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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 insulin signaling in human myotubes. Myotubes were treated with 100 μmol/L A769662 and/or 100 nmol/L insulin for 30 min before (A) assaying 2-deoxyglucose uptake or 100 nmol/L insulin for 10 min before (B) immunoblotting to assess the phosphorylation status of PKB/Akt and ACC and the total protein abundance of PKB/Akt. Values are mean ± SEM from five separate experiments; glucose uptake values were performed in triplicate. *P < 0.05 vs. control cells.
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Figure 7: Effect of pharmacological AMPK activation on insulin signaling in human myotubes. Myotubes were treated with 100 μmol/L A769662 and/or 100 nmol/L insulin for 30 min before (A) assaying 2-deoxyglucose uptake or 100 nmol/L insulin for 10 min before (B) immunoblotting to assess the phosphorylation status of PKB/Akt and ACC and the total protein abundance of PKB/Akt. Values are mean ± SEM from five separate experiments; glucose uptake values were performed in triplicate. *P < 0.05 vs. control cells.

Mentions: A769662 treatment had no additive effect on insulin-stimulated glucose uptake or PKB/Akt phosphorylation in any of the cell groups tested (Fig. 7A and B). As shown in Fig. 2, Ob-IGT and Ob-T2D myocytes show no response to insulin as measured by glucose uptake and PKB/Akt phosphorylation. It should be noted, however, that there was a trend toward increased basal glucose uptake in Ob-T2D myocytes (P = 0.065). Of interest, 4 h of A769662 treatment, which was sufficient to attenuate inflammation, did not increase insulin sensitivity of Ob-IGT or Ob-T2D myotubes (Fig. 6A). This finding was also confirmed at the level of PKB/Akt phosphorylation, which remained suppressed in Ob-IGT and Ob-T2D myocytes compared with Non-Ob and Ob-NGT myocytes (Fig. 7B).


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 insulin signaling in human myotubes. Myotubes were treated with 100 μmol/L A769662 and/or 100 nmol/L insulin for 30 min before (A) assaying 2-deoxyglucose uptake or 100 nmol/L insulin for 10 min before (B) immunoblotting to assess the phosphorylation status of PKB/Akt and ACC and the total protein abundance of PKB/Akt. Values are mean ± SEM from five separate experiments; glucose uptake values were performed in triplicate. *P < 0.05 vs. control cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Effect of pharmacological AMPK activation on insulin signaling in human myotubes. Myotubes were treated with 100 μmol/L A769662 and/or 100 nmol/L insulin for 30 min before (A) assaying 2-deoxyglucose uptake or 100 nmol/L insulin for 10 min before (B) immunoblotting to assess the phosphorylation status of PKB/Akt and ACC and the total protein abundance of PKB/Akt. Values are mean ± SEM from five separate experiments; glucose uptake values were performed in triplicate. *P < 0.05 vs. control cells.
Mentions: A769662 treatment had no additive effect on insulin-stimulated glucose uptake or PKB/Akt phosphorylation in any of the cell groups tested (Fig. 7A and B). As shown in Fig. 2, Ob-IGT and Ob-T2D myocytes show no response to insulin as measured by glucose uptake and PKB/Akt phosphorylation. It should be noted, however, that there was a trend toward increased basal glucose uptake in Ob-T2D myocytes (P = 0.065). Of interest, 4 h of A769662 treatment, which was sufficient to attenuate inflammation, did not increase insulin sensitivity of Ob-IGT or Ob-T2D myotubes (Fig. 6A). This finding was also confirmed at the level of PKB/Akt phosphorylation, which remained suppressed in Ob-IGT and Ob-T2D myocytes compared with Non-Ob and Ob-NGT myocytes (Fig. 7B).

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