Fig 2: Activation of mTORC2 engenders structural remodelling of nociceptive distal endings in epidermis.A-443654 (20 µL of 8 mM, intraplantar) was injected in ControltdTom (A) and Rictor-cKOtdTom (B) mice, and glabrous skin from the injected paw was collected 24 hours later. Representative images of the low (top) and high (bottom) magnification of nociceptive fibers in the epidermis, acquired using confocal microscopy with AiryScan. A-443654 increases main fiber length in ControltdTom but not Rictor-cKOtdTom mice (C, n = 5 mice per group). Similarly, A-443654 increases the number of branches per fiber in ControltdTom but not Rictor-cKOtdTom mice (D, n = 5 mice per group). ControltdTom (E) and Rictor-cKOtdTom (F) mice were injected with CFA (intraplantar), and glabrous skin from the injected paw was collected at day 1, day 3, day 7, and day 14 after injection. CFA increases main fiber length in ControltdTom but not Rictor-cKOtdTom mice at day 1 after CFA (G, n = 4–5 per group). Similarly, CFA increases the number of branches per fiber in ControltdTom but not Rictor-cKOtdTom mice (H, n = 4–5 per group). Statistics are based on 2-way ANOVA followed by Bonferroni’s post hoc comparison. All data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bars: 20 µm.

Fig 3: Rapamycin activates PI3K-Akt signaling, which in turn inhibits CTGF expression in HPCs. (A) LE/6 and WB-F344 cells were treated with rapamycin at indicated concentrations for 6 h. Lysates were subjected to Western blot analysis with antibodies against indicated proteins. ß-actin was used as a loading control; (B–D) LE/6 and WB-F344 cells were transfected with lentivirus carrying shRNA against mTOR, Raptor, Rictor, or scramble shRNA and Western blot analysis showed the expression of these proteins. ß-actin was used as a loading control; (E) LE/6 and WB-F344 cells were treated with rapamycin and LY294002 for 6 h. Lysates were subjected to Western blot analysis with antibodies against indicated proteins. ß-actin was used as a loading control; (F,G) LE-shmTOR#2, WB-shmTOR#2 and their scramble control (shScramble) were incubated with LY294002 or MK2206 at indicated concentrations for 6 h. Lysates were subjected to Western blot analysis with antibodies against indicated proteins. ß-actin was used as a loading control.

Fig 4: PPARd activation attenuated EtOH-induced fat accumulation and insulin resistance in mouse skeletal muscle. (A) TG levels in skeletal muscle of GW, GW plus EtOH, EtOH consumed mice (n = 6 muscles/group). (B,C) Blood glucose disposal capacities were measured using the intra-peritoneal glucose tolerance test (IPGTT) (n = 6/group). (D–H) AKT signaling and glucose uptakes were measured in EV or PPARd overexpressing myotubes treated with saline or EtOH (n = 6 myotubes/group). (I,J) Activation of Rictor and Raptor was measured time dependently in EtOH and PPARd overexpressing myotubes (n = 6 myotubes/group). (K) GLUT4 was measured in EV or PPARd overexpressing myotubes treated with saline or EtOH (n = 6 myotubes/group). Values are means ± SE. Significance was determined using two-way ANOVA with Tukey’s correction. *p < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: mTORC1 and mTORC2 inactivation have opposite effects on asynchronous synaptic vesicle release.(A1) Example traces of normalized EPSCs evoked at 0.1 Hz from Raptor-Con (black) and Raptor-KO (red) neurons. The gray shaded area represents the area under the curve of the synchronous component of synaptic vesicle (SV) release, and the pink shaded area represents the asynchronous component. (A2) Plot showing the rate constants for asynchronous SV release of Raptor-Con (black) and Raptor-KO (red) neurons. (B1) Example traces of normalized EPSCs evoked at 0.1 Hz from Rictor-Con (black) and Rictor-KO (blue) neurons shaded to highlight the synchronous (gray) and asynchronous (pink) components of SV release. (B2) Plot showing the rate constants for asynchronous SV release of Rictor-Con (black) and Rictor-KO (blue) neurons. (C1) Example traces of normalized EPSCs at the end of a 10 Hz train from Raptor-Con (black) and Raptor-KO (red) neurons. The gray shaded area represents the area under the curve of the synchronous component of SV release and the pink shaded area represents the asynchronous component. (C2) Plot showing the rate constants for asynchronous SV release at the end of a 10 Hz train from Raptor-Con (black) and Raptor-KO (red) neurons. (D1) Example traces of normalized EPSCs at the end of a 10 Hz train from Rictor-Con (black) and Rictor-KO (blue) neurons shaded to highlight the synchronous (gray) and asynchronous (pink) components of SV release. (D2) Plot showing the rate constants for asynchronous SV release at the end of a 10 Hz train from Rictor-Con (black) and Rictor-KO (blue) neurons. In the dot plots, each dot represents the mean value from one neuron obtained from three independent cultures per genotype, and the bars show the estimated marginal means and s.e.m. *=p < 0.05, **=p < 0.01, and n.s. = p > 0.05, as tested with Generalized Estimating Equations.