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. Author manuscript; available in PMC: 2013 Apr 4.
Published in final edited form as: Cell Metab. 2012 Apr 4;15(4):505–517. doi: 10.1016/j.cmet.2012.03.008

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

Sharon M Blättler 1,2, John T Cunningham 1,2,4, Francisco Verdeguer 1,2,4, Helen Chim 1,2, Wilhelm Haas 2, Huifei Liu 2, Klaas Romanino 3, Markus A Rüegg 3, Steven P Gygi 2, Yang Shi 2, Pere Puigserver 1,2,*
PMCID: PMC3324784  NIHMSID: NIHMS364800  PMID: 22482732

SUMMARY

Rapamycin and 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 leads to insulin resistance with suppression of insulin/IGF signaling and genes associated within this pathway, such as IGFs, IRS1/2 and AKTs. 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 increases in genes in this cascade that, in contrast to wild-type mice, were 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 tri-methylation leading to decreases in gene expression and insulin signaling. These results have implications for rapamycin action in human diseases and biological processes, such as longevity.

INTRODUCTION

mTOR is a serine/threonine protein kinase that receives inputs from multiple signals, such as growth factors and nutrients, and plays a central role in the regulation of cell growth, size and survival (Sengupta et al., 2010b). mTOR is part of two protein complexes, named mTORC1 and mTORC2. A specific component of mTORC1 is Raptor that is required for most of the signaling that controls protein synthesis and metabolism (Gingras et al., 2004; Polak and Hall, 2009). Rictor, which is part of mTORC2, is involved in cytoskeleton dynamics and feedback signaling to AKT (Jacinto et al., 2004; Sarbassov et al., 2005). Downstream effectors of mTORC1 regulate protein synthesis by phosphorylating key translational regulators, such as ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E binding protein (Dowling et al., 2010; Fingar et al., 2002; Sengupta et al., 2010b). Rapamycin is a small molecule that binds to FKBP12 and specifically inhibits mTORC1 activity by changing its conformation and preventing substrate binding (Kim et al., 2002; Sabatini et al., 1994; Yip et al., 2010). The biological activities of this drug, including the effects on life span, are driven through inhibition of mTOR (Bjedov et al., 2010; Harrison et al., 2009; Schieke and Finkel, 2006). In humans, rapamycin is a drug commonly prescribed to suppress the immune system in patients after solid organ transplantation (Monaco, 2009), and rapamycin analogs are currently in clinical trials for treatment of several cancers and tuberous sclerosis complex disease (Guertin and Sabatini, 2007; Meric-Bernstam and Gonzalez-Angulo, 2009; Petroulakis et al., 2006; Plas and Thomas, 2009; Sampson, 2009). Importantly, contrary to what would be predicted based on the mTOR activated S6K negative feedback on insulin signaling (Haruta et al., 2000; Sarbassov et al., 2005; Um et al., 2004), a percentage of patients treated with mTOR inhibitors develop a variety of diabetic-like symptoms, including glucose intolerance, insulin resistance and dyslipidemia (Bodziak and Hricik, 2009; Cole et al., 2008; Johnston et al., 2008; Roland et al., 2008). Interestingly, some of these metabolic defects have also been observed in rodent models treated with rapamycin (Fraenkel et al., 2008; Houde et al.), but the molecular mechanisms and target tissues by which this drug causes pro-diabetic effects are unknown.

Although upstream mechanisms and proteins regulating mTOR have been elucidated in detail (Hay and Sonenberg, 2004), its downstream effectors, particularly those regulating transcription, are not completely understood. mTOR controls lipid biosynthesis by modulating SREBP-1 and its target genes (Brown et al., 2007; Duvel et al., 2010; Mauvoisin et al., 2007; Peng et al., 2002; Porstmann et al., 2008). Furthermore, mTOR regulates glycolysis through hypoxia inducible factor 1 alpha (HIF1α) (Duvel et al., 2010) and ketogenesis through peroxisome proliferator-activated receptor alpha (PPARα), possibly by regulating subcellular localization of the corepressor NCoR1 (Sengupta et al., 2010a). Moreover, our previous work established that mTOR regulates mitochondrial gene expression through the transcription factor Yin Yang 1 (YY1) and the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α) (Cunningham et al., 2007). However, whether and how transcriptional regulatory mechanisms are involved in the rapamycin-induced diabetic-like effects on glucose and lipid metabolism and insulin action is unknown.

