Preventing β-cell loss and diabetes with calcium channel blockers

Abstract

Although loss of functional β-cell mass is a hallmark of diabetes, no treatment approaches that halt this process are currently available. We recently identified thioredoxin-interacting protein (TXNIP) as an attractive target in this regard. Glucose and diabetes upregulate β-cell TXNIP expression, and TXNIP overexpression induces β-cell apoptosis. In contrast, genetic ablation of TXNIP promotes endogenous β-cell survival and prevents streptozotocin (STZ)- and obesity-induced diabetes. Finding an oral medication that could inhibit β-cell TXNIP expression would therefore represent a major breakthrough. We were surprised to discover that calcium channel blockers inhibited TXNIP expression in INS-1 cells and human islets and that orally administered verapamil reduced TXNIP expression and β-cell apoptosis, enhanced endogenous insulin levels, and rescued mice from STZ-induced diabetes. Verapamil also promoted β-cell survival and improved glucose homeostasis and insulin sensitivity in BTBR ob/ob mice. Our data further suggest that this verapamil-mediated TXNIP repression is conferred by reduction of intracellular calcium, inhibition of calcineurin signaling, and nuclear exclusion and decreased binding of carbohydrate response element-binding protein to the E-box repeat in the TXNIP promoter. Thus, for the first time, we have identified an oral medication that can inhibit proapoptotic β-cell TXNIP expression, enhance β-cell survival and function, and prevent and even improve overt diabetes.

Figures

FIG. 1.

Effects of calcium channel blockers on β-cell TXNIP expression. A: INS-1 cells were incubated for 24 h with diltiazem (50 μmol/L), verapamil (50 μmol/L), NiCl2 (500 μmol/L), or the calcium chelator EGTA (500 μmol/L), and TXNIP expression was assessed by quantitative RT-PCR. B: INS-1 cells were incubated for 48 h in the presence (V) or absence (C) of verapamil (50 μmol/L), and effects on TXNIP protein levels were assessed by Western blotting and corrected for β-actin. C: Isolated human islets were incubated for 24 h at the designated concentrations of verapamil, and TXNIP expression was assessed by quantitative RT-PCR. D: C57BL/6 mice received verapamil (100 mg/kg/day) in their drinking water for 3 weeks, and their islet TXNIP expression was assessed by quantitative RT-PCR. Bars represent means ± SEM of at least three independent experiments.

FIG. 2.

Verapamil effects on TXNIP promoter activity. INS-1 cells were grown in 12-well plates and transfected with different TXNIP promoter reporter plasmids (0.4 μg/well) together with pRL-TK control plasmid (20 ng/well) using DharmaFECT Duo transfection reagent (1 μL/well). Verapamil was added to the medium with a final concentration of 100 μmol/L. At 24 h after transfection, cells were harvested and luciferase activity was determined by Dual Luciferase assay kit. Bars represent mean luciferase activity ± SEM of at least three independent experiments as compared with no verapamil control.

FIG. 3.

ChIP analysis of ChREBP binding to the TXNIP promoter in response to verapamil. INS-1 cells were treated with verapamil for the designated times, and ChIP assays were performed as described previously (11) using 4 μg ChREBP antibody (A) or normal rabbit IgG (B), and real-time PCR was performed using primers flanking the E-boxes. Effects of ChREBP overexpression on verapamil-mediated inhibition of TXNIP expression. INS-1 cells were grown in 6-well plates and transfected with control LacZ (C) or ChREBP plasmids (2 μg/well) (D). At 24 h after transfection, verapamil was added to the medium with a final concentration of 50 μmol/L; 48 h after transfection, cells were harvested for RNA extraction and analyzed by quantitative RT-PCR. Bars represent means ± SEM of at least three independent experiments.

FIG. 4.

Changes in nuclear ChREBP in response to verapamil and CyA. INS-1 cells were incubated in the presence or absence of verapamil (50 μmol/L) for 24 h, and ChREBP levels were analyzed by Western blotting in whole cell extracts (A) or nuclear fractions (B) as detailed in Research Design and Methods. C: INS-1 cells were transfected with the FL-TXNIP promoter reporter plasmid (0.4 μg/well) together with pRL-TK control plasmid (20 ng/well) using DharmaFECT Duo transfection reagent (1 μL/well) and incubated in the presence or absence of the calcineurin inhibitor CyA (5 μmol/L) for 24 h. Bars represent luciferase activity (mean ± SEM) of three independent experiments. D: INS-1 cells were incubated in the presence or absence of CyA (5 μmol/L) for 24 h, and ChREBP levels were assessed in the nuclear fractions (n = 3).

FIG. 5.

Verapamil effects on STZ-induced diabetes and β-cell apoptosis. Male C57BL/6 mice received multiple low-dose STZ injections (40 mg/kg/day) for 5 days and then half the mice received verapamil (V) in their drinking water (100 mg/kg/day). A: Blood glucose (mean ± SEM) in STZ- and STZ + verapamil–treated mice (n = 3 mice per group). B: Verapamil effects on islet TXNIP protein levels in STZ-induced diabetic mice. S, STZ. C: Comparison of pancreas cross-section of STZ- and STZ + verapamil–treated mice (blue = insulin staining). D and E: β-Cell apoptosis as assessed by TUNEL in response to verapamil treatment in STZ-induced diabetic mice. pos, positive. White arrows point at apoptotic nuclei. In total, >2,000 β-cell nuclei were analyzed per group; bars represent means ± SEM. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

Verapamil (V) effects on glucose homeostasis and β-cell function and survival in overtly diabetic mice. Male C57BL/6 mice received multiple low-dose STZ injections (40 mg/kg/day) for 5 days. At day 15, overtly diabetic mice (blood glucose >250 mg/dL) were selected to receive verapamil in their drinking water at a regular dose of 1 mg/mL or at a 50% lower dose of 0.5 mg/mL or to serve as controls. A: Blood glucose (mean ± SEM) in the three experimental groups. P values designate level of significance compared with the STZ control group (n = 4–8 mice per group). B: Serum insulin levels (mean ± SEM) in STZ-induced diabetic mice and STZ + verapamil–treated (0.5 mg/mL) mice (n = 6–7). C: Comparison of pancreas cross-section of STZ- and STZ + verapamil–treated mice (blue = insulin staining). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

Verapamil (V) effects on obesity-induced diabetes and β-cell survival. BTBRlepob/ob mice, as a model of severe obesity, insulin resistance, and diabetes, received verapamil (1 mg/mL) in their drinking water starting at age 4 weeks. A: Blood glucose (mean ± SEM) in verapamil-treated and control ob/ob mice (n = 8–10 mice per group). W, weeks. B: ITTs were performed as described in research design and methods. Blood glucose (mean ± SEM) in verapamil-treated and control ob/ob mice is shown (n = 4). ■, verapamil-treated mice; ○, control ob/ob mice. C: Serum insulin levels (mean ± SEM) in 8-week-old ob/ob (white bar) and verapamil-treated ob/ob (black bar) mice (n = 4–5). D and E: β-Cell apoptosis as assessed by TUNEL in response to verapamil treatment in obesity-induced BTBR ob/ob mice. pos, positive. White arrows point at apoptotic nuclei. In total, >3,000 β-cell nuclei were analyzed per group; bars represent means ± SEM. (A high-quality digital representation of this figure is available in the online issue.)