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Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase.

Green CJ, Pedersen M, Pedersen BK, Scheele C - Diabetes (2011)

Bottom Line: This correlated to a significant increase in tumor necrosis factor-α concentration in cell culture media.This work provides solid evidence that differentiated human muscle precursor cells maintain in vivo phenotypes of inflammation and insulin resistance and that obesity alone may not be sufficient to establish inflammation in these cells.It is important that we demonstrate an anti-inflammatory role for AMPK in these human cells.

View Article: PubMed Central - PubMed

Affiliation: Centre of Inflammation and Metabolism, Department of Infectious Diseases, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. cjgreen30@gmail.com

ABSTRACT

Objective: To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes.

Research design and methods: Muscle precursor cells were isolated from four age-matched subject groups: 1) nonobese, normal glucose tolerant; 2) obese, normal glucose tolerant; 3) obese, impaired glucose tolerant; and 4) obese, type 2 diabetes (T2D). The level of inflammation (nuclear factor-κB [NF-κB] signaling) and effect of pharmacological AMPK activation was assessed by Western blots, enzyme-linked immunosorbent assay, and radioactive assays (n = 5 for each subject group).

Results: NF-κB-p65 DNA binding activity was significantly elevated in myocytes from obese T2D patients compared with nonobese control subjects. This correlated to a significant increase in tumor necrosis factor-α concentration in cell culture media. In addition, insulin-stimulated glucose uptake was completely suppressed in myocytes from obese impaired glucose tolerant and T2D subjects. It is interesting that activation of AMPK by A769662 attenuated NF-κB-p65 DNA binding activity in obese T2D cells to levels measured in nonobese myocytes; however, this had no effect on insulin sensitivity of the cells.

Conclusions: This work provides solid evidence that differentiated human muscle precursor cells maintain in vivo phenotypes of inflammation and insulin resistance and that obesity alone may not be sufficient to establish inflammation in these cells. It is important that we demonstrate an anti-inflammatory role for AMPK in these human cells. Despite attenuation of NF-κB activity by AMPK, insulin resistance in obese T2D cells remained, suggesting factors in addition to inflammation may contribute to the insulin resistance phenotype in muscle cells.

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Effects of pharmacological AMPK activation on AMPK signaling in human myotubes from Non-Ob and obese subjects. Myotubes were treated with 100 μmol/L A769662 for 4 h before (A) immunoprecipitation with a specific antibody against AMPK-α2 before measurement of AMPK activity or (C) immunoblotting to assess the phosphorylation status of ACC and the total protein abundance of AMPK-α1/α2 and ACC (streptavidin). B: Effect of A769662 on AMPK-α2 activity was expressed as a fold change from lean controls. D: Effect of A769662 on ACC phosphorylation and total protein abundance of ACC (streptavidin) and AMPK-α1/α2 was quantified and expressed as a fold change from untreated, Non-Ob cells. Values are mean ± SEM from five separate experiments. *P < 0.05 vs. Non-Ob.
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Figure 5: Effects of pharmacological AMPK activation on AMPK signaling in human myotubes from Non-Ob and obese subjects. Myotubes were treated with 100 μmol/L A769662 for 4 h before (A) immunoprecipitation with a specific antibody against AMPK-α2 before measurement of AMPK activity or (C) immunoblotting to assess the phosphorylation status of ACC and the total protein abundance of AMPK-α1/α2 and ACC (streptavidin). B: Effect of A769662 on AMPK-α2 activity was expressed as a fold change from lean controls. D: Effect of A769662 on ACC phosphorylation and total protein abundance of ACC (streptavidin) and AMPK-α1/α2 was quantified and expressed as a fold change from untreated, Non-Ob cells. Values are mean ± SEM from five separate experiments. *P < 0.05 vs. Non-Ob.

Mentions: Obesity had no effect on the level of AMPK-α1/α2 protein expressed in the cells. However, in the Ob-T2D myocytes, there was a significant increase in the total protein amount of downstream AMPK target, ACC (Fig. 4A and B). It is interesting that obesity was associated with a trend of increased basal AMPK-α2 activity, independent of glucose tolerance of the cells. Figure 4C shows that in obese myocytes, there is a threefold increase in basal AMPK activity compared with that observed in Non-Ob myocytes. Treatment of cultured human myocytes with A769662 for 4 h significantly increased AMPK-α2 activity to equal extents in all cell groups (Fig. 5A). Of interest, AMPK activation in Ob-IGT and Ob-T2D myocytes led to a significantly higher level of ACC phosphorylation compared with Non-Ob and Ob-NGT control subjects. The latter observation cannot be fully accounted for by increased protein expression levels of ACC (Fig. 5C and D). A769662 treatment did not alter the protein expression of AMPK-α1/α2.


Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase.

