Dexmedetomidine alleviates CoCl2-induced hypoxic cellular damage in INS-1 cells by regulating autophagy
Article information
Abstract
Background
Ischemia-reperfusion (I/R) injury is inevitable during the perioperative period. The pancreas is susceptible to I/R injury. Autophagy, a self-digestion process, is upregulated during I/R injury and strongly induced by hypoxia. This study aims to determine whether dexmedetomidine can decrease pancreatic β-cell damage by regulating autophagy under hypoxia.
Methods
INS-1 rat insulinoma cells were cultured in dexmedetomidine before being exposed to cobalt chloride (CoCl2)-induced hypoxia. Cell viability and the expression of autophagy-related proteins (light chain 3B [LC3B]-II, p62, and ATGs) were assessed. The expression of apoptosis-related proteins (BCL-2 and P-BAD) were also evaluated. CoCl2-treated INS-1 cells were pretreated with the autophagosome formation inhibitor, 3-methyladenine (3-MA), to compare its effects with those of dexmedetomidine. Bafilomycin-A1 (Baf-A1) that inhibits autophagosome degradation was used to confirm the changes in autophagosome formation induced by dexmedetomidine.
Results
Dexmedetomidine attenuated the increased expression of autophagic proteins (LC3B-II, p62, and ATGs) and reversed the CoCl2-induced reduction in the proliferation of INS-1 cells after hypoxia. Dexmedetomidine also alleviated the decreased expression of the anti-apoptotic protein (BCL-2) and the increased expression of apoptotic protein (BAX). Dexmedetomidine reduces the activation of autophagy through inhibiting autophagosome formation, as confirmed by a decrease in LC3B-II/I ratio, a marker of autophagosome formation, in LC3B turnover assay combined with Baf-A1.
Conclusions
Dexmedetomidine alleviates the degree of cellular damage in INS-1 cells against CoCl2-induced hypoxia by regulating autophagosome formation. These results provide a basis for further studies to confirm these effects in clinical practice.
Introduction
Protecting major organs from ischemia-reperfusion (I/R) injury is a major concern for anesthesiologists. Hemodynamic instability, inflammatory responses, and blood transfusions all lead to oxygen depletion and further cellular damage, increasing the risk of I/R injury [1]. As with other major organs, the pancreas is also highly sensitive to I/R injury [2]. I/R injury disrupts pancreatic microcirculation and is crucial in the pathogenesis of acute pancreatitis that can be lethal if it occurs during the perioperative period [3–5]. Nevertheless, relatively little has been known about pancreatic I/R injury and its prevention as the incidence of critical pancreatic damage is lower than that of other major organs. The occurrence of acute pancreatitis in relation to pancreatic I/R injury, however, can lead to high mortality, which supports the need for research on pancreatic protection [6].
Many studies have explored methods to prevent or reduce I/R injury, notably using pharmacologic agents or remote ischemic preconditioning techniques; however, there are currently no known methods to definitely prevent it [7–9]. Autophagy, one of three major forms of cell death along with apoptosis and necrosis, is considered to be a therapeutic target for I/R injury [10,11]. Autophagy is a self-digestion process that might function as a pro-survival pathway because it is upregulated during I/R injury and maintains homeostasis by degrading or recycling cellular proteins and damaged organelles [10,12–15]. The double-edged nature of autophagy that is often induced in stressful conditions such as hypoxia may lead to cell death in association with the destruction of cellular structure [16]. Therefore, excessive autophagy can cause cell death distinct from apoptosis that is defined as autophagic cell death [17,18].
Dexmedetomidine is a highly selective α2-adrenoreceptor agonist, and intraoperative administration reduces the sympathetic response caused by surgical stimulation. Numerous previous studies have reported that dexmedetomidine protects the lungs and kidneys from I/R injury by regulating cell apoptosis, necrosis, and autophagy [19,20]. Furthermore, dexmedetomidine was revealed to protect the brain by inhibiting autophagy in cerebral I/R injury [21]. However, the effects of dexmedetomidine on autophagy in pancreatic cells remain unknown.
This study was designed to examine the hypothesis that dexmedetomidine can decrease cellular damage in pancreatic β-cells by regulating autophagy under hypoxic conditions.
Materials and Methods
Cell culture and treatment
INS-1 rat insulinoma cells, the cell model most widely used in pancreatic β-cell research [22], were cultured in RPMI 1640 medium containing 11 mM glucose supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin.