Here we show that chronic inhibition of mTOR by rapamycin leads to insulin resistance and lipid dysregulation associated with defective insulin/IGF signaling and suppression of key genes of this pathway, such as IGFs, IRS1/2 and AKTs. Importantly, skeletal muscle-specific YY1 knockout (YY1mKO) mice were protected against the rapamycin-induced diabetic-like effects including insulin resistance, dyslipidemia and suppression of insulin signaling. YY1mKO mice exhibited hyperactivation of insulin/IGF signaling and increased expression of the genes of this pathway. In contrast to wild-type mice, rapamycin did not suppress insulin/IGF signaling genes in YY1mKO mice. Upon rapamycin treatment YY1 functioned as a transcriptional repressor of insulin signaling genes through interaction with the polycomb protein Pc2 and recruitment at the promoters of these genes which was associated with H3K27 tri-methylation. Moreover, mTOR regulated the interaction between YY1 and Pc2 through YY1 phosphorylation at T30 and S365. In summary, we show that YY1 mediates suppression of insulin signaling genes through polycomb proteins and epigenetic changes in response to mTOR inhibition and this accounts, at least in part, for the pro-diabetic effects of rapamycin in vivo.

RESULTS

Chronic rapamycin treatment in mice causes insulin resistance and lipid dysregulation

Given the clinical relevance of rapamycin-induced diabetic effects, we used an experimental mouse model to investigate the molecular mechanisms. Figure 1 shows that mice treated for two weeks with rapamycin developed significant glucose intolerance (Figure 1A) and decreased insulin sensitivity (Figure 1B), as measured by glucose and insulin tolerance tests. These parameters correlated with changes in HOMA-IR (Homeostasis Model Assessment of Insulin Resistance), a surrogate of insulin sensitivity (Figure S1A). Interestingly, these metabolic effects were detected, although with smaller magnitude, with a single dose of rapamycin (Figure S1B). Moreover, mice chronically treated with rapamycin displayed hyperinsulinemia without changes in the insulin/C-peptide ratio (Figure 1C). To determine the tissues involved in the rapamycin-induced whole body insulin resistance, we performed hyperinsulinemic-euglycemic clamps (Figure S1C–D). Rapamycin-treated mice required lower glucose infusion rates to maintain blood glucose compared to vehicle-treated mice (Figure 1D). These effects were linked to decreases in glucose disposal in different tissues, including skeletal muscle (Figure 1E). Hepatic insulin resistance was also observed in rapamycin-treated mice as detected by augmented endogenous glucose production, measured in the clamp experiments (Figure S1E). Consistent with the clinical side effects of mTOR inhibitors in humans, mice treated with rapamycin displayed hypertriglyceridemia and hypercholesterolemia (Figure 1F). Furthermore, increased intracellular lipids in skeletal muscle (Figure 1G), which are often associated with insulin resistance (Erion and Shulman, 2010; Savage et al., 2007), were detected in mice treated with rapamycin. In these conditions, rapamycin did not affect the ability of the pancreas to secrete insulin as assessed by blood insulin levels after glucose administration by oral gavage (Figure S1F). Interestingly, all these metabolic changes were observed in the absence of significant differences in body weight (Figure S2A), adiposity, as analyzed by MRI, (Figure S2B) or food intake (Figure S2C). These results indicate that chronic rapamycin treatment in mice, similar to humans, causes whole-body insulin resistance associated with dyslipidemia and accumulation of lipids in skeletal muscle. Moreover, these data suggest that skeletal muscle plays a major role in the development of rapamycin-induced diabetic symptoms.

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

Figure 1

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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.

Next, we tested whether the metabolic effects of rapamycin in mice were occurring specifically through mTORC1 using skeletal muscle-specific Raptor knockout mice (RamKO) (Bentzinger et al., 2008) and a different mTOR chemical inhibitor, Torin 2 (Liu et al., 2011). RamKO mice were resistant to the rapamycin metabolic effects on glucose tolerance (Figure S2D) and increases in intramyocellular triglycerides (Figure S2E). In addition, chronic treatment with Torin 2 also caused glucose intolerance, though the effects were not as pronounced as with rapamycin (Figure S2F), suggesting different efficacies of these inhibitors and/or independent mTORC2 contributions. As predicted, these results suggest that mTOR is the target of rapamycin to cause these pro-diabetic symptoms in mice.

