Growth factor-dependent and independent regulation of skeletal muscle mass - Is IGF-1 necessary for skeletal muscle hypertrophy? -

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1 J Phys Fitness Sports Med, 2(1): (2013) JPFSM: Short Review Article Growth factor-dependent and independent regulation of skeletal muscle mass - Is IGF-1 necessary for skeletal muscle hypertrophy? - Mitsunori Miyazaki Department of Physical Therapy, School of Rehabilitation Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu-cho, Ishikari-gun, Hokkaido , Japan Received: October 17, 2012 / Accepted: December 17, 2012 Abstract Pituitary growth hormone (GH) and insulin-like growth factor-1 (IGF-1) play a critical role in the regulation of postnatal organ growth and overall body size. IGF-1 has been indicated as a very effective anabolic agent, and thus considered a critical regulator of skeletal muscle hypertrophy in response to increased workload such as resistance exercise. In contrast, recent studies using a genetic model of IGF-1/IGF-1 receptor have indicated that functional IGF-1-dependent mechanisms are not an absolute requirement for growth/hypertrophy in mature skeletal muscle. In this brief review, classic and recent aspects in hormonal/growth factordependent regulation of skeletal muscle mass are discussed. This review will particularly focus on 1) functional requirements of IGF-1 regulation in skeletal muscle hypertrophy and 2) cellular mechanisms in the regulation of protein synthesis in skeletal muscle. Keywords : growth hormone, insulin-like growth factor-1, protein synthesis, mammalian target of rapamycin, mechanical load Introduction Skeletal muscle comprises the largest organ system in the human body, consisting of more than 30-40% of body weight. The maintenance of skeletal muscle mass is critically important, since a decrease in muscle mass and power output is highly associated with functional impairment/disability which results in the loss of independence and increased risk of morbidity and mortality 1,2). Skeletal muscle mass is generally determined by the dynamic balance between protein synthesis and protein degradation. Following an increase in workload, such as resistance exercise, the rate of protein synthesis is enhanced relative to protein degradation such that there is a net positive balance in cellular protein content, leading to skeletal muscle hypertrophy 3). Pituitary growth hormone (GH) and insulin-like growth factor-1 (IGF-1) play a key role in the regulation of postnatal organ growth and overall body size. The anabolic effects of IGF-1 have been clearly indicated in skeletal muscle both in vitro and in vivo models, including transgenic overexpression, viral-mediated delivery, direct infusion, and plasmid injection 4-8). However, recent studies using a genetic model of an IGF-1/IGF-1 receptor have indicated that functional IGF-1-dependent mechanisms are not an absolute requirement for growth/hypertrophy in mature skeletal muscle. This review begins with the basic aspects of the GH/IGF-1 axis and the cellular mechanisms Correspondence: mmiyazaki@hoku-iryo-u.ac.jp regulating skeletal muscle mass in adults. The experimental evidence challenging the functional requirements of GH/IGF-1 dependent regulation for muscle growth/ hypertrophy are also discussed. Is pituitary growth hormone necessary for skeletal muscle growth/hypertrophy? GH deficiency or GH insensitivity leads to reduced growth during childhood and adolescence resulting in diminished body size, whereas supraphysiological GH results in pituitary gigantism, acromegaly or organ enlargement 9,10). The presence of pituitary GH is absolutely necessary for the normal growth of the body. In contrast, it is still controversial whether the presence of pituitary GH is required or not for the growth of the mature organ system. To assess this question, a combination of hypophysectomy and compensatory tissue growth models has been used to examine the physiological role of pituitary hormones in adaptational growth of tissues/organs in adults. In cardiac muscle, hypophysectomy prevented compensatory growth in response to induced coarctation of the aorta 11). It was also reported that unilateral nephrectomy, which usually causes compensatory growth of the remaining kidney, did not show cell growth/hyperplasia in hypophysectomized animals 12). These observations indicated that, in addition to postnatal growth, the presence of GH is required for compensatory growth in the mature heart or kidney. In contrast, this scenario was not true in adult skeletal muscle. Following tenotomy of the gastrocnemius muscle

