Evaluating the antidiabetic effects of R-verapamil in type 1 and type 2 diabetes mellitus mouse models

Abstract

The global incidence of diabetes mellitus (DM) is increasing. Types 1 and 2 DM are associated with declining β-cell function. Verapamil (50% S-verapamil and 50% R-verapamil) can treat DM by downregulating thioredoxin-interacting protein (TXNIP), which induces islet β-cell apoptosis. However, it may also induce cardiovascular side effects as S-verapamil is negatively inotropic. In contrast, R-verapamil only weakly induces adverse cardiac effects. In this study, we aimed to determine the antidiabetic efficacy and cardiovascular safety of R-verapamil. We examined R- and S-verapamil binding through in vitro studies. Streptozotocin-induced type 1 and db/db type 2 DM mouse models were used to assess the antidiabetic efficacy of verapamil. IL-6, blood glucose (BG), Txnip expression, and β-cells were evaluated in streptozotocin-induced diabetic mice, while body weight, BG, and serum insulin were measured in the db/db mice. In the type 1 DM study, 100 mg/kg/day R-verapamil and racemic verapamil lowered BG, downregulated Txnip expression, and reduced β-cell apoptosis. In the type 2 DM study, the optimal R-verapamil dosage was 60 mg/kg/day and it lowered BG and raised serum insulin. However, efficacy did not increase with R-verapamil dosage. R-verapamil combined with metformin/acarbose improved BG and serum insulin more effectively than metformin/acarbose alone or verapamil combined with acarbose. R-verapamil had weaker cardiovascular side effects than S-verapamil. R-verapamil was 9.0× and 3.4× less effective than S-verapamil at inhibiting atrial inotropy and ileal contractility, respectively. It was also 8.7× weaker than S-verapamil as an agonist of somatostatin receptor type 2 (SSTR2), inhibiting ileal neurogenic contraction. Hence, R-verapamil may be an optimal DM treatment as it is safe, improves glycemic control, and preserves β-cell function both as monotherapy and in combination with metformin or acarbose. R-Verapamil has potential for delaying or arresting DM progression and improving patients' quality of life.

Fig 1. Scheme of STZ-induced T1DM mice administered racemic verapamil or R-Vera.

Fig 2. Scheme of db/db mice administered verapamil or R-Vera with or without metformin for 6 wks.

Fig 3. Scheme of db/db mice administered verapamil and R-Vera combined with acarbose.

Fig 4. Fasting BG of STZ-induced mice treated with Verapamil (V) and R-Vera (RV) from days 6–15.

Relative BG improved for the racemic and R-Vera groups. Bars are means ± SD. N = 8 mice per vehicle, V-100, RV-100, and RV-50 groups. *** P<0.001, ****P < 0.0001, significant difference from vehicle, using two-way ANOVA with Tukey’s multiple comparison; ^#P < 0.05, significant difference between V-100 and RV-100 on day 11 and between V-100 or RV-100 and RV-50 on day 15, using an unpaired student’s t-test.

Fig 5. Proinflammatory cytokine (IL-6) and TXNIP expression and TUNEL assay for STZ-induced mice treated with Verapamil (V) and R-Vera (RV).

(A) STZ-induced mice administered 100 mg/kg/day racemic or R-Vera exhibited significantly reduced serum IL-6 expression compared to vehicle mice whereas those administered 50 mg/kg/day R-Vera did not. **P < 0.01 indicates significant difference from vehicle using one-way ANOVA. N = 8 in each group. (B) STZ-induced mice showed highly upregulated islet tissue TXNIP compared to normal C57BL/6J mice. TXNIP was downregulated after racemic or R-Vera administration compared to vehicle. ^# P < 0.05 indicated significant difference between normal and vehicle; *P < 0.05 indicated significant difference to vehicle using one-way ANOVA. N = 8 in each group. (C) β-cell apoptosis assessed using TUNEL staining. Deep staining spots in images are apoptotic β-cells (200×). (D) TUNEL-positive cell counts show that STZ-induced mice administered 100 mg/kg/day R-Vera had significantly reduce β-cell apoptosis. *P < 0.05 indicated a significant difference between vehicle and RV-100 using one-way ANOVA. The sample numbers of 5, 7, 5, and 9 indicate the section numbers from the Vehicle, V-100, RV-100, and RV-50 groups, respectively (rather than the number of mice). Each data point represents the percentage of TUNEL–positive cells per slide in each group.

Fig 6. Effects of Verapamil (V) and R-Vera (RV) on BG and serum insulin in db/db mice.

(A) BG and (B) serum insulin levels were measured on weeks 0 (before treatment), 2, and 4 and on day 6 in mice fasted 6 h. *P < 0.05 compared to vehicle (db/db); ^#P < 0.05 compared to metformin; ^$P < 0.05 compared to RV-30; two-way ANOVA. N = 5 per group except for n = 3 and n = 2 in V-60 group on weeks 2 and weeks 4 and 6, respectively, because of animal health issues. Data are expressed as means ± SD.

Fig 7. db/db mice treated with 30, 60, 100, and 150 mg/kg/day R-Vera (RV) for 7 weeks.

db/db mice receiving metformin or RV showed (A) reductions in BG level and (B) slight improvements in serum insulin level. Nevertheless, no obvious dose-dependent effects on BG and serum insulin levels were found. N = 5 per group. *P < 0.05 compared to vehicle (db/db) group with two-way ANOVA.

Fig 8. Effects of RV-mono and RVM-combination treatments in db/db mice.

(A) The RV-100+Met and metformin groups displayed significant body weight reduction relative to the vehicle group (*P < 0.05; two-way ANOVA). (B) The RVM-combination treatment had significant glucose-lowering efficacy compared to the vehicle (*P < 0.05, two-way ANOVA) and the metformin treatment (^#P<0.05, two-way ANOVA). (C) The serum insulin levels in the RVM-combination group were slightly higher than those in the metformin and RV-mono groups. N = 8 per group. Statistical analysis was performed using two-way ANOVA.

Fig 9. Effects of VA-combination, RVA-combination, and Acarbose-mono on db/db mouse body weight and BG.

(A) Body weight changes (B) fasting BG levels (C) non-fasting BG levels. N = 6 in each group. *P < 0.05 indicates significant difference relative to vehicle; ^#P < 0.05 and ^##P < 0.01 indicate significant difference from day 0 in same group; ^$$P < 0.01 indicates significant differences among groups.