Abstract
BackgroundAbnormal 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.Methodology/Principal FindingsThe 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 (PPARα, LXRα, LXRβ and RXRα).Conclusions/SignificanceCollectively, 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.
Highlights
The basic defect in cystic fibrosis (CF) is caused by mutations in the epithelial chloride channel, known as the cystic fibrosis transmembrane conductance regulator (CFTR)
The mechanisms involved in the regulation of fatty acid composition (FA) metabolism by CFTR are largely unknown despite recurrent evidences of abnormal FA composition and lipid profile in CF patients and heterozygous carriers of CFTR mutations [5,7]
If numerous studies have examined the FA status in various cell types derived from CF patients, very few investigations have focused on the functional role of CFTR [3,6]
Summary
The basic defect in cystic fibrosis (CF) is caused by mutations in the epithelial chloride channel, known as the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation remained the deletion of a phenylalanine residue at position 508 (DF508), which causes inappropriate folding of CFTR, followed by its proteolytic degradation in the endoplasmic reticulum This mutation is present in nearly 70% CF patients while 4% of the general population is estimated to be heterozygous carriers of DF508. Decreased levels of linoleic acid (LA; 18:2n-6), docosahexaenoic acid (DHA; 22:6n-3) and normal to increased levels of arachidonic acid (AA; 20:4n-6) have frequently been reported in CF plasma, cells and tissues [5,6,7] These abnormalities occur irrespective of the amount of energy and fat ingested or the pancreatic status, which argues against a nutritional origin and appears more suggestive of abnormalities in FA metabolism [8,9]. The mechanism(s) underlying these abnormalities remained, poorly understood despite the potentially CFTR contributing role
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