International Journal of Molecular Sciences

Review GH/IGF-1 Abnormalities and Muscle Impairment: From Basic Research to Clinical Practice

Betina Biagetti * and Rafael Simó *

Diabetes and Metabolism Research Unit, Vall d’Hebron Research Institute and CIBERDEM (ISCIII), Universidad Autónoma de Barcelona, 08193 Bellaterra, Spain * Correspondence: [email protected] (B.B.); [email protected] (R.S.); Tel.: +34-934894172 (B.B.); +34-934894172 (R.S.)

Abstract: The impairment of skeletal muscle function is one of the most debilitating least understood co-morbidity that accompanies acromegaly (ACRO). Despite being one of the major determinants of these patients’ poor quality of life, there is limited evidence related to the underlying mechanisms and treatment options. Although growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels are associated, albeit not indisputable, with the presence and severity of ACRO myopathies the precise effects attributed to increased GH or IGF-1 levels are still unclear. Yet, cell lines and animal models can help us bridge these gaps. This review aims to describe the evidence regarding the role of GH and IGF-1 in muscle anabolism, from the basic to the clinical setting with special emphasis on ACRO. We also pinpoint future perspectives and research lines that should be considered for improving our knowledge in the field.

Keywords: acromegaly; myopathy; review; growth hormone; IGF-1




 1. Introduction Acromegaly (ACRO) is a rare chronic disfiguring and multisystem disease due to Citation: Biagetti, B.; Simó, R. non-suppressible growth hormone (GH) over-secretion, commonly caused by a pituitary GH/IGF-1 Abnormalities and Muscle tumour [1]. The autonomous production of GH leads to an increase in the synthesis Impairment: From Basic Research to and secretion of insulin-like growth factor-1 (IGF-1), mainly by the liver, which results in Clinical Practice. Int. J. Mol. Sci. 2021, 22, 415. https://doi.org/10.3390/ somatic overgrowth, metabolic changes, and several comorbidities [1]. ijms22010415 One of the significant disabling co-morbidities accompanying ACRO is myopathy and its related musculoskeletal symptoms [2,3], which are significant determinants of the low

Received: 4 December 2020 quality of life (QoL) of these patients and may persist despite disease remission [2,4–6]. Accepted: 28 December 2020 However, we do not know the exact underlying mechanisms involved in its develop- Published: 2 January 2021 ment. Although the duration of the disease and serum GH/IGF-1 levels were shown to be independent predictors of overall mortality and co-morbidities of ACRO [7–11], their spe- Publisher’s Note: MDPI stays neu- cific role in the pathogenesis of myopathy remains to be elucidated. Besides, both GH tral with regard to jurisdictional clai- and IGF-1 levels are not always directly and unequivocally associated with patient QoL ms in published maps and institutio- and the presence and severity of co-morbidities [4,12]. For this reason, numerous efforts nal affiliations. are currently being made to detect active disease, pointing to other variables apart from GH/IGF-1 [13,14]. Experimental research using cell lines and animal models is necessary to understand better the mechanisms involved in the myopathy induced by ACRO. In this regard, there is Copyright: © 2021 by the authors. Li- some evidence regarding the effects of the GH/IGF-1 axis in the muscular system and the censee MDPI, Basel, Switzerland. This article is an open access article mechanisms by which different conditions could impact on muscle performance. distributed under the terms and con- This review aims to describe the evidence regarding the role of GH and IGF-1 in ditions of the Creative Commons At- muscle anabolism, from the basic to the clinical setting, taking ACRO as the leading tribution (CC BY) license (https:// paradigm in the clinical setting. The new perspectives and scientific gaps in this field to be creativecommons.org/licenses/by/ covered are also underlined. 4.0/).

Int. J. Mol. Sci. 2021, 22, 415. https://doi.org/10.3390/ijms22010415 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18

This review aims to describe the evidence regarding the role of GH and IGF‐1 in muscle anabolism, from the basic to the clinical setting, taking ACRO as the leading par‐ adigm in the clinical setting. The new perspectives and scientific gaps in this field to be Int. J. Mol. Sci. 2021, 22, 415 2 of 18 covered are also underlined.

2. GH/IGF‐1 Axis in Humans Pituitary2. synthesis GH/IGF-1 and Axis secretion in Humans of GH is stimulated by the episodic hypothalamic secretion of GH releasingPituitary factor synthesis and mainly and secretion inhabited of GHby somatostatin is stimulated [15]. by theGH episodicstimulates hypothalamic the synthesissecretion of IGF‐1 ofmostly GH releasing by the liver, factor and and both mainly circulating inhabited GH by and somatostatin IGF‐1 inhibit [15 ].GH GH stimulates secretion by thea negative synthesis loop of IGF-1at both mostly hypothalamic by the liver, and and pituitary both circulating levels. In addition, GH and IGF-1 age, inhibit GH gender, pubertalsecretion status, by food, a negative exercise, loop fasting, at both insulin, hypothalamic sleep and and body pituitary composition levels. play In addition, age, important regulatorygender, pubertal roles in the status, GH/IGF food,‐1 exercise, axis [15]. fasting, insulin, sleep and body composition play important regulatory roles in the GH/IGF-1 axis [15]. 2.1. GH Circulation, GH Receptor and Intracellular Signalling 2.1. GH Circulation, GH Receptor and Intracellular Signalling In the circulation, GH binds to growth hormone binding protein (GHBP) which is a In the circulation, GH binds to growth hormone binding protein (GHBP) which soluble truncated form of the extracellular domain of the GH receptor (GHR) [16]. There‐ is a soluble truncated form of the extracellular domain of the GH receptor (GHR) [16]. fore, bound and free GH both exist in the circulation, and GH bioavailability depends on Therefore, bound and free GH both exist in the circulation, and GH bioavailability depends the pulsatile pattern of its secretion. on the pulsatile pattern of its secretion. GHR belongs to the so‐called “cytokine receptor family”, specifically part of the class GHR belongs to the so-called “cytokine receptor family”, specifically part of the class I cytokine receptor family. Cytokine receptors lack intrinsic protein tyrosine kinase (PTK) I cytokine receptor family. Cytokine receptors lack intrinsic protein tyrosine kinase (PTK) activity and therefore rely on binding non‐receptor PTKs for their signal transduction, the activity and therefore rely on binding non-receptor PTKs for their signal transduction, so‐called Janus Kinase (JAK) (Figure 1). The dimerisation induced by the binding of GHR the so-called Janus Kinase (JAK) (Figure1). The dimerisation induced by the binding of is the critical step in activating the signalling pathway JAK/STAT (Janus kinase/signal GHR is the critical step in activating the signalling pathway JAK/STAT (Janus kinase/signal transducer and activator of transcription), and binding to GHR brings two JAK2 mole‐ transducer and activator of transcription), and binding to GHR brings two JAK2 molecules cules close enough to initiate their trans‐phosphorylation by recruiting STAT[17,18]. Phos‐ close enough to initiate their trans-phosphorylation by recruiting STAT [17,18]. Phosphory- phorylated STATSs,lated STATSs, translocate translocate to the to nucleus, the nucleus, binding binding to specific to specific DNA DNA sequences sequences and and induce induce the transcriptionthe transcription of specific of specific GH‐dependent GH-dependent genes, genes, thus thuspromoting promoting not only not onlythe the synthe- synthesis of sisIGF of‐1, IGF-1, IGF‐1 IGF-1 binding binding protein protein (IGFBP) (IGFBP) acid‐ acid-labilelabile sub‐units sub-units (ALS), (ALS), but also but also directly directly stimulatingstimulating proliferative proliferative and andmetabolic metabolic actions actions (Figure (Figure 1). 1).

Figure 1. Growth hormone (GH) receptor. The GH receptor’s dimerisation is the critical step in activating the signalling Figure 1. Growth hormone (GH) receptor. The GH receptor’s dimerisation is the critical step in pathway JAK/STAT.activating Phosphorylated the signalling STATS pathway translocate JAK/STAT. to the Phosphorylated nucleus, inducing STATS the transcription translocate to of the specific nucleus, GH-dependent genes, promotinginducing mainly the the transcription synthesis ofof IGF-1,specific IGFBP GH‐dependent acid-labile genes, sub-units, promoting but also, mainly stimulating the synthesis other of pathways (i.e., IRIS and SHC)IGF that‐1, regulateIGFBP acid cellular‐labile proliferation, sub‐units, but growth also, stimulating and metabolic other events. pathways In addition,(i.e., IRIS newlyand SHC) synthesised that IGF-1 promotes metabolicregulate and cellular cellular proliferation, events. IGF-1: growth insulin-like and metabolic growth events. factor, In GH: addition, growth newly hormone synthesised IGFBP3: IGF IGF-1‐1 binding protein type 3,promotes JAK/STAT: metabolic Janus kinase/signal and cellular events. transducer IGF‐ and1: insulin transcription‐like growth activator factor, IRS: GH: insulin growth receptor hormone substrate; PI3K: phosphatidylinositolIGFBP3: 3-kinase; IGF‐1 binding SHC: (adaptor protein type proteins). 3, JAK/STAT: Janus kinase/signal transducer and transcription activator IRS: insulin receptor substrate; PI3K: phosphatidylinositol 3‐kinase; SHC: (adaptor pro‐ teins). GHR is widely distributed throughout the body. Although GHR expression is particu- larly important in the liver, GH receptors are found in other tissues like muscle, fat, heart, kidney, brain and pancreas [19]. The actions of GH depend not only on the receptor but also on the organ it stimulates; specifically, GH actions in the muscle are controversial and depend, among other factors, on GH quantity, the time of exposition and the condition of the subjects. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 18

GHR is widely distributed throughout the body. Although GHR expression is par‐ ticularly important in the liver, GH receptors are found in other tissues like muscle, fat, heart, kidney, brain and pancreas [19]. The actions of GH depend not only on the receptor but also on the organ it stimulates; specifically, GH actions in the muscle are controversial Int. J. Mol. Sci. 2021and, 22 depend,, 415 among other factors, on GH quantity, the time of exposition and the condi‐ 3 of 18 tion of the subjects.

