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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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Effects of chronic palmitate exposure on mitochondrial protein expression(A) and (B) C2C12 myotubes were treated with fatty-acid-free BSA 4.8% in DMEM (lanes 1 and 2) or with 4.8% BSA and palmitate (0.2 mM) for 24 h (lanes 3 and 4). Cell lysates were obtained and separated by SDS/PAGE transferred to nitrocellulose filters and immunoblotted with the specific proteins as indicated. A representative experiment is shown. (C) Quantification of the effects of chronic saturated fatty acid exposure on OXPHOS complex protein expression. Data from three to five experiments were normalized to actin. Non-treated cells were used as reference. Statistical analysis: ANOVA with Bonferroni post-hoc test (*P<0.05).
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Figure 8: Effects of chronic palmitate exposure on mitochondrial protein expression(A) and (B) C2C12 myotubes were treated with fatty-acid-free BSA 4.8% in DMEM (lanes 1 and 2) or with 4.8% BSA and palmitate (0.2 mM) for 24 h (lanes 3 and 4). Cell lysates were obtained and separated by SDS/PAGE transferred to nitrocellulose filters and immunoblotted with the specific proteins as indicated. A representative experiment is shown. (C) Quantification of the effects of chronic saturated fatty acid exposure on OXPHOS complex protein expression. Data from three to five experiments were normalized to actin. Non-treated cells were used as reference. Statistical analysis: ANOVA with Bonferroni post-hoc test (*P<0.05).

Mentions: To further investigate the effects of palmitate on mitochondrial function and dynamics, we examined the expression of the OXPHOS protein complex subunits by Western blot in control (untreated cells) or cells exposed to palmitate. Fatty acid exposure elevated the expression of protein components of Complex IV and V, without affecting the expression levels of the proteins of Complex I–III (Figures 8A and 8C). Interestingly, we found that expression of porin or Mfn-2 were unaltered in fatty-acid-treated cells in comparison with controls (Figure 8B).


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)

Effects of chronic palmitate exposure on mitochondrial protein expression(A) and (B) C2C12 myotubes were treated with fatty-acid-free BSA 4.8% in DMEM (lanes 1 and 2) or with 4.8% BSA and palmitate (0.2 mM) for 24 h (lanes 3 and 4). Cell lysates were obtained and separated by SDS/PAGE transferred to nitrocellulose filters and immunoblotted with the specific proteins as indicated. A representative experiment is shown. (C) Quantification of the effects of chronic saturated fatty acid exposure on OXPHOS complex protein expression. Data from three to five experiments were normalized to actin. Non-treated cells were used as reference. Statistical analysis: ANOVA with Bonferroni post-hoc test (*P<0.05).
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Related In: Results  -  Collection

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Figure 8: Effects of chronic palmitate exposure on mitochondrial protein expression(A) and (B) C2C12 myotubes were treated with fatty-acid-free BSA 4.8% in DMEM (lanes 1 and 2) or with 4.8% BSA and palmitate (0.2 mM) for 24 h (lanes 3 and 4). Cell lysates were obtained and separated by SDS/PAGE transferred to nitrocellulose filters and immunoblotted with the specific proteins as indicated. A representative experiment is shown. (C) Quantification of the effects of chronic saturated fatty acid exposure on OXPHOS complex protein expression. Data from three to five experiments were normalized to actin. Non-treated cells were used as reference. Statistical analysis: ANOVA with Bonferroni post-hoc test (*P<0.05).
Mentions: To further investigate the effects of palmitate on mitochondrial function and dynamics, we examined the expression of the OXPHOS protein complex subunits by Western blot in control (untreated cells) or cells exposed to palmitate. Fatty acid exposure elevated the expression of protein components of Complex IV and V, without affecting the expression levels of the proteins of Complex I–III (Figures 8A and 8C). Interestingly, we found that expression of porin or Mfn-2 were unaltered in fatty-acid-treated cells in comparison with controls (Figure 8B).

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