Adult male Wistar rats, weighing 240–260 g, were purchased from Beijing Weitong Lihua experimental animal center (Beijing, PR China). Five-week-old male diabetic db/db mice (BKS - Lepr^em2Cd479/Gpt, stock number T002407) were obtained from GemPharmatech Co., Ltd (Nanjing, China). Rats and mice were maintained in specific-pathogen-free surroundings, with a 12 h-light/dark cycles under controlled temperature (22 ± 2°C) and humidity (55–60%) conditions, and with free access to water and food. All animal care and experimental procedures conformed to the ethical guidelines for animal research at Shanxi Medical University and were approved by the Animal Care and Use Committee of Shanxi Medical University (Taiyuan, China). Telmisartan, valsartan, GW9662 were purchased from MedChemExpress (Shanghai, China), irbesartan was purchased from Aladdin (Shanghai, China).

The rat pancreas was isolated following injection of 1 mg/ml collagenase P (Roche, Indianapolis, IN, United States) through the common bile duct. After digestion at 37°C for 11 min and density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, United States) for 23 min, the expanded pancreas was dispersed, and islets remaining in the supernatant separated from the sediment. The islets were hand-collected under a dissection microscope, and single islet cells were obtained from islets using Dispase II (Roche, Indianapolis, United States) digestion. The db/db mouse islets were similarly obtained, although the pancreas was injected with 1 mg/ml collagenase V (Roche, Indianapolis, United States), then digested for 16 min and centrifuged twice with Hanks Balanced Salt Solution. Isolated islets and cells were cultured in RPMI 1640 (Hyclone, Thermo Scientific, Waltham, MA, United States) medium containing 11.1 mM glucose, supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin at 37°C in a humidified atmosphere of 5% CO₂, 95% air.

Chinese hamster ovary (CHO) cells were obtained from the National Infrastructure of Cell Line Resource (Beijing, China). Lentivirus vectors overexpressing voltage-dependent potassium (Kv) 2.1 channels were constructed (Shanghai Genechem Co., Ltd., Shanghai, China) to transfect CHO cells, and the CHO-Kv2.1 cell line was established. CHO-Kv2.1 cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, Thermo Scientific, Waltham, MA, United States) containing 4500 mg/L glucose in addition to 10% fetal bovine serum, 1% penicillin and streptomycin and 0.5 μg/ml puromycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). CHO cells were cultured under similar conditions except for puromycin selection.

Handpicked separated islets were cultured for 1–2 days before the experiment. A total of five islets per tube were pre-incubated in Krebs Ringer bicarbonate- HEPES (KRBH) buffer under 2.8 mM glucose conditions for 30 min. The KRBH buffer contained 128.8 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 5 mM NaHCO₃, and 10 mM HEPES, adjusted to pH 7.4 with NaOH prior to the addition of 2% bovine serum albumin. Islets were then treated with different drugs and glucose conditions as indicated, and supernatant liquid was collected at the end of every 30 min incubation, and stored at −20 C for insulin concentration measurement. Insulin secretion was determined using an Iodine [¹²⁵I] Insulin Radioimmunoassay Kit (North Biological Technology Research Institute of Beijing).

Calcium imaging was carried out at 28–30°C using the calcium-sensitive dye Fura 2-AM (Dojindo Laboratories, Kumamoto, Japan), using an OLYMPUS IX71 inverted microscope and Meta Fluor software 7.8 (Molecular Devices, Sunnyvale, CA, United States). Islet cells were cultured on coverslips coated with adhesion reagent for 6–10 h, then were loaded with 2 μM Fura 2-AM in KRBH buffer with addition of 2.8 mM glucose for 30 min at 37°C. Ca²⁺-free KRBH with the same composition as KRBH except NaCl was 132.2 mM, CaCl₂ was omitted, and 0.1 mM EGTA was added. Subsequently, the loading buffer was removed, and cells were washed twice with KRBH solution to remove excessive fluorescent dye. Fura-2 was excited at 340 and 380 nm wavelengths in 1 s intervals with fluorescence emission detected at 510–520 nm wavelengths. The ratio of fluorescence intensity (F340/F380) was recorded to measure intracellular Ca²⁺ concentrations.