2.2. IGF‐1 Circulation,2.2. IGF-1 IGF‐1 Circulation, Receptor and IGF-1 Intracellular Receptor Signalling and Intracellular Signalling The majority of TheIGF majority‐1 circulates of IGF-1 in the circulates serum in as the a serumcomplex as awith complex the insulin with the‐like insulin-like growth growth factor‐bindingfactor-binding protein IGFBP protein and IGFBP ALS. and The ALS. function The of function ALS is to of prolong ALS is to the prolong half‐ the half-life of life of the IGF‐I‐IGFBPthe IGF-I-IGFBP-‐ binary complexes, binary complexes, thus regulating thus regulating IGF‐1 bioactivity IGF-1 bioactivity [20]. [20]. IGF‐I receptors (IGFIGF-I‐1R) receptors are highly (IGF-1R) homologous are highly to homologous the insulin receptor. to the insulin For this receptor. rea‐ For this reason, son, at high concentrationsat high concentrations insulin cross‐ insulinreacts with cross-reacts the IGF‐1R with and the vice IGF-1R versa and[21]. vice In cells versa [21]. In cells expressing both expressinginsulin and bothIGF‐I insulin receptors and as IGF-Iin the receptors myocyte, asthe in receptors the myocyte, undergo the het receptors‐ undergo ero‐dimerisationhetero-dimerisation (hybrid receptors), as (hybrid well as receptors), homo‐dimerisation. as well as In homo-dimerisation. skeletal muscle IGF In‐ skeletal muscle I receptors are mainlyIGF-I receptors present as are hybrids, mainly together present aswith hybrids, an excess together of classical with an insulin excess re of‐ classical insulin ceptors regardingreceptors other mammalian regarding other tissue. mammalian Hybrid receptors tissue. Hybridbind both receptors insulin bind and bothIGF‐I, insulin and IGF-I, although they havealthough a lower they insulin have affinity a lower than insulin classical affinity insulin than classicalreceptors insulin [21,22]. receptors [21,22]. In contrast to GHR,In contrast the IGF to‐1R GHR, is a member the IGF-1R of the is a receptor member tyrosine of the receptor kinase (RTK) tyrosine kinase (RTK) family. This typefamily. of receptor’s This type common of receptor’s characteristic common is characteristicto have an extracellular is to have andomain extracellular domain for ligand binding,for a ligand single binding,transmembrane a single domain transmembrane and an intracellular domain and domain an intracellular with ty‐ domain with rosine kinase activity,tyrosine which kinase is activated activity, which after the is activated ligand binds after to the the ligand extracellular binds to domain, the extracellular domain, thus triggering thethus phosphorylation triggering the phosphorylation of Insulin Receptor of Insulin Substrates Receptor (IRS). Substrates Once the IRS (IRS). is Once the IRS is phosphorylated,phosphorylated, it activates different it activates pathways different such as pathways phosphatidylinositol such as phosphatidylinositol 3‐kinase or 3-kinase or RAS (Figure 2). RAS (Figure2).

Figure 2. FigureIGF-1 receptor, 2. IGF‐1 receptor, insulin receptor insulin andreceptor hybrid and receptor. hybrid receptor. The insulin The receptor insulin receptor (IS-R), the (IS insulin-like‐R), the in‐ growth factor receptor typesulin I‐like (IGF-1-R) growth and factor the receptor hybrid receptor type I (IGF belong‐1‐R) to and the the family hybrid of receptor receptor belong tyrosine to kinases.the family The of figure shows a receptor tyrosine kinases. The figure shows a certain degree of functional overlap. After binding certain degree of functional overlap. After binding ligands, the receptors stimulate the phosphorylation of tyrosine residues, ligands, the receptors stimulate the phosphorylation of tyrosine residues, which then induces which then induces phosphorylation of the Insulin Receptor Substrate (IRS) and SHC, which dock proteins for activating phosphorylation of the Insulin Receptor Substrate (IRS) and SHC, which dock proteins for activat‐ the phosphatidylinositoling the phosphatidylinositol 3-kinase (PI3K-Akt) 3‐kinase pathway (PI3K‐Akt) or the pathway Ras/MAPK or the pathway,Ras/MAPK respectively. pathway, respec The PI3K-Akt‐ pathway is predominantlytively. The involved PI3K‐Akt in metabolicpathway is actions predominantly (glucose involved uptake, insulin in metabolic sensitivity) actions and (glucose cell proliferation, uptake, whereas the RAS/MAPKinsulin pathway, sensitivity) is primarily and cell involved proliferation, in mitogenic whereas effects the RAS/MAPK (proliferation, pathway, growth). is primarily The number involved of receptors alone is not the onlyin mitogenic determining effects factor (proliferation, in IGF-1 function. growth). In this The regard, number the of assemblyreceptors andalone intracellular is not the only machinery deter‐ disposition are also involved.mining factor in IGF‐1 function. In this regard, the assembly and intracellular machinery disposi‐ tion are also involved. The number of IGF-1Rs in each cell is tightly regulated by several systemic and tissue factors, including circulating GH, thyroxine, platelet-derived growth factor, and fibroblast growth factor. Therefore, in vivo, the number of receptors alone is not the only determining factor in IGF-1 function [20] (Figure2). Despite the ubiquity of IGF-1R, the in vitro biological effects of IGF-1 are relatively weak and often are not demonstrable except in the presence of other hormones or growth factors, thus suggesting that IGF-1 acts as a permissive factor augment the signals of other factors [22]. Furthermore, IGF-1 and insulin share common signalling pathways Int. J. Mol. Sci. 2021, 22, 415 4 of 18

and functional overlap. However, IGF-1 exerts a more decisive action than insulin in activating the RAS/MAP kinase pathway, which is related to cell growth, protein synthesis, and apoptosis. By contrast, insulin (and in lower grade IGF-1) regulates the metabolism of carbohydrates, lipids and proteins via PI-3K/Akt, [20,22] (Figure2).

3. The Skeletal Muscle: A Central Link between Structural and Metabolic Function The skeletal muscle is one of the three significant muscle tissues in the human body, along with cardiac and smooth muscles [23]. It is crossed with a regular red and white line pattern, giving the muscle a distinctive striated appearance, leading to it being called striated muscle. There are three types of muscle fibres, which differ in the composition of contractile proteins, oxidative capacity, and substrate preference for ATP production. Type I fibres or slow-twitch fibres have low fatigability (better for endurance), and display slow oxidative ATP, primarily from oxidative phosphorylation (a preference for fatty acids as substrate) [24,25]. Type II or fast-twitch fibres have the highest fatigability, the highest contraction strength, while glucose is the preferred substrate [24]. They are further divided into Type IIa and IIb fibres. Type IIa is used for intermediate endurance contraction and, in general, relies on aerobic/oxidative metabolism. Both types I and IIa fibres are considered red fibres because of their high amounts of myoglobin. They also contain high numbers of mitochondria, Type IIb fibres, (also called IIX/d) or fast glycolytic fibres, are the fastest twitching fibres and are more insulin resistant [26]. They are also white fibres with low levels of myoglobin and a high concentration of glycolytic enzymes and glycogen stores. This is because they produce ATP primarily from anaerobic/glycolysis [27,28]. Thus, the fibre type composition of skeletal muscle impacts systemic energy consumption and vice versa. Endurance or aerobic exercise increases mechanical and metabolic demand on skeletal muscle, resulting in a switch from a fast-twitch to a slow-twitch fibre type [24]. In summary, skeletal muscle’s structural function is the most obvious one. It gives structural support, helps maintain the body’s posture, and exerts the muscle contraction leading to movements. Furthermore, skeletal muscle is also an important site of the intermediary metabolism: it is a storage site for amino acids, plays a central role in maintaining thermogenesis, acts as an energy source during starvation and, along with the liver and adipose tissue, is crucial in the development of insulin resistance in type 2 diabetes mellitus [29].

4. GH/IGF-1 Actions in the Muscular System The muscle is a primary target of GH and IGF-1 [30,31]. The final contribution of GH and IGF-1 in any effect on the muscular system is not easy to establish in-vivo. Through its receptors, GH action can be both direct and indirect through the induced production of IGF-I. Furthermore, IGF-I is also produced locally in tissues and can act in a paracrine/autocrine manner [32,33]. As we commented above, the muscle is much more than an organ of support and contraction; it is a main metabolic organ. The fibre type composition of skeletal muscle impacts systemic energy consumption and vice versa [24]. Both hormones act in the muscle, modifying its metabolic function and structure. For example, Bramnert et al. (2003) [34] studied both the short-term (1 week) and long-term (6 months) effects of a low-dose (9.6 µg/kg body weight/d) GH replacement therapy or placebo on whole-body glucose and lipid metabolism and muscle composition in 19 GH-deficient adult subjects. GH therapy resulted in glucose metabolism deterioration and an enhanced lipid oxidation rate in both short and long-term treatment groups, reflecting a fuel use switch from glucose to lipids. Furthermore, there was a shift toward more insulin-resistant type II X fibres in the biopsied muscles during GH therapy. These studies significantly increased our knowledge regarding the relationship between skeletal muscle and metabolism’s fibre type composi- tion. However, whether this muscular fibre shift represents the cause or the consequence of insulin resistance requires further investigation. Int.Int. J.J. Mol.Mol. Sci.Sci. 2021,, 22,, xx FORFOR PEERPEER REVIEWREVIEW 5 of 18

