Just Accepted by Free Radical Research
Fructose-induced ROS generation impairs glucose utilization in L6 skeletal muscle cells Natasha Jaiswal, Chandan K. Maurya, Jyotsana Pandey, Amit K. Rai, Akhilesh K. Tamrakar Doi: 10.3109/10715762.2015.1031662
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
Abstract High fructose consumption has implicated in insulin resistance and metabolic syndrome. Fructose is a highly lipogenic sugar that has intense metabolic effects in liver. Recent evidences suggest that fructose exposure to other tissues has substantial and profound metabolic consequences predisposing towards chronic conditions such as type 2 diabetes. Since skeletal muscle is the major site for glucose utilization, in the present study we define the effects of fructose exposure on glucose utilization in skeletal muscle cells. Upon fructose exposure, the L6 skeletal muscle cells displayed diminished glucose uptake, GLUT4 translocation and impaired insulin signaling. The exposure to fructose elevated reactive oxygen species (ROS) production in L6 myotubes, accompanied by activation of the stress/ inflammation markers c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase 1/2 (ERK1/2), and degradation of inhibitor of NFκB (IκBα). We found that fructose caused impairment of glucose utilization and insulin signaling through ROS-mediated activation of JNK and ERK1/2 pathways, which was prevented in presence of antioxidants. In conclusion, our data demonstrate that exposure to fructose induces cell-autonomous oxidative response through ROS production leading to impaired insulin signaling and attenuated glucose utilization in skeletal muscle cells.
© 2015 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
7Fructose-induced ROS generation impairs glucose utilization in L6 skeletal muscle cells
Natasha Jaiswal, Chandan K. Maurya, Jyotsana Pandey, Amit K. Rai, Akhilesh K. Tamrakar Division of Biochemistry, CSIR-Central Drug Research Institute, Sector-10, Jankipuram
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
Extension, Sitapur Road, Lucknow-226031, U.P., India
Corresponding author: Dr. Akhilesh K Tamrakar, Division of Biochemistry, CSIRCentral Drug Research Institute, Lucknow-226031, India. Tel: +91-0522-2772550 Ext. 4635. Fax: +91-0522-2771941 E-mail: [email protected]
Short title: Fructose and insulin resistance Abstract High fructose consumption has implicated in insulin resistance and metabolic syndrome. Fructose is a highly lipogenic sugar that has intense metabolic effects in liver. Recent evidences suggest that fructose exposure to other tissues has substantial and profound metabolic consequences predisposing towards chronic conditions such as type 2 diabetes. Since skeletal muscle is the major site for glucose utilization, in the present study we define the effects of fructose exposure on glucose utilization in skeletal muscle cells. Upon fructose exposure, the L6 skeletal muscle cells displayed diminished glucose uptake, GLUT4 translocation and impaired insulin signaling. The exposure to fructose elevated reactive oxygen species (ROS) production in L6 myotubes, accompanied by activation of the stress/inflammation markers c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase 1/2 (ERK1/2), and degradation of inhibitor of NFκB (IκBα). We found that fructose caused impairment of glucose utilization and insulin signaling through ROS-mediated activation of JNK and ERK1/2 pathways, which was prevented in presence of antioxidants. In conclusion, our data demonstrate that exposure to fructose induces cell-autonomous oxidative response through ROS production leading to impaired insulin signaling and attenuated glucose utilization in skeletal muscle cells.
Keywords: Insulin resistance, Nutrient signal, Stress kinase, Oxidative stress.
