Ozone Triggers Insulin Resistance via Muscle JNK Activation
Ozone Triggers Insulin Resistance via Muscle JNK Activation
All chemicals and culture media were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) when no other origin is specified. Recombinant human insulin (100 IU/mL; Actrapid) was from Novo Nordisk (La Défense, France). Anti-phospho-Akt 1/2/3 rabbit IgG (catalog #7985R) and anti-Akt 1/2/3 rabbit IgG (catalog #8312) antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), anti-tubulin mouse IgG antibody was from Sigma-Aldrich, and anti-mouse IgG and anti-rabbit IgG antibodies were from Bio-Rad (Marnes-la-Coquette, France). Endoplasmic reticulum (ER) stress antibodies were all included in the ER Stress Antibody Sampler Kit from Cell Signaling Technology (Saint-Aubin, France). Super Signal West Pico Chemiluminescent Substrate and Restore Western Blot Stripping Buffer were obtained from Thermo Scientific (distributed by Perbio Sciences France, Brebières, France).
Animal experiments were performed under authorization 69–266–0501 (INSA-Lyon, Direction Départementale de la Protection des Populations-Services Vétérinaires du Rhône), according to the ethical guidelines laid down by the French Ministère de l'Agriculture (87–848) and the European Union Council Directive for the Care and Use of Laboratory Animals of November 24th, 1986 (86/609/EEC). Authors A.G. (license 69266332) and C.O.S. (license 69266257) hold special licenses to experiment on living vertebrates that were issued by the French Ministry of Agriculture and Veterinary Service Department. Wistar rats weighing 400–450 g (Janvier SA, Le Genest-Saint-Isle, France) were housed in an air-conditioned room at 24 ± 1°C with a 12-h light/dark cycle (light on at 6:30 a.m.) with free access to food (13.4 kJ/g, 65% carbohydrates, 11% fat, 24% proteins, weight for weight; diet A03; SAFE, Augy, France) and water. Great care was taken to minimize animal discomfort and suffering during the experimental protocol.
Rats were kept within a Plexiglas hermetic environmental chamber (width 0.35 m, length 0.70 m, height 0.40 m, surface 0.25 m, volume 0.1 m) supplied with a constant airflow (6 m/h) and subjected to 1,570 μg/m O3, corresponding to 0.8 parts per million (ppm) for 16 h, as previously described. O3 was generated by passing filtered air across a UV light, and concentration inside the cage was controlled by adjusting the inlet flow of air. The O3 concentration was continuously measured using a calibrated UV photometric O3 monitor (range ± 0.001 ppm; catalog #41M; Environement SA, Poissy, France) connected to the outlet line of the chamber. Control exposure was performed in a similar chamber provided with filtered room air at the same flow rate. All the exposures were performed during the dark phase (i.e., the active phase for the rats). Rats were fasted during the exposure but had free access to water. The temperature and relative humidity measured in the chambers during the exposure were 25.3 ± 0.4°C and 75%, respectively. Ammonia and CO2 concentrations remained undetectable. The mean O3 concentration inside the exposure chamber was 0.800 ± 0.05 ppm. Of note, the O3 concentration in the control chamber remained undetectable (<0.001 ppm). Rats were exposed during nighttime, namely, during their active phase, because O3 pollution peaks generally take place during the day (i.e., during human active phase).
Pretreatment With N-Acetylcysteine or 4-Phenylbutyric Acid. Some rats were pretreated for 10 days with N-acetylcysteine (NAC) prior to O3 exposure. Rats were given NAC orally in drinking water (10 mmol/L). The water intake was monitored daily to calculate the daily NAC intake. The mean daily intake of NAC was 225 ± 10 mg/kg/day. Another independent set of rats was gavaged daily with 4-phenylbutyric acid (PBA; 150 mg/kg) for 4 days prior to O3 exposure.
