Fluorescence microscopy-based quantitation of GLUT4 translocation.

Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.

Alternative model organisms for toxicological fingerprinting of relevant parameters in food and nutrition.

In the field of (food) toxicology, there is a strong trend of replacing animal trials with alternative methods for the assessment of adverse health effects in humans. The replacement of animal trials is not only driven by ethical concerns but also by the number of potential testing substances (food additives, packaging material, contaminants, and toxicants), which is steadily increasing. In vitro 2D cell culture applications in combination with in silico modeling might provide an applicable first response. However, those systems lack accurate predictions of metabolic actions. Thus, alternative in vivo models could fill the gap between cell culture and animal trials. In this review, we highlight relevant studies in the field and spotlight the applicability of alternative models, including C. elegans, D. rerio, Drosophila, HET-CAM and Lab-on-a-chip.

FODMAP Fingerprinting of Bakery Products and Sourdoughs: Quantitative Assessment and Content Reduction through Fermentation.

Fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) are associated with digestive disorders and with diseases such as irritable bowel syndrome. In this study, we determined the FODMAP contents of bread, bakery products, and flour and assessed the effectiveness of sourdough fermentation for FODMAP reduction. The fermentation products were analyzed to determine the DP 2-7 and DP >7 fructooligosaccharide (FOS) content of rye and wheat sourdoughs. FOSs were reduced by Acetobacter cerevisiae, Acetobacter okinawensis, Fructilactobacillus sanfranciscensis, and Leuconostoc citreum to levels below those in rye (-81%; -97%) and wheat (-90%; -76%) flours. The fermentation temperature influenced the sourdough acetic acid to lactic acid ratios (4:1 at 4 °C; 1:1 at 10 °C). The rye sourdough contained high levels of beneficial arabinose (28.92 g/kg) and mannitol (20.82 g/kg). Our study contributes in-depth knowledge of low-temperature sourdough fermentation in terms of effective FODMAP reduction and concurrent production of desirable fermentation byproducts.

Acute, reproductive, and developmental toxicity of essential oils assessed with alternative in vitro and in vivo systems.

Essential oils (EOs) have attracted increased interest for different applications such as food preservatives, feed additives and ingredients in cosmetics. Due to their reported variable composition of components, they might be acutely toxic to humans and animals in small amounts. Despite the necessity, rigorous toxicity testing in terms of safety evaluation has not been reported so far, especially using alternatives to animal models. Here, we provide a strategy by use of alternative in vitro (cell cultures) and in vivo (Caenorhabditis elegans, hen's egg test) approaches for detailed investigation of the impact of commonly used rosemary, citrus and eucalyptus essential oil on acute, developmental and reproductive toxicity as well as on mucous membrane irritation. In general, all EOs under study exhibited a comparable impact on measured parameters, with a slightly increased toxic potential of rosemary oil. In vitro cell culture results indicated a concentration-dependent decrease of cell viability for all EOs, with mean IC50 values ranging from 0.08 to 0.17% [v/v]. Similar results were obtained for the C. elegans model when using a sensitized bus-5 mutant strain, with a mean LC50 value of 0.42% [v/v]. In wild-type nematodes, approximately tenfold higher LC50 values were detected. C. elegans development and reproduction was already significantly inhibited at concentrations of 0.5% (wild-type) and 0.1% (bus-5) [v/v] of EO, respectively. Gene expression analysis revealed a significant upregulation of xenobiotic and oxidative stress genes such as cyp-14a3, gst-4, gpx-6 and sod-3. Furthermore, all three EOs under study showed an increased short-time mucous membrane irritation potential, already at 0.5% [v/v] of EO. Finally, GC-MS analysis was performed to quantitate the relative concentration of the most prominent EO compounds. In conclusion, our results demonstrate that EOs can exhibit severe toxic properties, already at low concentrations. Therefore, a detailed toxicological assessment is highly recommended for each EO and single intended application.

CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis.

Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag® with lipid membrane permeable dye, JaneliaFluor® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.