1
Introduction Insulin resistance is a major pathogenic factor in the development of type 2 diabetes mellitus and obesity related disturbance of glucose and lipid metabolism [1, 2]. Insulin resistance is characterized by complex interaction among genetic determinants, nutritional factors and lifestyle [3]. The main
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
driving force for the increased prevalence of insulin resistance is modern diets and associated obesity. Consumption of diets rich in saturated fats have been shown to induce weight gain, insulin resistance, and hyperlipidemia in humans and animals [4- 6]. It has been suggested that a high intake of refined carbohydrates may also increase the risk of insulin resistance [7]. In addition, diets specifically rich in fructose have been shown to contribute to metabolic disturbance in animal models resulting in weight gain, hyperlipidemia [8], and hypertension [9]. Fructose is readily absorbed and rapidly metabolized by liver. Westernization of diets has resulted in significant increase in daily consumptions of fructose [10]. The exposure of the liver to increased quantity of fructose leads to rapid stimulation of lipogenesis and triglyceride (TG) accumulation, which in turn
contributes
to
reduced
insulin
sensitivity
and
hepatic
insulin
resistance/glucose intolerance [11]. Fructose-induced insulin resistance is commonly characterized by a profound metabolic dyslipidemia, suggested to result from hepatic and intestinal overproduction of atherogenic lipoprotein particles [10, 12]. These lipoprotein particles are released from the liver destined toward peripheral tissues for storage in both fat and muscle cells, eventually
2
resulting in impaired insulin signaling and dyslipidemia [11]. Furthermore, excessive production of lipoproteins induces inflammatory response and adipocyte insulin resistance. The consequent elevation in circulating fatty acids and inflammatory cytokines causes insulin resistance in peripheral tissues leading to whole body insulin resistance [3, 13, 14]. Increasing evidences suggest that oxidative stress, results from increased
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
content of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) plays major role in the pathogenesis of insulin resistance [15]. ROS can function as a signaling molecule to activate a number of cellular stress-sensitive pathways linked to the insulin resistance [16]. Skeletal muscle is the major determinant of whole body insulin resistance. Insulin resistance in skeletal muscle is characterized by decreased insulin-stimulated glucose uptake, the rate-limiting step in glucose utilization [17, 18]. Although, insulin response in skeletal muscle is affected by circulating factors secreted from adipose or liver, evidences suggested that skeletal muscle has cell-autonomous response to external stimuli that might be capable of eliciting insulin resistance. Exposure to free fatty acids or ligands of innate immune systems modulate substrate metabolism and render muscle cells in culture resistant to insulin [19-21]. Indeed, whilst glucose is the major hexose substrate for skeletal muscle, several studies have demonstrated the significant utilization of fructose by this tissue [22], suggesting that fructose can influence carbohydrate metabolism in skeletal muscle cells. The objective of this research was to determine whether exposure to fructose influence glucose utilization and insulin-induced signaling in skeletal muscle cells, in a cellautonomous fashion. If so what are the mechanisms underlying the effect. We
3
demonstrate that exposure to fructose attenuate the glucose utilization and inhibit insulin response in skeletal muscle cells. These effects of fructose are associated with elevated ROS production, accompanied by activation of the cellular stresssensitive pathways including c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase 1/2 (ERK1/2), and nuclear factor-κB (NFκB), underlying the effect of fructose. Our study provides new insight towards the mechanisms of
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
fructose-induced insulin resistance through direct effect of fructose exposure on skeletal muscle glucose metabolism. Methods Materials Fetal bovine serum (FBS), trypsin, antibiotic/antimycotic solution and trizol reagent were from Gibco, USA. DMEM, glucose free DMEM, D-fructose, cytochalasin B, 2-deoxyglucose, protease inhibitor cocktail, monoclonal antiactinin-1 and all other chemicals unless otherwise noted were from Sigma Chemical (St. Louis, MO). 2-Deoxy-D-[3H]-glucose (2-DG) was from PerkinElmer, USA. Antibodies to GLUT4 (IF8), GLUT1 and AKT were from Santa Cruz Biotechnology, Inc (USA) and antibodies to phospho-AKT (Ser473), IRS-1, phospho-Tyr, phospho-IRS-1 (Ser-307), phospho-IRS-1 (Ser-612), Phospho-JNK (Thr-183/Tyr-185), JNK, Phospho-ERK1/2 (Thr-202/Tyr-205), ERK1/2, and IκBα were from Cell Signaling Technology (USA). Cell culture Wild type L6 skeletal muscle cells and L6 cells stably expressing rat GLUT4 with a myc epitope (L6-GLUT4myc) were kind gift of Dr. Amira Klip, Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada.
