Marys Medicine

Mg53-induced irs-1 ubiquitination negatively regulates skeletal myogenesis and insulin signalling

Received 29 Jan 2013 Accepted 25 Jul 2013 Published 22 Aug 2013 DOI: 10.1038/ncomms3354 MG53-induced IRS-1 ubiquitination negativelyregulates skeletal myogenesis and insulin signalling Jae-Sung Yi1, Jun Sub Park1, Young-Mi Ham1, Nga Nguyen1, Na-Rae Lee1, Jin Hong1, Bong-Woo Kim1, Hyun Lee1, Chang-Seok Lee1, Byung-Cheon Jeong1, Hyun Kyu Song1, Hana Cho1, Yoon Ki Kim1, Jae-Seon Lee2, Kyong Soo Park3, Haksub Shin4, Inho Choi4, Seung Hee Lee5, Woo Jin Park5, Shi-Young Park6, Cheol Soo Choi6,7, Peihui Lin8, Malith Karunasiri8, Tao Tan8, Pu Duann8, Hua Zhu8, Jianjie Ma8 & Young-Gyu Ko1 Mitsugumin 53 (MG53) negatively regulates skeletal myogenesis by targeting insulin receptor substrate 1 (IRS-1). Here, we show that MG53 is an ubiquitin E3 ligase that induces IRS-1 ubiquitination with the help of an E2-conjugating enzyme, UBE2H. Molecular manipulations that disrupt the E3-ligase function of MG53 abolish IRS-1 ubiquitination and enhance skeletal myogenesis. Skeletal muscles derived from the MG53  /  mice show an elevated IRS-1 level with enhanced insulin signalling, which protects the MG53  /  mice from developing insulin resistance when challenged with a high-fat/high-sucrose diet. Muscle samples derived from human diabetic patients and mice with insulin resistance show normal expression of MG53, indicating that altered MG53 expression does not serve as a causative factor for the development of metabolic disorders. Thus, therapeutic interventions that target the interaction between MG53 and IRS-1 may be a novel approach for the treatment of metabolic diseases that are associated with insulin resistance.
1 Department of Life Sciences, Korea University, Seoul 136-701, Korea. 2 Division of Radiation Cancer Research, Korea Institute of Radiological and MedicalSciences, Seoul 139-706, Korea. 3 Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-799, Korea. 4 Division ofBiological Science and Technology, Yonsei University, Gangwon-Do, Wonju 220-710, Korea. 5 College of Life Sciences, Gwangju Institute of Science andTechnology, Gwangju 500-712, Korea. 6 Korea Mouse Metabolic Phenotyping Center, Lee Gil Ya Cancer and Diabetes Institute, Incheon 406-840, Korea.
7 Department of Internal Medicine, Gil Medical Center, Gachon University, Incheon 406-840, Korea. 8 Division of Surgical and Biomedical Sciences,Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio 43210, USA. Correspondence and requests formaterials should be addressed to Y.-G.K. (email: NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 Skeletal muscle comprises about 40% of the human body reportofitsfunctionasanE3ligaseineitherskeletalmyogenesis mass and is a major organ that is necessary for locomotion or membrane repair. Here, we demonstrate that MG53 induces and glucose homeostasis. Adult skeletal muscle mass is IRS-1 ubiquitination with the help of the E2 enzyme UBE2H plastically regulated by recruiting satellite cells to preexisting during skeletal myogenesis by examining MG53-disrupted muscle fibres under hypertrophic conditions such as resistance skeletal muscle cells and tissues. We find that knocking out and endurance exercise. Skeletal muscle differentiation, the MG53 gene in mice ameliorates high-fat/high-sucrose diet- hypertrophy and atrophy are tightly regulated by a variety of induced insulin resistance by increasing the IRS-1 expression hormones, growth factors and cytokines. In particular, insulin- level and insulin signalling in skeletal muscle. We also find that like growth factor-1 (IGF-1) has a key role in the regulation of MG53 protein level is not upregulated in the muscle samples skeletal muscle size. IGF-1 knockout mice exhibit muscle hypo- derived from mice and human patients with metabolic disorders, plasia and die shortly after birth due to impaired respiration.
suggesting that MG53 upregulation is not a causative factor In vivo injection of IGF-1 in skeletal muscle or skeletal muscle- for the development of insulin resistance. Based on these specific overexpression of IGF-1 results in larger muscle fibres observations, we propose that therapeutic interventions that with enhanced muscle Interestingly, IGF-1 target direct interaction between MG53 and IRS-1 without production and secretion are increased by muscle hypertrophy- disruption of membrane repair function for MG53 may be a inducing hormones (for example, catecholamine and leptin) but novel approach for the treatment of metabolic disorders that are decreased by muscle atrophy-inducing hormones (for example, associated with insulin resistance.
TGFb, myostatin and glucocorticoid).
IGF-1 leads to the consecutive activation of IGF receptor (IGFR), insulin receptor substrate (IRS), phosphatidylinositol MG53 RING domain regulates skeletal myogenesis. MG53 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR) has been hypothesized to be an E3 ligase because it has an and S6 kinase (S6K), which together orchestrate skeletal myo- genesis and hypertrophyStudies with skeletal muscle-specific Fig. To elucidate the role of the MG53 RING knockout and transgenic mice for Akt, mTOR or S6K show that domain in skeletal myogenesis, we prepared two human MG53 the Akt-mTOR-S6K signalling axis is essential for skeletal muscle mutants: MG53 C14A, in which the critical cysteine at position 14 hypertrophy and regenerationIGF-1-mediated Akt activation was mutated to alanine and DR, a MG53 mutant devoid of the blocks the transcription factor forkhead box family proteins RING domain (Supplementary Fig. S1b). C2C12 myoblasts were (FOXO1 and FOXO3A) by phosphorylating and sequestering infected with adenovirus harbouring MG53 or the C14A or DR them in the cytoplasmBecause the target genes for FOXO mutant and then were differentiated to myotubes for 4 days.
proteins are E3 ubiquitin ligases such as atrogin-1 and muscle- Myogenesis was determined by immunofluorescence for myosin upregulated RING finger-1 (MURF-1) in the skeletal muscle, heavy chain (MyHC), the myogenic index and immunoblotting for myogenic marker proteins including MyHC, caveolin-3 proteasome system (UPS), which is essential for the degradation and myogenin. As shown in myogenesis was blocked by of myofibrillar, metabolic and transcriptional protei.
