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

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.

Figure 1. CFTR gene and protein knockdown following lentivirus infection of Caco 2/15 cells.

After lentivirus infection, Caco 2/15 cells were differentiated for a period of 14 days before being assessed for CFTR gene and protein expression by RT-PCR and Western Blot, respectively. Cells that were not infected with lentivirus served as controls and represent 100%. Cells infected with the empty vector (indicated as Mock Cells on the graph) exhibited comparable CFTR expression relatively to noninfected cells. Results represent mean± SEM of n = 5 independent cell preparations and are illustrated as % of the noninfected cells after the data have been calculated as densitometric ratio of CFTR to GAPDH or β-actin. *p<0,01.

Figure 2. 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.

Figure 3. Influence of pharmacological inhibition of CFTR on fatty acid concentrations in Caco-2/15 cells.

Treatment with 20 µM CFTR inh-172 at 12 days of differentiation was initiated and maintained for 3 days. Cells treated with DMSO (vehicle) served as controls. Cells were collected and subjected to direct trans-esterification and injected into a gas chromatograph. Result represent the means ± SEM of n = 3 to 4 separate wells originating from 3 independent experiments and are illustrated as µg/mg of cellular protein. *p<0.05 vs DMSO-treated cells. SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA; polyunsaturated fatty acids.

Figure 4. 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.

Figure 5. Free fatty acid (FFA) content in lipoproteins secreted by Caco-2/15 cells.

Cells infected with empty vector plko.1 or infected with lentivirus carrying CFTR-shRNAi were differentiated for 12 days and incubated with [¹⁴C]-oleic acid for 24 h. Thereafter, lipoproteins were isolated by ultracentrifugation according to their specific densities. Lipoprotein lipids were further extracted using a mixture of chloroform/methanol, spreaded onto TLC plates and the band corresponding to the FFA fraction was scraped off the plates. Radioactivity incorporated was further determined. Data were analyzed as dpm/mg of total protein but were reported as percent difference relative to plko.1-infected values representing 100%. Data represented means ± SEM of n = 3 independent experiments. *p<0.05 vs mock cells. CM: chylomicron; HDL: high density lipoprotein; LDL: low density lipoprotein; VLDL: very-low density lipoprotein.

Figure 6. 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.

Figure 7. Influence of CFTR knockdown on de novo lipogenesis in Caco-2/15 cells.

After 12 days of differentiation, cells were incubated with [¹⁴C]-acetate for 24 h. Cells (A) and media (B) were further collected and lipid-extracted in chloroform/methanol. The extracts were separated on silica plates and bands corresponding to TG, PL and CE were excised. Radioactivity incorporated into each fraction was counted. The discrepancy between synthesis of newly formed lipids and their secretion became evident when expressed as a medium/cell ratio (C). Data represented means ± SEM of n = 12 wells from two independent experiments. *p<0.05 vs mock cells. TG: triglycerides; PL: phospholipids; CE: cholesteryl ester.

Figure 8. Impact of CFTR knockdown on newly synthesized (A), secreted (B) free fatty acid (FFA) and on the medium to cell ratio (C).

After a 24 h-incubation with [¹⁴C]-acetate, cells and media were collected, lipids were extracted in chloroform/methanol and separated by TLC. The FFA band was scraped off the plate and counted for the amount of radioactivity incorporated. Data represented means ± SEM of n = 12 wells from two independent experiments. *p<0.05 vs mock cells.

Figure 9. 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.

Figure 10. Expression of sterol regulatory element binding protein-1c (SREBP-1c) in response to CFTR knockdown in Caco-2/15 cells.

PCR analysis was performed on cells differentiated for a period of 15 days. Y-axis represents SREBP-1c expression normalized to GAPDH expression. Results are expressed as means ± SEM of n = 5 independent experiments. *p<0.05 vs mock cells.

Figure 11. Effect of CFTR knockdown on peroxisome proliferators-activated receptors PPAR(α,β, γ) gene expression.

Transcript levels were determined 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.

Figure 12. 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.

Figure 13. Schematic representation of the different cellular changes induced by CFTR knockdown in Caco-2/15 cells.

CFTR depletion led to an increase in FA uptake mostly by L-FABP (and to a lesser extent by CD36) and channeled to the lipoprotein assembly pathway, resulting in increased output of FFA-enriched lipoproteins. Furthermore, activation of the lipogenic pathway through stimulation of SREBP-1c expression and ACC (dephosphorylation and activation) caused cellular FA accretion, especially SFA which were converted to MUFA due to increased desaturase activity. These newly synthesized FA were incorporated into PL, TG and CE and efficiently secreted by the cell. ACC: acetyl-CoA carboxylase; Apo: apolipoprotein; CE: cholesteryl ester; CM: chylomicron; FFA: free fatty acid; HDL: high-density lipoprotein; L-FABP: liver fatty-acid binding protein; LDL: low-density lipoprotein; LXR: Liver X receptor; MTP: microsomal transfer protein; MUFA: monounsaturated fatty acid; PPAR: Peroxisome proliferators-activated receptor; PL: phospholipids; RXR: Retinoid X receptor; SFA: saturated fatty acid; SREBP-1c: Sterol regulatory element-binding protein-1c; TG: triglycerides; VLDL: very low-density lipoprotein.