4
Cells were maintained in DMEM supplemented with 5 mM D-glucose, 10% FBS, and 1% antibiotic/antimycotic solution in a humidified atmosphere of air and 5% CO2 at 370C. Differentiation was induced by switching confluent cells to medium supplemented with 2% FBS. Experiments were performed in differentiated myotubes 6-7 days after seeding. To access the effect of fructose, cells were incubated in glucose free medium containing indicated concentration
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
of fructose for specified time duration. Assessment of cell viability and necrosis Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay [23]. After indicated treatments, the cells were incubated with MTT solution (5 mg/mL in PBS) and the absorbance was measured at 540 nm using an ELISA plate reader (Bioteck, USA). The activity of lactate dehydrogenase present in the incubation medium at the beginning and end of the treatment was evaluated as an index of necrosis. Lactate dehydrogenase activity was determined by incubating 100 µl of supernatant in assay mixture containing 50 mM lithium lactate, 0.9 mg phenazine methosulfate (PMS), 3.3 mg iodonitrotetrazolium (INT) and 8.6 mg NAD in 200 mM tris buffer. The reaction mixture was incubated for 5 min followed by end point absorbance measurement at 490 nm. Glucose uptake measurement Determination of 2-deoxyglucose (2-DG) uptake in L6 myotubes was performed as described previously [20]. Briefly L6 myotubes were incubated in culture medium containing indicated concentrations of fructose or glucose for different time period and 2-DG uptake was assessed for 5 min in HEPES-
5
buffered saline containing 10 µM 2-DG (0.5 µCi/ml 2-[3H] DG) at room temperature. To quantify the radioactivity incorporated, cells were lysed with 0.05 N NaOH and lysates were counted with scintillation fluid in a β-counter. Nonspecific uptake was determined in the presence of cytochalasin B (50 µM) during the assay, and these values were subtracted from all other values. Glucose uptake measured in triplicate and normalized to total protein, was expressed as
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
fold induction with respect to unstimulated cells. GLUT4 translocation measurement GLUT4 translocation to cell surface was determined in L6-GLUT4myc myotubes by measuring the cell surface level of GLUT4myc by an antibodycoupled colorimetric assay as previously described [20]. The fraction of GLUT4myc at the cell surface, measured in triplicate, was expressed as fold induction with respect to unstimulated cells. ROS production Cytoplasmic ROS level in L6 myotubes was assayed using 2′, 7′dichlorofluorescin diacetate (DCF-DA) by fluorometric assay. Cells grown in 24-well plates were treated as indicated and trypsinized with 0.1% trypsin. H2O2 (100 μM) was used as positive control. Harvested cells were adjusted to a concentration of 1x106 cells/ml in phosphate buffer saline (PBS) in FACS tubes. For probe loading, cells were incubated with the DCF-DA (1 μM) for 15 min at 37°C and washed twice in PBS. ROS levels in individual living cells were determined by measuring their fluorescence intensity on FACS Calibur (Becton Dickinson, USA). Data were analyzed by CellQuest Software (Becton-
6
Dickinson, San Diego, CA) and mean ROS values were evaluated for cell populations. Superoxide and hydrogen peroxide production Superoxide level was measured using the superoxide-sensitive dye DHE by flourimetric assay [24]. After indicated treatments, cells were incubated with DHE (5 µg/ml) for 30 min. Superoxide level was assayed by detecting
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
