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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 insulin(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 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: Student's t test (*P<0.05, **P<0.01 and ***P<0.001) (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 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 (μmol/μg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for four independent experiments each measured in triplicate. (C) Mitochondrial membrane potential (Δψ). C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 h before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Material 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; significance was analysed using a Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 h. 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). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed using a Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with insulin (100 nM, 48 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. The means±S.E.M. of fluorescence for n=41–59 cells are shown, analysed by Student's t test (***P<0.001).
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Figure 2: Mitochondrial function in C2C12 myotubes chronically treated with insulin(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 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: Student's t test (*P<0.05, **P<0.01 and ***P<0.001) (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 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 (μmol/μg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for four independent experiments each measured in triplicate. (C) Mitochondrial membrane potential (Δψ). C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 h before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Material 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; significance was analysed using a Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 h. 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). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed using a Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with insulin (100 nM, 48 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. The means±S.E.M. of fluorescence for n=41–59 cells are shown, analysed by Student's t test (***P<0.001).

Mentions: Next, we examined whether the mitochondrial function was impaired in chronically insulin-treated cells. To this end, we examined the OCR and ATP levels in cells chronically treated with insulin for 2 days. Insulin-treated cells showed a higher OCR and higher maximal respiratory capacity [in the presence of the uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone)] than the control untreated cells (Figure 2A). Despite this, the ATP levels of the cells were not different than controls (Figure 2B).


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 insulin(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 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: Student's t test (*P<0.05, **P<0.01 and ***P<0.001) (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 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 (μmol/μg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for four independent experiments each measured in triplicate. (C) Mitochondrial membrane potential (Δψ). C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 h before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Material 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; significance was analysed using a Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 h. 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). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed using a Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with insulin (100 nM, 48 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. The means±S.E.M. of fluorescence for n=41–59 cells are shown, analysed by Student's t test (***P<0.001).
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Figure 2: Mitochondrial function in C2C12 myotubes chronically treated with insulin(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 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: Student's t test (*P<0.05, **P<0.01 and ***P<0.001) (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 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 (μmol/μg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for four independent experiments each measured in triplicate. (C) Mitochondrial membrane potential (Δψ). C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 h before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Material 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; significance was analysed using a Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 h. 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). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed using a Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with insulin (100 nM, 48 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. The means±S.E.M. of fluorescence for n=41–59 cells are shown, analysed by Student's t test (***P<0.001).
Mentions: Next, we examined whether the mitochondrial function was impaired in chronically insulin-treated cells. To this end, we examined the OCR and ATP levels in cells chronically treated with insulin for 2 days. Insulin-treated cells showed a higher OCR and higher maximal respiratory capacity [in the presence of the uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone)] than the control untreated cells (Figure 2A). Despite this, the ATP levels of the cells were not different than controls (Figure 2B).

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