Euglycemic-hyperinsulinemic (EH) clamps were performed essentially as previously described. In brief, Wistar rats were implanted with indwelling catheters in the left carotid artery and right jugular vein. The rats were allowed to recover from the surgery for 5 days. Prior to the EH clamp, the rats were fasted overnight and a standard 2-h EH clamp was conducted using a primed and continuous infusion of human insulin at a rate of 6 mU/kg/min coupled with a variable infusion of 25% (weight for volume) glucose to maintain blood glucose concentrations at ~6 mmol/L. The glucose infusion rate (GIR) was calculated as the amount of glucose perfused to maintain euglycemia during the second hour of the clamp and was expressed as milligrams of glucose per kilogram per minute.
After an overnight fast, Wistar rats with indwelling catheters were injected intravenously with 1 g/kg l-arginine in saline solution. Blood was withdrawn from an arterial catheter and centrifuged (1 min, 3,500g), and plasma samples were snap frozen in liquid nitrogen and stored at −20°C until insulin assay.
After overnight fast, animals were injected intraperitoneally with 0.50 IU/kg body wt recombinant human insulin. Blood glucose was measured before, and 15, 30, 60, 90, and 120 min after insulin injection. Blood glucose values were determined from a drop of blood sampled from the terminal portion of the tail using a glucometer. As previously described, the glucose disappearance rate for an insulin tolerance test (ITT) (KITT; reported as the percentage per minute) was calculated as follows: KITT = 0.693 × 100/t1/2, where t1/2 was the half-life calculated from the slope of the plasma glucose concentration, considering an exponential decrement of glucose concentration during the 30 min after insulin administration.
Rats were anesthetized with sodium pentobarbital (35 mg/kg), and a PE-240 tracheal catheter was inserted into the trachea. Bronchoalveolar lavages were performed twice with 1 mL of sterilized Dulbecco's PBS. An aliquot fraction of bronchoalveolar lavage fluid (BALF) was removed for cellular numeration and leukocyte count while the BALF was centrifuged (8,000g, 2 min, 4°C) to pellet cells. Supernatants were removed and kept at −80°C. Total proteins in the BALF were determined by the Bradford protein assay. Lipid peroxidation was measured spectrophotometrically by the thiobarbituric acid method using 1.1.3.3-tetramethoxipropane as the standard. Cells were stained by the May-Grünwald Giemsa method for leukocyte counts, and 500 leukocytes were randomly counted in a Malassez cell under an optical microscope (original magnification ×100).
To study insulin signaling in skeletal muscle, liver, or adipose tissue, rats were injected with insulin (Actrapid 0.75 IU/kg i.p.) or saline solution at the end of O3 exposure. Thirty minutes after insulin injection, rats were deeply anesthetized with sodium pentobarbital (60 mg/kg i.p.). Lungs, gastrocnemius or tibialis muscles, retroperitoneal white adipose tissue, and liver were rapidly dissected out, snap frozen in liquid nitrogen, and stored at −80°C.
Plasma total cholesterol, triacylglycerol, and nonesterified fatty acid (NEFA) levels were measured with commercial kits Cholesterol RTU (bioMérieux, Marcy-l'Etoile, France), Triglyceride PAP (bioMérieux), and NEFA-C (WAKO). Levels of plasma insulin (SPIBio, Montigny-le-Bretonneux, France), corticosterone (Cayman Chemicals), tumor necrosis factor-α, and interleukin-1β (eBioscience, Paris, France) were determined with enzyme immunoassays according to manufacturer's recommendations. Plasma aldehydes (i.e., 4-hydroxy-2-nonenal [HNE] and 4-hydroxy-2-hexenal [HHE]) were measured using gas chromatography–mass spectrometry as previously described. Plasma malondialdehyde (MDA), reduced glutathione (GSH), and oxidized glutathione (GSSG) levels were measured by high-performance liquid chromatography coupled to fluorescence detection. Carbonyl groups on proteins were determined using 2.4-dinitrophenylhydrazine as described previously.
Total RNAs from gastrocnemius muscle samples (80–100 mg) were extracted using TRI Reagent (Sigma-Aldrich). Total RNA quantities and qualities were assessed using the Model 2100 Bioanalyzer and RNA 6000 LabChip Kit (Agilent Technologies, Massy, France). Reverse Transcriptions (RNAse H, Takara enzyme) were performed on 1 μg of total RNA. Real-time PCR assays for ER stress genes (Grp78/Bip, Atf4, Atf6, Ire1-α, Perk, total and unspliced Xbp1, and Eif2) were performed using a Rotor-GeneTM 6000 (QIAGEN). Values were normalized to TBP gene expression. The full list of genes and corresponding primer sequences is shown in Supplementary Table 1 http://diabetes.diabetesjournals.org/content/64/3/1011/suppl/DC1.
