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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

Effect of CFTR knockdown on gene expression of the nuclear receptors (A) retinoid X receptor (RXR) and (B) liver X receptor (LXR).Transcript levels were assessed by RT-PCR using RNA extracted from Caco-2/15 cells differentiated for a period of 15 days. Results are expressed as means ± SEM of n = 5 independent experiments. *p<0.05 vs mock cells.
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pone-0010446-g012: Effect of CFTR knockdown on gene expression of the nuclear receptors (A) retinoid X receptor (RXR) and (B) liver X receptor (LXR).Transcript levels were assessed by RT-PCR using RNA extracted from Caco-2/15 cells differentiated for a period of 15 days. Results are expressed as means ± SEM of n = 5 independent experiments. *p<0.05 vs mock cells.

Mentions: In an attempt to elucidate potential mechanisms linking CFTR to FA metabolism, we determined the status of several transcription factors that govern the expression of a variety of genes associated with lipid and cholesterol metabolism. Data regarding the gene expression of members of the PPAR family are presented in Figure 11. CFTR disruption did not influence the expression of PPAR-β and PPAR-γ whereas a 2-fold reduction was observed in PPAR-α mRNA levels. Consistent with these data, a significant transcript decline characterized RXR-α (acting as a partner of PPAR-α), while the gene expression of RXR-β remained unaltered (Figure 12A). The most striking observation, in response to CFTR knockdown, was the downregulation of LXR-α and LXR-β mRNA (Figure 12B).


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 gene expression of the nuclear receptors (A) retinoid X receptor (RXR) and (B) liver X receptor (LXR).Transcript levels were assessed by RT-PCR using RNA extracted from Caco-2/15 cells differentiated for a period of 15 days. Results are expressed as means ± SEM of n = 5 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-g012: Effect of CFTR knockdown on gene expression of the nuclear receptors (A) retinoid X receptor (RXR) and (B) liver X receptor (LXR).Transcript levels were assessed by RT-PCR using RNA extracted from Caco-2/15 cells differentiated for a period of 15 days. Results are expressed as means ± SEM of n = 5 independent experiments. *p<0.05 vs mock cells.
Mentions: In an attempt to elucidate potential mechanisms linking CFTR to FA metabolism, we determined the status of several transcription factors that govern the expression of a variety of genes associated with lipid and cholesterol metabolism. Data regarding the gene expression of members of the PPAR family are presented in Figure 11. CFTR disruption did not influence the expression of PPAR-β and PPAR-γ whereas a 2-fold reduction was observed in PPAR-α mRNA levels. Consistent with these data, a significant transcript decline characterized RXR-α (acting as a partner of PPAR-α), while the gene expression of RXR-β remained unaltered (Figure 12A). The most striking observation, in response to CFTR knockdown, was the downregulation of LXR-α and LXR-β mRNA (Figure 12B).

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