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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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Phenotypical insulin sensitivity is retained in human myotubes in culture. Myotubes were treated with 100 nmol/L insulin for 30 min before (A) assaying 2-deoxyglucose uptake or for 10 min before (B) immunoblotting to assess the phosphorylation status of PKB/Akt and extracellular signal–related kinase-1/2 and the total protein abundance of PKB/Akt. C: Effect of insulin on PKB/Akt phosphorylation was quantified and expressed as a fold change from untreated Non-Ob cells. Values are mean ± SEM from five separate experiments; glucose uptake values were performed in triplicate. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob; ***P < 0.0005 vs. Non-Ob.
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Figure 3: Phenotypical insulin sensitivity is retained in human myotubes in culture. Myotubes were treated with 100 nmol/L insulin for 30 min before (A) assaying 2-deoxyglucose uptake or for 10 min before (B) immunoblotting to assess the phosphorylation status of PKB/Akt and extracellular signal–related kinase-1/2 and the total protein abundance of PKB/Akt. C: Effect of insulin on PKB/Akt phosphorylation was quantified and expressed as a fold change from untreated Non-Ob cells. Values are mean ± SEM from five separate experiments; glucose uptake values were performed in triplicate. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob; ***P < 0.0005 vs. Non-Ob.

Mentions: To relate the inflammatory status with glucose tolerance in the cultured myocytes, it was important not only to refer to the subjects from whom cells were isolated (as shown in Table 1) but also to confirm that impaired insulin sensitivity was retained in cultured myocytes. To ascertain this, insulin-stimulated glucose uptake and PKB/Akt phosphorylation of myocytes were measured. Both Non-Ob and Ob-NGT myocytes had comparable levels of basal and insulin-stimulated glucose uptake, with a significant increase in glucose uptake (approximately twofold) in response to insulin (Fig. 3A). It is important that both Ob-IGT and Ob-T2D myocytes had no increase in glucose uptake when challenged with insulin. This lack of insulin sensitivity in these cells was reflected in the loss of insulin-stimulated PKB/Akt Ser 473 phosphorylation (Fig. 3B and C). Hence, the in vivo glucose tolerance status of the donors is reflected in cultured myocytes.


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)

Phenotypical insulin sensitivity is retained in human myotubes in culture. Myotubes were treated with 100 nmol/L insulin for 30 min before (A) assaying 2-deoxyglucose uptake or for 10 min before (B) immunoblotting to assess the phosphorylation status of PKB/Akt and extracellular signal–related kinase-1/2 and the total protein abundance of PKB/Akt. C: Effect of insulin on PKB/Akt phosphorylation was quantified and expressed as a fold change from untreated Non-Ob cells. Values are mean ± SEM from five separate experiments; glucose uptake values were performed in triplicate. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob; ***P < 0.0005 vs. Non-Ob.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Phenotypical insulin sensitivity is retained in human myotubes in culture. Myotubes were treated with 100 nmol/L insulin for 30 min before (A) assaying 2-deoxyglucose uptake or for 10 min before (B) immunoblotting to assess the phosphorylation status of PKB/Akt and extracellular signal–related kinase-1/2 and the total protein abundance of PKB/Akt. C: Effect of insulin on PKB/Akt phosphorylation was quantified and expressed as a fold change from untreated Non-Ob cells. Values are mean ± SEM from five separate experiments; glucose uptake values were performed in triplicate. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob; ***P < 0.0005 vs. Non-Ob.
Mentions: To relate the inflammatory status with glucose tolerance in the cultured myocytes, it was important not only to refer to the subjects from whom cells were isolated (as shown in Table 1) but also to confirm that impaired insulin sensitivity was retained in cultured myocytes. To ascertain this, insulin-stimulated glucose uptake and PKB/Akt phosphorylation of myocytes were measured. Both Non-Ob and Ob-NGT myocytes had comparable levels of basal and insulin-stimulated glucose uptake, with a significant increase in glucose uptake (approximately twofold) in response to insulin (Fig. 3A). It is important that both Ob-IGT and Ob-T2D myocytes had no increase in glucose uptake when challenged with insulin. This lack of insulin sensitivity in these cells was reflected in the loss of insulin-stimulated PKB/Akt Ser 473 phosphorylation (Fig. 3B and C). Hence, the in vivo glucose tolerance status of the donors is reflected in cultured myocytes.

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