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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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Effect of CFTR knockdown on protein expression of CD36 (A) and L-FABP (B).After 15 days of differentiation, Caco-2/15 cells were lysed and protein expression determined by Western Blot. β-actin served as loading control and was used for normalization. Data represented means ± SEM of three to four independent experiments *p<0.05 vs mock cells.
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pone-0010446-g006: Effect of CFTR knockdown on protein expression of CD36 (A) and L-FABP (B).After 15 days of differentiation, Caco-2/15 cells were lysed and protein expression determined by Western Blot. β-actin served as loading control and was used for normalization. Data represented means ± SEM of three to four independent experiments *p<0.05 vs mock cells.

Mentions: To elucidate the mechanisms underlying the significant raise of FA upon reduction in CFTR expression, we assessed the expression of two proteins involved in FA intestinal uptake and transport. First, the protein expression of FAT/CD36, a transporter involved in FA uptake, displayed a 1,5-fold increase in CFTR-depleted cells (Figure 6A) although this difference did not reach the significance threshold (p<0.09). Conversely, the protein expression of L-FABP that participates mainly in intracellular FA trafficking was increased by almost 2-fold in CFTR knockdown cells (Figure 6B).


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)

Effect of CFTR knockdown on protein expression of CD36 (A) and L-FABP (B).After 15 days of differentiation, Caco-2/15 cells were lysed and protein expression determined by Western Blot. β-actin served as loading control and was used for normalization. Data represented means ± SEM of three to four 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-g006: Effect of CFTR knockdown on protein expression of CD36 (A) and L-FABP (B).After 15 days of differentiation, Caco-2/15 cells were lysed and protein expression determined by Western Blot. β-actin served as loading control and was used for normalization. Data represented means ± SEM of three to four independent experiments *p<0.05 vs mock cells.
Mentions: To elucidate the mechanisms underlying the significant raise of FA upon reduction in CFTR expression, we assessed the expression of two proteins involved in FA intestinal uptake and transport. First, the protein expression of FAT/CD36, a transporter involved in FA uptake, displayed a 1,5-fold increase in CFTR-depleted cells (Figure 6A) although this difference did not reach the significance threshold (p<0.09). Conversely, the protein expression of L-FABP that participates mainly in intracellular FA trafficking was increased by almost 2-fold in CFTR knockdown cells (Figure 6B).

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