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18

(9.6 μg/kg body weight/d) GH replacement therapy or placebo on whole-body glucose (9.6and μg /lipidkg bod metabolismy weight/d and) GH muscle replacement composition therapy in 19 or GH placebo-deficient on wholeadult subjects.-body glucose GH ther- andapy lipid resulted metabolism in glucose and muscle metabolism composition deterioration in 19 GH and-deficient an enhanced adult lipid subjects. oxidation GH ther- rate in apyboth resulted short in an glucosed long -metabolismterm treatment deterioration groups, reflecting and an enhanced a fuel use lipid switch oxidation from glucoserate in to bothlipids.lipids. short and long-term treatment groups, reflecting a fuel use switch from glucose to lipids. Furthermore, there was a shift toward more insulin-resistant type II X fibres in the biopsiedFurthermore, muscles there during was a shift GH toward therapy. more These insulin studies-resistant significantly type II X fibres increased in the our Int. J. Mol. Sci. 2021, 22, 415 biopsiedknowledge muscles regarding during the GH relationship therapy. betweenThese studies skeletal significantly muscle and metabolism increased ’’ ours fibre 5 of 18 knowledgetype composition. regarding However, the relationship whether between this muscular skeletal fibre muscle shift andrepresents metabolism the cause’s fibre or the typeconsequence composition. of However,insulin resistance whether requires this muscular further fibre investigation. shift represents the cause or the Moreover, GH/IGF1 levels have been related to ageing. In particular, IGF-1 produc- consequenceMoreover,Moreover, of insulin GH GH/IGF1/resistanceIGF1 levels levelsrequires have have been further been related investigation. related to ageing. to ageing. In particular, In particular, IGF IGF-1-1 produc- produc- tion by the skeletal muscle is impaired in older humans and linked with aged related sar- tionMoreover,tion by the by skeletal theGH skeletal/IGF1 muscle levels muscle is haimpairedve is been impaired inrelated older in to olderhumans ageing. humans and In linkedparticular, and linkedwith IGF aged with-1 produc-related aged sar- related copenia [30]. Therefore, GH/IGF-1 actions in the muscle have structural, metabolic and tioncopenia bysarcopenia the skeletal [30]. Therefore, [30 muscle]. Therefore, is GH/IGF impaired GH/IGF-1-1 inactions older actions inhumans the in muscle the and muscle linked have have structural,with structural, aged relatedmetabolic metabolic sar- and and ageing consequences and vice versa. copeniaageingageing [30] consequences. Therefore, consequences GH/IGFand and vice vice- 1versa. actions versa. in the muscle have structural, metabolic and ageing consequences and vice versa. 4.1. 4.1.GH GH 4.1. GH TheThe first firstreports reports on the on anabolic the anabolic growth growth hormone hormone effects effectsdated back dated to back1948 and to 1948 sug- and gestedThesuggested first that reports GH that inhibits on GH the proteolysis inhibitsanabolic proteolysis growth during hormone fasting during [35] effects fasting.. SubsequentSubsequent dated [35]. back studiesstudies Subsequent to 1948 havehave and expandedexpanded studies sug- have gestedthis thatexpanded concept GH inhibits[36,37] this concept.. proteolysis [36,37 during]. fasting [35]. Subsequent studies have expanded this conceptGH GH is[36,37] the is the primary. primary hormone hormone in in the the fast fast state; state; from anan evolutionaryevolutionary point point of of view, view, when whenGHfood isfood the is scarce,is primary scarce, GH GH hormone changes changes fuelin fuel the consumption consumptionfast state; from from carbohydratesan carbohydrates evolutionary and pointand protein protein of view, to theto the use of whenuse food lipids,of lipids, is scarce, allowing allowing GH for changes for the the conservation fuelconservation consumption of vital of vital proteinfrom protein carbohydrates stores stores [38]. [38]Humans’ and.. HumansHumans protein nitrogen’’ to nitrogen thebalance usebalance of lipids,must must be allowing kept be kept stable for stable tothe maintain conservation to maintain the pool theof vital ofpool essential protein of essential amino stores amino acids [38]. (EAA) Humansacids ( involvedEAA’ nitrogen) involvedinvolved in protein balanceinin proteinproteinformation. must beformation.formation. kept Considering stable ConsideringConsidering to maintain this balance, thisthis the balance,balance, pool different of essentialdifferentdifferent EAA radiolabelling aminoEAAEAA radiolabellingradiolabelling acids (EAA techniques) involved techniquestechniques have been in proteinhaveused been formation. to used evaluate to Consideringevaluate the effects the of thiseffects GH balance, and of GH IGF-1 different and on IGF protein EAA-1 on metabolism.radiolabelling protein metabolism. Table techniques1 summarises Table 1 havesummarises beenthe resultsused theto of evaluate results studies of assessingthe studies effects assessing protein of GH anabolismand protein IGF- 1anabolism usingon protein radioisotopes. using metabolism. radioisotopes. The Table action 1The of GH summarisesactionon of muscle GHthe resultson tissue muscle of is studies mainlytissue is assessing anabolic mainly anabolic andprotein has littleandanabolism has effect little using on effect proteolysis. radioisotopes. on proteolysis. Still, The the Still, effects actionthe ofeffectscould GH be oncould differentmuscle be different tissue depending is depending mainly on the anabolic circumstances,on the circumstanand has doselittleces, andeffect dose time on and proteolysis. of time exposition. of exposition. Still, The acute the Theeffectsinfusion acute could infusion of be GH different onof GH amino depending on acidamino metabolism onacid the metabolism circumstan results inces,results a dose profound in and a profound time reduction of exposition. reduction in amino in acid Theamino acuteoxidation acidinfusion oxidation [36 of,37 GH,39 [36,37,39 –on41 ].amino Some–41] acid studies.. SomeSome metabolism studiesstudies demonstrated demonstrateddemonstratedresults thatin a profound acute thatthat GH acuteacute reduction infusion GHGH infusioninfusion increasedin aminoincreasedincreased whole-bodyacid oxidation wholewhole protein-body [36,37,39 protein synthesis–41] synthesis. Some without studies without any significant demonstrated any significantignificant changes that changeschanges in acute the synthetic GHinin thethe infusion syntheticsynthetic rate of mus- increasedratecle of whole proteins.muscle-body proteins. By protein contrast, By contrast,synthesis other studies other without usingstudies any higher usingsignificant doses higher changes of doses GH found ofin GHthe lower foundsynthetic proteolysis lower rateproteolysis of rates.muscle In proteins.rates. general, In general, By acute contrast, GH acute infusion other GH infusionstudies exerted using anexerted anabolic higher an anabolic effectdoses on of effect whole-bodyGH foundon whole lower amino-acid-body proteolysisamino(AA)-acid rates. metabolism, (AA In) general, metabolism, evidenced acute evidenced GH in reducedinfusion in leucine reducedexerted oxidation, an leucine anabolic oxidation, increased effect on nonoxidativeincreased whole-body nonox- leucine aminoidativeidative-disposal,acid leucineleucine (AA an) metabolism, disposal,disposal, increased anan amino evidenced increasedincreased acid disappearance aminoinamino reduced acidacid leucinedisappearancedisappearance rate (Rd) oxidation, (all indexesraterate increased (Rd of) ( proteinall nonox-indexes synthesis) of and less effect on protein breakdown (a lower rate of amino acid appearance (Ra)) probably idativeprotein leucine synthesis disposal,) and an less increased effect on amino protein acid breakdown disappearance (a lower rate (rateRd) of(all amino indexes acid of ap- related with dosage. Regarding chronic GH exposure, it seems more consistent than acute proteinpearance synthesis (Ra))) probablyand less effect related on with protein dosage. breakdown Regarding (a lower chronic rate GH of exposure,amino acid it ap-seems administration in promoting an increase in whole-body protein synthesis [42–44]. Similar pearancemore consistent(Ra)) probably than acuterelated administration with dosage. Regardingin promoting chronic an increase GH exposure, in whole it- bodyseems pro- results have been reported after GH administration in patients with growth hormone moretein consistent synthesis than [42– acute44].. SimilarSimilar administration resultsresults havehave in promoting beenbeen reportedreported an increase afterafter GHGH in administrationadministration whole-body pro- inin pa-pa- deficiency (GHD) [45,46], and after GH administration in malnourished patients under teintients synthesis with [42 growth–44]. Similar hormone results deficiency have been (GHD reported) [45,46] after,, andand GH afterafter administration GHGH administrationadministration in pa- inin haemodialysis [47]. tientsmalnourished with growth patients hormone under deficiency haemodialysis (GHD) [47][45,46].. , and after GH administration in Tablemalnourished 1. Most relevant patients studies under with GH haemodialysis evaluating protein [47].anabolism using radioisotopes. Table 1. Most relevant studies with GH evaluating protein anabolism using radioisotopes. Table 1. Most relevant studies with GH evaluating protein anabolism using radioisotopes. AuthorAuthor Subjects Subjects Design Design GH GHDose Dose Effects Effects Conclusions Conclusions 0.014 μg/kg/min Rd PHe Locally infused GH sti- Author Subjects Design 0.014GH μg0.014 Dose/kg/minµg/kg/min Rd EffectsPHeRd PHe LocallyLocallyConclusions infused infused GH GH sti- FrybergFryberg BrachialBrachial artery infusion artery 7 healthy7 healthy 0.014To μg rise/kg/minTo locally rise locally not notRd PHeBCAAs Locallymulatesstimulates infused skeletal GH skeletal sti-muscle muscle Fryberg[39][39] [39] Brachialno placebo arteryinfusion infusion no placebo BCAAs 7 healthy To risesystemic locallysystemic not BCAAsrelease release mulatesproteinprotein skeletal synthesis. synthesis. muscle [39] no placeboBrachial artery infusion GH 0.014 μg/kg/min 3 h Rd Brachial artery infusionsystemic GH 0.014 GH μg/kg/min release 3 h Rd proteinGH synthesis.blunted the action of Fryberg 3 h GH and then 3 h Insulin 0.02 mU/kg/min 6 h Rd GH blunted the action of Fryberg 7 healthy Brachial3 h GH arteryBrachial and infusion then artery 3 h GH Insulin0.014 μg0.014 0./02kg/min µmUg/kg/min/kg/min 3 h Rd 6 h Rd 3 h Rd insulin [40] 7 healthy GH + insulin/no pla- To rise locally not syste- Ra GH insulinbluntedGH blunted the action the actionof of Fryberg[40] Fryberg 3 h GH and+ insulin/noinfusion then 3 h 3 pla- h GHInsulin andTo rise 0.02Insulin locally mU/kg/min 0.02not syste- 6 h RdRa 6 h Rd to suppress proteolysis 7 healthy 7 healthy insulinto insulinsuppress proteolysis [40] [40] GH cebo+ insulin/no then 3 pla- h GH +To risemic locally mU/kg/min not syste- Ra BCAAs Ra to suppressto suppress proteolysis proteolysis cebo insulin/no placebomic To rise locally notBCAAs Similar muscle size, Yarasheski 18 healthy Resistance training GH 40 μg/d proteinBCAAs synthesis Yarasheski 18 healthy Resistance training GH 40systemic μg/d strength and protein synt- [44] man GH/placebo for 12 weeks Leu. Ox Similarstrength muscle and size, protein synt- Yarasheski[44] 18 healthyman ResistanceGH/placebo training GH for40 μg12 /weeksd proteinLeu. synthesis Ox hesis protein strengthhesis and protein synt- Leu. Ox Similar muscle size, [44] Yarasheskiman 18 healthyGH/placeboResistance trainingfor 12 weeksGH 40 µg/d synthesis hesis strength and protein [44] man GH/placebo for 12 weeks Leu. Ox synthesis

Leu Ra in 0.018 IU/kg/day for either the Double-blind, GH action in adults with Russell-Jones 1 month followed by placebo or GH- 18 adults GHD placebo-controlled GHD is due to an increase [45] 0.036 IU/kg/day for treated trial in protein synthesis. 1 month In GH group Leu Rd Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18