fluorescence produced, based on the appearance and disappearance of ethidium (535/615) and dihydroethidium (370/460), respectively. Hydrogen peroxide (H2O2) level was assayed by H2O2-mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari [25]. After indicated treatments, cell lysate was added to reaction mixture containing phenol red (0.28 nmol), horse radish peroxidase (8.5 units), and dextrose (5.5 nmol) in phosphate buffer (0.05 mol; pH 7.0), and were incubated for 60 min at 370C. The reaction was stopped by the addition of NaOH (10 N) and then centrifuged at 800×g for 5 min. The absorbance of the supernatant was recorded at 610 nm against a reagent blank. The quantity of H2O2 produced was expressed as nmol H2O2/min/mg protein based on the standard curve of H2O2 oxidized phenol red. Protein extraction and western blot analysis After indicated treatments, myotubes were washed with ice cold normal saline, scraped and solubilized in RIPA buffer supplemented with protease and phosphatase inhibitors. Whole cell lysates were cleared by centrifugation at 10000 rpm for 10 min at 40C and protein content was measured by the BCA assay. For western blotting, proteins were boiled in Laemmli buffer, separated
7
by SDS-PAGE and transferred onto PVDF membrane. Membranes were then blotted using primary antibodies (4°C overnight), washed and peroxidasecoupled secondary antibody was applied for 1 h at room temperature. Membranes were developed using enhanced chemiluminescence (ECL, Millipore), and analyzed using NIH Image J software. For insulin receptor substrate -1 (IRS-1) phosphorylation, cells exposed
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
to fructose were stimulated with 100 nM insulin for 5 min at 370C, lysed, and IRS-1 immunoprecipitated as described [20]. Briefly, whole cell lysates in IP buffer (50 mM Tris, 120 mM NaCl, 10 mM MgCl2, 2.5 mM EGTA, pH 7.5) were incubated with IRS-1-specific antibodies at 4°C overnight under constant rotation, and the antibody-antigen complex was pulled down by protein ASepharose beads. The pelleted protein was resolved by SDS-PAGE, and tyrosine phosphorylation of IRS-1 was detected by immunoblotting with monoclonal anti-phosphotyrosine antibody. Blots were stripped and reprobed with IRS-1 antibody to calculate relative phosphorylation. Gene expression analysis For gene expression analysis, RNA was extracted from myotubes exposed to 15 mM concentration of fructose or glucose for indicated time period. Semi-quantitative RT-PCR was performed using 200 ng total RNA. PCR cycles were titrated for each gene specific primer pair target to ensure linearity. Primers used were: IRS-1: FWD, 5’-tggacgtcacaggcagaat-3’ and REV, 5’gggatgcatcgtaccatctac-3’; PI-3-K: FWD, 5’-tcaccgagatgggaaatacg-3’ and REV, 5’-tacagagcaggcatagcagc-3’; AKT: FWD, 5’-ccgctattatgccatgaagat-3’ and REV, 5’-tgtgggcgacttcatcct-3’; GLUT4: FWD, 5’-gcagtgcctgagtcttcttt-3’ and REV, 5’-
8
ccagtcactcgctgctga-3’; GLUT1: FWD, 5’-gaggagctcttccaccctct-3’ and REV, 5’tctggagccatcaaagtcct-3’; GLUT5: FWD, 5’-cagcaaaattgccaaatcct-3’ and REV, 5’-agagctgaggaaccactcca-3’; 18SrRNA: FWD, 5’-aaacggctaccacatccaag-3’ and REV, 5’-ccctcttaatcatggcctca-3’. Statistical analysis Values are given as mean ± SE. Analysis of statistical significance of
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differences in measurements between samples was done by one-way ANOVA with Dunnet’s post hoc test (GraphPad Prism version 3). P < 0.05 was considered statistically significant. Results Fructose exposure diminishes 2-DG uptake in L6 myotubes To determine the effect of fructose on glucose metabolism in skeletal muscle cells, we studied its effect on 2-DG uptake in L6 myotubes. In control cells insulin induced 1.75-fold (p