MG53 overexpression but was enhanced with the C14A or Indeed, knockout mice for either atrogin-1 or MURF-1 are DR mutant. Transient non-viral overexpression of Flag-MG53, resistant to atrophy C14A or DR in C2C12 cells also provided similar results during The UPS for protein degradation is necessary for the regulation myogenesis (Supplementary Fig. S1c).
of skeletal muscle differentiation, hypertrophy and .
Next, we examined IGF-1 signalling after adenoviral infection Ubiquitin and E1 ubiquitin activating, E2-conjugating and E3 of MG53, C14A or DR in C2C12 myoblasts or myotubes. As ubiquitin-ligase enzymes are required for polyubiquitinating a shown in and Supplementary Fig. S2a–c, MG53 specific target protein. The E3 ligases have two major types, overexpression reduced IGF-1-elicited IRS-1 tyrosine phosphor- HECT (homologous to E6AP carboxyl terminus) ligases and ylation and Akt phosphorylation without affecting IGF-1-elicited RING (really interesting new gene) finger ligasesThe tripartite IGFR tyrosine phosphorylation and ERK1/2 phosphorylation. It motif-containing (TRIM) superfamily is composed of 77 human should be noted that IGF-1 activates ERK1/2 through an IGFR- proteins with a RING finger domain, one or two B-boxes and Ras-Raf pathway but not through an IGFR-IRS-1 .
one or two coiled-coil domainsTRIM proteins have a plethora These data suggest the possibility that IRS-1 could be a molecular of biological functions related to cellular signal transduction target of MG53. Interestingly, the IGF-1-elicited IRS-1 and Akt and differentiation, transcriptional and cell cycle regulation and activation was increased in C2C12 myotubes following transient innate anti-viral . Due to their RING finger domain with infection with C14A or DR. These results suggest that the RING E3-ligase activity, the TRIM superfamily proteins are essential domain of MG53 is necessary for the inhibition of IGF-1-elicited for the ubiquitination of their specific target protein prior to executing their cellular function.
MG53, also known as TRIM72, was identified in C2C12 myotubes by comparative two-dimensional electrophoresis of MG53 is a ubiquitin E3 ligase that targets IRS-1. Co-immuno- detergent-resistant lipid rafts, which work as a signal transduction precipitation studies revealed that MG53 was physically interacted MG53 expression gradually increases during the with IRS-1 and mutations in the RING domain of MG53 did not myogenesis of C2C12 and satellite cells, because its promoter affect the binding of MG53 to IRS-1 in HEK 293 cells overexpressing contains two E-boxes and a MEF-binding sites for the myogenic IRS-1 and either MG53, C14A or DR (Supplementary Fig. S3a). In transcription factors, MyoD and MEF, MG53 is contrast, the IRS-1 expression level was decreased about 60% by recruited to lipid rafts, where it associates with and inactivates MG53 but not by C14A or DR in both C2C12 myoblasts and IRS-1, leading to the negative feedback regulation of skeletal myotubes Supplementary Fig. S2d). Although the IRS-1 myogenesis. In addition, MG53 acts as a major regulator for messenger RNA (mRNA) level was slightly increased, its protein membrane repair by interacting with dysferlin-1, caveolin-3 and level gradually decreased during C2C12 myogenesis when MG53 cavin-1 Indeed, the muscle fibres of MG53  /  expression was induced ), whereas the protein levels of mice show membrane repair Although MG53 might be insulin receptor b (IRb) and IGFR did not appear to change during a putative E3 ligase due to its RING domain, there has been no C2C12 myogenesis.
NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 LacZ MG53C14A ΔR Myogenic index (%) 0 2 5 IGF-1 (min) Figure 1 The RING domain of MG53 is required to negatively regulate myogenesis. (a–c) C2C12 myoblasts were infected with an adenovirus containingb-galactosidase (LacZ) or the MG53, C14A or DR gene and differentiated to myotubes. Myogenesis was assessed by MyHC immunofluorescence andDAPI staining (a); myogenic index (b); and immunoblotting for MG53, MyHC, Cav-3 and Mgn, using actin as a loading control (c). Statistical data wereobtained from three independent experiments; t-test; *Po0.01 and **Po0.05. All data are means±s.d. (d) C2C12 myoblasts were infected withan adenoviral LacZ or MG53, C14A or DR and then further incubated (for myoblasts, left panel) or differentiated (for myotubes, right panel). After serumstarvation, the myoblasts or myotubes were elicited by IGF-1 treatment for the indicated times. MG53, pAkt, total Akt, pERK1/2, total ERK1/2and IRS-1 were analysed by immunoblotting, using actin as a loading control. Tyrosine phosphorylation (pY) of IGFR and IRS-1 was determined byimmunoprecipitation. IP, immunoprecipitation; WCL, whole-cell lysates.
To examine the mechanisms underlying the MG53-mediated induce ubiquitination even in the presence of MG132 downregulation of IRS-1, we used the transient expression of Because the lysis buffer for IRS-1 immunoprecipitation contained IRS-1 and MG53 in HEK 293 cells. The IRS-1 protein level was a strong detergent, the ubiquitination signal was from IRS-1 decreased by MG53 in a concentration-dependent manner but not from MG53. Next, we tested whether the RING (Supplementary Fig. S3b) and was restored by the addition of domain of MG53 is indispensable for IRS-1 ubiquitination in MG132, a proteasome inhibitor Supplementary Fig. S3c).
