2,3,7,8-Tetrachlorodibenzo- p -Dioxin (TCDD) Induces Calcium Influx Through T-type Calcium Channel and Enhances Lysosomal Exocytosis and Insulin Secretion in INS-1 Cells

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been associated with diabetes in several epidemiological studies. However, the diabetogenic action of TCDD on pancreatic cells is unclear. Here, we investigated the direct toxic effects of TCDD on a rat insulin-secreting beta cell line. We found that TCDD enhances exocytosis of MTT formazan and lysosomal proteins such as β-hexosaminindase and Lamp-1. This TCDD-induced exocytosis was abrogated by T-type calcium channel blockers (mibefradil, flunarizine) but not by an aryl hydrocarbon receptor antagonist (α-naphtoflavone). Indeed, cytosolic calcium levels were increased by TCDD. Furthermore, TCDD stimulated insulin secretion, which was inhibited by flunarizine. Taken together, our results suggest that TCDD-induced calcium influx via T-type channels regulates vesicular trafficking, such as lysosomal and secretory granule exocytosis, and that TCDD might exert adverse effects on beta cells by continuous insulin release followed by beta cell exhaustion. This could contribute to the link between TCDD exposure and the risk of developing diabetes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a byproduct of many industrial processes, is highly lipophilic and resistant to chemical and biological degradation processes. It readily accumulates in human and animal tissue and has a long half-life of 7 to 9 years. ^1,2 Therefore, it is one of the most potent toxic environmental pollutants because of its bioaccumulation potential. TCDD induces a broad spectrum of toxic responses in experimental animals including neurotoxicity, liver damage, immunotoxicity, reproductive toxicity, carcinogenicity, and wasting syndromes. ^3–5 Several studies have indicated positive associations between TCDD exposure and glucose metabolism disorders such as insulin resistance and hyperinsulinemia. ^6–10 Marked reductions in glucose uptake are caused by downregulation of glucose transporters, and drastic decreases in lipoprotein lipase activity and tumor necrosis factor-alpha upregulation ^11–13 have been identified as contributors to the mechanisms of TCDD-associated diabetogenic toxicity. However, mechanisms underlying TCDD-induced beta cell toxicity remain to be elucidated. Therefore, we examined the effects of TCDD on viability, and in particular, insulin release, in pancreatic beta cells.

In this study, MTT assay, a typical cell viability assay, informed us that MTT reduction can be used as an index of effects on exocytosis induced by TCDD independently of effects on cell viability. Based on the decrease of MTT reduction, we demonstrated that TCDD promotes exocytosis of secretory granule-containing insulin, as well as lysosomes. Consequently, we present the ability of TCDD to stimulate insulin secretion in INS-1 cells and provide evidence that this effect is a result of calcium influx through T-type calcium channels.

Materials and Methods

Materials

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was purchased from Cambridge Isotope Laboratories Inc (Andover, Massachusetts). MTT [3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide], dimethylsufoxide (DMSO), pluronic acid, diazoxide, ionomycin, α-naphthoflavone, nifedipine, mibefradil, flunarizine, EGTA, and 4-methylumbelliferyl-N-acetyl-β-D-glucosamide were purchased from Sigma Laboratories (St Louis, Missouri). CCK-8 was obtained from Dojindo Molecular Technologies (Gaithersburg, Maryland). Verapamil and Lamp-1 antibody were purchased from Calbiochem (Darmstadt, Germany). Fluo4-AM, probenecid, and Alexa 488 goat antimouse antibody were purchased from Molecular Probes (Eugene, Oregon).

Cell Culture

Rat insulinoma INS-1 cells were cultured in RPMI 1640 (Gibco BRL, Rockville, Maryland) supplemented with 10% fetal bovine serum, antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 10 mM HEPES. Cells were maintained in a T-25 tissue culture flask at 37°C and 5% CO₂ in a humidified atmosphere and routinely subcultured every 4 to 5 days by gentle trypsinization. Cell cultures were initiated at 10,000 cells per well in a 96-well plate for the cell proliferation assay and treated with TCDD and inhibitors after incubation for 2 days.

