Skeletal Muscle and Fetal Alcohol Spectrum Disorder

Page 1 of 32
1
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.
1 Skeletal muscle and fetal alcohol spectrum disorder
2 Semone B. Myriea,b, *, Mark A. Pindera,b
a
4 Department of Human Nutritional Sciences, bRichardson Centre for Functional Foods and
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by CORNELL UNIV on 11/02/17
5 Nutraceuticals, University of Manitoba, Winnipeg, MB, Canada, R3T 2N2
7 *Corresponding author. Tel: +012044947290; fax: +012044747593.
8 E-mail address: myrie@cc.umanitoba.ca
9 Running title: FASD and skeletal muscle
10
Page 2 of 32
2
11 Abstract
12 Skeletal muscle is critical for mobility and many metabolic functions integral to survival and
13 long-term health. Alcohol can affect skeletal muscle physiology and metabolism, which will
14 have immediate and long-term consequences on health. While skeletal muscle abnormalities,
15 including morphological, biochemical and functional impairments are well documented in adults
16 consuming excessive alcohol, there is a scarcity of information about skeletal muscle in the
17 prenatal alcohol exposure (PAE) offspring. This review examines the available studies
18 addressing skeletal muscle abnormalities due to PAE. Growth restriction, fetal alcohol myopathy
19 and abnormalities in the neuromuscular system, which contributes to deficits in locomotion, are
20 some direct, immediate consequences of PAE on skeletal muscle morphology and function.
21 Long-term health consequences of PAE-related skeletal abnormalities include impaired skeletal
22 muscle glucose metabolism, resulting in glucose intolerance and insulin resistance, leading to an
23 increased risk of type 2 diabetes. In general, there is limited information on morphological,
24 biochemical and functional features of skeletal abnormalities in PAE offspring. There is a need
25 to understand how PAE affects muscle growth and function at the cellular level during early
26 development in order to improve the immediate and long-term health of PAE offspring.
27
28 Keywords: muscle fiber; fetal alcohol myopathy; cytoskeleton proteins; amino acids; insulin
29 resistance
Page 3 of 32
3
30 Introduction
31 Skeletal muscle, comprising about 30% of the body mass in the neonate, is critical for
32 mobility and many metabolic functions integral to survival and long-term health (Columbus et al.
33 2015; Davis and Fiorotto 2009). Skeletal muscle abnormalities, including morphological,
34 biochemical and functional impairments, are well documented as common features associated
35 with chronic binge and excessive alcohol (ethanol, ETOH) consumption in adults (Fernandez-
36 Sola et al. 2007; Simon et al. 2014; Steiner and Lang 2015; Urbano-Marquez and Fernandez-
37 Sola 2004). However, while growth deficits are common, and form key diagnostic characteristics
38 in fetal alcohol spectrum disorders (FASD) (Del Campo and Jones 2017; Landgraf et al. 2013;
39 Riley et al. 2011), there is a scarcity of research on skeletal muscle abnormalities in prenatal
40 alcohol exposure (PAE) offspring. Given that growth rate is highest during the neonatal period
41 compared to at any other stage of postnatal development and that the majority of this growth is
42 skeletal muscle (Columbus et al. 2015; Davis and Fiorotto 2009), it is important to understand
43 the effects of ethanol exposure during this period on muscle metabolism and function. Impaired
44 muscle function during early development could have many consequences including increased
45 risks for future metabolic conditions such as type 2 diabetes, metabolic syndrome and
46 cardiovascular diseases (Beauchamp and Harper 2015; Brown 2014; Dunlop et al. 2015).
47 This review explores the existing body of knowledge available regarding the teratogenic
48 effects of ethanol exposure on skeletal muscle in the fetus and neonate. Research questions
49 addressed to this end include: what skeletal muscle abnormalities at the morphological,
50 functional and biochemistry levels have been observed in PAE offspring, and are there any
51 known long-term health consequences related to early life abnormalities in skeletal muscle.
52
Page 4 of 32
4
53 Morphological impairments in skeletal muscle associated with PAE
54 1. Growth restriction
55 Growth deficits seen in fetal alcohol syndrome (FAS) and other forms of FASD include low
56 birthweight, which affects stature as presented by abnormalities in projected body weight and
57 height (Carter et al. 2016; Del Campo and Jones 2017; Landgraf et al. 2013; Riley et al. 2011).
58 Inability to gain weight and low body mass index have been frequently reported in FASD
59 children, which have been suggested to be due to hyperactive behaviors, decreased cell mass and
60 increased metabolic rates (Del Campo and Jones 2017), however, these purported underlying
61 causes need to be studied. Growth deficits have also been reported in many animal models of
62 PAE (Chaudhuri 2000; Lee and Leichter 1983; Probyn et al. 2012; Sylvain et al. 2010). Some
63 research suggests that this physical growth restriction persists during the postnatal life (i.e., no
64 catch-up growth) despite adequate growth hormones (Aros et al. 2011; Castells et al. 1981;
65 Hellstrom et al. 1996; Tze et al. 1976) and nutrition (Lancaster et al. 1984). While the
66 mechanisms are not fully understood, one suggestion is that such alcohol-induced growth
67 restriction may be due in part to an allometric reduction in growth of skeletal muscles
68 (Ihemelandu 1984; Nwaogu and Ihemelandu 1999). Using a mouse model, Ihemelandu (1984)
69 found that body weight, and weights of specific muscle groups (soleus, biceps brachii, long
70 digital extensor and cranial tibial muscles) were significantly smaller in offspring exposed to
71 30% ETOH (v/v) during gestation. Similarly, using a rat model, Nwaogu and Ihemelandu (1999)
72 also found that the degree of growth retardation varied among different muscles, with more
73 active muscle groups like the tibialis anterior muscle of the rat showing less weight reduction
74 compared to the extensor digitorum longus muscle. The more active muscle groups have been
75 suggested to have a greater ability to regenerate and recover faster (David and Subramaniam
Page 5 of 32
5
76 2005). Although not directly assessed, the researchers from these two studies suggested that
77 retardation of muscle growth postnatally was due to suppression of normal myogenesis, resulting
78 in reduced hyperplasia and hypertrophy of muscle fiber. Later, using a rat model, David and
79 Subramaniam (2005) found that rat dams given 1 ml of 20% ETOH/100 g body weight
80 intraperitoneally during gestation resulted in significant reduction (p<0.05) in muscle weights,