Chronic rapamycin treatment decreases insulin signaling in skeletal muscle and liver

In order to determine whether rapamycin-induced insulin resistance correlated with defects in insulin signaling, we analyzed phosphorylation of different components of this transduction pathway as an index of activity. Consistent with the whole-body insulin resistance and reduction in glucose disposal in skeletal muscle, rapamycin significantly decreased insulin-stimulated phosphorylation of the insulin receptor, IRS1, AKT, and downstream targets involved in both glucose uptake (AS160) and insulin-dependent gene expression (FoxO1) (Figure 2A). The decrease in phosphorylation of insulin signaling proteins correlated in some cases with a reduced protein level, including IRS1/2 and AKT1/2/3. A similar pattern of defective insulin signaling was detected in liver (Figure 2B), which could account for the hepatic insulin resistance and increased rates of hepatic glucose production detected by hyperinsulinemic-euglycemic clamps in mice treated with rapamycin (Figure S1E). Consistent with the decreased insulin-stimulated signaling and levels of different proteins in this pathway, rapamycin treatment significantly repressed expression of the genes encoding for these proteins. In fact, IGF1/2, IRS1/2 and AKT1/2/3 mRNA levels were significantly lower in skeletal muscle of rapamycin-treated mice (Figure 2C). These results indicate that chronic rapamycin treatment in mice causes resistance to insulin signaling and action and represses insulin signaling gene expression, which is reflected in the reduction of their protein levels.

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

Figure 2

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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.

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

We have previously identified that part of the transcriptional response regulated by mTOR signals through the transcription factor YY1 (Cunningham et al., 2007). Because one of the major metabolic effects of rapamycin is decreased insulin-dependent peripheral glucose disposal that largely occurs in skeletal muscle (Figure 1E), we generated a skeletal muscle-specific YY1 knockout mouse (YY1mKO) to determine whether YY1 mediates the diabetic-like symptoms induced by rapamycin in vivo. To this purpose we crossed a floxed YY1 allele (Affar el et al., 2006) with a muscle-specific myogenin CRE driver (Li et al., 2005) to generate YY1mKO mice. YY1 mRNA levels were almost undetectable in skeletal muscle, but were not decreased in other tissues such as liver, white or brown fat (Figure S3A). YY1mKO mice were born at normal mendelian rate, and their body weight was comparable to their wild-type littermates (Figure S3B). Notably, these mice showed improved glucose tolerance and insulin sensitivity compared to the wild-type littermates (Figure 3A and 3B). Importantly, Figure 3C and 3D show that while control mice (fl/fl) treated with rapamycin developed glucose intolerance and insulin resistance, YY1mKO mice (cre-fl/fl) were protected and displayed similar glucose and insulin tolerance curves as vehicle-treated YY1mKO mice. Moreover, YY1mKO mice were also protected from increases in serum levels of cholesterol and triglycerides (Figure 3E), insulin (Figure 3F) and intramyocellular triglycerides (Figure 3G) caused by chronic rapamycin treatment. Interestingly, serum triglycerides were not increased in YY1mKO mice upon rapamycin treatment, but basal level were higher in these mice compared to wild-type littermates. This is in agreement with the observation that YY1mKO mice have decreased expression of fatty acid oxidation genes in skeletal muscle (unpublished data). These data indicate that skeletal muscle YY1 is required for the development of diabetic-like symptoms caused by rapamycin in mice.

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

Figure 3

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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.

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

Although YY1mKO mice were resistant to rapamycin-induced glucose intolerance, insulin resistance, hyperinsulinemia and hyperlipidemia, it still remained unclear whether YY1 directly mediated the rapamycin-induced diabetic symptoms by targeting core components of the insulin/IGF signaling cascade. We therefore investigated whether differences exist in insulin/IGF signaling activity in YY1mKO mice. Interestingly, Figure 4A shows that when compared to wild-type mice, vehicle-treated YY1mKO mice displayed increased basal and insulin-stimulated phosphorylation of AKT and downstream components of the insulin/IGF signaling, such as AS160, FoxO1, GKS3 and ribosomal S6. Remarkably, in contrast to wild-type mice that have decreased insulin-stimulated phospho-AKT in the presence of rapamycin, YY1mKO mice responded similarly to insulin in the presence and absence of rapamycin (Figure 4A). This insulin signaling pattern is consistent with the rapamycin-induced metabolic effects observed in glucose and insulin tolerance tests (Figures 3C and 3D). Next, we further analyzed additional components of the insulin/IGF signaling in YY1mKO mice in response to insulin stimulation. YY1mKO mice exhibited increased insulin-induced phosphorylation of the IGF1 and insulin receptors, AKT, mTOR, S6K and S6 ribosomal proteins, as well as downstream targets such as FoxO1, GSK3 and AS160 (Figure 4B). Notably, we found that levels of some of these proteins, especially IRS1/2 and AKTs, were strongly upregulated in skeletal muscle of YY1mKO mice, suggesting that YY1 might control expression of these insulin/IGF signaling proteins at the transcriptional level. It further suggests that YY1 could mediate the rapamycin suppressive effects on this signaling genes observed in wild-type mice. To test this, we measured expression of different genes involved in this pathway in wild-type and YY1mKO mice. Consistent with the metabolic and signaling parameters Figure 4C shows that YY1mKO mice have increased expression of insulin/IGF signaling genes such as IGF1, IGF2, IRS1, IRS2 and AKTs indicating that YY1 mediates transcriptional repression on these genes in vivo. Importantly, rapamycin did not repress skeletal muscle gene expression of these transcripts related to insulin/IGF signaling in YY1mKO mice, suggesting that YY1 is required for rapamycin mediated suppression effects on these genes (Figure 4C). These data indicate that increased expression of selective insulin signaling genes and their insensitivity to repression by rapamycin in YY1mKO mice could account for their protection against rapamycin-induced diabetic-like symptoms.