2 102 JPFSM: Miyazaki M (compensatory growth model of synergistic muscles), hypertrophy of remaining plantaris and soleus muscles occurred to the same extent in hypophysectomized rats compared to control 13). These experimental observations clearly indicate that pituitary GH is not necessary for workload-induced growth of mature skeletal muscle, even though normal developmental growth has ceased. Is IGF-1 necessary for skeletal muscle growth/hypertrophy? IGF-1, also known as somatomedin-c, is a 70 aminoacid peptide with diverse functions including stimulation of cellular proliferation, survival, and differentiation. Most IGF-1 in circulation ( 75%) originates in the liver, and has historically been thought to mediate the anabolic effects of pituitary GH (this theory is called the somatomedin hypothesis). According to the somatomedin hypothesis, pituitary GH stimulates whole body growth by enhancing liver production of IGF-1, which, in turn, stimulates body growth in an endocrine manner 14). The contribution of circulating GH and IGF-1, however, to skeletal muscle growth in mature animals has been called into question. As shown above, skeletal muscle hypertrophy occurres to the same extent in the absence of circulating pituitary hormones 13). Exogenous administration of GH or IGF-1 into the hypophysectomized animal did not appear to stimulate skeletal muscle growth 15). In addition, mice with liver-specific gene deletion of IGF-1 showed no significant difference in whole body size, bone length or skeletal muscle mass, and were able to fully respond to a hypertrophic stimulus despite a 75-85% reduction in the concentration of circulating IGF ). These observations clearly indicate that liver-derived serum IGF-1 does not play a fundamental role in mediating the growth/ hypertrophy of mature skeletal muscle. IGF-1 is also synthesized locally in extra-hepatic tissues including skeletal muscle, brain, and kidney, where it acts in an autocrine/paracrine manner 19). Currently, it is thought that this autocrine/paracrine regulation of IGF-1 may have a critical role in supporting normal development and postnatal growth. In mature skeletal muscle, locally produced IGF-1 was upregulated, both at mrna and protein levels, following an increased workload condition, such as acute muscle damage 20), mechanical overload 21) or exercise training 22), with no changes in circulating IGF- 1. The autocrine/paracrine action of IGF-1, as an anabolic agent, was clearly demonstrated in both an in vitro cell culture model and in vivo animal model. The addition of exogenous IGF-1 to cultured myotubes 23,24) or delivered into skeletal muscle fiber via localized infusion 4), showed a robust increase in cell size and protein content. Muscle-specific overexpression of a locally acting IGF-1 isoform also resulted in a pronounced muscle hypertrophic phenotype 8). Collectively, these studies show that the addition of exogenous IGF-1 can effectively induce skeletal muscle hypertrophy. Recently, the functional requirement of IGF-1 for skeletal muscle hypertrophy was directly challenged by Spangenburg et al. 25). They developed a transgenic mouse model in which the kinaseinactive form of IGF-1 receptor was overexpressed in a muscle-specific manner, acting as a dominant negative form of the IGF-1 receptor in skeletal muscle. Interestingly, no difference in mechanical load-induced skeletal muscle hypertrophy was observed between wild-type and transgenic mice, even though IGF-1/IGF-1 receptordependent mechanisms were completely blocked. These findings clearly contradict the previous hypothesis about the necessity of IGF-1, and also indicate that activation of the IGF-1 receptor is not necessary for the induction of skeletal muscle growth