All cell culture reagents, including media, antibiotics, fetal bovine serum (FBS) and phosphate-buffered saline (PBS), were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Mouse monoclonal anti-GLUT-1, rabbit monoclonal anti-GLUT-3, mouse monoclonal anti-HIF-1α and mouse monoclonal anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Cell culture plastics were purchased from Corning Incorporated (Corning, NY, USA). TRIzol reagent, SuperScript III reverse transcriptase and Platinum SYBR-Green qPCR SuperMix were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). A cytotoxicity detection kit was purchased from Roche Diagnostics (Indianapolis, IN, USA) and an Amplex Red Glucose Assay kit was purchased from Life Technologies (Thermo Fisher Scientific, Inc). An ATP assay kit was purchased from Merck Millipore (Darmstadt, Germany). The pyruvate assay kit and the glycogen assay kit were purchased from BioVision, Inc. (Milpitas, CA, USA). The remaining chemicals used were purchased from Sigma-Aldrich (St. Louis, MO, USA).

A total of 22, 1-day-old BALB/c mice, weighing 2±0.3 g were obtained from the Experimental Animal Centre of Zhengzhou University (Zhengzhou, China), housed in a pathogen-free environment, maintained on a 12-h light-dark cycle and fed with commercial pellets. All experiments were approved by the Ethics Committee of Life Sciences, Zhengzhou University (Zhengzhou, China).

The primary astrocyte culture was prepared as described previously (22). Briefly, the neonatal mice were sacrificed by decapitation following anesthesia with 30 mg/kg Zoletil 100 (Virbac Laboratories, Carros, France), cerebral cortices were removed, and tissue was transferred to complete Dulbecco's modified Eagle's medium (DMEM; supplemented with 10% FBS, 25 mM glucose, 50 U/ml penicillin and 50 mg/ml streptomycin-sulfate) and dissociated with 0.0025% trypsin/ethylenediaminetetraacetic acid. Cells were seeded at a density of 3×10⁴ cells/cm² in complete DMEM at 37°C in a humidified atmosphere with 5% CO₂. The medium was renewed on day in vitro (DIV) 1, DIV 5 and DIV 7. On DIV 9, microglia were discarded using the shake-off method (23). Astrocytes were harvested with trypsin and reseeded at a density of 3×10⁴ cells/cm² in complete DMEM/F12 medium. The homogeneity of astrocytes was 90–95% by detection of glial fibrillary acidic protein with 5–10% of cells being B4 isolectin⁺ microglia. Astrocytes were reseeded at a density of 5×10⁴ cells/cm² and incubated at 37°C with 5% CO₂. Experiments were performed on cells 10–18 days after plating at which point they formed an incomplete monolayer of stellate- and flat-shaped astrocytes. cells were exposed to 100 µM CoCl₂ and tested at distinct time points.

To examine GLUT-1 and GLUT-3 gene expression induced by CoCl₂ treatment according to the time courses, astrocytes were harvested following incubation with 100 µM CoCl₂ at different time points (0, 2, 4, 6, 8, 10, 12, 18 and 24 h). Total RNA was extracted by using TRIzol reagent, according to the manufacturer's protocol, and subjected to DNase treatment using a FastQuant RT kit (Tiangen Biotech Co., Ltd., Beijing, China). RNA quantity and quality was determined spectrophotometrically at 260 and 280 nm using an EU-2200R Ultraviolet spectrophotometer (Shanghai Onlab Instruments Co., Ltd., Shanghai, China). Reverse transcription of 20 ng RNA was performed using a SuperScript III reverse transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.) in preparation for qPCR.

GLUT-1, GLUT-3, HIF-1α and β-actin expression levels were evaluated by qPCR using Platinum SYBR-Green qPCR SuperMix. The running conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. qPCR was performed using the ABI 7500 sequence detector (Applied Biosystems; Thermo Fisher Scientific, Inc.). The design of the specific primers is presented in Table I.

The quantities of gene-specific mRNA expression were determined by the quantification cycle (Cq) method and the Cq value for β-actin was used as an internal calibrator. The comparative Cq method, or 2^−ΔΔCq, was used for relative quantization (25) using the following equation: ^ΔΔCq = (Cq_target − Cq_calibrator)_experimental − (Ct_target − Cq_calibrator)_control. Data are presented as ratios over control (time point, 0 h).