C2C12 myoblasts (Mus musculus) (catalog #CRL-1772; ATCC, LGC Standards, Molsheim, France) were grown and differentiated to myotubes as described previously. C2C12 myoblasts were cultured in DMEM supplemented with 10% heat-inactivated FBS (South America Origin; Biowest), 2 mmol/L l-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. When reaching 90% confluence, differentiation was induced by shifting to DMEM supplemented with 2% heat-inactivated horse serum (South America Origin; Biowest). Experiments were performed when >80% of cells had formed myotubes. Cells were serum starved for 2 h prior to the experiments. Cells were incubated for 30 min with 10% (volume for volume [v/v]) BALF diluted in cell culture media (either from O3 or from control rats). The cell culture medium containing 10% (v/v) BALF was discarded, and cells were further incubated in a fresh medium containing 100 nmol/L insulin for 20 min. Then, cell culture medium was discarded and cells were frozen. LBA cytotoxicity was determined measuring cell viability with an MTT assay (Roche).
Pretreatment for 1 h with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 μmol/L) or PBA (10 mmol/L) was performed prior to BALF incubation. An UltraLinkHydrazide Resin (Thermo Scientific, Illkirch, France) was used as an affinity support for immobilizing reactive lipid aldehydes present in BALF. C2C12 myotubes were incubated with 10% (v/v) of aldehyde-depleted BALF, as described above.
C2C12 myotubes were incubated for 20 min with 100 nmol/L insulin. Glucose uptake was measured using 2-deoxy-d-[H]-glucose (747 GBq/mmol; PerkinElmer, Courtaboeuf, France), as described previously.
Muscle tissue and C2C12 cells were lysed on ice in Standard Lysis Buffer (20 mmol/L Tris, 138 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 5% glycerol, and 1% NP40, supplemented extemporaneously with 5 mmol/L EDTA, 1 mmol/L Na3VO4, 20 mmol/L NaF, 1 mmol/L dithiothreitol, and a protease inhibitor cocktail) and centrifuged (13,000g, 15 min, 4°C). Protein concentration was determined with a Bradford protein assay. Sixty micrograms of protein lysate from skeletal muscle or 30 μg protein lysate from C2C12 cells were separated on SDS-polyacrylamide gels (10%) after heat denaturation (95°C, 5 min) and then transferred onto a nitrocellulose membrane (Hybond-ECL; GE Healthcare, Meylan, France). After transfer, the membranes were blocked with 5% BSA in Tris-buffered saline with Tween for 2 h. Blots were probed overnight with specific primary antibodies at 4°C followed by 1-h incubation at room temperature with secondary antibodies. Protein bands were detected with an enhanced chemiluminescence substrate kit (SuperSignal West Pico; Perbio Sciences France) using an Image Master VDS-CL camera (Amersham Pharmacia, Orsay, France) and quantified by Quantity One (Bio-Rad) or open-source ImageJ (http://rsbweb.nih.gov/ij/index.html) software. Protein phosphorylation levels were normalized to the matching densitometric values of total proteins. Results were expressed as arbitrary units (a.u.).
Data are expressed as the mean ± SEM. All data were analyzed using GraphPad Prism version 5.0 software (GraphPad Software, La Jolla, CA). Multiple comparisons were performed using ANOVA followed, when appropriate, by post hoc Fisher protected least significant differences tests. The results of ITTs, arginine tests, and clamps were compared by two-way ANOVA (time and treatment). Simple comparisons were performed using the Student t test. When appropriate, Welch correction for inequality of variances was applied. Differences were considered significant at the P < 0.05 level.
