Yin Yang 1 deficiency in skeletal muscle protects against rapamycin-induced diabetic-like symptoms through activation of insulin/IGF signaling

Abstract

Rapamycin and its derivatives are mTOR inhibitors used in tissue transplantation and cancer therapy. A percentage of patients treated with these inhibitors develop diabetic-like symptoms, but the molecular mechanisms are unknown. We show here that chronic rapamycin treatment in mice led to insulin resistance with suppression of insulin/IGF signaling and genes associated within this pathway, such as Igf1-2, Irs1-2, and Akt1-3. Importantly, skeletal muscle-specific YY1 knockout mice were protected from rapamycin-induced diabetic-like symptoms. This protection was caused by hyperactivation of insulin/IGF signaling with increased gene expression in this cascade that, in contrast to wild-type mice, was not suppressed by rapamycin. Mechanistically, rapamycin induced YY1 dephosphorylation and recruitment to promoters of insulin/IGF genes, which promoted interaction with the polycomb protein-2 corepressor. This was associated with H3K27 trimethylation leading to decreased gene expression and insulin signaling. These results have implications for rapamycin action in human diseases and biological processes such as longevity.

Figure 1. Chronic rapamycin treatment causes insulin resistance and lipid dysregulation

Mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days. (A) Glucose tolerance test. (B) Insulin tolerance test. (C) Serum insulin level and insulin/C-peptide ratio. (D) Glucose infusion rate during hyperinsulinemic-euglycemic clamps. (E) Glucose uptake rate under basal and clamp condition, and index of tissue glucose uptake (Rg). (F) Serum triglycerides and cholesterol. (G) Intramyocellular triglycerides. All values are presented as mean ± SD. n=6–10, *P<0.05, **P<0.01 and ***P<0.001. See also Figure S1 and S2.

Figure 2. Chronic rapamycin treatment decreases insulin signaling in skeletal muscle and liver

Mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days, fasted for 12h and then injected with vehicle or 0.6 U/kg insulin 10 min before sacrifice. Insulin signaling in (A) skeletal muscle and (B) liver. (C) Gene expression in the soleus from fed mice. All values are presented as mean ± SD. n=6–10, *P<0.05 and **P<0.01.

Figure 3. Skeletal muscle-specific YY1 knockout mice are protected from rapamycin-induced diabetic-like effects

Glucose tolerance test (A) and insulin tolerance test (B) in YY1mKO mice and wild-type littermates. (C) Wild-type or YY1mKO mice were treated with vehicle or 2.5 mg/kg rapamycin 14 days. Glucose tolerance test and (D) insulin tolerance test. (E) Serum cholesterol and triglycerides. (F) Serum insulin levels. (G) Intramyocellular triglycerides. All values are presented as mean ± SD. n=6–10, *P<0.05, **P<0.01 and ***P<0.001. See also Figure S3.

Figure 4. Skeletal muscle-specific YY1 knockout mice display hyperactivation of insulin/IGF signaling and increased gene expression that are insensitive to rapamycin treatment

(A) and (B) Wild-type or YY1mKO mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days, fasted for 12h and then injected with vehicle or 0.6 U/kg insulin 10 min before sacrifice. Insulin signaling in skeletal muscle. (C) Wild-type or YY1mKO mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days. Gene expression in the soleus of fed mice. (D) Gene expression in C₂C₁₂ myotubes infected with scrambled shRNA or YY1 shRNA for 72h (E) Gene expression in C₂C₁₂ myotubes infected with GFP or Flag-YY1 for 48h. All values are presented as mean ± SD. n=6–10, *P<0.05, **P<0.01 and ***P<0.001. See also Figure S4.

Figure 5. Rapamycin suppresses insulin/IGF signaling genes by promoting interaction between YY1 and the polycomb corepressor Pc2

(A) Coimmunoprecipitation and western blot analysis in HEK-293 cells. (B) YY1 protein cartoon. (C) Coimmunoprecipitation and western blot analysis in HEK-293 cells. (D) Luciferase assay in HEK-293 cells with a Gal4-YY1 luciferase construct and the indicated polycomb proteins. (E) Coimmunoprecipitation of HA-YY1 and Flag-Pc2 in HEK-293 cells treated with vehicle or rapamycin for 2h. (F) Endogenous interaction between YY1 and Pc2 was detected in skeletal muscle from mice treated with vehicle or rapamycin for 2h. (G) Gene expression in C₂C₁₂ myotubes infected with GFP or Flag-Pc2 for 48h. (H) Coimmunoprecipitation and western blot analysis of HEK-293 cells treated with vehicle or rapamycin for 2h. (I) Gene expression in C₂C₁₂ myotubes infected with GFP, wild-type YY1, YY1-AA or YY1–DD for 48h. All values are presented as mean ± SD. n=6, *P<0.05, **P<0.01 and ***P<0.001. See also Figure S5 and S6.

Figure 6. Rapamycin induces recruitment of YY1 and Pc2 to promoters of insulin/IGF signaling genes that is associated with H3K27 tri-methylation

Mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days. ChIP was performed in whole tissue extracts from skeletal muscle of refed mice using specific antibodies for (A) YY1, (B) Pc2 and (C–D) H3K27 tri-methylation. All values are presented as mean ± SEM. n=4, *P<0.05. IGF2 neg is a negative control in the IGF2 promoter, which does not contain a YY1 binding site.

Figure 7. YY1 suppresses insulin/IGF signaling genes through the polycomb proteins Pc2 and Ezh2

(A) Mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days. ChIP was performed in whole tissue extracts from skeletal muscle of refed mice using specific antibodies for Ezh2. All values are presented as mean ± SEM. n=4. (B) Coimmunoprecipitation and western blot analysis of HEK-293 cells treated with vehicle or rapamycin for 2h. (C) shScrambled-, shPc2- or shEzh2-stable C₂C₁₂ myotubes were infected with GFP, wild-type YY1, YY1-AA or YY1-DD for 48h and gene expression was measured. All values are presented as mean ± SD. n=6, *P<0.05, **P<0.01 and ***P<0.001. (D) Active mTORC1 induces YY1 phosphorylation at T30 and S365 resulting in displacement of Pc2, thereby activating insulin/IGF signaling gene transcription. Conversely, inactive mTORC1, such as in the presence of rapamycin, results in YY1 dephosphorylation at T30 and S365 permitting recruitment of Pc2 and consequently the polycomb repressor complex (PRC) to inhibit expression of insulin/IGF signaling genes. The recruitment of YY1 and Pc2 at these promoters correlates with an increased level of H3K27 tri-methylation produced by Ezh2, which is a marker of transcriptional repression. In the absence of YY1, the suppression on these genes is relieved leading to their hyperactivation and rapamycin-insensitivity. Ac, acetylation; Me, methylation; TAC, transcriptional activator complex.