Our primary interest was investigating the effect of dexmedetomidine on autophagy activity following hypoxia. A cobalt chloride (CoCl2)-induced hypoxia model for INS-1 cells was established to investigate whether dexmedetomidine reduces hypoxia-induced autophagy. The CoCl2-induced hypoxia model was used due to the difficulty of controlling the extent of cellular damage in the hypoxia-reperfusion model that is commonly used in cellular experiments related I/R injury. In addition, since hypoxia can induce both autophagy and apoptosis, the effects of dexmedetomidine on apoptosis after CoCl2-induced hypoxia was also investigated. However, excessive cell death, whether through necrosis or apoptosis, can complicate the evaluation of autophagy activity. Therefore, we aimed to perform the experiment in conditions where cell proliferation is not significantly affected, while the expressions of autophagic and apoptotic proteins induced by hypoxia are significantly impacted.
Cell proliferation assessment with a cell counting kit-8 (CCK-8) assay
To investigate whether CoCl2 and dexmedetomidine individually affect cell proliferation, each agent was independently applied to INS-1 cells, and changes in proliferation were assessed. INS-1 cells were grown in 96-well plates to a density of 5 × 104 cells/ml before treatment with CoCl2 or dexmedetomidine in complete media (n = 6). CoCl2 was applied to INS-1 cells for one day at concentrations of 0, 10, and from 50 to 400 µM in increments of 50 µM. Dexmedetomidine was applied for the same duration at concentrations of 0, 0.1, 0.5, 1, 5, 10, and 20 µg/ml. Cell proliferation was assessed with a CCK-8 assay according to the manufacturer’s protocol (CCK-8®, Dojindo Molecular Technologies). After 10 μl of CCK-8 solution was added and incubated in a high humidity environment at 37°C and 5% CO2 for 1 h, the optical density (OD) was read at 450 nm with a microplate reader. The relative values for proliferation rates were presented.
Expression of autophagic and apoptotic proteins by hypoxia
First, the expression of hypoxia-inducible factor (HIF)-1α and HIF-2 was confirmed to determine whether hypoxia was induced by CoCl2 in INS-1 cells. In addition, to determine the appropriate doses of CoCl2 and dexmedetomidine, the expression of autophagy- and apoptosis-related proteins was evaluated using Western blot analysis. Purified INS-1 cells were incubated at 37°C and treated with various concentrations of CoCl2 (0, 10, 50, 100, 150, 200, and 300 μM) for one day. To determine the dose of dexmedetomidine, INS-1 cells were cultured in 1, 5, 10, and 20 μg/ml dexmedetomidine for 2 h before being treated with CoCl2 for one day.
The autophagy inhibitor, 3-Methyladenine (3-MA) (Sigma-Aldrich) at 5 mM, and the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK) (Sigma-Aldrich) at 20 μM, were pretreated for 2 h before treating INS-1 cells with CoCl2. Both inhibitors block the autophagosome formation and apoptosis, respectively.
Western blot analysis
Treated cells were lysed in protein lysis buffer (1% sodium dodecyl sulfate (SDS) in 25 mmol/L Tris-HCl [pH 7.4], 1 mmol/L EDTA, 100 mmol/L NaCl, 1 mmol/L PMSF, 10 μg/ml leupeptin, and 10 μg/ml pepstatin). Cell lysates were centrifuged at 12,000 × g for 10 min at 4°C to pelletize the insoluble material. The protein concentration was measured using the Bicinchoninic acid protein assay. The total protein (30 μg) from each sample was separated on a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked in 5% non-fat milk and incubated with each antibody overnight. Following extensive washing, the immunocomplexes were detected using a Western blotting ECL solution. The relative expression level of each protein was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell-Signaling Technology). The following primary antibodies were used: light chain 3B (LC3B) and p62 were purchased from Novus, and HIF-1α, HIF-2, BCL-2, P-BAD, BAX, ATG3, ATG5, ATG7, BECLIN-1, and 14-3-3 were purchased from Cell Signaling Technology. The Western blots were quantified using the Gel Analysis Tool in ImageJ (National Institutes of Health, https://imagej.net). All experiments were performed in triplicate to ensure the reproducibility of results. Detailed information about antibodies used in the study is presented in Supplementary Table 1.