81 muscle fiber numbers and size in the offspring. However, this study also did not examine
82 myogenesis process at the molecular level.
83 Myofiber ontogenesis begins very early during embryonic development (Picard et al.
84 2002). In utero insults to the fetus are known to alter the number of fibers that are formed during
85 myogenesis, resulting in reduction in total skeletal muscle mass in the animal (Fahey et al. 2005;
86 Yates et al. 2012). Unfortunately, there is no research available that have assessed skeletal
87 muscle at the molecular level due to PAE, thus there is no information available on myogenesis
88 or other skeletal muscle-related subcellular biochemical processes that may affect muscle growth
89 in the PAE offspring. However, research on other types of stem cells indicate that ethanol alters
90 prenatal stem cell differentiation during early development, with almost all the studies to date
91 focusing on the central nervous system (CNS) development (Kim et al. 2016; Krishnamoorthy et
92 al. 2013). Additionally, the study by Hipp et al. (2010) showed that when amniotic fluid-derived
93 stem cells are exposed to ethanol during the uncommitted stage of differentiation this results in
94 commitment to osteogenic differentiation. In regards to muscle size, Mantha et al. (2014)
95 reported that mice exposed to ethanol during the second-trimester (period of muscle fiber
96 formation) had altered genetic networks and cellular processes in the fetal brain including
97 differentially expressed genes involved in skeletal and muscular formation, thus affecting
98 hypertrophy of skeletal muscle. However, this study was an exploratory assessment of gene
Page 6 of 32
6
99 expressions, thus there is also a need for more direct assessment of phenotype/function. Because
100 growth rate during the neonatal period is known to be a critical stage of development, with the
101 majority of mass accretion being skeletal muscle (Brown 2014; Brown and Hay 2016; Columbus
102 et al. 2015; Davis and Fiorotto 2009; Suryawan and Davis 2011), there is a need to study this
103 area in fetus/offspring exposed to ethanol during development as it will have immediate and life-
104 long consequences.
105 2. Fetal alcohol myopathy
106 Adult alcoholic skeletal myopathy is one of the most common pathologies associated
107 with chronic alcohol consumption (Fernandez-Sola et al. 2007; Steiner and Lang 2015; Urbano-
108 Marquez and Fernandez-Sola 2004). Conversely, although characteristics of FAS may also
109 include symptoms of myopathy in the neonate there is a scarcity of information. Fetal alcohol
110 myopathy, which differs from congenital myopathies, is a dysgenetic phenomenon that occurs
111 when developing myocytes are exposed to ethanol (Adickes and Mollner 1986; Adickes and
112 Shuman 1983). Studies in human and other animal models found that fetal alcohol myopathy is
113 in part characterized by morphological alterations of sarcomere structure such as disrupted Z-
114 lines organization (Figure 1) (Adickes and Mollner 1986, 1987; Adickes and Shuman 1983).
115 The work of Adickes and Shuman (1983) showed that PAE can lead to fetal alcohol myopathy,
116 which is characterized by a unique constellation of skeletal muscles morphologic features and
117 presents as hypotonicity and flaccidity in infants with FAS (Adickes and Shuman 1983). In their
118 study of three neonates with FAS, muscle biopsies from the vastus lateralis muscle in these
119 children showed generalized hypotrophy of myocytes, dominance of type II fibers (over 70% in
120 two cases, compared to the normal 50%) and central nuclei in 5%-10% of the myocytes (normal
121 <3%) (Adickes and Shuman 1983). The constellation of skeletal muscle structural modifications
Page 7 of 32
7
122 observed included that the centrally placed sarcolemmal nuclei were surrounded by poorly
123 demarcated sarcoplasm, and there was marked sarcomeric dysplasia at the ultrastructural level,
124 wherein there was variability in the length and diameters of sarcomeres; Z-band material with
125 actin persisted in a coagulum of granulofilamentous debris; the number of filaments was
126 decreased (Adickes and Shuman 1983). Normally, the filament proteins of muscle cytoskeleton
127 in the sarcomere are aligned in a configuration in which actin-containing filaments are attached
128 to the Z-band (Z-disc/Z-line) and interdigitated with myosin filaments (Figure 1) (Adickes and
129 Mollner 1986; Luther 2009; Vigoreaux 1994; Wang et al. 2005). For more on muscle Z-line/Z-
130 disc/Z-band, readers are referred to reviews elsewhere (Luther 2009; Sanger and Sanger 2008;
131 Vigoreaux 1994).
132 Adickes and Shuman (1983) believed that the pattern of muscle damage observed in
133 these neonates was due to dysplasia rather than degeneration as they found no inflammatory or
134 fibrotic response to the muscle damage. The damage seems to affect the contractile myofibrils
135 without inducing lysosomal activation and leaves nearby cellular components structurally
136 unaltered (Adickes and Shuman 1983). Overall, these researchers believed that hypotonia in FAS
137 is better attributed to an ethanol-induced dysplasia of muscle rather than to CNS malformation,
138 as previously thought (Schapiro et al. 1984). While there are morphological differences in adult
139 and fetal alcohol myopathy, it is believed that the mechanism of alcoholic damage to myocytes
140 may be similar between both, and any observed morphologic differences may be due to the
141 plasticity of growth in the neonate, which is lacking in adults (Adickes and Shuman 1983). Thus,
142 adult muscle shows a rapid progression to myonecrosis while infant muscle are more resilient to
143 cell death, but the surviving cell may suffer permanent damage to the subcellular apparati,
144 producing high quantities of disheveled proteins (Adickes and Shuman 1983). Animal models of
Page 8 of 32
8
145 PAE have also shown such structural damages to myoctes. Using Spargue-Dawley pregnant rats
146 given alcohol (12.5 and 20.0 g ETOH/kg body weight), Adickes and Mollner (1986) were able to
147 replicate that ethanol was teratogenic to developing myocytes resulting in disorganization of
148 sarcomeres and myofilaments. The nuclei in the damaged myocytes were either central or
149 subsarcolemmal. Myofibrillar damage manifested as Z-band streaming and disorganization of

150 cytoskeletal proteins such as actin and myosin (Adickes and Mollner 1986). The cytoskeletal
151 proteins, including actin, myosin and α-actinin, are important for cell shape and focal adhesion
152 (Ni et al. 1992; Vigoreaux 1994; Wang et al. 2005).
153 Besides major disruption of Z-line organization in the sarcomere, other structural
154 damages have been noted in fetal alcohol myopathy animal and cell culture models. Using a
155 guinea pig model, Nyquist-Battie et al. (1987) found that ethanol exposure (8g/kg body weight;
156 30% ETOH v/v) during the second half of gestation resulted in the presence of vacuolated
157 sarcoplasmic reticula and enlarged lipid droplets, mitochondrial abnormalities (included irregular
158 shape) and decreased interfibrillar glycogen in gastrocnemius muscle (Nyquist-Battie et al.
159 1987). Moreover, these researchers found that the increased fatty deposits and the vacuolization
160 of the sarcoplasmic reticulum occurred in separate sarcomeres and was possible in different
161 muscle fiber types, likely the slow twitch fiber (type I fiber) and fast twitch fiber (type II fiber),
162 respectively (Nyquist-Battie et al. 1987). Guinea pig gastrocnemius muscle has predominately
163 fast twitch fibers (~88%), which these researchers believed represents the major site for ethanol-
164 induced sarcoplasmic reticulum vacuolization, conversely the slow twitch fibers are known to
165 contain much higher quantities of mitochondria and lipid droplets compared to fast twitch fibers
166 (Nyquist-Battie et al. 1987). Overall, based on the results observed and available literature,
167 Nyquist-Battie et al. (1987) concluded that ethanol exposure during the second half of gestation
Page 9 of 32
9
168 can cause changes in skeletal muscle organelles similar to those seen in adult skeletal muscle
169 exposed to chronic ethanol. This would suggest that maturational state once embryogenesis is
170 complete may not play the main role in the type of damage ethanol produces in muscle; however
171 the developing muscle in the young may be more sensitive. Using a chick embryo model of FAS,
172 Chaudhuri (2004) reported skeletal muscle damage, as evident by neutrophil infiltration, fatty
173 degeneration and necrosis in chick embryos exposed to 10% or 15% ETOH. Chaudhuri (2004)
174 speculated that the fetal muscle damage induced by ethanol exposure could be due to a direct
175 fetotoxic effect of ethanol to cause generation of free oxygen radicals, or formation of aldehydes
176 such as 4-hydroxynonenal, because the fetus lacks an effective protective mechanism against
177 these disruptive processes. A derangement of maternal functions was also speculated as a cause
178 for the muscle damage and as well as growth retardation (Chaudhuri 2004). Ethanol teratogenic
179 effects on developing myocytes, resulting in disorganization of sarcomeres and myofilaments,
180 have also been observed in cardiac myocytes (Adickes and Mollner 1987; Adickes et al. 1990;
181 Ni et al. 1992; Uphoff et al. 1984).
182 In general, while there is limited research, the available evidence indicates that PAE can
183 cause myopathy through a variety of mechanisms, including disruption of myofibrillar structure
184 via alterations of cytoskeleton proteins and their disorganization in the sarcomere Z-band,
185 abnormal myofiber type distribution (dominance of type II myofiber), decreased myocyte size,
186 mitochondrial abnormalities, and enlarged lipid droplets. These features are all reminiscent of
187 observations in adult alcoholic myopathy (Rubin et al. 1976; Sunnasy et al. 1983; Urbano-
188 Marquez and Fernandez-Sola 2004). However, in contrast to adult alcoholic myopathy, these
189 modifications in the neonate are believed to be due to general disruption of myocte subcellular
190 structure rather than myonecrosis (Adickes and Shuman 1983). The limited animal in vivo and in
Page 10 of 32
10
191 vitro studies suggest that the mechanisms may vary depending on the severity of ethanol
192 exposure during fetal development and other factors (Nyquist-Battie et al. 1987), but should be
193 further explored. Future research should investigate if these damages are treatable or even
194 reversible if treated early. Studies in adult alcoholic myopathy suggested that myofiber damages
195 are mediated in part by direct ethanol effect, not necessarily nutritional deficiencies (Nicolas et
196 al. 2003; Preedy and Peters 1990). Adult alcoholic myopathy has been shown to be partially
197 reversible (Estruch et al. 1998).
198
199 Functional impairments in skeletal muscle activities associated with PAE
200 Peripheral neuropathy due to abnormalities in the neuromuscular system contributes to
201 locomotion deficits
202 While PAE is well known to be associated with CNS defects (de la Monte and Kril 2014; Del
203 Campo and Jones 2017; Fontaine et al. 2016), abnormalities to the peripheral nervous system
204 (PNS) may also occur with FASD (David and Subramaniam 2005; de la Monte and Kril 2014).
205 For instance, children exposed to heavy (dose not defined) PAE can present with locomotion
206 deficits (Simmons et al. 2012), which is due in part to abnormal maturation of the neuromuscular
207 system (David and Subramaniam 2005; Sylvain et al. 2010). Alcohol-related peripheral
208 neuropathies are also known as alcoholic polyneuropathy as it may involve damage to sensory,
209 autonomic and motor nerve cells, and the pathophysiology is primarily characterized by
210 degeneration of the axons and a reduction in myelination of the nerve fibers (David and
211 Subramaniam 2005; de la Monte and Kril 2014). Symptoms include numbness, sensory changes,
212 muscle weakness, muscle cramps, and tingling. It is believed that individuals afflicted with FAS
213 also suffer from alcoholic polyneuropathy as they often have muscle weakness, muscle wasting,
Page 11 of 32
11
214 and atrophy (David and Subramaniam 2005). A rat model of PAE found that there were
215 alterations in the myelination of sciatic nerve fibers and in the connectivity of myelin sheath, as
216 well as the neuromuscular junction in the alcohol-exposed animals. The PAE animals showed a
217 higher proportion of endplates polyneuronal innervation compared to control animals, suggesting
218 delay in the maturation process of the neuromuscular junction system of skeletal muscles (David
219 and Subramaniam 2005). Increased motoneuronal activity is critical for regression of
220 polyneuronal innervation; however, the adverse effect of alcohol on muscle fiber may cause
221 muscle to lose some of this functional abilities (David and Subramaniam 2005). Assessment of
222 electromyographic activity in the tricep surae and anterior tibialis muscles of children exposed to
223 alcohol showed delayed motor development as measured by increased long-latency responses,
224 which are thought to involve a transcortical pathway between peripheral somatosensory nerve
225 fibers and the CNS (Roebuck et al. 1998).
226 Sylvain et al. (2010) used a zebrafish embryo model to investigate the effects of ethanol on
227 cells involved in locomotion. Their results showed that ethanol exposure at 1.5% v/v or higher
228 affected motor neuron axon and muscle fiber morphology in a dose-dependent manner, where
229 3% ETOH (v/v) exhibited a mortality rate of more than 50%. In general, alcohol exposure
230 resulted in aberrant innervation, fewer axonal branches in primary and secondary motor neurons,
231 and smaller slow, tonic (red) and fast, twitch (white muscle) fibers, resulting in weaker,
232 uncoordinated movements during swimming in the zebrafish (Sylvain et al. 2010).
233 In the study by Adickes and Shuman (1983) the hypotonic state in the FAS neonate was
234 shown to be partly related to the altered distribution of myofiber types, with a dominance of type
235 II fiber. Type I fibers (slow-twitch myofibers), involved in sustained, tonic contractile events, are
236 required for maintenance of posture, whereas type II fibers (fast-twitch myofibers) are for quick
Page 12 of 32
12
237 contractions and fatigue rapidly (Bassel-Duby and Olson 2006). The pattern of motor nerve
238 stimulation is well-known to influence skeletal muscle fiber type, where tonic motor neuron
239 activity at low frequency promotes type I fiber (slow fibers), whereas phasic motor neuron firing
240 at high frequency (100-150 Hz) results in type II fibers (fast fibers) (Bassel-Duby and Olson
241 2006). However, this needs to be studied in the context of PAE.

