Metabolites produced by probiotic Lactobacilli rapidly increase glucose uptake by Caco-2 cells

<p>Abstract</p> <p>Background</p> <p>Although probiotic bacteria and their metabolites alter enterocyte gene expression, rapid, non-genomic responses have not been examined. The present study measured accumulation of tracer (2 μM) glucose by Caco-2 cells after exposure for 10 min or less to a chemically defined medium (CDM) with different monosaccharides before and after anaerobic culture of probiotic <it>Lactobacilli</it>.</p> <p>Results</p> <p>Growth of <it>L. acidophilus </it>was supported by CDM with 110 mM glucose, fructose, and mannose, but not with arabinose, ribose, and xylose or the sugar-free CDM. Glucose accumulation was reduced when Caco-2 cells were exposed for 10 min to sterile CDM with glucose (by 92%), mannose (by 90%), fructose (by 55%), and ribose (by 16%), but not with arabinose and xylose. Exposure of Caco-2 cells for 10 min to bacteria-free supernatants prepared after exponential (48 h) and stationary (72 h) growth phases of <it>L. acidophilus </it>cultured in CDM with 110 mM fructose increased glucose accumulation by 83% and 45%, respectively; exposure to a suspension of the bacteria had no effect. The increase in glucose accumulation was diminished by heat-denaturing the supernatant, indicating the response of Caco-2 cells is triggered by as yet unknown heat labile bacterial metabolites, not by a reduction in CDM components that decrease glucose uptake. Supernatants prepared after anaerobic culture of <it>L. gasseri, L. amylovorus, L. gallinarum</it>, and <it>L. johnsonii </it>in the CDM with fructose increased glucose accumulation by 83%, 32%, 27%, and 14%, respectively.</p> <p>Conclusion</p> <p>The rapid, non-genomic upregulation of SGLT1 by bacterial metabolites is a heretofore unrecognized interaction between probiotics and the intestinal epithelium.</p

Low Temperature and Chemical Rescue Affect Molecular Proximity of F508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Epithelial Sodium Channel (ENaC)

An imbalance of chloride and sodium ion transport in several epithelia is a feature of cystic fibrosis (CF), an inherited disease that is a consequence of mutations in the cftr gene. The cftr gene codes for a Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Some mutations in this gene cause the balance between Cl(-) secretion and Na(+) absorption to be disturbed in the airways; Cl(-) secretion is impaired, whereas Na(+) absorption is elevated. Enhanced Na(+) absorption through the epithelial sodium channel (ENaC) is attributed to the failure of mutated CFTR to restrict ENaC-mediated Na(+) transport. The mechanism of this regulation is controversial. Recently, we have found evidence for a close association of wild type (WT) CFTR and WT ENaC, further underscoring the role of ENaC along with CFTR in the pathophysiology of CF airway disease. In this study, we have examined the association of ENaC subunits with mutated ΔF508-CFTR, the most common mutation in CF. Deletion of phenylalanine at position 508 (ΔF508) prevents proper processing and targeting of CFTR to the plasma membrane. When ΔF508-CFTR and ENaC subunits were co-expressed in HEK293T cells, we found that individual ENaC subunits could be co-immunoprecipitated with ΔF508-CFTR, much like WT CFTR. However, when we evaluated the ΔF508-CFTR and ENaC association using fluorescence resonance energy transfer (FRET), FRET efficiencies were not significantly different from negative controls, suggesting that ΔF508-CFTR and ENaC are not in close proximity to each other under basal conditions. However, with partial correction of ΔF508-CFTR misprocessing by low temperature and chemical rescue, leading to surface expression as assessed by total internal reflection fluorescence (TIRF) microscopy, we observed a positive FRET signal. Our findings suggest that the ΔF508 mutation alters the close association of CFTR and ENaC

MicroRNA-Mediated Dynamic Bidirectional Shift between the Subclasses of Glioblastoma Stem-like Cells

Large-scale transcriptomic profiling of glioblastoma (GBM) into subtypes has provided remarkable insight into the pathobiology and heterogeneous nature of this disease. The mechanisms of speciation and inter-subtype transitions of these molecular subtypes require better characterization to facilitate the development of subtype-specific targeting strategies. The deregulation of microRNA expression among GBM subtypes and their subtype-specific targeting mechanisms are poorly understood. To reveal the underlying basis of microRNA-driven complex subpopulation dynamics within the heterogeneous intra-tumoral ecosystem, we characterized the expression of the subtype-enriched microRNA-128 (miR-128) in transcriptionally and phenotypically diverse subpopulations of patient-derived glioblastoma stem-like cells. Because microRNAs are capable of re-arranging the molecular landscape in a cell-type-specific manner, we argue that alterations in miR-128 levels are a potent mechanism of bidirectional transitions between GBM subpopulations, resulting in intermediate hybrid stages and emphasizing highly intricate intra-tumoral networking