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

Figure 4

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(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.

We next determined whether the effects of YY1 on insulin/IGF signaling gene expression were cell autonomous by using YY1 shRNA (Figure S4A) and YY1 overexpression (Figure S4B) in C2C12 myotubes. Depletion of YY1 mimicked the gene expression effects observed in YY1mKO mice (Figure 4D), whereas YY1 overexpression decreased the levels of insulin/IGF signaling genes (Figure 4E). These experiments show that modulation of YY1 affects insulin/IGF signaling gene expression in vivo leading to dysregulated protein expression, and consequently, signaling. Furthermore, our experiments show that the effect of YY1 on these genes is cell autonomous.

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

We have previously shown that YY1 physically interacts with mTORC1 (Cunningham et al., 2007), although the YY1 domains involved in this interaction were unknown. To mechanistically investigate how rapamycin controls YY1 transcriptional activity and represses insulin signaling gene expression, the Raptor (a component of the mTORC1 complex) specific interaction domain on YY1 was mapped. Figure 5A shows that Raptor specifically interacted with the REPO (REcruitment of POlycomb) domain of YY1 (Figure 5B). Since Raptor is known to bind to Tor Signaling (TOS) motifs in mTORC1 substrates (Schalm and Blenis, 2002), we searched the REPO domain of YY1 to determine if this was the mode of binding. Figure 5C shows that when the conserved phenylalanine of a putative TOS motif in YY1 (Figure S5A) is mutated to alanine, binding between YY1 and Raptor is abolished. Since the YY1 REPO domain is responsible for recruiting polycomb group proteins to DNA in Drosophila (Wilkinson et al., 2006), we hypothesized that changes in mTORC1 activity could be associated with docking of polycomb corepressors to YY1 as a mechanism to suppress expression of target genes. For this reason, we first tested whether any of the mammalian polycomb group proteins could affect YY1 transcriptional activity. By using a luciferase-based Gal4-YY1 transcriptional assay, several of the polycomb homologs (and one HP homolog - Cbx5) were screened to determine which, if any, control YY1 activity. Figure 5D shows that, of all the mammalian polycomb homologs, only Pc2/Cbx4 was able to significantly repress YY1 transcriptional activity. In addition, the activity of Gal4-YY1 was inhibited by treatment with rapamycin (Figure 5D, right panel). These results suggested that Pc2-mediated repression could be associated with its binding to YY1 and possibly be regulated by mTORC1 activity. Indeed, the binding of Pc2 to YY1 was strongly dependent upon mTORC1 activity, as Pc2, in contrast to other YY1 corepressors such as CtBP1 (Figure S5B), was recruited to YY1 upon rapamycin treatment in HEK-293 cells (Figure 5E). Notably, rapamycin promoted the interaction between endogenous YY1 and Pc2 in mouse skeletal muscle (Figure 5F). Consistent with these data, ectopic expression of Pc2 in C2C12 myotubes (Figure S5C) mimicked rapamycin treatment on insulin signaling gene expression (Figure 5G). These results indicate that inactivation of mTORC1 leads to Pc2 functioning as a negative regulator of YY1 transcriptional activity through direct physical recruitment, providing a molecular basis by which rapamycin represses YY1 target gene expression.

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

Figure 5

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(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.