The astrocytes were exposed to 100 µM CoCl₂ for the aforementioned periods of time, then the cells were collected and solubilized in lysis buffer (60 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM KCl, 5 mM Na₃ EDTA, 3 mM EGTA, and 1% Triton X-100) with protease inhibitors. Following centrifugation at 14,000 × g for 15 min at 4°C, the supernatant was collected and solubilized in loading buffer. Samples were normalized to protein concentration and proteins were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a running voltage of 200 v for 30 min and electrophoretically transferred to polyvinylidene difluoride membranes at 100 V for 1 h. The membranes were blocked with 5.0% fat-free milk in PBS with 0.05% Tween-20 (PBST) for 1 h at room temperature, and incubated separately with mouse monoclonal anti-GLUT-1 (cat. no. sc-377228; dilution 1:1,000), rabbit GLUT-3 (cat. no. sc-74399; dilution 1:1,000), mouse monoclonal anti-HIF-1α (cat. no. sc-71247; dilution 1:1,000) or mouse β-actin (cat. no. sc-47778; dilution 1:2,000; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Subsequently, the membranes were washed with PBST three times for 10 min each. Following incubation with Alexa Fluor^® 488 donkey anti-rabbit IgG (cat. no. R37118) and Alexa Fluor^® 680 rabbit anti-mouse IgG (cat. no. A-21065; Thermo Fisher Scientific, Inc.; dilution 1:10,000) for 60 min at room temperature in the dark, blots were detected on an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and relative density units were estimated from the mean pixel density using Image Studio Lite version 4.0 (LI-COR Biosciences) and normalized to β-actin. Data are presented as ratios over control (time point: 0 h).

Astrocytes were seeded at a density of 5×10⁴ cells/cm² in a 12 well plate and exposed to 100 µM CoCl₂ for the aforementioned time periods when cells were confluent. In the final 2 h of each time course, astrocytes were incubated in glucose-free medium (25 mM mannose) for 1 h, and then in 25 mM glucose medium for 1 h. During the incubation in medium containing 25 mM mannose and 25 mM glucose, astrocytes of the CoCl₂ treatment group were still treated with 100 µM CoCl₂. Subsequently, cells were rapidly chilled on ice and washed four times with ice-cold PBS, then they were collected and lysed in 500 µl of ice-cold lysis buffer (10 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1 mM benzamidine, 0.5% Triton X-100) with protease inhibitors (Sigma-Aldrich). The intracellular glucose concentration in cell lysates was determined using the Amplex Red Glucose Assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Subsequently, 50 µl cell lysate was mixed with 50 µl reaction mixture and incubated for 30 min at room temperature in the dark. The absorbance was then measured at 560 nm using an SP-Max 2300A2 microplate reader (Shanghai Flash Spectrum Biological Technology Co., Ltd., Shanghai, China). Intracellular glucose concentration was determined from a standard curve generated using the various concentrations normalized to the protein concentration. Data are presented as the percentage of glucose concentration compared with control (time point of CoCl₂ treatment, 0 h), which is expressed as 100%.

Total glycogen concentration was determined using a colorimetric glycogen assay kit (BioVision, Inc.), according to the manufacturer's protocol. The astrocytes were homogenized with dH₂O on ice and the homogenates were boiled for 5 min to inactivate the enzymes present. The boiled samples were spun at 15,000 × g at 4°C for 5 min to remove insoluble material, then the supernatant was collected for assaying optical density, which was measured at 570 nm using a microplate reader (SP-Max 2300A2; Shanghai Flash Spectrum Biological Technology Co., Ltd.). Glycogen concentration was determined from a standard curve generated using various concentrations of glycogen and normalized to protein concentration. Data are presented as the percentage glycogen concentration compared with control (time point, 0 h), which is expressed as 100%.

Pyruvate was extracted from the astrocytes using 4 volumes of the pyruvate assay buffer. The cells were centrifuged (10,000 × g; 10 min; 4°C) to remove insoluble material and collect the supernatant. A total of 2–50 µl supernatant was added into a 96-well plate and the volume adjusted to 50 µl/well with pyruvate assay buffer. Next, the 50 µl reaction mixture (containing 46 µl pyruvate assay buffer, 2 µl pyruvate probe and 2 µl enzyme mix) was added. Following incubation for 30 min at room temperature in the dark, intracellular pyruvate was measured by colorimetric assay using a pyruvate assay kit (BioVision, Inc.). Optical density was measured at 570 nm using a microplate reader (SP-Max 2300A2; Shanghai Flash Spectrum Biological Technology Co., Ltd.). Intracellular pyruvate was determined from a standard curve generated using various concentrations of pyruvate and normalized to protein concentration. Data are presented as the percentage pyruvate concentration compared with the control (time point of CoCl₂ treatment=0 h), which is expressed as 100%.

Astrocytes were seeded at a density of 5×10⁴ cells/cm² in a 12-well plate and were exposed to 100 µM CoCl₂ for testing at distinct time points. Cells of the control group were generated at the same time points without CoCl₂ treatment. Following the aforementioned cell preparations, intracellular ATP content was determined by bioluminescence assay using a commercial ATP assay kit (Merck Millipore), according to the manufacturer's protocol. The astrocytes were treated with 100 µl nuclear releasing reagent for 5 min at room temperature while gently shaking, then 1 µl ATP monitoring enzyme was added to the cell lysate. Bioluminescence of intracellular ATP content was determined from a standard curve generated using various concentrations of ATP and normalized to protein concentration.

Astrocyte cell viability was demonstrated at distinct time points following CoCl₂ treatment using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, according to the manufacturer's protocol. Astrocytes of the control group were cultured without CoCl₂ treatment and generated at the same time points. MTT solution (5.0 mg/ml) was added to the cells and incubated at 37°C for 4 h. The culture medium was aspirated, and 1 ml dimethyl sulfoxide was added and thoroughly mixed for 10 min. MTT absorbance was measured at 570 and 630 nm using a microplate reader (SP-Max 2300A2; Shanghai Flash Spectrum Biological Technology Co., Ltd.).