(9.6 μg/kg body weight/d) GH replacement therapy or placebo on whole-body glucose and lipid metabolism and muscle composition in 19 GH-deficient adult subjects. GH ther- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18 apy resulted in glucose metabolism deterioration and an enhanced lipid oxidation rate in both short and long-term treatment groups, reflecting a fuel use switch from glucose to Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18 lipids. (9.6 μg/kg body Furthermore,weight/d) GH there replacement was a shift therapy toward or moreplacebo insulin on whole-resistant-body type glucose II X fibres in the and lipid metabolismbiopsied and muscles muscle during composition GH therapy.in 19 GH -Thesedeficient studies adult subjects. significantly GH ther- increased our apy resulted(9.6knowledge in μgglucose/kg bod metabolismregardingy weight/d the deterioration) GH relationship replacement and between an therapy enhanced skeletal or lipidplacebo muscle oxidation on and whole metabolismrate- bodyin glucose’s fibre both short andantyped lipidlong composition. -metabolismterm treatment However, and groups,muscle whether compositionreflecting this muscular a fuel in 19 use GH fibre switch-deficient shift from represents adult glucose subjects. the to cause GH ther-or the lipids. apyconsequence resulted in of glucose insulin metabolism resistance requires deterioration further and investigation. an enhanced lipid oxidation rate in Furthermore,both shortMoreover, there an dwas long GH a shift-/termIGF1 toward treatmentlevels hamoreve groups, beeninsulin related reflecting-resistant to ageing. a type fuel II useIn X particular, fibresswitch in from the IGF glucose-1 produc- to biopsied muscleslipids.tion by the during skeletal GH muscle therapy. is impaired These in studies older humans significantly and linked increased with aged our related sar- knowledge regardingcopeniaFurthermore, [30] the. Therefore, relationship there was GH/IGF betweena shift-1 towardactions skeletal morein musclethe insulin muscle and-resistant have metabolism structural, type’s II fibre X metabolic fibres in theand type composition.biopsiedageing However, consequences muscles whether during and this vic GHe muscularversa. therapy. fibre These shift represents studies significantly the cause or increasedthe our consequenceknowledge of insulin regardingresistance requires the relationship further investigation. between skeletal muscle and metabolism’s fibre Moreover,type4.1. GHcomposition.GH/ IGF1 levels However, have been whether related this to ageing.muscular In fibreparticular, shift represents IGF-1 produc- the cause or the tion by the consequenceskeletalThe muscle first of reports insulinis impaired on resistance the in anabolic older requires humans growth further and hormone linked investigation. effects with aged dated related back tosar- 1948 and sug- copenia [30]gested. Therefore,Moreover, that GH GH/IGF GH inhibits/IGF1-1 proteolysisactions levels hain vethe during been muscle relatedfasting have [35]to structural, ageing.. Subsequent In metabolicparticular, studies and IGFhave -1 expanded produc- ageing consequencestionthis by concept the and skeletal [36,37] vice muscleversa.. is impaired in older humans and linked with aged related sar- copeniaGH [30] is .the Therefore, primary GH/IGF hormone-1 actionsin the fast in thestate; muscle from havean evolutionary structural, metabolic point of view,and 4.1. GH ageingwhen foodconsequences is scarce, GHand changesvice versa. fuel consumption from carbohydrates and protein to the The firstuse reports of lipids, on the allowing anabolic for growth the conservation hormone effects of vital dated protein back stores to 1948 [38] and. Humans sug- ’ nitrogen 4.1. GH gested that GHbalance inhibits must proteolysis be kept stable during to fastingmaintain [35] the. Subsequent pool of essential studies amino have expandedacids (EAA ) involved this concept in[36,37] proteinThe. first formation. reports on Considering the anabolic this growth balance, hormone different effects EAA dated radiolabelling back to 1948 techniques and sug- GH is gestedthehave primary beenthat GHused hormone inhibits to evaluate proteolysisin the the fast effects state;during offrom fasting GH an and [35]evolutionary IGF. Subsequent-1 on protein point studies ofmet view, abolism.have expanded Table 1 when food thisissummarises scarce, concept GH [36,37] changesthe results. fuel of consumption studies assessing from carbohydratesprotein anabolism and proteinusing radioisotopes. to the The use of lipids,action allowingGH of is GH thefor on theprimary muscle conservation hormone tissue is of mainly invital the protein fastanabolic state; stores and from [38]has an .little Humans evolutionary effect’ nitrogen on proteolysis. point of view, Still, balance mustwhenthe be effects kept food stable iscould scarce, to be maintain differentGH changes thedepending poolfuel consumptionof essentialon the circumstan amino from acidscarbohydratesces, (doseEAA and) involved and time protein of exposition. to the in protein formation.useThe of acute lipids, Consideringinfusion allowing of for GHthis the on balance, conservation amino differentacid metabolism of vitalEAA protein radiolabelling results stores in a[38] techniquesprofound. Humans reduction ’ nitrogen in have been usedbalanceamino to evaluatemustacid oxidation be keptthe effectsstable [36,37,39 toof maintainGH–41] and. Some IGFthe -poolstudies1 on of protein essentialdemonstrated met aminoabolism. that acids Tableacute (EAA 1GH ) involved infusion summarisesin increasedthe protein results formation.whole of studies-body Considering assessingprotein synthesis protein this balance, withoutanabolism different any using significant EAA radioisotopes. radiolabelling changes Thein the techniques synthetic action of GHhaverate on ofmusclebeen muscle used tissue proteins. to evaluateis mainly By contrast,the anabolic effects other and of GH has studies andlittle IGFusing effect-1 higheron on proteinproteolysis. doses met ofabolism. GHStill, found Table lower 1 the effects couldsummarisesproteolysis be different rates.the results depending In general, of studies on acute the assessingcircumstan GH infusion proteinces, exerted dose anabolism and an timeanabolic using of exposition. effect radioisotopes. on whole - bodyThe The acute infusionactionamino of- acidof GH GH (onAA on muscle) metabolism,amino tissue acid ismetabolism evidenced mainly anabolic inresults reduced and in a hasleucine profound little oxidation, effect reduction on proteolysis.increased in nonox- Still, amino acidthe oxidationidative effects leucine could [36,37,39 disposal,be different–41]. Somean depending increased studies aminoondemonstrated the circumstanacid disappearance thatces, acute dose GH rateand infusion time(Rd) of(all exposition. indexes of increased wholeTheprotein acute-body synthesis infusion protein) ofsynthesisand GH less on effect withoutamino on acid proteinany metabolism significant breakdown changesresults (a lowerin in a the profound rate synthetic of amino reduction acid ap-in rate of muscleaminopearance proteins. acid ( Raoxidation By)) contrast,probably [36,37,39 otherrelated –studies41] with. Some usingdosage. studies higher Regarding demonstrated doses ofchronic GH foundthat GH acute exposure, lower GH infusion it seems proteolysis increasedrates.more Inconsistent general, whole- bodythanacute acuteprotein GH infusion administration synthesis exerted without inan promoting anabolic any significant effect an increase on changes whole in- body wholein the - syntheticbody pro- amino-acidrate (teinAA of )synthesis metabolism,muscle proteins. [42– evidenced44]. SimilarBy contrast, in results reduced other have leucinestudies been reportedoxidation,using higher after increased doses GH administration of nonox- GH found lower in pa- idative leucineproteolysistients disposal, with rates. growth an increasedIn general, hormone amino acute deficiency acidGH infusiondisappearance (GHD exerted) [45,46] rate an, and (anabolicRd )after (all indexeseffectGH administration on of whole -body in protein synthesisaminomalnourished)- acidand (lessAA patients )effect metabolism, on under protein evidencedhaemodialysis breakdown in reduced [47](a lower. leucine rate of oxidation, amino acid increased ap- nonox- Int. J. Mol. Sci.pearance2021, 22, 415 (Raidative)) probably leucine related disposal, with an d increasedosage. Regarding amino acid chronic disappearance GH exposure, rate it(Rd seems) (all indexes of 6 of 18 moreTable consistent 1. Mostprotein relevant than synthesis acutestudies administration) with and GH less evaluating effect in on promoting protein protein anabolism breakdown an increase using ( ain radioisotopes. lower whole rate-body of pro-amino acid ap- pearance (Ra)) probably related with dosage. Regarding chronic GH exposure, it seems Author teinSubjects synthesis [42–44].Design Similar results have GHbeen Dose reported after GHEffects administration inConclusions pa- more consistent than acute administration in promoting an increase in whole-body pro- tients with growth hormone deficiency0.014 (μgGHD/kg/min) [45,46] , andRd afterPHe GH administrationLocally infused in GH sti- Fryberg teinBrachial synthesis artery [42 infusion–44]. Similar resultsTable 1. haveCont. been reported after GH administration in pa- 7 healthymalnourished patients under haemodialysisTo rise locally [47] .not BCAAs mulates skeletal muscle [39] tientsno placebowith growth hormone deficiency (GHD) [45,46], and after GH administration in Author Subjects Designsystemic GH Doserelease Effectsprotein synthesis. Conclusions Table 1. Most relevant studiesmalnourished with GH evaluatingpatients under protein haemodialysis anabolism using [47] radioisotopes.. Brachial artery infusion GH 0.014 μg/kg/min 3 h Rd Acute stimulation of Fryburt and Systemic brachial 0.06 µg/kg/min for GH blunted the action of Fryberg 3 h GH and then 3 h Insulin 0.02 mU/kg/min 6 h Rd Rd muscle but not Author SubjectsBarret 7 healthyTable 1. 8 Most healthyDesign relevant studiesGH infusion/nowithGH GH Dose evaluating 6 protein h Effects anabolism using radioisotopes.Conclusionsinsulin [40] GH + insulin/no pla- To rise locally not syste- Ra whole-body protein [36] placebo0.014 μg/kg/min systemicRd PHe IGF-1 LocallyRa infusedto suppressGH sti- proteolysis Fryberg Brachial artery infusion synthesis 7Author healthy Subjects cebo DesignTo rise locallymic not GH DoseBCAAs BCAAsEffectsmulates skeletal muscleConclusions [39] no placebo 0.014 μg/kg/min Rd PHe LocallySimilar Acuteinfused muscle GH GHsize, did sti- not affect FrybergYarasheski 18 healthy BrachialResistance artery systemictraining infusion GH 40 μg/drelease protein proteinsynthesis synthesis. 7 healthy To rise locally not BCAAs mulatesstrengthmuscle skeletal and protein protein muscle synt- synthesis [44] Copelandman Brachial arteryGH/placebo infusion Infusion/SSAGH 0.014 μg/kg/minfor +/ 12− weeks 2 3µ g/kg/hh Rd Leu. Ox Ra [39] 15 healthyno placebo GH blunted thehesis action despite of enhanced protein Fryberg [37] 3 h GH and then 3 h GH/controlInsulin 0.02 mUsystemic/kg/min for 6 h 3.5 Rd h release protein synthesis. 7 healthy insulinleu Ox synthesis in non-muscle [40] GH + insulin/noBrachial pla- arteryTo riseinfusion locally GH not 0.014 syste- μg/Rakg/min 3 h Rd to suppress proteolysisGH bluntedtissue the action of Fryberg cebo 3 h GH and micthen 3 h Insulin 0.02 mUBCAAs/kg/min 6 h Rd 7 healthy insulin [40] GH + insulin/noProspective pla- crossTo rise locally not syste- Ra Similar muscle size,Increased muscle protein Yarasheski 18 healthyGaribotto Resistance training overGH 40 trial μg 6-week/d run 5protein mg 3 times synthesis a week to suppresssynthesis proteolysis and 6 Dialysiscebo mic BCAAs strength Rd and protein synt- [44] man [47] GH/placebo andfor 12 6-week weeks forLeu. 6 weeks. Ox decreased negative muscle hesisBCAAs Similar muscle size, Yarasheski 18 healthy Resistance trainingwashout/no GH control 40 μg/d protein synthesis protein balance. strength and protein synt- [44] man GH/placebo for 12 weeks Leu. Ox 4.5 IU GH hesis (1) basal suppression (2) after 40 h of Suppression of GH during Systemic Ra 8 healthy basal fasting fasting leads to a 50% Norrelund infusion/pancreatic ———– or after 40 h (3) after 40 h of increase in urea-nitrogen [48] clamp/controlled GH fasting fasting + SSAs excretion and increased replacement (4) after 40 h of proteolysis. fasting with SSAs + Ra GH replacement. BCAAs Lowering FFA with Acipimox increased whole Nielsen Subcutaneous GH replacement 7 GHD Ra body and forearm protein [46] GH/controlled +/− acipimox Rd breakdown, and protein synthesis Randomised Short GH (150 g/h; 2,1 ± After GH synthetic rate of 9 healthy crossover design Ra [43] 0.1 g/kg h) GH/FS Rd muscle proteins A 1-h iv infusion of Administration of GH Buijs 6 NW Crossover design SSAs and GH (12 blunted the rise in Leu Ox [41] 6 OB placebo mU/kg/h or Leu Ox similarly in both NW and placebo OB Open-label No effect in protein Gibney 12 hypopit. randomised breakdown, suppressing 0.5 mg daily 6 weeks [42] man crossover protein ox and stimulating testosterone and GH protein synthesis Adapted from: Jørgensen JOL, Christiansen JS. Growth Hormone Deficiency in Adults. Book. Karger Medical and Sci-entific Publishers; 2005. Decrease Increase, not change. Ins: insulin, Leu: leucine, Ox: oxidation, BCAAs: branched-chain amino acids, Phe: phenylalanine, NW: normal weight, OB: obese, wk week. Units: Kg: kilogram, h: hours, IU: international units, min: minutes, mg: milligram, mU: primary units, µg: microgram. Rd: (Phe/Leu disappearance rate) represents protein synthesis. Ra: (rate of appearance of Phe/Leu) represents proteolysis. Hypopit: hypopituitarism. Ox—oxidation.

