Keywords

9.1 Introduction

Long-term maternal nutrient restriction (NR) during pregnancy impairs fetal growth, leading to intrauterine growth restriction (IUGR) or small for gestational age (SGA) offspring. In human medicine, intrauterine growth restriction (IUGR) has been defined as the offspring placed below the tenth percentile of fetal weight distribution at birth, and is typically associated to asymmetric growth (Goldenberg and Cliver 1997). In livestock species, maternal nutrient restriction is also a prevalent cause for IUGR, which have been defined as an impairment in gestational development of a fetus or its parts (Wu et al. 2006). A similar concept is small for gestational age (SGA) offspring, which is a broader classification and refers to fetuses that are smaller than expected for the species at a given gestational age (Goldenberg and Cliver 1997). IUGR or SGA offspring present a higher perinatal mortality and increased risk of metabolic syndrome during postnatal life (Barker et al. 1989).

Skeletal muscle, which represents about 40−45% of body weight in the young and adult, respectively (Wu 2018), is among the most sensitive tissues to maternal NR (Desai et al. 1996), and it plays an essential role in metabolic dysregulation due to its prominence in glucose and oxidative metabolism (Brown 2014). In addition, skeletal muscle is the major site for initiating the catabolism of branched-chain amino acids to synthesize glutamate, alanine and glutamine in mammals (Hou and Wu 2018; Wu 2013). Both alanine and glutamine participate in the inter-organ metabolism of nitrogen and carbons. Particularly, alanine is a major glucogenic precursor in the liver, whereas glutamine is used by the small intestine of many mammals (including sheep, swine and humans) to synthesize citrulline (Wu and Morris Jr. 1998). The latter is either converted locally into arginine in enterocytes or taken up by extra-intestinal tissues and cells (e.g., kidneys, endothelial cells, and macrophages) to generate arginine, the nitrogenous precursor of nitric oxide (a major vasodilator, a neurotransmitter, a signaling molecule, and a killer of pathogens), creatine (crucial for energy metabolism), and polyamines (essential for DNA and protein syntheses) in animals (Dai et al. 2013; Wang et al. 2014; Wu et al. 2016, 2018). Therefore, skeletal muscle plays an important role in both growth and health of individuals.

Using animal models to understand the effects of maternal undernutrition in skeletal muscle growth and metabolism provides valuable information for translational research as well as agricultural performance. This chapter will discuss insights from the sheep, pig, and rodent models regarding the effect of maternal nutrient restriction (NR) on fetal skeletal muscle and its potential implications for postnatal health.

9.2 Maternal Undernutrition and SGA Offspring

Worldwide estimations indicate that around 821 million people are undernourished. Famine and undernutrition cases have continuously increased since 2014 and are a public health concern primarily in developing and low-income countries (FAO 2017) with the majority of cases occurring in Africa and Asia, followed by Latin America and The Caribbean (FAO 2017). The consequences of undernutrition are ample and include a higher predisposition for disease, and in extreme situations, death. This scenario becomes particularly challenging during pregnancy, when the female experiences a physiological increase in nutrient requirements to support herself as well as the needs of her developing fetus and placenta. Maternal undernutrition during pregnancy results in SGA offspring, with more than 20 million cases reported annually (UNICEF 2004).

Individuals born as IUGR or SGA are more prone to suffer perinatal mortality and experience increased risk for hypertension (Gennser et al. 1988), obesity (Fernandez-Twinn and Ozanne 2006), type 2 diabetes (Rich-Edwards et al. 1999), heart disease (Barker et al. 1989) and metabolic syndrome (McMillen and Robinson 2005). Epidemiological studies in the field of fetal programming have suggested the thrifty phenotype hypothesis (Hales and Barker 1992), which suggests that early life nutrient deficiency leads to a programming effect that would support immediate survival. However, in a postnatal scenario of normal or excessive nutrient availability, these adaptations would lead to type 2 diabetes, obesity, and other dysregulations associated with metabolic syndrome (Gluckman et al. 2005; Symonds et al. 2009; Hyatt et al. 2011).

