Background. Abnormalities of fatty acid (FA) turnover associated with low levels of linoleic (LA) and docosahexaenoic (DHA) acid are observed in plasma and tissues of cystic fibrosis (CF) patients, possibly due to alterations in basal lipid metabolism. Objective. To study in CF and non CF cell lines the metabolism of n-6 and n-3 FA and to elucidate mechanisms possibly responsible of the altered FA status in CF, focusing on FA desaturase enzymes. In addition cell lines are treated with LA-BSA and DHA-BSA complexes in order to investigate whether these FA may affect processes modulating the severity of complications in CF patients. Methods. 16HBE14o- cells (a human bronchial epithelial cell line) were used, with CF phenotype (16HBE14o-AS3) and without CF phenotype (16HBE14o-S1). The two cell lines were incubated with EMEM without FBS for 24h, for the analysis of total FA profile by GC and of the expression of FA desaturases by Real-time PCR. In addition cells were incubated with [1-14C] linoleic or alpha-linolenic acid (-LNA) (1Ci/plate), respectively precursors of n-6 and n-3 FA, for the analysis of labelled FA in total lipids and their incorporation into different lipid classes by HPLC coupled with a radiodetector. Cells are also treated with increasing concentrations of LA-BSA and DHA-BSA complexes and the incorporation of FA in total lipids and the expression of the desaturases were assessed. Results. CF cells show low levels of LA, -LNA and DHA in comparison with control cells. Both [1-14C] LA and -LNA total conversion are increased in CF cells versus controls, associated to enhanced 6 and 5 desaturase activities. This is due to a higher desaturase expression in CF versus control cells. The incorporation of [1-14C] LA and -LNA in phospholipids in CF cells is increased while it is lower in triglycerides and cholesterol esters than in control cells. CF cells treated with increasing concentration of LA-BSA complex have low levels of LA in comparison with control cells at any concentration used and show a higher total conversion associated to a higher desaturase expression. In contrast, when cells are treated with increasing concentration of DHA-BSA complex, at the highest concentration used CF cells have lower level of DHA than control cells and desaturase expression is lower in CF cells than in controls. Conclusion. The metabolism of [1-14C] LA and -LNA to their derivatives is higher in CF cells than in controls, with enhanced formation of arachidonic and eicosapentaenoic acid respectively. This is associated to a higher desaturase activity in CF than in control cells, due to a higher desaturase expression. Supplementation with DHA could be a safe and effective treatment to reduce the severity of complications in CF patients, since DHA is the substrate for the synthesis of D-series resolvins, compactins and maresins, compounds with an anti-inflammatory action.

ALTERAZIONI DEL METABOLISMO DEGLI ACIDI GRASSI E LORO SUPPLEMENTAZIONE NELLA FIBROSI CISTICA / S. Ghezzi ; tutor: C. Galli ; coordinatore: G. Franceschini. - : . Università degli Studi di Milano, 2012 Feb 08. ((24. ciclo, Anno Accademico 2011. [10.13130/ghezzi-silvia_phd2012-02-08].

ALTERAZIONI DEL METABOLISMO DEGLI ACIDI GRASSI E LORO SUPPLEMENTAZIONE NELLA FIBROSI CISTICA

S. Ghezzi
2012-02-08

Abstract

Background. Abnormalities of fatty acid (FA) turnover associated with low levels of linoleic (LA) and docosahexaenoic (DHA) acid are observed in plasma and tissues of cystic fibrosis (CF) patients, possibly due to alterations in basal lipid metabolism. Objective. To study in CF and non CF cell lines the metabolism of n-6 and n-3 FA and to elucidate mechanisms possibly responsible of the altered FA status in CF, focusing on FA desaturase enzymes. In addition cell lines are treated with LA-BSA and DHA-BSA complexes in order to investigate whether these FA may affect processes modulating the severity of complications in CF patients. Methods. 16HBE14o- cells (a human bronchial epithelial cell line) were used, with CF phenotype (16HBE14o-AS3) and without CF phenotype (16HBE14o-S1). The two cell lines were incubated with EMEM without FBS for 24h, for the analysis of total FA profile by GC and of the expression of FA desaturases by Real-time PCR. In addition cells were incubated with [1-14C] linoleic or alpha-linolenic acid (-LNA) (1Ci/plate), respectively precursors of n-6 and n-3 FA, for the analysis of labelled FA in total lipids and their incorporation into different lipid classes by HPLC coupled with a radiodetector. Cells are also treated with increasing concentrations of LA-BSA and DHA-BSA complexes and the incorporation of FA in total lipids and the expression of the desaturases were assessed. Results. CF cells show low levels of LA, -LNA and DHA in comparison with control cells. Both [1-14C] LA and -LNA total conversion are increased in CF cells versus controls, associated to enhanced 6 and 5 desaturase activities. This is due to a higher desaturase expression in CF versus control cells. The incorporation of [1-14C] LA and -LNA in phospholipids in CF cells is increased while it is lower in triglycerides and cholesterol esters than in control cells. CF cells treated with increasing concentration of LA-BSA complex have low levels of LA in comparison with control cells at any concentration used and show a higher total conversion associated to a higher desaturase expression. In contrast, when cells are treated with increasing concentration of DHA-BSA complex, at the highest concentration used CF cells have lower level of DHA than control cells and desaturase expression is lower in CF cells than in controls. Conclusion. The metabolism of [1-14C] LA and -LNA to their derivatives is higher in CF cells than in controls, with enhanced formation of arachidonic and eicosapentaenoic acid respectively. This is associated to a higher desaturase activity in CF than in control cells, due to a higher desaturase expression. Supplementation with DHA could be a safe and effective treatment to reduce the severity of complications in CF patients, since DHA is the substrate for the synthesis of D-series resolvins, compactins and maresins, compounds with an anti-inflammatory action.
GALLI, CLAUDIO
FRANCESCHINI, GUIDO
acidi grassi ; fibrosi cistica ; desaturasi ; fatty acids ; cystic fibrosis ; human desaturases
Settore BIO/14 - Farmacologia
ALTERAZIONI DEL METABOLISMO DEGLI ACIDI GRASSI E LORO SUPPLEMENTAZIONE NELLA FIBROSI CISTICA / S. Ghezzi ; tutor: C. Galli ; coordinatore: G. Franceschini. - : . Università degli Studi di Milano, 2012 Feb 08. ((24. ciclo, Anno Accademico 2011. [10.13130/ghezzi-silvia_phd2012-02-08].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/170508
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