Fura 2-AM-loaded islet cells on coverslips were transferred to a glass chamber containing KRBH buffer with appropriate glucose conditions. Between each test, the reagent was dripped onto the coverslip and F340/F380 data points were acquired to monitor the changes of intracellular Ca²⁺ level. The average value during 30 s F340/F380 spikes (15 s before and after the peak of F340/F380) for each test was used to compare the change of Ca²⁺ concentrations under different treatments, unless otherwise stated.

Whole-cell recording patch-clamp technology was applied to detect voltage-activated currents and record action potentials using an EPC-10 amplifier and PULSE software from HEKA Electronik (Lambrecht, Germany) at room temperature. Islet cells were cultured on glass coverslips coated with cell adherent reagent (Applygen Technologies Inc., Beijing, China).

In voltage-clamp mode, to record Kv currents, patch pipettes (5–8 MΩ) were loaded with intracellular solution containing 10 mM NaCl, 1 mM MgCl₂, 0.05 mM EGTA, 140 mM KCl, 0.3 mM Mg-ATP, and 10 mM HEPES, pH 7.25 adjusted with KOH. Cells were transferred to a recording chamber containing extracellular solution consisting of 138 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl₂·6H₂O, 2.6 mM CaCl₂, 11.1 mM glucose, and 5 mM HEPES (pH 7.4 adjusted with NaOH). The β-cells were identified by cell capacitance (>7 pF) (Göpel et al., 1999) and were clamped to a holding potential of −70 mV, then test potentials were elicited by ranging from −70 to 80 mV in 10 mV steps for 400 ms.

For voltage-gated Ca²⁺channel (VGCC) currents, the intracellular solution contained: 120 mM CsCl, 20 mM TEA (Sigma-Aldrich), 5 mM MgATP, 1 mM MgCl₂, 0.05 mM EGTA, and 10 mM HEPES (pH 7.25 adjusted with CsOH). The extracellular solution consisted of: 100 mM NaCl, 20 mM TEA, 20 mMBaCl₂, 4 mM CsCl, 1 mM MgCl₂, 5 mM HEPES, and 3 mM glucose (pH 7.4 adjusted with NaOH). Ca²⁺ was replaced with Ba²⁺ as the charge carrier in the extracellular solution to eliminate Ca²⁺-dependent inactivation of the VGCCs. β-cells were clamped to a holding potential of −70 mV, and then elicited by test potentials of −50 to 30 mV in 10 mV steps for 50 ms.

In current-clamp mode, β-cells were elicited by 4 ms currents of 150 pA to record action potentials. The time between the initiation and the point where membrane potential returned to within 10 mV of the resting membrane potential, was considered to be the measurement of action potential duration.

At the age of 8 wk, the mice were given fasting glucose teste to ensure that diabetes models were successfully established. Given that a therapeutic doses of telmisartan are 40–80 mg/day in humans, the conversion for mice was approximately 8.2–16.4 mg/kg of body weight (Nair and Jacob, 2016). In our experiment, the mice were administered acute oral telmisartan treatment at 15 mg/kg of body weight. At 2 h following drug intake, when the onset of action of telmisartan reached a maximum, the oral glucose tolerance test (OGTT) was performed (Gohlke et al., 2001).

At the age of 8 wk, following overnight fasting (14 h), the mice were randomly divided into groups receiving treatment with telmisartan (in drinking water containing 0.5% carboxymethyl cellulose sodium salt) or vehicle by gavage. For OGTTs, groups of mice were fed with glucose at 1.5 g/kg body weight orally, then a blood sample was collected from the tail vein and glucose levels were assessed using a Sinocare Glucometer (Changsha, China) at baseline (0 min) and after 15, 30, 60, 90, and 120 min. At the age of 11 wk, the mice were treated as described above and additional blood samples (50 μl) were obtained in a heparinized microhematocrit tube at 0, 15, 30, 60, 90, and 120 min. After centrifugation, the plasma was collected for insulin concentration measurement using the Mercodia Mouse Insulin ELISA (stock number 10-1247-01, Uppsala, Sweden).

