Fig 2: Expression levels of FTO, AS160 Ser588 phosphorylation, and phosphorylated AMPK in the vastus lateralis muscle. a Representative Western Blots of pAS160Ser588, total AS160, pAMPK, total AMPK, FTO and Actin for each genotype measured during pre-exercise rest (R), and at 10 mins and 90 mins during passive recovery following HI intensity exercise. b & c Relative expression of FTO protein when normalised to Actin following HI and LO intensity exercise respectively. d Relative expression of AS160 Ser588 phosphorylation (pAS160Ser588) relative to total AS160 following HI intensity exercise. e Relative expression of phosphorylated AMPK relative to total AMPK following HI intensity exercise. Data are expressed as mean ± SEM of the fold change at 10 and 90 mins compared to pre-exercise measurements (set arbitrarily to 1). ‘a’ represents significant main effect for time (p < 0.05)

Fig 3: LPS induces inflammation and inhibits insulin signaling in human myotubes in a time-dependent manner.Human myotubes were either left untreated or treated with 100 ng/ml LPS for a varying length of time. The time course of I?Ba protein degradation (A), JNK phosphorylation (B), and insulin-stimulated IRS-1-tyr612 (E), Akt (F), and AS160 (G) phosphorylation following LPS treatment was determined by Western blotting. The time course of MCP-1 (C) and IL-6 (D) mRNA expression following LPS treatment was determined by quantitative real time PCR. All values are the mean ± SD of triplicates. *P<0.05; #P<0.05 compared to basal.

Fig 4: TAK-242 prevents LPS-induced inflammation and insulin resistance in human myotubes.Human myotubes were either left untreated or treated with 100 ng/ml LPS for 12 h. Prior to LPS exposure, cells were pre-treated with 1 µM TAK-242 or vehicle control for 1h. JNK phosphorylation (A), insulin-stimulated IRS-1-tyr612 (E), Akt (F), and AS160 (G) phosphorylation were determined by Western blotting. MCP-1 (B) and IL-6 (C) mRNA expression was determined by real time PCR. Glucose transport (D) was determined by measuring 3H-2-DG uptake. The absolute basal glucose transport rates were 43.2±3.2, 46.4±4.1, 46.7±5.6 and 46.5±6.8 pmol/mg,min in cells without LPS, cells with LPS, TAK-242-pretreated cells without LPS, and TAK-242-pretreated cells with LPS, respectively. Results for glucose transport are expressed as the mean ± SEM of data obtained from 5 subjects. All the rest of the values are the mean ± SD of triplicate determinations. Similar experiments were repeated 4 times using cells isolated from different subjects, and representative results are shown. *P<0.05; #P<0.05 compared to basal.

Fig 5: HSP70 knockdown (KD) attenuated the protective effect of Nar on HG-induced alterations of endothelial function. The siRNA technique was used to silence HSP70 in HG-treated HUVECs. The cells were then exposed to NG (5.5 mM glucose) or HG (30 mM glucose) media with or without the 50 mM of Nar for 36 h. To observe the effect of Nar on insulin signaling, the cells were treated with or without 100 nM of insulin for 10 min. (a) Effect of HSP70 KD on ROS formation by DHE staining. (b) Quantification of relative DHE fluorescence in (a). (c) Effect of HSP70 KD on intracellular MDA concentration. (d) Effect of HSP70 KD on NF-?B activity. (e) Effect of HSP70 KD on IL-6 concentration in the culture media. (f) Effect of HSP70 KD on mRNA levels of cell adhesion molecules ICAM-1 and VCAM-1. (g) Effect of HSP70 KD on insulin-stimulated phosphorylation of Akt and its downstream AS160. (h) Quantification of phosphorylated Akt and AS160 in (g). (i) Effect of HSP70 KD on insulin-stimulated 2-DG uptake. Data are expressed as means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus the indicated group.