Flow cytometry analysis
The apoptosis analysis was conducted using flow cytometry according to the standard procedure [23]. INS-1 cells were gained by centrifugation, washed with cold phosphate-buffered saline (PBS), and stained with propidium iodide solution and Annexin V-fluorescein isothiocyanate solution (ThermoFisher ScientificTM) consisting of 20 μg/ml RNase A (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich). Following 0.5 h incubation in the dark, the stained cells were detected in a BD FACSymphonyTM A5 flow cytometer (Becton Dickinson Biosciences). The distribution of cells in the different cell apoptosis phases was analyzed using Multicycle software.
Autophagic flux assay
To determine whether the reduction in autophagy resulted from decreased formation of autophagosomes or inhibition of autophagosome degradation, we used bafilomycin-A1 (Baf-A1) (Sigma-Aldrich) that inhibits autophagosome-lysosome fusion and degradation, the final stage of autophagy [24]. Baf-A1 at 50 nM was added to the CoCl2-treated INS-1 cells for the last 3 h of each treatment condition. Then they were washed twice with cold PBS and lysed in radio-immunoprecipitation assay buffer containing a protease inhibitors cocktail (Roche). The Western blot analysis was performed for LC3B and GAPDH antibodies.
Statistics
The results are expressed as mean ± standard deviations. The statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni correction using GraphPad Prism® (GraphPad Software, Inc.). Values of P < 0.05 were considered significant.
Results
Effects of CoCl2 and dexmedetomidine on the proliferation of INS-1 cells
INS-1 cell proliferation was significantly reduced in one day at a concentration above 200 μM of CoCl2, as demonstrated by the CCK-8 assay (Fig. 1B). Thus, a CoCl2 at concentration of 150 μM, the highest concentration that minimally affected cell proliferation, was selected as the suitable concentration for the main study. Dexmedetomidine itself did not influence the proliferation of INS-1 cells (Fig. 1C).
Effects of CoCl2 and dexmedetomidine on the proliferation of INS-1 cells. Cell viability was examined by CCK-8 assays. (A) A schematic representation of the experimental protocol. (B) INS-1 cells were treated with various concentrations of CoCl2 (0, 10, 50, 100, 150, 200, 250, 300, 350, and 400 μM). (C) INS-1 cells were treated with various concentration of dexmedetomidine (0, 0.1, 0.5, 1, 5, 10, and 20 μg/ml). Data are presented as mean ± standard deviation from six independent experiments. The experimental data were normalized by setting the control group’s mean value to 1. One-way analysis of variance (ANOVA) followed by Bonferroni correction was used. *P < 0.05 compared to the control group. CCK-8: cell counting kit-8, CoCl2: cobalt chloride, DEX: dexmedetomidine, INS-1 cell: rat insulinoma cell line.
CoCl2 induced hypoxia in INS-1 cells
Because HIF-1α and HIF-2 are activated in hypoxic conditions, their expression in CoCl2-treated INS-1 cells was evaluated. HIF-1α and HIF-2 expression in INS-1 cells increased markedly in a concentration-dependent manner after one day of treatment with 0–300 μM CoCl2 (Figs. 2B–D). These results indicate that CoCl2 induced hypoxia in INS-1 cells.
Effects of CoCl2-induced hypoxia in INS-1 cells. CoCl2 induced the expression of HIF-1α, HIF-2, and autophagic and apoptotic proteins. INS-1 cells were treated with various concentrations of CoCl2 (0, 10, 50, 100, 150, 200, 250, and 300 μM). (A) A schematic representation of the experimental protocol. (B) Western blot and (C, D) densitometric analysis of HIF-1α and HIF-2 expression. (E) Western blot and (F–K) densitometric analysis of ATGs, LC3B-II, BECLIN-1, and 14-3-3. (L) Western blot and (M–O) densitometric analysis of BCL-2, P-BAD, and BAX expression. Data are presented as mean ± standard deviation from three independent experiments. The experimental data were normalized by setting the control group’s mean value to 1. One-way analysis of variance (ANOVA) followed by Bonferroni correction was used. *P < 0.05 compared to the control group (no CoCl2). ATG: autophagy-related, CoCl2: cobalt chloride, DEX: dexmedetomidine, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, HIF: hypoxia-inducible factor, INS-1 cell: rat insulinoma cell line, LC3B: light chain 3B.
