Verapamil Restores β-Cell Mass and Function in Diabetogenic Stress Models via Proliferation and Mitochondrial Respiration

Abstract

Diabetes remains a global health challenge, characterized by persistent hyperglycemia and gradual depletion or impairment of pancreatic β-cells. Current treatments focus on managing glycemic control, but do not mitigate β-cell mass. Verapamil, an FDA-approved calcium channel blocker for hypertension, has shown potential therapeutic action towards β-cells in the context of diabetes. In this study, we investigated the cytoprotective and metabolic efficacy of verapamil on mouse-derived MIN6 β-cells under metabolic and diabetogenic stressors like high glucose, toxins, and an inflammatory cytokine cocktail, as well as investigated a zebrafish model. At safe, non-toxic doses, verapamil elevated the levels of cholecystokinin (CCK), an incretin associated with β-cell preservation and enhanced mitochondrial respiration. Notably, pretreatment and co-treatment of verapamil in the presence of stressors offered substantial protection and preserved mitochondrial function, whereas post-treatment effects were moderate and model dependent. In the zebrafish model, verapamil promoted β-cell recovery and regeneration before, during, and after targeted ablation. The drug seemed to work in several ways: inducing proliferation, reducing stress on β cells, boosting their energy production, and activating survival signals. Together, our data aligned with earlier human clinical trials showing that verapamil administration preserved β-cell mass and function in patients with recent-onset type 1 diabetes. The high efficacy, affordability, and broad mechanisms of action make verapamil a desirable therapeutic candidate for diabetes. Nevertheless, further mechanistic studies and long-term clinical trials are warranted to establish its utility in diabetes management.

Keywords: T1D; T2D; diabetes; verapamil.

Figure 1

Dose response and proliferative effect of verapamil in MIN6 cells. To determine the proliferative effect of verapamil, (A) MIN6 cells were treated with various concentrations of verapamil (μM) for 24 and 48 h in high glucose supplemented DMEM media as described in Section 2. Cell viability percentages were calculated relative to the untreated controls. (B) MIN6 cells were cultured in the high glucose supplemented DMEM media in the presence of verapamil (1–50 μM for 24 h), and relative cell viability percentages were calculated for untreated cells. (C,D) MIN6 cells were cultured on coverslips in high-glucose DMEM media supplemented with 2% free fatty acid BSA. The cells were treated with 50 μM of verapamil for 24 h. Then Ki67 immunostaining was conducted as described in Section 2. Cell compartments were stained as nuclei (blue—DAPI), actin filaments (red—phalloidin), and Ki67 positive proliferating cells (green). Nuclear co-localization is shown by yellow arrows (bar: 50 μm, n = 2). The corrected total cell fluorescence (CTCF) was calculated from 10 fields per sample. (E,F) Western blotting experiment using proteins extracted from MIN6 cells cultured in high-glucose DMEM media with 2% free fatty acid BSA, treated with 50 μM of verapamil for 24 h to detect the level of histone H3 protein expression as another proliferation marker compared to untreated cells. The level of histone H3 protein was normalized to β-Aactin (n = 2). These data showed no significant changes in relative cell viability, Ki67, and histone H3 protein expressions. (G,H) MIN6 cells cultured in high-glucose DMEM media with 2% free fatty acid BSA treated with 50 μM of verapamil for 24 h showed significantly higher CCK and PCNA protein expressions relative to β-Actin detected by Western blotting assay (n = 3). (I) MIN6 cells were cultured in DMEM media supplemented with FFA-free 2% BSA for 24 h. Then, the cells were treated with verapamil (50 μM) for 1, 2, 3, or 4 h, or left untreated as control. Harvested single cells were fixed with ice-cold 70% ethanol, and after centrifugation, the cells were treated at room temperature (RT) with RNase (100 µg/mL) for 30 min, followed by incubation with PI (40 µg/mL) for another 30 min. DNA content and cell cycle were analyzed using BD FACSCant Flow Cytometer, as described in detail in Section 2. Flow cytometry analysis tracked verapamil’s time-dependent effect on cell cycle phases: dead cells (black), G0/G1 (blue), S-phase (gray), and G2/M (red) cell populations (n = 4 independent experiments). (J) MIN6 cells were cultured at a concentration of 5 × 10⁴ cells/well in high-glucose media. The growth curve was plotted by cell counting every 24 h for 7 days, using trypan blue due exclusion assay on the cells treated with verapamil (50 μM, red line) versus untreated cells (black line). Data are presented as mean ± SEM using one-way ANOVA with Tukey’s multiple comparisons test. ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control.