Fructose-induced ROS generation impairs glucose utilization in L6 skeletal muscle cells Natasha Jaiswal, Chandan K. Maurya, Jyotsana Pandey, Amit K. Rai, Akhilesh K. Tamrakar Doi: 10.3109/10715762.2015.1031662
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
Abstract High fructose consumption has implicated in insulin resistance and metabolic syndrome. Fructose is a highly lipogenic sugar that has intense metabolic effects in liver. Recent evidences suggest that fructose exposure to other tissues has substantial and profound metabolic consequences predisposing towards chronic conditions such as type 2 diabetes. Since skeletal muscle is the major site for glucose utilization, in the present study we define the effects of fructose exposure on glucose utilization in skeletal muscle cells. Upon fructose exposure, the L6 skeletal muscle cells displayed diminished glucose uptake, GLUT4 translocation and impaired insulin signaling. The exposure to fructose elevated reactive oxygen species (ROS) production in L6 myotubes, accompanied by activation of the stress/ inflammation markers c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase 1/2 (ERK1/2), and degradation of inhibitor of NFκB (IκBα). We found that fructose caused impairment of glucose utilization and insulin signaling through ROS-mediated activation of JNK and ERK1/2 pathways, which was prevented in presence of antioxidants. In conclusion, our data demonstrate that exposure to fructose induces cell-autonomous oxidative response through ROS production leading to impaired insulin signaling and attenuated glucose utilization in skeletal muscle cells.
© 2015 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
7Fructose-induced ROS generation impairs glucose utilization in L6 skeletal muscle cells
Natasha Jaiswal, Chandan K. Maurya, Jyotsana Pandey, Amit K. Rai, Akhilesh K. Tamrakar Division of Biochemistry, CSIR-Central Drug Research Institute, Sector-10, Jankipuram
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
Extension, Sitapur Road, Lucknow-226031, U.P., India
Corresponding author: Dr. Akhilesh K Tamrakar, Division of Biochemistry, CSIRCentral Drug Research Institute, Lucknow-226031, India. Tel: +91-0522-2772550 Ext. 4635. Fax: +91-0522-2771941 E-mail: [email protected]
Short title: Fructose and insulin resistance Abstract High fructose consumption has implicated in insulin resistance and metabolic syndrome. Fructose is a highly lipogenic sugar that has intense metabolic effects in liver. Recent evidences suggest that fructose exposure to other tissues has substantial and profound metabolic consequences predisposing towards chronic conditions such as type 2 diabetes. Since skeletal muscle is the major site for glucose utilization, in the present study we define the effects of fructose exposure on glucose utilization in skeletal muscle cells. Upon fructose exposure, the L6 skeletal muscle cells displayed diminished glucose uptake, GLUT4 translocation and impaired insulin signaling. The exposure to fructose elevated reactive oxygen species (ROS) production in L6 myotubes, accompanied by activation of the stress/inflammation markers c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase 1/2 (ERK1/2), and degradation of inhibitor of NFκB (IκBα). We found that fructose caused impairment of glucose utilization and insulin signaling through ROS-mediated activation of JNK and ERK1/2 pathways, which was prevented in presence of antioxidants. In conclusion, our data demonstrate that exposure to fructose induces cell-autonomous oxidative response through ROS production leading to impaired insulin signaling and attenuated glucose utilization in skeletal muscle cells.
Keywords: Insulin resistance, Nutrient signal, Stress kinase, Oxidative stress.