Cell Viability Assay

MTT reduction assay was performed for 3 hours at 37°C after addition of MTT (0.5 mg/mL) to each well and terminated by adding 100 μL of solubilization solution containing 50% N, N-dimethylformamide, and 20% sodium dodecyl sulfate (SDS). Absorbance values were determined at 570 nm with a microplate reader. CCK-8 assay (CCK-8, Dojindo, Maryland), another tetrazolium salt-based viability assay like MTT assay, is based on the cleavage of the tetrazolium salt WST-8 in viable cells. CCK-8 solution (10 μL) was directly added to each well, and WST-8 reduction was allowed to proceed for 2 hours. Absorbance at 450 nm was measured according to the manufacturer’s instructions. Alternatively, cells were treated with 0.5 μCi/well of [³H]-thymidine (New England Nuclear, PerkinElmer, Boston, Massachusetts) for an additional 4 hours in the [³H]-thymidine incorporation assay. The cells were collected and lysed using a semiautomatic cell harvester (SKATRON instruments, Lier, Norway), and the radioactivity retained on the dried glass fiber filters was measured by liquid scintillation (LKB, Stockholm, Sweden). Viable cells were also counted by the trypan blue dye exclusion assay with a hemocytometer.

Cytotoxicity Assay

Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH) into the medium using the CytoTox96 LDH assay kit (Promega, Madison, Wisconsin). Culture supernatant (50 μL) was incubated with an equal volume of LDH substrate solution in dark conditions for 30 minutes. The reaction was stopped with 50 μL of 1 M acetic acid, and the absorbance was determined at 492 nm.

Western Blot Analysis

Cells were directly lysed in SDS-PAGE sample buffer after treatment with 100 nM TCDD for 6 hours. Protein samples were heated at 100°C for 5 minutes, separated on 10% SDS-PAGE, and transferred to polyvinylidence difluoride (PVDF) membranes (Millipore, Bedford, Massachusetts). The membranes were blocked with phosphate buffered saline (PBS) containing 0.1% Tween-20 and 5% skim milk for 30 minutes and incubated with CYP1A1 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, California) overnight at 4°C. The blots were washed 3 times with PBS containing 0.1% Tween-20 for 20 minutes and then incubated in horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories, South San Francisco, California) on a shaker for 1 hour. Peroxidase activity was detected by chemiluminescence with a West Pico Western blot detection reagent in accordance with the manufacturer’s instructions (Pierce, Rockford, Illinois).

Intracellular Calcium Measurements

Cytosolic calcium levels in INS-1 cells were monitored with fluo-4AM, a fluorescent calcium indicator. Cells were seeded on poly-L-ornithine-coated coverslips placed in 35-mm culture dishes for 3 days. The cells were loaded with 2 μM fluo-4AM, 2.5 mM probenecid, and 0.01% pluronic acid at 37°C in a CO₂ incubator for 30 minutes. Cells were washed 3 times after incubation with KRBH buffer (130 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.0 mM CaCl₂, 5 mM NaHCO₃, 10 mM HEPES, pH 7.4, 0.1% BSA) containing 2.5 mM probenecid. Calcium levels were measured with a LSM-510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany), and a single increment in fluorescence intensity indicated an increase in the cytosolic calcium level. Cells were incubated for 5 minutes at room temperature with 2 μM flunarizine before the addition of 100 nM TCDD in some experiments.

β-Hexosaminidase Activity Assay

INS-1 cells were treated with TCDD or ionomycin (3 μM) in PBS with 1 mM CaCl₂. Cells were incubated for 10 minutes and pelleted, and the supernatant was assayed for β-hexosaminidase activity. The total enzyme activity in the cell pellet was determined after solubilization with 1% NP-40. Supernatants (350 μL) or cell lysates were incubated for 15 minutes at 37°C with 50 μL of 6 mM 4-methyl-umbelliferyl-N-acetyl-b-D-glucosamide in 40 mM sodium citrate and 88 mM Na₂PO₄ (pH 4.5). The reaction was stopped by the addition of 100 μL of 2 M Na₂CO₃ and 1.1 M glycine. The fluorescence was measured in a spectro-fluorometer (KONTRON Instruments, Watford, United Kingdom) at an excitation of 365 nm and emission of 450 nm.

