Gluc-HET, a complementary chick embryo model for the characterization of antidiabetic compounds.

Insulin resistance and β cell failure are the main causes of elevated blood glucose levels in Type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease. Several medications to treat or reduce the symptoms of T2DM are used, including the injection of insulin and the application of insulin sensitizing or glucose production reducing drugs. Furthermore, the use of phytochemicals has attracted increasing attention for the therapy and prevention of T2DM. In order to identify and characterize antidiabetic compounds, efficient test systems are required. Here we present a modified chick embryo model (hens egg test, HET), which has originally been developed to determine the potential irritancy of chemicals, as a versatile tool for the characterization of phytochemicals with antidiabetic properties. We termed this modified assay variation Gluc-HET. More precisely, we determined the influence of variations in the incubation time of the fertilized eggs and studied the effects of different buffer parameters, such as the temperature, composition and volume, used for drug application. In addition, we tested several putative antidiabetic plant extracts, which have been identified in an in-vitro primary screening procedure, for their effectiveness in reducing blood glucose levels in-ovo. Taken together, our Gluc-HET model has proven to be a reliable and manageable system for the characterization of antidiabetic compounds

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

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