1
Introduction Insulin resistance is a major pathogenic factor in the development of type 2 diabetes mellitus and obesity related disturbance of glucose and lipid metabolism [1, 2]. Insulin resistance is characterized by complex interaction among genetic determinants, nutritional factors and lifestyle [3]. The main
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
driving force for the increased prevalence of insulin resistance is modern diets and associated obesity. Consumption of diets rich in saturated fats have been shown to induce weight gain, insulin resistance, and hyperlipidemia in humans and animals [4- 6]. It has been suggested that a high intake of refined carbohydrates may also increase the risk of insulin resistance [7]. In addition, diets specifically rich in fructose have been shown to contribute to metabolic disturbance in animal models resulting in weight gain, hyperlipidemia [8], and hypertension [9]. Fructose is readily absorbed and rapidly metabolized by liver. Westernization of diets has resulted in significant increase in daily consumptions of fructose [10]. The exposure of the liver to increased quantity of fructose leads to rapid stimulation of lipogenesis and triglyceride (TG) accumulation, which in turn
contributes
to
reduced
insulin
sensitivity
and
hepatic
insulin
resistance/glucose intolerance [11]. Fructose-induced insulin resistance is commonly characterized by a profound metabolic dyslipidemia, suggested to result from hepatic and intestinal overproduction of atherogenic lipoprotein particles [10, 12]. These lipoprotein particles are released from the liver destined toward peripheral tissues for storage in both fat and muscle cells, eventually
2
resulting in impaired insulin signaling and dyslipidemia [11]. Furthermore, excessive production of lipoproteins induces inflammatory response and adipocyte insulin resistance. The consequent elevation in circulating fatty acids and inflammatory cytokines causes insulin resistance in peripheral tissues leading to whole body insulin resistance [3, 13, 14]. Increasing evidences suggest that oxidative stress, results from increased
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
content of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) plays major role in the pathogenesis of insulin resistance [15]. ROS can function as a signaling molecule to activate a number of cellular stress-sensitive pathways linked to the insulin resistance [16]. Skeletal muscle is the major determinant of whole body insulin resistance. Insulin resistance in skeletal muscle is characterized by decreased insulin-stimulated glucose uptake, the rate-limiting step in glucose utilization [17, 18]. Although, insulin response in skeletal muscle is affected by circulating factors secreted from adipose or liver, evidences suggested that skeletal muscle has cell-autonomous response to external stimuli that might be capable of eliciting insulin resistance. Exposure to free fatty acids or ligands of innate immune systems modulate substrate metabolism and render muscle cells in culture resistant to insulin [19-21]. Indeed, whilst glucose is the major hexose substrate for skeletal muscle, several studies have demonstrated the significant utilization of fructose by this tissue [22], suggesting that fructose can influence carbohydrate metabolism in skeletal muscle cells. The objective of this research was to determine whether exposure to fructose influence glucose utilization and insulin-induced signaling in skeletal muscle cells, in a cellautonomous fashion. If so what are the mechanisms underlying the effect. We
3
demonstrate that exposure to fructose attenuate the glucose utilization and inhibit insulin response in skeletal muscle cells. These effects of fructose are associated with elevated ROS production, accompanied by activation of the cellular stresssensitive pathways including c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase 1/2 (ERK1/2), and nuclear factor-κB (NFκB), underlying the effect of fructose. Our study provides new insight towards the mechanisms of
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
fructose-induced insulin resistance through direct effect of fructose exposure on skeletal muscle glucose metabolism. Methods Materials Fetal bovine serum (FBS), trypsin, antibiotic/antimycotic solution and trizol reagent were from Gibco, USA. DMEM, glucose free DMEM, D-fructose, cytochalasin B, 2-deoxyglucose, protease inhibitor cocktail, monoclonal antiactinin-1 and all other chemicals unless otherwise noted were from Sigma Chemical (St. Louis, MO). 2-Deoxy-D-[3H]-glucose (2-DG) was from PerkinElmer, USA. Antibodies to GLUT4 (IF8), GLUT1 and AKT were from Santa Cruz Biotechnology, Inc (USA) and antibodies to phospho-AKT (Ser473), IRS-1, phospho-Tyr, phospho-IRS-1 (Ser-307), phospho-IRS-1 (Ser-612), Phospho-JNK (Thr-183/Tyr-185), JNK, Phospho-ERK1/2 (Thr-202/Tyr-205), ERK1/2, and IκBα were from Cell Signaling Technology (USA). Cell culture Wild type L6 skeletal muscle cells and L6 cells stably expressing rat GLUT4 with a myc epitope (L6-GLUT4myc) were kind gift of Dr. Amira Klip, Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada.