C2C12 myoblasts after adenoviral infection of MG53, C14A However, the proteasomal degradation of IRS-1 was not induced or DR. As shown in IRS-1 ubiquitination was again by the C14A and DR mutants, even in the absence of MG132 induced by MG53 but not by C14A or DR in the presence suggesting that the RING domain of MG53 is essential of MG132. Moreover, IRS-1 ubiquitination and degradation for the proteasomal degradation of IRS-1.
were abolished by MG53 knockdown in C2C12 myotubes We also determined the stability of the IRS-1 protein in C2C12 myotubes following short interfering RNA (siRNA)-mediated Because IRS-2 also mediates IGF-1 signalling in skeletal knockdown of MG53 by pulse-chase labelling. As shown in muscle, we investigated MG53-dependent IRS-2 ubiquitination.
the specific siRNA could downregulate more than 90% As shown in Supplementary Fig. S3d,e, the IRS-2 expression level and ubiquitination were not changed by MG53 overexpression in half-life time of the IRS-1 protein. To study MG53-mediated myoblasts or MG53 knockdown in myotubes. These data indicate IRS-1 ubiquitination, we co-expressed IRS-1 ubiquitin with that MG53 is a true E3-ligase enzyme for the ubiquitination of MG53, C14A or DR and found that neither C14A nor DR could IRS-1 and not IRS-2.
NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 Differentiation days – – + + – – + + Flag-IRS-1 Differentiation days – + – + – + – + HA-MG53 – – + + Flag-IRS-1 – + – + HA-ΔR Differentiation days S-labelled IRS-1 0.2 Relative abundance of + – + – Si-Control – – MG53 ΔR– – + – + Si-MG53 Figure 2 MG53 is an E3-ligase enzyme inducing IRS-1 ubiquitination. (a) RT–PCR analysis of IRS-1 during C2C12 myogenesis, using actin as a loadingcontrol. C2C12 myoblasts were differentiated to myotubes for the indicated times. (b) Quantitative RT–PCR of IRS-1 during C2C12 myogenesis for theindicated times. IRS-1 mRNA was normalized to actin mRNA. Statistical data were obtained from three independent experiments. All data are means±s.d.
(c) Immunoblotting analysis of MG53, Mgn, MyHC, Cav-3, IRb, IGFR and IRS-1 during C2C12 myogenesis, using actin as a loading control. (d–f) IRS-1 isdegraded by MG53 but not by C14A or DR. Flag-IRS-1 was co-transfected with HA-MG53 (d), C14A (e) or DR (f) into HEK 293 cells. With or withoutMG132 treatment, the expression levels of IRS-1 and MG53 were determined by immunoblotting. (g,h) The degradation rate of IRS-1 protein wasdetermined in si-control or si-MG53-treated C2C12 myotubes by pulse-chase analysis with [35S]methionine and [35S]cysteine. The IRS-1 protein wasimmunoprecipitated at the indicated times, separated by SDS–PAGE and detected by autoradiography (g). The relative abundance of labelled IRS-1 proteinwas calculated from three independent experiments (h). t-test; Po0.05 (control versus si-MG53). All data are means±s.d. (i) MG53 induces IRS-1ubiquitination in HEK 293 cells. Flag-IRS-1, His-Ubiquitin and HA-MG53, C14A or DR were co-transfected into HEK 293 cells in the indicated combinations.
After MG132 treatment, IRS-1 ubiquitination was determined by immunoprecipitation with an anti-Flag antibody. (j) C2C12 myoblasts were infected withadenoviral LacZ or MG53, C14A or DR. After MG132 treatment, IRS-1 ubiquitination was determined by immunoprecipitation. (k) C2C12 myoblasts weretreated with si-MG53 (200 nM) and differentiated to myotubes. After MG132 treatment, IRS-1 ubiquitination was determined by immunoprecipitation.
C14A and DR mutants of MG53 do not degrade IRS-1. Further (Supplementary Fig. S4d). These data demonstrate that these studies showed the C14A and DR mutants function as a RING domain-disrupted MG53 mutants (C14A and DR) func- dominant-negative form of MG53 in IRS-1 degradation. Myc- tion as a dominant-negative form of MG53 via their oligomer MG53 was shown to be associated with Flag-MG53, C14A and formation with MG53 and then enhance skeletal myogenesis by DR by reciprocal exogenous immunoprecipitation of HEK 293 abrogating MG53-induced IRS-1 ubiquitination cells (Supplementary Fig. S4a,b). Moreover, MG53, C14A and DR To further confirm the effect of MG53 on myogenesis and appeared to be oligomerized in the presence of glutaraldehyde, a IRS-1 ubiquitination, mouse embryonic fibroblasts (MEFs) were protein cross-linker (Supplementary Fig. S4c), suggesting obtained from MG53 þ / þ and MG53  /  mouse embryos and that the RING domain is not necessary for MG53 oligomeriza- were differentiated to myotubes after adenoviral MyoD over- tion. We found that MG53-induced IRS-1 degradation was expression. The MG53 protein level increased, along with completely abolished by C14A or DR mutant in HEK 293 cells other myogenic marker proteins such as MyHC, caveolin-3 and NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 Differentiation days +/+ –/– +/+ –/– Myogenic index (%) 10 Figure 3 IRS-1 ubiquitination is abrogated in MyoD-driven myotubes of MEFs. (a) The MG53 protein level is increased during MyoD-drivenmyogenesis of MEFs. MEFs were isolated from wild-type mice embryos, infected with adenovirus with MyoD and differentiated to myotubes for theindicated times. The expression levels of MG53, MyoD, MyHC, Cav-3, Mgn, IRS-1 and IGFR were monitored by immunoblotting, using actin as aloading control. (b–d) MG53 knockout enhances the MyoD-driven myogenesis of MEFs. MG53 þ / þ and MG53  /  MEFs were infected with adenoviralMyoD and differentiated to myotubes for 4 days. Myogenesis was assessed by MyHC immunofluorescence and DAPI staining (b); the myogenic index (c);and immunoblotting for MG53, MyoD, MyHC, Cav-3, Mgn, IRS-1 and actin (d). The myogenic index in the MyHC-stained cells was calculatedat least from the six different fields. t-test; *Po0.01. All data are means±s.d. (e) MG53 knockout abolishes IRS-1 ubiquitination in MyoD-driven myotubesof MEFs. Myotubes of MyoD-overexpressing MG53 þ / þ and MG53  /  MEFs were treated with or without MG132. IRS-1 ubiquitination was determinedby immunoprecipitation.
