Fig 2: Western blots of total AS160 protein content (A) and basal (open bars) and insulin-stimulated (filled bars) phosphorylation of AKT at serine 318 (B) and serine 588 (C) from red gastrocnemius muscle. Data are expressed relative to basal WT-Sed group, mean ± SE, n = 8–10. Experimental groups not sharing a letter are significantly different, P < 0.05. WT-Sed, wild-type+no exercise; WT-Ex, wild-type+exercise training; AdKO-Sed, Ad knockout+no exercise; Ad-Ex, Ad knockout+exercise training.

Fig 3: Rab14 recruitment to chlamydial inclusions is hindered by Akt/AS160 inhibition. (A) HeLa cells were infected with Ct (MOI 1) for 24 h. Immunodetection of endogenous AS160 with rabbit antibody followed by Cy5-coupled secondary antibodies. Detection of endogenous Rab14 was performed with mouse antibody followed by FITC-coupled secondary antibodies. Propidium iodide (PI) stained DNA. (B) Infected cells (MOI 1) were incubated with DMSO or increasing concentrations of iAkt (2, 5, and 10 µM) from 2 h pi until fixation at 24 h pi. Subcellular distribution of endogenous Rab14 was observed by confocal microscopy using mouse primary antibody followed by Cy3-conjugated anti-mouse secondary antibody. Inclusion membrane was detected using rabbit anti-CT529 antibody followed by FITC-conjugated anti-rabbit IgG. DAPI was used to stain DNA. Bars represent 10 µm. Images are representative of three independent experiments.

Fig 4: Disruption of the Akt/AS160 pathway causes retention of lipids at the Golgi apparatus. (A) Equal amounts of lipids extracted from infected cells incubated with DMSO, 2 µM or 10 µM iAkt were resolved by thin layer chromatography (TLC). A representative TLC from two independent experiments is shown. (B) HeLa cells were transfected with either siRNA-Luc or siRNA-AS160 and, 48 h later, infected with GFP-expressing C. trachomatis (MOI of 1). AS160-silenced and control cells were incubated with DMSO or increasing concentrations of iAkt (2 or 10 µM). After 24 h of infection and before fixation, cells were incubated for 30 min with BODIPY TR ceramide at 4°C in serum-free DMEM. Then, cells were washed with PBS and incubated with DMEM supplemented with FBS for 30 min at 37°C. Golgi apparatus was immunodetected in fixed cells with mouse anti-GM130 monoclonal antibody followed by Cy5 coupled anti-mouse secondary antibody. Bars show the average fluorescence intensity of lipids at the Golgi apparatus. ImageJ was used for this analysis. (C) Representative images of two independent experiments performed as indicated in (B). In the upper panels, subcellular distribution of sphigolipids appears in red. GFP expressing bacteria are shown in green. Golgi was stained with mouse anti-GM130 followed by anti-mouse Cy5-coupled secondary antibody (white). In the lower panel, fire scale represents the fluorescence intensity associated to sphingolipids. White lines demarcate Golgi structures. Nuclei were stained with DAPI. Asterisks indicate inclusions.

Fig 5: Effect of BBR on regulating the PI3K/Akt/AS160/GLUT1 pathway. Protein was extracted from cultured GMCs. GAPDH was used as internal controls. Intensity of PI3K, AKT, p-AKT, AS160, p-AS160, Total GLUT1 and m-GLUT1 was quantified, normalized to internal control and expressed as mean ± SEM. (A,B) The levels of PI3K, Akt, p-Akt, AS160, p-AS160, Total GLUT1 and m-GLUT1 were detected in each group; (C) RT-qPCR analyses were performed to detect the mRNA expression of PI3K, Akt, AS160 and GLUT1 in each group. Relative expression of PI3K, Akt, AS160 and GLUT1. Data are expressed as Mean ± SEM, n = 3. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. HG group. HG: high glucose; BBR: HG + BBR (60 µmol/L); LY294002 (HG + LY294002); Metformin: HG + metformin (5 mmol/L)