Endocrine regulation of fetal skeletal muscle growth: impact on future metabolic health

Abstract

Establishing sufficient skeletal muscle mass is essential for lifelong metabolic health. The intrauterine environment is a major determinant of the muscle mass that is present for the life course of an individual, because muscle fiber number is set at the time of birth. Thus, a compromised intrauterine environment from maternal nutrient restriction or placental insufficiency that restricts development of muscle fiber number can have permanent effects on the amount of muscle an individual will live with. Reduced muscle mass due to fewer muscle fibers persists even after compensatory or “catch up” postnatal growth occurs. Furthermore, muscle hypertrophy can only partially compensate for this limitation in fiber number. Compelling associations link low birth weight and decreased muscle mass to future insulin resistance, which can drive the development of the metabolic syndrome and type 2 diabetes, and risk for cardiovascular events later in life. There are gaps in knowledge about the origins of reduced muscle growth at the cellular level and how these patterns are set during fetal development. By understanding the nutrient and endocrine regulation of fetal skeletal muscle growth and development, we can direct research efforts towards improving muscle growth early in life in order to prevent the development of chronic metabolic disease later in life.

Introduction

Epidemiological studies have demonstrated that lower birth weight for a given gestational age increases an individual’s risk for developing obesity (Ravelli et al. 1999; Valdez et al. 1994), coronary heart disease (Barker et al. 1993; Barker et al. 2010), glucose intolerance (Hales et al. 1991; McKeigue et al. 1998; Phipps et al. 1993), and type 2 diabetes (Curhan et al. 1996; Rich-Edwards et al. 1999) later in life. Small for gestational age (SGA) status at the time of birth, defined arbitrarily as birth weight less than the 10% on standard pediatric growth curves (Battaglia & Lubchenco 1967), can result from many causes, one of which is placental insufficiency (Platz & Newman 2008). Placental insufficiency is defined as a smaller than normal placenta, with or without specific transporter deficiencies, that restricts nutrient flow from mother to fetus and uniquely produces intrauterine growth restriction (IUGR) (Marconi et al. 2006; Marconi & Paolini 2008; Molteni et al. 1978; Regnault et al. 2013). Fetal IUGR leads to increased perinatal and neonatal morbidity and mortality (Pollack & Divon 1992; Tuuli et al. 2011), as well as the later life pathologies noted above. While nearly every fetal organ system is affected in IUGR, skeletal muscle growth is particularly vulnerable because blood flow and nutrient supplies are preferentially shunted to vital organs in response to decreasing fetal oxygenation (Tchirikov et al. 1998; Yajnik 2004b). As a result, skeletal muscle growth is preferentially restricted (Beltrand et al. 2008; Larciprete et al. 2005; Padoan et al. 2004; Yau & Chang 1993).

Skeletal muscle serves several important metabolic functions. First, resting energy expenditure varies considerably based on the amount of lean mass that an individual possesses (Mifflin et al. 1990; Nelson et al. 1992; Taguchi et al. 2011). Based on estimates for the energy required to maintain the muscle fractional protein synthetic rate (Tipton et al. 2003; Waterlow 1984), it has been proposed that greater muscle mass and increased energy expenditure from muscle protein turnover may contribute to the prevention of obesity (Newsholme 1978; Wolfe 2006). Second, skeletal muscle accounts for 80% of whole body insulin-stimulated glucose uptake; thus muscle maintains whole body insulin sensitivity (DeFronzo et al. 1981). Third, several muscle secretory products or “myokines” improve insulin sensitivity (Basaria & Bhasin 2012) and stimulate energy consumption within adipose tissue (Bostrom et al. 2012). Finally, sarcopenia, or the degenerative loss of skeletal muscle mass and function, affects 30% of adults over age 65 and is a large contributor to morbidity and mortality (Doherty 2003). Thus, low muscle mass impacts adult health and has important implications for quality of life, excess weight gain, and risk for development of insulin resistance and type 2 diabetes.

Reduced fetal skeletal muscle growth is not fully compensated after birth, as individuals who are born with low birth weight have lower muscle mass in adulthood (Gale et al. 2001; Kensara et al. 2005; Yliharsila et al. 2007). Since skeletal myofiber number is set at the time of birth (Rowe & Goldspink 1969; Wigmore & Stickland 1983), it is possible that disruptions in myofiber formation during fetal life may not be fully recovered (Widdowson et al. 1972). In sheep models of maternal undernutrition or placental insufficiency, skeletal muscle growth is preferentially sacrificed and skeletal muscle mass is reduced at birth (Du et al. 2010). Under such circumstances, compensatory or “catch up” postnatal growth favors fat deposition and not muscle development (De Blasio et al. 2007; Ford et al. 2007; Louey et al. 2005). In humans, compelling associations link low birth weight and decreased muscle mass to future insulin resistance (Srikanthan & Karlamangla 2011), development of the metabolic syndrome and type 2 diabetes (Atlantis et al. 2009; Barker et al. 2002; Whincup et al. 2008), and risk for cardiovascular events later in life (Basaria & Bhasin 2012). Thus, suppressed development of muscle in the IUGR fetus could be a major contributor to their increased risk of later life sarcopenia, obesity, and diabetes.