Norreland et al. (2001) [48] evaluated the action of GH on fasting longer than overnight. They found that GH suppression led to increased proteolysis and with GH replacement they found a significant decrease in branched-chain amino acid levels, consistent with decreased proteolysis. Nevertheless, in studies designed to recover the muscle loss in the elderly, the over- all impact of GH administration has been far lower than expected [44,49,50]. Little is known about GH uses in fitness [51]. However, recently, in patients in need of reparative knee surgery Mendias et al. (2020) [52] found that rhGH compared to placebo improved quadriceps strength and reduced MMP3 (a subrogated marker of cartilage degradation). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18

(9.6 μg/kg body weight/d) GH replacement therapy or placebo on whole-body glucose and lipid metabolism and muscle composition in 19 GH-deficient adult subjects. GH ther- (9.6 μg/kg body weight/d) GH replacement therapy or placebo on whole-body glucose apy resulted in glucose metabolism deterioration and an enhanced lipid oxidation rate in and lipid metabolism and muscle composition in 19 GH-deficient adult subjects. GH ther- both short and long-term treatment groups, reflecting a fuel use switch from glucose to apy resulted in glucose metabolism deterioration and an enhanced lipid oxidation rate in lipids. both short and long-term treatment groups, reflecting a fuel use switch from glucose to Furthermore, there was a shift toward more insulin-resistant type II X fibres in the Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW lipids. 5 of 18 biopsied muscles during GH therapy. These studies significantly increased our Furthermore, there was a shift toward more insulin-resistant type II X fibres in the knowledge regarding the relationship between skeletal muscle and metabolism’s fibre biopsied muscles during GH therapy. These studies significantly increased our type composition. However, whether this muscular fibre shift represents the cause or the (9.6 μg/kg body weight/d) GH replacementknowledge therapy orregarding placebo theon whole relationship-body glucose between skeletal muscle and metabolism’s fibre consequence of insulin resistance requires further investigation. and lipid metabolism and muscle compositiontype in composition. 19 GH-deficient However, adult subjects.whether GHthis ther-muscular fibre shift represents the cause or the Moreover, GH/IGF1 levels have been related to ageing. In particular, IGF-1 produc- apy resulted in glucose metabolism deteriorationconsequence and an of enhanced insulin resistance lipid oxidation requires rate further in investigation. tion by the skeletal muscle is impaired in older humans and linked with aged related sar- Int. J. Mol. Sci. 2021both, 22 ,short 415 and long-term treatment groups, reflectingMoreover, a fuel GH use/IGF1 switch levels from have glucose been related to to ageing.7 of 18In particular, IGF-1 produc- copenia [30]. Therefore, GH/IGF-1 actions in the muscle have structural, metabolic and lipids. tion by the skeletal muscle is impaired in older humans and linked with aged related sar- ageing consequences and vice versa. Furthermore, there was a shift towardcopenia more insulin[30]. Therefore,-resistant typeGH/IGF II X-1 fibres actions in inthe the muscle have structural, metabolic and biopsied muscles during GH therapy. These studies significantly increased our Altogether, these experiments4.1.ageing GH consequences support the and anabolic vice versa. action of GH with less effect on knowledge regarding the relationship between skeletal muscle and metabolism’s fibre proteolysis, depending on timeThe lengthfirst reports and the on contextthe anabolic of GH growth exposition. hormone These effects effects dated back to 1948 and sug- type composition.probably However, become whether more this evident muscular4.1. GH with fibre more shift prolonged represents GH the exposure cause or or the in a pathological gested that GH inhibits proteolysis during fasting [35]. Subsequent studies have expanded consequence of insulinstate as resistance acromegaly requires accompanied furtherThe byinvestigation. first elevated reports insulin on the and anabolic IGF-I growth levels. hormone effects dated back to 1948 and sug- this concept [36,37]. Moreover, GH/IGF1 levels have been gestedrelated that to ageing. GH inhibits In particular, proteolysis IGF during-1 produc- fasting [35]. Subsequent studies have expanded GH is the primary hormone in the fast state; from an evolutionary point of view, tion by the skeletal4.2. muscle IGF-1 is impaired in olderthis concept humans [36,37] and linked. with aged related sar- when food is scarce, GH changes fuel consumption from carbohydrates and protein to the copenia [30]. Therefore,IGF-1 GH/IGF is one- of1 actions the most in important theGH muscle is the signals haveprimary instructural, muscle hormone anabolism metabolic in the andfast and repairsstate; from [30,53 an,54 evolutionary]. point of view, use of lipids, allowing for the conservation of vital protein stores [38]. Humans’ nitrogen ageing consequencesAs mentioned and vice versa. above, IGF-1when is secreted food is scarce, by the GH liver changes after GH fuel stimuli consumption and directly from carbohydrates from and protein to the balance must be kept stable to maintain the pool of essential amino acids (EAA) involved skeletal muscle in an autocrine/paracrineuse of lipids, allowing manner [for33 ].the IGF-1 conservation mediates beneficialof vital protein outcomes stores [38]. Humans’ nitrogen in protein formation. Considering this balance, different EAA radiolabelling techniques 4.1. GH of physical activity [55] andbalance can prevent must be chronic kept stable disease to [maintain56]. the pool of essential amino acids (EAA) involved have been used to evaluate the effects of GH and IGF-1 on protein metabolism. Table 1 The first reports Theon the first anabolic reports growth of IGF-1in hormoneprotein date back formation. effects to 1957 dated as Considering “sulphation back to 1948 this factor”, and balance, sug- when different Salomon EAA et al. radiolabelling techniques summarises the results of studies assessing protein anabolism using radioisotopes. The gested that GH inhibitsdescribed proteolysis its ability during to stimulate fastinghave been 35-sulphate[35] .used Subsequent to incorporationevaluate studies the have effects into expanded rat of cartilage. GH and Later,IGF-1 IGF-1on protein metabolism. Table 1 action of GH on muscle tissue is mainly anabolic and has little effect on proteolysis. Still, this concept [36,37](called. somatomedin-C) wassummarises assumed the to beresults a mediator of studies of anabolicassessing and protein mitogenic anabolism GH using radioisotopes. The the effects could be different depending on the circumstances, dose and time of exposition. GH is the primaryactivity [hormone57], but this in conceptthe fastaction drasticallystate; of GHfrom on changed an muscle evolutionary after tissue verification is point mainly of of anabolicview, the local and production has little effect on proteolysis. Still, The acute infusion of GH on amino acid metabolism results in a profound reduction in when food is scarce,and GH action changes of IGF-1 fuel [33 consumption,58].the effects from could carbohydrates be different dependingand protein on to the the circumstan ces, dose and time of exposition. amino acid oxidation [36,37,39–41]. Some studies demonstrated that acute GH infusion use of lipids, allowingImmediately for the conservation after IGF-1The of biosynthesisvitalacute protein infusion stores [59 of], aGH [38] new on. Humans challenge amino ’acid nitrogen in clinicalmetabolism research results was in a profound reduction in increased whole-body protein synthesis without any significant changes in the synthetic balance must be openedkept stable up. First,to maintain Guler etthe al.amino pool (1987) ofacid [essential60 ]oxidation compared amino [36,37,39 eight acids healthy (–EAA41]. adult)Some involved volunteers,studies demonstrated the short- that acute GH infusion rate of muscle proteins. By contrast, other studies using higher doses of GH found lower in protein formation.term metabolicConsidering effects this ofbalance, IFG-1increased anddifferent insulin.whole EAA-body Next, radiolabelling protein Laron etsynthesis al. techniques (1990) without [61,62 ]any investigated significant changes in the synthetic proteolysis rates. In general, acute GH infusion exerted an anabolic effect on whole-body have been used IGF-1to evaluate impact the on effects GHRH of in GHrate healthy andof muscle adultsIGF-1 proteins. andon protein children By met contrast, withabolism. primary other Table IGF-1 studies 1 deficiency using higher (GH doses of GH found lower amino-acid (AA) metabolism, evidenced in reduced leucine oxidation, increased nonox- summarises the insensitivity)results of studies or Laron’s assessing Syndromeproteolysis protein (LS).anabolism rates. Laron In general, using et al. wereradioisotopes. acute also GH the infusion first The to exerted introduce an anabolic the effect on whole-body idative leucine disposal, an increased amino acid disappearance rate (Rd) (all indexes of action of GH on longmuscle term tissue administration is mainly anabolic ofamino biosynthetic- andacid has(AA little IGF-1) metabolism, effect in LS on [63 proteolysis. evidenced,64]. Some inStill, of reduced the most leucine relevant oxidation, increased nonox- protein synthesis) and less effect on protein breakdown (a lower rate of amino acid ap- the effects could studiesbe different in humans, depending with on systemic idativethe circumstan leucine IGF-1 administration ces,disposal, dose and an increasedtime and of its exposition. impact amino on acid protein disappearance and the rate (Rd) (all indexes of pearance (Ra)) probably related with dosage. Regarding chronic GH exposure, it seems The acute infusionmuscular of GH system,on amino is summarisedacid metabolismprotein in synthesis Table results2. ) andin a lessprofound effect onreduction protein inbreakdown (a lower rate of amino acid ap- more consistent than acute administration in promoting an increase in whole-body pro- amino acid oxidation [36,37,39–41]. Some pearancestudies demonstrated (Ra)) probably that related acute with GH dinfusionosage. Regarding chronic GH exposure, it seems Table 2. Most relevant studies in humans,tein synthesis with systemic [42–44] IGF-1. Similar and muscle results actions. have been reported after GH administration in pa- increased whole-body protein synthesis withoutmore consistent any significant than acutechanges administration in the synthetic in promoting an increase in whole-body pro- tients with growth hormone deficiency (GHD) [45,46], and after GH administration in rate of muscle proteins. By contrast, other teinstudies synthesis using higher[42Muscle–44] doses. Similar of GH resultsMuscle found have lower been reported after GH administration in pa- Author Subjects Design Dosemalnourished patients under haemodialysis [47].Conclusions proteolysis rates. In general, acute GH infusiontients exerted with growth anAnabolism anabolic hormone effect deficiency onFunction whole -(bodyGHD ) [45,46], and after GH administration in amino-acid (AA) metabolism,Table evidencedrhGH: 1. Most 0.05 inrelevantmalnourished mg/kg reduced studies Withleucine withpatients IGF-1 oxidation, GH serum evaluatingunder increasedhaemodialysis protein nonox-anabolism [47]. using radioisotopes. 2 weeks IGF-1 reversed the idative leucine disposal, an increased*6 d or amino iv infusion acid disappearanceurea nitrogen rate (Rd) (all indexes of Clemmons calorically catabolism caused protein6 healthy synthesisAuthor) and less effectSubjectsTable on protein1. Mostof relevant breakdownDesign studiesthe reduction ( a with lower GH was rate evaluating GHof amino DoseNA protein acid anabolism ap- Effects using radioisotopes. Conclusions [65] restricted by a restriction pearance (Ra)) probably related withrhIGF-I dosage. 12 µg/kg Regardinggreater chronic than0.014 with GH μg exposure,/kg/min it seemsRd PHe Locally infused GH sti- FrybergAuthor Subjects Brachial arteryDesign infusion GH Dose of theEffects diet. Conclusions 7 healthy IBW *16 h GH To rise locally not BCAAs mulates skeletal muscle more consistent[39] than acute administrationno in placebo promoting an increase0.014 in μgwhole/kg/min-body pro-Rd PHe Locally infused GH sti- FrybergAcute IGF-I dosesBrachial artery infusionsystemic release protein synthesis. tein synthesis [42–44]. Similar7 healthy results have been reportedDose-dependent after GHTo administration rise locally not in pa-BCAAs mulates skeletal muscle Turkalj 19 healthy [39]randomised 5, 7.5, 15Brachial andno placebo artery infusion GH 0.014 μg/kg/min GF-13 h Rd proteolysis tients with growth hormone deficiency (GHD) [45,46], anddecrease after systemicGH administration inrelease GHprotein blunted synthesis. the action of [66] man Fryberg rhIGF-1 30 µg/kg/h3 h GH and then 3 h Insulin 0.02 mUNA/kg/min 6 hIGF-1 Rd protein malnourished patients under7 healthy haemodialysis Brachial[47]. arteryof infusion leucine GH 0.014 μg/kg/min 3 h Rd insulin [40] ascending doses (n = 4) andGH saline + insulin/no pla- To rise locally not syste- Rasynthesis GH blunted the action of Fryberg 3 h GH and thenoxidation. 3 h Insulin 0.02 mU/kg/min 6 h Rd to suppress proteolysis vs. saline. 7 healthycontrol (ncebo = 3). mic BCAAs insulin Table 1. Most relevant studies[40] with GH evaluating protein anabolismGH + insulin/no using radioisotopes. pla- To rise locally not syste- Ra IGF-1 Similarto suppress muscle proteolysis size, Yarasheski 18 healthy Resistancecebo training GHmic 40 μg /d proteinBCAAs synthesis Author Subjects 5 healthy DesignRandom order of GH3-h Dose IV infusion of Effects Ra Conclusions IGF-1 increases strength and protein synt- Russell- adequate [44] both IGF-1 orman IGF-1: 43.7GH/placebo for 12 weeks proteinLeu. Ox synthesis in Similar muscle size, Yarasheski 0.01418 μg healthy/kg/min ResistanceRd PHe trainingRd LocallyGH 40 infusedμg/d GH sti- protein synthesis hesis Fryberg Jones substrateBrachial arteryinsulin infusion 1 week pmol*kg/min or NA contrast to insulin, strength and protein synt- 7 healthy To rise locally not BCAAs Insulin mulates skeletal muscle Leu. Ox [39] [67] supplyno placebo [44] apart + AA man Insulin 3.4GH/placebo for 12 weeks which acts to systemic release protein synthesis. hesis condition), infusion pmol/kg/min Ra reduce proteolysis. Brachial artery infusion GH 0.014 μg/kg/min 3 h Rd Rd GH blunted the action of Fryberg 3 h GH and then 3 h Insulin 0.02 mU/kg/min 6 h Rd 7 healthy IV infusion of insulin [40] GH + insulin/no pla- To rise locally not syste- Ra 2 weeks rhIGF-1 12 µg/kg to suppress proteolysis Combination of GH cebo caloricallymic IBW *16 h *5 dBCAAs and IGF-I was Nitrogen retention Kupfer restricted or Similar muscle size, substantially more Yarasheski 18 healthy 7Resistance healthy training GH 40 μg/d proteinwas synthesis 2.4-fold greater NA [68] (20 kcal/kg IBW rhIGF-1 (same strength and protein synt- anabolic [44] man GH/placebo for 12 weeks Leu. Ox in combination per d), with 1 g doses above) + hesis than either GH or protein/kg IBW. rhGH: 0.05 mg/kg IGF-I alone. *5 d

