Enhancement of cellular glucose uptake by reactive species: a promising approach for diabetes therapy

Abstract

It is generally known that antidiabetic activity is associated with an increased level of glucose uptake in adipocytes and skeletal muscle cells. However, the role of exogenous reactive oxygen and nitrogen species (RONS) in muscle development and more importantly in glucose uptake is largely unknown. We investigate the effect of RONS generated by cold atmospheric plasma (CAP) in glucose uptake. We show that the glucose uptake is significantly enhanced in differentiated L6 skeletal muscle cells after CAP treatment. We also observe a significant increase of the intracellular Ca⁺⁺ and ROS level, without causing toxicity. One of the possible reasons for an elevated level of glucose uptake as well as intracellular ROS and Ca⁺⁺ ions is probably the increased oxidative stress leading to glucose transport.

Fig. 1. Schematic diagram of the μs-DBD device. Nitrogen is used as a feeding gas with a flow rate of 1 SLM.

Fig. 2. Measurements of pH (a) and temperature (b) after plasma treatment of the cell media. All values are expressed as ±SD in triplicates.

Fig. 3. Measurements of the RONS content after plasma treatment of the cell media. Determination of (a) OH, (b) H₂O₂, (c) NO and (d) NO₂⁻ concentrations in 2 ml media after μs-DBD plasma exposure at different times. All values are expressed as ±SD in triplicates.

Fig. 4. Analysis of L6 cell cytotoxicity (a), proliferation (b), differentiation (c) and total protein content (d). The cell differentiation analysis was performed using light microscopy, whereas the measurement of the total protein content was carried out on day 2, 4 and 8 after exposure to μs-DBD. Comparison between all groups showed significant differences by one-way ANOVA (* denotes P < 0.05, ** denotes P < 0.01 and *** denotes P < 0.001).

Fig. 5. Cellular differentiation analysis through actin, tubulin and nuclear immunofluorescence images of L6 cells on day 8 after exposure to the μs-DBD.

Fig. 6. Glucose uptake (a) and (b), cytosolic Ca⁺⁺ (c) and (d) and intracellular ROS (e) analysis after treatment with the μs-DBD for 90 s as well as with insulin (see (a) and (b)), chelator BAPTA (see (c) and (d)) and trolox (see (e)), measured on day 2, 4 and 8. Fluorescence images of 2NBDG uptake (b) and Ca⁺⁺ (d) were taken on day 8. Comparison between all groups showed significant differences by one-way ANOVA (* denotes P < 0.05, ** denotes P < 0.01 and *** denotes P < 0.001).