Influence of the buffer parameters on the Gluc-HET performance.

<p>(A) Comparison of different buffer systems. Eggs were incubated for 11 days and treated with different buffer formulations for 120 min. The drop of the blood glucose level was determined via a blood glucose meter. Error bars are based on the standard error of the mean. (B) Influence of the buffer temperature on the assay performance. Eggs were incubated for 11 days and treated either with HBSS buffer stored at 38°C or room temperature (23°C). Blood glucose levels were determined by a blood glucose meter. Error bars are based on the standard error of the mean. (C) Influence of the buffer volume on the assay performance. Eggs were incubated for 11 days and treated with either 100 μL or 300 μL of HBSS with or without NovoRapid (3.3 U/mL) for up to 3 hours. Blood glucose levels were determined by a blood glucose meter. Error bars are based on the standard error of the mean. *P < 0.05, ***P < 0.001 and ****P < 0.0001, significant difference between 100 μL and 300 μL.</p

Description of the Gluc-HET model.

<p>(A) Important steps of a Gluc-HET experiment. From top to bottom: opening and removal of the eggshell (1–2); equilibration and removal of the eggshell membrane with the chorioallantoic membrane exposed (3); prepared vessel on a pH strip (4); and collection of blood and measurement of the glucose concentration by a glucose meter (5). The enlarged images (red and green rectangles) highlight the nature and handling of the blood vessels. (B) Comparison of two methods to quantitate blood glucose levels. Eggs were incubated without (control) or with KRPH buffer for different incubation times, and the blood glucose was determined by HPLC or with a blood glucose meter. Error bars are based on the standard error of the mean.</p

Biomolecular Characterization of Putative Antidiabetic Herbal Extracts

<div><p>Induction of GLUT4 translocation in the absence of insulin is considered a key concept to decrease elevated blood glucose levels in diabetics. Due to the lack of pharmaceuticals that specifically increase the uptake of glucose from the blood circuit, application of natural compounds might be an alternative strategy. However, the effects and mechanisms of action remain unknown for many of those substances. For this study we investigated extracts prepared from seven different plants, which have been reported to exhibit anti-diabetic effects, for their GLUT4 translocation inducing properties. Quantitation of GLUT4 translocation was determined by total internal reflection fluorescence (TIRF) microscopy in insulin sensitive CHO-K1 cells and adipocytes. Two extracts prepared from purslane (<i>Portulaca oleracea</i>) and tindora (<i>Coccinia grandis</i>) were found to induce GLUT4 translocation, accompanied by an increase of intracellular glucose concentrations. Our results indicate that the PI3K pathway is mainly responsible for the respective translocation process. Atomic force microscopy was used to prove complete plasma membrane insertion. Furthermore, this approach suggested a compound mediated distribution of GLUT4 molecules in the plasma membrane similar to insulin stimulated conditions. Utilizing a fluorescent actin marker, TIRF measurements indicated an impact of purslane and tindora on actin remodeling as observed in insulin treated cells. Finally, <i>in-ovo</i> experiments suggested a significant reduction of blood glucose levels under tindora and purslane treated conditions in a living organism. In conclusion, this study confirms the anti-diabetic properties of tindora and purslane, which stimulate GLUT4 translocation in an insulin-like manner.</p></div

Quantitation of GLUT4 translocation and glucose uptake in 3T3-L1 cells.

<p>(A) TIRF microscopy based quantitation of GLUT4 translocation in 3T3-L1 GLUT4-GFP adipocytes. Cells were grown in 96-well plates (20,000 cells/well) for 24 hours and then starved overnight in serum-free medium. Images before and 10 minutes after stimulation with indicated substances are shown. Scale bar = 20 μm. (B) GLUT4-GFP signal intensity increase was analyzed after 10 and 30 minutes of stimulation. Error bars are based on the standard error of the mean. **P < 0.01, ***P < 0.001 and ****P < 0.0001, significant increase with respect to KRPH treated cells. (C) For glucose uptake measurements 3T3-L1 cells were grown in 96-well plates (2,000 cells/well) and differentiated to adipocytes. Cells were starved in serum-free medium overnight, glucose deprived in KRPH buffer for 40 minutes, and then stimulated with the indicated substances for 20 minutes followed by addition of 2-DG (20 minutes). Cell extracts were prepared and 2-DG uptake was measured after a colorimetric reaction using a plate reader device. Samples were measured in duplicates at least in three individual experiments. Error bars are based on the standard error of the mean. *P < 0.05, **P < 0.01 and ****P < 0.0001, significant increase with respect to KRPH (Ins, PP60, PUR) or 0.25% DMSO in KRPH (TIN, GIN, BIL, JIA, MT) treated cells.</p

HET-CAM experiments for the determination of blood glucose decreasing properties of selected herbal compounds <i>in-ovo</i>.