An understanding of how fetal skeletal muscle growth adapts to nutrient availability is important for determining how deficits in muscle growth contribute to metabolic disease in adulthood. Therefore, the objectives of this review are to (1) review the fundamentals of fetal myogenesis; (2) review the experimental studies showing that fetal undernutrition from maternal dietary restriction or placental insufficiency influences skeletal muscle growth; (3) highlight the epidemiological studies showing that low birth weight is associated with reduced muscle mass in adulthood; and (4) discuss how insufficient muscle mass as a result of IUGR might influence long term metabolic health. Finally, potential therapeutic approaches to improving muscle mass in the IUGR fetus and research needs in this area will be addressed.

Skeletal muscle development

Proliferation and differentiation of myoblasts and myofibers

Myoblasts are mononuclear cells that have the capacity to proliferate and differentiate into skeletal myofibers (Gerrard & Grant 2003; Zammit et al. 2006). Myoblasts, as well as adipocytes and fibroblasts, differentiate from the multipotent mesenchymal stem cell (MSC) population in the developing human embryo (Pittenger et al. 1999). Because both myocytes and adipocytes share a common progenitor, the milieu of nutrients and growth factors in early embryonic and fetal life could affect MSC commitment to either a myogenic or adipogenic lineage (Du et al. 2013). Indeed, exposure of C2C12 myoblasts to adipogenic inducers in vitro has been shown to convert the differentiation pathway of myoblasts into that of adipoblasts (Teboul et al. 1995).

Once differentiated, myoblasts are classified as embryonic, fetal, or adult (Gerrard & Grant 2003). Embryonic myoblasts fuse to form primary myofibers by 20% of the length of gestation in sheep (Russell & Oteruelo 1981). Primary myofibers provide the scaffolding for the proliferation and differentiation of fetal myoblasts into secondary myofibers (Beermann et al. 1978). Secondary myofibers comprise the majority of myofibers and are highly nutrient responsive (Dwyer et al. 1994; Ward & Stickland 1991; Zhu et al. 2004). Secondary myogenesis occurs between 20% and 70% of gestation (Russell & Oteruelo 1981) and involves proliferation of fetal myoblasts followed by expression of muscle regulatory factors (MRFs) (Figure 1 a, b). MRFs are a set of helix-loop-helix transcription factors, including MYF5, MYOD, MRF4, and myogenin, that are expressed in a sequential manner during the differentiation process (Berkes & Tapscott 2005; Braun & Gautel 2011). Targets of the MRFs include proteins that regulate the switch from proliferation to differentiation, including retinoblastoma protein (RB), which is an inhibitor of cell cycle progression. Cyclin D1 and cyclin dependent kinase-4 (CDK4) phosphorylate and inhibit RB to induce proliferation (Spiller et al. 2002; Weinberg 1995). As differentiation occurs, MYOD binds myostatin, which results in withdrawal of myoblasts from the cell cycle (Spiller et al. 2002). In addition, P21 inhibits CDK4 so that RB remains dephosphorylated to reduce cell cycle activity (Guo et al. 1995). Cell cycle withdrawal is concomitant with the expression of MYOD, MYF5, and myogenin (Sabourin & Rudnicki 2000) (Figure 1c).

Figure 1. Key regulatory genes and proteins involved in fetal myogenesis.

a) Schematic diagram showing the differentiation of proliferating myoblasts into multinucleated myotubes and maturation into myofibers during fetal life. b) Expression of muscle regulatory factors (MRF) during myogenesis: Pax7 and MYF5 are expressed in the myoblast. The progression of differentiation is marked by the temporal expression of MYOD, MRF4 and myogenin. Desmin is the major intermediate filament expressed in mature muscle and its expression increases during gestation. c) Targets of MRF regulate the switch from proliferation to differentiation: Expression of cyclin D1 and CDK4 induce myoblast proliferation by maintaining the inhibitor retinoblastoma protein (RB) in its phosphorylated and inactive state. As differentiation is activated, MYOD binds myostatin and myoblasts withdraw from the cell cycle. In addition, P21 inhibits CDK4 which results in the dephosphorylation of RB to reduce cell cycle activity. This process results in withdrawal of myoblasts from the cell cycle and subsequent fusion into myotubes. Myotube maturation and hypertrophy is stimulated by growth factors, amino acids, and stretch/load activity. (Gaster et al. 2001; Molkentin & Olson 1996; Yang & Makita 1996; Zammit et al. 2006).

Myogenesis is nearly complete by the end of gestation, as a full complement of myofibers has been observed at the time of birth in both mice and piglets (Rowe & Goldspink 1969; Wigmore & Stickland 1983). Postnatal muscle growth occurs primarily by myofiber hypertrophy, as has been demonstrated in mice (White et al. 2010). Muscle satellite cells (or adult myoblasts) reside between the basal lamina and myofiber membrane (Yin et al. 2013). During late fetal and early postnatal life, myofiber growth is accompanied by proliferation and fusion of satellite cells with existing myofibers (Moss & Leblond 1971; White et al. 2010). During later stages of postnatal life and into adult life, increases in myofiber cross-sectional area occur without significant change in myonuclear number (White et al. 2010). In response to extreme mechanical loading, injury, inflammation, and/or anabolic hormone stimulation, satellite cells serve as bone fide stem cells that can proliferate and differentiate to create new muscle (Ten Broek et al. 2010; Yin et al. 2013). Thus, satellite cells retain plasticity and regenerative capacity during postnatal life. However, the postnatal satellite cell population is vulnerable to fetal undernutrition. When pregnant mice were undernourished during the last week of gestation, pups at 7 weeks postnatal age had reduced muscle mass, a 33% decrease in skeletal muscle precursor cells, and reduced regenerative capacity in response to muscle injury in vivo (Woo et al. 2011). Whether the satellite cell population is permanently compromised during pregnancies complicated by IUGR in humans is an important question when considering long term effects of the compromised intrauterine environment.

