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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 skeletal muscle from Non-Ob and obese subjects. Muscle biopsies from vastus lateralis were taken from Non-Ob (BMI 23.4 ± 2.4), Ob-NGT, Ob-IGT, and Ob-T2D (BMI 35.1 ± 3.2) volunteers. Total protein lysate from biopsies was then used for (A) SDS-PAGE (20 μg total protein lysate) and immunoblotted for IκB-α and β-tubulin or (C) 10 μg total protein lysate to measure NF-κB-p65 subunit DNA binding activity in an ELISA assay. B: The effect of obesity (filled bars) on IκB-α abundance was quantified and expressed as a fold change from Non-Ob cells (open bar). Values shown are mean ± SEM from five individuals for each group. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob.
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Figure 1: NF-κB signaling in human skeletal muscle from Non-Ob and obese subjects. Muscle biopsies from vastus lateralis were taken from Non-Ob (BMI 23.4 ± 2.4), Ob-NGT, Ob-IGT, and Ob-T2D (BMI 35.1 ± 3.2) volunteers. Total protein lysate from biopsies was then used for (A) SDS-PAGE (20 μg total protein lysate) and immunoblotted for IκB-α and β-tubulin or (C) 10 μg total protein lysate to measure NF-κB-p65 subunit DNA binding activity in an ELISA assay. B: The effect of obesity (filled bars) on IκB-α abundance was quantified and expressed as a fold change from Non-Ob cells (open bar). Values shown are mean ± SEM from five individuals for each group. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob.

Mentions: The clinical characteristics of the subjects used in this study are shown in Table 1. All subjects were age-matched (54 ± 1 years) from a mixture of males and females. All obese subjects were matched for BMI (34.2 ± 0.9), which was statistically higher than Non-Ob control subjects (P < 0.05) (Table 1). We have previously shown that plasma markers of inflammation (IL-6, TNF-α, and C-reactive protein) were significantly upregulated in both obese and diabetic subjects compared with healthy nonobese control subjects (19,20). However, inflammation in skeletal muscle isolated from these subjects was not investigated. Here we show that skeletal muscle from Ob-T2D patients has significantly lower protein expression of IκB-α (∼70% lower) when compared with skeletal muscle from Non-Ob subjects (Fig. 1A and B). It is important that this lower expression of IκB-α in Ob-T2D muscle was sufficient to significantly increase NF-κB (p65 subunit) DNA binding activity (Fig. 1C). There was no significant difference in NF-κB-p65 subunit DNA binding activity between skeletal muscle from Non-Ob and either Ob-NGT or Ob-IGT muscle.


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 skeletal muscle from Non-Ob and obese subjects. Muscle biopsies from vastus lateralis were taken from Non-Ob (BMI 23.4 ± 2.4), Ob-NGT, Ob-IGT, and Ob-T2D (BMI 35.1 ± 3.2) volunteers. Total protein lysate from biopsies was then used for (A) SDS-PAGE (20 μg total protein lysate) and immunoblotted for IκB-α and β-tubulin or (C) 10 μg total protein lysate to measure NF-κB-p65 subunit DNA binding activity in an ELISA assay. B: The effect of obesity (filled bars) on IκB-α abundance was quantified and expressed as a fold change from Non-Ob cells (open bar). Values shown are mean ± SEM from five individuals for each group. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: NF-κB signaling in human skeletal muscle from Non-Ob and obese subjects. Muscle biopsies from vastus lateralis were taken from Non-Ob (BMI 23.4 ± 2.4), Ob-NGT, Ob-IGT, and Ob-T2D (BMI 35.1 ± 3.2) volunteers. Total protein lysate from biopsies was then used for (A) SDS-PAGE (20 μg total protein lysate) and immunoblotted for IκB-α and β-tubulin or (C) 10 μg total protein lysate to measure NF-κB-p65 subunit DNA binding activity in an ELISA assay. B: The effect of obesity (filled bars) on IκB-α abundance was quantified and expressed as a fold change from Non-Ob cells (open bar). Values shown are mean ± SEM from five individuals for each group. *P < 0.05 vs. Non-Ob. **P < 0.005 vs. Non-Ob.
Mentions: The clinical characteristics of the subjects used in this study are shown in Table 1. All subjects were age-matched (54 ± 1 years) from a mixture of males and females. All obese subjects were matched for BMI (34.2 ± 0.9), which was statistically higher than Non-Ob control subjects (P < 0.05) (Table 1). We have previously shown that plasma markers of inflammation (IL-6, TNF-α, and C-reactive protein) were significantly upregulated in both obese and diabetic subjects compared with healthy nonobese control subjects (19,20). However, inflammation in skeletal muscle isolated from these subjects was not investigated. Here we show that skeletal muscle from Ob-T2D patients has significantly lower protein expression of IκB-α (∼70% lower) when compared with skeletal muscle from Non-Ob subjects (Fig. 1A and B). It is important that this lower expression of IκB-α in Ob-T2D muscle was sufficient to significantly increase NF-κB (p65 subunit) DNA binding activity (Fig. 1C). There was no significant difference in NF-κB-p65 subunit DNA binding activity between skeletal muscle from Non-Ob and either Ob-NGT or Ob-IGT muscle.

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