Effects of Intestinal Microbiota on Brain Development in Humanized Gnotobiotic Mice

Poor growth in the Neonatal Intensive Care Unit is associated with an increased risk for poor neurodevelopmental outcomes for preterm infants, however the mechanism is unclear. The microbiome has increasingly been recognized as a modifiable environmental factor to influence host development. Here we explore the hypothesis that the microbiome influences both growth phenotype and brain development. A germ free mouse transfaunation model was used to examine the effects of preterm infant microbiotas known to induce either high growth or low growth phenotypes on postnatal brain development. The microbiome which induced the low growth phenotype was associated with decreases in the neuronal markers NeuN and neurofilament-L as well as the myelination marker MBP when compared to the microbiome associated with the high growth phenotype. Additionally, poor growth phenotype-associated microbiota was associated with increased neuroinflammation marked by increased Nos1, as well as alteration in IGF-1 pathway including decreased circulating and brain IGF-1, decreased circulating IGFBP3, and increased Igfbp3 brain mRNA expression. This study suggests that growth-associated microbiota can influence early neuron and oligodendrocyte development and that this effect may be mediated by effects on neuroinflammation and circulating IGF-1

The Peroxisome Deficient Arabidopsis Mutant sse1 Exhibits Impaired Fatty Acid Synthesis

The Arabidopsis Shrunken Seed 1 (SSE1) gene encodes a homolog of the peroxisome biogenesis factor Pex16p, and a loss-of-function mutation in this gene alters seed storage composition. Two lines of evidence support a function for SSE1 in peroxisome biogenesis: the peroxisomal localization of a green fluorescent protein-SSE1 fusion protein and the lack of normal peroxisomes in sse1 mutant embryos. The green fluorescent protein-SSE1 colocalizes with the red fluorescent protein (RFP)-labeled peroxisomal markers RFP-peroxisome targeting signal 1 and peroxisome targeting signal 2-RFP in transgenic Arabidopsis. Each peroxisomal marker exhibits a normal punctate peroxisomal distribution in the wild type but not the sse1 mutant embryos. Further studies reported here were designed toward understanding carbon metabolism in the sse1 mutant. A time course study of dissected embryos revealed a dramatic rate decrease in oil accumulation and an increase in starch accumulation. Introduction of starch synthesis mutations into the sse1 background did not restore oil biosynthesis. This finding demonstrated that reduction in oil content in sse1 is not caused by increased carbon flow to starch. To identify the blocked steps in the sse1 oil deposition pathway, developing sse1 seeds were supplied radiolabeled oil synthesis precursors. The ability of sse1 to incorporate oleic acid, but not pyruvate or acetate, into triacylglycerol indicated a defect in the fatty acid biosynthetic pathway in this mutant. Taken together, the results point to a possible role for peroxisomes in the net synthesis of fatty acids in addition to their established function in lipid catabolism. Other possible interpretations of the results are discussed

Ethanol administration to cystic fibrosis knockout mice results in increased fatty acid ethyl ester production.

BACKGROUND: Fatty acid ethyl esters (FAEE) are nonoxidative ethanol metabolites shown to produce toxic effects in the liver and pancreas in vivo and in vitro. Because alcohol-induced chronic pancreatitis is associated with mutations in the gene responsible for cystic fibrosis (CFTR), we hypothesized that CFTR dysfunction leads to increased levels of these toxic nonoxidative ethanol metabolites following alcohol administration. METHODS: Cystic fibrosis (CF) and wild-type (WT) mice were injected intraperitoneally with 1, 2, or 3 g/kg of 50% ethanol. Mice were sacrificed and the liver and pancreas removed for FAEE analysis. RESULTS: The mean FAEE concentration (pmol/g) detected in the liver of cftr mice following injection with 2 g/kg of ethanol was significantly greater than the amount detected in WT (p \u3c 0.005). A similar trend in FAEE concentration was seen in the pancreas, but the difference was not statistically different. In both the liver and pancreas, analysis of individual FAEE species demonstrated a selective increase in ethyl oleate. CONCLUSION: These data show an association between CFTR dysfunction and qualitative and quantitative changes in FAEE in liver and pancreas upon ethanol exposure