myogenin, but the IRS-1 protein level decreased during MyoD- Clearly, assessment of myogenesis by immunofluorescence for driven myogenesis in MG53 þ / þ MEFs As evidenced HA and MyHC and myogenic index revealed that the MG53- by MyHC immunofluorescence, myogenic index and immuno- induced inhibition of myogenesis was abolished by UBE2H blotting, MG53 knockout enhanced myogenesis by increasing the knockdown In addition, the MG53-induced IRS-1 level of IRS-1 expression We also found that MG53 ubiquitination was further increased by UBE2H overexpression in knockout abolished IRS-1 ubiquitination in the MyoD-driven HEK 293 cells (Supplementary Fig. S5e). These data suggest that myotubes of MEFs supporting the concept that MG53 is UBE2H is a genuine E2-conjugating enzyme for MG53.
an E3 ligase for IRS-1.
MG53 expression affects insulin signalling in cultured C2C12myotubes. To test whether the MG53-mediated control of IRS-1 UBE2H is involved in MG53-mediated IRS-1 ubiquitination.
expression may contribute to insulin signalling in skeletal muscle, Because a specific ubiquitin-conjugating E2 enzyme transfers we monitored insulin-elicited IRS-1, Akt and ERK1/2 phos- ubiquitin to a specific target protein through E3 ligaseMG53- phorylation in MG53-overexpressing C2C12 myoblasts. As mediated IRS-1 ubiquitination should have an E2 enzyme shown in and Supplementary Fig. S6, insulin-elicited partner. To identify the ubiquitin-conjugating E2 enzyme for IRS-1, Akt and ERK1/2 activation was dramatically decreased MG53, we examined the molecular association of MG53 with by MG53 overexpression. In contrast to IGF-1 signalling, IRS-1 various E2 enzymes. As shown in in vitro binding assay is required for insulin-elicited ERK1/2 . These data (Supplementary Fig. S5a; MG53 strongly interacted indicate that MG53 negatively regulates insulin signalling.
with UBE2H. The UBE2H mRNA and protein levels gradually Because the IRS-1 protein level was decreased during myo- increased along with MG53 expression during C2C12 myogenesis genesis insulin signalling might also be (Supplementary Fig. S5b–d). The physical association of UBE2H downregulated in myotubes, compared with myoblasts. Indeed, with MG53 was further confirmed by exogenous co-immuno- myotubes had less insulin-elicited phosphorylation of IRS-1, Akt precipitation studies in HEK 293 cells that overexpress UBE2H and ERK1/2 than myoblasts did, whereas both cells had no and MG53 and by reciprocal endogenous immunoprecipitation difference in insulin-elicited IRb phosphorylation in C2C12 myotubes Thus, UBE2H appears to be a However, myotubes had greater insulin-induced glucose uptake good candidate for the E2-conjugating enzyme for MG53.
than myoblasts did (Supplementary Fig. S7) because of the higher The role for UBE2H in myogenesis was tested in C2C12 cells expression level of GLUT4 in the myotubes following siRNA-mediated knockdown. As shown in C2C12 myogenesis was enhanced by UBE2H knockdown, wherethe IRS-1 expression level was increased about threefold and Systemic MG53 disruption improves muscle insulin signalling.
IRS-1 ubiquitination was abolished in C2C12 myotubes Insulin signalling was also analysed in the soleus and gastro- To test whether the MG53 inhibition of myogenesis cnemius-plantaris (GP) muscles of 8-week-old MG53  /  mice is reversed by UBE2H knockdown, HA-MG53 was transiently after the intravenous injection of insulin. In both muscles, the transfected along with si-control or si-UBE2H (200 nM) in increase of insulin-elicited IRS-1, Akt and ERK1/2 activation was C2C12 myoblasts, which were further differentiated to myotubes.
observed along with an elevation in the IRS-1 expression level in NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 Myogenic index (%) Si-Contro Si-UBE2H in HA-positive cells The number of nuclei Figure 4 UBE2H is an E2 enzyme for MG53-mediated IRS-1 ubiquitination. (a) In vitro binding assay of MG53 with various E2 enzymes. The mixtures ofMBP-MG53 and various His-E2 enzymes were immobilized on an amylose resin, and the interaction was analysed by immunoblotting. (b) Myc-MG53and Flag-UBE2H were co-transfected into HEK 293 cells in different combinations. The molecular interaction between MG53 and UBE2H was monitored byreciprocal immunoprecipitation. (c) The molecular association of MG53 with UBE2H by reciprocal endogenous immunoprecipitation from differentiatedC2C12 myotubes. (d–f) C2C12 myoblasts were treated with si-control or si-UBE2H (200 nM) and then differentiated to myotubes. Myogenesis wasassessed by MyHC immunofluorescence and DAPI staining (d), the myogenic index (e) and immunoblotting (f). The myogenic index in the MyHC-stainedcells was calculated at least from the six different fields. t-test; *Po0.01. All data are means±s.d. (g) After UBE2H knockdown, C2C12 myotubeswere treated with MG132, and IRS-1 ubiquitination was determined by endogenous immunoprecipitation. (h,i) HA-MG53 was transiently co-transfectedalong with si-control or si-UBE2H (200 nM) in C2C12 myoblasts. The myoblasts were further differentiated into myotubes and analysed by MyHC and HAimmunofluorescence (h). The nuclei were stained with DAPI. The number of nuclei in HA-positive cells was calculated in three independent microscopicfields (i). t-test; *Po0.01. All data are means±s.d.