242
243 Metabolic effects in PAE that could directly impact skeletal muscle
244 Amino acids availability and protein synthesis in PAE
245 Amino acids are the building blocks for proteins and key regulators of metabolic
246 pathways that are crucial processes in development and growth (Davis and Fiorotto 2009; Lin et
247 al. 2014). During developmental periods, inadequate amino acids supplies for muscle protein
248 synthesis and accretion will lead to retarded growth. Alteration of protein synthesis has been
249 suggested as partial explanation of the mechanism of FAS (Schenker et al. 1990; Shibley and
250 Pennington 1997). Maternal alcohol consumption can impact nutrients utilization, metabolism
251 and transport by the mother as well as the fetus (Weinberg 1984). Indeed, PAE has been shown
252 to alter amino acids bioavailability and metabolism in the fetus (Marquis et al. 1984;
253 Padmanabhan et al. 2002; Ramadoss et al. 2008; Sawant et al. 2015; Shibley and Pennington
254 1997; Washburn et al. 2013). For instance, high dose of alcohol (0.29-0.33 g/100g body weight;
255 47.5% ETOH) to pregnant rats reduced protein synthesis in the brains of the fetus due to a
256 significant decrease in leucine uptake (Fisher et al. 1981). Henderson et al. (1982) showed that
257 high dose of ETOH (4 g/kg; 20% v/v) administered on the 20th day of gestation 2 hr before
258 sacrifice in rats decreased placental uptake of alanine, leucine and lysine. Marquis et al. (1984)
259 showed that rat dams given 20% ETOH (v/v) ad libitum for 4 wks prior to mating + 30% ETOH
Page 13 of 32
13
260 (v/v) during gestation produced fetus with significantly low plasma levels of proline and aspartic
261 acid compared to control fetus. Maternal acute alcohol exposure (single dose of 0.03 ml/g; 25%
262 ETOH v/v) in a mouse model resulted in decreased plasma concentrations of threonine, serine,
263 glutamine, glycine, alanine and methionine in both the dam and fetus (Padmanabhan et al. 2002).
264 Maternal alcohol exposure in sheep models resulted in acidemia in the mother and fetus, which
265 reduced maternal plasma glutamine concentrations resulting in decreased circulating levels of
266 glutamine-related amino acids such as citrulline, arginine, glycine and asparagine in the fetus
267 (Ramadoss et al. 2008; Sawant et al. 2013; Sawant et al. 2015; Washburn et al. 2013). Using an
268 ovine model of maternal alcohol binge drinking (1.75-2.25 g/kg body weight; 40% ETOH w/v)
269 during the third trimester, Sawant et al. (2015) found that glutamine supplementation (100 mg/kg
270 body weight, 3 times daily) during this period was able to mitigate ethanol-induced growth
271 restriction, which was due in part to enhanced amino acids bioavailability to the fetus. Results
272 from their study indicated that maternal alcohol exposure during the third trimester significantly
273 decreased the bioavailability of asparagine, glutamine, histidine and threonine in the fetal plasma
274 as well as asparagine, glutamine, glycine, threonine, citrulline, alanine and leucine in fetal
275 aminiotic fluid (Sawant et al. 2015). Glutamine is involved in many crucial cellular processes,
276 including regulating gene expression, cell signaling and antioxidative responses (Wu 2014).
277 Unfortunately, almost all the aforementioned studies have focused on the effect of low amino
278 acids bioavailability on neurological functions in the fetus/offspring. For instance, glutamine is
279 found in high concentrations in the CNS and known to be important to neurochemical
280 distribution (Albrecht et al. 2010), and thus, the neurological pathophysiology of FASD
281 (Howells et al. 2016; Valenzuela et al. 2008). There is a clear need to also understand the impact
Page 14 of 32
14
282 of diminished amino acids bioavailability on muscle metabolism as amino acids such as leucine
283 is known to be a major regulatory of muscle mass in the neonate (Columbus et al. 2015).
284 Adult alcohol skeletal myopathy; the loss of muscle protein, may result from decreased
285 muscle protein synthesis and/or accelerated muscle degradation (Fernandez-Sola et al. 2007;
286 Simon et al. 2014; Steiner and Lang 2015). Although available evidences suggest that protein
287 degradation might be minimally impacted in adult alcohol skeletal myopathy compared to
288 regulation of protein synthesis (Steiner and Lang 2015). Given the rapid rate of development of
289 the fetus and neonate, one must wonder if a similar phenomenon occurs in ethanol-exposed
290 neonates. Unfortunately, the underlying metabolic mechanisms for fetal ethanol myopathy have
291 not been properly investigated to date in ethanol-exposed neonate models. Adickes and Mollner
292 (1986) showed that PAE can cause fetal alcohol myopathy. They proposed that ethanol
293 teratogenic effects on developing myocytes lead to structural alteration of cytoskeletal proteins
294 such as actin and myosin, which in turn disrupt sarcomere structure via Z-band disorganization.
295 However, although their study with rats showed structural damages to skeletal muscle, the
296 damage was not mitigated by dietary protein manipulation as measured by comparing low–
297 protein (10% protein-calories) and high-protein (25% protein-calories) diets. Though this work is
298 just one animal study and therefore, more studies are required to understand the impact of protein
299 and/or amino acid supplementations on muscle in the PAE offspring.
300
301 Long-term consequences of PAE associated with skeletal muscle abnormalities
302 Increased metabolic diseases such as type 2 diabetes due to altered regulation of skeletal
303 muscle glucose metabolism

