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.

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  • 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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    Vincamine promotes β-cell survival. (A) Chemical structure of vincamine. (B) In the absence of STZ, INS-832/13 cells were incubated with vincamine (5, 10, 20 μM) for 24 h, and then MTT assay was applied. (C) MTT assay was performed after incubation with STZ (0.4 mM) and vincamine (5, 10, 20 μM) in INS-832/13 cells for 24 h. EC50 of vincamine was fitted by GraphPad Prism. (D) INS-832/13 cells treated as C were stained with Annexin V-FITC, followed by the determination of flow cytometry. (E) CyQUANT GR dye was used to detect 480/520 nm fluorescence in INS-832/13 cells treated with Liraglutide (0.32 nM) or vincamine (5, 10, 20 μM) for 24 h. Data were shown as means ± s.e.m. with three independent experimental replicates. Significant differences between groups are represented as **P < 0.01 and ***P < 0.001, and P > 0.05 stood for no significance (ns). (Vin, as vincamine.)

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    Vincamine protects β-cells through IRS2/PI3K/Akt pathway. (A) After incubation with STZ (0.4 mM) and vincamine (0.1, 1, 5, 10, 20 μM) in INS-832/13 cells for 24 h, the level of p-Akt was determined by AlphaLISA assay. Vincamine-EC50 of p-Akt recovery was fitted by GraphPad Prism. (B) INS-832/13 cells with or without STZ treatment were incubated with vincamine (5, 10, 20 μM) for 24 h, and then p-Akt was detected by Western blot. (C) In the presence of wortmannin (2 μΜ), vincamine-induced β-cell survival was measured as in Fig. 1C. (D) The level of p-Akt was detected by Western blot in INS-832/13 cells incubated with STZ (0.4 mM) and vincamine (20 μM) for 24 h in the presence of wortmannin (2 μM). (F, G and H) INS-832/13 cells were incubated with STZ (0.4 mM) and vincamine (5, 10, 20 μM) for 6 h, and then the activity of Caspase 9 (F) and Caspase 3 (G) were examined using corresponding assay kits and the expression of cleaved Caspase 3 (H) was detected by Western blot. (E, I, J, K and L) INS-832/13 cells were treated with STZ (0.4 mM) and vincamine (5, 10, 20 μM) for 24 h. IRS2/PI3K/Akt pathway-related proteins (E, L) were then assessed by Western blot, and the mRNA expressions of p21 (I), Bim (J) and IRS2 (K) were detected by quantitative real-time PCR (qRT-PCR). Data were shown as means ± s.e.m. 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). (Vin, as vincamine; Wort, as wortmannin.)

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    cAMP and Ca2+ contribute to vincamine-induced β-cell protection. (A, B, C and D) Cytosolic Ca2+ signals in INS-832/13 cells were measured using Fluo-4 AM. Vincamine (5, 10, 20 μM) was added into INS-832/13 cells automatically, and Ca2+ signal in the first 20 s of detection was set as the baseline signal. Ca2+ signal assay was carried out in the presence (A) or absence (C) of extracellular Ca2+, and data were presented as AUC form. (B and D) B and D were the real-time Ca2+ dynamics of A and C respectively. (E) After pre-incubation with nifedipine (10 μM), Ca2+ signal was detected as in A. (F) In the presence of nifedipine (10 μM), the viability of INS-832/13 cells was examined as in Fig. 1C. (G and J) The expressions of p-Akt and IRS2 were detected as in Fig. 2B under the treatment with nifedipine (10 μM) (G) and H89 (10 μM) (J). (H) INS-832/13 cells were treated with FSK (10 μM) or vincamine (5, 10, 20 μM) for 1 h, and concentration of intracellular cAMP was detected using assay kit. (I) Viability of INS-832/13 cells was examined as in Fig. 1C in the presence of H89 (10 μM) and MDL-12,330A hydrochloride (MDL, 5 μM). (K) Ca2+ signal was detected as in A in the presence of MDL (5 μM). Data were shown as means ± s.e.m. 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). (EC-Ca2+, as Extracellular Ca2+; Nif, as Nifedipine; Vin, as vincamine.)

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    Vincamine induces GSIS through cAMP/Ca2+/CaMKII pathway. (A) INS-832/13 cells were stimulated with 2.8 or 16.8 mM glucose in the presence of glibenclamide (1 μM) or vincamine (5, 10, 20 μM) for 2 h, and the supernatant of cells was then harvested to detect insulin content. (B, C and D) Insulin content was analyzed as in A when INS-832/13 cells were incubated with nifedipine (1, 10 μM) (B), MDL (5, 20 μM) (C) and KN62 (20 μM) (D). Data were shown as means ± s.e.m. 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). (Glib, as glibenclamide; Nif, as nifedipine; Vin, as vincamine.)

