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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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NF-κB signaling in human myotubes from Non-Ob and obese subjects. Myoblasts isolated from Non-Ob (n = 5), Ob-NGT (n = 5), Ob-IGT (n = 5), and Ob-T2D (n = 5) patients were grown in culture until mature myotubes were formed. A: Lysates were immunoblotted to assess the phosphorylation status of IKK-α/β and total protein abundance of IκB-α and the p65 subunit of NF-κB. Equal gel loading was ascertained by immunoblotting with an antibody against β-tubulin. B: The effect of obesity (filled bars) on IKK-α/β phosphorylation and IκB-α abundance was quantified and expressed as a fold change from Non-Ob cells (open bars). C: IκB-α protein abundance in skeletal muscle tissue was compared with abundance in muscle precursor cells from the same individuals. D: Lysates were used to measure NF-κB-p65 subunit DNA binding activity in an ELISA assay. E and F: Cell culture medium was collected 4 h after addition to cells, and concentration of TNF-α and IL-6 was assessed by Meso Scale Discovery ELISA assay. Values shown are mean ± SEM from muscle precursor cells from five individuals for each group. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob.
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Figure 2: NF-κB signaling in human myotubes from Non-Ob and obese subjects. Myoblasts isolated from Non-Ob (n = 5), Ob-NGT (n = 5), Ob-IGT (n = 5), and Ob-T2D (n = 5) patients were grown in culture until mature myotubes were formed. A: Lysates were immunoblotted to assess the phosphorylation status of IKK-α/β and total protein abundance of IκB-α and the p65 subunit of NF-κB. Equal gel loading was ascertained by immunoblotting with an antibody against β-tubulin. B: The effect of obesity (filled bars) on IKK-α/β phosphorylation and IκB-α abundance was quantified and expressed as a fold change from Non-Ob cells (open bars). C: IκB-α protein abundance in skeletal muscle tissue was compared with abundance in muscle precursor cells from the same individuals. D: Lysates were used to measure NF-κB-p65 subunit DNA binding activity in an ELISA assay. E and F: Cell culture medium was collected 4 h after addition to cells, and concentration of TNF-α and IL-6 was assessed by Meso Scale Discovery ELISA assay. Values shown are mean ± SEM from muscle precursor cells from five individuals for each group. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob.

Mentions: We wanted to assess whether this increased inflammation was conserved in our system of cultured muscle cells isolated from the same subjects. To assess this, we measured the phosphorylation of IKK-α/β and the associated loss in its downstream target, IκB-α, as a readout for NF-κB activation. We show that myocytes from Ob-T2D have threefold higher IKK-α/β phosphorylation and concomitant loss in IκB-α protein abundance of 45% (Fig. 2A and B) compared with Non-Ob myocytes. Importantly, we found that the level of IκB-α protein expression in skeletal muscle tissue and muscle precursor cells from the same individual had a significant correlation (P = 0.0034) with an r2 value of 0.5 (Fig. 2C). To assess whether this loss in IκB-α protein was sufficient to upregulate NF-κB activity, the ability of the p65 subunit of NF-κB to bind to DNA was measured. Figure 2D shows that Ob-T2D myocytes have a significantly higher level of p65 DNA binding compared with Non-Ob myocytes. It is important that Ob-NGT myocytes had comparable levels of NF-κB signaling with Non-Ob control subjects, whereas Ob-IGT myocytes seemed to have an intermediate level of NF-κB signaling (between that of Non-Ob and Ob-T2D). To fully elucidate the level of inflammation in these cells, the concentrations of TNF-α and IL-6 in cell culture media were measured. TNF-α concentrations were significantly higher in Ob-T2D compared with Non-Ob control subject concentrations (Fig. 2E). Of interest, concentrations of IL-6 in cell culture media from Ob-IGT and Ob-T2D was significantly lower than that in Non-Ob cell media (Fig. 2F). This latter finding supports the idea that IL-6 exerts an inhibitory effect on TNF-α in muscle (21).


