Volatile anesthetics suppress glucose-stimulated insulin secretion in MIN6 cells by inhibiting glucose-induced activation of hypoxia-inducible factor 1

Reagents

Isoflurane was obtained from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan) and sevoflurane was from Maruishi Pharmaceutical Co., Ltd. (Osaka, Japan). Oxygen (Taiyo Nippon Sanao, Wakayama, Japan), and nitrogen (Taiyo Nippon Sanso, Tokyo, Japan) were also used. An inhibitor of HIF, 5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol (YC-1), the HIFα hydroxylase inhibitor, dimethyloxaloylglycine (DMOG), the selective K_ATP (Kir6 subunit) blocker, glibenclamide, and the activator, diazoxide, were all obtained from Abcam (Cambridge, MA, USA). n-Propyl gallate (nPG; 3,4,5-trihydroxybenzoic acid propyl ester), the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), sucrose, and maltose were all obtained from Sigma Aldrich (St. Louis, MO, USA).

Cells and cell culture

The mouse insulinoma MIN6 and MIN7 cell lines were a gift from Dr. J Miyazaki (Osaka University) (Miyazaki et al., 1990). MIN6 and MIN7 cells were maintained at 37 °C under 5% CO₂ and 95% air in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) containing 450 mg/dl glucose, 10% fetal bovine serum (FBS), penicillin, streptomycin, and 50 µM β-mercaptoethanol.

Treatment with volatile anesthetics

Cells were maintained in an airtight chamber or work-station (AS-600P; AsOne, Osaka, Japan) perfused with mixed air (MODEL RK120XM series; Kofloc, Kyotanabe, Japan) with or without each test anesthetic, delivered by a specialized vaporizer within the open circuit. The concentrations of gases and anesthetics in the incubator were monitored during each treatment using an anesthetic gas monitor (Type 1304; Blüel & Kjær, Nærum, Denmark) that was calibrated with a commercial standard gas (47% O₂, 5.6% CO₂, 47% N₂O, 2.05% sulfur hexafluoride) (Itoh et al., 2001). The anesthetic concentration in the medium was measured by gas chromatography (5890A; Hewlett Packard, Palo Alto, CA, USA), as described previously (Namba et al., 2000).

Measurement of insulin concentration

Insulin secretion into the culture medium from MIN6 cells was measured using the Mouse Insulin H-type™ enzyme-linked immunosorbent assay kit (Shibayagi Co. Ltd., Shibukawa, Japan), according to the manufacturer’s protocol (Matsumoto et al., 2012). Briefly, MIN6 cells were cultured for 30 min in Krebs-Ringer bicarbonate HEPES (KRBH) buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH₂PO₄, 0.5 mM MgSO₄, 1.5 mM CaCl₂, 2 mM NaHCO₃, 10 mM HEPES, 0.1% bovine serum albumin) containing 40 mg/dl glucose. Then, the KRBH buffer was changed to one containing the indicated concentration of glucose, and the cells were cultured for 10 min or 1 h. The KRBH buffer was collected and subjected to insulin measurement. The cells on the dish were washed once with phosphate-buffered saline, lysed, and collected by scraping. The cells were sonicated and the protein concentration was determined using a protein assay kit (BioRad Laboratories, Hercules, CA, USA), with bovine serum albumin as the standard. The insulin concentration of the buffer was normalized to the total cell protein level. The results were normalized to the concentration of control samples of each independent experiment and the normalized values were demonstrated as insulin secretion ratio.

Cytotoxicity assay

Changes in the cellular viability of MIN6 cells were determined by the CellTiter 96™ AQueous One Solution cell proliferation assay (Promega, Madison, WI, USA) (Kai et al., 2012). The assay uses a colorimetric method to determine the number of viable cells in cytotoxicity assays. MIN6 cells were seeded in 96-well plates at a density of 2.0 × 10⁴ per well (in 100 µl medium). After 24 h, cells were exposed to isoflurane or sevoflurane for 8 h. MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]/phenazine ethosulfate (PES) solution was added and incubation was continued for 30 min. The absorbance of individual wells was then measured at a wavelength of 490 nm corrected to 650 nm using a Thermo Max™ microplate reader (Molecular Devices, Sunnyvale, CA, USA). Assays were performed at triplicate at least twice. Data were expressed as mean ± standard deviation (SD).