Immunofluorescence Imaging

INS-1 cells were seeded on poly-L-ornithine-coated coverslips placed in 12-well Costar plates (Corning, New York) and grown for 2 days at 37°C in a CO₂ incubator. Cells were incubated in 100 nM TCDD and/or 2 μM flunarizine for 10 minutes, washed with ice cold PBS, and incubated with the Lamp-1 (LY1C6 mAb) antibody dissolved in PBS with 1% BSA for 30 minutes at 4°C. Cells were washed 3 times and were fixed with 3.7% paraformaldehyde at 4°C for 20 minutes and incubated with the Alexa 488 goat antimouse antibody for 30 minutes at room temperature. Images were generated on a Zeiss Axiovert 200 inverted microscope using an Axiocam MRc5 digital camera (Carl Zeiss Microscopy).

Insulin Secretion Assay

INS-1 cells were transferred onto 12-well Costar plates and grown to 80% confluence. Cells were washed with fresh medium, and 1 mL of glucose-free KRBH buffer was added to each well for 1 hour at 37°C. The cells were then washed twice and incubated with TCDD (10–100 nM) for 30 minutes at 37°C in fresh KRBH buffer containing 2.8 mM glucose. Media were transferred to Eppendorf tubes and spun at 2000g at 4°C for 1 minute. Supernatants were collected and placed on ice. Insulin was measured by radioimmunoassay (Linco Research Inc, St Charles, Missouri) according to the instructions of the manufacturer using rat insulin as a standard.

Statistical Analysis

Statistical significance of the differences between groups was evaluated by 1-way analysis of variance (ANOVA), followed by the Duncan multiple range test using SAS version 9.1 (SAS Institute Inc, Cary, North Carolina). A P value of less than .05 was regarded as statistically significant. Values are presented as mean ± SEM.

Results

TCDD Inhibits Cellular Reduction of MTT Independently of Cell Viability in INS-1 Cells

To investigate the direct toxic effects of TCDD on pancreatic beta cells, we evaluated cell viability using MTT, [³H]-thymidine incorporation, and trypan blue exclusion assay in INS-1 cells. Cells treated with TCDD for 24 hours showed a significant decrease in MTT reduction in a dose-dependent manner (Figure 1A). Interestingly, however, the DNA synthesis rate and the number of viable cells were not decreased as shown in MTT assay (Figure 1A). To confirm this unexpected finding, we compared MTT findings over a short period of time with the CCK-8 assay, another tetrazolium salt-based viability assay, and found that 30 minutes of transient TCDD exposure even induces a dose-dependent decrease in MTT reduction but not in WST-8 reduction in the CCK-8 assay (Figure 1B). TCDD inhibited MTT reduction by 20% after 10 minutes and by 60% after 6 hours, while the reduction of WST-8 was not affected. Moreover, there was no LDH release into culture media from TCDD-treated cells, indicating that cells were not damaged, at least, by TCDD (Figure 1C). These results suggest that TCDD interferes in MTT reduction without affecting cell viability in INS-1 cells.

Inhibition of MTT Reduction by TCDD Is Independent of Aryl Hydrocarbon Receptor (AhR) Activation