All experimental data are presented as the means ± SEM. p < 0.05 was considered to indicate statistical significance. Shapiro–Wilk tests were used to analyze the normality of the data. Upon normal distribution, the means of numerical variables were compared using the Student’s t test or one-way analysis of variance (ANOVA), whereas data with non-normal distribution were analyzed using the Mann–Whitney Rank Sum Test or Kruskal–Wallis one-way ANOVA on Ranks. If any statistically significant difference was detected among three or more groups, the Student–Newman–Keuls method or Tukey test was performed for post hoc comparisons, unless otherwise stated.

To examine the effects of ARBs on insulin secretion, isolated rat islets were treated with various doses of telmisartan. As shown in Figure 1A, telmisartan (10 and 50 μM) potentiated insulin secretion under 8.3 mM glucose conditions but had no effect under 2.8 mM glucose. Furthermore, the data in Figure 1B confirmed that telmisartan-induced insulin secretion was glucose-dependent. Next, the functions of other ARBs were evaluated. Notably, no promotion of insulin secretion was observed following treatment with valsartan and irbesartan under 8.3 (data not shown) and 16.7 mM glucose conditions (Figures 1C,D). Telmisartan, valsartan and irbesartan are clinically available ARBs owing to their high specificity for AT1 receptors (Michel et al., 2013), and the antihypertensive bioactivity of valsartan and irbesartan has been confirmed by us, therefore our results suggested that telmisartan-mediated insulinotropic effect was independent of AT1 receptors.

Within β-cells, eliciting an increase in [Ca²⁺]_i causes insulin granule exocytosis; therefore, the elevation in [Ca²⁺]_i level is essential to induce insulin secretion (Sabatini et al., 2019). To verify whether the insulinotropic effect of telmisartan was related to the change in [Ca²⁺]_i, we applied the calcium-sensitive dye Fura 2-AM to detect changes in fluorescence intensity. Telmisartan (10 and 50 μM) induced an acute increase in fluorescence intensity dose-dependently. Moreover, the elevation only occurred under high (11.1 and 16.7 mM) (Figures 2C–F) but not low (2.8 mM) (Figures 2A,B) glucose conditions. In addition, in the calcium imaging experiment, neither valsartan (Figures 2G,H) nor irbesartan (Figures 2I,J) increased the [Ca²⁺]_i concentration of β-cells under high glucose conditions (16.7 mM).

Telmisartan and irbesartan have also been reported to function as a partial agonist of PPARγ (Schupp et al., 2004, 2005). In view of the absence of changes in insulin secretion and [Ca²⁺]_i levels with irbesartan, we speculated that PPARγ might not be responsible for the effects of telmisartan on these measures. We therefore performed the insulin secretion assay and calcium imaging experiment using GW9662, a selective PPARγ antagonist. As shown in Figure 3, GW9662 alone had no effect on GSIS and [Ca²⁺]_i concentration, and the addition of GW9662 did not influence the effects of telmisartan on insulin secretion or [Ca²⁺]_i level. However, we found GW9662 could inhibit rosiglitazone-induced PPARγ activation (data not shown). Therefore, our results demonstrated that PPARγ was not involved in telmisartan-induced insulin secretion and elevation of [Ca2+]_i levels.

[Ca²⁺]_i levels of β-cells are tightly maintained through the regulation of extracellular Ca²⁺ influx and the movement of Ca²⁺ within intracellular stores (Sabatini et al., 2019). We thus examined the concentration of [Ca²⁺]_i in the absence of extracellular Ca²⁺ to study the pathway by which telmisartan increases [Ca²⁺]_i concentration. Calcium imaging (Figures 4A,B) showed that telmisartan-induced elevation of [Ca²⁺]_i levels was reversed in Ca²⁺-free KRBH medium, although the [Ca²⁺]_i level was considerably increased upon intracellular Ca²⁺ mobilization via thapsigargin.

