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Mitochondrial dysfunction in insulin resistance: differential contributions of chronic insulin and saturated fatty acid exposure in muscle cells.

Yang C, Aye CC, Li X, Diaz Ramos A, Zorzano A, Mora S - Biosci. Rep. (2012)

Bottom Line: The expression of mitochondrial OXPHOS (oxidative phosphorylation) subunits or Mfn-2 (mitofusin 2) were not significantly altered in comparison with untreated cells, whereas expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) and UCPs (uncoupling proteins) were reduced.In contrast, saturated fatty acid exposure caused insulin resistance, reducing PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) activation while increasing activation of stress kinases JNK (c-Jun N-terminal kinase) and p38.Palmitate-treated cells also showed a reduced glycolytic rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, U.K.

ABSTRACT
Mitochondrial dysfunction has been associated with insulin resistance, obesity and diabetes. Hyperinsulinaemia and hyperlipidaemia are hallmarks of the insulin-resistant state. We sought to determine the contributions of high insulin and saturated fatty acid exposure to mitochondrial function and biogenesis in cultured myocytes. Differentiated C2C12 myotubes were left untreated or exposed to chronic high insulin or high palmitate. Mitochondrial function was determined assessing: oxygen consumption, mitochondrial membrane potential, ATP content and ROS (reactive oxygen species) production. We also determined the expression of several mitochondrial genes. Chronic insulin treatment of myotubes caused insulin resistance with reduced PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) signalling. Insulin treatment increased oxygen consumption but reduced mitochondrial membrane potential and ROS production. ATP cellular levels were maintained through an increased glycolytic rate. The expression of mitochondrial OXPHOS (oxidative phosphorylation) subunits or Mfn-2 (mitofusin 2) were not significantly altered in comparison with untreated cells, whereas expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) and UCPs (uncoupling proteins) were reduced. In contrast, saturated fatty acid exposure caused insulin resistance, reducing PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) activation while increasing activation of stress kinases JNK (c-Jun N-terminal kinase) and p38. Fatty acids reduced oxygen consumption and mitochondrial membrane potential while up-regulating the expression of mitochondrial ETC (electron chain complex) protein subunits and UCP proteins. Mfn-2 expression was not modified by palmitate. Palmitate-treated cells also showed a reduced glycolytic rate. Taken together, our findings indicate that chronic insulin and fatty acid-induced insulin resistance differentially affect mitochondrial function. In both conditions, cells were able to maintain ATP levels despite the loss of membrane potential; however, different protein expression suggests different adaptation mechanisms.

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Mitochondrial function in C2C12 myotubes chronically treated with palmitate(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 0.2 mM palmitate for 24 h. Oxygen consumption was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: oligomycin A 10 μg/ml, FCCP 10 μM,). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed by Student's t test (*P<0.05 and **P<0.01). (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with 0.2 mM palmitate for 24 h. ATP content was measured using a luciferin/luciferase kit (Sigma) and following the manufacturer's instructions. The concentrations of ATP were normalized for the total protein concentrations (μM ATP/mg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for three independent experiments, n=6–13 per group. (C) Mitochondrial membrane potential (ΔΨm). C2C12 myotubes were left untreated or treated with 0.2 mM palmitate before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Materials and methods section. Acute treatment of insulin was for 30 min. Cells were trypsinized and analysed by flow cytometry. Data are presented as the percentage of the control and significance was analysed by Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 0.2 mM palmitate. Proton excretion as a measure of glycolysis was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: Oligomycin A 10 μg/ml, FCCP 10 μM). Statistical analysis: data were analysed by Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with 0.2 mM palmitate for 24 h. Cells were washed in PBS and stained with DCF-DA as detailed in the Materials and methods section. Cells were visualized in a Leica DMI6000 microscope under 488 nm excitation filter and fluorescence was quantified over a 5 min time intervals with Leica LAF software. Means±S.E.M. fluorescence for n=48–186 cells are shown. Analysed by one-way ANOVA (***P<0.001).
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Figure 6: Mitochondrial function in C2C12 myotubes chronically treated with palmitate(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 0.2 mM palmitate for 24 h. Oxygen consumption was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: oligomycin A 10 μg/ml, FCCP 10 μM,). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed by Student's t test (*P<0.05 and **P<0.01). (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with 0.2 mM palmitate for 24 h. ATP content was measured using a luciferin/luciferase kit (Sigma) and following the manufacturer's instructions. The concentrations of ATP were normalized for the total protein concentrations (μM ATP/mg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for three independent experiments, n=6–13 per group. (C) Mitochondrial membrane potential (ΔΨm). C2C12 myotubes were left untreated or treated with 0.2 mM palmitate before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Materials and methods section. Acute treatment of insulin was for 30 min. Cells were trypsinized and analysed by flow cytometry. Data are presented as the percentage of the control and significance was analysed by Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 0.2 mM palmitate. Proton excretion as a measure of glycolysis was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: Oligomycin A 10 μg/ml, FCCP 10 μM). Statistical analysis: data were analysed by Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with 0.2 mM palmitate for 24 h. Cells were washed in PBS and stained with DCF-DA as detailed in the Materials and methods section. Cells were visualized in a Leica DMI6000 microscope under 488 nm excitation filter and fluorescence was quantified over a 5 min time intervals with Leica LAF software. Means±S.E.M. fluorescence for n=48–186 cells are shown. Analysed by one-way ANOVA (***P<0.001).

