Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press The glucose-sensing transcription factor MLX promotes myogenesis via myokine signaling Liam C. Hunt,1 Beisi Xu,2 David Finkelstein,2 Yiping Fan,2 Patrick A. Carroll,3 Pei-Feng Cheng,3 Robert N. Eisenman,3 and Fabio Demontis1 1Department of Developmental Neurobiology, Division of Developmental Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA; 2Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA; 3Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA Metabolic stress and changes in nutrient levels modulate many aspects of skeletal muscle function during aging and disease. Growth factors and cytokines secreted by skeletal muscle, known as myokines, are important signaling factors, but it is largely unknown whether they modulate muscle growth and differentiation in response to nutrients. Here, we found that changes in glucose levels increase the activity of the glucose-responsive transcription factor MLX (Max-like protein X), which promotes and is necessary for myoblast fusion. MLX promotes myogenesis not via an adjustment of glucose metabolism but rather by inducing the expression of several myokines, including insulin- like growth factor 2 (IGF2), whereas RNAi and dominant-negative MLX reduce IGF2 expression and block myo- genesis. This phenotype is rescued by conditioned medium from control muscle cells and by recombinant IGF2, which activates the myogenic kinase Akt. Importantly, MLX-null mice display decreased IGF2 induction and di- minished muscle regeneration in response to injury, indicating that the myogenic function of MLX is manifested in vivo. Thus, glucose is a signaling molecule that regulates myogenesis and muscle regeneration via MLX/IGF2/Akt signaling. [Keywords: MLX; IGF2; myokine; glucose; myogenesis; regeneration; skeletal muscle] Supplemental material is available for this article. Received July 6, 2015; revised version accepted October 26, 2015. Unicellular and multicellular organisms have evolved occur even at low glucose concentrations that are nor- several homeostatic adaptive strategies to regulate cell mally not permissive (Fulco et al. 2008). Despite these and organismal behavior in the face of fluctuating nutri- studies, the mechanisms and signaling pathways sensing ent levels. Skeletal muscle is one of the tissues that are glucose and responsible for glucose-induced adaptive most profoundly remodeled in response to changes in myogenesis are largely unknown. the levels of glucose and other nutrients. Many aspects A class of proteins that have recently gained attention of skeletal muscle homeostasis are modulated by glu- for their many signaling roles is myokines; i.e., growth fac- cose, including myogenesis, the process by which mono- tors and cytokines secreted by skeletal muscle in response nucleated progenitors (myoblasts) fuse into syncytial to a range of different stimuli (Pedersen and Febbraio 2012; muscle cells during development and regeneration. Sev- Demontis et al. 2013a). Myokines can act locally on the eral lines of evidence indeed point to glucose as an muscle itself as well as systemically on other tissues to important mediator of myogenesis. First, high glucose regulate many physiological functions ranging from met- levels promote myogenesis (Nedachi et al. 2008), and abolic homeostasis to aging and disease-related processes the expression of genes involved in glucose metabolism (Boström et al. 2012; Demontis et al. 2014). In addition to is up-regulated during myogenesis in vitro (Webster endocrine functions, there are many examples of auto- et al. 1990). Second, glucose metabolism via glycolysis crine and paracrine signaling of myokines on the muscle, is necessary for myogenesis in the Drosophila embryo including the modulation of myogenesis (Rai and Demon- (Tixier et al. 2013). Last, glucose restriction inhibits tis 2015). For example, leukemia inhibitory factor (LIF) is a myogenesis by activating AMPK kinase and the sirtuin SIRT1, and partial loss of SIRT1 allows myogenesis to © 2015 Hunt et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue pub- lication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). Af- Corresponding author: [email protected] ter six months, it is available under a Creative Commons License Article published online ahead of print. Article and publication date are (Attribution-NonCommercial 4.0 International), as described at http:// online at http://www.genesdev.org/cgi/doi/10.1101/gad.267419.115. creativecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 29:2475–2489 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/15; www.genesdev.org 