SGLT2 inhibitors act from the extracellular surface of the cell membrane

Abstract

SGLT2 inhibitors are a new class of drugs that have been recently developed to treat type II diabetes. They lower glucose levels by inhibiting the renal Na(+)/glucose cotransporter SGLT2, thereby increasing the amount of glucose excreted in the urine. Pharmacodynamics studies have raised questions about how these inhibitors reach SGLT2 in the brush border membrane of the S1 and S2 segments of the renal proximal tubule: are these drugs filtered by the glomerulus and act extracellularly, or do they enter the cell and act intracellularly? To address this question we expressed hSGLT2 in HEK-293T cells and determined the affinity of a specific hSGLT2 inhibitor, TA-3404 (also known as JNJ-30980924), from the extra- and intracellular side of the plasma membrane. Inhibition of SGLT2 activity (Na(+)/glucose currents) by TA-3404 was determined using the whole-cell patch clamp that allows controlling the composition of both the extracellular and intracellular solutions. We compared the results to those obtained using the nonselective SGLT inhibitor phlorizin, and to the effect of TA-3404 on hSGLT1. Our results showed that TA-3404 is a potent extracellular inhibitor of glucose inward SGLT2 transport (IC50 2 nmol/L) but it was ineffective from the intracellular compartment at both low (5 mmol/L) and high (150 mmol/L) intracellular NaCl concentrations. We conclude that TA-3404 only inhibits SGLT2 from the extracellular side of the plasma membrane, suggesting that it is filtered from the blood through the glomerulus and acts from within the tubule lumen.

Keywords: Inhibitors; SGLT2; type II diabetes.

Figure 1.

Chemical structure of TA‐3404. Chemical structure of JNJ‐30980924 (TA‐3404) or Chloro‐3‐(5‐(6‐fluoro‐3‐pyridyl)‐2‐thienylmethyl)‐1‐(β‐D‐glucopyranosyl) benzene.

Figure 2.

Inhibitors effect on [¹⁴C]‐αMDG uptake mediated by hSGLT1 and hSGLT2. [¹⁴C]‐αMDG uptake was measured in HEK‐293T cells expressing hSGLT1 or hSGLT2 in control conditions (white bars), in presence of 100 μmol/L phlorizin (Pz, light gray bars) and 300 nmol/L (hSGLT1) or 2 nmol/L (hSGLT2) TA‐3404 (TA, dark gray bars). Uptake was measured at 37°C and expressed as quantity of tracer (pmol) per minute per μg protein. Bars are means ± SE, n = 4 wells.

Figure 3.

TA‐3404 effect on glucose‐induced currents. (A) Current recording obtained from cells expressing hSGLT2 at a V_h = −60 mV and at 37°C. Glucose‐dependent current was induced by adding 100 mmol/L glucose to the extracellular solution. The time course of inhibition of glucose‐induced current was monitored upon application of TA‐3404 (TA) and upon removal of the inhibitor, the time course of current recovery. (B) Current recording obtained from cells expressing hSGLT1 at a V_h = −60 mV and at 22°C. Glucose‐dependent current was induced by adding 0.5 mmol/L glucose to the extracellular solution. The time course of inhibition of glucose‐induced current was monitored upon application of TA‐3404 (TA) and upon removal of the inhibitor, the time course of current recovery.

Figure 4.

Inhibition of hSGLT1 and hSGLT2 by extracellular TA‐3404. (A) [¹⁴C]‐αMDG (50 μmol/L) uptake was measured in cells expressing hSGLT2 in control condition and in presence of 1, 3, 5, 10, 100, and 500 nmol/L TA‐3404. Bars are means ± SE, n = 4 wells. (B) Current induced by 0.5 mmol/L glucose was recorded from cells expressing hSGLT1 in control condition and in presence of 100, 300, 500, and 1000 nmol/L TA‐3404. Bars are means ± SE, n = 4 wells.

Figure 5.

Sidedness of TA‐3404 interaction for hSGLT2 and hSGLT1. Inward glucose currents were measured in cells expressing hSGLT2 (A and B) and hSGLT1 (C and D) as a function of the intracellular inhibitors (phlorizin and TA‐3404) in 5 or 150 mmol/L NaCl). All cells were incubated in standard 150 mmol/L NaCl buffer at 37°C in the presence and absence of glucose (100 mmol/L for hSGLT2, and 10 mmol/L for hSGLT1) at a holding potential of −60 mV. (A) hSGLT2 inward glucose currents measured in the presence of 5 mmol/L NaCl intracellular buffers before and after the addition of 10 μmol/L phlorizin, 200 μmol/L phlorizin or 200 nmol/L TA‐3404. Bars are means ± SE, n = 4 cells. (B) hSGLT2 inward glucose currents measured in the presence of 150 mmol/L NaCl intracellular buffers before and after adding the addition of 10 μmol/L phlorizin, 200 μmol/L phlorizin or 200 nmol/L TA‐3404. Bars are means ± SE, n = 4. (C) hSGLT1 inward glucose currents measured in the presence of 5 mmol/L NaCl intracellular buffers before and after the addition of 10 μmol/L phlorizin, 200 μmol/L phlorizin or 200 nmol/L TA‐3404. Bars are means ± SE, n = 4 cells. (D) hSGLT1 inward glucose currents measured in the presence of 150 mmol/L NaCl intracellular buffers before and after adding the addition of 10 μmol/L phlorizin, 200 μmol/L phlorizin or 200 nmol/L TA‐3404. Bars are means ± SE, n = 4.