<p>(A) The chorioallantoic membrane of chicken embryos was incubated with the tested substances for 3 hours. After removal of the egg membrane (1), blood was collected from a main vessel (2), and the concentration of glucose in prepared plasma samples was measured by HPLC. (B) Normalized glucose concentration after incubation at indicated time points. Error bars are based on the standard error of the mean. *P < 0.05, ***P < 0.001 and ****P < 0.0001, significant decrease with respect to KRPH (NovoRapid, PUR) or DMSO (TIN) treated cells.</p

GLUT4 translocation induction by putative insulin mimetic herbal compounds quantitated by TIRF microscopy.

<p>(A) Schematic illustration of insulin or herbal compound induced GLUT4 translocation. Putative insulin mimetic substances stimulating the insulin receptor (IR), other plasma membrane or cytosolic effector proteins (1) induce signal transduction events (2) that lead to the translocation of GLUT4 containing vesicles (3) and their subsequent fusion with the PM (4). This transfer is linked to an increase of the GFP-signal intensity in the evanescent field. (B) GLUT4-GFP signal in CHO-K1 hIR/GLUT4-myc-GFP cells before and after stimulation with indicated substances. Cells were seeded in 96-well plates (35,000 cells/well) and grown over night followed by 3 hours of starvation in HBSS buffer. Cells were then stimulated by the indicated substances for 10 min and the GFP-signal was recorded. Scale bar = 20 μm. (C) The GLUT4-GFP signal intensity increase in the evanescent field was quantitated after 10 and 60 minutes. Error bars are based on the standard error of the mean. At least 60 cells were analyzed for each substance. Data were collected from the same cells before and after substance application. ***P < 0.001 and ****P < 0.0001, significant increase with respect to KRPH (Ins, PP60, PUR) or 0.25% DMSO in KRPH (TIN, GIN, BIL, JIA, MT) treated cells, respectively.</p

Effects of herbal compounds on actin remodeling.

<p>CHO-K1 hIR/GLUT4-myc-GFP cells were transiently transfected with the F-actin marker Lifeact-tdTomato, seeded in 96-well plates (100,000 cells/well), grown over night, and then starved for 3 hours in HBSS buffer. (A) GLUT4-GFP and Lifeact-tdTomato signals recorded at 488 and 561 nm before and after stimulation (15 minutes). For a better visualization of changes in cell size upon compound treatment, additional color adjustment (blue and red images, respectively) of the Lifeact-tdTomato channel is shown. Signal oscillations and sustained intensity increases in the cell periphery are marked by blue arrows, while changes in cell size are indicated by yellow arrows. Scale bar = 20 μm. (B) Quantitation of compound induced change of cell size. Fluorescence images in the Lifeact-tdTomato channel (561 nm excitation) were recorded before and after (5, 15, and 25 minutes, respectively) compound treatment. Size of individual cells was quantitated (n > 30) at the different time points. Error bars are based on the standard error of the mean. **P < 0.01 and ****P < 0.0001, significant increase with respect to mock control.</p

Effects of PI3K, MAPK, and AMPK inhibitors on GLUT4 translocation in PUR, TIN, and GIN stimulated cells measured by TIRF microscopy.

<p>CHO-K1 hIR/GLUT4-myc-GFP cells were seeded in 96-well plates (35,000 cells/well), grown over night and then starved for 3 hours in HBSS buffer followed by addition of insulin or the herbal compounds in combination with different inhibitors (CC, SB, wort) for 1 hour. Response curves were generated by measuring the increase of the GFP-signal at 488 nm excitation after application of insulin (A), PUR (B), TIN (C) or GIN (D) at indicated time points. Error bars are based on the standard error of the mean.</p