To measure lactate dehydrogenase (LDH) release in the astrocyte supernatant, the supernatant was collected by centrifugation at 250 × g at 4°C for 10 min and transferred into a 96-well plate following exposure to CoCl₂ treatment for the indicated time points. Astrocytes of the control group were cultured without CoCl₂ treatment and the supernatant was generated at the same time points. LDH was determined using a cytotoxicity detection kit (Roche Diagnostics) according to the manufacturer's protocol, with Triton X-100 (2.0%) serving as a positive control. Absorbance was measured using a microplate reader (SP-Max 2300A2; Shanghai Flash Spectrum Biological Technology Co., Ltd.) at a test wavelength of 490 nm and a reference wavelength of 630 nm.

Statistical analyses was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Data from multiple experiments were processed and are expressed as the mean ± standard deviation. Statistical differences between the different time points were determined using one-way analysis of variance followed by a Student-Newman-Keuls test. Statistical analyses of intracellular ATP content, cell viability and LDH release at all time points between two groups were performed using the independent-samples t-test. P<0.05 was considered to indicate a statistically significant difference.

The time course analysis using RT-qPCR indicated that GLUT-1 and GLUT-3 mRNA expression levels in astrocytes changed in a time-dependent manner following CoCl₂ administration. Expression levels of GLUT-1 and GLUT-3 mRNA increased immediately and significantly in each time interval during the first 12 h time period. Throughout the rest of the recording period, the expression levels for GLUT-1 and GLUT-3 mRNAs remained at ~4.5- and 3.5-fold higher than that of the control (P<0.05 vs. control; Fig. 1A and B).

HIF-1α mRNA expression in astrocytes was also analyzed as an indication of hypoxic stress. HIF-1α mRNA expression exhibited time-dependent alterations similar to those observed with GLUT-1 and GLUT-3. Following exposure to CoCl₂ treatment, the astrocytes exhibited a ~2.9-fold increase in HIF-1α mRNA expression during the first 12 h time course, and the increase remained constant throughout the remaining recording period (Fig. 1C).

Protein expression levels of GLUT-1, GLUT-3 and HIF-1α were determined by western blot analysis (Fig. 2A). Quantitative analysis demonstrated that treatment withCoCl₂ in the first 2 h resulted in no significant differences in GLUT-1 and GLUT-3 protein expression levels in astrocytes (P>0.05 vs. control; Fig. 2B and C); however, a significant increase in the protein expression of GLUT-1 and GLUT-3 occurred from the 4 h time point throughout the experiments (P<0.05 vs. control; Fig. 2B and C). In particular, 6.3- and 3.2-fold increases for GLUT-1 and GLUT-3, respectively, were observed from the 12 h time point throughout the remaining recording period (Fig. 2B and C).

Additionally, to investigate whether CoCl₂ treatment induced GLUT-1 and GLUT-3 activity, and whether HIF-1α behaved in an independent manner, protein expression levels for HIF-1α were determined. As a reflection of hypoxic stress, HIF-1α protein expression behaved similarly to that of GLUT-1 and GLUT-3. There was no statistically significant alteration within the initial 4 h, however a significant increase was identified at the 6 h time point. The increase reached ~4.0-fold at the 12 h time point and remained at that level throughout remaining recording period (P<0.05 vs. control; Fig. 2D).

To determine the effects of CoCl₂ treatment on intracellular glucose concentrations and glycogen storage at different time points, intracellular glucose and glycogen concentrations were determined. When astrocytes were exposed to CoCl₂ treatment, intracellular glucose concentration immediately fell by >25% between 0 and 2 h (P<0.05). Although an increase occurred between the 4–8 h time points, intracellular glucose and glycogen concentrations were significantly reduced compared with the control by the 24 h time point (P<0.05; Fig. 3A and B).

Intracellular pyruvate in astrocytes (Fig. 4A) immediately increased by ~1.2-fold within the first 2 h (P<0.05 vs. control), but remained steady at a level of ~1.6-fold increase relative to the control between 4 and 8 h (P<0.05 vs. control). Subsequently, the intracellular pyruvate concentration further increased from the 12 h time point to ~2.5-fold at the 24 h time point (P<0.05; Fig. 4A).

By contrast, there were no statistically significant differences between the two groups in terms of intracellular ATP content in the first 8 h subsequent to exposure of astrocytes to 100 µM of CoCl₂ (P>0.05; Fig. 4B); however, intracellular ATP content significantly reduced in the hypoxia group each subsequent time interval (Fig. 4B).

To further investigate the effects of CoCl₂ treatment on cell viability and cytotoxicity in astrocytes, cell viability and LDH release activity were determined. Compared with the control group, cell viability and LDH release activity in the hypoxia group remained at the baseline level for the first 8 h of CoCl₂ treatment (P>0.05 vs. control; Fig. 5A and B); however, cell viability reduced significantly following exposure to 100 µM CoCl₂ for >12 h, while LDH release activity continued to increase in each subsequent time interval.