CoCl2-induced hypoxia increased the expression of autophagy markers in INS-1 cells
CoCl2 also increased autophagy markers: ATG3, 5, 7; LC3B-II, BECLIN-1; and 14-3-3 (Figs. 2E–K). These results indicate that CoCl2-induced hypoxia is associated with autophagic cell damage in INS-1 cells.
CoCl2-induced hypoxia decreased the expression of anti-apoptotic proteins and increased the expression of apoptotic proteins in INS-1 cells
CoCl2 reduced the expression of the anti-apoptotic protein BCL-2 and increased the expression of P-BAD and BAX that are apoptotic proteins (Figs. 2L-O). These results indicate that CoCl2-induced hypoxia caused apoptosis in INS-1 cells.
Dexmedetomidine attenuated CoCl2-induced autophagy and apoptosis in INS-1 cells
To investigate the effects of dexmedetomidine on CoCl2-induced INS-1 cell injury, INS-1 cells were treated with 150 μM of CoCl2 and 1, 5, 10, and 20 μg/ml dexmedetomidine. Dexmedetomidine decreased the expression of autophagic markers (ATG3, 5, 7; LC3B-II; BECLIN-1; and 14-3-3) in a dose-dependent manner (Figs. 3B–H). Dexmedetomidine increased the expression of BCL-2, an anti-apoptotic protein, and decreased the expression of P-BAD and BAX apoptotic proteins, in a dose-dependent manner (Figs. 3I–L). The concentration of dexmedetomidine was selected based on the results of Fig. 3. At a concentration of 10 μg/ml, the expressions of ATG5, LC3B-II, BECLIN-1, 14-3-3, and BAX were reduced compared to those at a concentration of 5 μg/ml, while increasing the concentration of dexmedetomidine to 20 μg/ml did not significantly alter protein expression. Therefore, dexmedetomidine at a concentration of 10 μg/ml was selected as the suitable concentration for further study.
Dexmedetomidine attenuated the expression of autophagic and apoptotic proteins in INS-1 cells after CoCl2-induced hypoxia. INS-1 cells were treated with 150 μM CoCl2 and various concentration of dexmedetomidine (0, 1, 5, 10, and 20 μg/ml). (A) A schematic representation of the experimental protocol. (B) Western blot and (C–H) densitometric analysis of ATGs, LC3B-II, BECLIN-1, and 14-3-3. (I) Western blot and (J–L) densitometric analysis of BCL-2, P-BAD, and BAX. Data are presented as mean ± standard deviation from three independent experiments. The experimental data were normalized by setting the control group’s mean value to 1. One-way analysis of variance (ANOVA) followed by Bonferroni correction was used. *P < 0.05 compared to the control group (150 μM CoCl2 and no dexmedetomidine), †P < 0.05 compared to the 150 μM CoCl2 and 5 μg/ml of the dexmedetomidine group. ATG: autophagy-related, CoCl2: cobalt chloride, DEX: dexmedetomidine, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, INS-1 cell: rat insulinoma cell line, LC3B: light chain 3B.
In the experiment investigating the effects of dexmedetomidine on cell proliferation after CoCl2-induced hypoxia, dexmedetomidine reversed the CoCl2-induced reduction in cell proliferation, while the effect was not large (Fig. 4B).
Effects of dexmedetomidine on cell proliferation and the expression of autophagic and apoptotic proteins. Dexmedetomidine reversed CoCl2-induced reduction in cell proliferation. Dexmedetomidine attenuated the activation of autophagy and apoptosis after CoCl2-induced hypoxia in INS-1 cells. INS-1 cells were treated with combination of CoCl2 and dexmedetomidine, or 3-MA, or z-VAD-FMK. (A) A schematic representation of the experimental protocol. (B) Cell viability was examined by CCK-8 assays. (C) A schematic representation of the experimental protocol. (D) Western blot and (E–H) densitometric analysis of LC3B-II, p62, BCL-2, and BAX. Data are presented as mean ± standard deviation from six (B) and three (E–H) independent experiments, respectively. The experimental data were normalized by setting the control group’s mean value to 1. One-way analysis of variance (ANOVA) followed by Bonferroni correction was used. *P < 0.05 compared to the control group (no CoCl2); †P < 0.05 compared to the CoCl2 only group. ATG: autophagy-related, CoCl2: cobalt chloride, CCK-8: cell counting kit-8, DEX: dexmedetomidine, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, INS-1 cell: rat insulinoma cell line, LC3B: light chain 3B, z-VAD-FMK: benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, 3-MA: 3-methyladenine.