Page 15 of 32
15
304 Skeletal muscle is a major site of glucose metabolism, accounting for over 80% of whole
305 body glucose uptake (DeFronzo et al. 1981). Alterations in skeletal muscle play an important
306 role in many chronic diseases, including type 2 diabetes mellitus (Dunlop et al. 2015; Wolfe
307 2006). Glucose transporter-4 (Glut4) is a key molecular regulator of glucose uptake by
308 myocytes, which is stimulated through insulin signal pathways (Figure 2). Glucose taken up by
309 muscle can be oxidized via glycolysis or stored as glycogen. Glucose intolerance and insulin
310 resistance have been reported in the offspring prenatally and/or postnatally exposed to ethanol in
311 various animal models (Gardebjer et al. 2015; Ting and Lautt 2006; Vaiserman 2015; Yao et al.
312 2014), although the evidence is scarce in human (Castells et al. 1981).
313 Although the mechanisms underlying glucose intolerance and insulin resistance in the
314 prenatal- and/or postnatal-ethanol exposed offspring are not fully elucidated, studies have shown
315 that ethanol may impair insulin action at various regulatory steps (Ting and Lautt 2006),
316 including in skeletal muscle insulin signal pathway activation of glucose transporter, Glut4
317 (Figure 2). Models of adult rat offspring prenatally exposed to ethanol have been shown to alter
318 skeletal muscle insulin signaling pathways due in part to decreased levels of activated insulin
319 receptor substrate-1 (IRS-1) (phosphorylated at tyrosine residue) (Chen et al. 2005),
320 downregulation of phosphatidylinositol -3-kinase (P13K) expression (Chen et al. 2005; Yao and
321 Gregoire Nyomba 2007), increased phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase
322 (PTEN) protein expression (Yao and Gregoire Nyomba 2007; Yao and Nyomba 2008), and
323 inhibits protein kinase B (Akt) activation (Gardebjer et al. 2015; Yao and Gregoire Nyomba
324 2007), and atypical protein kinase C (aPKC) (Chen et al. 2005), which decreases expression and
325 translocation of skeletal muscle Glut4 to the cell membrane, resulting in lower Glut4 levels
326 (Chen and Nyomba 2003a, b), which may cause insulin resistance. These insulin signaling
Page 16 of 32
16
327 impairments observed in early ethanol exposed animal models are similar to modifications noted
328 in various animal and human models of altered in utero environment (Dunlop et al. 2015). In
329 addition to the aforementioned mechanisms, PAE or early postnatal ethanol exposure may alter
330 histone deacetylase (HDAC) enzymes, which have key roles in regulating glucose homeostasis
331 (Zhao et al. 2010). HDACs are a family of enzymes that catalyze the deacetylation of lysine
332 residues in the histone proteins, which are important for post-translation modification. Ethanol
333 exposure is known to elevate markers of endoplasmic reticulum (ER) stress (Nammi et al. 2007;
334 Yao and Nyomba 2008) with research showing that ER stress can lead to elevation in the levels
335 of some HDACs (Kahali et al. 2012; Kawaguchi et al. 2003). Glut4 expression has been shown
336 to be hindered by deacetylation of core histones by HDAC (Raichur et al. 2012; Raychaudhuri et
337 al. 2008; Takigawa-Imamura et al. 2003) and insulin resistance has been observed resulting from
338 HDAC deacetylation of Akt inhibitor proteins PTEN and tribbles homolog3 (Trb3) (Yao and
339 Nyomba 2008).
340 Interestingly, the work of Yao et al. (2014) suggested that early treatment with
341 tauroursodeoxycholic acid (TUDCA) may reverse glucose intolerance in the prenatally ethanol
342 exposed rat offspring. TUDCA is a soluble bile acid that can modulate endoplasmic reticulum
343 (Yao et al. 2013). Studies of human offspring have previously proposed that glucose intolerance
344 (Castells et al. 1981) and obesity, particularly in females (Werts et al. 2014), can result from
345 PAE. Using a rat model, the researchers provided the dams with moderate ethanol (2 g/kg body
346 weight, 36% ETOH v/v) during the last week of pregnancy, and the adult female offspring (13
347 weeks old) were treated with TUDCA (15 mg/kg) for 3 weeks. This resulted in normalized
348 responses in skeletal muscle tissue to both glucose tolerance test (GTT) and insulin tolerance test
349 (ITT), restoration of PTEN and Trb3 levels to normal, reduction of nuclear HDACs to near
Page 17 of 32
17
350 control levels and elevation of cytosolic HDACs to normal concentrations. Glut4 mRNA and
351 protein in the muscle were returned to normative ranges with TUDCA treatment and
352 translocation of Glut4 to the cellular membrane was enhanced in response to insulin, while
353 TUDCA alone did not alter association of Glut4 with the membrane (Yao et al. 2014). This
354 suggests that enhanced Glut4 expression alone is not corrective of impaired muscle glucose
355 metabolism (as measured by GTT and ITT) resulting from ethanol exposure, and that other
356 factors relying on insulin are required. One suggested factor is the inhibition of nuclear PTEN
357 and/or Trb3 allowing for upregulation of Akt activation by insulin (Yao et al. 2014). HDACs in
358 the muscle cell nucleus, but not the cyotosol were elevated by PAE and deacetylation of PTEN
359 and Trb3 in the nucleus would lead to cytoplasmic translocation where these proteins would
360 block Akt activation by insulin and translocation of Glut4 to the cellular membrane (Yao et al.
361 2014). Through a reduction in nuclear HDACs, the levels of PTEN and Trb3 found in the
362 nucleus are lowered, resulting in restoration of normal reaction to insulin for Akt and Glut4
363 translocation. It is also known that deacetylation of IRS-1 in the liver caused by HDAC2 can
364 hinder the insulin response of the P13K pathway (Kaiser and James 2004). Though Yao et al.
365 (2014) did not study HDAC2 or IRS proteins, they speculated that TUDCA restored acetylation
366 of IRS-1 and impacted the phosphorylation of Akt and Glut4 translocation in the presence of
367 insulin. Overall, because skeletal muscle is a major site for glucose disposal and insulin
368 sensitivity, a decreased muscle mass and other early muscle alterations will predispose the PAE
369 offspring to glucose intolerance and insulin resistance, greatly elevating the risk of diabetes in
370 the long-term.
371 Conclusions
Page 18 of 32
18
372 It has been stated that there tends to be an underappreciation for the role of skeletal
373 muscle in health and disease states in adults (Wolfe 2006), and a similar situation may also apply
374 to neonatal conditions such as FASD. Growth restriction, fetal alcohol myopathy and deficits in
375 locomotion, due in part to peripheral neuropathy, are some direct, immediate consequences of
376 PAE on skeletal muscle morphology and function. Long-term health consequences of PAE-
377 related skeletal abnormalities include impaired skeletal muscle glucose metabolism, resulting in
378 glucose intolerance and insulin resistance, leading to an increased risk of type 2 diabetes. While
379 altered protein synthesis and the reduced availability of amino acids to the fetus are known to be
380 partially responsible for FASD, the research in this area focuses almost exclusively on the impact
381 on the CNS leaving a need for research targeting the effects on the peripheral nervous system
382 and skeletal muscle development and function. In general, there is limited information on
383 morphological, biochemical and functional features of skeletal abnormalities in PAE offspring.
384 There is a need to understand how PAE affects muscle growth (including effect on muscle
385 protein synthesis and protein accretion/mass) and function at the cellular level during early
386 development in order to improve the immediate and long-term health of PAE offspring.
387
388 Acknowledgements
389 The authors declare no conflicts of interest relevant to this manuscript.