MG53  /  mice, compared with MG53 þ / þ mice The cross-sectional area was increased in the soleus muscle but was However, there was no difference in IRb protein expression level similar in the GP and TA of MG53  /  mice (Supplementary and insulin-elicited IRb activation between skeletal muscles Fig. S10b–e), compared with those of MG53 þ / þ . In addition, obtained from MG53 þ / þ and MG53  /  , indicating that IRb is higher muscle fatigue resistance was shown in the soleus but not in not a substrate of E3 ligase MG53.
extensor digitorum longus (EDL) of MG53  /  mice, compared The 8-week-old MG53  /  mice did not show pathological with those of MG53 þ / þ (Supplementary Fig. S11).
cardiac hypertrophy and alterations in total body weight, We also analysed muscular hypertrophy and insulin signalling in 38-week-old MG53  /  mice. The older MG53  /  mice (Supplementary Figs S8,S9). Because MG53 has been reported did not show muscular hypertrophy any more (Supplementary to be highly expressed in soleus muscle compared with other Fig. S12a,b). However, IRS-1 expression level and insulin-elicited skeletal , we speculated that the change of muscle fibre IRS-1, Akt and ERK1/2 phosphorylation were still increased in size by MG53 knockout might appear in the soleus. Indeed, only the skeletal muscles of MG53  /  mice, compared with those of soleus muscle of MG53  /  mice was B27% heavier than that of MG53 þ / þ mice (Supplementary Fig. S12c,d).
MG53 þ / þ mice, whereas there were no differences in GP and The increased insulin signalling in skeletal muscle might lead to tibialis anterior (TA) muscle weight (Supplementary Fig. S10a).
insulin-sensitive phenotypes in MG53  /  mice. Although there NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 Figure 5 Insulin signalling is increased in the skeletal muscles of MG53 knockout mice. (a,b) MG53 was adenovirally overexpressed in C2C12myoblasts. The myoblasts were then serum-starved and elicited by insulin (100 nM) for the indicated times. MG53, pAkt, total Akt, pERK1/2, total ERK1/2and actin were monitored by immunoblotting. The tyrosine phosphorylation of IRS-1 was determined by immunoprecipitation (a). The pIRS-1, pAktand pERK1/2 levels were statistically determined (n ¼ 3 for each condition) (b). t-test; *Po0.05 and **Po0.01 (LacZ versus MG53). All data aremeans±s.d. (c) C2C12 myoblasts were differentiated to myotubes for 4 days. The myoblasts and myotubes were serum-starved and elicited by insulin forindicated times. IRS-1, pAkt, total Akt, pERK1/2, total ERK1/2, Cav-3 and actin were monitored by immunoblotting. The tyrosine phosphorylation ofIRS-1 was determined by immunoprecipitation. (d,e) After intravenous injection of insulin, both the soleus (Sol) and gastrocnemius-plantaris (GP)were isolated from 8-week-old male MG53 þ / þ and MG53  /  mice. Insulin signalling was analysed by immunoblotting and immunoprecipitation (d).
The IRS-1, pIRS-1 and pAkt levels were statistically assessed (n ¼ 5 for each group). t-test; *Po0.05 and **Po0.01. All data are means±s.d. (e).
was no difference in whole body weight, fat mass, food intake, inflammation status by measuring mRNA levels of inflammatory energy expenditure and locomotor activity between high-fat diet cytokines (IL1b, PAI and TNF-a) and by immunofluorescence of (HFD)-fed MG53 þ / þ and MG53  /  mice (Supplementary macrophage infiltration markers (F4/80 and MCP-1) in the Fig. S13), the serum levels of triacylglycerol, free fatty acid, total epididymal adipose tissue obtained from HF/HS-fed MG53 þ / þ cholesterol, insulin and leptin were dramatically lower in HF/HS- and MG53  /  mice. As shown in Supplementary Fig. S14a, there fed MG53  /  mice, compared with HF/HS-fed MG53 þ / þ mice was no difference in the mRNA levels between HF/HS-fed . Glucose disposal rates during the glucose and insulin tolerance test were improved from MG53  /  mice fed with both MG53 þ / þ and MG53  /  mice had similar macrophage infiltra- of regular and HF/HS diet compared with regular and HF/HS tion into adipose tissue (Supplementary Fig. S14b). These data diet-fed MG53 þ / þ mice, respectively .
indicate that MG53 disruption ameliorates HF/HS-induced insulin As inflammation in adipose tissues induces insulin resis, resistance without preventing fat mass increase and macrophage we next monitored the effect of MG53 disruption on the infiltration in adipose tissue from HF/HS-fed mice.
NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 Triacylglycerol (mM) Free fatty acid (mM) Plasma insulin (ng ml Plasma leptin (ng ml Total cholesterol ( MG53–/–, HF/HS MG53–/–, HF/HS Plasma glucose (mg dl Figure 6 Insulin resistance does not develop in MG53  /  mice. Four-week-old male MG53 þ / þ and MG53  /  mice (n ¼ 6 for each group) were feda regular diet or a HF/HS diet for 10 weeks. (a–e) Serum was obtained from overnight-fasted mice. The concentration of triacylglycerol (a), free fattyacid (b), total cholesterol (c), insulin (d) and leptin (e) were measured. R, regular diet. t-test; *Po0.01. (f,g) Glucose tolerance (f) and insulin tolerance(g) tests were then performed in the R- and the HF/HS-fed MG53 þ / þ and MG53  /  mice (n ¼ 6 for each group). AUC indicates the area underthe curve. t-test; *Po0.05 and **Po0.01 (MG53  /  HF/HS versus MG53 þ / þ HF/HS) and #Po0.05 (MG53  /  R versus MG53 þ / þ R). All data aremeans±s.d.