4
Cells were maintained in DMEM supplemented with 5 mM D-glucose, 10% FBS, and 1% antibiotic/antimycotic solution in a humidified atmosphere of air and 5% CO2 at 370C. Differentiation was induced by switching confluent cells to medium supplemented with 2% FBS. Experiments were performed in differentiated myotubes 6-7 days after seeding. To access the effect of fructose, cells were incubated in glucose free medium containing indicated concentration
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
of fructose for specified time duration. Assessment of cell viability and necrosis Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay [23]. After indicated treatments, the cells were incubated with MTT solution (5 mg/mL in PBS) and the absorbance was measured at 540 nm using an ELISA plate reader (Bioteck, USA). The activity of lactate dehydrogenase present in the incubation medium at the beginning and end of the treatment was evaluated as an index of necrosis. Lactate dehydrogenase activity was determined by incubating 100 µl of supernatant in assay mixture containing 50 mM lithium lactate, 0.9 mg phenazine methosulfate (PMS), 3.3 mg iodonitrotetrazolium (INT) and 8.6 mg NAD in 200 mM tris buffer. The reaction mixture was incubated for 5 min followed by end point absorbance measurement at 490 nm. Glucose uptake measurement Determination of 2-deoxyglucose (2-DG) uptake in L6 myotubes was performed as described previously [20]. Briefly L6 myotubes were incubated in culture medium containing indicated concentrations of fructose or glucose for different time period and 2-DG uptake was assessed for 5 min in HEPES-
5
buffered saline containing 10 µM 2-DG (0.5 µCi/ml 2-[3H] DG) at room temperature. To quantify the radioactivity incorporated, cells were lysed with 0.05 N NaOH and lysates were counted with scintillation fluid in a β-counter. Nonspecific uptake was determined in the presence of cytochalasin B (50 µM) during the assay, and these values were subtracted from all other values. Glucose uptake measured in triplicate and normalized to total protein, was expressed as
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
fold induction with respect to unstimulated cells. GLUT4 translocation measurement GLUT4 translocation to cell surface was determined in L6-GLUT4myc myotubes by measuring the cell surface level of GLUT4myc by an antibodycoupled colorimetric assay as previously described [20]. The fraction of GLUT4myc at the cell surface, measured in triplicate, was expressed as fold induction with respect to unstimulated cells. ROS production Cytoplasmic ROS level in L6 myotubes was assayed using 2′, 7′dichlorofluorescin diacetate (DCF-DA) by fluorometric assay. Cells grown in 24-well plates were treated as indicated and trypsinized with 0.1% trypsin. H2O2 (100 μM) was used as positive control. Harvested cells were adjusted to a concentration of 1x106 cells/ml in phosphate buffer saline (PBS) in FACS tubes. For probe loading, cells were incubated with the DCF-DA (1 μM) for 15 min at 37°C and washed twice in PBS. ROS levels in individual living cells were determined by measuring their fluorescence intensity on FACS Calibur (Becton Dickinson, USA). Data were analyzed by CellQuest Software (Becton-
6
Dickinson, San Diego, CA) and mean ROS values were evaluated for cell populations. Superoxide and hydrogen peroxide production Superoxide level was measured using the superoxide-sensitive dye DHE by flourimetric assay [24]. After indicated treatments, cells were incubated with DHE (5 µg/ml) for 30 min. Superoxide level was assayed by detecting
Free Radic Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/15/15 For personal use only.
