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CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

Mailhot G, Rabasa-Lhoret R, Moreau A, Berthiaume Y, Levy E - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Research Centre, CHU Sainte-Justine, Université de Montréal, Montreal, Quebec, Canada.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

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Related in: MedlinePlus

Total, cellular and medium free fatty acid (FFA) content of Caco-2/15 cells.Mock-infected and knockdown cells were differentiated for 12 days and incubated with [14C]-oleic acid for 24 h. Lipids of cell homogenates and medium were extracted with chloroform-methanol, isolated by TLC and the radioactivity incorporated into FFA fraction determined. Results were analyzed as dpm/mg of total protein but were reported as a proportion of mock-infected values representing 100%. Data represented means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells.
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pone-0010446-g004: Total, cellular and medium free fatty acid (FFA) content of Caco-2/15 cells.Mock-infected and knockdown cells were differentiated for 12 days and incubated with [14C]-oleic acid for 24 h. Lipids of cell homogenates and medium were extracted with chloroform-methanol, isolated by TLC and the radioactivity incorporated into FFA fraction determined. Results were analyzed as dpm/mg of total protein but were reported as a proportion of mock-infected values representing 100%. Data represented means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells.

Mentions: To verify whether changes in FA concentration might be accounted for by alterations in FA uptake and output by the intestinal cell, we incubated cells with a common dietary FA, [14C]-oleic acid, and determined the amount of radioactivity present in various cellular and secreted lipid fractions. CFTR knockdown cells displayed a 4-fold increase in the amount of FFA exported to the basolateral medium whereas no changes were recorded in the cellular amount of FFA (Figure 4). Subsequently, we measured the amount of FA transported by the lipoprotein classes: CM, VLDL, LDL and HDL isolated from CFTR knockdown cells. They displayed higher levels of FFA compared to control mock-infected cells: 186%, 153%, 143%, and 719%, respectively (Figure 5).


CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

Mailhot G, Rabasa-Lhoret R, Moreau A, Berthiaume Y, Levy E - PLoS ONE (2010)

Total, cellular and medium free fatty acid (FFA) content of Caco-2/15 cells.Mock-infected and knockdown cells were differentiated for 12 days and incubated with [14C]-oleic acid for 24 h. Lipids of cell homogenates and medium were extracted with chloroform-methanol, isolated by TLC and the radioactivity incorporated into FFA fraction determined. Results were analyzed as dpm/mg of total protein but were reported as a proportion of mock-infected values representing 100%. Data represented means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864762&req=5

pone-0010446-g004: Total, cellular and medium free fatty acid (FFA) content of Caco-2/15 cells.Mock-infected and knockdown cells were differentiated for 12 days and incubated with [14C]-oleic acid for 24 h. Lipids of cell homogenates and medium were extracted with chloroform-methanol, isolated by TLC and the radioactivity incorporated into FFA fraction determined. Results were analyzed as dpm/mg of total protein but were reported as a proportion of mock-infected values representing 100%. Data represented means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells.
Mentions: To verify whether changes in FA concentration might be accounted for by alterations in FA uptake and output by the intestinal cell, we incubated cells with a common dietary FA, [14C]-oleic acid, and determined the amount of radioactivity present in various cellular and secreted lipid fractions. CFTR knockdown cells displayed a 4-fold increase in the amount of FFA exported to the basolateral medium whereas no changes were recorded in the cellular amount of FFA (Figure 4). Subsequently, we measured the amount of FA transported by the lipoprotein classes: CM, VLDL, LDL and HDL isolated from CFTR knockdown cells. They displayed higher levels of FFA compared to control mock-infected cells: 186%, 153%, 143%, and 719%, respectively (Figure 5).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Research Centre, CHU Sainte-Justine, Université de Montréal, Montreal, Quebec, Canada.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH
Related in: MedlinePlus