Page 19 of 32
19
390 References
391 Adickes, E.D., and Mollner, T.J. 1986. Ethanol-induced cytoskeletal dysgenesis with dietary protein
392 manipulations. Alcohol Alcohol 21(4): 347-355.
393 Adickes, E.D., and Mollner, T.J. 1987. Ethanol-Induced Cytoskeletal Dysgenesis in Developing Skeletal
394 and Cardiac Myocytes. Ann Ny Acad Sci 492: 344-346. doi: DOI 10.1111/j.1749-6632.1987.tb48690.x.
395 Adickes, E.D., Mollner, T.J., and Lockwood, S.K. 1990. Ethanol induced morphologic alterations during
396 growth and maturation of cardiac myocytes. Alcoholism, clinical and experimental research 14(6): 827-
397 831.
398 Adickes, E.D., and Shuman, R.M. 1983. Fetal alcohol myopathy. Pediatric pathology / affiliated with the
399 International Paediatric Pathology Association 1(4): 369-384.
400 Albrecht, J., Sidoryk-Wegrzynowicz, M., Zielinska, M., and Aschner, M. 2010. Roles of glutamine in
401 neurotransmission. Neuron glia biology 6(4): 263-276. doi: 10.1017/S1740925X11000093.
402 Aros, S., Mills, J.L., Iniguez, G., Avila, A., Conley, M.R., Troendle, J., Cox, C., and Cassorla, F. 2011.
403 Effects of prenatal ethanol exposure on postnatal growth and the insulin-like growth factor axis. Hormone
404 research in paediatrics 75(3): 166-173. doi: 10.1159/000319706.
405 Bassel-Duby, R., and Olson, E.N. 2006. Signaling pathways in skeletal muscle remodeling. Annual
406 review of biochemistry 75: 19-37. doi: 10.1146/annurev.biochem.75.103004.142622.
407 Beauchamp, B., and Harper, M.E. 2015. In utero Undernutrition Programs Skeletal and Cardiac Muscle
408 Metabolism. Frontiers in physiology 6: 401. doi: 10.3389/fphys.2015.00401.
409 Brown, L.D. 2014. Endocrine regulation of fetal skeletal muscle growth: impact on future metabolic
410 health. The Journal of endocrinology 221(2): R13-29. doi: 10.1530/JOE-13-0567.
411 Brown, L.D., and Hay, W.W., Jr. 2016. Impact of placental insufficiency on fetal skeletal muscle growth.
412 Molecular and cellular endocrinology 435: 69-77. doi: 10.1016/j.mce.2016.03.017.
413 Carter, R.C., Jacobson, J.L., Molteno, C.D., Dodge, N.C., Meintjes, E.M., and Jacobson, S.W. 2016. Fetal
414 Alcohol Growth Restriction and Cognitive Impairment. Pediatrics 138(2). doi: 10.1542/peds.2016-0775.
Page 20 of 32
20
415 Castells, S., Mark, E., Abaci, F., and Schwartz, E. 1981. Growth retardation in fetal alcohol syndrome.
416 Unresponsiveness to growth-promoting hormones. Developmental pharmacology and therapeutics 3(4):
417 232-241.
418 Chaudhuri, J.D. 2000. Alcohol and the developing fetus--a review. Medical science monitor :
419 international medical journal of experimental and clinical research 6(5): 1031-1041.
420 Chaudhuri, J.D. 2004. Effect of a single dose of ethanol on developing skeletal muscle of chick embryos.
421 Alcohol 34(2-3): 279-283.
422 Chen, L., and Nyomba, B.L. 2003a. Effects of prenatal alcohol exposure on glucose tolerance in the rat
423 offspring. Metabolism: clinical and experimental 52(4): 454-462. doi: 10.1053/meta.2003.50073.
424 Chen, L., and Nyomba, B.L. 2003b. Glucose intolerance and resistin expression in rat offspring exposed
425 to ethanol in utero: modulation by postnatal high-fat diet. Endocrinology 144(2): 500-508. doi:
426 10.1210/en.2002-220623.
427 Chen, L., Yao, X.H., and Nyomba, B.L. 2005. In vivo insulin signaling through PI3-kinase is impaired in
428 skeletal muscle of adult rat offspring exposed to ethanol in utero. J Appl Physiol (1985) 99(2): 528-534.
429 doi: 10.1152/japplphysiol.01098.2004.
430 Columbus, D.A., Fiorotto, M.L., and Davis, T.A. 2015. Leucine is a major regulator of muscle protein
431 synthesis in neonates. Amino acids 47(2): 259-270. doi: 10.1007/s00726-014-1866-0.
432 David, P., and Subramaniam, K. 2005. Prenatal alcohol exposure and early postnatal changes in the
433 developing nerve-muscle system. Birth defects research. Part A, Clinical and molecular teratology 73(11):
434 897-903. doi: 10.1002/bdra.20190.
435 Davis, T.A., and Fiorotto, M.L. 2009. Regulation of muscle growth in neonates. Current opinion in
436 clinical nutrition and metabolic care 12(1): 78-85. doi: 10.1097/MCO.0b013e32831cef9f.
437 de la Monte, S.M., and Kril, J.J. 2014. Human alcohol-related neuropathology. Acta neuropathologica
438 127(1): 71-90. doi: 10.1007/s00401-013-1233-3.

Page 21 of 32
21
439 DeFronzo, R.A., Jacot, E., Jequier, E., Maeder, E., Wahren, J., and Felber, J.P. 1981. The effect of insulin
440 on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous
441 catheterization. Diabetes 30(12): 1000-1007.
442 Del Campo, M., and Jones, K.L. 2017. A review of the physical features of the fetal alcohol spectrum
443 disorders. European journal of medical genetics 60(1): 55-64. doi: 10.1016/j.ejmg.2016.10.004.
444 Dunlop, K., Cedrone, M., Staples, J.F., and Regnault, T.R. 2015. Altered fetal skeletal muscle nutrient
445 metabolism following an adverse in utero environment and the modulation of later life insulin sensitivity.
446 Nutrients 7(2): 1202-1216. doi: 10.3390/nu7021202.
447 Estruch, R., Sacanella, E., Fernandez-Sola, J., Nicolas, J.M., Rubin, E., and Urbano-Marquez, A. 1998.
448 Natural history of alcoholic myopathy: a 5-year study. Alcoholism, clinical and experimental research
449 22(9): 2023-2028.
450 Fahey, A.J., Brameld, J.M., Parr, T., and Buttery, P.J. 2005. Ontogeny of factors associated with
451 proliferation and differentiation of muscle in the ovine fetus. Journal of animal science 83(10): 2330-
452 2338.
453 Fernandez-Sola, J., Preedy, V.R., Lang, C.H., Gonzalez-Reimers, E., Arno, M., Lin, J.C., Wiseman, H.,
454 Zhou, S., Emery, P.W., Nakahara, T., Hashimoto, K., Hirano, M., Santolaria-Fernandez, F., Gonzalez-
455 Hernandez, T., Fatjo, F., Sacanella, E., Estruch, R., Nicolas, J.M., and Urbano-Marquez, A. 2007.
456 Molecular and cellular events in alcohol-induced muscle disease. Alcoholism, clinical and experimental
457 research 31(12): 1953-1962. doi: 10.1111/j.1530-0277.2007.00530.x.
458 Fisher, S.E., Barnicle, M.A., Steis, B., Holzman, I., and Van Thiel, D.H. 1981. Effects of acute ethanol
459 exposure upon in vivo leucine uptake and protein synthesis in the fetal rat. Pediatric research 15(4 Pt 1):
460 335-339. doi: 10.1203/00006450-198104000-00009.
461 Fontaine, C.J., Patten, A.R., Sickmann, H.M., Helfer, J.L., and Christie, B.R. 2016. Effects of pre-natal
462 alcohol exposure on hippocampal synaptic plasticity: Sex, age and methodological considerations.
463 Neuroscience and biobehavioral reviews 64: 12-34. doi: 10.1016/j.neubiorev.2016.02.014.