Research Design and Methods
Chemicals, Reagents, and Antibodies
All chemicals and culture media were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) when no other origin is specified. Recombinant human insulin (100 IU/mL; Actrapid) was from Novo Nordisk (La Défense, France). Anti-phospho-Akt 1/2/3 rabbit IgG (catalog #7985R) and anti-Akt 1/2/3 rabbit IgG (catalog #8312) antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), anti-tubulin mouse IgG antibody was from Sigma-Aldrich, and anti-mouse IgG and anti-rabbit IgG antibodies were from Bio-Rad (Marnes-la-Coquette, France). Endoplasmic reticulum (ER) stress antibodies were all included in the ER Stress Antibody Sampler Kit from Cell Signaling Technology (Saint-Aubin, France). Super Signal West Pico Chemiluminescent Substrate and Restore Western Blot Stripping Buffer were obtained from Thermo Scientific (distributed by Perbio Sciences France, Brebières, France).
Animal Care
Animal experiments were performed under authorization 69–266–0501 (INSA-Lyon, Direction Départementale de la Protection des Populations-Services Vétérinaires du Rhône), according to the ethical guidelines laid down by the French Ministère de l'Agriculture (87–848) and the European Union Council Directive for the Care and Use of Laboratory Animals of November 24th, 1986 (86/609/EEC). Authors A.G. (license 69266332) and C.O.S. (license 69266257) hold special licenses to experiment on living vertebrates that were issued by the French Ministry of Agriculture and Veterinary Service Department. Wistar rats weighing 400–450 g (Janvier SA, Le Genest-Saint-Isle, France) were housed in an air-conditioned room at 24 ± 1°C with a 12-h light/dark cycle (light on at 6:30 a.m.) with free access to food (13.4 kJ/g, 65% carbohydrates, 11% fat, 24% proteins, weight for weight; diet A03; SAFE, Augy, France) and water. Great care was taken to minimize animal discomfort and suffering during the experimental protocol.
O3 Exposure
Rats were kept within a Plexiglas hermetic environmental chamber (width 0.35 m, length 0.70 m, height 0.40 m, surface 0.25 m, volume 0.1 m) supplied with a constant airflow (6 m/h) and subjected to 1,570 μg/m O3, corresponding to 0.8 parts per million (ppm) for 16 h, as previously described. O3 was generated by passing filtered air across a UV light, and concentration inside the cage was controlled by adjusting the inlet flow of air. The O3 concentration was continuously measured using a calibrated UV photometric O3 monitor (range ± 0.001 ppm; catalog #41M; Environement SA, Poissy, France) connected to the outlet line of the chamber. Control exposure was performed in a similar chamber provided with filtered room air at the same flow rate. All the exposures were performed during the dark phase (i.e., the active phase for the rats). Rats were fasted during the exposure but had free access to water. The temperature and relative humidity measured in the chambers during the exposure were 25.3 ± 0.4°C and 75%, respectively. Ammonia and CO2 concentrations remained undetectable. The mean O3 concentration inside the exposure chamber was 0.800 ± 0.05 ppm. Of note, the O3 concentration in the control chamber remained undetectable (<0.001 ppm). Rats were exposed during nighttime, namely, during their active phase, because O3 pollution peaks generally take place during the day (i.e., during human active phase).
Pretreatment With N-Acetylcysteine or 4-Phenylbutyric Acid. Some rats were pretreated for 10 days with N-acetylcysteine (NAC) prior to O3 exposure. Rats were given NAC orally in drinking water (10 mmol/L). The water intake was monitored daily to calculate the daily NAC intake. The mean daily intake of NAC was 225 ± 10 mg/kg/day. Another independent set of rats was gavaged daily with 4-phenylbutyric acid (PBA; 150 mg/kg) for 4 days prior to O3 exposure.
Euglycemic-hyperinsulinemic Clamps
Euglycemic-hyperinsulinemic (EH) clamps were performed essentially as previously described. In brief, Wistar rats were implanted with indwelling catheters in the left carotid artery and right jugular vein. The rats were allowed to recover from the surgery for 5 days. Prior to the EH clamp, the rats were fasted overnight and a standard 2-h EH clamp was conducted using a primed and continuous infusion of human insulin at a rate of 6 mU/kg/min coupled with a variable infusion of 25% (weight for volume) glucose to maintain blood glucose concentrations at ~6 mmol/L. The glucose infusion rate (GIR) was calculated as the amount of glucose perfused to maintain euglycemia during the second hour of the clamp and was expressed as milligrams of glucose per kilogram per minute.