Assessment of apoptosis in MIN6 cells

Apoptosis was measured with the Apo-ONE™ Homogeneous Caspase-3/7 Assay (Promega) according to the manufacturer’s protocol. The assay contains proflourescent rhodamin 110 (Z-DEVD-R110) which serves as a substrate for both Caspase-3 and -7. MIN6 cells were seeded in 96-well plates at a density of 2.0 × 10⁴ per well (in 100 µl medium). After 24 h, cells were exposed to isoflurane or sevoflurane. After an additional 8 h of incubation, fluorescence was measured with Ensopire™ plate reader (PerkinElmer, Waltham, MA, USA) to determine the Caspase-3/7 activity. Assays were performed at triplicate at least twice. Data were expressed as mean ± standard deviation (SD).

Measurement of total cellular O₂ consumption (OCR)

The total OCR was measured as described previously (Kai et al., 2012; Zhang et al., 2007). Cells were trypsinized and suspended at 1 × 10⁷ cells per ml in DMEM containing 10% FBS and 25 mM HEPES buffer. For each experiment, equal numbers of cells suspended in 1 ml were pipetted into the chamber of an Oxytherm electrode unit (Hansatech Instruments, Norfolk, United Kingdom), which uses a Clark-type electrode to monitor the dissolved O₂ concentration in the sealed chamber over time. The data were exported to a computerized chart recorder (Oxygraph; Hansatech Instruments) that calculated the OCR. The temperature was maintained at 25 °C during measurement. The O₂ concentration in 1 ml of DMEM medium without cells was also measured over time to provide background values. O₂ consumption experiments were repeated at least thrice. Data were expressed as mean ± standard deviation (SD) (Kai et al., 2012). MIN6 cells were pre-exposed to the volatile anesthetics isoflurane and sevoflurane for 1 h. Cells were harvested and OCR measurement was performed in a working chamber. CCCP was added just before OCR measurement.

Immunoblot assays

Whole-cell lysates were prepared as described previously (Goto et al., 2015; Tanaka et al., 2010). In brief, these were prepared using ice-cold lysis buffer (0.1% SDS, 1% Nonidet P-40 [NP-40], 5 mM EDTA, 150 mM NaCl, 50 mM Tris-Cl [pH 8.0], 2 mM DTT, 1 mM sodium orthovanadate, and Complete Protease Inhibitor™ (Roche Diagnostics, Tokyo, Japan)) using a protocol described previously (Kai et al., 2012). Samples were centrifuged at 10,000 × g to sediment the cell debris, and the supernatant was used for subsequent immunoblotting experiments. For HIF-1α and HIF-1β determinations, 100 µg of protein was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5% gel), transferred to membranes and immunoblotted using primary antibodies at a dilution of 1:1,000. Horseradish peroxidase-conjugated to sheep anti-mouse IgG (GE Healthcare, Piscataway, NJ, USA) was used as a secondary antibody at a dilution of 1:1,000. The signal was developed using enhanced chemiluminescence reagent (GE Healthcare, Little Chalfont, UK). Experiments were repeated at least two times and the representative blots were demonstrated.

Measurement of [ATPi]

MIN6 cells were plated in a 96-well tissue culture plate. After the indicated treatments, [ATPi] was determined using a Cellno ATP Assay Kit (TOYO BNet, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, 100 µl of the lysis/assay solution provided by the manufacturer was added to the cells. After shaking for 1 min and incubating for 10 min at 23 °C, the luminescence of an aliquot of the solution was measured in a luminometer (ExSpire™, Perkin Emler, Waltham, MA, USA) (Koyanagi et al., 2011).

Quantitative reverse transcriptase-PCR analysis

RNA was purified using RNeasy™ (Qiagen, Valencia, CA, USA) and treated with DNase. First-strand synthesis and real-time PCR were performed using the QuantiTect SYBR green PCR kit (Qiagen), according to the manufacturer’s protocol. PCR primers were purchased from Qiagen. PCR and detection were performed using a 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The relative change in expression of each target mRNA relative to 18S rRNA was calculated (Suzuki et al., 2013).