To further investigate the effects of dexmedetomidine on the homeostasis of autophagy and apoptosis, INS-1 cells were pretreated with dexmedetomidine, 3-MA, or z-VAD-FMK before being exposed to CoCl2-induced hypoxia. The CoCl2-induced upregulation of LC3B-II expression was attenuated by dexmedetomidine (Figs. 4D and E), and the CoCl2-induced downregulation of p62 expression was also reversed by dexmedetomidine (Figs. 4D and F). In the experiment to evaluate the potency of dexmedetomidine on autophagosome formation, dexmedetomidine showed effects on the expression of LC3B-II and p62 that is in the same direction as the effect of 3-MA, a well-known inhibitor of autophagosome formation (Figs. 4D–F).
Dexmedetomidine also reversed the CoCl2-induced decrease in the expression of the anti-apoptotic protein BCL-2 (Figs. 4D and G), and reduced the CoCl2-induced increase in the expression of apoptotic proteins (BAX) (Figs. 4D and H). These results were comparable to those observed with z-VAD-FMK, a pan-caspase inhibitor (Figs. 4D, G and H).
The changes in the protein expression of autophagy and apoptosis markers suggest that dexmedetomidine reduces the activation of autophagy through the inhibition of autophagosome formation and apoptosis in CoCl2-induced INS-1 cell injury.
Dexmedetomidine reduced apoptosis in CoCl2-treated INS-1 cells
Flow cytometry showed that the percentage of apoptotic cells increased significantly in CoCl2-treated INS-1 cells compared with non-treated INS-1 cells, and that increase was significantly reduced by dexmedetomidine. The percentage of apoptotic cells is presented in right upper and right lower quadrant (Fig. 5).
Dexmedetomidine reduced CoCl2-induced apoptosis in INS-1 cells. CoCl2 increased apoptosis in INS-1 cells compared to the control group, whereas co-treatment with dexmedetomidine decreased it. (A) A schematic representation of the experimental protocol. (B) Flow cytometry plots using annexin V-FITC/PI staining for apoptosis. (C) The percentage of apoptotic cells was statistically compared. Data are presented as mean ± standard deviation from four independent experiments. Percentage of apoptotic cell was presented in right upper and right lower quadrant. One-way analysis of variance (ANOVA) followed by Bonferroni correction was used. *P < 0.05 compared to the control group (no CoCl2); †P < 0.05 compared to the CoCl2 only group. CoCl2: cobalt chloride, DEX: dexmedetomidine, FITC: fluorescein isothiocyanate, INS-1 cell: rat insulinoma cell line; PI: propidium iodide.
Dexmedetomidine decreased autophagic flux in CoCl2-treated INS-1 cells
CoCl2 increased the expression of LC3B-II, and that increase was attenuated by dexmedetomidine. When INS-1 cells were treated with Baf-A1 that inhibits autophagosome degradation, LC3B-II expression was increased. In that condition, CoCl2 further increased the expression of LC3B-II and dexmedetomidine ameliorated that increase. The increased LC3B-II/I ratio by CoCl2 was also attenuated by dexmedetomidine in INS-1 cells treated with Baf-A1 (Fig. 6). These data indicate that the dexmedetomidine decreased autophagosome formation in CoCl2-treated INS-1 cells.
Dexmedetomidine decreased autophagosome formation in CoCl2-treated INS-1 cells. INS-1 cells were treated or not treated with CoCl2 for one day. Dexmedetomidine was pretreated or not in CoCl2-treated INS-1 cells. Baf-A1 at 50 nM was added to the INS-1 cells for the last 3 h of each treatment condition. (A) A schematic representation of the experimental protocol. (B) Western blot, (C) densitometric analysis of LC3B-II, and (D) ratio of LC3B-II and LC3B-I. Data are presented as mean ± standard deviation from three independent experiments. The experimental data were normalized by setting the control group’s mean value to 1. One-way analysis of variance (ANOVA) followed by Bonferroni correction was used. *P < 0.05 compared to the control group (no CoCl2 and no dexmedetomidine); †P < 0.05 compared to the CoCl2 only group; ‡P < 0.05 compared to the Baf-A1 only group; §P < 0.05 compared to the Baf-A1 and CoCl2 group. Baf-A1: bafilomycin A1, CoCl2: cobalt chloride, DEX: dexmedetomidine, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, INS-1 cell: rat insulinoma cell line, LC3B: light chain 3B.