fluorescence produced, based on the appearance and disappearance of ethidium (535/615) and dihydroethidium (370/460), respectively. Hydrogen peroxide (H2O2) level was assayed by H2O2-mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari [25]. After indicated treatments, cell lysate was added to reaction mixture containing phenol red (0.28 nmol), horse radish peroxidase (8.5 units), and dextrose (5.5 nmol) in phosphate buffer (0.05 mol; pH 7.0), and were incubated for 60 min at 370C. The reaction was stopped by the addition of NaOH (10 N) and then centrifuged at 800×g for 5 min. The absorbance of the supernatant was recorded at 610 nm against a reagent blank. The quantity of H2O2 produced was expressed as nmol H2O2/min/mg protein based on the standard curve of H2O2 oxidized phenol red. Protein extraction and western blot analysis After indicated treatments, myotubes were washed with ice cold normal saline, scraped and solubilized in RIPA buffer supplemented with protease and phosphatase inhibitors. Whole cell lysates were cleared by centrifugation at 10000 rpm for 10 min at 40C and protein content was measured by the BCA assay. For western blotting, proteins were boiled in Laemmli buffer, separated
7
by SDS-PAGE and transferred onto PVDF membrane. Membranes were then blotted using primary antibodies (4°C overnight), washed and peroxidasecoupled secondary antibody was applied for 1 h at room temperature. Membranes were developed using enhanced chemiluminescence (ECL, Millipore), and analyzed using NIH Image J software. For insulin receptor substrate -1 (IRS-1) phosphorylation, cells exposed
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to fructose were stimulated with 100 nM insulin for 5 min at 370C, lysed, and IRS-1 immunoprecipitated as described [20]. Briefly, whole cell lysates in IP buffer (50 mM Tris, 120 mM NaCl, 10 mM MgCl2, 2.5 mM EGTA, pH 7.5) were incubated with IRS-1-specific antibodies at 4°C overnight under constant rotation, and the antibody-antigen complex was pulled down by protein ASepharose beads. The pelleted protein was resolved by SDS-PAGE, and tyrosine phosphorylation of IRS-1 was detected by immunoblotting with monoclonal anti-phosphotyrosine antibody. Blots were stripped and reprobed with IRS-1 antibody to calculate relative phosphorylation. Gene expression analysis For gene expression analysis, RNA was extracted from myotubes exposed to 15 mM concentration of fructose or glucose for indicated time period. Semi-quantitative RT-PCR was performed using 200 ng total RNA. PCR cycles were titrated for each gene specific primer pair target to ensure linearity. Primers used were: IRS-1: FWD, 5’-tggacgtcacaggcagaat-3’ and REV, 5’gggatgcatcgtaccatctac-3’; PI-3-K: FWD, 5’-tcaccgagatgggaaatacg-3’ and REV, 5’-tacagagcaggcatagcagc-3’; AKT: FWD, 5’-ccgctattatgccatgaagat-3’ and REV, 5’-tgtgggcgacttcatcct-3’; GLUT4: FWD, 5’-gcagtgcctgagtcttcttt-3’ and REV, 5’-
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ccagtcactcgctgctga-3’; GLUT1: FWD, 5’-gaggagctcttccaccctct-3’ and REV, 5’tctggagccatcaaagtcct-3’; GLUT5: FWD, 5’-cagcaaaattgccaaatcct-3’ and REV, 5’-agagctgaggaaccactcca-3’; 18SrRNA: FWD, 5’-aaacggctaccacatccaag-3’ and REV, 5’-ccctcttaatcatggcctca-3’. Statistical analysis Values are given as mean ± SE. Analysis of statistical significance of
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differences in measurements between samples was done by one-way ANOVA with Dunnet’s post hoc test (GraphPad Prism version 3). P < 0.05 was considered statistically significant. Results Fructose exposure diminishes 2-DG uptake in L6 myotubes To determine the effect of fructose on glucose metabolism in skeletal muscle cells, we studied its effect on 2-DG uptake in L6 myotubes. In control cells insulin induced 1.75-fold (p