Potential utility of plasma fatty acid analysis in the diagnosis of cystic fibrosis

BACKGROUND: An altered distribution of fatty acids in cells and tissues is found in patients with cystic fibrosis (CF). In this study, we assessed the potential role of plasma fatty acid analysis in the diagnosis of CF. METHODS: In this 2-part study, we first used gas chromatography-mass spectrometry to analyze fatty acids in plasma from 13 CF patients and 11 controls without CF. We then used the fatty acid distribution data to identify the fatty acids or multiple fatty acid calculations most effective in identifying CF patients. Part 2 of the study was a blinded analysis of 10 CF patients and 9 controls to directly test the effectiveness of the diagnostic parameters for CF identified from the plasma fatty acid analysis. RESULTS: In the nonblinded trial, the multiplication product of (18:2 n-6) x (22:6 n-3) (each as percentage of total plasma fatty acid) was the most effective indicator for distinguishing patients with CF from controls (P = 0.0003). In part 2 (the blinded trial), this multiplication product was also the most effective indicator for distinguishing CF patients from controls (P = 0.0008). CONCLUSIONS: The product of (18:2 n-6) x (22:6 n-3) is effective for distinguishing CF patients from persons without CF. This diagnostic marker may have value as an alternative to the sweat chloride test in selected patients being evaluated for CF

Linoleic acid supplementation results in increased arachidonic acid and eicosanoid production in CF airway cells and in cftr−/− transgenic mice

Cystic fibrosis (CF) patients display a fatty acid imbalance characterized by low linoleic acid levels and variable changes in arachidonic acid. This led to the recommendation that CF patients consume a high-fat diet containing >6% linoleic acid. We hypothesized that increased conversion of linoleic acid to arachidonic acid in CF leads to increased levels of arachidonate-derived proinflammatory metabolites and that this process is exacerbated by increasing linoleic acid levels in the diet. To test this hypothesis, we determined the effect of linoleic acid supplementation on downstream proinflammatory biomarkers in two CF models: 1) in vitro cell culture model using 16HBE14o− sense [wild-type (WT)] and antisense (CF) human airway epithelial cells; and 2) in an in vivo model using cftr−/− transgenic mice. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS), and IL-8 and eicosanoids were measured by ELISA. Neutrophils were quantified in bronchoalveolar lavage fluid from knockout mice following linoleic acid supplementation and exposure to aerosolized Pseudomonas LPS. Linoleic acid supplementation increased arachidonic acid levels in CF but not WT cells. IL-8, PGE2, and PGF2α secretion were increased in CF compared with WT cells, with a further increase following linoleic acid supplementation. cftr−/− Mice supplemented with 100 mg of linoleic acid had increased arachidonic acid levels in lung tissue associated with increased neutrophil infiltration into the airway compared with control mice. These findings support the hypothesis that increasing linoleic acid levels in the setting of loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to increased arachidonic acid levels and proinflammatory mediators

A mechanism accounting for the low cellular level of linoleic acid in cystic fibrosis and its reversal by DHA*

Specific fatty acid alterations have been described in the blood and tissues of cystic fibrosis (CF) patients. The principal alterations include decreased levels of linoleic acid (LA) and docosahexaenoic acid (DHA). We investigated the potential mechanisms of these alterations by studying the cellular uptake of LA and DHA, their distribution among lipid classes, and the metabolism of LA in a human bronchial epithelial cell model of CF. CF (antisense) cells demonstrated decreased levels of LA and DHA compared with wild type (WT, sense) cells expressing normal CFTR. Cellular uptake of LA and DHA was higher in CF cells compared with WT cells at 1 h and 4 h. Subsequent incorporation of LA and DHA into most lipid classes and individual phospholipids was also increased in CF cells. The metabolic conversion of LA to n-6 metabolites, including 18:3n-6 and arachidonic acid, was upregulated in CF cells, indicating increased flux through the n-6 pathway. Supplementing CF cells with DHA inhibited the production of LA metabolites and corrected the n-6 fatty acid defect. In conclusion, the evidence suggests that low LA level in cultured CF cells is due to its increased metabolism, and this increased LA metabolism is corrected by DHA supplementation