Figure 2

Verapamil protects MIN6 cells against diabetogenic stressors STZ and T1D-cytomix. To detect the protective role of verapamil in MIN6 cells challenged with diabetogenic stressors, MTT assay was conducted on the cells cultured in a high-glucose medium in the presence or absence of stressors (STZ or T1D-cytokine cocktail with different timing of different doses of verapamil (1, 5, 10, and 50 μM) for 24 h under pre-, post-, or co-treatment conditions. (A,B) Verapamil pre-treatment condition for 24 h, then challenging with STZ or TD-cytomix stressors, respectively. Pre-treatment with verapamil significantly improved cell survival dose-dependently. (C,D) Verapamil post-treatment enhanced cell recovery moderately after challenge with STZ (C) but significantly improved cell survival post-T1D-cytomix cocktail (D) treatments for 24 h. (E,F) Verapamil and stressor co-treatment for 24 h showed significant protection against STZ (E) and T1D-cytomix (F). Data is presented as the ratio of cell viability of verapamil-treated cells to verapamil-untreated cells. All experiments included >5 biological replicates. Data are presented as mean ± SEM and were analyzed by two-way analysis of variance (ANOVA), and p < 0.05 was considered a significant difference. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns: non-significant.

Figure 3

Verapamil enhances mitochondrial function in stressed MIN6 cells. To assess mitochondrial function, MIN6 cells were grown in a high-glucose medium and exposed to either diabetogenic stressors (STZ or a T1D-cytokine mix) with different timings of 50 µM verapamil treatment under pre-, post-, or co-treatment conditions. The cellular oxygen consumption rate (OCR) was measured by metabolic flux analysis following sequential treatment with 1 µM oligomycin, 2 µM FCCP, and 0.5 µM rotenone/antimycin A. (A–C) Verapamil alone: 50 μM verapamil (24 h) increased both basal (B) and maximal (C) OCR compared to the untreated controls. (D–F) Pre-treatment: cells pretreated with verapamil before stress exposure maintained higher basal respiration (E) and maximal respiration (F). (G–I) Post-treatment: verapamil administration after stress improved basal OCR (H) and maximal OCR (I). (J–L) Co-treatment: simultaneous verapamil + stressor treatment preserved basal respiration (K) and maximal respiration (L). The OCR data were normalized to the total protein extract measurements. The experimental groups were compared using two-way analysis of variance (ANOVA), with a statistically significant set at p-values < 0.05 considered statistically significant, and n = 4 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 4

Verapamil treatments overcome the cytotoxic effect of MTZ in zebrafish larvae. (A) Experimental timeline and treatment groups. Larvae (3 dpf) were randomly divided into five groups: Group 1 (untreated), normal developmental control; Group 2 (MTZ alone), larvae exposed to 10 mM MTZ for 24 h (to damage β-cells); Group 3 (Verapamil → MTZ), pre-treatment with 10 µM verapamil for 24 h before 10 mM MTZ exposure for 48 h; Group 4 (MTZ → Verapamil), post-treatment with 10 µM verapamil for 48 h after 10 mM MTZ insult for 24 h; Group 5 (MTZ + Verapamil), co-treatment with both 10 µM verapamil and 10 mM MTZ for 72 h. mCherry fluorescence reporter protein (ChFP) expressed in the larvae’s β-cells was imaged at 4 dpf (T0) and 6 dpf (T1). The change in ChFP intensity between T0 and T1 was measured. (B) Representative images of β-cells (red fluorescence) at T0 post-MTZ damage and T1 at recovery phase. Inserts show the magnified ChFP area. (C) Quantification of β-cell fluorescence (ChFP) at T1. Untreated Group 1 showed the physiological development of β-cells. The MTZ-alone group, Group 2, showed severe loss of fluorescence at T1, whereas the verapamil-treated groups 3–5 showed an improved β-cell preservation or recovery. Images were taken using Stereo Discovery 1.2 ZIESS microcopy. Experiments were performed in three independent trials (n = 20–30 larvae/group). Data are presented as mean ± SEM values. * p = 0.016, ** p = 0.01, *** p = 0.0004.