in response to increased mechanical load in vivo. Thus, while it is very clear that the addition of exogenous IGF-1 to muscle cells shows robust anabolic effects, IGF-1-dependent mechanisms are not an absolute requirement for growth/hypertrophy in mature skeletal muscle. mtor signaling regulating protein synthesis in skeletal muscle To date, numerous studies have shown that the mammalian (or mechanistic) target of rapamycin (mtor) plays a critical role in regulating the rate of protein synthesis and cell hypertrophy in skeletal muscle 26-29). mtor is a serine/threonine kinase of the phosphatidylinositol kinaserelated kinase family, and is highly conserved from yeast to mammals 30). In skeletal muscle cells, mtor serves as a central integrator of a wide range of signals including growth factors, nutrients, energy status and mechanical stresses, and promotes protein synthesis through the phosphorylation of downstream effectors such as eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and p70 ribosomal S6 kinase 1 (S6K1) 3). The most well characterized mechanism regulating mtor activity in skeletal muscle is the insulin-like growth factor-1 (IGF-1)/insulindependent pathway 28,29). Stimulation of muscle cells with insulin/igf-1, leads to activation of phosphoinositide 3-kinase (PI3K) and its downstream effector Akt, triggering multiple downstream signaling events including mtor activation 31). Akt contols mtor-dependent signaling through regulation of tuberous sclerosis complex (TSC) 1/TSC2, a heterodimeric protein complex which functions as a negative regulator of mtor 32,33). Together with its partner TSC1, TSC2 functions as a GTPaseactivating protein (GAP) for a small G protein named Ras homolog enriched in brain (Rheb), a direct activator of mtor. Akt directly phosphorylates TSC2 on multiple residues (at least two sites, Ser939 and Thr1462) and modulates subcellular localization of the TSC1/ TSC2 complex 34). Altered distribution of the TSC1/TSC2 complex results in the negative regulation of its GAP activity, thereby allowing Rheb to accumulate in its active GTP-bound form 35-37). This leads to well-established

3 JPFSM: Is IGF-1 necessary for skeletal muscle hypertrophy? 103 A B phos-s6k1 (T389) phos-s6k1 (T421/S424) C -S6K1 0 Arbitrary Unit (fold changes to ) D T389 T421/S424 E phos-akt (T308) phos-akt (S473) -Akt sham sham 0 sham Arbitrary Unit (fold changes to ) T308 S473 phos (T389) phos (T421/S424) S6K1 phos (T308) phos (S473) Akt phos-gsk3 (S21) in vitro Akt kinase assay DMSO Wortmannin Rapamycin Fig. 1 Inhibition of PI3K signaling did not prevent mtor activation in response to mechanical overload. A) Representative images of S6K1 showing phosphorylation status of T389 and T421/S424 and S6K1 protein. B) The relative (normalized to OV- 0) phosphorylation level of T389 and T421/S424 sites and protein level of S6K1 at each time point were quantified (n=4-5/ group). Significant differences; : quantification of S6K1-T389 phosphorylation compared to the control () group, p < *: quantification of S6K1-T421/S424 phosphorylation compared to the control () group, p < C) Representative images showing Akt phosphorylation (S473 and T308 sites) states and Akt protein. D) The relative phosphorylation (S473 and T308 sites) and protein levels of Akt at each time point were quantified (n=4-5/group). Significant differences; : quantification of Akt-S473 phosphorylation, compared to the control () group, p < *: quantification of Akt-T308 phosphorylation, compared to the control () group, p < : quantification of Akt-, compared to the control () group, p < All results are expressed as the mean ± SE. The phosphorylation state of S6K1 at T389 was significantly increased 8.5-fold at day 1 after overload, and remained significantly elevated for the entire 10 days of study (Fig. 1A-B). In contrast, a significant change in the phosphorylation state of Akt was not detected until (S473 site, 1.8-fold) or (T308 site, 5.3-fold) following mechanical overload of the plantaris muscle. E) The effect of wortmmanin or rapamycin treatment on mechanical overload-induced phosphorylation of Akt and S6K1 and in vitro Akt kinase activity in the plantaris