in CaCo-2 cells by upregulating SR-BI via a PI3K, AKT, and mTOR-dependent pathway

Marcela Fuentes; Nicolás Santander; Víctor Cortés

doi:10.1002/jcb.27410

Insulin increases cholesterol uptake, lipid droplet content, and apolipoprotein B secretion in CaCo-2 cells by upregulating SR-BI via a PI3K, AKT, and mTOR-dependent pathway

J Cell Biochem. 2019 Feb;120(2):1550-1559. doi: 10.1002/jcb.27410. Epub 2018 Oct 2.

Authors

Marcela Fuentes¹, Nicolás Santander¹, Víctor Cortés¹

Affiliation

¹ Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.

PMID: 30278109
DOI: 10.1002/jcb.27410

Abstract

The actions of insulin on intestinal cholesterol absorption and lipoprotein secretion are not well understood. Herein, we determined the effects of insulin on the levels of cholesterol transporter scavenger receptor, class B, type I (SR-BI), cellular cholesterol uptake, intracellular lipid accumulation, and lipoprotein secretion in a cellular model of human intestinal epithelium.

Methods: CaCo-2 cells were cultured to postconfluency in Transwell filters and stimulated with glucose (25 mM) in the presence or absence of insulin (100 nM) at their basolateral surface. SR-BI mRNA and protein levels were quantified by quantitative reverse transcription-PCR and immunoblot, respectively. Polarized localization of SR-BI was determined by cell surface proteins biotinylation and streptavidin precipitation. Activities of PI3K, AKT, mTOR, and SR-BI were pharmacologically antagonized. Cholesterol uptake was assessed by NBD-cholesterol incorporation. Apolipoprotein (apo) B concentration was quantified by ELISA. Subcellular localization of neutral lipids (BODIPY) and SR-BI (immunofluorescence) was determined by confocal microscopy.

Results: In polarized CaCo-2 cells, insulin increased SR-BI at the mRNA and protein levels. SR-BI was exclusively present at apical cell surface, as indicated by biotinylation and confocal microscopy analysis. Glucose did not modify SR-BI abundance or subcellular localization. Effects of insulin on SR-BI levels were abrogated by PI3K, AKT, or mTOR pharmacological antagonism. Cholesterol uptake, neutral lipid abundance, and apo B secretion were increased by insulin in CaCo-2 cells, and these effects were prevented by SR-BI pharmacological antagonism with block lipid transport-1.

Conclusions: insulin promotes cholesterol uptake, intracellular lipid store, and apo B-containing lipoproteins secretion by SR-BI-dependent mechanisms in a model of human intestinal epithelium.

Keywords: CaCo-2 cells; cholesterol; insulin; intestine; lipoprotein; scavenger receptor, class B, type I (SR-BI).

Abstract

Grants and funding