Our observation that YY1 binds constitutively to mTORC1 through the REPO domain, and that rapamycin leads to suppression of YY1 target gene expression, suggested that YY1 phosphorylation might be involved in this transcriptional regulatory mechanism. We therefore tested whether mTORC1 activity could affect YY1 phosphorylation status using a combination of qualitative and semi-quantitative analysis with mass spectrometry. As detailed in supplemental materials and methods, the analysis of digested YY1 peptides in qualitative analysis led to the detection of phospho-T30 and phosho-S365 only in YY1 immunoprecipitated from cells treated with vehicle, but these two residues were not detected phosphorylated in YY1 immunoprecipitated from cells treated with rapamycin. Interestingly, in semi-quantitative analysis peptide amounts containing phospho-T30 were significantly less abundant in YY1 from rapamycin treated cells (Figure S5D–G). Because mTORC1 inactivation resulted in decreased phosphorylation of YY1, next we determined whether T30 and S365 phosphorylation could modulate the physical interaction with Pc2. The double T30A/S365A YY1 mutant (YY1-AA) showed selectively enriched interaction with Pc2 that, in contrast to wild-type YY1, was not increased by rapamycin (Figure 5H), suggesting that dephosphorylation at both sites is needed for efficient Pc2 recruitment after rapamycin treatment. In fact, the T30D/S365D YY1 mutant (YY1-DD) did not bind to Pc2 efficiently (Figure 5H), further supporting that phosphorylation of these two residues mediates the interaction between YY1 and Pc2. In keeping with the effects of YY1- and rapamycin/Pc2-dependent suppression of insulin/IGF signaling genes, the proximal region of the IRS2 promoter fused to luciferase was strongly repressed by YY1 overexpression as well as by rapamycin (Figure S6A). In addition, the YY1-AA mutant, which mimics the dephosphorylation of YY1 induced by rapamycin, inhibited IRS2 promoter-driven luciferase expression to a greater extent than wild-type YY1. Overexpression of Pc2 further increased the repression of both YY1 wild-type and YY1-AA mutant. Notably, YY1-AA mutant was insensitive to rapamycin. Consistent effects were also observed using YY1 alleles on endogenous insulin/IGF signaling genes in C2C12 myotubes. In fact, YY1 dephospho-mimetic mutant acted as a potent suppressor of these genes compared to YY1 wild-type or YY1 phospho-mimetic mutant (Figure 5I and Figure S6B). Together, these results indicate that rapamycin controls the physical interaction between YY1 and Pc2 corepressor through phosphorylation of YY1 residues T30 and S365, and leads to transcriptional repression of YY1 targeted insulin/IGF signaling genes.

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

Although modulation of YY1 expression and rapamycin treatment affected insulin/IGF signaling gene expression in skeletal muscle, it remained unclear if YY1 is able to bind directly to the promoters of these genes. For this reason, we decided to perform chromatin immunoprecipitation (ChIP) analysis using skeletal muscle from vehicle- and rapamycin-treated mice. Figure 6 shows that recruitment of YY1 (Figure 6A) and Pc2 (Figure 6B) was significantly increased to promoter regions of genes encoding insulin/IGF signaling proteins such as IGF2, IRS1, IRS2 and AKT2 after chronic treatment with rapamycin. Interestingly, these are the same set of genes that were decreased by rapamycin (Figure 2C) and increased in YY1-deficient skeletal muscle (Figure 4C), providing a possible mechanism of transcriptional repression. Pc2 is part of the PRC1 polycomb repressor complex that is sequentially recruited to chromatin promoter regions after the PRC2 complex, which contains the methyl-transferase Ezh2 and catalyzes tri-methylation of H3K27, an epigenetically repressive histone marker (Hansen et al., 2008; Kagey et al., 2003; Kuzmichev et al., 2002; Levine et al., 2004). In fact, Pc2 contains a chromodomain that specifically interacts with tri-methylated H3K27 (Bernstein et al., 2006). In order to determine if recruitment of YY1 and Pc2 to the promoters of insulin/IGF signaling genes correlated with increases in H3K27 tri-methylation, we performed ChIP analysis using antibodies that specifically recognize this histone modification. Consistent with recruitment of Pc2, the level of H3K27 tri-methylation was significantly increased at the promoters of insulin/IGF signaling genes from skeletal muscle of mice chronically treated with rapamycin. As a control, H3K27 tri-methylation at the smooth actin promoter did not change upon rapamycin treatment (Figure 6D). These results indicate that upon rapamycin treatment, YY1 and Pc2 are directly recruited to the promoters of insulin/IGF signaling genes increasing the level of H3K27 tri-methylation.

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

Figure 6

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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.

