EHT 1864, a small molecule inhibitor of Ras-related C3 botulinum toxin substrate 1 (Rac1), attenuates glucose-stimulated insulin secretion in pancreatic β-cells

Abstract

Glucose-stimulated insulin secretion (GSIS) in the pancreatic β-cells entails a variety of signaling mechanisms including activation of small GTP-binding proteins (G-proteins). Previous studies from our laboratory in human islets, rodent islets and clonal β-cells have demonstrated that G-proteins (e.g., Arf6, Cdc42 and Rac1) play novel roles in cytoskeletal remodeling, which is a critical step in the trafficking of insulin-laden secretory granules for fusion with plasma membrane and release of insulin. To further understand regulatory roles of Rac1 in GSIS, we utilized, herein, EHT 1864, a small molecule inhibitor, which attenuates Rac1 activation by retaining the G-protein in an inert/inactive state, thereby preventing activation of its downstream effector proteins. We demonstrate that EHT 1864 markedly attenuated GSIS in INS-1 832/13 cells. In addition, EHT 1864 significantly reduced glucose-induced activation and membrane targeting of Rac1 in INS-1 832/13 cells. This Rac1 inhibitor also suppressed glucose-induced activation of ERK1/2 and p53, but not Akt. Lastly, unlike the inhibitors of protein prenylation (simvastatin), EHT 1864 did not exert any significant effects on cell morphology (cell rounding) under the conditions it attenuated Rac1-sensitive signaling steps leading to GSIS. Based on these findings, we conclude that EHT 1864 specifically inhibits glucose-induced Rac1 activation and membrane association and associated downstream signaling events culminating in inhibition of GSIS.

Glucotoxicity promotes aberrant activation and mislocalization of Ras-related C3 botulinum toxin substrate 1 [Rac1] and metabolic dysfunction in pancreatic islet β-cells: reversal of such metabolic defects by metformin

“…Data from our current studies have provided evidence to suggest that Rac1 activation is upstream to CD36 expression since EHT1864, a known inhibitor of Rac1 [31, 32], attenuated HG-induced CD36 expression in INS-1 832/13 cells. Our findings are also compatible with recent observations of Elumalai and associates demonstrating regulatory roles for Rac1-Nox2 signaling axis promotes CD36 expression in INS-1 cells under the duress of glucotoxic conditions [19].…”

“…However, there was a 50% reduction in nuclear association of N17 mutant in cells under the same conditions [Figure 2; Panels A and B]. In the second approach, we utilized EHT1864, a known inhibitor of Rac1 [31, 32] to functionally inactivate endogenous Rac1 and then assessed nuclear association of Rac1 under LG and HG conditions. Data in Figure 2 [Panels C and D] demonstrate decreased localization of Rac1 in INS-1 832/13 cells exposed to EHT1864.…”

Coenzyme Q10 protects against β‐cell toxicity induced by pravastatin treatment of hypercholesterolemia

“…Previous studies using ex vivo (rat islets) and in vitro (INS1E cells (Metz et al, 1993;Sidarala, Veluthakal, Syeda, & Kowluru, 2015) and overall cell cycle progression and proliferation (Garcia-Ruiz, (Sacher, Weigl, Werner, Szegedi, & Hohenegger, 2005). Rat soleus muscle fiber biopsies incubated with simvastatin exhibited an inhibition of mitochondrial respiration and an increase in the rate of hydrogen peroxide production and reduction of CoQ 10 levels.…”

Up‐regulation of Rac1 in the bronchial smooth muscle of murine experimental asthma

“…In the presence of atropine (1 μmol/L), force development was observed in response to all constriction‐inducing agents administered, with the exception of CCh. The Rac1 inhibitor EHT1864 reportedly inhibits Rac1 activation selectively in vitro, and 10 μmol/L EHT1864 is used by many studies . Therefore, 10 μmol/L EHT1864 was used in this study.…”