Discussion
In this study investigating the effects of dexmedetomidine on autophagy in INS-1 cells under hypoxia, CoCl2-induced hypoxia increased autophagy, as evidenced by an accumulation of autophagic markers (including ATGs and LC3B-II proteins), and dexmedetomidine treatment significantly mitigated those changes. The dexmedetomidine-induced inhibition of autophagy was associated with the inhibition of autophagosome formation during the process of autophagic flux, as confirmed by co-treatment with Baf-A1 that inhibits autophagosome-lysosome fusion and degradation.
Like other major organs, the pancreas is vulnerable to I/R injury, which is considered a critical factor for acute pancreatitis [3–5]. Considering the controversies over diagnostic indicators and the high incidence of asymptomatic hyperamylasemia [4], the actual incidence of postoperative acute pancreatitis reported in previous studies may vary depending on diagnostic criteria and type of surgeries. Further, patients with diabetes or hyperglycemia are more susceptible than other patients to acute pancreatitis [25,26], with pancreatic I/R injury as a key mechanism. As there is no effective treatment for acute pancreatitis [4], efforts are needed to reduce its occurrence. In this context, we focus on the strategies to reduce pancreatic I/R injury, as autophagy plays a certain role in pancreatic β-cell death or damage [27,28], with hypoxia affecting the homeostasis of pancreatic β-cells by regulating autophagy [29].
Autophagy acts as a defense mechanism against hypoxia and helps maintain cellular homeostasis and survival [30]. The activation of autophagy is important for maintaining cell viability [29,31]; however, excessive induction of autophagy can cause autophagic cell death that contributes to pancreatic β-cell mass loss in type 2 diabetes [29]. These dual roles of autophagy in cell survival and death are a crucial part of pancreatic β-cell adaptation to stress [18,29].
Dexmedetomidine, a highly selective α2-agonist, has been shown to have anti-inflammatory and anti-apoptotic effects, as well as has protective effects against diverse stress conditions, such as hypoxia or I/R injury in the lung, brain, kidney, and heart [19–21,32–34]. A growing body of evidence indicates that autophagy-modulating properties might play a certain role in the protection conferred by dexmedetomidine against organ injury [33]. Oh et al. [35] reported that dexmedetomidine reduced cardiac dysfunction and suppressed autophagy in streptozotocin-induced rats by regulating the impaired activation of the ERK and AKT signaling pathways. Luo et al. [21] showed that dexmedetomidine protects mouse brains against I/R injury by inhibiting autophagy through the upregulation of HIF-1α. So far, the effects of dexmedetomidine on autophagy in β-cells under hypoxia remain unclear.
In this study, a CoCl2-induced hypoxia model was used because this is stable and enabled us to control the extent of cellular damage. INS-1 cell viability decreased as the concentration of CoCl2 increased. We found that CoCl2, a well-known hypoxia-mimetic agent [36], significantly activated the expression of HIF-1α and HIF-2, key transcriptonal regulators of the hopoxic response [37], that indicates that CoCl2 induced hypoxia and hypoxic cell injury to INS-1 cells. In this study, dexmedetomidine treatment suppressed autophagy and apoptosis in INS-1 cells exposed to CoCl2-induced hypoxia, which is consistent with the results of previous studies showing that dexmedetomidine increased cell survival by inhibiting autophagy and apoptosis in a cerebral injury model [21,38].
When autophagy is activated, an accumulation of LC3B-II and a decrease in p62 can be observed because p62 is degraded after interacting with autophagosomes [29]. The hypoxia induced autophagy in CoCl2-treated INS-1 cells, confirmed by increase in LC3B-II and a decrease in p62, was likely to be attenuated with dexmedetomidine treatment through autophagosome formation, as observed with 3-MA, an autophagosome formation inhibitor (Fig. 4).
To determine how dexmedetomidine reduces autophagic activity, we performed the LC3B turnover assay combined with Baf-A1 that blocks autophagosome-lysosome fusion and degradation. During the process of autophagy, LC3B-I is converted into LC3B-II by ATG7 and ATG3 enzymes, and then LC3-II is recruited to the autophagosomal membrane [39]. Therefore, LC3B conversion (LC3B-I to LC3B-II) reflects the level of autophagic activity, and the LC3B turnover assay measures the accumulation of autophagosomes. Because accumulation of autophagosome can result from both increased production and inhibited clearance, the LC3B turnover assay combined with Baf-A1 is used to distinguish between these [40]. The decrease in the expression of LC3B-II upon co-treatment with Baf-A1 indicates a decrease in autophagosome formation [24]. In this study, LC3B turnover assay with co-treatment of Baf-A1, expressed by the changes in LC3B-II/I ratio, showed that dexmedetomidine decreased autophagic flux mainly due to decreased autophagosome formation rather than increased autophagosome clearance in CoCl2-treated INS-1 cells (Fig. 6).