Mentions: Having established a saturated fatty-acid-induced in vitro model of insulin resistance we next sought to examine the mitochondrial function in these cells. First, we determined the OCR and ATP levels in cells treated with 0.2 mM palmitate and control untreated cells. We observed that the OCR and maximal respiratory capacity (in the presence of FCCP) were decreased in the fatty-acid-treated cells in comparison with controls (Figure 6A). The mitochondrial membrane potential was also significantly reduced in cells treated with palmitate for 24 h (Figure 6C), concomitantly with a reduced formation of ROS (Figure 6E). However, the ATP content in cells was maintained at the same time that glycolytic rate (ECAR) was reduced (Figures 6B and 6D, respectively), suggesting a preference for fatty acid utilization over glucose in the palmitate-exposed cells.


Mitochondrial dysfunction in insulin resistance: differential contributions of chronic insulin and saturated fatty acid exposure in muscle cells.

Yang C, Aye CC, Li X, Diaz Ramos A, Zorzano A, Mora S - Biosci. Rep. (2012)

Mitochondrial function in C2C12 myotubes chronically treated with palmitate(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 0.2 mM palmitate for 24 h. Oxygen consumption was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: oligomycin A 10 μg/ml, FCCP 10 μM,). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed by Student's t test (*P<0.05 and **P<0.01). (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with 0.2 mM palmitate for 24 h. ATP content was measured using a luciferin/luciferase kit (Sigma) and following the manufacturer's instructions. The concentrations of ATP were normalized for the total protein concentrations (μM ATP/mg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for three independent experiments, n=6–13 per group. (C) Mitochondrial membrane potential (ΔΨm). C2C12 myotubes were left untreated or treated with 0.2 mM palmitate before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Materials and methods section. Acute treatment of insulin was for 30 min. Cells were trypsinized and analysed by flow cytometry. Data are presented as the percentage of the control and significance was analysed by Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 0.2 mM palmitate. Proton excretion as a measure of glycolysis was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: Oligomycin A 10 μg/ml, FCCP 10 μM). Statistical analysis: data were analysed by Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with 0.2 mM palmitate for 24 h. Cells were washed in PBS and stained with DCF-DA as detailed in the Materials and methods section. Cells were visualized in a Leica DMI6000 microscope under 488 nm excitation filter and fluorescence was quantified over a 5 min time intervals with Leica LAF software. Means±S.E.M. fluorescence for n=48–186 cells are shown. Analysed by one-way ANOVA (***P<0.001).
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Figure 6: Mitochondrial function in C2C12 myotubes chronically treated with palmitate(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 0.2 mM palmitate for 24 h. Oxygen consumption was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: oligomycin A 10 μg/ml, FCCP 10 μM,). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed by Student's t test (*P<0.05 and **P<0.01). (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with 0.2 mM palmitate for 24 h. ATP content was measured using a luciferin/luciferase kit (Sigma) and following the manufacturer's instructions. The concentrations of ATP were normalized for the total protein concentrations (μM ATP/mg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for three independent experiments, n=6–13 per group. (C) Mitochondrial membrane potential (ΔΨm). C2C12 myotubes were left untreated or treated with 0.2 mM palmitate before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Materials and methods section. Acute treatment of insulin was for 30 min. Cells were trypsinized and analysed by flow cytometry. Data are presented as the percentage of the control and significance was analysed by Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 0.2 mM palmitate. Proton excretion as a measure of glycolysis was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: Oligomycin A 10 μg/ml, FCCP 10 μM). Statistical analysis: data were analysed by Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with 0.2 mM palmitate for 24 h. Cells were washed in PBS and stained with DCF-DA as detailed in the Materials and methods section. Cells were visualized in a Leica DMI6000 microscope under 488 nm excitation filter and fluorescence was quantified over a 5 min time intervals with Leica LAF software. Means±S.E.M. fluorescence for n=48–186 cells are shown. Analysed by one-way ANOVA (***P<0.001).
Mentions: Having established a saturated fatty-acid-induced in vitro model of insulin resistance we next sought to examine the mitochondrial function in these cells. First, we determined the OCR and ATP levels in cells treated with 0.2 mM palmitate and control untreated cells. We observed that the OCR and maximal respiratory capacity (in the presence of FCCP) were decreased in the fatty-acid-treated cells in comparison with controls (Figure 6A). The mitochondrial membrane potential was also significantly reduced in cells treated with palmitate for 24 h (Figure 6C), concomitantly with a reduced formation of ROS (Figure 6E). However, the ATP content in cells was maintained at the same time that glycolytic rate (ECAR) was reduced (Figures 6B and 6D, respectively), suggesting a preference for fatty acid utilization over glucose in the palmitate-exposed cells.