Myofiber hypertrophy

Myofiber hypertrophy, or an increase in fiber diameter and length with or without satellite cell fusion, occurs as a net increase in balance between protein synthesis and degradation. When the rate of protein synthesis exceeds that of protein breakdown, the end result is net protein accretion and myofiber hypertrophy. Nutrients and growth factors are primary regulators of net protein balance and myofiber hypertrophy. However, stretching and loading of muscle also regulate muscle mass and protein synthesis, even during fetal life (Racca et al. 2013).

Nutrients and growth factors coordinate net protein accretion in skeletal muscle through the mammalian target of rapamycin (mTOR). Two multiunit complexes constitute mTOR: mTORC1 and mTORC2. The mTORC1 complex senses both intracellular and extracellular cues, such as growth factors, amino acids, energy status, and oxygen availability to either stimulate or inhibit cell growth. Comprehensive reviews of mTOR signaling and its coordination with cell growth in skeletal muscle are available (Dodd & Tee 2012; Goodman et al. 2011; Laplante & Sabatini 2012; Weigl 2012).

Under conditions of nutrient sufficiency (Figure 2), growth factors such as insulin and IGF1 bind their respective tyrosine kinase receptors, which phosphorylate insulin receptor substrate 1 (IRS1). IRS1 activates phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) to stimulate mTORC1 (Takahashi et al. 2002). Amino acids such as leucine can stimulate mTORC1 independently of insulin or IGF1 by binding to leucyl-tRNA synthetase and activating RAG GTPase proteins, thus bringing RHEB (Ras homolog enriched in brain) to the surface of the lysosome (Han et al. 2012). Based on these positive inputs, mTORC1 then activates two major downstream effectors, ribosomal protein S6 kinase (S6K) and 4E-binding protein 1 (4EBP1). mTORC1 phosphorylates the translation initiation repressor 4EBP1, which then releases eukaryotic initiation factor 4E (eIF4E) and enables it to form the translation initiation complex.

Figure 2. Major signaling pathways that regulate myofiber growth.

Under conditions of nutrient sufficiency (depicted in green), nutrients (leucine) and growth factors (insulin, IGF1) coordinate net protein accretion in skeletal muscle through the mammalian target of rapamycin complex 1 (mTORC1). Leucine binds its respective leucyl-tRNA synthetase and activates RAG GTPase proteins, thus bringing RHEB (Ras homolog enriched in brain) to the surface of the lysosome. Insulin/IGF1 bind their respective tyrosine kinase receptors, which phosphorylate insulin receptor substrate 1 (IRS1). IRS1 activates phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) to stimulate mTORC1. mTORC1 activates ribosomal protein S6 kinase (S6K) and phosphorylates the translation initiation repressor 4EBP1, which then releases eukaryotic initiation factor 4E (eIF4E) and enables it to form the translation initiation complex. Under conditions of oxygen and energy restriction (depicted in pink), hypoxia inducible factor (HIF), regulated in development and DNA damage responses 1 (REDD1), and 5′ adenosine monophosphate-activated protein kinase (AMPK) decrease rates of protein synthesis by activating tuberous sclerosis complex (TSC) to suppress mTORC1 activity. When amino acids are not available, uncharged tRNAs activate the protein general control nonrepressed 2 (GCN2) to phosphorylate eIF2A and suppress mRNA translation. Under conditions of cellular stress (depicted in red), forkhead box transcription factor 3 (FOXO3) in its dephosphorylated state is translocated into the nucleus to upregulate ubiquitin mediated proteolysis markers atrogin-1 and MURF1, as well as the autophagy marker LC3. Dashed lines represent lack of signal.

Under conditions of nutrient and energy restriction (Figure 2), rates of protein synthesis are decreased by activation of the tuberous sclerosis complex (TSC) and suppression of mTORC1 activity. Limited oxygen and energy availability to the cell are sensed by three key proteins, all of which can activate TSC: hypoxia inducible factor (HIF), regulated in development and DNA damage responses 1 (REDD1), and 5′ adenosine monophosphate-activated protein kinase (AMPK) (Hardie et al. 2012; Liu et al. 2012). AMPK, which is activated by an increased AMP:ATP ratio, also inhibits eukaryotic elongation factor 2 (eEF2) and peptide chain elongation (Leprivier et al. 2013). When amino acids are not available, uncharged tRNAs activate the protein general control nonrepressed 2 (GCN2) to phosphorylate eIF2A and suppress mRNA translation (Dong et al. 2000; Saad et al. 2013).

