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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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Effect of pharmacological AMPK activation on NF-kB signaling in human myotubes from Non-Ob and obese subjects. Myotubes were treated with 100 μmol/L A769662 for 4 h before (A) immunoblotting to assess the phosphorylation status of IKK-α/β and total protein abundance of IκB-α and the p65 subunit of NF-κB. Equal loading was ascertained by immunoblotting with an antibody against β-tubulin, (C) measurement of NF-κB-p65 subunit DNA binding activity by ELISA assay, or concentration of (D) TNF-α and (E) IL-6 in culture media by Meso Scale Discovery ELISA assay. B: Effect of A769662 on IKK-α/β phosphorylation and total protein abundance of IκB-α 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 cells. **P < 0.005 vs. untreated Ob-T2D. #P < 0.05 vs. untreated Ob-T2D.
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Figure 6: Effect of pharmacological AMPK activation on NF-kB signaling in human myotubes from Non-Ob and obese subjects. Myotubes were treated with 100 μmol/L A769662 for 4 h before (A) immunoblotting to assess the phosphorylation status of IKK-α/β and total protein abundance of IκB-α and the p65 subunit of NF-κB. Equal loading was ascertained by immunoblotting with an antibody against β-tubulin, (C) measurement of NF-κB-p65 subunit DNA binding activity by ELISA assay, or concentration of (D) TNF-α and (E) IL-6 in culture media by Meso Scale Discovery ELISA assay. B: Effect of A769662 on IKK-α/β phosphorylation and total protein abundance of IκB-α 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 cells. **P < 0.005 vs. untreated Ob-T2D. #P < 0.05 vs. untreated Ob-T2D.

Mentions: It has been reported that AMPK is able to antagonize the effects of a number of proinflammatory markers in a variety of cell lines (12–15). However, its role as a modulator of inflammation has not been well established in human skeletal muscle. Therefore, we assessed the effect of pharmacological activation of AMPK using the specific AMPK activator, A769662, on NF-κB signaling in Non-Ob and obese myocytes. It is important that activation of AMPK using A769662 attenuated IKK phosphorylation to a level that was not significantly different from Non-Ob control subject levels. The loss of IκB-α protein expression was also attenuated in cells from Ob-T2D (Fig. 6A and B). In line with this, A769662 treatment of Ob-T2D cells attenuated the level of NF-κB-p65 DNA binding activity to that comparable with Non-Ob myocytes (Fig. 6C). Because levels of TNF-α were robustly increased in the cell culture media from basal Ob-T2D myocytes, we measured concentrations of this cytokine after 4 h of AMPK activation. A769662 suppressed TNF-α in the cell culture media to a level that was not significantly different from that measured in media from Non-Ob cells (Fig. 6D). Although A769662 did not significantly increase IL-6 concentrations in Ob-IGT and Ob-T2D media, there was a trend to increase IL-6 concentrations after A769662 treatment in these groups (Fig. 6E).


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)

Effect of pharmacological AMPK activation on NF-kB signaling in human myotubes from Non-Ob and obese subjects. Myotubes were treated with 100 μmol/L A769662 for 4 h before (A) immunoblotting to assess the phosphorylation status of IKK-α/β and total protein abundance of IκB-α and the p65 subunit of NF-κB. Equal loading was ascertained by immunoblotting with an antibody against β-tubulin, (C) measurement of NF-κB-p65 subunit DNA binding activity by ELISA assay, or concentration of (D) TNF-α and (E) IL-6 in culture media by Meso Scale Discovery ELISA assay. B: Effect of A769662 on IKK-α/β phosphorylation and total protein abundance of IκB-α 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 cells. **P < 0.005 vs. untreated Ob-T2D. #P < 0.05 vs. untreated Ob-T2D.
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Figure 6: Effect of pharmacological AMPK activation on NF-kB signaling in human myotubes from Non-Ob and obese subjects. Myotubes were treated with 100 μmol/L A769662 for 4 h before (A) immunoblotting to assess the phosphorylation status of IKK-α/β and total protein abundance of IκB-α and the p65 subunit of NF-κB. Equal loading was ascertained by immunoblotting with an antibody against β-tubulin, (C) measurement of NF-κB-p65 subunit DNA binding activity by ELISA assay, or concentration of (D) TNF-α and (E) IL-6 in culture media by Meso Scale Discovery ELISA assay. B: Effect of A769662 on IKK-α/β phosphorylation and total protein abundance of IκB-α 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 cells. **P < 0.005 vs. untreated Ob-T2D. #P < 0.05 vs. untreated Ob-T2D.
Mentions: It has been reported that AMPK is able to antagonize the effects of a number of proinflammatory markers in a variety of cell lines (12–15). However, its role as a modulator of inflammation has not been well established in human skeletal muscle. Therefore, we assessed the effect of pharmacological activation of AMPK using the specific AMPK activator, A769662, on NF-κB signaling in Non-Ob and obese myocytes. It is important that activation of AMPK using A769662 attenuated IKK phosphorylation to a level that was not significantly different from Non-Ob control subject levels. The loss of IκB-α protein expression was also attenuated in cells from Ob-T2D (Fig. 6A and B). In line with this, A769662 treatment of Ob-T2D cells attenuated the level of NF-κB-p65 DNA binding activity to that comparable with Non-Ob myocytes (Fig. 6C). Because levels of TNF-α were robustly increased in the cell culture media from basal Ob-T2D myocytes, we measured concentrations of this cytokine after 4 h of AMPK activation. A769662 suppressed TNF-α in the cell culture media to a level that was not significantly different from that measured in media from Non-Ob cells (Fig. 6D). Although A769662 did not significantly increase IL-6 concentrations in Ob-IGT and Ob-T2D media, there was a trend to increase IL-6 concentrations after A769662 treatment in these groups (Fig. 6E).

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