Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy

Abstract

Background and PurposeInsulin stimulates the transport of glucose in target tissues by triggering the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Resistance to insulin, the major abnormality in type 2 diabetes, results in a decreased GLUT4 translocation efficiency. Thus, special attention is being paid to search for compounds that are able to enhance this translocation process in the absence of insulin.Experimental ApproachTotal internal reflection fluorescence (TIRF) microscopy was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein.Key ResultsUsing our approach, we demonstrated GLUT4 translocation modulatory properties of selected substances and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake. Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR.Conclusions and ImplicationsTaken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs.

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

“…Our data indicate the potential of guava leaf and fruit extracts to inhibit GLUT2 and SGLT1 and therefore reduce intestinal glucose transport in vitro and in vivo. Important strategies for the prevention and regulation of hyperglycemia include the exploration for insulin mimetic substances and direct manipulation of the major blood glucose regulator GLUT4, as well as the exploration for compounds that reduce glucose uptake in the small intestine by inhibiting transporters such as the SGLT1 and GLUT2 . Our results indicate that consumption of not only guava leaf but also fruit extracts and functionalized beverages might be a potential source for dietary polyphenols and other bioactive compounds that are able to effectively inhibit glucose transport in the small intestine.…”

“…Therefore, developing new strategies for treatment and prevention of these health problems is of great importance. One possible strategy is the direct manipulation of blood glucose levels via insulin mimetic substances and enhanced glucose transporter 4 (GLUT4) activities . We have recently developed an in ovo model to test the efficacy of such compounds also in a living organism .…”

Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores

“…In order to prove the practicability of the setup described above, we characterized the well-known stimulus-dependent translocation of GLUT4 from intracellular storage compartments to the plasma membrane. For this purpose, insulin as well as insulin-mimetic phytochemicals such as tannic acid and an extract of Bellis perennis were used as active agents [ 27 , 36 ]. The potential of SIM in comparison with conventional wide-field microscopy is shown in Figure 2 for a Chinese Hamster Ovary (CHO) cell transfected with GFP-tagged GLUT4 (CHO-K1-hIR-myc-GLUT4-GFP) without further stimulation.…”

“…In this regard, insulin mimetic substances, which induce GLUT4 translocation independently of insulin, may represent a novel therapeutic strategy for insulin-resistant T2DM patients. Therefore, recent work of the authors has focused on TIRF microscopy to quantitate GLUT4 translocation [ 27 , 28 ]. SIM and TIRF-SIM, as reported in this paper, will permit a further increase in resolution and improve the visibility of GLUT4 translocation upon stimulation by insulin or insulin-mimetic drugs.…”

Cell growth stimulating effect of Ganoderma lucidum spores and their potential application for Chinese hamster ovary K1 cell cultivation

“…Sampling was conducted at the 0, 48th, and 84th h for EGFP fluorescence intensity measurement. The production of INF-a protein was detected by measuring the fluorescence of GFP signal in CHO cell re-suspended in PBS solution, and relative fluorescence units (RFU) were compared to determine the effects of Gls [46]. The fluorescence reading of PBS blank solution was used to subtract background interference.…”