Gene silencing using short interfering RNA (siRNA)

siRNAs corresponding to mouse HIF-1α were from Qiagen Inc. MIN6 cells were transfected by 100 nM siRNA using HiPer-Fect™ Transfection Reagent (Qiagen) following a protocol provided by the manufacturer (Oda et al., 2008).

Statistical analysis

All experiments were repeated on at least two occasions in triplicate. Data were expressed as the mean ± SD and analyzed by one-way analysis of variance, followed by Turkey’s multiple comparisons test. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University), which is a graphical user interface for R (The R Foundation for Statistical Computing, version 3.1.3) (Kanda, 2013). More precisely, it is a modified version of R commander (version 1.6–3) and includes statistical functions that are frequently used in biostatistics. A P-value of <0.05 was considered statistically significant.

Establishment of the GSIS experimental system in MIN6 cells

First, we investigated the insulin secretion response of MIN6 and MIN7 cells to stimulation by extracellular glucose. MIN6 and MIN7 cells were maintained with 40 mg/dl glucose and then exposed to a range of glucose concentrations (40–400 mg/dl) for 1 h. Concentration-dependent GSIS was observed in response to 100–400 mg/dl glucose in MIN6 cells (Fig. 1A). GSIS was not observed in MIN7 cells (Fig. 1A) (Miyazaki et al., 1990). The time profile of the GSIS of MIN6 cells was also examined. The cells were exposed to medium containing 400 mg/dl glucose and the insulin concentrations were measured at 10, 20, 40, 60, and 120 min (Fig. 1B). Insulin secretion reached its maximum point at 40 min. The insulin secretion response to the polysaccharides, sucrose and maltose, was investigated at molar concentrations corresponding to 400 mg/dl glucose (Fig. 1C). Insulin secretion was only observed in the presence of glucose in MIN6 cells.

Reversible inhibition of GSIS by isoflurane and sevoflurane

We examined the effect of isoflurane and sevoflurane on GSIS in MIN6 cells. The cells were incubated with the indicated dose of anesthetic and 400 mg/dl glucose for 1 h prior to determination of the insulin concentrations of the culture supernatants. Isoflurane and sevoflurane inhibited GSIS significantly in a concentration-dependent manner between 0.6% and 2.4% for isoflurane (Fig. 2A), and between 0.8% and 3.6% for sevoflurane (Fig. 2B).

To examine whether this suppression of GSIS was reversible, MIN6 cells were exposed to 1.2% isoflurane or 1.8% sevoflurane with 400 mg/dl glucose for 1 h; they were then incubated under 40 mg/dl glucose without isoflurane or sevoflurane for 6 h, prior to re-exposure to 400 mg/dl glucose. No statistically significant differences were observed in the GSIS of MIN6 cells pretreated with volatile anesthetics and those that were not exposed to these compounds (Fig. 2C).

The effect of volatile anesthetics on molecular aspects of GSIS in MIN6 cells

To investigate the molecular mechanisms underlying volatile anesthetic-mediated GSIS inhibition, cell proliferation and cell death were examined in MIN6 cells. Exposure to these volatile anesthetics (isoflurane: 2.4%, sevoflurane: 3.6%) for 8 h did not induce caspase 3/7 activation in MIN6 cells (Fig. 3A) and 8-h exposure periods did not affect the cell proliferation rate (Fig. 3B). This indicated that exposure to these volatile anesthetics did produce any statistically significant effects on MIN6 cell death or proliferation.

The expression of proteins involved in GSIS was investigated. mRNA expression of the glucose transporter 2 (GLUT2), the Kir6.2 subunit of K_ATP, and the voltage-dependent calcium channel, Cav1.2, was not affected by glucose or by the volatile anesthetics within 1 h (Fig. 3C).