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

The increase in H3K27 tri-methylation by rapamycin treatment at insulin/IGF signaling gene promoters containing YY1 binding sites suggested that the methyl-transferase Ezh2 might be involved in the repression of these genes. We therefore assessed the presence of Ezh2 at these promoters using ChIP analysis from skeletal muscle of vehicle or rapamycin-treated mice. Figure 7A shows that Ezh2 was detected at the promoters of insulin/IGF signaling genes, but this enrichment was not sensitive to rapamycin treatment. Consistent with the recruitment of Ezh2 at these promoters, YY1 interacted with Ezh2 in a rapamycin-independent manner (Figure 7B), suggesting that mechanisms other than increases in Ezh2 methyl transferase occupancy might control the levels of H3K27 tri-methylation at promoters of these genes upon rapamycin treatment. Finally, to determine the contribution of the PRC1 and PRC2 polycomb protein complexes to the YY1-dependent suppression on insulin/IGF signaling genes, we depleted C2C12 cells of Pc2 or Ezh2 using specific shRNA lentiviral constructs against these proteins (Figure S7). These stable cell lines were used to express the different YY1 alleles, including wild-type and phospho-mutants (Figure 7C). As expected, in shRNA control cells YY1-AA exhibited strong repression of insulin/IGF genes. This repression however, was completely blunted in cells depleted of Pc2 or Ezh2. Interestingly, the degree of de-repression caused by Pc2 or Ezh2 was slightly different within the genes analyzed suggesting that some specific factors might contribute to these effects. A similar gene expression pattern, although less pronounced, was also observed using YY1 wild-type, whereas the YY1-DD mutant did not affect the gene expression. This data indicates that suppression of insulin/IGF signaling genes driven by YY1 depends on Pc2 and Ezh2, members of the PRC1 and PRC2 polycomb complexes, respectively. These results also suggest that the recruitment of Pc2 upon rapamycin treatment could account for the transcriptional suppression effects of this drug. Furthermore, the Pc2 enrichment at YY1 binding sites of insulin/IGF signaling promoter genes might be necessary for Ezh2 to increase the levels of the transcriptional repression mark H3K27 tri-methylation.

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

Figure 7

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(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.

DISCUSSION

In these studies we have delineated a signaling/transcriptional pathway by which rapamycin, a drug prescribed in post-transplantation and cancer therapies (Guertin and Sabatini, 2007; Meric-Bernstam and Gonzalez-Angulo, 2009; Monaco, 2009; Plas and Thomas, 2009; Sampson, 2009), causes diabetic-like symptoms and insulin resistance. We show that rapamycin controls phosphorylation of YY1, allowing for binding of the polycomb corepressor Pc2 and recruitment to the chromatin promoters of insulin/IGF genes. Furthermore, rapamycin treatment leads to an increase in the histone repressive modification H3K27 tri-methylation, which is consistent with this drug suppressing transcription of these genes. The rapamycin-dependent regulatory role of YY1 is demonstrated using YY1mKO mice that are protected against the pro-diabetic effects of this drug due to elevated expression of insulin/IGF signaling genes. Importantly, while rapamycin suppressed these genes and caused insulin resistance in wild-type mice, YY1mKO mice were completely refractory to the effects of rapamycin and maintained normal insulin sensitivity. In summary, we propose a model in which YY1 mediates rapamycin-induced suppression of insulin/IGF signaling genes through polycomb proteins and epigenetic changes that lead to the pro-diabetic effects of rapamycin in vivo (Figure 7D).

Our studies show that skeletal muscle is a key target tissue of rapamycin and that it mediates, at least in part, the metabolic dysregulation caused by this drug. This is supported by several pieces of evidence. First, the clamp experiments show that one of the major effects of rapamycin on glucose metabolism is through substantial decreases in glucose disposal, particularly in skeletal muscle (Figure 1E). Second, skeletal muscle YY1 and Raptor deficient mice were entirely protected against rapamycin-induced whole body diabetic effects (Figure 3). Despite the fact that our data show that skeletal muscle is a major tissue targeted by rapamycin and mediates its metabolic effects, it is likely that other tissues exacerbate the diabetic symptoms. For example, the liver would contribute through hepatic insulin resistance to uncontrolled higher rates of hepatic glucose production, as shown using hyperinsulinemic-euglycemic clamps in Figure S1E and Figure S2B and as previously suggested in rats (Houde et al., 2010). Unlike other reports (Fraenkel et al., 2008; Houde et al., 2010), our data does not suggest a defect in beta cell function and insulin secretion upon rapamycin treatment. The reason for this discrepancy is not clear at this point but the use of another animal model might be a possible explanation. These results have important clinical implications and support the use of anti-diabetic drugs as therapeutic strategies to prevent diabetes in patients treated with mTOR inhibitors, particularly drugs that target glucose disposal, such as AMPK activators (Cuthbertson et al., 2007; Musi et al., 2002).