In catabolic states such as starvation, cancer, and burn injury, proteolytic pathways are activated in skeletal muscle for the purpose of supplying amino acids to organs such as the heart, liver, and brain (Biolo et al. 1995; Biolo et al. 2002; Kadar et al. 2000). Two proteolytic systems are active within skeletal muscle: ubiquitin-proteosome pathway (mediated by ubiquitin ligases atrogin-1 and muscle RING-finger protein-1 [MURF1]) and the autophagy-lysosome pathway. These pathways can modulate one another and are under coordinated control with protein synthetic pathways to maintain proper cell size (Bonaldo & Sandri 2013). AKT and Forkhead box transcription factors (FOXO) play a crucial role in the regulation of this process (Figure 2). The translocation of FOXO into the nucleus in its dephosphorylated state is required for the upregulation of atrogin-1 and MURF1, as well as for the transcription of autophagy-related genes, including LC3 and BNIP3 (Mammucari et al. 2007; Zhao et al. 2007). With growth factor stimulation, AKT phosphorylates FOXO, promoting its export from the nucleus, which thereby suppresses proteolysis when conditions favor protein synthesis. A recent report also showed that autophagy in skeletal muscle of starved rats is regulated by insulin via mTORC1-mediated inhibition of UNC-51 like kinase (ULK1) (Naito et al. 2013).

Our laboratory and others have shown that the AKT-mTORC1 signaling pathway is active in the skeletal muscle of fetal sheep in response to a variety of anabolic stimuli such as amino acids, insulin, and IGF1 (Anderson et al. 2005; Brown et al. 2009; Shen et al. 2002). However, it is not known whether the human IUGR fetus slows protein synthetic rates in muscle via adaptation to reduced nutrients and growth factors, or whether it activates protein breakdown as a result of cellular stress. Likely, these processes are not mutually exclusive; the fetus might develop a slower growth rate in response to decreased nutrient supply early in the course of placental insufficiency, but then might activate catabolic pathways in the setting of worsening hypoxia and increased catecholamine and cortisol production as nutrient restriction progresses. This is a fundamental area of future investigation, as treatments to improve muscle growth will vary based on whether growth is slowed because of decreased anabolism, or increased catabolism.

Nutrient and growth factor regulation of fetal skeletal muscle growth

Growth factors

Skeletal muscle growth is regulated by several growth factors, including IGF1, insulin, basic fibroblast growth factor (bFGF), and transforming growth factor-B (TGF-B) (Allen & Rankin 1990; Frost & Lang 2012).

Several studies in humans, transgenic animals, and cell lines have demonstrated that IGF1 regulates both myoblast proliferation and myofiber hypertrophy. Igf1 heterozygous knockouts in mice demonstrate reduced muscle mass (Powell-Braxton et al. 1993), whereas homozygous knockouts have severe muscle hypoplasia from both decreased myocyte number and myofiber cross-sectional area (Liu et al. 1993; Mavalli et al. 2010). Similarly, mutations in the IGF1 and IGF1 receptor genes in humans cause both intrauterine and postnatal growth restriction (Abuzzahab et al. 2003; Woods et al. 1996). Conversely, excessive IGF1 results in increased muscle mass and hypertrophy in postnatal life, as demonstrated by overexpressing Igf1 in a transgenic mouse or by direct intravenous infusion into rats (Adams & McCue 1998; Coleman et al. 1995). In subconfluent myoblasts in vitro, IGF1 acts initially to promote proliferation (Rosenthal & Cheng 1995). In differentiated myotubes, IGFI promotes protein synthesis (Coleman et al. 1995; Harper et al. 1987).

Insulin also functions as a potent fetal skeletal muscle growth factor. Pancreatectomy in fetal sheep results in growth restricted fetuses with decreased upper and lower extremity limb length (Fowden et al. 1989). These experiments confirm lack of insulin as the cause of growth restriction in cases of pancreatic agenesis in humans (Lemons et al. 1979). Conversely, studies in catheterized fetal sheep have shown that both insulin and IGF1 infusions promote whole fetal and muscle-specific protein synthesis (Boyle et al. 1998; Milley 1994; Shen et al. 2003). Furthermore, insulin infusion into the neonatal piglet stimulates skeletal muscle protein synthesis (Davis et al. 2002). High doses of insulin (at least 0.1 μM) increase protein synthesis and suppress protein breakdown in both primary cultured and immortalized myotubes in vitro (Cassar-Malek et al. 1999; Gulve & Dice 1989; Shen et al. 2005), including myotubes harvested and cultured directly from fetal sheep (Harper et al. 1987).

Other growth factors, including basic fibroblast growth factor (bFGF) and transforming growth factor-B (TGF-B), upregulate cyclin D1 levels in myoblasts that then stimulate proliferation and myogenesis (Rao & Kohtz 1995). Myostatin is a member of the TGF-B family of regulators and a potent inhibitor of myogenesis. Double-muscled cattle carry an inactivating mutation in the myostatin gene and have 20% more muscle than normal-muscled cattle (Grobet et al. 1997). Myostatin null mice have 2–3 times greater muscle mass than wild types (McPherron et al. 1997). These animals also show less insulin resistance and fat deposition, demonstrating the important role of muscle in regulating adipose tissue balance and insulin sensitivity (Guo et al. 2012). Follistatin is an inhibitor of myostatin and works through activation of the IGF1 receptor. Mice over-expressing follistatin have a 3-fold greater increase in myofiber diameter than mice over-expressing follistatin with a non-functional IGF1 receptor (Kalista et al. 2012).