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18

(9.6 μg/kg body weight/d) GH replacement therapy or placebo on whole-body glucose and lipid metabolism and muscle composition in 19 GH-deficient adult subjects. GH ther- apy resulted in glucose metabolism deterioration and an enhanced lipid oxidation rate in both short and long-term treatment groups, reflecting a fuel use switch from glucose to lipids. Furthermore, there was a shift toward more insulin-resistant type II X fibres in the biopsied muscles during GH therapy. These studies significantly increased our knowledge regarding the relationship between skeletal muscle and metabolism’s fibre type composition. However, whether this muscular fibre shift represents the cause or the consequence of insulin resistance requires further investigation. Moreover, GH/IGF1 levels have been related to ageing. In particular, IGF-1 produc- tion by the skeletal muscle is impaired in older humans and linked with aged related sar- copenia [30]. Therefore, GH/IGF-1 actions in the muscle have structural, metabolic and ageing consequences and vice versa.

4.1. GH The first reports on the anabolic growth hormone effects dated back to 1948 and sug- gested that GH inhibits proteolysis during fasting [35]. Subsequent studies have expanded this concept [36,37]. GH is the primary hormone in the fast state; from an evolutionary point of view, when food is scarce, GH changes fuel consumption from carbohydrates and protein to the use of lipids, allowing for the conservation of vital protein stores [38]. Humans’ nitrogen balance must be kept stable to maintain the pool of essential amino acids (EAA) involved in protein formation. Considering this balance, different EAA radiolabelling techniques have been used to evaluate the effects of GH and IGF-1 on protein metabolism. Table 1 summarises the results of studies assessing protein anabolism using radioisotopes. The action of GH on muscle tissue is mainly anabolic and has little effect on proteolysis. Still, the effects could be different depending on the circumstances, dose and time of exposition. The acute infusion of GH on amino acid metabolism results in a profound reduction in amino acid oxidation [36,37,39–41]. Some studies demonstrated that acute GH infusion increased whole-body protein synthesis without any significant changes in the synthetic rate of muscle proteins. By contrast, other studies using higher doses of GH found lower proteolysis rates. In general, acute GH infusion exerted an anabolic effect on whole-body amino-acid (AA) metabolism, evidenced in reduced leucine oxidation, increased nonox- idative leucine disposal, an increased amino acid disappearance rate (Rd) (all indexes of protein synthesis) and less effect on protein breakdown (a lower rate of amino acid ap- pearance (Ra)) probably related with dosage. Regarding chronic GH exposure, it seems more consistent than acute administration in promoting an increase in whole-body pro- tein synthesis [42–44]. Similar results have been reported after GH administration in pa- tients with growth hormone deficiency (GHD) [45,46], and after GH administration in Int. J. Mol. Sci. 2021malnourished, 22, 415 patients under haemodialysis [47]. 8 of 18

Table 1. Most relevant studies with GH evaluating protein anabolism using radioisotopes.

Author Subjects Design GH DoseTable 2. Cont. Effects Conclusions 0.014 μg/kg/min Rd PHe Locally infused GH sti- Fryberg Brachial artery infusion 7 healthy To rise locallyHigh not doses BCAAs mulates skeletal muscle High doses of [39] no placebo (30 µg/kg/h IGF-I Ra Randomisedsystemic and release protein synthesis. IGF-1 compared to or 0.23 nmol/kg/h Rd Brachial artery pairedinfusion three GH 0.014 μg/kg/min 3 h Rd insulin decreased Lagger 24 healthy insulin); ———— GH blunted the action of Fryberg 3 h GH and then 3groups h Insulin 0.02 mU/kg/min 6 h Rd NA more proteolysis. 7 healthy[69] male low doses Any changeinsulin [40] GH + insulin/noIGF-1-insulin pla- To rise locally not syste- Ra Unexpected (5 µg/kg/h IGF-I compared to to suppress proteolysis inhibition of cebo mic or 0.04 nmol/kg/hBCAAs control Similar muscle size, protein synthesis Yarasheski 18 healthy Resistance training GH 40 μg/d insulin) protein synthesis strength and protein synt- [44] man GH/placebo for 12 weeks Leu. Ox Boys with DMD. 6 Did not hesis months of rhIGF-1 Boys with Randomised, GC-treated + IGF-1 observe a therapy did not Rutter DMD rhIGF-1 vs. (n = 17) vs. controls change in NA change motor [70] 21 control placebo. (GC-therapy only n functional function but did 17 IGF1 6 month = 21) motor improve linear outcomes growth. rhIGF-I/rhIGFBP-3 administration for Low dose Significant 28 days improved Randomised, rhIGF-I/rhIGFBP-3 increase in Nishan Fifty-six aerobic double-blind, (30 mg/d), or high maximal Guha recreational NA performance in placebo- dose oxygen con- [71] athletes recreational controlled rhIGF-I/rhIGFBP-3 sumption athletes, with no (60 mg/d) for 28 d effects on body composition. DMD = Duchenne muscular dystrophy EA: essential amino acid IBW: ideal body weight. Ins: insulin, Leu: leucine, Ox: oxidation, BCAAs: branched-chain amino acids, Phe: phenylalanine, wk week. NA: not available. (*) during. Units: d. day, kg: kilogram, h: hours, IU: international units, min: minutes, mg: milligram, mU: primary units, µg: microgram nmol: nanomole pmol: picomole, Rd: (Phe/Leu disappearance rate or nonoxidative AA disposal) represents protein synthesis. Ra: (rate of appearance of Phe/Leu) represents proteolysis.

