Figure 7. T1R3 depletion induces autophagy.

(A) H9C2 cells in complete growth medium or in KRBH were permeabilized and LC3 was detected by immunofluorescence. (C) H9C2 cells transfected with either control non target siRNA1 or T1R3 siRNA1 were incubated in complete growth medium and LC3 was detected as above. (B and D) LC3 punctae in Z-stack images from (A and C, respectively) were quantitated as described in Extended Experimental Procedures and are the mean +/− SEM of LC3 punctae/cell. (E) Lysates of H9C2 cells that had been transfected with the indicated siRNA were incubated in KRBH for 2 hrs before lysis. Lysates were immunoblotted to detect the indicated proteins. (F) Quantitation of immunoblots is graphed as ratios of the means +/− SEM of LC3-II/GAPDH of a total of 4 samples from three independent experiments. (G) H9C2 cells were transfected with the indicated siRNA and cultured in complete growth medium before harvest. (H) Quantitation was as in (F) and is graphed as the ratio +/− SEM of the amounts of indicated proteins divided by the amount of S6. (I) C57BL/6 wild-type or T1R3 knockout mice were fasted for 12 hrs before sacrifice. The indicated proteins were immunoblotted. (J) Model. Amino acids are sensed directly via T1R1/T1R3. PLCβ is then activated and calcium influx increases, activating ERK1/2 and Rsk. These kinases aid in activation of mTORC1 by phosphorylating Raptor and TSC2. Growth factors feed into this pathway at multiple levels. T1R1/T1R3 may also positively regulate mTORC1 by affecting the localization of Rag proteins.