CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.
BACKGROUND
Abnormal fatty acid composition (FA) in plasma and tissue lipids frequently occurs in homozygous and even in heterozygous carriers of cystic fibrosis transmembrane conductance regulator (CFTR) mutations. The mechanism(s) underlying these abnormalities remained, however, poorly understood despite the potentially CFTR contributing role.


RESULTS
The aim of the present study was to investigate the impact of CFTR depletion on FA uptake, composition and metabolism using the intestinal Caco-2/15 cell line. shRNA-mediated cftr gene silencing induced qualitative and quantitative modifications in FA composition in differentiated enterocytes as determined by gas-liquid chromatography. With the cftr gene disruption, there was a 1,5 fold increase in the total FA amount, largely attributable to monounsaturated and saturated FA compared to controls. The activity of delta-7 desaturase, estimated by the 16:1(n-7)/16:0, was significantly higher in knockdown cells and consistent with the striking elevation of the n-7 FA family. When incubated with [14C]-oleic acid, CFTR-depleted cells were capable of quick incorporation and export to the medium concomitantly with the high protein expression of L-FABP known to promote intracellular FA trafficking. Accordingly, lipoprotein vehicles (CM, VLDL, LDL and HDL), isolated from CFTR knockdown cells, exhibited higher levels of radiolabeled FA. Moreover, in the presence of [14C]-acetate, knockdown cells exhibited enhanced secretion of newly synthesized phospholipids, triglycerides, cholesteryl esters and free FA, thereby suggesting a stimulation of the lipogenic pathway. Conformably, gene expression of SREBP-1c, a key lipogenic transcription factor, was increased while protein expression of the phosphorylated and inactive form of acetylCoA carboxylase was reduced, confirming lipogenesis induction. Finally, CFTR-depleted cells exhibited lower gene expression of transcription factors (PPARalpha, LXRalpha, LXRbeta and RXRalpha).


CONCLUSIONS
Collectively, our results indicate that CFTR depletion may disrupt FA homeostasis in intestinal cells through alterations in FA uptake and transport combined with stimulation of lipogenesis that occurs by an LXR/RXR-independent mechanism. These findings exclude a contributing role of CFTR in CF-associated fat malabsorption.
Mailhot
Geneviève
G
Research Centre, CHU Sainte-Justine, Université de Montréal, Montreal, Quebec, Canada.
Rabasa-Lhoret
Rémi
R
Moreau
Alain
A
Berthiaume
Yves
Y
Levy
Emile
E
D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000378:metabolism; D018938:Caco-2 Cells; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000172:deficiency* / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D016086:Lentivirus / Q000378:metabolism; D050155:Lipogenesis; D026901:Membrane Transport Proteins / Q000378:metabolism; D047492:Peroxisome Proliferator-Activated Receptors / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1 / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D005227:Fatty Acids; D026901:Membrane Transport Proteins; D047492:Peroxisome Proliferator-Activated Receptors; D011956:Receptors, Cell Surface; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000103:Acetyl-CoA Carboxylase
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