MG53 in animal models and humans with metabolic disorders.
top). Second, to test whether development of the mice can A recent study by Song et al.showed that MG53 protein level is impact the effect of HFD on MG53 expression in skeletal muscle, upregulated in the skeletal muscles obtained from multiple we treated mice with HFD for 1 week starting at the adolescent animal models for metabolic disorders. In particular, they age (8-week old). Again, there were no significant changes in reported that skeletal muscle derived from the db/db mice at 25 MG53 expression between regular chow diet- and HFD-treated weeks of age exhibited greater than threefold upregulation of mice middle). Third, when the adolescent mice MG3 protein when compared with the lean mice and feeding of were subjected to 4 weeks of HFD treatment, there were no mice with HFD led to several-fold upregulation of MG53, as early significant changes in MG53 expression in skeletal muscle as 1 week following HFD treatment. These were the main experimental evidence supporting their conclusion that MG53 Overall, our data show that MG53 is not upregulated in the upregulation serves as a causative factor for metabolic disorders.
skeletal muscles from animals and human patients with metabolic We performed immunoblotting with skeletal muscle derived disorders, arguing against the claim made by Song et al.
from db/db and their littermate lean control mice and found nosignificant difference in MG53 expression in both slow-twitchsoleus and fast-twitch EDL at 22–29 weeks of age Similar protein levels for MG53 were also observed between the Here, we provide multiple lines of evidence to show that MG53 is ob/ob, db/db and age-matched lean control mice at 8-week age a negative feedback regulator of myogenesis and insulin signalling when the db/db mice already developed metabolic disorder in skeletal muscle. First, we showed that MG53 overexpression We next assayed the expression of MG53 using skeletal blocked myogenesis, whereas RNA interference-mediated silen- muscle biopsies obtained from the type 2 diabetic human cing of MG53 enhanced myogenesis by modulating IGF-1- patients. As shown in while the level of MG53 protein elicited IRS-1, PI3K and Akt . Second, we found that may vary from different patients, immunoblotting revealed no overexpression of RING domain-disrupted MG53 mutants significant changes in MG53 expression among the different age (C14A and DR) led to disruption of MG53-mediated IRS-1 and gender groups between the healthy people and diabetic ubiquitination and consequently the robust muscle differentiation patients. Thus, our human muscle data do not reveal a correlation Such dominant-negative effect of the MG53 between changes in MG53 expression and diabetic disorder.
mutants provide an attractive avenue for pharmacological or We also found negligible changes in expression of MG53 in molecular manipulations that can be used to target the inter- skeletal muscle following three different paradigms of HFD molecular interactions among MG53 and its target molecules for treatment First, when the wild-type mice were treated treatment of metabolic diseases linked to muscle dysfunctions.
with HFD or regular chow diet for 1 week starting right after Third, we provide conclusive evidence that links myogenesis their weaning age (25 days after birth), such acute HFD treatment enhancement with increased level of IRS-1 in MyoD-driven did not alter the expression for MG53 in skeletal muscle MG53  /  myotubes and the MG53  /  mice NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 Figure 7 MG53 protein in animals and humans with metabolic disorders. (a) Immunoblots of soleus (Sol) and EDL derived from littermatesof db/db and lean control mice at ages of 22 and 29 weeks. (b) Study with ob/ob, db/db and lean mice purchased from Jackson Lab also did not revealsignificant changes in MG53 expression in extensor digitorum longus (EDL), gastrocnemius (Gast) and quadriceps (Quad) at 8-week age. (c) Skeletalmuscle (vastus lateralis) biopsies were obtained from human patients with type 2 diabetes (T2D) and healthy (H) controls. Protein expression levels wereanalysed by immunoblotting using actin as a loading control. T2D was diagnosed using American Diabetes Association ‘Young' refers to agesbetween 28 and 55 years, ‘old' indicates ages between 65 and 82. (d) Sol, EDL and Gast obtained from littermates of wild-type mice (C57B/6J) following1-week treatment of regular diet (RD) or high fat diet (HFD) starting from 25 days after birth (top panel) or 8 weeks after birth (middle panel). Separatetrials were performed with 8-week-old mice following 4-week treatment with HFD (bottom panel). MG53 protein levels in the different types of skeletalmuscles shown in a–d were statistically not different between control and animal models with metabolic disorders or human diabetes patients.
Together, our data indicate that MG53 is a real E3-ligase enzyme Because IRS-1 is a convergent signal molecule for IGF-1 that is required for IRS-1 ubiquitination to negatively regulate and insulin signalling, IRS-1 deficiency abolishes skeletal myo- skeletal muscle myogenesis and insulin signalling.
genesis and insulin signalling. Lee et al.showed that MG53 Based on the above findings, we propose the following model knockdown-induced myogenesis enhancement is completely for MG53 as a negative feedback regulator of IGF-1 signalling abrogated by IRS-1 knockdown, indicating that IRS-1 is a during skeletal myogenesis In myoblasts that do not downstream signalling molecule of MG53 that is required for the express MG53 left panel), IGF-1 initiates myogenesis via negative regulation of myogenesisIn addition, IRS-1 the IGFR-IRS-PI3K-Akt-MyoD signalling pathway. IGF-1- knockout mice show insulin resistance and no IGF-1-induced elicited Akt activation leads to phosphorylation of p300, causing muscle . These findings indicate that IRS-1 is essential an association of MyoD with p300 and PCAF .
for insulin signalling and IGF-1-mediated muscle growth.
The activated transcriptional complex binds to the MG53 To investigate MG53-dependent myogenesis inhibition and promoter with two E-boxes and a MEF-binding sites and then insulin signalling, we monitored C2C12 differentiation and initiates MG53 transcriptionMG53 protein begins to insulin signalling after MG53 overexpression. IGF-1 and insulin accumulate in the sarcolemma and associates with and signalling and myogenesis were decreased after MG53 over- polyubiquitinates IRS-1 with the help of UBE2H. The expression Thus, our data suggest that both polyubiquitinated IRS-1 is degraded by the UPS. Thus, in fully MG53-dependent myogenesis inhibition and insulin insensitivity differentiated myotubes with forced expression of MG53 are tightly related with IRS-1.
right panel), the IGF-1-elicited Akt activation is weakened due to IRS-1 is polyubiquitinated and degraded by SOCS1/3, Cbl-b, the low IRS-1 level, which is caused by the UBE2H-MG53- Cul7 and SCF-Fbxo40. SOCS1/3 is activated by inflammation proteasome system. However, IGF-1-elicited Erk1/2 activation is and might induce insulin resistance in the liver by polyubiquiti- not influenced by MG53 in either myoblasts or myotubes because nating IRS-1 and IRS-2 Cbl-b targets IRS-1 during the Ras-Raf-Erk1/2 pathway is diverted from IGFRwhose muscular atrophy such as unloadingCul7-induced IRS-1 expression level and activity are not changed by MG53.
polyubiquitination might contribute to the regulation of NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 disruption of the membrane repair function for MG53 might bedeveloped as drug candidates for the treatment of insulin resistance.
