Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals.

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

Elder (Sambucus nigra), identified by high-content screening, counteracts foam cell formation without promoting hepatic lipogenesis.

Cholesterol deposition in intimal macrophages leads to foam cell formation and atherosclerosis. Reverse cholesterol transport (RCT), initiated by efflux of excess cholesterol from foam cells, counteracts atherosclerosis. However, targeting RCT by enhancing cholesterol efflux was so far accompanied by adverse hepatic lipogenesis. Here, we aimed to identify novel natural enhancers of macrophage cholesterol efflux suitable for the prevention of atherosclerosis. Plant extracts of an open-access library were screened for their capacity to increase cholesterol efflux in RAW264.7 macrophages trace-labeled with fluorescent BODIPY-cholesterol. Incremental functional validation of hits yielded two final extracts, elder (Sambucus nigra) and bitter orange (Citrus aurantium L.) that induced ATP binding cassette transporter A1 (ABCA1) expression and reduced cholesteryl ester accumulation in aggregated LDL-induced foam cells. Aqueous elder extracts were subsequently prepared in-house and both, flower and leaf extracts increased ABCA1 mRNA and protein expression in human THP-1 macrophages, while lipogenic gene expression in hepatocyte-derived cells was not induced. Chlorogenic acid isomers and the quercetin glycoside rutin were identified as the main polyphenols in elder extracts with putative biological action. In summary, elder flower and leaf extracts increase macrophage ABCA1 expression and reduce foam cell formation without adversely affecting hepatic lipogenesis.

Insulin-Mimetic Activity of Herbal Extracts Identified with Large-Scale Total Internal Reflection Fluorescence Microscopy.

Diabetes mellitus is a spreading global pandemic. Type 2 diabetes mellitus (T2DM) is the predominant form of diabetes, in which a reduction in blood glucose uptake is caused by impaired glucose transporter 4 (GLUT4) translocation to the plasma membrane in adipose and muscle cells. Antihyperglycemic drugs play a pivotal role in ameliorating diabetes symptoms but often are associated with side effects. Hence, novel antidiabetic compounds and nutraceutical candidates are urgently needed. Phytogenic therapy can support the prevention and amelioration of impaired glucose homeostasis. Using total internal reflection fluorescence microscopy (TIRFM), 772 plant extracts of an open-access plant extract library were screened for their GLUT4 translocation activation potential, resulting in 9% positive hits. Based on commercial interest and TIRFM assay-based GLUT4 translocation activation, some of these extracts were selected, and their blood glucose-reducing effects in ovo were investigated using a modified hen's egg test (Gluc-HET). To identify the active plant part, some of the available candidate plants were prepared in-house from blossoms, leaves, stems, or roots and tested. Acacia catechu (catechu), Pulmonaria officinalis (lungwort), Mentha spicata (spearmint), and Saponaria officinalis (common soapwort) revealed their potentials as antidiabetic nutraceuticals, with common soapwort containing GLUT4 translocation-activating saponarin.

Combined acid hydrolysis and fermentation improves bioactivity of citrus flavonoids in vitro and in vivo.

Many bioactive plant compounds, known as phytochemicals, have the potential to improve health. Unfortunately, the bioavailability and bioactivity of phytochemicals such as polyphenolic flavonoids are reduced due to conjugation with sugar moieties. Here, we combine acid hydrolysis and tailored fermentation by lactic acid bacteria (Lactiplantibacillus plantarum) to convert the biologically less active flavonoid glycosides hesperidin and naringin into the more active aglycones hesperetin and naringenin. Using a comprehensive approach, we identify the most effective hydrolysis and fermentation conditions to increase the concentration of the aglycones in citrus extracts. The higher cellular transport and bioactivity of the biotransformed citrus extract are also demonstrated in vitro and in vivo. Superior antioxidant, anti-inflammatory and cell migration activities in vitro, as well as intestinal barrier protecting and antioxidant activities in Drosophila melanogaster are identified. In conclusion, the presented biotransformation approach improves the bioactivity of flavonoids, clearly traced back to the increase in aglycone content.

Anti-Hyperglycemic Effects of Oils and Extracts Derived from Sea Buckthorn - A Comprehensive Analysis Utilizing In Vitro and In Vivo Models.

