Chloroquine Increases Glucose Uptake via Enhancing GLUT4 Translocation and Fusion with the Plasma Membrane in L6 Cells

Abstract

Background/Aims: Chloroquine can induce an increase in the cellular uptake of glucose; however, the underlying mechanism is unclear. Methods: In this study, translocation of GLUT4 and intracellular Ca²⁺ changes were simultaneously observed by confocal microscope in L6 cells stably over-expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM) was traced using HA-GLUT4-GFP. Glucose uptake was measured using a cell-based glucose uptake assay. GLUT4 protein was detected by Western blotting and mRNA level was detected by RT-PCR. Results: We found that chloroquine induced significant increases in glucose uptake, glucose transporter GLUT4 translocation to the plasma membrane (GTPM), GLUT4 fusion with the PM, and intracellular Ca²⁺ in L6 muscle cells. Chloroquine-induced increases of GTPM and intracellular Ca²⁺ were inhibited by Gallein (G_βγ inhibitor) and U73122 (PLC inhibitor). However, 2-APB (IP₃R blocker) only blocked the increase in intracellular Ca²⁺ but did not inhibit GTPM increase. These results indicate that chloroquine, via the G_βγ-PLC-IP₃-IP₃R pathway, induces elevation of Ca²⁺, and this Ca²⁺ increase does not play a role in chloroqui-ne-evoked GTPM increase. However, GLUT4 fusion with the PM and glucose uptake were significantly inhibited with BAPTA-AM. This suggests that Ca²⁺ enhances GLUT4 fusion with the PM resulting in glucose uptake increase. Conclusion: Our data indicate that chloroquine via G_βγ-PLC-IP₃-IP₃R induces Ca²⁺ elevation, which in turn promotes GLUT4 fusion with the PM. Moreover, chloroquine can enhance GLUT4 trafficking to the PM. These mechanisms eventually result in glucose uptake increase in control and insulin-resistant L6 cells. These findings suggest that chloroquine might be a potential drug for improving insulin tolerance in diabetic patients.

Antidiabetic Activity of Ergosterol from Pleurotus Ostreatus in KK‐Ay Mice with Spontaneous Type 2 Diabetes Mellitus

“…For example, we were not able to determine how PKC was activated. Based on our previous study, we hypothesize that G protein and phospholipase C (PLC) might be involved in the process. The results of the current study suggest that ERG may be a potential hypoglycemic agent for the treatment of T2DM.…”

Ethanolic Extract of Folium Sennae Mediates the Glucose Uptake of L6 Cells by GLUT4 and Ca2+

“…The glucose uptake stimulatory effect of FSE was determined in L6 cells by a previously reported method [ 60 ] with slight modifications [ 61 , 62 ]. Briefly, L6 cells were grown in 96-well culture plates at 37 °C under 5% CO 2 and cultured for 3–4 days until the cells were confluent.…”

Dandelion Chloroform Extract Promotes Glucose Uptake via the AMPK/GLUT4 Pathway in L6 Cells

“…Insulin-responsive aminopeptidase (IRAP) is a protein that strongly colocalizes with GLUT4 [ 15 , 16 ] and can be used as a reporter molecule to reflect the translocation of GLUT4 [ 17 , 18 ]. Stable expression of IRAP in the L6 cell line (L6 IRAP-mOrange) was determined as we previously described [ 19 ]. L6 cells were cultured in α -MEM with 10% FBS and supplemented with penicillin (100 U/mL) and streptomycin (100 μ g/mL) at 37°C in a 5% CO 2 environment.…”

“…The GV348-myc-GLUT4-mOrange L6 cells on circular glass slides were starved with serum-free medium for 2 hours and then treated with 30 μ g/mL DCE or 100 nM insulin for 30 minutes. Next, the cells were fixed with 3% paraformaldehyde, blocked in 2% (w/v) BSA in PBS, and incubated with mouse anti-myc antibody (1:200 dilution) [ 19 ]. Cells were subsequently labeled with anti-mouse-FITC-conjugated secondary antibody (1: 200 dilution).…”

“…Next, the cells were placed on ice, washed three times with cold PBS, and treated with a protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (Selleckchem, USA) at 4°C. Cells were then lysed as described previously [ 19 ]. Equal amounts of protein were separated by 10% (v/v) SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane.…”