Alternatively, molecular interventions that selectively abolish the E3- ligase function for MG53 without impacting the tissue repair functionfor MG53 could be an attractive avenue for development of MG53 asa therapeutic reagent for regenerative medicin.
Generation of MG53  /  mice. MG53  /  mice were generated as described previously. Briefly, exons 1, 2 and 3 of the TRIM72 gene were replaced with aPGK-neo sequence within the targeting vector by homologous recombination. Forscreening of recombination-positive embryonic stem cells, genomic DNA was digested with BamHI, and analysed by Southern blotting. To backcross the mice,MG53 þ /- mice were bred with C57BL/6 mice for seven generations. Mice werehoused in plastic cages under a 12:12-h light–dark photoperiod with free access towater and food. To induce insulin resistance, the 4-week-old male mice were fed with a HF/HS diet (35.8% (w/w) fat, 35.5% (w/w) carbohydrate and 23.0 (w/w)protein; D12331, Research Diet, New Brunswick, NJ) for 10 weeks. Animals werehandled according to the Principles of Laboratory Animal Care (NIH Publication No. 85-23, revised 1985), and the protocols were approved by the Institutional Animal Care and Use Committee of Korea University.
Cell culture. C2C12 cells were purchased from ATCC and grown in growth Figure 8 MG53-mediated negative feedback regulation of skeletal medium (Dulbecco's modified Eagle's medium supplemented with 2% penicillin/streptomycin and 10% fetal bovine serum) in a 5% CO myogenesis. MG53 transcription is initiated by an IGFR-IRS-1-Akt-MyoD 2 incubator at 37 °C.
Confluent C2C12 myoblasts were differentiated into myotubes by incubation with pathway during skeletal myogenesis. The MG53 protein interacts with, differentiation medium (DMEM supplemented with the same antibiotics as above ubiquitinates and degrades IRS-1, leading to the blockage of IGF and insulin and 2% horse serum). Every 48 h, the myotubes were fed with fresh differentiation signalling in fully differentiated myotubes.
medium. MEFs were obtained from MG53 þ / þ and MG53  /  embryos atembryonic day 12.5 according to Shim et al.To differentiate the cells intomyotubes, MEFs were transfected with adenoviral MyoD (5  109 VP/ml  1) for12 h and incubated with differentiation medium.
oncogene-induced . Skeletal muscle-specific Fbxo40interacts myogenesisHowever, there is no direct evidence to date that Adenoviral preparation and infection. Adenoviruses harbouring MG53, C14A connects these E3 ligases with insulin resistance.
and DR were produced according to a previously described Adenoviruscontaining MyoD was obtained from Cell Biolabs (San Diego, CA). To amplify the Recently, Song et al.claimed that MG53 protein expression virus, viral stocks were re-infected into AD293 cells and purified by double caesium level is highly increased in the skeletal muscle obtained from chloride-gradient ultracentrifugation. Infectious viral particles in the caesium HFD-fed and db/db mice, spontaneously hypertensive rats, chloride gradient (density ¼ B1.345) were collected, dialyzed against 10 mM Tris nonhuman primates with metabolic syndrome and human (pH 8.0), 2 mM MgCl2 and 5% sucrose solution and stored at  80 °C. C2C12myoblasts or MEFs were infected by adenovirus at a dosage of 5  109 VP ml  1.
obese patients, concluding that upregulation of MG53 serves asa universal causative factor for development of metabolic diseasesin skeletal . However, our data showed that MG53 Plasmids for transient transfection and luciferase assay. Human IRS-1, upregulation in skeletal muscle was not observed from ob/ob, ubiquitin and MG53, C14A and DR cDNA constructs were generated by PCR andcloned into the pCMV-Tag2b and pCMV-3Tag4a vectors. DNA transfection was db/db and HFD-fed mice and type 2 diabetes patients performed using Polyfect (Qiagen, Valencia, CA) or electroporation (Invitrogen, challenging the conclusion that MG53 upregulation serves as a Grand Island, NY) according to the manufacturer's protocol.
preceding factor for the development of metabolic disorders. Songet al.also claimed that IRb is ubiquitinated by MG53 because Antibody-based assays. Immunoblotting and immunofluorescence were that IRb ubiquitination and insulin-elicited IRb phosphorylation performed according to Yi et al.For immunoblotting, proteins were separated are increased in MG53 transgenic mice and abolished in MG53 on polyacrylamide gels and were transferred onto a PVDF membrane. The knockout . However, our data showed that IRb might not membranes were then blocked for 1 h at room temperature, and allowed to reactwith a sequence of primary and secondary antibodies. The antigen signals were be a substrate of E3-ligase MG53 because IRb protein level or visualized using ECL reagents. For immunofluorescence, cells were fixed with 3.7% insulin-elicited IRb phosphorylation were not changed during formaldehyde for 10 min, permeabilized with 0.1% TX-100 in PBS, washed three C2C12 myogenesis when MG53 protein level was gradually times with PBS and then blocked with 5% BSA in PBS for 1 h. After blocking, cells increased by MG53 overexpression in C2C12 myoblasts were incubated with primary antibodies and primary antibodies were detected or by systemic MG53 disruption These using fluorescence-conjugated secondary antibodies. Cells were observed with afluorescence microscope (Axioplan-2; Carl Zeiss, Oberkochen, Germany). For data challenge the MG53-induced IRb degradation observed by immunoprecipitation, the cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 20 mM NaF, 10 mM Na4P2O7, We showed that MG53 disruption increased insulin-elicited 1 mM Na3VO4, 1% NP-40, 1 mM PMSF and a protease inhibitor cocktail (Roche, IRS-1 activation with an elevated IRS-1 protein level in mouse Mannheim, Germany). The whole-cell lysates (500 mg protein) were incubated withspecific antibodies for 90 min and then with 50 ml of a Protein A-Agarose bead skeletal muscle, inducing skeletal muscle hypertrophy at least in the (Roche, Mannheim, Germany) slurry for 90 min. The immunoprecipitates were soleus, improving glucose tolerance even in regular diet-fed mice and analysed by immunoblotting. For immunofluorescence, C2C12 cells were washed ameliorating HF/HS diet-induced insulin resistance ( briefly with PBS. Supplementary Table S1 and S2 show the information of anti- Thus, we can conclude that the inhibition of MG53-mediated IRS-1 bodies, which were used for immunoblotting, immunoprecipitation and immu-nofluorescence. Full-length immunoblots are shown in Supplementary Figure S15.
