J Physiol 586.

10 (2008) pp 2651–2664 2651

AMP-activated protein kinase signalling pathways are

down regulated and skeletal muscle development
impaired in fetuses of obese, over-nourished sheep
Mei J. Zhu1 , Bin Han2 , Junfeng Tong1 , Changwei Ma2 , Jessica M. Kimzey1 , Keith R. Underwood1 , Yao Xiao1 ,
Bret W. Hess1 , Stephen P. Ford1 , Peter W. Nathanielsz3 and Min Du1
1
Department of Animal Science and Interdepartmental Molecular and Cellular Life Sciences Program, University of Wyoming,
Laramie, WY 82071, USA
2
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China
3
Center for Pregnancy and Newborn Research, University of Texas, Health Sciences Center, San Antonio, TX 78229, USA

Maternal obesity and over-nutrition give rise to both obstetric problems and neonatal morbidity.
The objective of this study was to evaluate effects of maternal obesity and over-nutrition on
signalling of the AMP-activated protein kinase (AMPK) pathway in fetal skeletal muscle in
an obese pregnant sheep model. Non-pregnant ewes were assigned to a control group (Con,
fed 100% of NRC nutrient recommendations, n = 7) or obesogenic group (OB, fed 150%
of National Research Council (NRC) recommendations, n = 7) diet from 60 days before to
75 days after conception (term 150 days) when fetal semitendinosus skeletal muscle (St) was
sampled. OB mothers developed severe obesity accompanied by higher maternal and fetal
plasma glucose and insulin levels. In fetal St, activity of phosphoinositide-3 kinase (PI3K)
associated with insulin receptor substrate-1 (IRS-1) was attenuated (P < 0.05), in agreement
with the increased phophorylation of IRS-1 at serine 1011. Phosphorylation of AMP-activated
protein kinase (AMPK) at Thr 172, acetyl-CoA carboxylase at Ser 79, tuberous sclerosis 2 at
Thr 1462 and eukaryotic translation initiation factor 4E-binding protein 1 at Thr 37/46 were
reduced in OB compared to Con fetal St. No difference in energy status (AMP/ATP ratio) was
observed. The expression of protein phosphatase 2C was increased in OB compared to Con fetal
St. Plasma tumour necrosis factor α (TNFα) was increased in OB fetuses indicating an increased
inflammatory state. Expression of peroxisome proliferator-activated receptor γ (PPARγ) was
higher in OB St, indicating enhanced adipogenesis. The glutathione : glutathione disulphide
ratio was also lower, showing increased oxidative stress in OB fetal St. In summary, we have
demonstrated decreased signalling of the AMPK system in skeletal muscle of fetuses of OB
mothers, which may play a role in altered muscle development and development of insulin
resistance in the offspring.
(Received 10 December 2007; accepted after revision 21 March 2008; first published online 27 March 2008)
Corresponding author M. Du, Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA.
Email: mindu@uwyo.edu

According to the latest National Health and Nutrition
Examination Survey (1999–2002), 26% of non-pregnant
women 20–39 years of age are overweight and 29%
are obese (Hedley et al. 2004). More importantly, the
shift towards higher gestational weight gain appears
evident (Siega-Riz et al. 2006), showing excessive nutrient
intake during gestation in affluent countries. High
energy diets combined with maternal obesity represent
a special problem because of both adverse effects on
maternal health and fetal development that can result
in harmful, persistent effects in offspring, including
predisposition to obesity and diabetes (Barker, 2002;
Fowden et al. 2006; Nathanielsz, 2006). Many animal
studies on malnutrition during gestation, and some
just now becoming available on maternal obesity and
over-nutrition during gestation, demonstrate that both
a deficient and excessive maternal diet can predispose
offspring to obesity and insulin resistance (Nishina
et al. 2003; Symonds et al. 2003; Bispham et al. 2005;
Fernandez-Twinn et al. 2005, 2006; Zambrano et al. 2006;
Cleal et al. 2007; Nathanielsz et al. 2007). However,
cellular mechanisms linking maternal nutrition and
insulin resistance and obesity of offspring remain poorly
defined.