Moreover, telmisartan-induced effects on [Ca²⁺]_i levels were monitored following the application of azelnidipine, an L-type voltage-gated Ca²⁺channel (VGCC) blocker. We observed that the increase in [Ca²⁺]_i levels with telmisartan was completely blocked by azelnidipine, supporting that telmisartan enhances extracellular calcium influx through L-type VGCCs. Conversely, significant elevation remained upon thapsigargin addition (Figures 4C,D), confirming the lack of telmisartan effect on intracellular calcium stores.

Pancreatic β-cells are electrically excitatory. Previous studies have demonstrated that Kv channels play an important role in GSIS and glucose-stimulated increase of [Ca²⁺]_i (Roe et al., 1996; MacDonald and Wheeler, 2003; Herrington et al., 2006); therefore, we applied patch-clamp techniques to explore the effects of telmisartan on the Kv channels of β-cells. Figures 5A,B illustrate that telmisartan decreased the Kv channel currents compared with that of controls.

Kv channels participate in the repolarization of action potentials of β-cells, so that inhibition of Kv channels delays the repolarization, thus prolonging the APD, namely extending the duration of extracellular Ca²⁺ influx (MacDonald and Wheeler, 2003; Herrington et al., 2006; Jacobson and Philipson, 2007). Therefore, we next recorded the action potentials in current-clamp mode to observe the effect of telmisartan on APD. As presented in Figure 5C, comparison of APDs with or without telmisartan indicated that telmisartan extended APD.

As the Kv2.1 channel constitutes the main subtype among Kv families involved in the regulation of insulin release by β-cells (MacDonald et al., 2001; Jacobson et al., 2007; Li et al., 2013), we applied specific Kv2.1 channel blocker Guangxitoxin-1E (GxTX-1E) in patch-clamp experiments. As shown in Figures 5D,E, Kv currents had extremely reduced amplitudes when compared to the controls, moreover, telmisartan did not further significantly inhibit Kv current in the presence of GxTX-1E. Therefore, our results suggested that the inhibition of Kv2.1 was mainly responsible for the effect of telmisartan on Kv channels in β-cell.

We evaluated the effects of valsartan and irbesartan in the voltage-clamp experiment. Neither valsartan nor irbesartan exhibited similar effects on Kv channels as those observed with telmisartan treatment (Figures 6A,B). Moreover, GW9662 addition did not influence the telmisartan-induced inhibition of Kv channels (Figures 6C,D). The results indicated that telmisartan inhibited Kv channels independent of the AT1 receptor and PPARγ.

We therefore hypothesized that telmisartan might directly inhibit Kv channels. Considering the importance of Kv2.1 channels in β-cells, we carried out patch-clamp experiments to determine whether telmisartan directly inhibited Kv2.1 channels. Chinese hamster ovary (CHO) cells, which do not express any endogenous Kv channels (Yu and Kerchner, 1998), were utilized to establish the Kv2.1-overexpressing CHO-Kv2.1 cell line. Under whole-cell voltage-clamp mode, Kv2.1 channel currents and their suppression by telmisartan were both detected in CHO-Kv2.1 cells (Figures 6E,F), suggesting that telmisartan exerted direct inhibition on Kv2.1 channels.

To further confirm whether Kv channels are alone involved in mediating telmisartan-induced insulin secretion and increase of [Ca²⁺]_i levels, tetraethylammonium chloride (TEA), a potent inhibitor of Kv channels, was employed in pancreatic β-cells. Previous studies have shown that 20 mM TEA blocks the majority of Kv channels and causes calcium elevation (Roe et al., 1996; MacDonald et al., 2001). As shown in Figure 7A, TEA stimulated insulin secretion under 11.1 mM glucose conditions and telmisartan still significantly promoted insulin secretion in the presence of TEA, indicating that other factors may participate in telmisartan-stimulated insulin secretion. Consistent with this results, telmisartan also enhanced the [Ca²⁺]_i concentration in the presence of TEA under 11.1 mM glucose conditions (Figure 7B).