The use of animal models for the study of maternal NR on programming of fetal growth and metabolism has provided supporting evidence for the initial epidemiological studies. A decrease in fetal weight has been a seminal finding of these studies (Osgerby et al. 2002; Kwon et al. 2004; Gao et al. 2008; Lassala et al. 2010; Satterfield et al. 2010). Results from our group show that impairment of fetal growth is correlated to reduced concentration of polyamines and amino acids in maternal and fetal plasma, as well as fetal allantoic and amniotic fluids (Kwon et al. 2004). Lower plasma levels of insulin like growth factor 1 (IGF1) and insulin have also been reported in fetal plasma as a consequence of maternal NR (Osgerby et al. 2002), and both factors play an essential role in stimulation of fetal growth (Fowden et al. 1989; Baker et al. 1993).

Growth and metabolism in several fetal organs are affected by maternal NR (Osgerby et al. 2002; Vonnahme et al. 2003; Zhu et al. 2004, 2006; Costello et al. 2008; George et al. 2012; Lloyd et al. 2012; Satterfield et al. 2013; Shukla et al. 2014). Among them, skeletal muscle is particularly susceptible to maternal NR during fetal development because of nutrient prioritization to vital organs such as the brain (Desai et al. 1996)

9.3 Overview of Fetal Skeletal Muscle Development

Skeletal muscle development and growth during the fetal stage are accomplished by both cellular hyperplasia and fusion to originate myofibers (myogenesis), and hypertrophy, which continues postnatally. Myogenesis can be divided into primary and secondary myogenesis. During primary myogenesis, myoblasts fuse to form a primary myotube which will become a primary myofiber. A small percentage of myofibers are formed in this process, which starts during the first third of pregnancy (Maltin 2008). Secondary myogenesis occurs during the second third of pregnancy and accounts for the majority of myofiber formation (Maltin 2008).

Once the majority of myofibers are formed, fetal muscle growth continues through hypertrophy which starts around the second half of pregnancy and remains as an active process postnatally. In the sheep, it has been shown that myofiber area begins to increase around gestational day (GD) 85 (term ~147 days), which was caused by the addition of myonuclei between GD 85 and 100, while myoblast proliferation was completed by GD 100, and was followed by an increase in myofiber size, likely due to intracellular protein deposition (Wei et al. 2014). Since hypertrophy continues during postnatal life, prenatal alterations in myofiber size due to maternal NR have the potential to be compensated postnatally. However, persistent reductions in muscle mass and a tendency to increased adipose tissue have been demonstrated in adult sheep after prenatal NR (Ford et al. 2007), and similarly, decreased muscle mass persists until adulthood in low-birth-weight humans (Kensara et al. 2005).

Protein deposition is essential for muscle growth and hypertrophy (Yao et al. 2008) and is dependent upon an increase in the net balance of protein synthesis and protein degradation (Brown 2014). A central regulator of protein deposition is mechanistic target of rapamycin (MTOR), particularly MTOR complex 1, which is associated with regulatory associated protein of MTOR complex 1 (RPTOR) (Kim et al. 2002) and is activated by phosphorylation of its serine 2448 residue. Insulin, IGF1, AKT1, and amino acids (e.g., leucine, arginine, glutamine and glycine) have a stimulatory effect on MTOR complex 1 activity (Sun et al. 2016; Yao et al. 2008; Yoon 2017). In contrast, the activity of MTOR complex 1 is inhibited by glucocorticoids (Shimizu et al. 2011), protein kinase AMP-activated catalytic subunit alpha 2AMP-dependent kinase (PRKAA2) (aka AMPK), and myostatin (Rodriguez et al. 2014) (Fig. 9.1). As activation of MTOR is nutrient-dependent, severe prenatal undernutrition has the potential to decrease protein deposition in the fetus, and produce a reduction in lean mass content in the body.