2475 Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Hunt et al. myokine that regulates myoblast proliferation and differ- to glucose and that MLX is necessary for and promotes entiation and is produced in response to exercise (Hunt myogenic differentiation. However, rather than promot- et al. 2010, 2011; Broholm et al. 2011). However, despite ing glucose metabolism, MLX induces the expression of the important signaling roles of myokines, little is known many myokines in response to glucose, including insu- about the transcription factors that modulate their expres- lin-like growth factor 2 (IGF2), which recapitulates the ef- sion in response to glucose and other nutrients. fects of MLX on myogenic differentiation. These findings Max-like protein X (MLX) is a member of the Myc-Max indicate that MLX modulates glucose-induced myoblast family of basic helix–loop–helix (bHLH) transcription fac- fusion and differentiation via myokine signaling. tors (Billin et al. 1999) that, in response to high glucose levels, translocates from the cytoplasm to the nucleus (Peterson et al. 2010), where it regulates the expression Results of target genes with carbohydrate response element The transcription factor MLX induces target gene (CHORE) motifs in their promoter (Minn et al. 2005). expression in muscle cells via glucose-induced MLX is ubiquitously expressed across tissues and regu- DNA binding and histone H4 acetylation lates transcription by forming heterodimers with either MLX-interacting protein (MLXIP/MondoA) or MLXIP- To experimentally modulate MLX transcriptional activity like (MLXIPL/CHREBP) that are primarily expressed in in muscle cells, wild-type MLX (MLX-WT), dominant- the muscle (Billin et al. 2000) and liver (Yamashita et al. negative MLX (MLX-DN), MLX RNAi (MLX shRNA), 2001), respectively. Increased intracellular glucose leads and N-terminally truncated MLX (MLX-ΔN121) con- to nuclear accumulation of MLX (along with MLXIP and structs were generated and stably expressed in C2C12 MLXIPL) and increased DNA binding. Several lines of ev- mouse myoblasts via lentiviral-mediated transduction. idence support the hypothesis that glucose-6-phosphate is Overexpression of MLX variants led to an ∼60-fold in- the primary metabolite responsible for MLX activation. crease in Mlx mRNA levels compared with empty vector First, MLX translocation to the nucleus and transcription- control, whereas MLX shRNA led to an 80% decrease in al activity can be stimulated by 2-deoxyglucose, an analog endogenous Mlx mRNA levels compared with a nontar- of glucose that accumulates in cells following phosphory- geting control shRNA (Fig. 1A; Supplemental Fig. S1A). lation but cannot be further metabolized (Stoltzman et MLX-WT, MLX-DN, and MLX-ΔN121 protein variants al. 2008). Second, phosphorylation of glucose by hexoki- were detected by Western blot at the expected sizes nase is required for MLX activity (Stoltzman et al. 2008). (∼20 kDa for MLX-ΔN121 and ∼30 kDa for MLX-WT Last, phosphoglucoisomerase deficiency leads to glu- and MLX-DN) (Supplemental Fig. S1B) and by immunos- cose-6-phosphate buildup and enhances MLX activation taining of lentiviral-transduced cells that coexpress GFP (Stoltzman et al. 2011). The precise mechanisms by which (Supplemental Fig. S1C). Both MLX-WT and MLX-DN glucose-6-phosphate activates MLX are not completely were ubiquitously detected in the cells, whereas MLX- understood but perhaps entail direct binding to MLXIP/ ΔN121 localized to nuclei (Supplemental Fig. S1C), con- MLXIPL (McFerrin and Atchley 2012). In addition, glu- sistent with previous evidence that the N-terminal cose-dependent post-translational modifications can reg- domain of MLX harbors cytoplasmic retention sequences ulate MLXIPL (Guinez et al. 2011), which can also be (Billin et al. 2000). phosphorylated and inactivated by the energy-sensing ki- To assess the transcriptional activity of transgenic MLX nase AMPK (Kawaguchi et al. 2002). Thus, MLX and its variants, we examined changes in the expression of Txnip coactivators can sense glucose via multiple mechanisms. and Arrdc4, two genes known to be induced by glucose Furthermore, because MLX is part of a large interactive and MLX (Stoltzman et al. 2008; Yu et al. 2009). Both network of transcriptions factors, including Myc, Max, MLX-DN and MLX shRNA prevented glucose-induced Mxd, and MLXIP/MLXIPL, its transcriptional output expression of Txnip and Arrdc4, whereas MLX-WT signif- may be determined by the context-specific organization icantly enhanced their expression (Fig. 1B). Moreover of this network (Diolaiti et