muscle. Wortmannin was administered twice (immediately and 12 hours after synergist ablation surgery) and rapamycin was injected once (immediately after surgery). Shamoperated (sham) was served as a control. Muscle samples were collected 24 hrs after adminstration of DMSO or inhibitor. There was no change in Akt phosphorylation at this early time point (); but phosphorylation of S6K1 at T389 was blocked by rapamycin, but not wortmannin. The in vitro Akt kinase assay revealed no change in Akt activity confirming the Akt phosphorylation data. These data demonstrate that mtor activation in response to mechanical overload occurs independently of PI3K signaling in skeletal muscle. (Cited from Miyazaki et al. 41) )

4 104 JPFSM: Miyazaki M growth factor associated signaling through the PI3K-Akt- TSC1/2-Rheb-mTOR cascade. In addition to growth factor regulation, mechanical load is an important signal input in the activation of mtor and subsequent enhancement of protein synthesis in skeletal muscle. Recently, it was shown that mechanical loadinduced activation of mtor signaling in skeletal muscle is independent of IGF-1 regulation. Studies using a muscle-specific dominant negative form of the IGF-1 receptor showed that a functional IGF-1 receptor-dependent mechanism was not necessary for mtor activation and the induction of skeletal muscle hypertrophy in response to increased mechanical load 38) or a single bout of high frequency muscle contractions 39). Additionally, it was shown that PI3K/Akt-dependent regulation was not required for the activation of mtorc1 signaling following eccentric contractions in skeletal muscle under ex vivo conditions 40). Our laboratory has shown that activation of mtor in response to mechanical overload occurred independent of PI3K/Akt regulation in skeletal muscle in vivo 41). In re- sponse to mechanical overload, the phosphorylation states of S6K1 and its downstream effector, rps6, which show the functional activity of mtor signaling, were robustly increased following one day of overload; whereas no activation of PI3K/Akt signaling was observed. In addition, pharmacologic inhibition of PI3K signaling, by wortmannin administration, did not affect mechanical overloadinduced activation of mtor (Fig. 1). These findings provide strong evidence that activation of mtor signaling, in response to mechanical overload, occurs independently of PI3K/Akt signaling. Furthermore, we have also observed that, at the early phase of mechanical overload, increased activation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling coincides with the phosphorylation of TSC2 at S664 (ERK-specific phosphorylation site), a negative regulator of mtor signaling. Thus, we suggest that MEK/ ERK inhibition of TSC2 contributes to mtor activation during skeletal muscle hypertrophy in response to mechanical overload (Fig. 2). Mechanical Overload Growth Factors (Insulin, IGF-1) MEK U0126 S664 P TSC1 TSC2 P S939 T1462 PI3K Wortmannin ERK Rheb Akt mtorc1 Rapamycin S235/236 P P S6K1 P rps6 T389 S240/244 Protein Synthesis Fig. 2 Schematic representation of the molecular mechanisms in the regulation of mtor signaling via growth factors and mechanical overload in skeletal muscle. Growth factor-dependent regulation of mtor signaling is mediated through a PI3K-Akt- TSC1/2-Rheb-mTOR cascade, whereas the MEK/ ERK pathway, through TSC2 phosphorylation at S664, likely contributes to mtor activation in response to mechanical overload. References 1) Janssen I, Heymsfield SB and Ross R Low relative skeletal muscle mass (sarcopenia) in older persons is associated with functional impairment and physical disability. J Am Geriatr Soc 50: ) Doherty TJ Invited review: Aging and sarcopenia. J Appl Physiol 95: ) Miyazaki M and Esser KA Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals. J Appl Physiol 106: ) Adams GR and McCue SA Localized infusion of IGF- I results in skeletal muscle hypertrophy in rats. J Appl Physiol 84: ) Barton-Davis ER, Shoturma DI, Musaro A, Rosenthal N and Sweeney HL Viral mediated expression of insulinlike growth factor I blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci USA 95:

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