Negative regulation of AMPK signaling by high glucose via E3 ubiquitin ligase MG53

•High-glucose-induced ROS promotes phosphorylation of AMPKα at S485/491 by AKT•Phosphorylation is required for recruitment E3 ligase MG53•MG53 mediates skeletal muscle ubiquitination and subsequent degradation•High glucose also deactivates inducing disassociation from LKB1 As a master regulator metabolism, AMP-activated protein kinase (AMPK) activated upon energy shortage but suppressed overnutrition. Exaggerated negative regulation AMPK signaling nutrient overload plays crucial role in metabolic diseases. However, the mechanism underlying poorly understood. Here, we demonstrate that high represses via MG53 (also called TRIM72) E3-ubiquitin-ligase-mediated degradation deactivation. Specifically, high-glucose-stimulated reactive oxygen species (ROS) signals AKT to phosphorylate S485/491, which facilitates AMPKα. In addition, ROS-dependent suppression T172. 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Kristensen Højlund Intact network response male illumination recovery exercise.Diabetes. 2016; 65: 1219-1230Crossref Musi 2001Musi Fujii Ekberg Fröberg Ljungqvist Thorell during 2001; 50: 921-927Crossref (297) discrepancy might be attributed heterogeneity who could stages disease treated different medications. further evidenced failure two agonists, MK8722 A769662, induce uptake 12 weeks age (Figures 1B S1B). abundance AMPKα1 AMPKα2 were markedly 32 1C). Similar reductions diet (HFD)-treated mice, non-human primates (NHPs) spontaneous syndrome, 1C; Table S1). there no significant change mRNA level either HFD-treated S1C S1D), suggesting posttranscriptional level. Indeed, overtly 1D 1E), ubiquitin-proteasome-dependent may represent an important disease-associated Previous upregulation striated-muscle-enriched ligase, TRIM72; Figure S1E), triggers disorders targeting receptor (IR) IR substrate (IRS1) ubiquitin-dependent (Liu 2015Liu Song Guo Chen Huang C.Y. al.Upregulation induces cardiomyopathy through transcriptional peroxisome proliferation-activated α.Circulation. 2015; 131: 795-804Crossref (84) 2013Song Peng Lv Wu H.K. Cao Pi Jin al.Central ubiquitin disorders.Nature. 494: 375-379Crossref Yi 2013Yi J.S. Park Ham Y.M. Nguyen Lee N.R. Hong Kim B.W. Jeong B.C. al.MG53-induced IRS-1 negatively regulates myogenesis signalling.Nat. Commun. 4: 2354Crossref (98) Consistent reports, aforementioned models subjects, upregulated 1C S1F), IRβ IRS1 downregulated S1G). Notably, increase accompanied reduction isoforms fact, correlation between 1F 1G). Vice versa, MG53-deficient IRS1, amount significantly 1H). hypothesized act contributing metabolic-disorder-associated Using co-immunoprecipitation (coIP) assay, found interacted 2A). AMPKα2, dominant striated formed complex HEK293T when proteins overexpressed S2A). An vitro pull-down assay direct interaction S2B). Furthermore, coIP revealed C terminus 2B S2C). Similarly, pinpointed SPLa Ryanodine Receptors (SPRY) domain, substrate-binding domain (Esposito 2017Esposito Koliopoulos M.G. Rittinger Structural determinants TRIM function.Biochem. Soc. Trans. 183-191Crossref (77) 2010Park E.Y. Kwon O.B. Ko Y.G. Crystal structure PRY-SPRY TRIM72.Proteins. 