Because autophagy and apoptosis can occur in the same cell and interact with each other [41,42], the direct effect of dexmedetomidine on apoptosis in CoCl2-treated INS-1 cells was investigated. When comparing the effects of dexmedetomidine with those of z-VAD-FMK, a pan-caspase inhibitor, dexmedetomidine decreased the level of apoptosis (Fig. 4). Flow cytometry results showed that dexmedetomidine reduced cell apoptosis, which was in accordance with previous studies performed in H9c2 cells and SH-SY5Y cells as models of cardiac injury and brain injury, respectively [43,44]. All these results indicate that dexmedetomidine also has an anti-apoptotic effect against CoCl2-induced hypoxia.
The effects of autophagy in pancreatic β-cells seems to be two-sided. Fujimoto et al. [15] reported that inhibition of autophagy prolonged cell survival and delayed cell death in mouse insulinoma 6 cells under nutrient deprivation. On the other hand, Liang et al. [29] reported that inhibition of autophagy via chloroquine increased cell death in human islet β-cells under CoCl2-induced hypoxia, and Li et al. [45] presented that liraglutide protected INS-1 cells under palmitate-induced lipotoxicity by enhancing autophagy. These discrepancies among studies regarding the protective effect of autophagy in pancreatic β-cells under stressful conditions might result from differences in the type and intensity of the stress or thresholds for either response. In this study, dexmedetomidine alleviates the degree of cellular damage to a small extent by inhibition of triggered autophagy and apoptosis after 24 h of exposure to CoCl2-induced hypoxia. This suggests that proper regulation of autophagy is detrimental to β-cell survival after hypoxia exposure.
There are several limitations in this study. First, dexmedetomidine reduces both autophagy and apoptosis activity. There is crosstalk between autophagy and apoptosis because some proteins, such as BECLIN-1, modulate both autophagy and apoptosis [46]. Therefore, there might be an anti-apoptotic effect of autophagy induced by dexmedetomidine, or vice versa. Second, the dual roles of autophagy in cell survival and death are complex and may require further exploration to fully understand the mechanisms involved. Third, the impact of dexmedetomidine on autophagy in terms of cytotoxicity and cell function, as well as its long-term effects, was not assessed. Fourth, the concentration of dexmedetomidine used in this study was much higher than that used clinically, and the protective effect of dexmedetomidine on cell survival was not remarkable, although the hypoxia-induced decrease in cell proliferation was attenuated with dexmedetomidine treatment in the current study. Therefore, further research is needed to apply the results of this study to clinical practice.
In conclusion, dexmedetomidine alleviates the degree of cellular damage in INS-1 cells against CoCl2-induced hypoxia by regulating autophagy activity. Dexmedetomidine might attenuate autophagosome formation, as confirmed by a decrease in the LC3B-II/I ratio. Dexmedetomidine also reduced CoCl2-induced apoptosis and the expression of related proteins. These results provide a basis for further studies to confirm these effects in clinical practice.
Acknowledgements
The authors would like to thank Dr. Ji Hae Jun for her expert assistance in the laboratory.
Notes
Funding
This research was supported by grant No. KSA-2023-002 from the Korean Society of Anesthesiologists.
Conflicts of Interest
Young-Lan Kwak has been the Editor-in-Chief for the Korean Journal of Anesthesiology since 2016. However, she was not involved in any process of review for this article, including peer reviewer selection, evaluation, or decision-making. There were no other potential conflicts of interest relevant to this article.
Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Author Contributions
Jin Ha Park (Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Software; Visualization; Writing – original draft; Writing – review & editing)
Ju Eun Oh (Data curation; Investigation; Methodology; Software; Writing – original draft)
Namo Kim (Formal analysis; Investigation; Methodology; Software; Writing – original draft)
Young-Lan Kwak (Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Project administration; Supervision; Writing – original draft; Writing – review & editing)
Supplementary Material
Information about antibodies used in this study.