In healthy people, Clemmons et al. (1992) [65] found evidence that short term IGF-1 infusion, was able to reverse fasting diet-induced muscular catabolism. There is also some evidence indicating that the effects of the systemic administration of IGF-I on protein metabolism depend on environmental AA levels. In this regard, Turkalj et al. (1992) [66] showed that IGF-I administered without AA, suppressed proteolysis, while in the study by Russell-Jones DL, et al. (1994) [67]. IGF-1 administration in combination with AA infusion increased protein synthesis. Likewise, Kupfer et al. (1993) [68] found that GH and IGF-1 anabolic effects were enhanced when co-administered in 7 healthy calorically restricted patients. Lagger et al. (1993) [69], compared high and low doses of rhIGF-1 and found that high doses of IGF-1 decreased proteolysis more than insulin. However, it was accompanied by an unexpected inhibition of protein synthesis. More recently, some researchers have studied the effects of rhIGF-1 on muscle perfor- mance in special populations. Rutter et al. (2020) [70] investigated 21 boys with Duchenne muscular dystrophy and found that after 6 months of rhIGF-1 therapy, despite improve- ments in linear growth, muscular motor function did not change. Guha et al. (2020) [71] studied fifty-six recreational athletes and observed that muscular aerobic performance improved after 28 days of rhIGF-1/rhIGFBP-3 administration, but no effects on body com- position. It should be taken into account that circulating IGF-1 is probably less important in those tissues that can produce the hormone themselves, such as skeletal muscle [72] Focusing on ACRO, some degree of IGF-I receptor resistance to IGF1 action has recently been reported [73]. This important finding could explain why in the presence of high IGF1 levels, the metabolic GH effects are predominant in these patients. In addition, IGF-1 resistance might also be involved in other ACRO related muscle disturbances. In summary, these data suggest that the administration of IGF-1 inhibits proteolysis and improves muscle performance with less effect on protein synthesis. Other factors such as local IGF-1 secretion and/or IGF1R resistance could play an important role. The data is Int. J. Mol. Sci. 2021, 22, 415 9 of 18

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 18 still controversial, and the combined and additive effect of IGF-1 and GH co-administration remains a subject of debate.

5. The Key of mTOR5. The in Key Muscle of mTOR Structural in Muscle and StructuralMetabolic andFunction Metabolic Function Skeletal muscleSkeletal mass and muscle whole mass‐body and metabolism whole-body are closely metabolism related. are One closely of the related. ma‐ One of the jor players in coordinatingmajor players growth in coordinating with nutrient growth availability with nutrient is rapamycin’s availability mammalian is rapamycin’s mammalian target (mTOR) target[74]. There (mTOR) are two [74]. different There are mTOR two complexes, different mTOR mTORC1 complexes, and mTORC2. mTORC1 In and mTORC2. brief, mTORC1In senses brief, nutrients, mTORC1 oxygen, senses energy, nutrients, and oxygen,growth factors energy, promote and growth cell growth, factors promote cell regulates metabolicgrowth, processes, regulates and metabolic mTORC2 processes, responds and more mTORC2 specifically responds to growth more factors specifically to growth and regulates cellfactors survival and and regulates metabolism cell survival [75]. Specifically, and metabolism mTORC1 [75 ].plays Specifically, a key role mTORC1 in plays a muscle structure,key stimulating role in muscle postprandial structure, stimulatingand post‐exercise postprandial muscle andprotein post-exercise synthesis. muscle protein synthesis. This pathway is mainly activated by mechanical contraction (i.e., exercise), This pathway is mainly activated by mechanical contraction (i.e., exercise), which causes which causes paracrine IGF-1 secretion [72]. However, AA intake also participates in paracrine IGF‐1 secretion [72]. However, AA intake also participates in governing the governing the mTORC1 pathway. It has been shown that leucine is a potent stimulator of mTORC1 pathway. It has been shown that leucine is a potent stimulator of muscle protein muscle protein synthesis by triggering mTORC1 signalling in human skeletal muscle [76]. synthesis by triggering mTORC1 signalling in human skeletal muscle [76]. Recent work Recent work has found that during AA sufficiency, mTORC1 kinase activity is stimulated has found that during AA sufficiency, mTORC1 kinase activity is stimulated mainly in 3 mainly in 3 ways: (1) The lysosomal AA transporter SLC38A9 sensing arginine within the ways: (1) The lysosomal AA transporter SLC38A9 sensing arginine within the lysosome lysosome [77]; (2) The leucine sensor Sestrin2 [78]; (3) The arginine sensor [cellular arginine [77]; (2) The leucine sensor Sestrin2 [78]; (3) The arginine sensor [cellular arginine sensor sensor for mTORC1 (CASTOR1)] [79]. All these pathways ultimately allow mTORC1 to be for mTORC1 (CASTOR1)] [79]. All these pathways ultimately allow mTORC1 to be acti‐ activated (Figure3). vated (Figure 3).

Figure 3. TheFigure interplay 3. The ofinterplay GH, IGF-1, of GH, insulin, IGF‐1, hybrid insulin, receptor hybrid receptor and nutrients and nutrients in the muscle. in the muscle. Schematic Sche‐ and simplified matic and simplified signalling pathway and the interplay of GH, IGF‐1, insulin, hybrid receptor signalling pathway and the interplay of GH, IGF-1, insulin, hybrid receptor and nutrients in the muscle. AA (amino-acid) and nutrients in the muscle. AA (amino‐acid) stimulates mTOR kinase activity mainly in 3 ways: stimulates mTOR kinase activity mainly in 3 ways: (1) The leucine sensor Sestrin, (2) the lysosomal sensor SLC38A9, (1) The leucine sensor Sestrin, (2) the lysosomal sensor SLC38A9, (3) The arginine sensor [cellular (3) The argininearginine sensor sensor [cellular for mTORC1 arginine (CASTOR1)]. sensor for mTORC1 GH activates (CASTOR1)]. via IRIS and GH SHC activates protein via and IRIS mitogenic and SHC protein and mitogenic effects as as well well as as IGF IGF-1R/InsulinR‐1R/InsulinR and and HybridR. HybridR. GH GH also also stimulates stimulates via JAK/STAT via JAK/STAT local muscular local muscular IGF-1 secretion. InIGF addition,‐1 secretion. the metabolicIn addition, effects the metabolic of GH impact effects on of muscleGH impact anabolism. on muscle The anabolism. main metabolic The main action of GH is a lipolytic actionmetabolic on adipose action tissue, of GH releasing is a lipolytic free action fatty acids on adipose (FFA), whichtissue, favoursreleasing protein free fatty preservation, acids (FFA), as well as liver and kidney-enhancedwhich favours gluconeogenesis. protein preservation, as well as liver and kidney‐enhanced gluconeogenesis.

Similarly to IGFSimilarly‐1R resistance to IGF-1R described resistance in ACRO, described an “anabolic in ACRO, resistance” an “anabolic due to resistance” an due to impairment of anthe impairment “sensor” capacity of the of “sensor” the muscle capacity to metabolise of the muscle circulating to metabolise AA has been circulating AA has related to the sarcopeniabeen related that to occurs the sarcopenia in the elderly that [76]. occurs This in reduced the elderly ability [76 ].is probably This reduced ability is associated withprobably impairments associated in molecular with impairments signalling in such molecular as decreased signalling amino such asacid decreased amino transport, whichacid results transport, in reduced which mTORC results in activation. reduced mTORC activation. Recently, we have reported low circulating levels of branched‐chain amino acid in active ACRO [80]. This finding could contribute to the myopathy observed in patients with ACRO, but further basic and clinical research on this issue is needed. In this regard,

Int. J. Mol. Sci. 2021, 22, 415 10 of 18

Recently, we have reported low circulating levels of branched-chain amino acid in active ACRO [80]. This finding could contribute to the myopathy observed in patients with ACRO, but further basic and clinical research on this issue is needed. In this regard, to the best of our knowledge, there are no studies addressed to investigate the potential role on muscle performance of diet enriched with AA in ACRO.

6. Impact of GH/IGF-1 Axis Impairment on Muscle Metabolism and Function: Lessons from Basic Research The GH and IGF-1-mediated regulation of skeletal muscle structure and performance have recently been reviewed [54,81,82]. We will focus on the impact on the muscle of diseases with low or excess GH/IGF-1.

6.1. Primary Cell Cultures In vitro studies using immortalised the mouse myoblast cell line C2C12 has provided preliminary evidence regarding the action of IGF-I in muscle stimulating muscle protein synthesis, and also in suppressing proteolysis [83]. IGF-1 is also related to myoblast proliferation [84] and differentiation [54]. In addition, IGF-1 modulates the expression of L-type amino acid transporters in the muscles of spontaneous dwarf rats [85]. Mahendra et al. (2010) [86], in an elegant work using cell lines of mice lacking either the GH receptor or the IGF-1 receptor in skeletal muscle, found that GH mediates skele- tal muscle development by enhancing myoblast fusion in an IGF-1-dependent manner. They showed that GH treatment quickly increases IGF-1 mRNA and that this autocrine production of IGF-I leads to a significant increase in primary myoblast proliferation. By con- trast, disruption of the GH receptor in skeletal muscle produces marked alterations in muscle nutrient uptake and insulin sensitivity in an IGF-1-independent manner. Altogether, these findings indicate that the myoblast anabolic process is a complex action where both hormones are complementary. Nevertheless, metabolic dysfunctions seem to be more related to GH impairment.