78: 790-795PubMed binding region 2C S2D), implying MG53. It shown cullin-RING 4A (CRL4A) decreases Streptozotocin (STZ)-induced rats (Ko 2018Ko Seo D.Y. S.H. Kwak H.B. K.S. Rhee B.D. Han Aerobic training cereblon increases STZ-induced rats.Biochem. Biophys. 2018; 501: 448-453Crossref (10) bone marrow mast (Kwon 2019Kwon Deng Chang H.W. down-regulated CRL4A-CRBN axis polyubiquitination isoforms.FASEB 33: 6539-6550Crossref (17) CRL4A S2E). Moreover, knocking down did affect C2C12 myotubes cultured low S2F). WWP1 implicated (Lee 2013Lee J.O. S.K. J.H. You G.Y. Moon Jie S.J. Y.W. Kang H.J. al.E3 WWP1, interacts down-regulates cells.J. Chem. 288: 4673-4680Abstract Full Text PDF (24) HFD-induced S2G), weakening mediating under long-term overload, case T2D. primary especially conditions upregulated. Importantly, myotubes, endogenous D-glucose, L-glucose, co-localization 2D) co-immunoprecipitated 2E). concentration D-glucose promoted L-glucose had effect 2F, 2G, S2H). Knockdown attenuated 2H) maintained after high-glucose 2G). contrast, overexpression MG53, inactive mutant (C14A) (Song boosted 2I S2I). results indicate functions catalyze isoforms. Unlike AMPKα, enhance S2J) S2K), although manipulating altered S2K S2L). next examined whether high-glucose-enhanced, MG53-dependent accelerates turnover. dose-dependent manner S2M), subunits β1, β2, γ1 affected S2N). C14A failed decrease 2J S2O). fully blocked proteasome inhibitors clasto-lactacystin β-lactone 2J) MG132 constructed mutants (lysine 48 lysine 63 arginine [K48R K63R, respectively]) (Weissman, 2001Weissman A.M. Themes variations ubiquitylation.Nat. Cell 2: 169-178Crossref (1233) MG53-induced occurred K48-linked chains (for degradation) rather than K63-linked transduction trafficking) 2K), indicating MG53-mediated targets degradation. Mutation three potential sites (Wagner 2012Wagner Beli Weinert B.T. Schölz Kelstrup C.D. Young Nielsen M.L. Olsen J.V. Brakebusch Choudhary Proteomic analyses reveal divergent ubiquitylation site patterns murine tissues.Mol. Cell. Proteomics. 2012; 11: 1578-1585Abstract (202) showed 470 (K470R), 60 (K60R) 379 (K379R), potently 2L). Concomitantly, K470R, among mutants, resistant 2M S2P). above suggested K470 this modification stability. Taken together, stability regulated muscle-enriched conditions, such exaggerate noticed hyperglycemic 3A, 3B, expected, diminished ACC following S2Q). While liver B1 (LKB1) established upstream activates phosphorylating (Gowans 2013Gowans G.J. Ross F.A. AMP true physiological allosteric enhancing net phosphorylation.Cell 556-566Abstract (315) multiple kinases, S6K, inhibitory S485 S491 (Dagon 2012Dagon Hur Zheng Wellenstein Cantley L.C. Kahn B.B. p70S6 phosphorylates serine 491 leptin’s food intake.Cell 16: 104-112Abstract (166) Ning 2011Ning Xi Clemmons D.R. Suppression IGF-I hyperglycemia mediated vascular smooth cells.Endocrinology. 2011; 152: 3143-3154Crossref AKT, 3C). activate S3A). AKT-specific inhibitor, MK2206, abolished 3D S3B), highlighting cells, wild alanine (T172A S491A, respectively) comparable, even media S3C). With exogenous expressed, S491A mutation, T172A, 3E). Consistently, 3F) 3G S3D), high-glucose-induced, AMPKα2. enhanced, S491D (mimicking phosphorylated S491) able recruit S3E) sensitive S3F) S3G), required, sufficient, noteworthy stimuli experimental systems distinct that, turn, specific substrates. For instance, IGF1-induced minor serum-starved cancer (Hawley 2014Hawley Gowans Tibarewal Leslie Phosphorylation Akt within ST loop AMPK-α1 tumour cells.Biochem. 