Cell culture models demonstrate that CFTR dysfunction leads to defective fatty acid composition and metabolism

Abstract

Cystic fibrosis (CF) is associated with fatty acid alterations characterized by low linoleic and docosahexaenoic acid. It is not clear whether these fatty acid alterations are directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction or result from nutrient malabsorption. We hypothesized that if fatty acid alterations are a result of CFTR dysfunction, those alterations should be demonstrable in CF cell culture models. Two CF airway epithelial cell lines were used: 16HBE, sense and antisense CFTR cells, and C38/IB3-1 cells. Wild-type (WT) and CF cells were cultured in 10% fetal bovine serum (FBS) or 10% horse serum. Fatty acid levels were analyzed by GC-MS. Culture of both WT and CF cells in FBS resulted in very low linoleic acid levels. When cells were cultured in horse serum containing concentrations of linoleic acid matching those found in human plasma, physiological levels of linoleic acid were obtained and fatty acid alterations characteristic of CF tissues were then evident in CF compared with WT cells. Kinetic studies with radiolabeled linoleic acid demonstrated in CF cells increased conversion to longer and more-desaturated fatty acids such as arachidonic acid. In conclusion, these data demonstrate that CFTR dysfunction is associated with altered fatty acid metabolism in cultured airway epithelial cells.

Figures

Fig. 1.

Cystic fibrosis transmembrane conductance regulator (CFTR) expression in airway epithelial cell lines. Western blot analysis of CFTR was performed on cell lysates of 16HBE sense (S) and antisense (AS) CFTR cell lines and C38 and IB3-1 cell lines using a rabbit polyclonal anti-CFTR antibody. Thirty micrograms of protein was loaded per well. A mouse β-actin antibody was used as loading control. Data are representative of two different experiments.

Fig. 2.

Fatty acid profile of 16HBE cells cultured in different sera. 16HBE cells were cultured in (A) 10% FBS, (B) 10% horse serum lot A, or (C) 10% horse serum lot B. Fatty acids were extracted, methylated, and analyzed by GC-MS. The internal standard was 17:0. Data are expressed as the mean ± SEM of a minimum of three different experiments, with each condition tested in duplicate. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 3.

Fatty acid profile in C38/IB3-1 cells. C38 [wild-type (WT)] and IB3-1 (CF ΔF508/W1282×) cells were cultured in (A) 10% FBS, (B) 10% horse serum lot A, or (C) 10% horse serum lot B and incubated with and without sodium butyrate (2.5 mM) and G418 (150 μg/ml) for 48 h before harvest. Fatty acids were extracted, methylated, and analyzed by GC-MS. The internal standard was 17:0. Data are expressed as the mean ± SEM and are representative of a minimum of three different experiments, with each condition tested in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 4.

The effect of confluency on fatty acids in sense and antisense CFTR 16HBE cells. Cells were cultured in 10% horse serum lot B to 80%, 100% confluency, and 2 days and 4 days past confluency. Cells were scraped, and fatty acids were extracted, methylated, and analyzed by GC-MS. Data are expressed as mean ± SEM and are representative of two different experiments, with each condition tested in triplicate. A: Mole percent of linoleic acid. B: Mole percent of docosahexaenoic acid. Closed bars, WT; open bars, CF. ** P < 0.01, *** P < 0.001.

Fig. 5.

Analysis of fatty acid metabolism. For analysis of the n-6 pathway, 16HBE cells were incubated with [14C]linoleic acid (4.9 μM, 0.5 μCi) for 4 h in lipid-free cell culture media, washed twice, and incubated for 20 h in serum-containing media. Lipids were extracted and methylated. Fatty acids were then separated by HPLC, and radioactivity was quantified by scintillation counting, as described in the Methods section. Results are shown for cells cultured in 10% horse serum (A) and in 10% FBS (B). Data are representative of two experiments, with each condition tested in triplicate and expressed as mean ± SEM. C: Results from analysis of n-3 fatty acid synthesis in16HBE cells cultured in 10% horse serum, where cells were incubated with [3H]eicosapentaenoic acid (4.5 μM, 0.5 μCi) and analyzed as above. Data are expressed as mean ± SEM, n = 2. * P < 0.05, ** P < 0.01.

Fig. 6.

HPLC chromatograms of fatty acid metabolism in sense and antisense CFTR 16HBE cells. WT and cystic fibrosis (CF) cells were labeled with [14C]18:2n-6 or [3H]20:5n-3 in lipid-free cell culture media for 4 h and further incubated for 20 h. After extraction and methylation, fatty acids were separated by HPLC and radioactivity was counted for each peak. A: N-6 fatty acid metabolism in WT cells. B: N-6 fatty acid metabolism in CF cells. C: N-3 fatty acid metabolism in WT cells. D: N-3 fatty acid metabolism in CF cells.