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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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Influence of CFTR knockdown on total fatty acids (FA) content and FA classes in Caco-2/15 cells.At 15 days of differentiation, cells were collected, subjected to direct transesterification and injected into a gas chromatograph. Results represent the means ± SEM of 4 independent experiments and are illustrated as µg/mg of cellular protein. *p<0.05 vs mock cells. SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA; polyunsaturated fatty acids.
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pone-0010446-g002: Influence of CFTR knockdown on total fatty acids (FA) content and FA classes in Caco-2/15 cells.At 15 days of differentiation, cells were collected, subjected to direct transesterification and injected into a gas chromatograph. Results represent the means ± SEM of 4 independent experiments and are illustrated as µg/mg of cellular protein. *p<0.05 vs mock cells. SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA; polyunsaturated fatty acids.

Mentions: We used the shRNAi experimental approach to generate a model of Caco-2/15 cells deficient in CFTR. Infection of Caco-2/15 cells with lentivirus carrying CFTR-shRNAi caused a reduction in CFTR gene (61%) and protein (57%) expression when compared to non-infected cells (Figure 1). Mock-infected cells expressed a level of CFTR gene and protein, which was similar to non-infected cells and were, therefore, used as controls in subsequent experiments. Cell viability, integrity and differentiation state were not altered by genetic manipulation (data not shown). FA composition of cell homogenates were assessed by gas chromatography and comparisons were set up between control mock and knockdown cells. Total amount of FA increased by 1.5-fold upon CFTR gene disruption (Figure 2). This raised level was imputed to increased levels of specific FA species, including monounsaturated (50%), and saturated (49%) FA relatively to mock-infected cells. In particular, the total n-7 FA levels (236.5±7.0 µM/mg protein vs 416.8±60.4 µM/mg protein) experienced the highest increase (76% over mock values) whereas total n-9 FA levels (252.7±25.2 µM/mg protein vs 319.4±19.4 µM/mg protein) were only moderately raised (26% over control values). The proportions of individual FA are summarized in Table 1. CFTR knockdown resulted in a marked increase in the amounts of myristic (14:0) and palmitic (16:0) FA, while stearic acid (18:0) and longer saturated FAs displayed reduced values (P<0.05). As a consequence of CFTR genetic manipulation, n-7 FAs appeared elevated whereas n-3 and n-6 FAs remained unchanged. Proportions of n-9 FA were reduced to various degrees in knockdown cells with oleic acid (18:1n-9) exhibiting the highest, albeit non-significant, decrease compared to mock cell values.


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)

Influence of CFTR knockdown on total fatty acids (FA) content and FA classes in Caco-2/15 cells.At 15 days of differentiation, cells were collected, subjected to direct transesterification and injected into a gas chromatograph. Results represent the means ± SEM of 4 independent experiments and are illustrated as µg/mg of cellular protein. *p<0.05 vs mock cells. SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA; polyunsaturated fatty acids.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010446-g002: Influence of CFTR knockdown on total fatty acids (FA) content and FA classes in Caco-2/15 cells.At 15 days of differentiation, cells were collected, subjected to direct transesterification and injected into a gas chromatograph. Results represent the means ± SEM of 4 independent experiments and are illustrated as µg/mg of cellular protein. *p<0.05 vs mock cells. SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA; polyunsaturated fatty acids.
Mentions: We used the shRNAi experimental approach to generate a model of Caco-2/15 cells deficient in CFTR. Infection of Caco-2/15 cells with lentivirus carrying CFTR-shRNAi caused a reduction in CFTR gene (61%) and protein (57%) expression when compared to non-infected cells (Figure 1). Mock-infected cells expressed a level of CFTR gene and protein, which was similar to non-infected cells and were, therefore, used as controls in subsequent experiments. Cell viability, integrity and differentiation state were not altered by genetic manipulation (data not shown). FA composition of cell homogenates were assessed by gas chromatography and comparisons were set up between control mock and knockdown cells. Total amount of FA increased by 1.5-fold upon CFTR gene disruption (Figure 2). This raised level was imputed to increased levels of specific FA species, including monounsaturated (50%), and saturated (49%) FA relatively to mock-infected cells. In particular, the total n-7 FA levels (236.5±7.0 µM/mg protein vs 416.8±60.4 µM/mg protein) experienced the highest increase (76% over mock values) whereas total n-9 FA levels (252.7±25.2 µM/mg protein vs 319.4±19.4 µM/mg protein) were only moderately raised (26% over control values). The proportions of individual FA are summarized in Table 1. CFTR knockdown resulted in a marked increase in the amounts of myristic (14:0) and palmitic (16:0) FA, while stearic acid (18:0) and longer saturated FAs displayed reduced values (P<0.05). As a consequence of CFTR genetic manipulation, n-7 FAs appeared elevated whereas n-3 and n-6 FAs remained unchanged. Proportions of n-9 FA were reduced to various degrees in knockdown cells with oleic acid (18:1n-9) exhibiting the highest, albeit non-significant, decrease compared to mock cell values.

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