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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 gene expression in C2C12 myotubes exposed to chronic palmitateCells were treated with fatty-acid-free BSA (4.8% (w/v) in DMEM) or with BSA (4.8%) and palmitate (0.2 mM) for 24 h. The mRNA abundance of mitochondrial genes was determined by quantitative real-time PCR by ΔΔCT method. Data are means±S.E.M. for n=3 experiments, quantified in triplicate. Statistical analysis: two-way ANOVA with Bonferroni post-hoc test (*P<0.05).
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Figure 7: Mitochondrial gene expression in C2C12 myotubes exposed to chronic palmitateCells were treated with fatty-acid-free BSA (4.8% (w/v) in DMEM) or with BSA (4.8%) and palmitate (0.2 mM) for 24 h. The mRNA abundance of mitochondrial genes was determined by quantitative real-time PCR by ΔΔCT method. Data are means±S.E.M. for n=3 experiments, quantified in triplicate. Statistical analysis: two-way ANOVA with Bonferroni post-hoc test (*P<0.05).

Mentions: We next set to examine whether fatty acid treatment resulted in changes in the expression of transcripts coding for mitochondrial proteins or proteins associated with regulating mitochondrial biogenesis. Treatment of cells with 0.2 mM palmitate did not change PGC-1α and PGC-1β transcript levels, or indeed the levels of transcripts coding for the transcription factors NRF1 or Tfam (Figure 7). In contrast, fatty acid exposure markedly increased the transcripts coding for UCP2 and UCP3 (Figure 7). In addition, we detected a tendency to increase the transcript coding for ATP6 (ATP synthase subunit 6) (Figure 7).


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 gene expression in C2C12 myotubes exposed to chronic palmitateCells were treated with fatty-acid-free BSA (4.8% (w/v) in DMEM) or with BSA (4.8%) and palmitate (0.2 mM) for 24 h. The mRNA abundance of mitochondrial genes was determined by quantitative real-time PCR by ΔΔCT method. Data are means±S.E.M. for n=3 experiments, quantified in triplicate. Statistical analysis: two-way ANOVA with Bonferroni post-hoc test (*P<0.05).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475448&req=5

Figure 7: Mitochondrial gene expression in C2C12 myotubes exposed to chronic palmitateCells were treated with fatty-acid-free BSA (4.8% (w/v) in DMEM) or with BSA (4.8%) and palmitate (0.2 mM) for 24 h. The mRNA abundance of mitochondrial genes was determined by quantitative real-time PCR by ΔΔCT method. Data are means±S.E.M. for n=3 experiments, quantified in triplicate. Statistical analysis: two-way ANOVA with Bonferroni post-hoc test (*P<0.05).
Mentions: We next set to examine whether fatty acid treatment resulted in changes in the expression of transcripts coding for mitochondrial proteins or proteins associated with regulating mitochondrial biogenesis. Treatment of cells with 0.2 mM palmitate did not change PGC-1α and PGC-1β transcript levels, or indeed the levels of transcripts coding for the transcription factors NRF1 or Tfam (Figure 7). In contrast, fatty acid exposure markedly increased the transcripts coding for UCP2 and UCP3 (Figure 7). In addition, we detected a tendency to increase the transcript coding for ATP6 (ATP synthase subunit 6) (Figure 7).

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