ubiquitination could be used as a therapeutic strategy for thetreatment of muscular atrophy and insulin resistance. As MG53 hasdual functions as an E3 ligase that targets ubiquitination-mediated Measurement of the myogenic index. Differentiated C2C12 cells or MEFs were degradation of IRS-1 and an indispensable component of the cell stained with anti-MyHC antibody and DAPI and cell images were obtained under afluorescence microscope (Axioplan-2). The myogenic index was determined as the membrane repair machin, compounds that prevent the ratio of the nuclei within MyHC-positive myotubes to the total nuclear number in molecular association of MG53 with IRS-1 or UBE2H without the stained field.
NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS DOI: 10.1038/ncomms3354 RNA interference. siRNA oligomers targeting MG53 (si-MG53) or UBE2H exchange data. RQ is the ratio of VCO2 to VO2, which changes depending on the (si-UBE2H) and a scrambled oligomer (si-control) were obtained from Ambion.
energy source that the animal is using. Energy expenditure ¼ (3.815 þ 1.232 * RQ) The target sequence of MG53 was 50-AAGCACGCCUCAAGACACAGC-30, and * VO2. Activity was measured along the x- and z-axes using infrared beams to the target sequence of UBE2H was 50-CUAUGAUCUUACCAAUAUAtt-30. C2C12 count the number of beam breaks during the specified measurement period.
myoblasts were transfected with 100 nM of si-control, si-MG53 or si-UBE2H byelectroporation (Invitrogen) according to the manufacturer's protocol.
Glucose tolerance and insulin tolerance tests. For glucose tolerance tests,14-week-old male mice that had been fasted overnight received an intraperitoneal Chemical cross-linking. HEK 293 cells were harvested with 60 mM octyl-b- injection of D-glucose (2 g kg  1 body weight). For the insulin tolerance test, insulin D-glucopyranoside in PBS. Whole-cell lysates were mixed with the indicated (0.75 U kg  1 for mice fed a regular diet, 1.5 U kg  1 for mice fed a HF/HS) concentrations of glutaraldehyde and incubated at 37 °C for 20 min. The cross- were intraperitoneally injected into 14-week-old male mice that had been fed.
linking reaction was stopped by adding 1.5 M Tris-HCl (pH 7.4) and then the Blood was obtained from the tail and glucose levels were determined with proteins were separated on SDS–PAGE.
an automatic glucose monitor.
In vitro binding assay. Human MG53 (residues 7–470) was cloned into the Measurement of serum samples. Blood samples were collected from the tails of pMAL-c2x vector (NEB). Escherichia coli C41(DE3) was used as an expression host 14-week-old male mice that were fasted overnight. After centrifugation at 1,000  g strain for MBP-MG53. The expressed protein was purified using affinity column for 10 min, the supernatants of the blood samples were separated. The serum levels (amylose resin) followed by anion exchange chromatography (HiTrap Q Fast Flow of triacylglycerol, free fatty acids and total cholesterol were measured with column). Pull-down assays were carried out in 1 ml assay buffer (1X PBS and 1 mM colorimetric assay kits (BioVision, Mountain View, CA). The serum levels of DTT) containing 20 ml amylose resin, 50 mg MBP-MG53 and 100 mg His-tagged E2 insulin and leptin were determined with the Bio-Plex Pro mouse diabetes assay kits enzymes for 1 h at 4 °C. His-tagged E2 enzymes were purchased from Boston (Bio-Rad, Hercules, CA).
Biochem (Cambridge, MA) and LifeSensors (Malvern, PA). The interactionbetween MG53 and E2 enzymes was examined using immunoblotting withanti-His and anti-MBP antibodies.
Statistical analysis. Statistical values are presented as the mean±s.e.m.
A two-tailed Student's t-test was used to calculate the P values.
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NATURE COMMUNICATIONS 4:2354 DOI: 10.1038/ncomms3354 & 2013 Macmillan Publishers Limited. All rights reserved.



Neuroscience in Anesthesiology and Perioperative Medicine Section Editor: Gregory J. Crosby Cerebral Oxygen Desaturation Events Assessed byNear-Infrared Spectroscopy During Shoulder Arthroscopyin the Beach Chair and Lateral Decubitus Positions Glenn S. Murphy, MD,* Joseph W. Szokol, MD,* Jesse H. Marymont, MD,* Steven B. Greenberg, MD,*Michael J. Avram, PhD,† Jeffery S. Vender, MD,* Jessica Vaughn, BA,* and Margarita Nisman, BA*

Taylor, MC; Kelly, JM (2006) pTcINDEX: a stable tetracycline-regulated expression vector for Trypanosoma cruzi. BMC Biotechnol,6. p. 32. ISSN 1472-6750 DOI: 10.1186/1472-6750-6-32 Downloaded from: Please refer to usage guidelines at or alterna-tively contact Available under license: Research articlepTcINDEX: a stable tetracycline-regulated expression vector for Trypanosoma cruziMartin C Taylor* and John M Kelly