Whereas the signaling components of the mTOR pathway have been investigated in considerable detail (Sengupta et al., 2010b), the transcriptional targets and mechanisms that might mediate, at least in part, the mTOR metabolic response are not clear. Recently, several transcription factors including SREBP and HIF1α have been linked to mTOR activation (Brown et al., 2007; Duvel et al., 2010; Mauvoisin et al., 2007; Peng et al., 2002; Porstmann et al., 2008) but their involvement in the rapamycin-mediated metabolic dysregulation is unknown. While these transcription factors might play a role, we have identified that the transcription factor YY1 in skeletal muscle is controlled through mTOR activity, and it mediates rapamycin induction of diabetic-like symptoms. YY1 functions in different transcriptional protein complexes that activate or repress expression of genes and participates in different biological processes such as cancer progression (Castellano et al., 2009; Gordon et al., 2006; Shi et al., 1997). Our data indicate that the repressive function of YY1 on insulin/IGF signaling genes promotes the pro-diabetic effects of rapamycin. Among these genes we have found that IGF1, IGF2, IRS1, IRS2 as well as AKTs were suppressed by rapamycin and YY1 (Figure 2C and Figure 4E). Although the extent to which a specific decrease in these or other proteins within this pathway can account for the effects of rapamycin on insulin signaling is not entirely clear, the results suggest that overall decreases in different insulin signaling components leads to lower activity of this pathway after insulin stimulation. In fact, this is consistent with several genetic mouse models in which several of these components are deleted. For example, skeletal muscle-specific IRS1/IRS2 knockout mice display decreased insulin-stimulated glucose uptake in muscle and insulin resistance (Long et al., 2010). Furthermore, AKT2 deficient mice display high plasma insulin, insulin resistance and impaired ability to reduce glucose levels, a phenotype which is attributed to the impairment of insulin action in skeletal muscle and liver (Cho et al., 2001; Garofalo et al., 2003). YY1 mediates rapamycin-induced repression of insulin/IGF signaling genes in skeletal muscle, thus the absence of YY1 in this tissue results in increased expression of these genes and loss of rapamycin repression causing hyperactivation of insulin/IGF signaling and insulin sensitivity in YY1mKO mice. The specific and individual contribution of these or additional genes to insulin signaling effects is currently unclear. It is possible that upregulation of IGF proteins could be sufficient to explain protection to insulin resistance in YY1mKO mice.

Our data show that YY1 plays a pivotal role in the suppression of insulin/IGF signaling genes in response to rapamycin. Mechanistically, we found that rapamycin induced YY1 dephosphorylation and Pc2 corepressor recruitment to the promoters of these genes. Although YY1 phosphorylation depends on mTOR activity, it is unclear if mTOR directly phosphorylates YY1 at T30 and S365, or if other kinases are involved. Consistent with previous reports that recruitment of Pc2 to promoters of genes correlates with H3K27 tri-methylation (Levine et al., 2004), we found that rapamycin induced this histone modification in skeletal muscle and was associated with decreases in gene expression. In addition, we detected the presence of the methyl-transferase Ezh2, which catalyzes H3K27 tri-methylation, at promoters of the insulin/IGF signaling genes. In fact, Ezh2 physically interacted with YY1, even though this interaction was not rapamycin-sensitive. Importantly, YY1-mediated suppression on these genes depended on both Pc2 (PRC1 complex) and Ezh2 (PRC2 complex), suggesting that recruitment of Pc2 might be necessary to increase Ezh2 methyl-transferase activity at these promoters upon rapamycin treatment. To further clarify this mechanism, it will be necessary to determine the dynamics of recruitment of YY1 and polycomb protein complexes with associated enzymatic activities and how they affect different patterns of histone modifications. These results might have important implications that go beyond the rapamycin-induced diabetic-like effects and could provide one of the mechanisms by which nutrient changes control the epigenetic state of genes through mTOR pathway and could have an impact in metabolic adaptation and diseases.