The effect of volatile anesthetics on intracellular signaling processes involved in GSIS

[ATPi] has been reported to increase in response to high-glucose stimulation in pancreatic β-cells (Ashcroft, 2005). We investigated the effect of isoflurane and sevoflurane over intracellular ATP concentration at 40 min and 2 h after exposure to 400 mg/dl glucose. In MIN6 cells, the mitochondrial electric transfer chain inhibitor, rotenone (100 nM), suppressed the glucose-induced increase in [ATPi]. Exposure to 2.4% isoflurane or 3.6% sevoflurane suppressed the increase in [ATPi] observed in response to 400 mg/dl glucose stimulation at 40 min (Fig. 4A) and 2 h (Fig. 4B). But the effects were weaker than 100 nM rotenone, which suppressed GSIS (Fig. 4C).

Elevation of [ATPi] closes K_ATP and depolarizes the plasma membrane (Ashcroft, 2005; Seino, 2012). The channel opener, diazoxide, inhibited insulin secretion in pancreatic β-cells (Seino, 2012), while channel blockade by glibenclamide facilitated insulin secretion, even under low-glucose conditions. In MIN6 cells, glibenclamide induced insulin secretion in the presence of 400 mg/dl glucose, but even with 40 mg/dl glucose; conversely, diazoxide suppressed GSIS observed in the presence of 400 mg/dl glucose (Fig. 4D). Neither 2.4% isoflurane nor 3.6% sevoflurane affected glibenclamide-induced insulin secretion (Fig. 4E).

The effects of volatile anesthetics on the OCR

The elevated mitochondrial respiration observed in response to high-glucose simulation contributes to cellular hypoxia and HIF-1 activation in MIN6 cells (Kurokawa et al., 2015; Sato et al., 2011). We found that exposure to high levels of glucose significantly increased the OCR in MIN6 cells but to a lesser extent than the mitochondrial uncoupler, CCCP (5 µM) (Fig. 5). The mitochondrial electron transfer chain inhibitor, rotenone (100 nM), suppressed the OCR. Importantly, 1.2% isoflurane and 1.8% sevoflurane also suppressed the OCR, although to a lesser extent than rotenone.

The effects of volatile anesthetics on HIF-1 in GSIS

In order to elucidate the time course of HIF-1 activation, HIF-1α protein accumulation was investigated. As early as 1 h after exposure to 400 mg/dl glucose, HIF-1α protein accumulation was observed (Fig. 6A). Both of the tested volatile anesthetics significantly suppressed glucose-induced HIF-1α protein expression in MIN6 cells (Fig. 6B). Exposure to DMOG (100 µM) (Zhou et al., 2007) or nPG (100 µM) (Kimura et al., 2008) increased HIF-1α protein expression, which was not affected by isoflurane (Fig. 6C). The mRNA expression of HIF-1α was not affected by exposure to a high glucose level, volatile anesthetics, DMOG, or n-PG. In contrast, expression of downstream genes such as the glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF) were suppressed by isoflurane treatment. Importantly, this suppression was not observed by pretreatment with DMOG or n-PG (Fig. 6D).

Impact of HIF-1 inhibition on GSIS

To elucidate the involvement of HIF-1 in glucose-elicited insulin secretion, effect of inhibition of HIF-1 activity was investigated by adopting siRNA against hif1α and the HIF-1α transcription inhibitor YC-1. Expression of mRNA of HIF-1α was suppressed by treatment with anti-hif1α siRNA (Fig. 7A) and induction of the expression of HIF-1α protein was also suppressed by anti-hif1α siRNA (Fig. 7B). YC-1 treatment did not affect the expression of mRNA and protein of HIF-1α subunit (Figs. 7A and 7B).

Inhibition of HIF-1 activity by YC-1 treatment and RNA interference-mediated knockdown of HIF-1α expression significantly suppressed GSIS in MIN6 cells (Fig. 7C). To examine if the constitutive activity of HIF-1 could rescue the suppression of GSIS by volatile anesthetics, MIN6 cells were incubated with 100 µM DMOG or nPG for 8 h before treatment with the volatile anesthetics. This inhibited HIF-α prolyl and asparaginyl hydroxylases, which induced HIF-1 activation, even under normoxic conditions. This pre-activation of HIF-1 inhibited the isoflurane-mediated suppression of GSIS and reduction of [ATPi] (Figs. 7D and 7E).