Lung versus gut exposure to air pollution particles differentially affect metabolic health in mice.

BACKGROUND: Air pollution has emerged as an unexpected risk factor for diabetes. However, the mechanism behind remains ill-defined. So far, the lung has been considered as the main target organ of air pollution. In contrast, the gut has received little scientific attention. Since air pollution particles can reach the gut after mucociliary clearance from the lungs and through contaminated food, our aim was to assess whether exposure deposition of air pollution particles in the lung or the gut drive metabolic dysfunction in mice. METHODS: To study the effects of gut versus lung exposure, we exposed mice on standard diet to diesel exhaust particles (DEP; NIST 1650b), particulate matter (PM; NIST 1649b) or phosphate-buffered saline by either intratracheal instillation (30 µg 2 days/week) or gavage (12 µg 5 days/week) over at least 3 months (total dose of 60 µg/week for both administration routes, equivalent to a daily inhalation exposure in humans of 160 µg/m3 PM2.5) and monitored metabolic parameters and tissue changes. Additionally, we tested the impact of the exposure route in a "prestressed" condition (high-fat diet (HFD) and streptozotocin (STZ)). RESULTS: Mice on standard diet exposed to particulate air pollutants by intratracheal instillation developed lung inflammation. While both lung and gut exposure resulted in increased liver lipids, glucose intolerance and impaired insulin secretion was only observed in mice exposed to particles by gavage. Gavage with DEP created an inflammatory milieu in the gut as shown by up-regulated gene expression of pro-inflammatory cytokines and monocyte/macrophage markers. In contrast, liver and adipose inflammation markers were not increased. Beta-cell secretory capacity was impaired on a functional level, most likely induced by the inflammatory milieu in the gut, and not due to beta-cell loss. The differential metabolic effects of lung and gut exposures were confirmed in a "prestressed" HFD/STZ model. CONCLUSIONS: We conclude that separate lung and gut exposures to air pollution particles lead to distinct metabolic outcomes in mice. Both exposure routes elevate liver lipids, while gut exposure to particulate air pollutants specifically impairs beta-cell secretory capacity, potentially instigated by an inflammatory milieu in the gut.

Pathogen inactivation treatment of triple-dose apheresis platelets with amotosalen and ultraviolet a light.

BACKGROUND: A triple storage (TS) set allows for pathogen inactivation (PI) treatment of triple-dose apheresis platelet products with amotosalen + UVA. We evaluated the quality and metabolic parameters of platelet concentrates (PCs) pathogen inactivated and stored for 7 days. MATERIALS AND METHODS: Twelve triple-dose products collected with two different apheresis platforms were treated with amotosalen+UVA. Products were split into three single-dose units. Testing was made pretreatment, after splitting, at days 5 and 7 of storage. RESULTS: Single-dose PI PCs had a mean platelet content of 2.89 ± 0.35 x 1011 . From baseline to day 7, pH remained stable (7.1 ± 0.1 vs. 7.0 ± 0.1), pO2 increased (11.3 ± 2.4 vs. 18.3 ± 3.5 kPa) as did LDH (201 ± 119 vs. 324 ± 203 U/L) and lactate (3.6 ± 1.7 vs. 12.1 ± 1.5 mmol/L) (all p < 0.01); pCO2 decreased (4.1 ± 0.8 vs. 1.5 ± 0.7 mmHg; p < 0.01) and so did bicarbonate (6.6 ± 1.1 vs. 2.5 ± 1.4 mmol/L), glucose (5.6 ± 1.2 vs. 0.4 ± 0.4 mmol/L) and ATP (3.4 ± 0.9 vs. 2.5 ± 1.4 nmol/108 platelets) (all p < 0.05). CONCLUSION: Triple-dose PCs processed with the TS sets fulfilled the quality requirements and displayed metabolic changes of expected extent during 7-day storage.

Technical Considerations and Confounders for Urine CXCL10 Chemokine Measurement.

BACKGROUND: The urine C-X-C motif chemokine 10 (CXCL10) is a promising screening biomarker for renal allograft rejection. The aim of the study was to investigate important technical and biological aspects as well as potential confounders when measuring urine CXCL10. METHODS: We analyzed 595 urine samples from 117 patients, who participated in a randomized controlled trial investigating the clinical utility of urine CXCL10 monitoring for posttransplant management. Urine CXCL10 was measured by an immunoassay using electrochemiluminescence. RESULTS: Intraassay coefficient of variation was 2.5%, and interassay coefficient of variation was 10%. Urine CXCL10 remained stable (ie, <10% degradation) for 8 hours at 25°C or 37°C and for 3 days at 4°C. CXCL10 concentrations [pg/mL] strongly correlated with urine CXCL10/creatinine ratios [ng/mmol] (r2 = 0.98; P < 0.0001). Leucocyturia and active BK-polyomavirus infection are associated with higher CXCL10 concentrations, while allograft function, serum CRP, patient age, proteinuria, urine pH, hematuria, squamous epithelia cell count, and bacteriuria did not correlate with urine CXCL10 concentrations. In 145 paired samples obtained within 1-2 weeks, 80% showed a CXCL10/creatinine ratio change of < ±2 ng/mmol or ±50%, respectively. CONCLUSIONS: Urine CXCL10 measurement on the used platform is accurate and robust. Leucocyturia and active BK-polyomavirus infection are major confounders, which can be easily detected but represent important diagnostic "blind spots" when using urine CXCL10 to screen for allograft rejection. The intraindividual biological variability of urine CXCL10 within 1-2 weeks is mostly below ±50%, which is still much higher than the technical variability due to sample handling/processing (<20%).

