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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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Chronic saturated fatty acid exposure inhibits PI3K signalling in C2C12(A) C2C12 myotubes were treated with increasing concentrations of palmitate in DMEM/4.8% BSA or DMEM/4.8% BSA alone for 24 h as described in the Materials and methods section. Cells were then serum starved for 3–4 h and subsequently either left untreated or treated with insulin for 30 min as indicated. Cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (B) Quantification of the effects of fatty acid exposure on insulin signalling. Data for individual proteins were normalized to loading controls (Akt, ERK, JNK or actin). Control cells were used as reference. Data shown are means±S.E.M. for three to five experiments. Statistical analysis: ANOVA with Bonferroni post-hoc test (*or ◇P<0.05, ** or ◇◇P<0.01 and ◇◇◇P<0.001.
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Figure 5: Chronic saturated fatty acid exposure inhibits PI3K signalling in C2C12(A) C2C12 myotubes were treated with increasing concentrations of palmitate in DMEM/4.8% BSA or DMEM/4.8% BSA alone for 24 h as described in the Materials and methods section. Cells were then serum starved for 3–4 h and subsequently either left untreated or treated with insulin for 30 min as indicated. Cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (B) Quantification of the effects of fatty acid exposure on insulin signalling. Data for individual proteins were normalized to loading controls (Akt, ERK, JNK or actin). Control cells were used as reference. Data shown are means±S.E.M. for three to five experiments. Statistical analysis: ANOVA with Bonferroni post-hoc test (*or ◇P<0.05, ** or ◇◇P<0.01 and ◇◇◇P<0.001.

Mentions: Chronic treatment of cells with the saturated fatty acid palmitate induced insulin resistance in a dose-dependent manner, as evidenced by the attenuated Akt activation following an acute treatment of cells with insulin for 30 min (Figure 5). As the concentrations of the fatty acid increased, we observed a marked decline in the capacity of insulin to phosphorylate Akt and a decrease in the basal activation of Akt. Concomitantly with this, as the concentration of palmitate increased we observed increased basal activation of the stress kinases JNK and p38 (Figures 5A and 5B). In subsequent experiments, we chose to examine mitochondrial function at 0.2 mM palmitate, since at this concentration the activation of Akt was significantly reduced (to approx. 45% of that seen in control cells, i.e. insulin-treated in the absence of fatty acid, without compromising cell viability (cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and at this concentration of fatty acid it was not significantly different to control untreated (results not shown).


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)

Chronic saturated fatty acid exposure inhibits PI3K signalling in C2C12(A) C2C12 myotubes were treated with increasing concentrations of palmitate in DMEM/4.8% BSA or DMEM/4.8% BSA alone for 24 h as described in the Materials and methods section. Cells were then serum starved for 3–4 h and subsequently either left untreated or treated with insulin for 30 min as indicated. Cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (B) Quantification of the effects of fatty acid exposure on insulin signalling. Data for individual proteins were normalized to loading controls (Akt, ERK, JNK or actin). Control cells were used as reference. Data shown are means±S.E.M. for three to five experiments. Statistical analysis: ANOVA with Bonferroni post-hoc test (*or ◇P<0.05, ** or ◇◇P<0.01 and ◇◇◇P<0.001.
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Related In: Results  -  Collection

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Figure 5: Chronic saturated fatty acid exposure inhibits PI3K signalling in C2C12(A) C2C12 myotubes were treated with increasing concentrations of palmitate in DMEM/4.8% BSA or DMEM/4.8% BSA alone for 24 h as described in the Materials and methods section. Cells were then serum starved for 3–4 h and subsequently either left untreated or treated with insulin for 30 min as indicated. Cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (B) Quantification of the effects of fatty acid exposure on insulin signalling. Data for individual proteins were normalized to loading controls (Akt, ERK, JNK or actin). Control cells were used as reference. Data shown are means±S.E.M. for three to five experiments. Statistical analysis: ANOVA with Bonferroni post-hoc test (*or ◇P<0.05, ** or ◇◇P<0.01 and ◇◇◇P<0.001.
Mentions: Chronic treatment of cells with the saturated fatty acid palmitate induced insulin resistance in a dose-dependent manner, as evidenced by the attenuated Akt activation following an acute treatment of cells with insulin for 30 min (Figure 5). As the concentrations of the fatty acid increased, we observed a marked decline in the capacity of insulin to phosphorylate Akt and a decrease in the basal activation of Akt. Concomitantly with this, as the concentration of palmitate increased we observed increased basal activation of the stress kinases JNK and p38 (Figures 5A and 5B). In subsequent experiments, we chose to examine mitochondrial function at 0.2 mM palmitate, since at this concentration the activation of Akt was significantly reduced (to approx. 45% of that seen in control cells, i.e. insulin-treated in the absence of fatty acid, without compromising cell viability (cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and at this concentration of fatty acid it was not significantly different to control untreated (results not shown).

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