Amino acids

Amino acids are essential for muscle protein synthesis. In addition to forming the building blocks of protein, amino acids have important regulatory effects on mTORC1 activation and muscle protein synthesis. Amino acids increase skeletal muscle protein synthesis in adults, both under normal postprandial conditions as well as during catabolic states such as after trauma or sepsis (Drummond & Rasmussen 2008; Wolfe 2005). Increasing amino acid delivery positively affects net protein balance in infants born preterm or at term (Poindexter et al. 1997; Reynolds et al. 2008; Thureen et al. 2003). In the neonatal piglet, mixed amino acid or leucine supplementation increases muscle protein synthesis though mTORC1 dependent pathways (O’Connor et al. 2003b; O’Connor et al. 2003a; Suryawan et al. 2008; Suryawan et al. 2012).

Studies that address the effects of amino acids on skeletal muscle growth during fetal life are more limited. One study by de Boo et al. showed that a mixed amino acid infusion administered for 4 hours increased whole fetal protein accretion in fetal sheep studied during late gestation (de Boo et al. 2005). Our laboratory showed that a mixed amino acid infusion activated signaling through mTORC1 within the skeletal muscle of fetal sheep, but only when there was a concurrent rise in insulin concentrations (Brown et al. 2009). Further studies to determine the interactive roles of amino acids and growth factors in regulating fetal skeletal muscle development are needed.

Reduced fetal skeletal muscle growth: Insights from models of fetal undernutrition

Effects of fetal undernutrition on myoblast proliferation and myofiber number

Studies in mice and pigs have shown that myofiber number is set around the time of birth (Rowe & Goldspink 1969; White et al. 2010). Similar growth patterns have been observed in humans, as DNA content in the gastrocnemius muscle increases exponentially between weeks 15 and 25 of gestation and plateaus by term (Widdowson et al. 1972). Thus, conditions which deprive the fetus of nutrients and growth factors during myofiber formation can have a lasting impact on myofiber number.

Studies of maternal nutrient restriction during pregnancy in a variety of animal models show dramatic effects of reduced nutrient supply on the establishment of fetal myofiber number, with secondary myofibers being more vulnerable to restricted fetal nutrient supply than primary myofibers in sheep and pigs (Dwyer et al. 1994; Ward & Stickland 1991; Zhu et al. 2004). In rats receiving 30% of an ad libitum diet during gestation, secondary myofiber number within the fetal soleus and lumbrical muscles was decreased by 30% (Wilson et al. 1988). When pregnant sheep were diet restricted by 50% during early and mid gestation, which is coincident with the maximal period of myoblast proliferation (Fahey et al. 2005a), fetal myofiber number was decreased at mid gestation and this effect persisted when tested at 8 months of age (Zhu et al. 2004; Zhu et al. 2006). Guinea piglets born to mothers who had a 40% reduction in feed intake during the entirety of gestation had 25% less myofibers within glycolytic muscle types (Dwyer & Stickland 1992a). Similar reductions in myofiber number were observed when the duration of maternal diet restriction in guinea pigs was shortened, but still overlapped with the peak period of secondary myofiber formation (Dwyer et al. 1995). Runted piglets, or those piglets that weigh 60% of the mean litter weight, have persistent deficits in myofiber number and muscle mass as adults (Powell & Aberle 1980). They also became fatter and less insulin sensitive (Poore & Fowden 2004). Myofiber number in pigs positively correlated with average daily weight into early adulthood (70 to 130 days postnatal age), providing evidence that myofiber number influences postnatal muscle growth trajectory (Dwyer et al. 1993).

The mechanisms for restriction of muscle fiber number from fetal undernutrition are not understood, though there is evidence for the suppression of fetal myoblast cell cycle activity. In a model of placental insufficiency resulting from sheep bred to produce litters of multiple lambs of variable birthweight (from 2 to 5 kg), low birth weight lambs demonstrated less muscle DNA and decreased percentage of nuclei entering the S-phase of the cell cycle, indicative of fewer myonuclei per myofiber and decreased cell cycle activity compared to larger lambs (Greenwood et al. 1999; Greenwood et al. 2000). This is not unexpected, given that maternal nutrient restriction results in reductions in circulating fetal plasma IGF1 concentrations in rats (Straus et al. 1991), guinea pigs (Dwyer & Stickland 1992b), and sheep (Costello et al. 2008; Lee et al. 1997; Osgerby et al. 2002; Ward et al. 2008). However, it should be noted that total myofiber number was not different between small and large lambs in this study (Greenwood et al. 1999). There remain many unanswered questions about the interaction between chronic nutrient restriction and subsequent decreases in fetal growth factors on myoblast proliferation and the capacity for compensatory muscle growth.

Effects of fetal undernutrition on myofiber hypertrophy

Studies that extend maternal dietary restriction into late gestation to evaluate the effects on fetal myofiber hypertrophy are more limited. When pregnant sheep received 70% of a control diet beginning at day 26 of gestation, the fetal semitendinosus muscle weight was decreased by 20% on day 135 of gestation (Osgerby et al. 2002). When pregnant sheep were diet restricted by 50% during mid to late gestation (days 85–115 of a 145 day gestation), individual muscle weights of offspring at 2 weeks of life were decreased by 15–20% compared to controls (Fahey et al. 2005b). A shorter but more severe dietary restriction allowing 30% of ad libitum intakes for 7 days during late gestation in sheep resulted in decreased muscle weights compared to controls (Greenwood et al. 1999). In a model of placental insufficiency in sheep bred to produce multiple lambs per litter, the trajectory of muscle growth was decreased in small, runted lambs compared to large lambs, as measured by weight over time between 85 and 130 days gestation. The muscle protein to DNA ratio on day 130 of gestation in small lambs was also decreased (Greenwood et al. 1999).