TCDD initially binds to the aryl hydrocarbon receptor (AhR), a ligand-activated basic helix-loop-helix transcription factor, then translocates into the nucleus and induces expression of a variety of genes including CYP1A1. ⁴ Therefore, we examined whether TCDD inhibits MTT reduction through the AhR. TCDD-induced inhibition of MTT reduction was not reversed by pretreatment with α-naphthoflavone (α-NF, 100–500 nM), an AhR antagonist (Figure 2A). We assessed the CYP1A1 protein levels induced in HepG2 cells after TCDD treatment, with or without 100 nM α-NF. We could not detect the CYP1A1 protein by Western blot in INS-1 cells, and therefore HepG2 cells were used in this experiment with the same α-NF concentrations. CYP1A1 protein level was increased after 6 hours of TCDD treatment, and this was abolished by α-NF pretreatment (Figure 2B, blot). MTT reduction was similarly inhibited in HepG2 cells even with α-NF pretreatment as seen in INS-1 cells. Pretreatment of INS-1 cells with the protein synthesis inhibitor cycloheximide did not significantly restore TCDD-mediated suppression of MTT reduction (Figure 2C). These results indicate that the inhibition of MTT reduction by TCDD does not require new protein synthesis through AhR-dependent transcriptional activation.

TCDD Enhances MTT Formazan Exocytosis and Inhibits Its Subsequent Reduction

MTT cannot permeate the lipid membrane and enters the cell through endocytosis. MTT reduction to MTT formazan occurs in the endosomal and lysosomal compartments, and MTT formazan is transported to the cell surface through exocytosis. ¹⁴ The presence of MTT formazan on the cell surface inhibits further MTT uptake. Studies indicate that agents capable of enhancing MTT formazan exocytosis (beta-amyloid peptides, human amylin, and fibrillar insulin with a beta-pleated sheet structure) indirectly resulted in the inhibition of cellular MTT reduction without inhibiting cell growth. ¹⁵ Similarly, we investigated whether TCDD-mediated suppression of MTT reduction is caused by enhanced exocytosis of reduced MTT granules. INS-1 cells were grown in normal media incubated with 0.5 mg/mL MTT at 37°C and examined under a light microscope. Reduced MTT formazan granules were initially observed in the cytoplasmic compartment approximately 30 minutes after MTT addition. The granules became darker over time and, at 2 hours, formed needle-like formazan crystals which were transported to the cell surface (Figure 3Aa, b, c). In contrast, INS-1 cells treated with TCDD displayed the needle-like formazan crystals on the cell surface as early as 30 minutes after MTT addition. The needle-like formazan crystals at the cell surface were slightly increased as a function of MTT incubation time, and intracellular formazan granules were inversely reduced in the cytoplasmic compartment (Figure 3Ad, e, f). Therefore, the total amount of reduced MTT formazan in TCDD-treated cells was not increased after greater than 1 hour of incubation, but that of control cells was continuously increased with incubation time, reaching maximum levels in approximately 2 hours, and remained at high levels thereafter (Figure 3B). These results indicate that there is no difference in MTT reduction between control and TCDD-treated cells within the first 30 minutes of MTT incubation but that TCDD resulted in significant decreases in the total cellular MTT reduction after greater than 30 minutes of incubation (Figure 3A). Genistein, an inhibitor of lysosomal exocytosis, completely blocked the formation of needle-like formazan crystals at the cell surface without inhibiting the formation of intracellular formazan granules (data not shown) and abolished TCDD-mediated suppression of MTT reduction (Figure 3B). These data suggest that formation of needle-like formazan crystals at the cell surface results from exocytosis of intracellular MTT formazan granules, which is enhanced by TCDD treatment.

TCDD Increases Intracellular Ca²⁺ Level and Inhibits MTT Reduction Through Extracellular Ca²⁺ Influx

TCDD may increase intracellular calcium levels in various types of cells, ^16–19 although it did not induce Ca²⁺ influx in the human mammary epithelial cell line MCF-10A. ²⁰ MTT inhibition by β-amyloid treatment was recovered by calcium channel blockers such as nifedifine, which suggested that Ca²⁺ influx might play a role in MTT inhibition. ¹⁵ We loaded INS-1 cells with Fluo-4AM, a fluorescent calcium indicator, and monitored cytosolic calcium levels using a LSM-520 confocal microscope to investigate effects of TCDD treatments on cytosolic Ca²⁺ concentration. TCDD increased intracellular calcium levels for up to 10 minutes (Figure 4A). Cells were incubated with 100 nM TCDD for 30 minutes after pretreatment with the calcium chelator EGTA to examine the effect of calcium levels on TCDD-mediated suppression of MTT reduction. TCDD resulted in the inhibition of MTT reduction by approximately 40%. However, this inhibition was completely restored by the depletion of extracellular calcium with EGTA (Figure 4B). These data suggest that TCDD causes calcium mobilization from the extracellular into the cytosolic space and subsequently inhibits MTT reduction.