Fig. 9.1
figure 1

Major regulatory pathways in skeletal muscle protein deposition. Protein deposition depends on the rate of protein synthesis and degradation. Pathways that stimulate protein synthesis in skeletal muscle are shown in blue. Amino acids (primarily the branched-chain amino acid leucine, and arginine) induce translocation of MTOR complex 1 to the lysosome, where the complex is activated by RHEB. TSC has inhibitory activity over RHEB. Insulin and IGF1 activate AKT1 which activates RHEB by inducing its separation from the inhibitory factor TSC. Pathways that inhibit protein synthesis or stimulate protein degradation are shown in orange. Myostatin inhibits MTOR through inactivation of AKT1. Glucocorticoids bind to their receptor (NR3C1) to induce expression of KLF15. It is suggested that this decreases activation of MTOR through increased breakdown of branched-chain amino acids (BCAA) via BCAT2. KFL15 upregulation would also increase protein degradation through upregulation of the ubiquitin ligases FBXO32 and TRIM63. PRKAA2 (aka AMPK) also has an inhibitory effect on MTOR when AMP is increased in the cell (not shown) (Based on the data from Shimizu et al. 2011; Yoon 2017)

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9.4 Maternal Nutrient Restriction and Developmental Programming of Skeletal Muscle

9.4.1 Role of Skeletal Muscle in Whole-Body Metabolism

Skeletal muscle plays an essential role in locomotion and structural support, but it is also involved in several functions that regulate amino acid and energy metabolism. This tissue is highly abundant in free glutamine, glutamate, alanine, glycine, and taurine (Wu and Thompson 1990; Wu et al. 1991; Flynn and Wu 1996). Examples are, the capacity for oxidation of fatty acids, glucose, and some amino acids; storage of glycogen (Argilés et al. 2016), and support of gluconeogenesis in liver and kidney through the release of the amino acids, alanine and glutamine (Marliss et al. 1971; Garber and Missouri 1976). Skeletal muscle is essential in the regulation of glucose metabolism because about 80% of insulin-induced glucose uptake occurs in this tissue (Ferrannini et al. 1985; DeFronzo and Tripathy 2009). Skeletal muscle represents 45% of body mass in adult organisms (Janssen et al. 2000; Wu 2018), so any alteration in muscle mass or metabolism will significantly impact whole-body metabolism (Brown 2014). As example, it has been shown that insulin resistance at the skeletal muscle level is one of the primary metabolic alterations leading to type 2 diabetes in humans (DeFronzo and Tripathy 2009).

Solute carrier family 2 member 4 (SLC2A4) is the major glucose transporter in skeletal muscle, and its action is insulin-dependent. The abundance and activity of this transporter are essential for insulin-mediated glucose uptake (Scheepers et al. 2004). SLC2A4 proteins are stored in cytoplasmic vesicles and translocated to the plasma membrane by activation of the PI3K/AKT1 pathway after binding of insulin to insulin receptor (INSR) (Fig. 9.2) (Kohn et al. 1996; Taniguchi et al. 2006). IGF1 can also trigger the activation of this pathway by binding to its receptor or to INSR (Mora et al. 1995; Belfiore et al. 2009). Muscle contraction can also stimulate SLC2A4 translocation, which becomes relevant during postnatal life (Gao et al. 1994). Upregulation of SLC2A4 in skeletal muscle begins late in fetal life and continues postnatally when this glucose transporter reaches maximum functionality (Stuart et al. 2000).

Fig. 9.2
figure 2

Insulin-mediated SLC2A4 translocation. Around 80% of insulin-mediated glucose uptake occurs in skeletal muscle. Insulin binds to its receptor (INSR) to activate the downstream target AKT1 which will activate TBC1D4 to induce SLC2A4 translocation from cytoplasmic vesicles to the sarcolemma. Because of a high level of homology, IGF1 can also activate this pathway through its receptor (IGF1R) or binding to INSR (not shown)

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Another factor that influences the metabolic characteristics of skeletal muscle is myofiber type composition, which impacts glucose metabolism and fatty acid oxidation (Mortensen et al. 2010). Type I myofibers are primarily oxidative and more sensitive to insulin than type II myofibers. Thus, its proportion shows a positive correlation with fatty acids and glucose oxidation, and with insulin-mediated glucose transport and whole-body insulin sensitivity (Lillioja et al. 1987; Fisher et al. 2017). Several studies have demonstrated that maternal NR impairs skeletal muscle growth, insulin sensitivity, and energetic metabolism (Table 9.1), which in addition to other systemic alterations, leads to a phenotype of increased risk for metabolic syndrome.