SCOPE: Sea buckthorn (Hippophaes rhamnoides) is capable of ameliorating disturbed glucose metabolism in animal models and human subjects. Here, the effect of sea buckthorn oil as well as of extracts of fruits, leaves, and press cake on postprandial glucose metabolism is systematically investigated. METHODS AND RESULTS: Sea buckthorn did neither exert decisive effects in an in vitro model of intestinal glucose absorption nor did it alter insulin secretion. However, sea buckthorn stimulates GLUT4 translocation to the plasma membrane comparable to insulin, indicative of increased glucose clearance from the circulation. Isorhamnetin is identified in all sea buckthorn samples investigated and is biologically active in triggering GLUT4 cell surface localization. Consistently, sea buckthorn products lower circulating glucose by ≈10% in a chick embryo model. Moreover, sea buckthorn products fully revert hyperglycemia in the nematode Caenorhabditis elegans while they are ineffective in Drosophila melanogaster under euglycemic conditions. CONCLUSION: These data indicate that edible sea buckthorn products as well as by-products are promising resources for hypoglycemic nutrient supplements that increase cellular glucose clearance into target tissues.

Avens Root (Geum Urbanum L.) Extract Discovered by Target-Based Screening Exhibits Antidiabetic Activity in the Hen's Egg Test Model and Drosophila melanogaster.

Medicinal plant extracts are becoming increasingly important as an alternative for traditional drugs against diabetes mellitus (DM). For this reason, we initialized a target-based screening of 111 root extracts from an open access plant extract library (PECKISH) by ascertaining their in-vitro inhibitory efficacy on α-glucosidase. The two most active extracts Geum urbanum L. (roseroot) and Rhodiola rosea L. (avens root) were further tested for their antidiabetic activities in terms of their impact on different regulatory key points of glucose homeostasis. To this end, various enzyme- and cell culture-based in-vitro assays were employed including the determination of sodium-dependent glucose transporter 1 (SGLT1) activity in Caco-2 monolayers by Ussing chambers and of glucose transporter 4 (GLUT4) translocation in a GFP-reporter cell line. Subsequently, the antidiabetic potential of the root extracts were further evaluated in in-vivo models, namely hen's eggs test and the fruit fly Drosophila melanogaster. Avens root extract was found to be a more potent inhibitor of the enzymes α-glucosidase and dipeptidyl peptidase-4 (DPP4) than roseroot extract. Most importantly, only avens root extract exhibited antidiabetic activity in the two in-vivo models eliciting a reduced blood glucose level in the in-ovo model and a decline of the triglyceride level in a dietary starch-induced D. melanogaster obesity model. Analyses of the polyphenolic composition of the avens root extract by HPLC revealed a high content of ellagic acid and its derivatives as well as ellagitannins such as pedunculagin, stenophyllanin, stachyurin, casuarinin and gemin A. In conclusion, avens root extract represents a promising medicinal plant that should be considered in further in-vivo studies on hyperglycemia in laboratory rodents and humans.

A High-Content Screen for the Identification of Plant Extracts with Insulin Secretion-Modulating Activity.

Bioactive plant compounds and extracts are of special interest for the development of pharmaceuticals. Here, we describe the screening of more than 1100 aqueous plant extracts and synthetic reference compounds for their ability to stimulate or inhibit insulin secretion. To quantify insulin secretion in living MIN6 ß cells, an insulin-Gaussia luciferase (Ins-GLuc) biosensor was used. Positive hits included extracts from Quillaja saponaria, Anagallis arvensis, Sapindus mukorossi, Gleditsia sinensis and Albizia julibrissin, which were identified as insulin secretion stimulators, whereas extracts of Acacia catechu, Myrtus communis, Actaea spicata L., Vaccinium vitis-idaea and Calendula officinalis were found to exhibit insulin secretion inhibitory properties. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to characterize several bioactive compounds in the selected plant extracts, and these bioactives were retested for their insulin-modulating properties. Overall, we identified several plant extracts and some of their bioactive compounds that may be used to manipulate pancreatic insulin secretion.

Aqueous extracts of lingonberry and blackberry leaves identified by high-content screening beneficially act on cholesterol metabolism.

Decreasing circulating low-density lipoprotein (LDL) cholesterol levels leads to decreased risk of cardiovascular diseases. Natural compounds are capable of lowering LDL-cholesterol even on top of lifestyle modification or medication. To identify novel plant-derived compounds to lower plasma LDL cholesterol levels, we performed high-content screening based on the transcriptional activation of the promoter of the LDL receptor (LDLR). The identified hits were thoroughly validated in human hepatic cell lines in terms of increasing LDLR mRNA and protein levels, lowering cellular cholesterol levels and increasing cellular LDL uptake. By means of this incremental validation process in vitro, aqueous extracts prepared from leaves of lingonberries (Vaccinium vitis-idaea) as well as blackberries (Rubus fruticosus) were found to have effects comparable to lovastatin, a prototypic cholesterol-lowering drug. When applied in vivo in mice, both extracts induced subtle increases in hepatic LDLR expression. In addition, a significant increase in high-density lipoprotein (HDL) cholesterol was observed. Taken together, aqueous extracts from lingonberry or blackberry leaves were identified and characterized as strong candidates to provide cardiovascular protection.