Thioglycosides as inhibitors of hSGLT1 and hSGLT2: potential therapeutic agents for the control of hyperglycemia in diabetes

Abstract

The treatment of diabetes has been mainly focused on maintaining normal blood glucose concentrations. Insulin and hypoglycemic agents have been used as standard therapeutic strategies. However, these are characterized by limited efficacy and adverse side effects, making the development of new therapeutic alternatives mandatory. Inhibition of glucose reabsorption in the kidney, mediated by SGLT1 or SGLT2, represents a promising therapeutic approach. Therefore, the aim of the present study was to evaluate the effect of thioglycosides on human SGLT1 and SGLT2. For this purpose, stably transfected Chinese hamster ovary (CHO) cells expressing human SGLT1 and SGLT2 were used. The inhibitory effect of thioglycosides was assessed in transport studies and membrane potential measurements, using alpha-methyl-glucoside uptake and fluorescence resonance energy transfer, respectively. We found that some thioglycosides inhibited hSGLT more strongly than phlorizin. Specifically, thioglycoside I (phenyl-1'-thio-beta-D-glucopyranoside) inhibited hSGLT2 stronger than hSGLT1 and to a larger extent than phlorizin. Thioglycoside VII (2-hydroxymethyl-phenyl-1'-thio-beta-D-galacto-pyranoside) had a pronounced inhibitory effect on hSGLT1 but not on hSGLT2. Kinetic studies confirmed the inhibitory effect of these thioglycosides on hSGLT1 or hSGLT2, demonstrating competitive inhibition as the mechanism of action. Therefore, these thioglycosides represent promising therapeutic agents for the control of hyperglycemia in patients with diabetes.

Figure 1

Effect on sodium-dependent [¹⁴C]AMG-uptake obtained in hSGLT1 or hSGLT2 treated with thioglycosides (10 µM each) or phlorizin (10 µM). Results are expressed as percent of inhibition based on uptake in CHO cells expressing hSGLT1 or hSGLT2 not exposed to thioglycosides (control cells). Blue and red bars represent hSGLT1 and hSGLT2, respectively. Results are the mean of six different experiments. Error bars represents standard deviations. * p < 0.01 shows significantly higher inhibition of sodium-dependent AMG uptake in treated cells as compared to control cells. Control uptake in CHO cells expressing hSGLT1 was 735 pmol/mg/h ± 22 pmol/mg/h and in CHO cells expressing hSGLT2 was 342 pmol/mg/h ± 15 pmol/mg/h.

Figure 2

Effect of thioglycoside I and thioglycoside VII on sodium-dependent AMG uptake on CHO cells expressing hSGLT1 (A) and CHO cells expressing hSGLT2 (B) was determined by IC₅₀ assessment. Different concentrations of thioglycoside I and VII in log scale were plotted against [¹⁴C]AMG uptake as percentage of CHO control cells. The curves for hSGLT 1 and 2 on each cell type were constructed from results from eight different concentrations ranging from 10^-7 to 5x10^-4. The IC₅₀ values of phlorizin are shown as a known reference inhibitory effect.

Figure 3

Correlation of sodium-dependent AMG-uptake to sugar-induced cell membrane depolarization is shown. The correlation coefficient of 0.92 demonstrates a strong linear relationship between the two variables (p < 0.001).

Figure 4

Changes in cell membrane potential induced by D-glucose, thioglycosides I and VII (10 p < 0.01 shows significantly higher induction of cell membrane depolarization in treated cells as compared to control cells (not exposed to thioglycosides).