The Long Non-coding RNA HIF1A-AS2 Facilitates the Maintenance of Mesenchymal Glioblastoma Stem-like Cells in Hypoxic Niches

Long non-coding RNAs (lncRNAs) have an undefined role in the pathobiology of glioblastoma multiforme (GBM). These tumors are genetically and phenotypically heterogeneous with transcriptome subtype-specific GBM stem-like cells (GSCs) that adapt to the brain tumor microenvironment, including hypoxic niches. We identified hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) as a subtype-specific hypoxia-inducible lncRNA, upregulated in mesenchymal GSCs. Its deregulation affects GSC growth, self-renewal, and hypoxia-dependent molecular reprogramming. Among the HIF1A-AS2 interactome, IGF2BP2 and DHX9 were identified as direct partners. This association was needed for maintenance of expression of their target gene, HMGA1. Downregulation of HIF1A-AS2 led to delayed growth of mesenchymal GSC tumors, survival benefits, and impaired expression of HMGA1 in vivo. Our data demonstrate that HIF1A-AS2 contributes to GSCs’ speciation and adaptation to hypoxia within the tumor microenvironment, acting directly through its interactome and targets and indirectly by modulating responses to hypoxic stress depending on the subtype-specific genetic context

Glucose-Based Regulation of miR-451/AMPK Signaling Depends on the OCT1 Transcription Factor

In aggressive, rapidly growing solid tumors such as glioblastoma multiforme (GBM), cancer cells face frequent dynamic changes in their microenvironment, including the availability of glucose and other nutrients. These challenges require that tumor cells have the ability to adapt in order to survive periods of nutrient/energy starvation. We have identified a reciprocal negative feedback loop mechanism in which the levels of microRNA-451 (miR-451) are negatively regulated through the phosphorylation and inactivation of its direct transcriptional activator OCT1 by 5′ AMP-activated protein kinase (AMPK), which is activated by glucose depletion-induced metabolic stress. Conversely, in a glucose-rich environment, unrestrained expression of miR-451 suppresses AMPK pathway activity. These findings uncover miR-451 as a major effector of glucose-regulated AMPK signaling, allowing tumor cell adaptation to variations in nutrient availability in the tumor microenvironment

MicroRNA Signatures and Molecular Subtypes of Glioblastoma: The Role of Extracellular Transfer

Summary Despite the importance of molecular subtype classification of glioblastoma (GBM), the extent of extracellular vesicle (EV)-driven molecular and phenotypic reprogramming remains poorly understood. To reveal complex subpopulation dynamics within the heterogeneous intratumoral ecosystem, we characterized microRNA expression and secretion in phenotypically diverse subpopulations of patient-derived GBM stem-like cells (GSCs). As EVs and microRNAs convey information that rearranges the molecular landscape in a cell type-specific manner, we argue that intratumoral exchange of microRNA augments the heterogeneity of GSC that is reflected in highly heterogeneous profile of microRNA expression in GBM subtypes

Proteolytic Cleavage of Human Acid-sensing Ion Channel 1 by the Serine Protease Matriptase*

Acid-sensing ion channel 1 (ASIC1) is a H+-gated channel of the amiloride-sensitive epithelial Na+ channel (ENaC)/degenerin family. ASIC1 is expressed mostly in the central and peripheral nervous system neurons. ENaC and ASIC function is regulated by several serine proteases. The type II transmembrane serine protease matriptase activates the prototypical αβγENaC channel, but we found that matriptase is expressed in glioma cells and its expression is higher in glioma compared with normal astrocytes. Therefore, the goal of this study was to test the hypothesis that matriptase regulates ASIC1 function. Matriptase decreased the acid-activated ASIC1 current as measured by two-electrode voltage clamp in Xenopus oocytes and cleaved ASIC1 expressed in oocytes or CHO K1 cells. Inactive S805A matriptase had no effect on either the current or the cleavage of ASIC1. The effect of matriptase on ASIC1 was specific, because it did not affect the function of ASIC2 and no matriptase-specific ASIC2 fragments were detected in oocytes or in CHO cells. Three matriptase recognition sites were identified in ASIC1 (Arg-145, Lys-185, and Lys-384). Site-directed mutagenesis of these sites prevented matriptase cleavage of ASIC1. Our results show that matriptase is expressed in glioma cells and that matriptase specifically cleaves ASIC1 in heterologous expression systems