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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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Impact of CFTR knockdown on the protein expression of (A) AMP-activated protein kinase (AMPK) and (B) acetylCoA carboxylase (ACC) in Caco-2/15 cells.Phosphorylated AMPK (p-AMPK) and ACC (p-ACC) and total AMPK and ACC were analyzed by Western Blot and the ratios of phosphorylated form to total protein level were calculated. Results are expressed as means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells.
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pone-0010446-g009: Impact of CFTR knockdown on the protein expression of (A) AMP-activated protein kinase (AMPK) and (B) acetylCoA carboxylase (ACC) in Caco-2/15 cells.Phosphorylated AMPK (p-AMPK) and ACC (p-ACC) and total AMPK and ACC were analyzed by Western Blot and the ratios of phosphorylated form to total protein level were calculated. Results are expressed as means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells.

Mentions: The mechanisms for the modifications of de novo lipogenesis were further investigated by examining key regulatory protein players involved in this metabolic pathway. We focused on AMPK that currently comes into a spotlight as an important regulator for lipid metabolism by modulating phosphorylation and activity of the key ACC enzyme. To this end, we performed Western blot analysis to estimate AMPK and ACC mass and phosphorylation (p-AMPK and p-ACC) in control and CFTR knockdown cells. CFTR-depleted cells displayed lower AMPK levels than controls without significant changes in the p-AMPK form, thereby resulting in a higher, although non-significant, p-AMPK/AMPK ratio (Figure 9A). On the other hand, we observed a significant decrease in the amount of p-ACC upon CFTR genetic manipulation, which was coupled to an increased trend of total ACC, leading to a marked decrease in the calculated ratio of p-ACC/ACC (Figure 9B). These data are suggestive that CFTR knockdown leads to a stimulation of the active form of ACC at the cellular level, thereby providing a molecular explanation for the enhancement in de novo lipogenesis. Since the induction of ACC gene transcription is under the regulatory action of SREBP-1c that binds the sterol response elements located in the ACC promoter, we determined its gene expression. In line with the enhanced lipogenesis and the decreased phosphorylation state of ACC, CFTR knockdown resulted in a significant increase in SREBP-1c gene expression (Figure 10).


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)

Impact of CFTR knockdown on the protein expression of (A) AMP-activated protein kinase (AMPK) and (B) acetylCoA carboxylase (ACC) in Caco-2/15 cells.Phosphorylated AMPK (p-AMPK) and ACC (p-ACC) and total AMPK and ACC were analyzed by Western Blot and the ratios of phosphorylated form to total protein level were calculated. Results are expressed as means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells.
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Related In: Results  -  Collection

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

pone-0010446-g009: Impact of CFTR knockdown on the protein expression of (A) AMP-activated protein kinase (AMPK) and (B) acetylCoA carboxylase (ACC) in Caco-2/15 cells.Phosphorylated AMPK (p-AMPK) and ACC (p-ACC) and total AMPK and ACC were analyzed by Western Blot and the ratios of phosphorylated form to total protein level were calculated. Results are expressed as means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells.
Mentions: The mechanisms for the modifications of de novo lipogenesis were further investigated by examining key regulatory protein players involved in this metabolic pathway. We focused on AMPK that currently comes into a spotlight as an important regulator for lipid metabolism by modulating phosphorylation and activity of the key ACC enzyme. To this end, we performed Western blot analysis to estimate AMPK and ACC mass and phosphorylation (p-AMPK and p-ACC) in control and CFTR knockdown cells. CFTR-depleted cells displayed lower AMPK levels than controls without significant changes in the p-AMPK form, thereby resulting in a higher, although non-significant, p-AMPK/AMPK ratio (Figure 9A). On the other hand, we observed a significant decrease in the amount of p-ACC upon CFTR genetic manipulation, which was coupled to an increased trend of total ACC, leading to a marked decrease in the calculated ratio of p-ACC/ACC (Figure 9B). These data are suggestive that CFTR knockdown leads to a stimulation of the active form of ACC at the cellular level, thereby providing a molecular explanation for the enhancement in de novo lipogenesis. Since the induction of ACC gene transcription is under the regulatory action of SREBP-1c that binds the sterol response elements located in the ACC promoter, we determined its gene expression. In line with the enhanced lipogenesis and the decreased phosphorylation state of ACC, CFTR knockdown resulted in a significant increase in SREBP-1c gene expression (Figure 10).

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