Page 22 of 32
22
464 Gardebjer, E.M., Anderson, S.T., Pantaleon, M., Wlodek, M.E., and Moritz, K.M. 2015. Maternal alcohol
465 intake around the time of conception causes glucose intolerance and insulin insensitivity in rat offspring,
466 which is exacerbated by a postnatal high-fat diet. FASEB journal : official publication of the Federation
467 of American Societies for Experimental Biology 29(7): 2690-2701. doi: 10.1096/fj.14-268979.
468 Hellstrom, A., Jansson, C., Boguszewski, M., Olegard, R., Laegreid, L., and Albertsson-Wikland, K.
469 1996. Growth hormone status in six children with fetal alcohol syndrome. Acta Paediatr 85(12): 1456-
470 1462.
471 Henderson, G.I., Patwardhan, R.V., McLeroy, S., and Schenker, S. 1982. Inhibition of placental amino
472 acid uptake in rats following acute and chronic ethanol exposure. Alcoholism, clinical and experimental
473 research 6(4): 495-505.
474 Hipp, J.A., Hipp, J.D., Atala, A., and Soker, S. 2010. Ethanol alters the osteogenic differentiation of
475 amniotic fluid-derived stem cells. Alcoholism, clinical and experimental research 34(10): 1714-1722. doi:
476 10.1111/j.1530-0277.2010.01258.x.
477 Howells, F.M., Donald, K.A., Roos, A., Woods, R.P., Zar, H.J., Narr, K.L., and Stein, D.J. 2016. Reduced
478 glutamate in white matter of male neonates exposed to alcohol in utero: a (1)H-magnetic resonance
479 spectroscopy study. Metabolic brain disease 31(5): 1105-1112. doi: 10.1007/s11011-016-9850-x.
480 Ihemelandu, E.C. 1984. Effect of maternal alcohol consumption on pre- and post-natal muscle
481 development of mice. Growth 48(1): 35-43.
482 Kahali, S., Sarcar, B., Prabhu, A., Seto, E., and Chinnaiyan, P. 2012. Class I histone deacetylases localize
483 to the endoplasmic reticulum and modulate the unfolded protein response. FASEB journal : official
484 publication of the Federation of American Societies for Experimental Biology 26(6): 2437-2445. doi:
485 10.1096/fj.11-193706.
486 Kaiser, C., and James, S.R. 2004. Acetylation of insulin receptor substrate-1 is permissive for tyrosine
487 phosphorylation. BMC biology 2: 23. doi: 10.1186/1741-7007-2-23.

Page 23 of 32
23
488 Kawaguchi, Y., Kovacs, J.J., McLaurin, A., Vance, J.M., Ito, A., and Yao, T.P. 2003. The deacetylase
489 HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell
490 115(6): 727-738.
491 Kim, Y.Y., Roubal, I., Lee, Y.S., Kim, J.S., Hoang, M., Mathiyakom, N., and Kim, Y. 2016. Alcohol-
492 Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells. PloS
493 one 11(9): e0163812. doi: 10.1371/journal.pone.0163812.
494 Krishnamoorthy, M., Gerwe, B.A., Scharer, C.D., Sahasranaman, V., Eilertson, C.D., Nash, R.J., Usta,
495 S.N., Kelly, S., Rose, M., Peraza, R., Arumugham, J., Stewart, B., and Stice, S.L. 2013. Ethanol alters
496 proliferation and differentiation of normal and chromosomally abnormal human embryonic stem cell-
497 derived neurospheres. Birth defects research. Part B, Developmental and reproductive toxicology 98(3):
498 283-295. doi: 10.1002/bdrb.21063.
499 Lancaster, F.E., Phillips, S.M., Patsalos, P.N., and Wiggins, R.C. 1984. Brain myelination in the offspring
500 of ethanol-treated rats: in utero versus lactational exposure by crossfostering offspring of control, pairfed
501 and ethanol treated dams. Brain research 309(2): 209-216.
502 Landgraf, M.N., Nothacker, M., and Heinen, F. 2013. Diagnosis of fetal alcohol syndrome (FAS):
503 German guideline version 2013. European journal of paediatric neurology : EJPN : official journal of the
504 European Paediatric Neurology Society 17(5): 437-446. doi: 10.1016/j.ejpn.2013.03.008.
505 Lee, M., and Leichter, J. 1983. Skeletal development in fetuses of rats consuming alcohol during
506 gestation. Growth 47(3): 254-262.
507 Lin, G., Wang, X., Wu, G., Feng, C., Zhou, H., Li, D., and Wang, J. 2014. Improving amino acid
508 nutrition to prevent intrauterine growth restriction in mammals. Amino acids 46(7): 1605-1623. doi:
509 10.1007/s00726-014-1725-z.
510 Luther, P.K. 2009. The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling. Journal
511 of muscle research and cell motility 30(5-6): 171-185. doi: 10.1007/s10974-009-9189-6.
512 Mantha, K., Laufer, B.I., and Singh, S.M. 2014. Molecular changes during neurodevelopment following
513 second-trimester binge ethanol exposure in a mouse model of fetal alcohol spectrum disorder: from
Page 24 of 32
24
514 immediate effects to long-term adaptation. Developmental neuroscience 36(1): 29-43. doi:
515 10.1159/000357496.
516 Marquis, S.M., Leichter, J., and Lee, M. 1984. Plasma amino acids and glucose levels in the rat fetus and
517 dam after chronic maternal alcohol consumption. Biology of the neonate 46(1): 36-43.
518 Nammi, S., Dembele, K., and Nyomba, B.L. 2007. Increased 11beta-hydroxysteroid dehydrogenase type-
519 1 and hexose-6-phosphate dehydrogenase in liver and adipose tissue of rat offspring exposed to alcohol in
520 utero. American journal of physiology. Regulatory, integrative and comparative physiology 292(3):
521 R1101-1109. doi: 10.1152/ajpregu.00255.2006.
522 Ni, Y., Feng-Chen, K.C., and Hsu, L. 1992. A tissue culture model for studying ethanol toxicity on
523 embryonic heart cells. Cell biology and toxicology 8(1): 1-11.
524 Nicolas, J.M., Garcia, G., Fatjo, F., Sacanella, E., Tobias, E., Badia, E., Estruch, R., and Fernandez-Sola,
525 J. 2003. Influence of nutritional status on alcoholic myopathy. The American journal of clinical nutrition
526 78(2): 326-333.
527 Nwaogu, I.C., and Ihemelandu, E.C. 1999. Effects of maternal alcohol consumption on the allometric
528 growth of muscles in fetal and neonatal rats. Cells, tissues, organs 164(3): 167-173. doi: 16654.
529 Nyquist-Battie, C., Uphoff, C., and Cole, T.B. 1987. Maternal ethanol consumption: effect on skeletal
530 muscle development in guinea pig offspring. Alcohol 4(1): 11-16.
531 Padmanabhan, R., Ibrahim, A., and Bener, A. 2002. Effect of maternal methionine pre-treatment on
532 alcohol-induced exencephaly and axial skeletal dysmorphogenesis in mouse fetuses. Drug and alcohol
533 dependence 65(3): 263-281.
534 Picard, B., Lefaucheur, L., Berri, C., and Duclos, M.J. 2002. Muscle fibre ontogenesis in farm animal
535 species. Reproduction, nutrition, development 42(5): 415-431.
536 Preedy, V.R., and Peters, T.J. 1990. Alcohol and skeletal muscle disease. Alcohol Alcohol 25(2-3): 177-
537 187.
538 Probyn, M.E., Zanini, S., Ward, L.C., Bertram, J.F., and Moritz, K.M. 2012. A rodent model of low- to
539 moderate-dose ethanol consumption during pregnancy: patterns of ethanol consumption and effects on
Page 25 of 32
25
540 fetal and offspring growth. Reproduction, fertility, and development 24(6): 859-870. doi:
541 10.1071/RD11200.
542 Raichur, S., Teh, S.H., Ohwaki, K., Gaur, V., Long, Y.C., Hargreaves, M., McGee, S.L., and Kusunoki, J.
543 2012. Histone deacetylase 5 regulates glucose uptake and insulin action in muscle cells. Journal of
544 molecular endocrinology 49(3): 203-211. doi: 10.1530/jme-12-0095.