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    Vincamine ameliorates β-cell function and promotes GSIS by targeting GPR40. (A) GK enzymatic activity was tested with the treatment of vincamine (10, 20 μM). (B) The enzymatic activity of PDE was assessed when incubated with IBMX (500 μM) or vincamine (5, 10, 20 μM). (C) After treatment with NMDA (500, 1000 μM) and t-PDC (500 μM), the viability of INS-832/13 cells was detected as in Fig. 1C. (D) Vincamine and DHA in different concentrations were added into hGPR40-CHO cells pre-incubated with Fluo-4 AM, and Ca2+ signals were recorded. (E) CETSAs for INS-832/13 cell lysate treated with DMSO and vincamine (20 μM) or TAK-875 (20 μM) were conducted using Western blot to detect the interaction between vincamine and GPR40, and quantification of this data was also shown in E. (F) After incubation with GW1100 (20 μM), the viability of INS-832/13 cells was detected as in Fig. 1C. (G) Expressions of p-Akt and IRS2 were examined as in Fig. 2B in the presence of GW1100 (20 μM). (H) After transfection with non-targeting siRNA or GPR40-siRNA (100 pM), the viability of INS-832/13 cells was detected as in Fig. 1C. (I) Detection of GPR40, p-Akt and IRS2 expression was conducted by Western blot under the same condition as in H. (J and K) The insulin content was detected as in Fig. 4A after treatment with GW1100 (20 μM) (J) and transfection with non-target siRNA or GPR40-siRNA (100 pM) (K). (L) Intracellular cAMP concentration was determined as in Fig. 3H under GW1100 (20 μM) treatment. (M) Ca2+ mobilization was measured as in Fig. 3A in the presence of GW1100 (20 μM). Data were shown as means ± s.e.m. 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). ((−): with DMSO; Nt-siRNA, as non-targeting siRNA; (V): with vincamine; Vin, as vincamine; (T): with TAK-875; TAK, as TAK-875.)

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    Vincamine administration improves glucose tolerance in HFD/STZ and db/db male mice. (A and C) Fasting blood glucose levels of HFD/STZ (A) and db/db (C) male mice were measured weekly. (Black line, vehicle; blue line, 30 mg/kg/day vincamine; red line, 15 mg/kg/day vincamine) (B and D) HbA1c levels of HFD/STZ (B) and db/db (D) male mice were detected after vehicle or vincamine (15, 30 mg/kg/day) administration for 5–6 weeks. (E and H) Blood glucose levels of HFD/STZ (E) and db/db (H) male mice during i.g. glucose (1.0 g/kg) tolerance tests with vehicle or vincamine (15, 30 mg/kg/day) treatment. (F and I) F and I were the AUC form of E and H respectively. (G and J) Serum insulin levels of HFD/STZ (G) and db/db (J) male mice were determined during the same process as shown in E and H. Data were shown as means ± s.e.m. with nine mice in each group. Significant differences between groups are represented as *P < 0.05, **P < 0.01 and ***P < 0.001. (H, 30 mg/kg/day; L, 15 mg/kg/day; Vin, as vincamine.)

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    Vincamine administration ameliorates β-cell dysfunction and increases β-cell mass in HFD/STZ and db/db male mice. (A and B) Insulin IHC assays of pancreases in HFD/STZ (A) and db/db (B) male mice were displayed. (C) and (D) were quantitative results in insulin-positive area of pancreatic islets as shown in A and B respectively. Data were shown as means ± s.e.m. Significant differences between groups are represented as *P < 0.05, **P < 0.01 and ***P < 0.001. (H, 30 mg/kg/day; L, 15 mg/kg/day; Vin, as vincamine.)

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    Vincamine modulates IRS2/PI3K/Akt pathway in vivo. (A and B) Expressions of the related proteins in IRS2/PI3K/Akt pathway were assayed by Western blot in pancreatic homogenate of HFD/STZ (A) and db/db (B) male mice. (C and D) Quantitative results of A and B were displayed correspondingly in C and D. (E and F) Caspase 3 activity in pancreatic homogenate of HFD/STZ (E) or db/db (F) male mice was determined by assay kit. (G and H) IF assays for insulin (green) and IRS2 (red) were detected in pancreases of HFD/STZ (G) and db/db (H) male mice. (I and J) IF assays for insulin (green) and cleaved Caspase 3 (red) were detected in pancreases of HFD/STZ (I) and db/db (J) male mice. The intensity of IRS2 (K and L) and cleaved Caspase 3 (M and N)-positive signals in insulin-positive area was measured. Data were shown as means ± s.e.m. Significant differences between groups are represented as *P < 0.05, **P < 0.01 and ***P < 0.001. Bar = 50 μm. (H, 30 mg/kg/day; L, 15 mg/kg/day; Vin, as vincamine.)

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    Vincamine has no effect on insulin sensitivity in vivo and ex vivo. (A) Fasting blood glucose levels of db/db female mice were measured weekly. (Black line, vehicle; gray line, 30 mg/kg/day vincamine.) (B) HbA1c levels of db/db female mice were detected after vehicle or vincamine (30 mg/kg/day) administration for 6 weeks. (C) Blood glucose levels of db/db female mice during i.g. glucose (1.0 g/kg) tolerance tests with vehicle or vincamine (30 mg/kg/day) treatment. (D) was the AUC form of C. (E) Blood glucose levels of db/db female mice during i.p. insulin (1.5 U/kg) tolerance tests with vehicle or vincamine (30 mg/kg/day) treatment. (F) was the AUC form of E. (G) Mouse primary hepatocytes were incubated with vincamine (20 μM) or rosiglitazone (10 μM) for 24 h in the presence of insulin (10 nM) for another 15 min, and then the expressions of p-IR and p-Akt were detected by Western blot. Data were shown as means ± s.e.m. with nine mice in each group. Significant differences between groups are represented as *P < 0.05, **P < 0.01, ***P < 0.001 and P > 0.05 meant no significance (ns). (H, 30 mg/kg/day; Rosi, rosiglitazone; Vin, as vincamine.)

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    Schematic illustration of the vincamine-mediated β-cell protection and insulin secretion.

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