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

Verena Stadlbauer,Julian Weghuber,Alexander Stallinger,Tobias Aumiller,Marcus Iken,Cathrina Neuhauser

doi:10.3390/molecules26144346

Abstract

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.

Highlights

We have implemented and refined a total internal reflection fluorescence (TIRF) microscopy approach to quantify glucose transporter 4 (GLUT4) translocation in live cells [17,19]. We used this technique on a large scale and screened 643 aqueous plant extracts from the PECKISH library for their ability to induce GLUT4 translocation in CHO-K1 cells that are stably expressing the human insulin receptor and a GLUT4-myc-GFP fusion protein
Extracts prepared from Hoodia, Reetha A (Sapindus mukorossi), soap bark tree (Quillaja saponaria), poppy and chestnut
In our first study describing the suitability of TIRF microscopy for the quantitative analysis of GLUT4 translocation [19], we clearly demonstrated that the increase in the GFP signal in the evanescent field needs to be confirmed by effective plasma membrane insertion of GLUT4 proteins

Summary

Introduction

Diabetes mellitus is a metabolic disease with continually increasing numbers of patients globally. Since 1980, the number of adults suffering from diabetes has nearly quadrupled worldwide [1]. The disease leads to hyperglycemia for a prolonged period and is thought to contribute to one out of nine deaths in 20- to 79-year-old adults [2]. Two different dysfunctions cause two types of diabetes mellitus. With 90–95% of all diabetes mellitus patients having T2DM, it represents the most prevalent subtype [3]. Risk factors for T2DM include obesity, a change in cholesterol levels and a lack of physical exercise, which are modifiable issues, in addition to nonmodifiable factors, such as genetics or increasing age. Diagnosis of the disease often takes years because it develops gradually, and early symptoms are not severe [4].

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