Green CJ, Pedersen M, Pedersen BK, Scheele C - Diabetes (2011)

Effects of pharmacological AMPK activation on AMPK signaling in human myotubes from Non-Ob and obese subjects. Myotubes were treated with 100 μmol/L A769662 for 4 h before (A) immunoprecipitation with a specific antibody against AMPK-α2 before measurement of AMPK activity or (C) immunoblotting to assess the phosphorylation status of ACC and the total protein abundance of AMPK-α1/α2 and ACC (streptavidin). B: Effect of A769662 on AMPK-α2 activity was expressed as a fold change from lean controls. D: Effect of A769662 on ACC phosphorylation and total protein abundance of ACC (streptavidin) and AMPK-α1/α2 was quantified and expressed as a fold change from untreated, Non-Ob cells. Values are mean ± SEM from five separate experiments. *P < 0.05 vs. Non-Ob.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198079&req=5

Figure 5: Effects of pharmacological AMPK activation on AMPK signaling in human myotubes from Non-Ob and obese subjects. Myotubes were treated with 100 μmol/L A769662 for 4 h before (A) immunoprecipitation with a specific antibody against AMPK-α2 before measurement of AMPK activity or (C) immunoblotting to assess the phosphorylation status of ACC and the total protein abundance of AMPK-α1/α2 and ACC (streptavidin). B: Effect of A769662 on AMPK-α2 activity was expressed as a fold change from lean controls. D: Effect of A769662 on ACC phosphorylation and total protein abundance of ACC (streptavidin) and AMPK-α1/α2 was quantified and expressed as a fold change from untreated, Non-Ob cells. Values are mean ± SEM from five separate experiments. *P < 0.05 vs. Non-Ob.
Mentions: Obesity had no effect on the level of AMPK-α1/α2 protein expressed in the cells. However, in the Ob-T2D myocytes, there was a significant increase in the total protein amount of downstream AMPK target, ACC (Fig. 4A and B). It is interesting that obesity was associated with a trend of increased basal AMPK-α2 activity, independent of glucose tolerance of the cells. Figure 4C shows that in obese myocytes, there is a threefold increase in basal AMPK activity compared with that observed in Non-Ob myocytes. Treatment of cultured human myocytes with A769662 for 4 h significantly increased AMPK-α2 activity to equal extents in all cell groups (Fig. 5A). Of interest, AMPK activation in Ob-IGT and Ob-T2D myocytes led to a significantly higher level of ACC phosphorylation compared with Non-Ob and Ob-NGT control subjects. The latter observation cannot be fully accounted for by increased protein expression levels of ACC (Fig. 5C and D). A769662 treatment did not alter the protein expression of AMPK-α1/α2.

Bottom Line: This correlated to a significant increase in tumor necrosis factor-α concentration in cell culture media.This work provides solid evidence that differentiated human muscle precursor cells maintain in vivo phenotypes of inflammation and insulin resistance and that obesity alone may not be sufficient to establish inflammation in these cells.It is important that we demonstrate an anti-inflammatory role for AMPK in these human cells.

View Article: PubMed Central - PubMed

Affiliation: Centre of Inflammation and Metabolism, Department of Infectious Diseases, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. cjgreen30@gmail.com

ABSTRACT

Objective: To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes.

Research design and methods: Muscle precursor cells were isolated from four age-matched subject groups: 1) nonobese, normal glucose tolerant; 2) obese, normal glucose tolerant; 3) obese, impaired glucose tolerant; and 4) obese, type 2 diabetes (T2D). The level of inflammation (nuclear factor-κB [NF-κB] signaling) and effect of pharmacological AMPK activation was assessed by Western blots, enzyme-linked immunosorbent assay, and radioactive assays (n = 5 for each subject group).

Results: NF-κB-p65 DNA binding activity was significantly elevated in myocytes from obese T2D patients compared with nonobese control subjects. This correlated to a significant increase in tumor necrosis factor-α concentration in cell culture media. In addition, insulin-stimulated glucose uptake was completely suppressed in myocytes from obese impaired glucose tolerant and T2D subjects. It is interesting that activation of AMPK by A769662 attenuated NF-κB-p65 DNA binding activity in obese T2D cells to levels measured in nonobese myocytes; however, this had no effect on insulin sensitivity of the cells.

Conclusions: This work provides solid evidence that differentiated human muscle precursor cells maintain in vivo phenotypes of inflammation and insulin resistance and that obesity alone may not be sufficient to establish inflammation in these cells. It is important that we demonstrate an anti-inflammatory role for AMPK in these human cells. Despite attenuation of NF-κB activity by AMPK, insulin resistance in obese T2D cells remained, suggesting factors in addition to inflammation may contribute to the insulin resistance phenotype in muscle cells.

Show MeSH
Related in: MedlinePlus