Since telmisartan-stimulated insulin secretion was Ca²⁺ dependent, we performed patch-clamp experiments to observe the effects of telmisartan on VGCCs in pancreatic β-cells. As presented in Figures 7C,D, telmisartan increased voltage-dependent inward Ca²⁺ currents densities compared with those of controls. As telmisartan enhances extracellular calcium influx through L-type VGCCs, we further observed the effect of telmisartan on L-type VGCCs using azelnidipine. The results showed that the activation of VGCCs by telmisartan was blocked in the presence of azelnidipine (Figures 7E,F). In addition, no significant difference was observed when VGCC currents were recorded following treatment with valsartan or irbesartan (Figures 7G,H); telmisartan-induced activation of VGCCs was not inhibited by GW9662 co-administration (Figures 7I,J). The results thus demonstrated that telmisartan activated L-type VGCCs of β-cells; moreover, neither the AT1 receptor nor the PPARγ mediated this effect.

We applied db/db mice as T2DM model mice to determine whether telmisartan induced hypoglycemic effects in vivo. Male mice were administered with telmisartan (15 mg/kg) or vehicle once by gavage at the age of 8 and 11 wk, then OGTT was performed to observe the effects of telmisartan on glucose response.

In 8-wk-old mice, blood glucose levels monitoring revealed that glucose clearance at 30 min and thereafter was improved significantly in telmisartan-treated mice, and noticeable difference was observed when the glycemic response was measured via the area under the curve (AUC) compared with that of control (Figure 8A). However, although the time of peak blood glucose was similarly advanced to 15 min in 11-wk-old mice, and the AUC results showed significant difference between the groups, it was not until 90 and 120 min (approximately 1 h later than in 8-wk-old mice) that the blood glucose values were markedly lower than those of controls (Figure 8B). Furthermore, the levels of plasma insulin in the telmisartan-treated group were considerably higher than those in the control group at 15, 30, and 60 min with the AUC differing significantly between the groups (Figure 8C). Therefore, the glucose-lowering effect of telmisartan was accompanied by the increase in the levels of plasma insulin, suggesting that the hypoglycemic effects of telmisartan were a result of increased insulin secretion. We speculated that the glucose-lowering effect of telmisartan was delayed and weakened in 11-wk-old mice, possibly owing to the progression of insulin resistance and the deterioration of β-cell function in db/db mice. In addition, our data in vivo were not completely consistent with the previous results of telmisartan in regard to acute regulation of pancreatic islet microcirculation and glycaemia (Olverling et al., 2013). The reasons for this discrepancy might conceivably relate to differences in concentrations of drug administration in experiments, and different impaired β-cell function state caused by species and age of the animal model.

At the end of the experiment, islets were isolated from db/db mice and used for an ex vivo study. The results showed that telmisartan potentiated insulin secretion under 16.7 and 30 mM glucose conditions (Figure 8D). Consistent with the results of in vivo studies, telmisartan treatment similarly enhanced GSIS under the pathological condition of diabetes. However, GSIS in cultured islets only occurred under 30 mM glucose conditions whereas 2.8 and 16.7 mM glucose showed equivalent secretion. The bluntness of GSIS might be related to impaired β-cell function caused by long-term exposure to high glucose and lipids in the development of diabetes in the db/db mice (Olofsson et al., 2007).

In T2DM, high glucose and free fatty acids leads to adverse effects (including blunted GSIS and decreased cell viability) (Olofsson et al., 2007; Tan et al., 2013), with the modulation of Kv and voltage-gated Ca²⁺ channels by “glucolipotoxicity” also being involved (Hoppa et al., 2011; Lee et al., 2018). We next performed the patch-clamp experiments to ascertain whether telmisartan exerted similar electrophysiological effects on pathological β-cells. We observed that both the decreased Kv channel currents (Figures 8E,F) and increased VGCC currents (Figures 8G,H) remained in telmisartan-treated β-cells of db/db mice. Therefore, under the pathological condition of T2DM, telmisartan still served the function of an insulin secretagogue through its action on ion channels.