545 Ramadoss, J., Wu, G., and Cudd, T.A. 2008. Chronic binge ethanol-mediated acidemia reduces
546 availability of glutamine and related amino acids in maternal plasma of pregnant sheep. Alcohol
547 (Fayetteville, N.Y.) 42(8): 657-666. doi: 10.1016/j.alcohol.2008.08.008.
548 Raychaudhuri, N., Raychaudhuri, S., Thamotharan, M., and Devaskar, S.U. 2008. Histone code
549 modifications repress glucose transporter 4 expression in the intrauterine growth-restricted offspring. The
550 Journal of biological chemistry 283(20): 13611-13626. doi: 10.1074/jbc.M800128200.
551 Riley, E.P., Infante, M.A., and Warren, K.R. 2011. Fetal alcohol spectrum disorders: an overview.
552 Neuropsychology review 21(2): 73-80. doi: 10.1007/s11065-011-9166-x.
553 Roebuck, T.M., Simmons, R.W., Richardson, C., Mattson, S.N., and Riley, E.P. 1998. Neuromuscular
554 responses to disturbance of balance in children with prenatal exposure to alcohol. Alcoholism, clinical
555 and experimental research 22(9): 1992-1997.
556 Rubin, E., Katz, A.M., Lieber, C.S., Stein, E.P., and Puszkin, S. 1976. Muscle damage produced by
557 chronic alcohol consumption. The American journal of pathology 83(3): 499-516.
558 Sanger, J.M., and Sanger, J.W. 2008. The dynamic Z bands of striated muscle cells. Sci Signal 1(32):
559 pe37. doi: 10.1126/scisignal.132pe37.
560 Sawant, O.B., Ramadoss, J., Hogan, H.A., and Washburn, S.E. 2013. The role of acidemia in maternal
561 binge alcohol-induced alterations in fetal bone functional properties. Alcoholism, clinical and
562 experimental research 37(9): 1476-1482. doi: 10.1111/acer.12118.
563 Sawant, O.B., Wu, G., and Washburn, S.E. 2015. Maternal L-glutamine supplementation prevents
564 prenatal alcohol exposure-induced fetal growth restriction in an ovine model. Amino acids 47(6): 1183-
565 1192. doi: 10.1007/s00726-015-1945-x.

Page 26 of 32
26
566 Schapiro, M.B., Rosman, N.P., and Kemper, T.L. 1984. Effects of chronic exposure to alcohol on the
567 developing brain. Neurobehavioral toxicology and teratology 6(5): 351-356.
568 Schenker, S., Becker, H.C., Randall, C.L., Phillips, D.K., Baskin, G.S., and Henderson, G.I. 1990. Fetal
569 alcohol syndrome: current status of pathogenesis. Alcoholism, clinical and experimental research 14(5):
570 635-647.
571 Shibley, I.A., Jr., and Pennington, S.N. 1997. Metabolic and mitotic changes associated with the fetal
572 alcohol syndrome. Alcohol Alcohol 32(4): 423-434.
573 Simmons, R.W., Nguyen, T.T., Levy, S.S., Thomas, J.D., Mattson, S.N., and Riley, E.P. 2012. Children
574 with heavy prenatal alcohol exposure exhibit deficits when regulating isometric force. Alcoholism,
575 clinical and experimental research 36(2): 302-309. doi: 10.1111/j.1530-0277.2011.01625.x.
576 Simon, L., LeCapitaine, N., Berner, P., Vande Stouwe, C., Mussell, J.C., Allerton, T., Primeaux, S.D.,
577 Dufour, J., Nelson, S., Bagby, G.J., Cefalu, W., and Molina, P.E. 2014. Chronic binge alcohol
578 consumption alters myogenic gene expression and reduces in vitro myogenic differentiation potential of
579 myoblasts from rhesus macaques. American journal of physiology. Regulatory, integrative and
580 comparative physiology 306(11): R837-844. doi: 10.1152/ajpregu.00502.2013.
581 Steiner, J.L., and Lang, C.H. 2015. Dysregulation of skeletal muscle protein metabolism by alcohol.
582 American journal of physiology. Endocrinology and metabolism 308(9): E699-E712. doi:
583 10.1152/ajpendo.00006.2015.
584 Sunnasy, D., Cairns, S.R., Martin, F., Slavin, G., and Peters, T.J. 1983. Chronic alcoholic skeletal muscle
585 myopathy: a clinical, histological and biochemical assessment of muscle lipid. Journal of clinical
586 pathology 36(7): 778-784.
587 Suryawan, A., and Davis, T.A. 2011. Regulation of protein synthesis by amino acids in muscle of
588 neonates. Front Biosci (Landmark Ed) 16: 1445-1460.
589 Sylvain, N.J., Brewster, D.L., and Ali, D.W. 2010. Zebrafish embryos exposed to alcohol undergo
590 abnormal development of motor neurons and muscle fibers. Neurotoxicology and teratology 32(4): 472-
591 480. doi: 10.1016/j.ntt.2010.03.001.

Page 27 of 32
27
592 Takigawa-Imamura, H., Sekine, T., Murata, M., Takayama, K., Nakazawa, K., and Nakagawa, J. 2003.
593 Stimulation of glucose uptake in muscle cells by prolonged treatment with scriptide, a histone deacetylase
594 inhibitor. Bioscience, biotechnology, and biochemistry 67(7): 1499-1506.
595 Ting, J.W., and Lautt, W.W. 2006. The effect of acute, chronic, and prenatal ethanol exposure on insulin
596 sensitivity. Pharmacology & therapeutics 111(2): 346-373. doi: 10.1016/j.pharmthera.2005.10.004.

597 Tze, W.J., Friesen, H.G., and MacLeod, P.M. 1976. Growth hormone response in fetal alcohol syndrome.
598 Archives of disease in childhood 51(9): 703-706.
599 Uphoff, C., Nyquist-Battie, C., and Toth, R. 1984. Cardiac muscle development in mice exposed to
600 ethanol in utero. Teratology 30(1): 119-129. doi: 10.1002/tera.1420300116.
601 Urbano-Marquez, A., and Fernandez-Sola, J. 2004. Effects of alcohol on skeletal and cardiac muscle.
602 Muscle & nerve 30(6): 689-707. doi: 10.1002/mus.20168.
603 Vaiserman, A.M. 2015. Early-life exposure to substance abuse and risk of type 2 diabetes in adulthood.
604 Current diabetes reports 15(8): 48. doi: 10.1007/s11892-015-0624-3.
605 Valenzuela, C.F., Partridge, L.D., Mameli, M., and Meyer, D.A. 2008. Modulation of glutamatergic
606 transmission by sulfated steroids: role in fetal alcohol spectrum disorder. Brain research reviews 57(2):
607 506-519. doi: 10.1016/j.brainresrev.2007.04.009.
608 Vigoreaux, J.O. 1994. The muscle Z band: lessons in stress management. Journal of muscle research and
609 cell motility 15(3): 237-255.
610 Wang, J., Shaner, N., Mittal, B., Zhou, Q., Chen, J., Sanger, J.M., and Sanger, J.W. 2005. Dynamics of Z-
611 band based proteins in developing skeletal muscle cells. Cell motility and the cytoskeleton 61(1): 34-48.
612 doi: 10.1002/cm.20063.
613 Washburn, S.E., Sawant, O.B., Lunde, E.R., Wu, G., and Cudd, T.A. 2013. Acute alcohol exposure,
614 acidemia or glutamine administration impacts amino acid homeostasis in ovine maternal and fetal plasma.
615 Amino acids 45(3): 543-554. doi: 10.1007/s00726-012-1453-1.
616 Weinberg, J. 1984. Nutritional issues in perinatal alcohol exposure. Neurobehavioral toxicology and
617 teratology 6(4): 261-269.

Page 28 of 32
28
618 Werts, R.L., Van Calcar, S.C., Wargowski, D.S., and Smith, S.M. 2014. Inappropriate feeding behaviors
619 and dietary intakes in children with fetal alcohol spectrum disorder or probable prenatal alcohol exposure.
620 Alcoholism, clinical and experimental research 38(3): 871-878. doi: 10.1111/acer.12284.
621 Wolfe, R.R. 2006. The underappreciated role of muscle in health and disease. The American journal of
622 clinical nutrition 84(3): 475-482.