T-type Ca²⁺ Channels Are Involved in the Ca²⁺ Influx by TCDD

We determined that TCDD induces intracellular Ca²⁺ influx from the extracellular pool and examined calcium channel types involved in TCDD-induced Ca²⁺ influx. INS-1 cells express various types of voltage-dependent calcium channels including L-type and T-type channels, ²¹ and high-voltage activated L-type calcium channels are preferentially associated with exocytosis in pancreatic β cells. We treated the cells with L-type Ca²⁺ channel-selective inhibitors and followed by incubation of 100 nM TCDD for 30 minutes to determine whether L-type calcium channels are involved in TCDD-induced Ca²⁺ influx. Neither verapamil nor nifedifine as L-type selective antagonists abrogated the inhibition of MTT reduction caused by TCDD (Figure 5A). However, low-voltage activated T-type calcium channel blockers such as mibefradil and flunarizine completely abolished TCDD-mediated suppression of MTT reduction (Figure 5B). In addition, TCDD-mediated suppression of MTT reduction was not recovered by pretreatment with diazoxide, an ATP-sensitive K⁺ channel activator, which could switch off voltage-gated calcium channels by increasing K⁺ membrane permeability (Figure 5C). These data indicate that TCDD does not induce membrane depolarization by blocking ATP-sensitive K⁺ channels but can cause cells to mobilize Ca²⁺ influx, specifically by T-type but not by L-type channels.

TCDD-mediated Ca²⁺ Influx Triggers Exocytosis of Lysosomes

Increases in intracellular Ca²⁺ levels enhanced lysosome exocytosis, ^15,22,23 and TCDD increased intracellular Ca²⁺ levels (Figure 4A). Therefore, we examined TCDD-mediated Ca²⁺ influx and its ability to trigger lysosome exocytosis in INS-1 cells. Cells were treated with 100 nM TCDD or ionomycin as a positive control, and the activity of β-hexosaminidase, an enzyme in the lumen of lysosome, was increasingly detected in the culture media shortly after TCDD ionomycin treatment (Figure 6A). The release of cytosolic lactate dehydrogenase (LDH), an indicator of plasma membrane disruption, was not increased (data not shown). Immunoreactivity against Lamp-1, an abundant lysosomal membrane glycoprotein, was demonstrated on the INS-1 cell surface by TCDD (Figure 6Bc) or ionomycin (Figure 6Bb). Pretreatment with 2 μM flunarizine, a T-type Ca²⁺ channel blocker, prevented the lysosomal exocytosis (Figure 6Bd) as well as intracellular Ca²⁺ influx (Figure 6C). These data indicate that TCDD-induced Ca²⁺ influx through T-type Ca²⁺ channel increases lysosomal exocytosis.

TCDD Increases Insulin Secretion in INS-1 Cells

The evidence that TCDD enhanced lysosomal exocytosis by increasing intracellular Ca²⁺ concentration suggested that TCDD might enhance the exocytosis of secretory vesicles containing insulin. Insulin release as assessed by radioimmunoassay was dose-dependently increased in the media from cultures treated with TCDD as compared to control cells treated with DMSO solvent (Figure 7A). Insulin secretion enhanced by TCDD was not abrogated by the pretreatment with α-NF, an AhR pathway inhibitor (Figure 7B), and was prevented by treatment with the T-type channel blocker flunarizine (Figure 7C).

