Fig 2: Proposed model of FKBP51 as a regulator of glucose uptake. FKBP51 scaffolds Akt2, PHLPP1, and AS160. The associations between FKBP51, AKT2, PHLPP1, and AS160 may be either direct or indirect through additional intermediate proteins. In the presence of FKBP51, PHLPP1 phosphatase activity is directed towards AKT2 to favor inactive Akt2, decreased AS160 phosphorylation and reduced glucose uptake. By contrast, loss of FKBP51’s scaffolding function leads to increased AKT2. In the presence of SAFit2, a conformational change within FKBP51 disrupts its ability to form a complex with AS160, while simultaneously enhancing AKT2-AS160 binding. Ultimately, loss of FKBP51 and FKBP51 antagonism with SAFit2 both promote glucose uptake. “?” refers to possible unidentified intermediate proteins within the FKBP51 signaling complex. Curved arrows indicate PHLPP1-mediated dephosphorylation of AKT2 at Ser473. The green double arrow indicates enhanced binding between AKT2 and AS160. Green outlines reflect enhanced phosphorylation; red outlines reflect decreased phosphorylation. The width of the arrows correspond to the magnitude of downstream activation

Fig 3: FKBP51 affects insulin signaling and consequently glucose uptake. a, b Insulin signaling was enhanced in EDL (a) and soleus (b) skeletal muscles of 51KO mice compared to WT mice as assessed by pAKT2, pAS160, and pp70S6K protein expression. c Following subcellular fractionation to isolate the plasma membrane compartment, we observed increased GLUT4 expression in skeletal muscle membrane fractions of 51KO mice. d In primary EDL myotubes, loss of FKBP51 heightened glucose uptake under both no insulin and insulin-stimulated states. For quantification of phosphorylated protein, n = 6 per group. For GLUT4 membrane localization, n = 3 per group. For glucose uptake experiments, 3 wells for each condition were measured. The data are expressed as relative fold change compared to wild-type condition ± SEM. + P < 0.05, ++ P < 0.01, +++ P < 0.001, ## P < 0.01, two-tailed t tests for a–d, two-way ANOVA for e; + significant genotype effect, # significant insulin effect, T trend for genotype. Supplementary Fig. 12 shows uncropped gel images

Fig 4: FKBP51 antagonism parallels the metabolic effects resulting from genetic ablation of FKBP51. a A single application of a slow-release-formulated SAFit2 gel had no effect on glucose tolerance or (b) Body weight under control diet conditions. c Under HFD conditions, acute administration of SAFit2 gel significantly improved glucose tolerance. d The effects of SAFit2 on glucose tolerance under HFD conditions were not present in 51KO. e Despite the effects of acute SAFit2 on glucose tolerance under HFD conditions, there was no effect on body weight (f) 10-day SAFit2 treatment had no significant effect on body weight. g Despite no effect on body weight, SAFit2 treatment significantly improved glucose tolerance as reflected in the glucose area under the curve (AUC) for the GTT measured on treatment day 8. h At the experimental end point (following 30 days of treatment), mice treated with SAFit2 weighed significantly less than their diet counterparts. Nevertheless, mice fed with the HFD remained significantly heavier independent of treatment. i The extended SAFit2 treatment schedule furthermore protected against HFD-induced impaired glucose tolerance as reflected in the glucose AUC measured on day 25. For acute treatment schedule in C57BL6 n = 12 per treatment group; for acute treatment in 51KO n = 8 per treatment group. For the 10-day treatment schedule, n = 8 per treatment group. For 30-day treatment schedule n = 12 Vehicle-Control, n = 13 SAFit2-Control, n = 12 Vehicle-HFD, n = 13 SAFit2-HFD. The data are represented as mean ± SEM. + P < 0.05; # P < 0.05, two-tailed t test for a–g, two-way ANOVA for h, two-way ANOVA plus Bonferroni testing for i; + significant treatment effect; # significant diet effect

Fig 5: FKBP51 antagonism affects AKT2-AS160 signaling complex. Tissue lysates from 30-day vehicle-treated or SAFit2-treated mice exposed to HFD were immunoprecipitated with anti-AKT2 and anti-FKBP51 and then analyzed by Western blot using FKBP51, (p)AKT2, (p)AS160, and PHLPP1. a, b Immunoprecipitation reactions revealed that SAFit2 treatment increased binding between (p)AKT2 and (p)AS160 in soleus (a) and EDL (b) muscles, while simultaneously decreased binding between FKBP51 and AS160 in both muscle types. For co-immunoprecipitation experiments n = 3 per group. Data are expressed as relative fold change compared to vehicle condition ± SEM. + P < 0.05, two-tailed t tests for a, b; + significant SAFit2 treatment effect. Supplementary Fig. 15 shows uncropped gel images