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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 insulin treatment does not alter expression of OXPHOS protein complexes porin or Mfn-2(A) C2C12 myotubes were left untreated (lanes 1 and 2) or treated with insulin for 24 h (lanes 3 and 4) or for 48 h (lanes 5 and 6). Whole-cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (B) C2C12 myotubes were left untreated (lanes 1 and 2) or treated with insulin for 48 h (lanes 3 and 4). Whole-cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (C) Quantification of effects of chronic insulin treatment on OXPHOS complex protein expression [a.u. (arbitrary units)]. Data from three experiments were normalized to actin. Non-treated cells were used as reference. Statistical analysis: one-way ANOVA.
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Figure 4: Chronic insulin treatment does not alter expression of OXPHOS protein complexes porin or Mfn-2(A) C2C12 myotubes were left untreated (lanes 1 and 2) or treated with insulin for 24 h (lanes 3 and 4) or for 48 h (lanes 5 and 6). Whole-cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (B) C2C12 myotubes were left untreated (lanes 1 and 2) or treated with insulin for 48 h (lanes 3 and 4). Whole-cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (C) Quantification of effects of chronic insulin treatment on OXPHOS complex protein expression [a.u. (arbitrary units)]. Data from three experiments were normalized to actin. Non-treated cells were used as reference. Statistical analysis: one-way ANOVA.

Mentions: To investigate further the impact of chronic insulin on mitochondrial function we examined by Western blotting the expression of ETC (electron chain complex) protein subunits Mfn-2, a protein involved in mitochondrial fusion, and porin in these cells. Chronic insulin exposure did not significantly alter the expression of ETC protein Complexes I–V (Figure 4A) or the levels of Mfn-2 (Figure 4B). The expression levels of porin were also unchanged in insulin treated cells in comparison with controls (Figure 4B). In any case these data indicate that insulin-induced insulin resistance does not compromise the expression of mitochondrial OXPHOS proteins.


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 insulin treatment does not alter expression of OXPHOS protein complexes porin or Mfn-2(A) C2C12 myotubes were left untreated (lanes 1 and 2) or treated with insulin for 24 h (lanes 3 and 4) or for 48 h (lanes 5 and 6). Whole-cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (B) C2C12 myotubes were left untreated (lanes 1 and 2) or treated with insulin for 48 h (lanes 3 and 4). Whole-cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (C) Quantification of effects of chronic insulin treatment on OXPHOS complex protein expression [a.u. (arbitrary units)]. Data from three experiments were normalized to actin. Non-treated cells were used as reference. Statistical analysis: one-way ANOVA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475448&req=5

Figure 4: Chronic insulin treatment does not alter expression of OXPHOS protein complexes porin or Mfn-2(A) C2C12 myotubes were left untreated (lanes 1 and 2) or treated with insulin for 24 h (lanes 3 and 4) or for 48 h (lanes 5 and 6). Whole-cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (B) C2C12 myotubes were left untreated (lanes 1 and 2) or treated with insulin for 48 h (lanes 3 and 4). Whole-cell lysates were obtained, and separated by SDS/PAGE and immunoblotted with specific antibodies as indicated. A representative experiment is shown. (C) Quantification of effects of chronic insulin treatment on OXPHOS complex protein expression [a.u. (arbitrary units)]. Data from three experiments were normalized to actin. Non-treated cells were used as reference. Statistical analysis: one-way ANOVA.
Mentions: To investigate further the impact of chronic insulin on mitochondrial function we examined by Western blotting the expression of ETC (electron chain complex) protein subunits Mfn-2, a protein involved in mitochondrial fusion, and porin in these cells. Chronic insulin exposure did not significantly alter the expression of ETC protein Complexes I–V (Figure 4A) or the levels of Mfn-2 (Figure 4B). The expression levels of porin were also unchanged in insulin treated cells in comparison with controls (Figure 4B). In any case these data indicate that insulin-induced insulin resistance does not compromise the expression of mitochondrial OXPHOS proteins.

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