Discussion

We investigated TCDD-mediated calcium mobilization and subsequent exocytosis enhancement in INS-1 cells. The initial goal of this study was to examine the direct cytotoxicity of TCDD to insulin-secreting beta cells. To this end, we evaluated TCDD’s effect on INS-1 cell growth through cell viability assay including MTT assay. We found that there is no expected correlation between the rate of MTT reduction and the number of viable cells at early time points (about 30 minutes) after TCDD exposure. This suggested that TCDD caused inhibition of cellular reduction of MTT to MTT formazan. Further, we recognized that this was likely due to the blockade of further MTT uptake through the enhancement of MTT formazan exocytosis, as is similarly seen with amyloid beta peptide. ¹⁵

Exocytosis is the robust process by which cells excrete waste products or chemical transmitters. In multicellular organisms, there are 2 types of exocytosis: (1) Ca²⁺-triggered nonconstitutive and (2) non-Ca²⁺-triggered constitutive. Exocytosis in neuronal synapses is Ca²⁺ triggered and is responsible for interneuronal signaling. Constitutive exocytosis is involved in the release of components of the extracellular matrix or delivery of newly synthesized membrane proteins that are incorporated into the plasma membrane.

We investigated whether calcium is involved in the TCDD-facilitated MTT exocytosis and visually examined a significant rise in intracellular calcium ([Ca²⁺]_i) in the cytoplasm of INS-1 cells within a few minutes following TCDD exposure. Further, the enhancement of TCDD-mediated MTT exocytosis was antagonized by EGTA. These results indicate that the elevation in [Ca²⁺]_i results from the entry of extracellular calcium. Other studies have consistently shown that TCDD stimulated an increase of [Ca²⁺]_i in various types of cells. ^16–19,24 Therefore, the early increase of [Ca²⁺]_i may be a common response to TCDD exposure, regardless of the origin of the calcium.

In addition, we demonstrated that the TCDD-induced calcium influx also triggered lysosomal exocytosis. This was confirmed through measurement of protein activity and localization in the lysosomal lumen (β-hexosaminidase) and membrane (Lamp-1) and suggests reasons for TCDD-mediated enhancement of MTT formazan exocytosis.

We clarified the channels that are involved in TCDD-induced calcium entry through the use of specific calcium channel blockers. Flunarizine and mibefradil efficiently restored TCDD-mediated decreases in MTT reduction, indicating that T-type calcium channels are involved in the calcium influx. This may explain TCDD’s contribution to exocytosis in INS-1 cells.

T-type calcium channels are commonly found in cells undergoing rhythmic electrical behavior, such as pacemaker cells (sinoatrial and atrioventricular nodes) of the heart due to their rapid kinetics. TCDD administration has induced a ventricular standstill in zebrafish, ²⁵ and this may occur because T-type calcium currents enhanced by TCDD may deform action potentials. The cardiovascular system is also a main target of TCDD toxicity in humans. ^26–28

Exocytosis in the insulin-secreting beta cell is a calcium-mediated process, and elevated [Ca²⁺]_i is a principal trigger for secretion. Insulin was released by TCDD exposure in a dose-dependent manner in INS-1 cells without glucose stimulation, and this secretion was prevented by flunarizine, suggesting calcium entry through T-type channels contributed to the TCDD-induced insulin secretion. TCDD may impair the insulin response to glucose by exhaustion as a result of pertinent secretion. Piaggi and coworkers ²⁹ showed the impairment of glucose-stimulated insulin secretion by TCDD in INS-1E cells. Further, T-type calcium channels facilitate insulin secretion in INS-1 cells. ³⁰

In this study, TCDD induced calcium influx through low voltage-dependent T-type calcium channels and stimulated cellular exocytosis for MTT formazan or lysosomal proteins as well as secretory vesicles containing insulin. Our results suggest that TCDD exerts its direct cytotoxic effect on INS-1 cells by altering or impairing the normal Ca²⁺-regulated vesicular trafficking prior to affecting cellular viability via AhR-mediated gene expression and that the beta cell exhaustion from continuous insulin release could eventually result in a loss of beta cell mass, contributing to the onset of diabetes.