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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 induces insulin resistance and inhibits PI3K signalling(A) C2C12 myotubes were left untreated (lanes 1–4) or treated with 100 nM insulin for 24 h (lanes 5–8) or 48 h (lanes 9–12) before they were serum-deprived in DMEM for 4 h and either left untreated (lanes 1, 2, 5, 6, 9 and 10) or treated with insulin (100 nM) for 30 min (lanes 3, 4, 7, 8, 11 and 12). Whole-cell extracts were prepared and samples separated by SDS/PAGE and immunoblotted with the indicated antibodies. A representative experiment is shown. (B) Quantification of the effects of chronic insulin treatment on insulin signalling. Results are means±S.E.M. [AU (arbitrary units)] from three to five experiments. Data for individual proteins were normalized to loading controls (actin, Akt, ERK, JNK or p38). Non-treated cells were used as reference. Statistical analysis: one-way ANOVA (*P<0.05, **P<0.01 and ***P<0.001).
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Figure 1: Chronic insulin treatment induces insulin resistance and inhibits PI3K signalling(A) C2C12 myotubes were left untreated (lanes 1–4) or treated with 100 nM insulin for 24 h (lanes 5–8) or 48 h (lanes 9–12) before they were serum-deprived in DMEM for 4 h and either left untreated (lanes 1, 2, 5, 6, 9 and 10) or treated with insulin (100 nM) for 30 min (lanes 3, 4, 7, 8, 11 and 12). Whole-cell extracts were prepared and samples separated by SDS/PAGE and immunoblotted with the indicated antibodies. A representative experiment is shown. (B) Quantification of the effects of chronic insulin treatment on insulin signalling. Results are means±S.E.M. [AU (arbitrary units)] from three to five experiments. Data for individual proteins were normalized to loading controls (actin, Akt, ERK, JNK or p38). Non-treated cells were used as reference. Statistical analysis: one-way ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

Mentions: In order to investigate the role of impaired insulin signalling in mitochondrial function we first generated a model of insulin resistance in cultured C2C12 myotubes, by treating cells chronically with insulin (100 nM) for up to 2 days. As shown in Figure 1, chronic insulin treatment decreased the expression of the insulin receptor, which was evident following 24 h of insulin treatment. Quantification of three to five independent experiments showed a reduction of 60% of insulin receptor protein after 24 h insulin exposure and up to 75–80% after 48 h in comparison with untreated cells (Figure 1B). Concomitantly, 48 h of insulin treatment reduced the subsequent insulin-stimulated phosphorylation of the serine/threonine kinase Akt (Figure 1A, lanes 11 and 12 in comparison with control cells, lanes 3 and 4, and Figure 1B). Activation of ERKs (p44/p42) following an acute insulin treatment was also reduced in cells chronically treated with insulin for 48 h (Figure 1B). Basal activation of ERK p44/p42 was also increased after insulin treatment of cells. However, basal activation of the stress kinases p38 MAPK or JNK was not different in the insulin-treated group in comparison with control non-treated cells. We observed no changes in the phosphorylation levels of p38 MAPK in the insulin-treated cells in comparison with control cells. These data suggest that 48 h of chronic insulin treatment causes insulin resistance by reducing signalling through PI3K/Akt and p44/p42 MAPK without affecting p38 or JNK activation.


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 induces insulin resistance and inhibits PI3K signalling(A) C2C12 myotubes were left untreated (lanes 1–4) or treated with 100 nM insulin for 24 h (lanes 5–8) or 48 h (lanes 9–12) before they were serum-deprived in DMEM for 4 h and either left untreated (lanes 1, 2, 5, 6, 9 and 10) or treated with insulin (100 nM) for 30 min (lanes 3, 4, 7, 8, 11 and 12). Whole-cell extracts were prepared and samples separated by SDS/PAGE and immunoblotted with the indicated antibodies. A representative experiment is shown. (B) Quantification of the effects of chronic insulin treatment on insulin signalling. Results are means±S.E.M. [AU (arbitrary units)] from three to five experiments. Data for individual proteins were normalized to loading controls (actin, Akt, ERK, JNK or p38). Non-treated cells were used as reference. Statistical analysis: one-way ANOVA (*P<0.05, **P<0.01 and ***P<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475448&req=5

Figure 1: Chronic insulin treatment induces insulin resistance and inhibits PI3K signalling(A) C2C12 myotubes were left untreated (lanes 1–4) or treated with 100 nM insulin for 24 h (lanes 5–8) or 48 h (lanes 9–12) before they were serum-deprived in DMEM for 4 h and either left untreated (lanes 1, 2, 5, 6, 9 and 10) or treated with insulin (100 nM) for 30 min (lanes 3, 4, 7, 8, 11 and 12). Whole-cell extracts were prepared and samples separated by SDS/PAGE and immunoblotted with the indicated antibodies. A representative experiment is shown. (B) Quantification of the effects of chronic insulin treatment on insulin signalling. Results are means±S.E.M. [AU (arbitrary units)] from three to five experiments. Data for individual proteins were normalized to loading controls (actin, Akt, ERK, JNK or p38). Non-treated cells were used as reference. Statistical analysis: one-way ANOVA (*P<0.05, **P<0.01 and ***P<0.001).
Mentions: In order to investigate the role of impaired insulin signalling in mitochondrial function we first generated a model of insulin resistance in cultured C2C12 myotubes, by treating cells chronically with insulin (100 nM) for up to 2 days. As shown in Figure 1, chronic insulin treatment decreased the expression of the insulin receptor, which was evident following 24 h of insulin treatment. Quantification of three to five independent experiments showed a reduction of 60% of insulin receptor protein after 24 h insulin exposure and up to 75–80% after 48 h in comparison with untreated cells (Figure 1B). Concomitantly, 48 h of insulin treatment reduced the subsequent insulin-stimulated phosphorylation of the serine/threonine kinase Akt (Figure 1A, lanes 11 and 12 in comparison with control cells, lanes 3 and 4, and Figure 1B). Activation of ERKs (p44/p42) following an acute insulin treatment was also reduced in cells chronically treated with insulin for 48 h (Figure 1B). Basal activation of ERK p44/p42 was also increased after insulin treatment of cells. However, basal activation of the stress kinases p38 MAPK or JNK was not different in the insulin-treated group in comparison with control non-treated cells. We observed no changes in the phosphorylation levels of p38 MAPK in the insulin-treated cells in comparison with control cells. These data suggest that 48 h of chronic insulin treatment causes insulin resistance by reducing signalling through PI3K/Akt and p44/p42 MAPK without affecting p38 or JNK activation.

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