Normalizing dietary intake after early maternal nutrient restriction (days 30–70) in pregnant sheep, however, resulted in compensatory myofiber hypertrophy as evidenced by fewer myofibers but larger fiber cross-sectional area compared to controls (Fahey et al. 2005b; Zhu et al. 2006). The phenomenon of postnatal catch-up growth after fetal growth restriction has been well described in a variety of species, including in humans (Jimenez-Chillaron & Patti 2007; Tudehope et al. 2013). However, the extent to which muscle growth is able to fully compensate during postnatal life after nutrient restriction in utero is not entirely clear. Longer term follow-up of sheep into adolescence and adulthood after mid-gestation nutrient restriction shows accelerated fat deposition at the expense of lean mass growth (De Blasio et al. 2007; Ford et al. 2007; Louey et al. 2005). In the undernourished fetal rat, compensatory myofiber hypertrophy in the diaphragm occurs through postnatal day 21, though adult myofiber cross-sectional area is ultimately smaller (Prakash et al. 1993). These results, taken together, indicate decreased protein accretion and fetal myofiber hypertrophy as a result of fetal undernutrition, with partial, but not complete capacity for compensatory muscle growth during postnatal life.

Effects of fetal undernutrition on myofiber maturation

A complex schema exists for defining muscle fiber types in mammals based on a variety of features, including the predominant type of myosin heavy chain (MHC) expressed (type I, type IIa, type IIx and type IIb), contractile machinery and speed of contraction, distribution of oxidative and glycolytic enzymes, and mitochondrial density (Pette & Staron 2001; Schiaffino & Reggiani 2011). Fiber type composition of skeletal muscle can undergo changes based on environmental influences (Simoneau & Bouchard 1995), as myofibers can be affected by neuromuscular activity, exercise training, mechanical loading, and aging (Pette & Staron 2001). Additionally, maternal dietary restriction results in fiber type transitions in offspring, generally favoring increased type I fiber expression. For example, studies that have evaluated fiber type shifts in the late fetal or early neonatal period after maternal diet restriction in both sheep and rats found either a relative increase in type I oxidative fibers or a relative decrease in glycolytic type II fibers (Costello et al. 2008; Fahey et al. 2005b; Prakash et al. 1993). Runted piglets have more type I fibers than their appropriately grown littermates (Bauer et al. 2006; Wank et al. 2000). These findings are not uniformly consistent, as pups of undernourished pregnant mice demonstrated a shift from type I to type IIa and IIb fibers at 7 weeks of age (Woo et al. 2011). However, any adaptation in MHC expression that develops in response to fetal undernutrition does not appear to persist and, in fact, may shift during the lifespan. For example, when fiber type assessments were extended into the adolescent period in sheep, glycolytic type II fibers predominated (Daniel et al. 2007; Zhu et al. 2006). Low birth weight humans evaluated at 19 years of age had a decreased proportion of type IIa fibers compared to a control group with normal birth weights (Jensen et al. 2007). Further work in this area is required to determine the long term significance of fiber type shifts as a result of fetal undernutrition.

Skeletal muscle growth is particularly vulnerable in the fetus exposed to fetal undernutrition from placental insufficiency

Placental insufficiency is a condition whereby a poorly functioning placenta restricts nutrient supply to the fetus, preventing normal fetal growth (Figueras & Gardosi 2011). If placental insufficiency begins early in pregnancy, the entire process of myogenesis is at high risk for attenuation and/or disruption. Deficient skeletal muscle mass is a characteristic of the human fetus affected by IUGR (Beltrand et al. 2008; Larciprete et al. 2005; Padoan et al. 2004; Yau & Chang 1993). Placental insufficiency commonly begins early in pregnancy so that nutrient restriction to the fetus is chronic, progressive, and severe, often leading to preterm delivery when fetal well being is severely compromised (De Jesus et al. 2013). Fetal skeletal muscle growth is particularly vulnerable during placental insufficiency, because blood, oxygen, and nutrients are preferentially shunted to vital organs (Tchirikov et al. 1998; Yajnik 2004b). Consequently, at least in animal models of placental insufficiency, skeletal muscle weight is disproportionately reduced compared to body weight (Bauer et al. 2003; Desai et al. 1996; Greenwood et al. 2000).