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)

NF-κB signaling in human myotubes from Non-Ob and obese subjects. Myoblasts isolated from Non-Ob (n = 5), Ob-NGT (n = 5), Ob-IGT (n = 5), and Ob-T2D (n = 5) patients were grown in culture until mature myotubes were formed. A: Lysates were immunoblotted to assess the phosphorylation status of IKK-α/β and total protein abundance of IκB-α and the p65 subunit of NF-κB. Equal gel loading was ascertained by immunoblotting with an antibody against β-tubulin. B: The effect of obesity (filled bars) on IKK-α/β phosphorylation and IκB-α abundance was quantified and expressed as a fold change from Non-Ob cells (open bars). C: IκB-α protein abundance in skeletal muscle tissue was compared with abundance in muscle precursor cells from the same individuals. D: Lysates were used to measure NF-κB-p65 subunit DNA binding activity in an ELISA assay. E and F: Cell culture medium was collected 4 h after addition to cells, and concentration of TNF-α and IL-6 was assessed by Meso Scale Discovery ELISA assay. Values shown are mean ± SEM from muscle precursor cells from five individuals for each group. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob.
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Figure 2: NF-κB signaling in human myotubes from Non-Ob and obese subjects. Myoblasts isolated from Non-Ob (n = 5), Ob-NGT (n = 5), Ob-IGT (n = 5), and Ob-T2D (n = 5) patients were grown in culture until mature myotubes were formed. A: Lysates were immunoblotted to assess the phosphorylation status of IKK-α/β and total protein abundance of IκB-α and the p65 subunit of NF-κB. Equal gel loading was ascertained by immunoblotting with an antibody against β-tubulin. B: The effect of obesity (filled bars) on IKK-α/β phosphorylation and IκB-α abundance was quantified and expressed as a fold change from Non-Ob cells (open bars). C: IκB-α protein abundance in skeletal muscle tissue was compared with abundance in muscle precursor cells from the same individuals. D: Lysates were used to measure NF-κB-p65 subunit DNA binding activity in an ELISA assay. E and F: Cell culture medium was collected 4 h after addition to cells, and concentration of TNF-α and IL-6 was assessed by Meso Scale Discovery ELISA assay. Values shown are mean ± SEM from muscle precursor cells from five individuals for each group. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob.
Mentions: We wanted to assess whether this increased inflammation was conserved in our system of cultured muscle cells isolated from the same subjects. To assess this, we measured the phosphorylation of IKK-α/β and the associated loss in its downstream target, IκB-α, as a readout for NF-κB activation. We show that myocytes from Ob-T2D have threefold higher IKK-α/β phosphorylation and concomitant loss in IκB-α protein abundance of 45% (Fig. 2A and B) compared with Non-Ob myocytes. Importantly, we found that the level of IκB-α protein expression in skeletal muscle tissue and muscle precursor cells from the same individual had a significant correlation (P = 0.0034) with an r2 value of 0.5 (Fig. 2C). To assess whether this loss in IκB-α protein was sufficient to upregulate NF-κB activity, the ability of the p65 subunit of NF-κB to bind to DNA was measured. Figure 2D shows that Ob-T2D myocytes have a significantly higher level of p65 DNA binding compared with Non-Ob myocytes. It is important that Ob-NGT myocytes had comparable levels of NF-κB signaling with Non-Ob control subjects, whereas Ob-IGT myocytes seemed to have an intermediate level of NF-κB signaling (between that of Non-Ob and Ob-T2D). To fully elucidate the level of inflammation in these cells, the concentrations of TNF-α and IL-6 in cell culture media were measured. TNF-α concentrations were significantly higher in Ob-T2D compared with Non-Ob control subject concentrations (Fig. 2E). Of interest, concentrations of IL-6 in cell culture media from Ob-IGT and Ob-T2D was significantly lower than that in Non-Ob cell media (Fig. 2F). This latter finding supports the idea that IL-6 exerts an inhibitory effect on TNF-α in muscle (21).

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