2014; 459: 275-287Crossref (127) Nevertheless, here, high-glucose-activated elevates subunit. question what events link destabilization Since 2F) S2H), speculated metabolism must essentially involved. sensor, alterations AMP/ATP ADP/ATP ratio (Lin 2018Lin S.C. AMPK: status.Cell 27: 299-313Abstract (363) Salt 1998Salt I.P. Johnson Ashcroft cell lines derived pancreatic beta regulate release.Biochem. 1998; 335: 533-539Crossref (321) Counterintuitively, 30 min, ratios 4A), would expected trigger instead repression signaling. ATP switching consistent previous reports (Glaser 1980Glaser Giloh Kasir Gross Mager On glucose-induced catabolism ascites reversal pyruvate.Biochem. 1980; 192: 793-800Crossref (16) van Beek, 2018van Beek J.H.G.M. dynamic side Warburg effect: glycolytic intermediate storage buffer fluctuating O supply tumor cells.F1000Res. 7: 1177PubMed showing transient inefficiency late stage glycolysis NADH accumulation, slows flux fructose-1,6-bisphosphate (FBP) pyruvate overall content S4A). S4B S4G) FBP S4B–S4F). Meanwhile, generated (Ding 2015Ding Fang Shang Xiao Sun Hou Pan Zhou al.Mitoflash stress insulin-resistant muscle.J. Med. (Berl.). 93: 1119-1130Crossref (20) 2017Wang Jian Lu al.Mitochondrial flashes heart.eLife. e23908Crossref (35) High-glucose generation, indexed 2',7'-dichlorofluorescein (DCF) staining, pretreatment general scavenger, Tiron 4B S4H). eliminating effects glucose, activation, augmented phosphorylation, degradation, 4C S4I). led activating presence absence 4D), deactivation AMPK. summary, vivo vitro, degrades 4E). sequentially intracellular resulting suppresses T172, leading Under T2D, together repressed signaling, exacerbate disturbance. define open new avenues severe diseases, obesity, variety study provided evidence S491, Interestingly, T172A mutation can protect and, if so, interferes subsequently affects If case, then patients, beneficial attributed, least part, stabilizing reducing MG53-facilitated ubiquitination, since elevate (Kjøbsted 2018Kjøbsted Fentz Sanz M.N. Pehmøller Shum Marette Mounier Treebak J.T. al.AMPK function metabolism.FASEB 32: 1741-1777Crossref (149) study, it unclear evoked muscle. questions merit future investigation. Tabled 1REAGENT RESOURCESOURCEIDENTIFIERAntibodiesp-AMPKα T172Cell Signaling TechnologyCat# 2535; RRID:AB_331250p-AMPKα S485/491Cell 4185; RRID:AB_2169402AMPKαCell 2532; RRID:AB_330331p-ACC S79Cell 3661; RRID:330337ACCCell 3662; RRID:AB_2219400p-S6K T389Cell 9205; RRID:AB_330944S6KCell 9202; RRID:AB_331676p-AKT T308Cell 2965; RRID:AB_2255933p-AKT S473Cell 4060; RRID:AB_2315049AKTCell 9272; RRID:AB_329827UbiquitinCell 3936; RRID:AB_331292LKB1Cell 3047; RRID:AB_2198327AMPKα1Cell 2795; RRID:AB_560856AMPKα2AbcamCat# ab3760; RRID:AB_304055β-actinSigma-AldrichCat# A5441; RRID:AB_476744FlagSigma-AldrichCat# F1804; RRID:AB_262044MycSigma-AldrichCat# SAB4700447; RRID:AB_10895876IRβCell 3025; RRID:AB_2280448IRS1MilliporeCat# 06-248; RRID:AB_2127890IRS1Cell 2382; RRID:AB_330333CRL4AProteintechCat# 14851-1-AP; RRID:AB_2261175Bacterial virus strainsAdenovirus expressing: β-galSong ScholarN/AAdenovirus Myc-MG53Song