Figure 5.
AKT Suppresses and Foxo Stimulates atrogin-1 Expression and Foxo Activation Causes Marked Atrophy of Mouse Muscle Fibers
(A) AKT prevents induction of atrogin-1 expression by fasting. Left image: mouse tibialis anterior muscles were transfected by electroporation with the 3.5AT1 and pRL-TK. Seven days later, the mice were fed or fasted and then sacrificed. Firefly and Renilla luciferase activity was measured in muscle extracts. In situ hybridization on sections from muscles of fed and fasted mice (left image, insert). Scale bar is equal to 60 μm. Middle image: muscles were cotransfected with 3.5AT1, pRL-TK, and either c.a. HA-AKT or the parent vector. Seven days later, the mice were fasted for 24 hr and sacrificed. Firefly/Renilla luciferase activity was measured. Right image: serial cross-sections of these transfected muscles were processed for immunofluorescence with anti-HA antibody or for in situ hybridization with an atrogin-1 antisense probe. Scale bar is equal to 50 μm.
(B) Nuclear localization of c.a. FOXO3A. Sections of adult tibialis anterior muscles transfected with c.a. FOXO3A or FOXO3A were prepared and visualized with anti-HA antibodies (for Foxo) and Hoechst staining (for nuclei) four days after infection. Images were merged to demonstrate colocalization. Scale bars are equal to 20 μm for c.a. FOXO3A and 30 μm for FOXO3A.
(C) Foxo3 activates the atrogin-1 promoter in transfected muscle fibers. Muscles were cotransfected with 3.5AT1, pRL-TK, and with either FOXO3A or c.a. FOXO3A as described above. Similarly, a Foxo reporter (DBE promoter) was transfected in place of the atrogin-1 reporter. Luciferase activity was measured four days after transfection.
(D) Atrogin-1 mRNA is increased in muscle fibers overexpressing Foxo3. Cross-sections of tibialis anterior muscle transfected with c.a. FOXO3A were processed for immunofluorescence with anti-HA antibody or for in situ hybridization for atrogin-1 four days after transfection. Atrogin-1 transcripts are increased in Foxo3 overexpressing fibers, in close proximity to the Foxo3-positive nuclei (arrow) Scale bar is equal to 40 μm.
(E) siRNA-mediated knockdown of Foxo1-3 inhibits atrogin-1 promoter activity during fasting. Left image: adult skeletal muscle was cotransfected with pSUPER-vectors, pRL-TK and 3.5AT1. Seven days later, the mice were fasted and sacrificed. Firefly/renilla luciferase activity was measured as above. Right image: Foxo1 and 3 protein level detected by immunoblot in myotubes.
(F) Foxo3 expression for 8 or 14 days causes marked muscle fiber atrophy. Left images: adult tibialis anterior muscles were transfected with c.a. FOXO3A and mice were sacrificed after 8 or 14 days. Atrophic fibers expressing c.a. FOXO3A were detected in transverse sections stained with anti-HA (for Foxo) (asterix in eight day micrograph) For eight days, scale bar is equal to 50 μm. For 14 days, scale bar is equal to 20 μm. Right images: frequency histograms showing the distribution of cross-sectional areas (μm2) of fibers expressing c.a. FOXO3A for eight days (red bars) or 14 days (pink bars) and surrounding untransfected fibers (control fibers) (black bars).