CFTR Depletion Results in Changes in Fatty Acid Composition and Promotes Lipogenesis in Intestinal Caco 2/15 Cells

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.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).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

K+ channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

AbstractActive Na+ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and KATP K+ channel activities exerts sustained control in Na+ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K+ channels, and 2) to determine the physiological impact of K+ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and KATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to KATP activation by pinacidil. Conversely, pharmacological KvLQT1 and KATP inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K+ channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and KATP activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K+ channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K+ channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance

Increased Sugar Concentration with PM-Cutting and Wide Swathing Improves Alfalfa Silage Fermentation

Extensive protein degradation during silage fermentation reduces the efficiency of N utilization by ruminants and excess N is excreted in the environment. Forage nonstructural carbohydrates (NSC) represent the main source of readily fermentable energy for lactic bacteria during silage fermentation. Increasing forage NSC concentration can enhance silage fermentation, lactic acid production, and the decline in pH with an overall reduction in the extent of protein degradation. The NSC concentration increases during the day in alfalfa (Medicago sativa L.) to reach a maximum by the end of the afternoon. Under good wilting conditions, PM-cut alfalfa wilted in wide swaths had a greater NSC concentration than AM-cut alfalfa (Morin et al. 2012). Our objective was to study the effect of PM cutting and wide swathing on alfalfa silage quality attributes

High-Sugar Alfalfa for Dairy Cows

Alfalfa proteins are extensively degraded during wilting, silage fermentation, and in the rumen. To efficiently use alfalfa non protein N, rumen microbes need a readily available energy source such as nonstructural carbohydrates (NSC); otherwise, surplus N in the form of rumen ammonia is converted into urea and excreted in the environment. Increasing the NSC concentration of alfalfa was thus the focus of our research program. Our objectives were to assess the impact of high NSC alfalfa on digestibility and microbial protein synthesis measured in vitro, and on ingestion, rumen metabolism, N use efficiency, and dairy cow performance. Increasing NSC concentration of alfalfa significantly enhanced in vitro dry matter (DM) digestibility and decreased NH3-N concentration in rumen fluid. An increase of 23 g/kg in alfalfa NSC concentration can improve forage DM intake (+5 %) and energy corrected milk production (+8 %). Feeding high-NSC alfalfa led to a higher rumen pH, suggesting that sugars do not cause rumen acidosis, and to a lower milk urea N (MUN) indicating an improvement in N utilization. Increasing NSC concentration of alfalfa is a low-cost tool to improve its utilisation in dairy rations and potentially mitigate the environmental footprint of milk production

Improving Forage Nonstructural Carbohydrates through Management and Breeding

Nonstructural carbohydrates (NSC) are an important source of readily fermentable energy available to rumen microbes. Limited concentrations of readily available energy in forages combined with fast and intensive protein degradation contribute to poor N use efficiency by dairy cows and other ruminants. Increasing NSC in forages has been shown to improve intake, milk yield, and N use efficiency (Brito et al. 2009). We assessed several strategies to increase forage NSC accumulation, including PM-cutting, species selection and genetic improvement

Impact of Bacterial Toxins in the Lungs

Bacterial toxins play a key role in the pathogenesis of lung disease. Based on their structural and functional properties, they employ various strategies to modulate lung barrier function and to impair host defense in order to promote infection. Although in general, these toxins target common cellular signaling pathways and host compartments, toxin- and cell-specific effects have also been reported. Toxins can affect resident pulmonary cells involved in alveolar fluid clearance (AFC) and barrier function through impairing vectorial Na$^{+}$ transport and through cytoskeletal collapse, as such, destroying cell-cell adhesions. The resulting loss of alveolar-capillary barrier integrity and fluid clearance capacity will induce capillary leak and foster edema formation, which will in turn impair gas exchange and endanger the survival of the host. Toxins modulate or neutralize protective host cell mechanisms of both the innate and adaptive immunity response during chronic infection. In particular, toxins can either recruit or kill central players of the lung's innate immune responses to pathogenic attacks, i.e., alveolar macrophages (AMs) and neutrophils. Pulmonary disorders resulting from these toxin actions include, e.g., acute lung injury (ALI), the acute respiratory syndrome (ARDS), and severe pneumonia. When acute infection converts to persistence, i.e., colonization and chronic infection, lung diseases, such as bronchitis, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) can arise. The aim of this review is to discuss the impact of bacterial toxins in the lungs and the resulting outcomes for pathogenesis, their roles in promoting bacterial dissemination, and bacterial survival in disease progression

Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung disease

Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function, but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10−44) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases

A novel lung disease phenotype adjusted for mortality attrition for cystic fibrosis Genetic modifier studies

Genetic studies of lung disease in Cystic Fibrosis are hampered by the lack of a severity measure that accounts for chronic disease progression and mortality attrition. Further, combining analyses across studies requires common phenotypes that are robust to study design and patient ascertainment

Genome-wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2

A combined genome-wide association and linkage study was used to identify loci causing variation in CF lung disease severity. A significant association (P=3. 34 × 10-8) near EHF and APIP (chr11p13) was identified in F508del homozygotes (n=1,978). The association replicated in F508del homozygotes (P=0.006) from a separate family-based study (n=557), with P=1.49 × 10-9 for the three-study joint meta-analysis. Linkage analysis of 486 sibling pairs from the family-based study identified a significant QTL on chromosome 20q13.2 (LOD=5.03). Our findings provide insight into the causes of variation in lung disease severity in CF and suggest new therapeutic targets for this life-limiting disorder