Table 9.1 Summary of selected studies indicating the effect of maternal NR on fetal skeletal muscle features in different animal models
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9.4.2 Prenatal Programming of Skeletal Muscle and Consequences for Postnatal Health

9.4.2.1 Ovine Model

Decreased number of secondary myofibers has been found in longissimus dorsi at GD 78 after 50% maternal NR between GD 28 and 78. Similarly, a peri-conception treatment of 50% NR applied from 18 days before ovulation to 6 days after ovulation found a tendency for decreased myofiber number in sheep semitendinosus muscle at GD 75 (Quigley et al. 2005). The study of Zhu et al. (2004) also found a decrease in myofiber area in longissimus dorsi of fetuses from NR dams which was associated with a reduction in MTOR and RPS6KB1 protein phosphorylation. Another model of 50% NR from GD 85 to 115 found a decrease in weight of longissimus dorsi in 14-day-old lambs (Fahey et al. 2005). Decreased muscle mass can be partially compensated postnatally as hypertrophy continues as an active process. However, having a lower number of myofibers limits a complete compensation because myofiber formation is not an active process under normal postnatal conditions.

Impaired fetal growth after prenatal nutrient restriction is usually followed by compensatory growth during postnatal life (De Blasio et al. 2007). However, in the long term, this compensatory growth will favor adipose tissue deposition instead of muscle growth. For example, 280-day-old lambs born to dams that were subjected to 50% NR from GD 28 to 78 were heavier than controls, had increased renal and pelvic adipose tissue, and a tendency for decreased weight in longissimus dorsi and semitendinosus muscles. This study also found evidence of hyperglycemia and altered insulin secretion after a glucose tolerance test (Ford et al. 2007). Accordingly, 1-year-old offspring born to sheep under 50% NR from GD 110 to term, showed evidence of glucose intolerance as indicated by increased areas under the curve for glucose and insulin (Gardner et al. 2005).

Indicators of alterations in glucose and insulin metabolism have also been found in skeletal muscle at the fetal stage after maternal nutrient restriction. A sheep model of 50% NR from GD 104 to 127 produced upregulation of SLC2A4, INSR, and IGF1 mRNA in fetal triceps brachii muscle at GD127 (Costello et al. 2008). These results suggest a metabolic programming effect that could be partly responsible for the compensatory growth that SGA animals experience early in postnatal life. The authors also suggested that an initial upregulation in insulin receptor may play a role in the development of metabolic diseases later in postnatal life (Costello et al. 2008).

Maternal nutrient restriction has also been shown to alter myofiber type composition in skeletal muscle. A 50% NR from GD 28 to GD 78 has been associated with increased content of type IIb myofibers in longissimus dorsi of 8-month-old lambs (Zhu et al. 2006). This study also found decreased activity of the enzyme carnitine palmitoyltransferase-1, which is involved in fatty acid oxidation, and accordingly, intramuscular triglyceride (IMTG) content was increased. These findings suggest a metabolic programming in skeletal muscle that would impair oxidative capacity and insulin sensitivity, as type II myofibers are primarily glycolytic and less insulin sensitive than type I myofibers (He et al. 2001). Accumulation of IMTG has also been recognized as a cause for disruption in insulin signaling, and insulin resistance in skeletal muscle (Corcoran et al. 2007).

