Effect of GSK3 inhibitor CHIR99021 on IL-1β-induced pro-inflammatory cytokine IL-6 in adipose tissue and skeletal muscle.

<p>Human (<b>A,B</b>) omental adipose tissue and (<b>C,D</b>) skeletal muscle were incubated with 1 ng/ml IL-1β in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). (<b>A,C</b>) Gene expression for IL-6 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to IL-1β. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. IL-1β (one-way ANOVA). (<b>B,D</b>) The incubation medium was assayed for concentration of IL-6 release by ELISA. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. IL-1β (one-way ANOVA).</p

Effect of GSK3 inhibitor SB216763 on LPS-induced pro-inflammatory cytokines and chemokines in adipose tissue.

<p>Human omental adipose tissue was incubated with 10 µg/ml LPS in the absence or presence of 20 µM SB216763 (SB) for 20 h (n = 5 patients). (<b>A–E</b>) Gene expression for TNF-α, IL-1β, IL-6, IL-8 and MCP-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. LPS (one-way ANOVA). (<b>F–J</b>) The incubation medium was assayed for concentration of TNF-α, IL-1β, IL-6, IL-8 and MCP-1 release by ELISA. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. LPS (one-way ANOVA).</p

Effect of GSK3 inhibitor CHIR99021 on LPS-induced pro-inflammatory cytokines in skeletal muscle.

<p>Human skeletal muscle was incubated with 10 µg/ml LPS in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). (<b>A–C</b>) Gene expression for TNF-α, IL-1β and IL-6 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. LPS (one-way ANOVA). (<b>D–F</b>) The incubation medium was assayed for concentration of TNF-α, IL-1β and IL-6 release by ELISA. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. LPS (one-way ANOVA).</p

Effect of GSK3 inhibitor CHIR99021 on LPS-induced adhesion molecules in adipose tissue and skeletal muscle.

<p>Human (<b>A–D</b>) omental adipose tissue and (<b>E–H</b>) skeletal muscle were incubated with 10 µg/ml LPS in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). (<b>A,B,E,F</b>) Gene expression for VCAM-1 and ICAM-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. LPS (one-way ANOVA). (<b>C,D,G,H</b>) The incubation medium was assayed for concentration of sVCAM-1 and sICAM-1 release by ELISA. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. LPS (one-way ANOVA).</p

Phosphorylated GSKα/β expression in skeletal muscle from NGT and GDM women.

<p>Skeletal muscle was obtained from (<b>A–C</b>) non-obese and (<b>D–F</b>) obese women with NGT (n = 6 patients per group) and diet-controlled GDM (n = 6 patients per group) at the time of term Caesarean section. Phosphorylation of GSK3α at serine 21 (p-GSKα) and GSK3β at serine 9 (p-GSKβ) was analysed by immunoblotting and normalised to total GSK3β protein expression. The fold change was calculated relative to NGT and data is displayed as mean ±SEM. *<i>P</i><0.05 vs. NGT (Student's t-test). Representative Western blot from 3 NGT and 3 diet-controlled GDM patients is also shown.</p

Effect of GSK3 inhibitor CHIR99021 on IL-1β-induced adhesion molecules in adipose tissue and skeletal muscle.

<p>Human (<b>A–D</b>) omental adipose tissue and (<b>E–H</b>) skeletal muscle were incubated with 1 ng/ml IL-1β in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). (<b>A,B,E,F</b>) Gene expression for VCAM-1 and ICAM-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to IL-1β. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. IL-1β (one-way ANOVA). (<b>C,D,G,H</b>) The incubation medium was assayed for concentration of sVCAM-1 and sICAM-1 release by ELISA. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. IL-1β (one-way ANOVA).</p

Effect of GSK3 inhibitor CHIR99021 on LPS-induced pro-inflammatory chemokines in adipose tissue and skeletal muscle.