Sodium channel TRPM4 and sodium/calcium exchangers (NCX) cooperate in the control of Ca2+-induced mucin secretion from goblet cells.

Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles. Alterations in the quantity or quality of secreted mucins are therefore detrimental to airway and colon physiology. Based on various biochemical assays in several human cell lines, we report here that Na+/Ca2+ exchanger 2 (NCX2) works in conjunction with transient receptor potential cation channel subfamily M member 4 (TRPM4), and perhaps TRPM5, Na+ channels to control Ca2+-mediated secretion of both mucin 2 (MUC2) and MUC5AC from HT29-18N2 colonic cancer cells. Differentiated normal bronchial epithelial (NHBE) cells and tracheal cells from patients with cystic fibrosis (CFT1-LC3) expressed only TRPM4 and all three isoforms of NCXs. Blocking the activity of TRPM4 or NCX proteins abrogated MUC5AC secretion from NHBE and CFT1-LC3 cells. Altogether, our findings reveal that NCX and TRPM4/TRPM5 are both required for mucin secretion. We therefore propose that these two proteins could be potential pharmacological targets to control mucus-related pathologies such as cystic fibrosis.

TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells.

Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion. DOI:http://dx.doi.org/10.7554/eLife.00658.001.

MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening.

To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf-MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)-ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export.

p28, a novel ERGIC/cis Golgi protein, required for Golgi ribbon formation.

The mammalian Golgi apparatus consists of individual cisternae that are stacked in a polarized manner to form the compact zones of the Golgi. Several stacks are linked to form a ribbon via dynamic lateral bridges. The determinants required for maintaining the characteristic Golgi structure are incompletely understood. Here, we have characterized p28, a new gamma-subfamily member of p24 membrane proteins. p28 localized to endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and cis Golgi and accumulated in the ERGIC upon Brefeldin A treatment, typical for a protein cycling in the early secretory pathway. p28 interacted with a subset of p24 proteins. Its depletion by small interfering RNA (siRNA) led to fragmentation of the Golgi without affecting the overall organization of microtubules but considerably reducing the amount of acetylated tubulin. The distribution of COPI and tethers, including GM130, was not affected. At the ultrastructural level, the Golgi fragments appeared as mini-stacks with apparently unchanged cis-trans topology. Golgi fragmentation did not impair anterograde or retrograde traffic. Fluorescence recovery after photobleaching (FRAP) experiments revealed that silencing p28 prevents protein exchange between Golgi stacks during reassembly after Brefeldin A-induced Golgi breakdown. These results show that the formation of a Golgi ribbon requires the structural membrane protein p28 in addition to previously identified SNAREs, coat proteins and tethers.

The cargo receptors Surf4, endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53, and p25 are required to maintain the architecture of ERGIC and Golgi.

Rapidly cycling proteins of the early secretory pathway can operate as cargo receptors. Known cargo receptors are abundant proteins, but it remains mysterious why their inactivation leads to rather limited secretion phenotypes. Studies of Surf4, the human orthologue of the yeast cargo receptor Erv29p, now reveal a novel function of cargo receptors. Surf4 was found to interact with endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53 and p24 proteins. Silencing Surf4 together with ERGIC-53 or silencing the p24 family member p25 induced an identical phenotype characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus without effect on anterograde transport. Live imaging showed decreased stability of ERGIC clusters after knockdown of p25. Silencing of Surf4/ERGIC-53 or p25 resulted in partial redistribution of coat protein (COP) I but not Golgi matrix proteins to the cytosol and partial resistance of the cis-Golgi to brefeldin A. These findings imply that cargo receptors are essential for maintaining the architecture of ERGIC and Golgi by controlling COP I recruitment.

Profile-based data base scanning for animal L-type lectins and characterization of VIPL, a novel VIP36-like endoplasmic reticulum protein.

Consensus profiles were established to screen data bases for novel animal L-type lectins. The profiles were generated from linear sequence motifs of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 and by optimal alignment of the entire carbohydrate recognition domain of these proteins. The search revealed numerous orthologous and homologous L-type lectin-like proteins in animals, protozoans, and yeast, as well as the sequence of a novel family member related to VIP36, named VIPL for VIP36-like. Sequence analysis suggests that VIPL is a ubiquitously expressed protein and appeared earlier in evolution than VIP36. The cDNA of VIPL was cloned and expressed in cell culture. VIPL is a high-mannose type I membrane glycoprotein with similar domain organization as VIP36. Unlike VIP36 and ERGIC-53 that are predominantly associated with postendoplasmic reticulum (ER) membranes and cycle in the early secretory pathway, VIPL is a non-cycling resident protein of the ER. Mutagenesis experiments indicate that ER retention of VIPL involves a RKR di-arginine signal. Overexpression of VIPL redistributed ERGIC-53 to the ER without affecting the cycling of the KDEL-receptor and the overall morphology of the early secretory pathway. The results suggest that VIPL may function as a regulator of ERGIC-53.