Placental size and function, fetal growth, and fetal nutrient and growth factor availability have been well characterized in a sheep model of chronic and progressive placental insufficiency, which was developed to mimic a natural condition of placental and fetal growth restriction that occurs in sheep that carry their pregnancies in the hot summer months (PI-IUGR) (Bell et al. 1987) (Figure 3). The PI-IUGR model accurately reflects the characteristics that occur during human pregnancies affected by conditions that produce placental insufficiency and IUGR (Barry et al. 2008). Similar to maternal dietary restriction during pregnancy, fetal plasma insulin and IGF1 concentrations in the PI-IUGR fetus are 50% of normal values as early as 70% of the total length of pregnancy (Figure 3) (de Vrijer et al. 2006; Macko et al. 2013; Thorn et al. 2009). Other sheep models of chronic placental insufficiency produced by pre-pregnancy reduction of placental attachment sites or uteroplacental embolization also demonstrate decreased fetal insulin and IGF-I concentrations (Eremia et al. 2007; Owens et al. 1994), as do human IUGR fetuses (Iniguez et al. 2006; Lassarre et al. 1991; Leger et al. 1996; Nicolini et al. 1990). Leucine flux from mother to fetus is decreased by 90% of gestation in PI-IUGR sheep (Figure 3) (Brown et al. 2012; Regnault et al. 2013). Similarly, amino acid transport across the placenta is impaired in the third trimester of human IUGR pregnancy (Cetin et al. 1992; Paolini et al. 2001).

Figure 3. Progressive physiological changes in a model of placental insufficiency-induced IUGR (PI-IUGR) in relation to fetal myogenesis.

Pregnant sheep were housed in an environmental chamber with elevated ambient temperatures to restrict placental growth (PI-IUGR) beginning on day 40 for a maximum of 80 days during their 145 day gestation. Placental weights, fetal weights, and fetal lengths were compared between PI-IUGR sheep and sheep housed in thermo neutral conditions (controls) during early (day 55), mid (days 90 and 103), and late gestation (day 135). At mid and late gestation time points, surgery was performed in PI-IUGR and control sheep for placement of fetal umbilical and arterial sampling catheters to compare umbilical and fetal oxygenation, circulating fetal growth factors, and fetal glucose and amino acid (AA) concentrations. Arrows represent changes in parameters from PI-IUGR animals compared to controls; ↔ represents no change between PI-IUGR and control). A schematic showing the progression of fetal myogenesis during ovine gestation is shown at the top of the figure (Fahey et al. 2005a). (Arroyo et al. 2008; Arroyo et al. 2009; Brown et al. 2012; de Vrijer et al. 2006; Limesand et al. 2006; Macko et al. 2013; Regnault et al. 1999; Regnault et al. 2002; Regnault et al. 2007; Thorn et al. 2009; Ziebell et al. 2007).

Two of the distinguishing features that separate models of placental insufficiency from maternal dietary restriction are fetal hypoxemia and increased production of counter regulatory hormones. In the PI-IUGR sheep model, norepinephrine and cortisol are increased (Leos et al. 2010), and fetuses are severely hypoxemic (Leos et al. 2010; Regnault et al. 2007), consistent with evidence from severe IUGR in human pregnancies (Pardi et al. 1993). Oxygen sensors such as HIF and AMPK might play a role in regulating fetal muscle growth under conditions of placental insufficiency, as has been shown in placentas collected from human IUGR pregnancies (Cindrova-Davies et al. 2013).

Relationships between low birth weight and skeletal muscle mass in humans

Epidemiological evidence supports that reduced muscle mass in adulthood is, in part, due to environmental influences from placental insufficiency during fetal life. Such evidence has been generated by studies of infants with a birth weight <2500 grams at term gestation or who were documented as SGA. Two important studies using epidemiologic data from the third National Health and Nutrition Examination Survey (NHANES) cohort have identified low birth weight as an early predictor of reduced muscle growth in childhood. Hediger et al. first demonstrated a overall deficit in the amount of muscularity estimated by anthropometric measurements of upper arm circumference in SGA infants compared to appropriately sized for gestational age (AGA) infants up to 3 years of age (Hediger et al. 1998). Baker et al. further showed that while head circumference growth is maintained between 2 months and 8 years of age in SGA children compared to AGA children, muscle growth remains stunted (Baker et al. 2010). When body composition was measured by dual-energy X-ray absorptiometry (DXA) in adolescents, low birth weight was associated with decreased lean body mass independent of age, sex, pubertal stage, physical activity, and height (Singhal et al. 2003). Even as early as during the immediate neonatal period, lean body mass increased between 36 and 41 weeks in AGA neonates, but not in SGA neonates (Lapillonne et al. 1997).

The association between birthweight and muscle mass extends into adulthood. Several large population-based studies have found that low birth weight predicts lower adult muscle mass (measured by DXA) in men and women during young adulthood (Kahn et al. 2000), middle-ages (Kensara et al. 2005; Sayer et al. 2004; Yliharsila et al. 2007), and late adulthood (Gale et al. 2001). Monozygotic twin studies in both men and women found that the lower birth weight twin had less lean body mass and more subcutaneous fat than the heavier twin, supporting the consistent observation that the intrauterine environment, independent of genetic influences, predicts lean mass in adulthood (Loos et al. 2001; Loos et al. 2002). Even as late as the seventh decade of life, 25% of the variation in lean body mass was explained by birthweight (Gale et al. 2001). As obesity is generally associated with excess lean as well as fat mass, obesity found following low birth weight presents a unique mechanism that may not necessarily be due to increased energy intake that characterizes most cases of adult obesity.