<p>Human (<b>A–D</b>) omental adipose tissue and (<b>E–H</b>) skeletal muscle were incubated with 10 µg/ml LPS in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). (<b>A,B,E,F</b>) Gene expression for IL-8 and MCP-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. LPS (one-way ANOVA). (<b>C,D,G,H</b>) The incubation medium was assayed for concentration of IL-8 and MCP-1 release by ELISA. Data displayed as mean ±SEM. *<i>P</i><0.05 vs. LPS (one-way ANOVA).</p

Phosphorylated GSKβ expression in adipose tissue from NGT and GDM women.

<p>Omental adipose tissue was obtained from (<b>A,B</b>) non-obese and (<b>C,D</b>) obese women with NGT (n = 6 patients per group) and diet-controlled GDM (n = 6 patients per group) at the time of term Caesarean section. Phosphorylation of GSK3α at serine 21 (p-GSKα) was very low and thus not analysed further. Phosphorylation of GSK3β at serine 9 (p-GSKβ) was analysed by immunoblotting and normalised to total GSK3β protein expression. The fold change was calculated relative to NGT and data is displayed as mean ±SEM. *<i>P</i><0.05 vs. NGT (Student's t-test). Representative Western blot from 3 NGT and 3 diet-controlled GDM patients is also shown.</p

Anti-diabetic drugs inhibits thapsigargin-induced IL-1α and IL-1β secretion in tissues primed with LPS, poly(I:C) or TNF-α.

<p>Adipose tissue was incubated in the absence or presence of 0.5 mM metformin (Metf) or 25 μM glibenclamide (Glib)for 60 min prior to the addition of <b>(A-C)</b> 10 μg/ml LPS, <b>(D-F)</b> 20 μg/ml poly(I:C) or <b>(G-I)</b> 10 ng/ml TNF-α. After 18 h incubation, tissues were incubated with 30 μM thapsigargin (TG) for a further 2 h (n = 6 patients per treatment). The incubation medium was assayed for IL-1α, IL-1β and IL-6 concentration by ELISA. Each bar represents mean concentration ± SEM. *<i>P</i><0.05 vs. LPS (one way ANOVA); **<i>P</i><0.05 vs. LPS + TG (one way ANOVA); #<i>P</i><0.05 vs. poly(I:C) (one-way ANOVA); ##<i>P</i><0.05 vs. poly(I:C) + TG (one-way ANOVA); †<i>P</i><0.05 vs. TNF-α (one-way ANOVA); ††<i>P</i><0.05 vs. TNF-α + TG (one-way ANOVA).</p

ER stress is increased in adipose tissue from obese pregnant women and with GDM.

<p>Adipose tissue was obtained from NGT lean (n = 6 patients) and obese (n = 6 patients) pregnant women at the time of term Caesarean section. The protein abundance of <b>(A)</b> GRP78, <b>(B)</b> IRE1α and <b>(C)</b> XBP-1s was analysed by Western blot. Protein abundance was normalised to Ponceau S staining and the fold change was calculated relative to the lean group. Data is presented as mean ± SEM. *<i>P</i><0.05 vs. lean (Student’s t-test). Representative Western blot from six patients (3 obese and 3 lean) is also shown. Adipose tissue were also obtained from <b>(D-F)</b> lean and <b>(G-I)</b> obese pregnant women with NGT (n = 6 patients per group), diet-controlled GDM (n = 6 patients per group), insulin-controlled GDM (n = 6 patients) and combined GDM (n = 12 patients per group) at the time of term Caesarean section. The protein abundance of <b>(D-G)</b> GRP78, <b>(E-H)</b> IRE1α and <b>(F-I)</b> XBP-1s was analysed by Western blot. Protein abundance was normalised to Ponceau S staining and the fold change was calculated relative to the NGT group. Data is presented as mean ± SEM. *<i>P</i><0.05 vs. NGT (Student’s t-test). Representative Western blot from nine patients (3 NGT, 3 GDM diet and 3 GDM insulin) is also shown.</p