Contradictory results have been found in the longissimus dorsi of 14-day-old lambs in which an increase in type I myofibers was found after 50% NR from GD 30 to 70 (Fahey et al. 2005). The difference in offspring age at which these two studies were conducted may be a cause for these conflicting results. However; the results of Zhu et al. (2006) are supported by the study of Costello et al. (2008), in which a sheep model of 50% NR from GD 104 to 127 was shown to reduce type I myofiber content in fetal triceps brachii muscle at GD127. Myofiber type composition conserves a certain level of plasticity during postnatal life in response to some stimuli such as exercise. Thus, more research is needed to confirm the long-lasting effect of myofiber type programming during fetal development (Brown 2014).

Findings from our group showed that administration of arginine to 50% NR ewes was successful in increasing fetal weight (Lassala et al. 2010). Similarly, administration of sildenafil citrate from GD 28 to 115 to ewes under 50% NR was effective in increasing fetal weight and total amino acids and polyamines in amniotic and allantoic fluids, and fetal serum (Satterfield et al. 2010). Amino acids are building blocks for protein synthesis (Wu 2013), and particularly leucine and arginine stimulate MTOR activity (Yao et al. 2008; Davis et al. 2010), enhancing protein deposition and skeletal muscle growth. Arginine also stimulates myoblast proliferation (Kalbe et al. 2013) and fusion (Long et al. 2006), likely supporting myofiber formation. These results represent potential treatments to mitigate the effects of maternal NR on skeletal muscle in the sheep model (Fig. 9.3).

Fig. 9.3
figure 3

Suggested model for the effects of maternal NR on fetal skeletal muscle growth and metabolism and consequences for postnatal health. Three major pathways of programming have been described in studies from ovine, swine, and rodent models. They are decreased muscle mass (blue), decreased oxidative capacity (green), and accumulation of IMTG and decreased insulin sensitivity (yellow). The additive effect of those programming trajectories would lead to impaired glucose clearance capacity, insulin resistance at the skeletal muscle level, and progression to type 2 diabetes, obesity, and metabolic syndrome

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9.4.2.2 Swine Model

A model of restricted protein (6% dietary crude protein) throughout pregnancy led to decreased muscle mass and myofiber area, upregulated MSTN signaling, and downregulated MTOR signaling in longissimus dorsi muscle of 35-day-old piglets (Liu et al. 2015). Moreover, a model of maternal low-energy diet (11.24 MJ/Kg of digestible energy) from mating to GD 90 was found to downregulate mRNA expression of PPARGC1A, NRF1, TFAM, ATB5B, SIRT1, and CS, which are regulators of mitochondrial biogenesis (Zou et al. 2016). Mitochondrial DNA content was also reduced, indicating a negative programming in oxidative capacity of skeletal muscle after prenatal NR (Zou et al. 2016). Decreased oxidative capacity in skeletal muscle may lead to IMTG accumulation, which is associated with the onset of insulin resistance (Corcoran et al. 2007). Accordingly, a model of 75% NR throughout pregnancy, followed by a postnatal cafeteria feeding up to 6 weeks, resulted in an impaired ability to clear blood glucose and was associated with a downregulation of SLC2A4 mRNA in skeletal muscle (Wang et al. 2016).

In addition to dietary NR, the pig model presents naturally occurring IUGR fetuses which suffer nutrient restriction because of uterine crowding (Wu et al. 2006). Using this model, the skeletal muscle proteome of IUGR piglets showed higher content of proteasome, a major system involved in protein degradation in skeletal muscle, indicating an upregulation of ubiquitin-dependent protein degradation in these animals (Wang et al. 2008). Enhanced protein degradation would lead to decreased muscle mass, which, in addition to impaired oxidative metabolism, may enhance the postnatal risk of metabolic syndrome. Through genetic and epigenetic changes, underdevelopment of fetal skeletal muscle has negative impacts on the postnatal growth and health of offspring (Ji et al. 2016, 2017).