Effects of IUGR on skeletal muscle growth, function, and long term metabolic health

Populations around the world are faced with epidemic increases in the incidence of obesity and diabetes. In the United States, more than one-third of adults and 17% of children and adolescents are obese (Ogden et al. 2012). Projections show that one in three Americans will develop diabetes by 2050 (Boyle et al. 2010). Compelling associations link low birth weight and decreased muscle mass to development of the metabolic syndrome and type 2 diabetes (Atlantis et al. 2009; Whincup et al. 2008), and increased risk for cardiovascular events later in life (Basaria & Bhasin 2012). Thus, it is imperative that all major factors that contribute to diabetes and obesity risk be investigated, including the role of disproportionately reduced muscle mass.

The amount of muscle mass has major impact on fat deposition, insulin sensitivity, strength, and locomotion. Interactions between muscle mass, fat mass, and fat distribution (visceral versus subcutaneous) have been demonstrated, supporting the concept that reduced capacity for muscle growth favors accelerates visceral fat deposition and obesity (Kensara et al. 2005; Yajnik 2004a). For example, intrauterine growth patterns contribute to the “thin-fat” phenotype of Indian diabetic patients. This phenotype is characterized by less muscle mass but greater body fat and central obesity (Yajnik 2004a). Infants born in India compared to infants born in the United Kingdom are lighter, shorter, and thinner, but have similar subscapular skin fold thicknesses, indicating smaller muscle mass but preserved fat mass (Yajnik et al. 2003). There also are direct links between low birth weight and insulin resistance, with evidence that insulin signaling pathways within human skeletal muscle have been disrupted as a result of an IUGR pregnancy (Jensen et al. 2008; Ozanne et al. 2005). The effects of low birth weight also have been shown to affect muscle strength. Grip strength is a measure of muscle strength and predictor of quality of life, morbidity, and mortality (Rantanen et al. 2000). Epidemiological studies have shown a positive correlation between birth weight and grip strength in adulthood (Inskip et al. 2007), primarily through the association of lean mass composition (Ortega et al. 2009; Yliharsila et al. 2007). One study comprehensively evaluated grip strength, maximal isometric voluntary contraction (MVC) of the quadriceps femoris, and muscle fatigue before and after an 8 week training program in women who had a ponderal index (PI, a marker of thinness) recorded at the time of birth. Results showed that women born with a PI <10%, reflecting IUGR during the pregnancy, had 11% lower grip strength, 9–24% lower MCV, and a higher rate of muscle fatigue both pre- and post-training compared to women who had a normal PI at the time of birth (Brutsaert et al. 2011). In summary, these findings implicate structural and functional deficits in muscle as major contributors to increased risk for later life development of metabolic and cardiovascular disease in those born with low birth weight from IUGR (Barker et al. 2005; Barker 2006; Warner & Ozanne 2010).

Potential for improving fetal muscle growth in IUGR: Future research directions

Clinical attempts at increasing maternal nutrition during human pregnancy to improve fetal growth have been largely unsuccessful. Nutritional supplements with balanced energy and protein given to pregnant mothers at high risk of having an IUGR fetus increased birthweight, though whether this promoted fat deposition versus lean mass was not determined (Rush et al. 1980). Current postnatal nutritional interventions for the IUGR neonate designed to increase body weight favor fat deposition over muscle growth. The problem is magnified when preterm birth is considered. Almost all extremely low birth weight preterm infants experience postnatal growth restriction in the Neonatal Intensive Care Unit, even when they are born AGA (Dusick et al. 2003; Ehrenkranz et al. 2006). Preterm birth alone can disrupt normal skeletal muscle development, as preterm infants who were not IUGR have decreased lean mass and increased fat mass at the time of discharge compared to normal full term infants (Johnson et al. 2012).

Promisingly, however, specific nutrient and growth factor supplementation studies in sheep models of placental insufficiency have yielded encouraging results. Chronic, low dose IGF1 infusions into the IUGR sheep fetus, either by direct fetal intravenous infusion or by intra-amniotic supplements, improved fetal organ growth (Eremia et al. 2007). Additionally, short term amino acid infusion given directly to the PI-IUGR sheep fetus increased protein accretion rates by suppressing protein breakdown rates, although whether this affected skeletal muscle growth specifically has yet to be determined (Brown et al. 2012). The use of large animal models of chronic placental insufficiency, such as the PI-IUGR model, allows for the manipulation of fetal substrate and hormone concentrations during critical windows in muscle development. This strategy will provide critical information about the optimal timing and type of supplementation to improve muscle growth in the IUGR fetus, as well as determine cellular deficits that might explain how myoblast proliferation and myofiber hypertrophy become permanently impaired (Figure 4). Targeting strategies to promote fetal muscle development could improve the potential for postnatal muscle growth, thereby minimizing the risk for developing long term insulin resistance and chronic metabolic disease in the IUGR individual.

Figure 4. Proposed mechanisms for reduced skeletal muscle growth during conditions of fetal undernutrition.

During conditions of fetal undernutrition (either from maternal dietary restriction or placental insufficiency), nutrient delivery to the fetus (amino acids, glucose, and oxygen) and circulating fetal growth factors (insulin, IGF1) are restricted. The combination of decreased growth factors and nutrients leads to reduced rates of myoblast proliferation and myofiber hypertrophy, ultimately producing reductions in fetal skeletal muscle mass. The dashed line demonstrates pathways yet to be determined for how decreased fetal muscle mass contributes to increased risk of adult metabolic diseases such as obesity, coronary heart disease, and type 2 diabetes.