Insulin Secretion Test With l-Arginine
After an overnight fast, Wistar rats with indwelling catheters were injected intravenously with 1 g/kg l-arginine in saline solution. Blood was withdrawn from an arterial catheter and centrifuged (1 min, 3,500g), and plasma samples were snap frozen in liquid nitrogen and stored at −20°C until insulin assay.
Insulin Tolerance Test
After overnight fast, animals were injected intraperitoneally with 0.50 IU/kg body wt recombinant human insulin. Blood glucose was measured before, and 15, 30, 60, 90, and 120 min after insulin injection. Blood glucose values were determined from a drop of blood sampled from the terminal portion of the tail using a glucometer. As previously described, the glucose disappearance rate for an insulin tolerance test (ITT) (KITT; reported as the percentage per minute) was calculated as follows: KITT = 0.693 × 100/t1/2, where t1/2 was the half-life calculated from the slope of the plasma glucose concentration, considering an exponential decrement of glucose concentration during the 30 min after insulin administration.
Bronchoalveolar Lavage Fluid
Rats were anesthetized with sodium pentobarbital (35 mg/kg), and a PE-240 tracheal catheter was inserted into the trachea. Bronchoalveolar lavages were performed twice with 1 mL of sterilized Dulbecco's PBS. An aliquot fraction of bronchoalveolar lavage fluid (BALF) was removed for cellular numeration and leukocyte count while the BALF was centrifuged (8,000g, 2 min, 4°C) to pellet cells. Supernatants were removed and kept at −80°C. Total proteins in the BALF were determined by the Bradford protein assay. Lipid peroxidation was measured spectrophotometrically by the thiobarbituric acid method using 1.1.3.3-tetramethoxipropane as the standard. Cells were stained by the May-Grünwald Giemsa method for leukocyte counts, and 500 leukocytes were randomly counted in a Malassez cell under an optical microscope (original magnification ×100).
Sacrifice and Tissue Dissection
To study insulin signaling in skeletal muscle, liver, or adipose tissue, rats were injected with insulin (Actrapid 0.75 IU/kg i.p.) or saline solution at the end of O3 exposure. Thirty minutes after insulin injection, rats were deeply anesthetized with sodium pentobarbital (60 mg/kg i.p.). Lungs, gastrocnemius or tibialis muscles, retroperitoneal white adipose tissue, and liver were rapidly dissected out, snap frozen in liquid nitrogen, and stored at −80°C.
Biochemical Measurements
Plasma total cholesterol, triacylglycerol, and nonesterified fatty acid (NEFA) levels were measured with commercial kits Cholesterol RTU (bioMérieux, Marcy-l'Etoile, France), Triglyceride PAP (bioMérieux), and NEFA-C (WAKO). Levels of plasma insulin (SPIBio, Montigny-le-Bretonneux, France), corticosterone (Cayman Chemicals), tumor necrosis factor-α, and interleukin-1β (eBioscience, Paris, France) were determined with enzyme immunoassays according to manufacturer's recommendations. Plasma aldehydes (i.e., 4-hydroxy-2-nonenal [HNE] and 4-hydroxy-2-hexenal [HHE]) were measured using gas chromatography–mass spectrometry as previously described. Plasma malondialdehyde (MDA), reduced glutathione (GSH), and oxidized glutathione (GSSG) levels were measured by high-performance liquid chromatography coupled to fluorescence detection. Carbonyl groups on proteins were determined using 2.4-dinitrophenylhydrazine as described previously.
Analysis of Gene Expression in Muscle by Quantitative PCR
Total RNAs from gastrocnemius muscle samples (80–100 mg) were extracted using TRI Reagent (Sigma-Aldrich). Total RNA quantities and qualities were assessed using the Model 2100 Bioanalyzer and RNA 6000 LabChip Kit (Agilent Technologies, Massy, France). Reverse Transcriptions (RNAse H, Takara enzyme) were performed on 1 μg of total RNA. Real-time PCR assays for ER stress genes (Grp78/Bip, Atf4, Atf6, Ire1-α, Perk, total and unspliced Xbp1, and Eif2) were performed using a Rotor-GeneTM 6000 (QIAGEN). Values were normalized to TBP gene expression. The full list of genes and corresponding primer sequences is shown in Supplementary Table 1 http://diabetes.diabetesjournals.org/content/64/3/1011/suppl/DC1.