Emerging evidence shows that glycine enhances MTOR activity and inhibits expression of genes involved in ubiquitin-dependent protein degradation (FBXO32 and TRIM63) in C2C12 myoblasts (Sun et al. 2016). Because the content of glycine is low in all plant-source foods (Hou et al. 2019), endogenous synthesis from amino acids or dietary provision of glycine plays an important role in stimulating muscle protein synthesis and animal growth (Li and Wu 2018, 2020; Wu et al. 2019). Leucine supplementation in neonatal pigs receiving a low-protein diet has been effective to enhance protein synthesis in longissimus dorsi muscle (Yin et al. 2010). Arginine supplementation in neonatal pigs was effective in increasing MTOR signaling and protein synthesis in skeletal muscle (Yao et al. 2008). Also, arginine supplementation in adult pigs had a beneficial effect on metabolic profiles in skeletal muscle and adipose tissue (Tan et al. 2011). These insights from the pig model are promising treatment alternatives to enhance muscle growth and metabolic profiles. However, their efficiency in a prenatal NR context remain to be determined.

9.4.2.3 Rodent Models

A rat model of 50% protein restriction and isocaloric diet throughout pregnancy showed increased mRNA and protein levels of SLC2A4 within gastrocnemius muscle in 38-day-old female offspring. Histone epigenetic modifications in the promoter region of SLC2A4 were also found in female offspring from this study (Zheng et al. 2012). An early-life upregulation in insulin-responsive molecules has been suggested to happen before progression to metabolic diseases in postnatal life (Costello et al. 2008; Muhlhausler et al. 2009), and the discussed results indicate a potential sex-specific programming in glucose metabolism in skeletal muscle.

An upregulation in mRNA expression and protein content of Cebpb was found in gastrocnemius muscle of 38-day-old female offspring using a rat model of 50% protein restriction and isocaloric diet from GD 2 to term. An increase in histone acetylation was found at the promoter region of Cebpb in those females (Zheng et al. 2011). Cebpb is a transcription factor involved in the regulation of genes related to energy homeostasis, and one of its effects is the stimulation of adipogenesis by induction of fibroblast differentiation to adipocytes. These results support a programming effect towards increased intramuscular fat accumulation, which is correlated with insulin resistance and type 2 diabetes.

A mouse model of 50% NR from GD 12.5 to 18.5 reduced the mitochondrial content in tibialis anterior muscle and increased the levels of carcass adiposity in 14-week-old offspring (Beauchamp et al. 2015). This study also showed resistance to weight loss in offspring from NR dams, as these animals lost 50% of weight compared to control after a 40% caloric restriction from postnatal week 10−14. A decreased oxidative capacity and resistance to lose weight would further enhance the risk of metabolic disease. Interestingly, dietary supplementation with watermelon juice, which is a source of citrulline, increases arginine availability and reduced adipose tissue accretion, serum glucose concentrations and free fatty acids in a rat model of non-insulin dependent diabetes (Wu et al. 2007). Additionally, leucine supplementation to diet-induced obese mice was successful in activating genes involved in mitochondrial biogenesis and preventing mitochondrial dysfunction (Li et al. 2012). These results represent potential treatments to counteract the enhanced risk for metabolic disease once a stage of disease is already present. However, their effectiveness in individuals that have experienced maternal NR remains to be tested.

9.5 Concluding Remarks

Several studies have demonstrated that maternal nutrient restriction is a cause for SGA or IUGR offspring, which was epidemiologically correlated with increased risk of metabolic syndrome. Insights from diverse animal models have provided the molecular basis for the developmental trajectories that are induced by nutrient scarcity and lead to postnatal metabolic dysregulation. Seminal results collected from ovine, swine, and rodent models indicate that muscle mass, oxidative capacity, and insulin sensitivity are the major features affected by prenatal NR. These would contribute to the onset of insulin resistance at the skeletal muscle level, with progression to a whole-body effect and type 2 diabetes, and obesity. Current data provide promising treatment alternatives to counteract these effects and minimize the negative consequences of prenatal NR in postnatal health. However, the specific windows for intervention, and conclusive results from NR models are still needed.