Immunomodulatory Activities of Selected Essential Oils.

Recently, the application of herbal medicine for the prevention and treatment of diseases has gained increasing attention. Essential oils (EOs) are generally known to exert various pharmacological effects, such as antiallergic, anticancer, anti-inflammatory, and immunomodulatory effects. Current literature involving in vitro and in vivo studies indicates the potential of various herbal essential oils as suitable immunomodulators for the alternative treatment of infectious or immune diseases. This review highlights the cellular effects induced by EOs, as well as the molecular impacts of EOs on cytokines, immunoglobulins, or regulatory pathways. The results reviewed in this article revealed a significant reduction in relevant proinflammatory cytokines, as well as induction of anti-inflammatory markers. Remarkably, very little clinical study data involving the immunomodulatory effects of EOs are available. Furthermore, several studies led to contradictory results, emphasizing the need for a multiapproach system to better characterize EOs. While immunomodulatory effects were reported, the toxic potential of EOs must be clearly considered in order to secure future applications.

Ginseng Extract Ameliorates the Negative Physiological Effects of Heat Stress by Supporting Heat Shock Response and Improving Intestinal Barrier Integrity: Evidence from Studies with Heat-Stressed Caco-2 Cells, C. elegans and Growing Broilers.

Climatic changes and heat stress have become a great challenge in the livestock industry, negatively affecting, in particular, poultry feed intake and intestinal barrier malfunction. Recently, phytogenic feed additives were applied to reduce heat stress effects on animal farming. Here, we investigated the effects of ginseng extract using various in vitro and in vivo experiments. Quantitative real-time PCR, transepithelial electrical resistance measurements and survival assays under heat stress conditions were carried out in various model systems, including Caco-2 cells, Caenorhabditis elegans and jejunum samples of broilers. Under heat stress conditions, ginseng treatment lowered the expression of HSPA1A (Caco-2) and the heat shock protein genes hsp-1 and hsp-16.2 (both in C. elegans), while all three of the tested genes encoding tight junction proteins, CLDN3, OCLN and CLDN1 (Caco-2), were upregulated. In addition, we observed prolonged survival under heat stress in Caenorhabditis elegans, and a better performance of growing ginseng-fed broilers by the increased gene expression of selected heat shock and tight junction proteins. The presence of ginseng extract resulted in a reduced decrease in transepithelial resistance under heat shock conditions. Finally, LC-MS analysis was performed to quantitate the most prominent ginsenosides in the extract used for this study, being Re, Rg1, Rc, Rb2 and Rd. In conclusion, ginseng extract was found to be a suitable feed additive in animal nutrition to reduce the negative physiological effects caused by heat stress.

In Vitro and In Vivo Inhibition of Intestinal Glucose Transport by Guava (Psidium Guajava) Extracts.

SCOPE: Known pharmacological activities of guava (Psidium guajava) include modulation of blood glucose levels. However, mechanistic details remain unclear in many cases. METHODS AND RESULTS: This study investigated the effects of different guava leaf and fruit extracts on intestinal glucose transport in vitro and on postprandial glucose levels in vivo. Substantial dose- and time-dependent glucose transport inhibition (up to 80%) was observed for both guava fruit and leaf extracts, at conceivable physiological concentrations in Caco-2 cells. Using sodium-containing (both glucose transporters, sodium-dependent glucose transporter 1 [SGLT1] and glucose transporter 2 [GLUT2], are active) and sodium-free (only GLUT2 is active) conditions, we show that inhibition of GLUT2 was greater than that of SGLT1. Inhibitory properties of guava extracts also remained stable after digestive juice treatment, indicating a good chemical stability of the active substances. Furthermore, we could unequivocally show that guava extracts significantly reduced blood glucose levels (≈fourfold reduction) in a time-dependent manner in vivo (C57BL/6N mice). Extracts were characterized with respect to their main putative bioactive compounds (polyphenols) using HPLC and LC-MS. CONCLUSION: The data demonstrated that guava leaf and fruit extracts can potentially contribute to the regulation of blood glucose levels.