623 Wu, G. 2014. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein
624 nutrition. Journal of animal science and biotechnology 5(1): 34. doi: 10.1186/2049-1891-5-34.
625 Yao, X.H., and Gregoire Nyomba, B.L. 2007. Abnormal glucose homeostasis in adult female rat
626 offspring after intrauterine ethanol exposure. American journal of physiology. Regulatory, integrative and
627 comparative physiology 292(5): R1926-1933. doi: 10.1152/ajpregu.00822.2006.
628 Yao, X.H., Nguyen, H.K., and Nyomba, B.L. 2013. Prenatal ethanol exposure causes glucose intolerance
629 with increased hepatic gluconeogenesis and histone deacetylases in adult rat offspring: reversal by
630 tauroursodeoxycholic acid. PloS one 8(3): e59680. doi: 10.1371/journal.pone.0059680.
631 Yao, X.H., Nguyen, K.H., and Nyomba, B.L. 2014. Reversal of glucose intolerance in rat offspring
632 exposed to ethanol before birth through reduction of nuclear skeletal muscle HDAC expression by the
633 bile acid TUDCA. Physiological reports 2(12). doi: 10.14814/phy2.12195.
634 Yao, X.H., and Nyomba, B.L. 2008. Hepatic insulin resistance induced by prenatal alcohol exposure is
635 associated with reduced PTEN and TRB3 acetylation in adult rat offspring. American journal of
636 physiology. Regulatory, integrative and comparative physiology 294(6): R1797-1806. doi:
637 10.1152/ajpregu.00804.2007.
638 Yates, D.T., Macko, A.R., Nearing, M., Chen, X., Rhoads, R.P., and Limesand, S.W. 2012.
639 Developmental programming in response to intrauterine growth restriction impairs myoblast function and
640 skeletal muscle metabolism. Journal of pregnancy 2012: 631038. doi: 10.1155/2012/631038.
641 Zhao, S., Xu, W., Jiang, W., Yu, W., Lin, Y., Zhang, T., Yao, J., Zhou, L., Zeng, Y., Li, H., Li, Y., Shi, J.,
642 An, W., Hancock, S.M., He, F., Qin, L., Chin, J., Yang, P., Chen, X., Lei, Q., Xiong, Y., and Guan, K.L.
646
645
644
643
Page 29 of 32
327(5968): 1000-1004. doi: 10.1126/science.1179689.

2010. Regulation of cellular metabolism by protein lysine acetylation. Science (New York, N.Y.)
29
Page 30 of 32
30
647 Figure captions
648 Figure 1. Simplified view of myofibril and its associated sarcomere unit. A myofibril is made
649 up of myofilament proteins: actin (thin filament) and myosin (thick filament). Sacomere, a
650 repeating unit of a myofibril, is defined as the area between two Z-lines (Z-discs). Thin filaments
651 are tethered to the Z-lines and interdigitate with the thick filament.
652
653 Figure 2. Putative effects of prenatal ethanol exposure on skeletal muscle insulin signaling
654 pathways in adulthood. Ethanol exposed skeletal muscle may decrease insulin-stimulated
655 glucose uptake by the insulin signal pathway due to down-regulation in the expressions of
656 various components, including IRS-1 (insulin receptor substrate-1), PI3K (phosphatidylinositol-
657 3-kinase), Akt (protein kinase B) and aPKC (atypical protein kinase C). Increased expression of
658 PTEN (phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase) is also noted. See text for
659 references. Akt mediates many insulin-induced metabolic effects including glucose metabolism
660 (e.g., Akt can inhibit GLUT4 translocation to the membrane) and protein synthesis via mTOR
661 (mammalian target of rapamycin) complex 1. PDK1, 3-phosphoinositide-dependent protein
662 kinase-1; PIP2, phosphatidylinositol 4,5 bisphosphate; PIP3, phosphatidylinositol (3,4,5)-
663 trisphosphate; Trb3, tribbles homolog 3; GLUT4, glucose transporter-4; p70 ribosomal S6
664 protein kinase (p70S6K); Glc-6-P, glucose-6-phosphate.
665
666
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 31 of 32
Figure 1
254x190mm (300 x 300 DPI)

Figure 2
254x190mm (300 x 300 DPI)

Page 32 of 32

Skeletal Muscle and Fetal Alcohol Spectrum Disorder

Încărcat de

Informații document

Descriere originală:

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Skeletal Muscle and Fetal Alcohol Spectrum Disorder

Încărcat de

Drepturi de autor:

Formate disponibile

Page 1 of 32

1 Skeletal muscle and fetal alcohol spectrum disorder

2 Semone B. Myriea,b, *, Mark A. Pindera,b

5 Nutraceuticals, University of Manitoba, Winnipeg, MB, Canada, R3T 2N2

7 *Corresponding author. Tel: +012044947290; fax: +012044747593.

8 E-mail address: myrie@cc.umanitoba.ca

9 Running title: FASD and skeletal muscle

21 Long-term health consequences of PAE-related skeletal abnormalities include impaired skeletal

23 increased risk of type 2 diabetes. In general, there is limited information on morphological,

53 Morphological impairments in skeletal muscle associated with PAE

67 restriction may be due in part to an allometric reduction in growth of skeletal muscles

80 intraperitoneally during gestation resulted in significant reduction (p<0.05) in muscle weights,

82 myogenesis process at the molecular level.

94 commitment to osteogenic differentiation. In regards to muscle size, Mantha et al. (2014)

104 long consequences.

105 2. Fetal alcohol myopathy

113 in part characterized by morphological alterations of sarcomere structure such as disrupted Z-

131 Vigoreaux 1994).

149 subsarcolemmal. Myofibrillar damage manifested as Z-band streaming and disorganization of

152 (Ni et al. 1992; Vigoreaux 1994; Wang et al. 2005).

179 effects on developing myocytes, resulting in disorganization of sarcomeres and myofilaments,

181 Ni et al. 1992; Uphoff et al. 1984).

197 reversible (Estruch et al. 1998).

199 Functional impairments in skeletal muscle activities associated with PAE

200 Peripheral neuropathy due to abnormalities in the neuromuscular system contributes to

201 locomotion deficits

225 fibers and the CNS (Roebuck et al. 1998).

241 2006). However, this needs to be studied in the context of PAE.

244 Amino acids availability and protein synthesis in PAE

299 and/or amino acid supplementations on muscle in the PAE offspring.

301 Long-term consequences of PAE associated with skeletal muscle abnormalities

303 muscle glucose metabolism

321 Gregoire Nyomba 2007), increased phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase

339 Nyomba 2008).

370 the long-term.

389 The authors declare no conflicts of interest relevant to this manuscript.

392 manipulations. Alcohol Alcohol 21(4): 347-355.

399 International Paediatric Pathology Association 1(4): 369-384.

401 neurotransmission. Neuron glia biology 6(4): 263-276. doi: 10.1017/S1740925X11000093.

404 research in paediatrics 75(3): 166-173. doi: 10.1159/000319706.

406 review of biochemistry 75: 19-37. doi: 10.1146/annurev.biochem.75.103004.142622.

408 Metabolism. Frontiers in physiology 6: 401. doi: 10.3389/fphys.2015.00401.

410 health. The Journal of endocrinology 221(2): R13-29. doi: 10.1530/JOE-13-0567.

412 Molecular and cellular endocrinology 435: 69-77. doi: 10.1016/j.mce.2016.03.017.

416 Unresponsiveness to growth-promoting hormones. Developmental pharmacology and therapeutics 3(4):

421 Alcohol 34(2-3): 279-283.

429 doi: 10.1152/japplphysiol.01098.2004.

431 synthesis in neonates. Amino acids 47(2): 259-270. doi: 10.1007/s00726-014-1866-0.

434 897-903. doi: 10.1002/bdra.20190.

438 127(1): 71-90. doi: 10.1007/s00401-013-1233-3.

441 catheterization. Diabetes 30(12): 1000-1007.

446 Nutrients 7(2): 1202-1216. doi: 10.3390/nu7021202.

449 22(9): 2023-2028.

457 research 31(12): 1953-1962. doi: 10.1111/j.1530-0277.2007.00530.x.

460 335-339. doi: 10.1203/00006450-198104000-00009.

463 Neuroscience and biobehavioral reviews 64: 12-34. doi: 10.1016/j.neubiorev.2016.02.014.

473 research 6(4): 495-505.

481 development of mice. Growth 48(1): 35-43.

487 phosphorylation. BMC biology 2: 23. doi: 10.1186/1741-7007-2-23.

490 115(6): 727-738.

493 one 11(9): e0163812. doi: 10.1371/journal.pone.0163812.

498 283-295. doi: 10.1002/bdrb.21063.

501 and ethanol treated dams. Brain research 309(2): 209-216.