Cell Culture
C2C12 myoblasts (Mus musculus) (catalog #CRL-1772; ATCC, LGC Standards, Molsheim, France) were grown and differentiated to myotubes as described previously. C2C12 myoblasts were cultured in DMEM supplemented with 10% heat-inactivated FBS (South America Origin; Biowest), 2 mmol/L l-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. When reaching 90% confluence, differentiation was induced by shifting to DMEM supplemented with 2% heat-inactivated horse serum (South America Origin; Biowest). Experiments were performed when >80% of cells had formed myotubes. Cells were serum starved for 2 h prior to the experiments. Cells were incubated for 30 min with 10% (volume for volume [v/v]) BALF diluted in cell culture media (either from O3 or from control rats). The cell culture medium containing 10% (v/v) BALF was discarded, and cells were further incubated in a fresh medium containing 100 nmol/L insulin for 20 min. Then, cell culture medium was discarded and cells were frozen. LBA cytotoxicity was determined measuring cell viability with an MTT assay (Roche).
Pretreatment for 1 h with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 μmol/L) or PBA (10 mmol/L) was performed prior to BALF incubation. An UltraLinkHydrazide Resin (Thermo Scientific, Illkirch, France) was used as an affinity support for immobilizing reactive lipid aldehydes present in BALF. C2C12 myotubes were incubated with 10% (v/v) of aldehyde-depleted BALF, as described above.
2-Deoxyglucose Uptake
C2C12 myotubes were incubated for 20 min with 100 nmol/L insulin. Glucose uptake was measured using 2-deoxy-d-[H]-glucose (747 GBq/mmol; PerkinElmer, Courtaboeuf, France), as described previously.
Protein Isolation, Immunoprecipitation, and Immunoblotting
Muscle tissue and C2C12 cells were lysed on ice in Standard Lysis Buffer (20 mmol/L Tris, 138 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 5% glycerol, and 1% NP40, supplemented extemporaneously with 5 mmol/L EDTA, 1 mmol/L Na3VO4, 20 mmol/L NaF, 1 mmol/L dithiothreitol, and a protease inhibitor cocktail) and centrifuged (13,000g, 15 min, 4°C). Protein concentration was determined with a Bradford protein assay. Sixty micrograms of protein lysate from skeletal muscle or 30 μg protein lysate from C2C12 cells were separated on SDS-polyacrylamide gels (10%) after heat denaturation (95°C, 5 min) and then transferred onto a nitrocellulose membrane (Hybond-ECL; GE Healthcare, Meylan, France). After transfer, the membranes were blocked with 5% BSA in Tris-buffered saline with Tween for 2 h. Blots were probed overnight with specific primary antibodies at 4°C followed by 1-h incubation at room temperature with secondary antibodies. Protein bands were detected with an enhanced chemiluminescence substrate kit (SuperSignal West Pico; Perbio Sciences France) using an Image Master VDS-CL camera (Amersham Pharmacia, Orsay, France) and quantified by Quantity One (Bio-Rad) or open-source ImageJ (http://rsbweb.nih.gov/ij/index.html) software. Protein phosphorylation levels were normalized to the matching densitometric values of total proteins. Results were expressed as arbitrary units (a.u.).
Data Analysis
Data are expressed as the mean ± SEM. All data were analyzed using GraphPad Prism version 5.0 software (GraphPad Software, La Jolla, CA). Multiple comparisons were performed using ANOVA followed, when appropriate, by post hoc Fisher protected least significant differences tests. The results of ITTs, arginine tests, and clamps were compared by two-way ANOVA (time and treatment). Simple comparisons were performed using the Student t test. When appropriate, Welch correction for inequality of variances was applied. Differences were considered significant at the P < 0.05 level.