6.2. Transgenic Mice Several genetically modified animal models (giants and dwarfs) have been gener- ated and characterised in the last 30 years [87–89] and have been used to investigate the phenotype, consequences and mechanisms underlying the altered GH/IGF-1 axis [90] This review will focus on the most relevant ones: transgenic mice overexpressing GH and GH knockout mice. For comprehensive reviews, please see references [87,91–93]. High GH levels in transgenic mice have elevated GH and IGF-1 levels, increased body weight, and lean mass. The extreme elevation in circulating GH also results in hyperinsulinemia despite euglycemia, probably due to reduced insulin receptors or GLUT4 channels in skeletal muscle. There are some differences among the various GH transgenes species in mice (e.g., ovine GH, bovine GH or human GH). Human GH can bind to both GH and prolactin (PRL) receptors, whereas rat, bovine and ovine GH bind exclusively to GHR. Thus, overexpression of human GH in mice results in a physiological state in which both GH and PRL receptors are simultaneously activated in contrast with rat, bovine, or ovine where only GHR is activated [92]. Together with the different ages of the mice used in the experiments, these differences could explain the heterogeneity of the published results regarding body composition. Besides, the following aspects should be taken into account when using transgenic mice: (1) In ACRO, GH secretion is from a pituitary adenoma whereas, in GH transgenic mice, GH excess is due to transgene insertion resulting in ectopic GH secretion. (2) The level of circulating GH is often markedly greater in transgenic mice than is observed in ACRO. (3) Transgenic mice exposure to excessive GH begun immediately after birth mimics gigantism more than ACRO (4) in human transgenic mice, GH and PRL receptors are simultaneously activated, unlike in ACRO. In complete contrast to high GH level transgenic mice, there are the dwarfism models: The GH knockout mice (GHKO or GH−/−), completely lack GH due to a targeted deletion of the GH gene. These mice represent a model of human isolated GH deficiency. The GH Int. J. Mol. Sci. 2021, 22, 415 11 of 18

receptor gene disrupted mice (GHR−/−) are mice completely resistant to GH action; they also have a pronounced decrease in body size with extremely low circulating IGF-1 de- spite elevated GH levels and constitute the model for LS. Other types of dwarfism models are transgenic IGF-1 knockout mice. Although the animals are born alive, mortality rates range from 32% to 95% due to respiratory failure caused by impaired diaphragm develop- ment, severe muscle dystrophy and intercostal muscles. These mice also develop metabolic abnormalities such as mild hyperglycaemia (250 mg/dl) and decreased pancreatic beta cells [63,94]. Finally, another model of IGF-1 deficiency is a consequence of a deletion of the gene encoding the IGF-1 in the liver (only in the liver, not in other tissues), resulting in a 75% reduction of circulating plasma IGF-1 levels [95]. These animals usually grow and are fertile. It has been suggested that high levels of growth hormone and increased paracrine or tissue IGF-1 levels compensate for the lower plasma levels of IGF-1. Overall, these findings provide evidence that both GH and IGF-1, including locally muscular secreted IGF-1, have active implications for growth and muscle development.

6.3. Experimental Research Focused on the GH/IGF-1 System Effects on Muscle The effects of GH deficiency in muscle have been controversial. Ayling et al. (1989) [96] reported a 50% reduction in type I fibres after rat hypophysectomy, that recovered after GH therapy. Similar results were obtained by Loughna and Bates five years later (1994) [97]. By contrast, Yamaguchi et al. (1996) [98] also observed a significant increase in type I fibres after rat hypophysectomy. Some studies have also reported no change in the composition of type I or type II fibres after GH replacement in the same experimental model [99]. These divergent results could be explained by the different exposure to GHD, GH therapy duration, and the associated deficit of other hormones such as thyroxine, testosterone and glucocorticoids. Nielsen et al. (2014) [100] in an elegant work, measured the ultra-structure and collagen in the Achilles tendon, employing three groups of mice: (1) Giant transgenic mice that expressed bovine GH (bGH): the ACRO model; (2) Dwarf mice disrupted GH receptor GHR−/− (the LS model) and (3) A wild-type control group. They found that the mean collagen fibril diameter was significantly decreased in the ACRO and LS models. The ultra-structural pattern was more severely affected in the GHR−/− mouse model. Similarly, a recent study of Cinaforlini et al. (2020) [101] investigated muscle repair and found that GH administration accelerated the muscle healing process. Regarding the direct effect of IGF-1 on the muscle, Schoenle et al. in 1982 [102] reported that IGF-1 stimulated growth in hypophysectomised rats, thus providing evidence of independent IGF-1 action from GH. DeVol et al. in 1990 described for the first time the local skeletal muscle production of IGF-1 [58]. Using a rat model of compensatory hypertrophy of the soleus, the authors showed that muscle hypertrophy and local IGF-1 production occurred independently of GH. However, compensatory hypertrophy was not blunted in hypophysectomised rats. All this evidence reinforces the concept that although the actions of GH and IGF1 in the muscle, are independent, they are complementary.

7. The Muscle in GH/IGF1 Axis Deficiency Isolated GH deficiency (IGHD) either congenital or acquired, is the commonest pi- tuitary hormone deficiency [103]. First description and treatment date from 1963 [104]. During childhood and puberty, the classical phenotype of GHD is short stature with height ≤ −2 SDS, frontal bossing, mid-facial hypoplasia (“doll-like” facies) and truncal adipos- ity [105]. Adult-onset patients with GHD have increased total and visceral fat, low bone mass, reduced muscle strength and impaired anaerobic physical capacity, an unfavourable cardiovascular profile, and low quality of life [106,107]. There is conflicting data regarding body composition and muscle structure and perfor- mance after GH treatment. In a recent study, Andrade-Guimarães et al. (2019) [108] found Int. J. Mol. Sci. 2021, 22, 415 12 of 18

that individuals with congenital untreated IGHD had better muscle strength parameters adjusted for weight and fat-free mass than controls, and also exhibited greater peripheral resistance to fatigue. In adults with GHD, Díez et al. (2018) [109] and Jørgensen et al. (2018) [107] among others, argued in favour of the beneficial effects of GH administration on body composition, muscle exercise capacity, bone structure and serum lipids. By contrast, He and Barkan (2020) [110] critically reviewed the evidence of treatment benefits in adult-onset GHD. They found most of the data inconsistent, thus recommending that GH treatment should be a personalised decision. Regarding GH resistance (LS) syndrome, the main clinical characteristics are dwarfism, specific facial features such as a protruding forehead in the presence of a subnormal head circumference, sparse hair, skeletal and muscular underdevelopment, obesity and underdeveloped genitalia [111]. Long-term observations have shown that these individuals are protected from cancer [112,113]. In relation to the muscle, they have a reduced lean body mass and reduced muscle force and endurance [114]. Curiously, growth velocity obtained with IGF-I administration is smaller than that observed with hGH in children with congenital isolated GH deficiency [115]. Muscle benefit with IGF-1 therapy has not been fully established. Long-term treatment for adult patients living with LS aimed at strengthening the muscular and skeletal systems is not currently approved.

8. Muscle Impairment in Human Acromegaly Muscular weakness and pain represent one of the significant disabling co-morbidities in ACRO [5,116] and, as above mentioned, despite long-term remission of the disease, this co-morbidity may persist and deeply compromise QoL. Although musculoskeletal pain is reported in up to 90%, it is not usually recorded in the clinical history [116]. It should be noted that as a result of treatment improvement, life expectancy in people living with ACRO is currently comparable to that of the general population [117] and, therefore, the rate of musculoskeletal impairment is not expected to be reduced. Nevertheless, this is one of the least studied co-morbidities [118], and there are few studies focused on the evolution of myopathy after ACRO treatment. Mastaglia et al. in 1970 [119] reported for the first time on muscle impairment in ACRO. Mild proximal muscle weakness was present in six out of 11 cases. Histological changes in hypertrophy of both type-I and type-II muscle fibres were current in five out of nine biopsy specimens taken from a proximal limb muscle. However, electron microscopy showed a patchy myopathy process, thus accounting for these patients’ weakness and myalgia. By contrast, Nagulesparen et al. (1976) [120] studied 18 patients with acromegaly of varying degrees of severity; half of the specimens showed hypertrophy of type 1 fibres, and atrophy in type 2 fibres, although a direct correlation between muscle appearances and growth hormone levels was not observed. These findings suggest, firstly, a dissociation between muscle macro/microstructure and the IGF-1/GH level, and secondly an alteration in the damage repair mechanism in acromegaly. Thus, other factors independently of hormone levels could play a key role in ACRO myopathy, and treatments strengthening the muscle damage repair mechanism could improve this co-morbidity. Concerning energy consumption, Szendroedi et al. (2008) [121], found that patients with ACRO exhibited reduced muscular ATP synthesis and oxidative capacity that may persist despite the normalisation of GH secretion. Regarding strength, Füchtbauer et al. (2017) [5] studied ACRO patients at diagnosis (n = 48), one year after surgery (n = 29) and after long-term follow-up (median 11 years) (n = 24), compared to healthy subjects. They found reduced grip and strength in active ACRO and increased proximal muscle fatigue even after GH/IGF-1 normalisation. Although the most apparent muscular structural change in active ACRO seemed to be hypertrophy [122,123], more recent studies, have not confirmed this finding [124]. Recently Gokce et al. (2020) [125] using ultrasound found that the thicknesses of many Int. J. Mol. Sci. 2021, 22, 415 13 of 18

components of the quadriceps muscle were lower in ACRO patients than control subjects matched by age, sex and the body mass index. We have very little evidence on interventional programs to improve this devastating co-morbidity. However, Lima et al. (2019) [6] evaluated the effects of a functional home rehabilitation program in seventeen adults with ACRO that resulted in improvements in muscle function, functional capacity, general fatigue, body balance, and QoL. Accordingly, other therapeutic strategies apart from GH/IGF-1 normalisation, with a direct impact on muscle structure/performance should be followed to improve our patients QoL.

9. Concluding Remarks and Future Perspectives In ACRO, the sustained increase of GH and IGF-1 critically affects the skeletal, muscu- lar system with severe repercussions on life quality. At present, our knowledge regarding the underlying mechanisms and outcome of different treatments is scarce. In addition, there are no clinical guidelines or any structured recommendations for dealing with this debilitating co-morbidity. Therefore, there is a pressing need to promote interaction between basic and clinical research, improving our knowledge and permitting us to design therapeutic strategies. Nevertheless, we can remark on some important points: (1) Up to 90% of ACRO patients suffer from musculoskeletal pain. (2) This co-morbidity may persist despite biochemical control of the disease and deeply compromise QoL. (3) Other factors apart from GH/IGF-1 levels could have a significant role in ACRO myopathy’s underlying mechanism. (4) New biomarkers linking muscle mass and metabolism to diagnose better and phenotype myopathy and design the best therapeutic strategy are needed. Cell lines and transgenic mice give important insights into the many actions of GH and IGF-1. The degree of the contribution of GH or IGF-1 to myopathy in acromegaly remains to be further investigated. In the clinical setting, we need (1) tools that efficiently and reliably permit us to evaluate the muscular status of patients with ACRO, (2) collaborative, multicentre studies with larger samples to provide more accurate data, and (3) further studies that investigate treatments that directly influence muscle performance, even in patients in remission. Finally, it should be emphasised that the muscle should be seen as a key role organ, with mechanical and metabolic functions. Disturbances in metabolic processes (for ex- ample, hyperglycaemia, amino acid deficiency) can ultimately impact muscle structure and, vice versa, changes in fibre type can promote metabolic changes. In this regard, studies aimed at assessing the molecular link between structural changes and metabolic function are needed.

Author Contributions: Both authors equally contributed to the conception of the review. B.B. drafted the work and R.S. revised it critically for important intellectual content. B.B. and R.S. gave final approval of the version of the manuscript; and agree to be accountable for all aspects of the work. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

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