Fig 2: CDN1163 showed no changes in TBC1D4Thr642 phosphorylation. Human myotubes were pretreated with 0.1 ?µM CDN1163 for 5 days. Prior to harvesting, half of the samples were treated with 100 ?nM insulin. Proteins were isolated and expressions of total TBC1D4 and TBC1D4 phosphorylated at Thr642 were assessed by immunoblotting. Expression of pTBC1D4 was normalised to total TBC1D4 expression. Data are presented as mean ?± ?SEM from eight individual experiments (n ?= ?8). *Statistically significant compared to basal (unstimulated) cells (p ?< ?0.05, unpaired t-test).

Fig 3: Protein content of GSK3, Akt, AS160, and HKII in skeletal muscle of HIV-infected patients before and after 16 weeks of endurance (n = 8) and strength training (n = 10). Values are shown as posttraining values divided by pretraining values thereby expressing fold changes in response to training. Dotted line shows pretraining levels. Values are means ± SE. *P < 0.05 (vs. pretraining levels).

Fig 4: SBI-0206965 inhibits insulin-stimulated glucose transport. Quantification of (A) glucose transport and (B) Akt Thr308, (C) Akt Ser473, (D) TBC1D4 Thr642, and (E) ULK Ser757 phosphorylation in soleus muscles stimulated with or without insulin (60 nM, 20 min) and with or without 10 µM SBI-0206965 for 1 h. (F) Representative blots of quantified and total proteins. ANOVA main or interaction effects are indicated in the panels. **/*** p < 0.01/0.001 vs. Basal, #/##/### p < 0.05/0.01/0.001 vs. DMSO. n = 3. All values are shown as mean ± SEM.

Fig 5: DKI mice are glucose intolerant but maintain whole-body insulin sensitivity. (a) Blood glucose was assessed via tail tip bleed in male DKI and WT mice following a 6 h fast. Male mice, n = 9, 17–20 wk. (b) Blood was collected via retro-orbital bleed from DKI and WT mice following a 6 h fast, and serum samples were analyzed for insulin concentration. Male mice, n = 10–12, 20–27 wk. (c and inset) Following a 6 h fast, DKI and WT mice were injected intraperitoneally with glucose (1 g·kg-1 total lean mass), and blood glucose responses were measured over 2 h post-injection and area under the curve (AUC), with baseline as the mean blood glucose at time 0 for each genotype, was calculated to assess glucose handling. Male mice, n = 7–10, 17–20 wk. (d and inset) Following a 4 h fast, DKI and WT mice were injected intraperitoneally with insulin (0.5 units·kg-1 total body mass), and blood glucose responses were measured over 2 h and AUC was calculated. Male mice, n = 9–10, 17–20 wk. Skeletal muscle was collected from male DKI and WT mice in the fed state, and phosphorylation status and total content of proteins associated with skeletal muscle glucose transport were quantified. (e) Representative immunoblots and stain-free image and quantified results of (f) Akt p-S573, (g) total Akt, (h) AS160 p-T642, (i) total AS160, and (j) total GLUT4 content in DKI versus WT skeletal muscle. Protein quantification was performed by normalizing the band intensity of the protein of interest to total lane protein using stain-free imaging, and phosphorylation was expressed relative to respective total protein content. Fed male mice, n = 7–9, 20–32 wk. * p < 0.05, ** p < 0.01.