Fatty acids regulate endothelial lipase and inflammatory markers in macrophages and in mouse aorta: a role for PPARγ.

OBJECTIVE: Macrophage endothelial lipase (EL) is associated with increased atherosclerosis and inflammation. Because of their anti-inflammatory properties we hypothesized that n-3 fatty acids, in contrast to saturated fatty acids, would lower macrophages and arterial EL and inflammatory markers. METHODS AND RESULTS: Murine J774 and peritoneal macrophages were incubated with eicosapentaenoic acid or palmitic acid in the presence or absence of lipopolysaccaride (LPS). LPS increased EL mRNA and protein. Palmitic acid alone or with LPS dose-dependently increased EL mRNA and protein. In contrast, eicosapentaenoic acid dose-dependently abrogated effects of LPS or palmitic acid on increasing EL expression. EL expression closely linked to peroxisome proliferator activated receptor (PPAR)γ expression. Eicosapentaenoic acid blocked rosiglitazone (a PPARγ agonist)-mediated EL activation and GW9662 (a PPARγ antagonist)-blocked palmitic acid-mediated EL stimulation. Eicosapentaenoic acid alone or with LPS blunted LPS-mediated stimulation of macrophage proinflammatory interleukin-6, interleukin-12p40, and toll-like receptor-4 mRNA and increased anti-inflammatory interleukin-10 and mannose receptor mRNA. In vivo studies in low density lipoprotein receptor knockout mice showed that high saturated fat rich diets, but not n-3 diets, increased arterial EL, PPARγ, and proinflammatory cytokine mRNA. CONCLUSIONS: n-3 fatty acids, in contrast to saturated fatty acids, decrease EL in parallel with modulating pro- and anti-inflammatory markers, and these effects on EL link to PPARγ.

Proinflammatory phenotype and increased caveolin-1 in alveolar macrophages with silenced CFTR mRNA.

The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-kappaB, a positive regulator of IL-8 expression, and decreased expression of IkappaB-alpha, the inhibitory protein of NF-kappaB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is co-localized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1.

Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that affect protein structure and channel function. CFTR, localized in the apical membrane within cholesterol and sphingomyelin rich regions, is an ABC transporter that functions as a chloride channel. Here, we report that expression of defective CFTR (DeltaF508CFTR or decreased CFTR) in human lung epithelial cell lines increases sphingolipid synthesis and mass of sphinganine, sphingosine, four long-chain saturated ceramide species, C16 dihydroceramide, C22, C24, C26-ceramide, and sphingomyelin, and decreases mass of C18 and unsaturated C18:1 ceramide species. Decreased expression of CFTR is associated with increased expression of long-chain base subunit 1 of serine-palmitoyl CoA, the rate-limiting enzyme of de novo sphingolipid synthesis and increased sphingolipid synthesis. Overexpression of DeltaF508CFTR in bronchoalveolar cells that do not express CFTR increases sphingolipid synthesis and mass, whereas overexpression of wild-type CFTR, but not of an unrelated ABC transporter, ABCA7, decreases sphingolipid synthesis and mass. The data are consistent with a model in which CFTR functions within a feedback system that affects sphingolipid synthesis and in which increased sphingolipid synthesis could reflect a physiological response to sequestration of sphingolipids or altered membrane structure.

Decreased lipoprotein clearance is responsible for increased cholesterol in LDL receptor knockout mice with streptozotocin-induced diabetes.

OBJECTIVE: Patients with diabetes often have dyslipidemia and increased postprandial lipidmia. Induction of diabetes in LDL receptor (Ldlr(-/-)) knockout mice also leads to marked dyslipidemia. The reasons for this are unclear. RESEARCH DESIGN AND METHODS: We placed Ldlr(-/-) and heterozygous LDL receptor knockout (Ldlr(+/-)) mice on a high-cholesterol (0.15%) diet, induced diabetes with streptozotocin (STZ), and assessed reasons for differences in plasma cholesterol. RESULTS: STZ-induced diabetic Ldlr(-/-) mice had plasma cholesterol levels more than double those of nondiabetic controls. Fast-performance liquid chromatography and ultracentrifugation showed an increase in both VLDL and LDL. Plasma VLDL became more cholesterol enriched, and both VLDL and LDL had a greater content of apolipoprotein (apo)E. In LDL the ratio of apoB48 to apoB100 was increased. ApoB production, assessed using [(35)S]methionine labeling in Triton WR1339-treated mice, was not increased in fasting STZ-induced diabetic mice. Similarly, postprandial lipoprotein production was not increased. Reduction of cholesterol in the diet to normalize the amount of cholesterol intake by the control and STZ-induced diabetic animals reduced plasma cholesterol levels in STZ-induced diabetic mice, but plasma cholesterol was still markedly elevated compared with nondiabetic controls. LDL from STZ-induced diabetic mice was cleared from the plasma and trapped more rapidly by livers of control mice. STZ treatment reduced liver expression of the proteoglycan sulfation enzyme, heparan sulfate N-deacetylase/N-sulfotrasferase-1, an effect that was reproduced in cultured hepatocytyes by a high glucose-containing medium. CONCLUSIONS: STZ-induced diabetic, cholesterol-fed mice developed hyperlipidemia due to a non-LDL receptor defect in clearance of circulating apoB-containing lipoproteins.