Bottom Line: The expression of mitochondrial OXPHOS (oxidative phosphorylation) subunits or Mfn-2 (mitofusin 2) were not significantly altered in comparison with untreated cells, whereas expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) and UCPs (uncoupling proteins) were reduced.In contrast, saturated fatty acid exposure caused insulin resistance, reducing PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) activation while increasing activation of stress kinases JNK (c-Jun N-terminal kinase) and p38.Palmitate-treated cells also showed a reduced glycolytic rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, U.K.

ABSTRACT
Mitochondrial dysfunction has been associated with insulin resistance, obesity and diabetes. Hyperinsulinaemia and hyperlipidaemia are hallmarks of the insulin-resistant state. We sought to determine the contributions of high insulin and saturated fatty acid exposure to mitochondrial function and biogenesis in cultured myocytes. Differentiated C2C12 myotubes were left untreated or exposed to chronic high insulin or high palmitate. Mitochondrial function was determined assessing: oxygen consumption, mitochondrial membrane potential, ATP content and ROS (reactive oxygen species) production. We also determined the expression of several mitochondrial genes. Chronic insulin treatment of myotubes caused insulin resistance with reduced PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) signalling. Insulin treatment increased oxygen consumption but reduced mitochondrial membrane potential and ROS production. ATP cellular levels were maintained through an increased glycolytic rate. The expression of mitochondrial OXPHOS (oxidative phosphorylation) subunits or Mfn-2 (mitofusin 2) were not significantly altered in comparison with untreated cells, whereas expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) and UCPs (uncoupling proteins) were reduced. In contrast, saturated fatty acid exposure caused insulin resistance, reducing PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) activation while increasing activation of stress kinases JNK (c-Jun N-terminal kinase) and p38. Fatty acids reduced oxygen consumption and mitochondrial membrane potential while up-regulating the expression of mitochondrial ETC (electron chain complex) protein subunits and UCP proteins. Mfn-2 expression was not modified by palmitate. Palmitate-treated cells also showed a reduced glycolytic rate. Taken together, our findings indicate that chronic insulin and fatty acid-induced insulin resistance differentially affect mitochondrial function. In both conditions, cells were able to maintain ATP levels despite the loss of membrane potential; however, different protein expression suggests different adaptation mechanisms.

Show MeSH
Related in: MedlinePlus