Vincamine as a GPR40 agonist improves glucose homeostasis in type 2 diabetic mice

in Journal of Endocrinology

Correspondence should be addressed to J Chen or L Hu or X Shen: jingchen@simm.ac.cn or lhhu@njucm.edu.cn or xshen88@163.com
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Vincamine, a monoterpenoid indole alkaloid extracted from the Madagascar periwinkle, is clinically used for the treatment of cardio-cerebrovascular diseases, while also treated as a dietary supplement with nootropic function. Given the neuronal protection of vincamine and the potency of β-cell amelioration in treating type 2 diabetes mellitus (T2DM), we investigated the potential of vincamine in protecting β-cells and ameliorating glucose homeostasis in vitro and in vivo. Interestingly, we found that vincamine could protect INS-832/13 cells function by regulating G-protein-coupled receptor 40 (GPR40)/cAMP/Ca2+/IRS2/PI3K/Akt signaling pathway, while increasing glucose-stimulated insulin secretion (GSIS) by modulating GPR40/cAMP/Ca2+/CaMKII pathway, which reveals a novel mechanism underlying GPR40-mediated cell protection and GSIS in INS-832/13 cells. Moreover, administration of vincamine effectively ameliorated glucose homeostasis in either HFD/STZ or db/db type 2 diabetic mice. To our knowledge, our current work might be the first report on vincamine targeting GPR40 and its potential in the treatment of T2DM.

Supplementary Materials

    • Supplemental Figure 1 Vincamine inhibits β-cell apoptosis via GPR40/cAMP/Ca2+/IRS2/PI3K/Akt signaling pathway. (A-C) In the presence of wortmannin (2 μM) (A), GW1100 (20μM) (A), nifedipine (10 μM) (B), MDL (5 μM) (B), H89 (10 μM) (B) and GPR40-siRNA (100 pM) (C), vincamine-inhibited β-cell apoptosis was measured as in Fig. 1D. (D-F) D, E and F were quantitative results as shown in A, B and C respectively. Data were shown as means ± SEM with three independent experimental replicates. Significant differences between groups are represented as **p <0.01 and ***p <0.001, and p > 0.05 meant no significance (ns). (Vin, as vincamine; Wort, as wortmannin; Nif, as Nifedipine; Nt-siRNA, as Non-targeting siRNA)
    • Supplemental Figure 2 Vincamine treatment has no effect on PI3K enzyme activity and PI3K modulators, including LXRα/β/RXRα. (A) The enzymatic activity of PI3K was detected in the presence of wortmannin (2 μM) or vincamine (5, 10, 20 μM). (B, C) HEK 293T cells were co-transfected with RXRα, LXRα/β, LXRE-Luc and SV40, and then vincamine (5, 10, 20 μM) was added into the cells in the presence or absence of TO901317 (5 μM). Cell lysate was harvested to examine the transactivation activity of compounds against LXRα/RXRα (B) and LXRβ/RXRα (C) heterodimer. Data were shown as means ± SEM with three independent experimental replicates. Significant differences between groups are represented as ***p <0.001, and p > 0.05 meant no significance (ns) (Vin, as vincamine; Wort, as wortmannin; TO901317, as TO90)
    • Supplemental Figure 3 Vincamine promotes GSIS independently of IP3R. Insulin content was analyzed as in Fig. 4A when INS-832/13 cells were incubated with 2-APB (20 μM). Data were shown as means ± SEM with three independent experimental replicates. Significant differences between groups are represented as **p <0.01 and ***p <0.001. (Vin, as vincamine)
    • Supplemental Figure 4 Vincamine-induced β-cell protection is not related to insulin signaling. Insulin content was analyzed after incubation with STZ (0.4 mM) and vincamine (5, 10, 20 μM) in INS-832/13 cells. Data were shown as means ± SEM with three independent experimental replicates. No significance between groups is represented as ns (p > 0.05) (Vin, as vincamine)
    • Supplemental Figure 5 Vincamine has no effect on the enhancement of Akt and GSK3β protein stability. (A) CETSAs for INS-832/13 cell lysate with or without vincamine (20 μM) were performed using western blot to detect the interaction between vincamine and Akt or GSK3β. (B) Quantitative results of A were presented. Data were shown as means ± SEM with three independent experimental replicates. Significant differences between groups are represented as *p <0.05, and p > 0.05 meant no significance (ns). (Vin, as vincamine; -: without vincamine; +: with vincamine)
    • Supplemental Figure 6 The mRNA and protein level of GPR40 are knocked down efficiently by GPR40-siRNA in INS-832/13 cells. (A, B) INS-832/13 cells were transfected with non-targeting siRNA or GPR40-siRNA (100 pM) and the expression of GPR40 was then determined by qRT-PCR (A) and western blot (B). Data were shown as means ± SEM with three independent experimental replicates. Significant differences between groups are represented as ***p <0.001. (Nt-siRNA, as Non-targeting siRNA)
    • Supplemental Figure 7 GPR40 overexpression triggers events in INS-832/13 cells. (A) INS-832/13 cells were transfected with empty vector and GPR40 plasmid and the expression of GPR40 was then determined by western blot. (B) After transfection with empty vector and GPR40 plasmid, the viability of INS-832/13 cells was examined with or without STZ (0.4 mM). (C, D) After transfection with empty vector and GPR40 plasmid, the Caspase 3 activity (C) and cleaved-Caspase 3 expression (D) of INS-832/13 cells were detected with or without STZ (0.4 mM). (E) After transfection, insulin content was detected as in Fig. 4A. (F) Intracellular cAMP concentration was determined as in Fig. 3H under plasmid transfection. (G) Ca2+ signal was detected as in Fig. 3A after transfection. Data were shown as means ± SEM with three independent experimental replicates. Significant differences between groups are represented as *p <0.05, **p <0.01 and ***p <0.001, and p > 0.05 meant no significance (ns).
    • Supplemental Figure 8 Vincamine has no effect on basal insulin secretion in HFD/STZ and db/db male mice. (A, B) Serum insulin level of HFD/STZ (A) and db/db (B) male mice was detected after overnight fasting. Data were shown as means ± SEM with nine mice in each group. No significance between groups is represented as ns (p > 0.05). (Vin, as vincamine; H, 30 mg/kg/d; L, 15 mg/kg/d)

 

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