Finally, our results, in addition to supporting the use of anti-diabetic drugs in patients treated with rapamycin, point towards the need to consider the pro-diabetic effects of this drug in the context of the life span promoting effects recently reported (Harrison et al., 2009) and evaluate the long-term benefits and side effects of rapamycin treatment

EXPERIMENTAL PROCEDURES

Animal Experiments

All experiments and protocols were approved by the Institutional Animal Care and Use Committees of Dana-Farber Cancer Institute or Beth Israel Deaconess Medical Center. 6 weeks old male C57BL/6 mice were injected daily intraperitoneally at 5 PM with 2.5 mg/kg rapamycin or vehicle (0.1% DMSO into sterile PBS). The dose of rapamycin was chosen in order to mimic a typical therapeutic concentration in humans (McAlister et al., 2002). Glucose tolerance test was performed on mice injected with rapamycin for 11 days and fasted for 12h prior to 2 g/kg dextrose intraperitoneal injections. Insulin tolerance test was performed on mice injected with rapamycin for 11 days and fasted for 5h prior to 0.6 U/kg insulin intraperitoneal injections. For the short-term rapamycin treatment, mice were injected with 2.5 mg/kg rapamycin or vehicle (0.1% DMSO into sterile PBS) and 12h later they were fasted overnight. Glucose tolerance test was performed in the morning after 12h fasting. HOMA-IR was calculated by the formula: (fasted insulin [μU/ml] × fasted glucose [mg/dL])/405.

Generation of YY1mKO mice

YY1mKO mice were generated by breeding animals harboring a floxed YY1 allele (Affar el et al., 2006) with mice that transgenically express CRE recombinase under the control of the myogenin promoter and the MEF2C enhancer (Li et al., 2005). Wild-type littermates carrying the floxed allele but not CRE were used as control group. These mice have a mixed background.

Cell culture and treatments

C2C12 myoblasts were cultured in DMEM containing 10% fetal bovine serum. Myoblasts were differentiated in DMEM with 2% horse serum for 72h before infection with the indicated adenoviral constructs. 48–72h infected C2C12 myotubes were treated with 20 nM rapamycin or vehicle for 16h as indicated.

Chromatin IP

Mice (n=4) were treated with vehicle or 2.5 mg/kg rapamycin for 2 weeks and fasted for 16h/re-fed for 6h before sacrifice. Two gastrocnemius muscles of each mouse were dissected, cut in pieces and fixed in 1% formaldehyde/PBS for 15 min at room temperature. The fixation was quenched by adding a final concentration of 0.150 M glycine and washed twice in cold PBS. The tissue was homogenized in cold PBS (containing protease inhibitors) in a motor pestle. The homogenate was filtered in 100 μm cell strainers and pelleted by centrifugation at 5 min, 4000rpm and 4°C. The pellet was resuspended in 500 μL SDS lysis buffer from Milipore EZ-ChIP kit. Samples were sonicated in Diagenode Bioruptor during 5 cycles of a duty of 30″ on/30″ off and finally centrifuged for 15 min at 10000g. 50 μL of each sample were diluted 1:10 in dilution buffer (from EZ-ChIP kit) and incubated overnight at 4°C with 2 μg of the antibodies YY1 (Santa Cruz, sc-281X), Pc2 (Alexis, ALX-210-570), H3K27me3 (Abcam, ab60021), Ezh2 (Active Motif) and IgG, respectively. Immunocomplexes were recovered with Protein A Dynabeads (Invitrogen, Dynabeads 100-02D) and DNA was amplified with specific primers for the indicated genes by quantitative real-time PCR.

Statistics

Data were analyzed by one-way ANOVA followed by an appropriate Post hoc test for the comparison between two groups. In particular, comparisons between two groups of mice at different time points were performed by ANOVA followed by Bonferroni test, whereas comparisons between two groups of mice at single time points were performed by ANOVA followed by Tukey test. Significance was defined as P < 0.05. Data are presented throughout as means ± SD unless otherwise indicated.

Supplementary Material

01

HIGHLIGHTS.

  • YY1 deficiency in skeletal muscle protects from rapamycin-induced diabetes

  • YY1 mediates rapamycin-induced repression of insulin/IGF signaling genes

  • Rapamycin induces YY1 dephosphorylation at residues T30 and S365

  • Rapamycin promotes binding between YY1 and Pc2 to increase H3K27 tri-methylation

Acknowledgments

We would like to thank members of the Puigserver lab for advice and fruitful discussions, Christine Chin for technical assistance, Benjamin Szlyk for assistance in the creation of the figures, Eric Olson for the myogenin CRE mice, Markus Schubert for the IRS2-driven luciferase construct, Nathanael Gray for Torin 2 and Owen McGuinness for interpretation of the clamp experiments. These studies were supported by a postdoctoral fellowship from the Swiss National Science Foundation (SMB) and by the NIH/NIDDK RO1 DK081418 grant and Muscular Dystrophy Association MDA202237 (PP).

Footnotes

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Supplementary Materials

01
NIHMS364800-supplement-01.doc (5.9MB, doc)

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