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2652 M. J. Zhu and others J Physiol 586.10

Skeletal muscle is the main peripheral tissue responsible Table 1. Composition of the diet fed to ewes throughout the
for glucose and fatty acid oxidation (Selak et al. 2003; study
Lowell & Shulman, 2005; Ozanne et al. 2005). The fetal Ingredients (%) Analysed composition4
period is crucial for skeletal muscle development, because Ground bromegrass hay1 14.02 DM (%) 88.54
no net increase in the number of muscle fibres occurs Ground corn 63.89 NDF (% of DM) 24.09
after birth (Greenwood et al. 2000; Nissen et al. 2003). Soybean meal 13.30 ADF (% of DM) 9.99
Impaired fetal skeletal muscle growth will impair the Liquid molasses 5.60 CP (% of DM) 17.39
metabolism of glucose and fatty acids in response to insulin Limestone 2.24 IVDMD (%) 93.92
stimulation and, thus, predispose offspring to diabetes and Ammonium chloride 0.50
obesity in later life (Stannard & Johnson, 2004; Zambrano Mineralized salt2 0.24
et al. 2005). On the other hand, enhanced muscle growth Magnesium chloride 0.10
renders animals resistant to diet-induced obesity and ADE premix3 0.10
insulin resistance (Zhao et al. 2005; Yang & Zhao, 2006). Rumensin 80 0.02
1 Mean particle length = 2.54 cm. 2 Contained 13% NaCl, 10% Ca,
Insulin-like growth factor-1 (IGF-1) and insulin share
a common signalling pathway which stimulates skeletal 10% P, 2% K, 1.5% Mg, 0.28% Fe, 0.27% Zn, 0.12% Mn, 0.01%
muscle growth and development, mainly through the I, 35 p.p.m. Se, and 20 p.p.m. Co. 3 Contained 110 000 IU kg−1
phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) vitamin A, 27 500 IU kg−1 vitamin D and 660 IU kg−1 vitamin
E. 4 DM, dry matter; NDF, neutral detergent fibre; ADF, acid
signalling pathway via insulin receptor substrate-1 (IRS-1)
detergent fibre; CP, crude protein; IVDMD, in vitro dry matter
(Bush et al. 2003; Latres et al. 2005; Park et al. 2005; Song
digestibility..
et al. 2005; Subramaniam et al. 2005; Vary, 2006). Activated
Akt enhances skeletal muscle growth and development
by enhancing protein synthesis and inhibiting protein result of the provision of excessive energy (ATP) for cells,
degradation (Lai et al. 2004). increase fetal TNFα and the level of oxidative stress, and
AMPK is a heterotrimeric enzyme with α, β and affect fetal muscle development.
γ subunits (Hardie, 2004; Kim et al. 2004). The
α subunit is the catalytic unit, the γ subunit has
a regulatory function, and the β subunit provides Methods
anchorage sites for the α and γ subunits (Sambandam &
Care and use of animals
Lopaschuk, 2003). AMPK is switched on by an increase
in the AMP/ATP ratio, leading to phosphorylation of All animal procedures were approved by the University
AMPK at Thr 172 by AMPK kinases (Hardie, 2004; of Wyoming Animal Care and Use Committee. From
Kim et al. 2004). Once activated, AMPK promotes 60 days before conception to day 75 of gestation (day of
glucose uptake and fatty acid oxidation, and inhibits mating, day 0; term 148 days), multiparous Rambouillet/
lipid synthesis in cells (Hardie & Hawley, 2001; Fujii Columbia ewes were fed either a highly palatable diet
et al. 2006). AMPK mediates IGF-1/insulin signalling at 100% (control, Con) of National Research Council
through several possible mechanisms (Jakobsen et al. (NRC) recommendations or 150% (obesogenic, OB) of
2001; Steinberg et al. 2006). Inhibition of AMPK NRC recommendations on a metabolic body weight (BW)
promotes lipogenesis and adipogenesis (Giri et al. basis (BW0.75 ) (Table 1). All ewes were weighed at weekly
2006). intervals and rations adjusted for weight gain, and body
Obesity commonly leads to a low degree inflammation condition was scored at monthly intervals to evaluate
response (Steinberg, 2007), indicated by increased changes in fatness. A body condition score of 1 (emaciated)
production of tumour necrosis factor-α (TNFα). Recently, to 9 (obese) was assigned by two trained observers after
TNFα has been shown to reduce AMPK activity palpation of the transverse and vertical processes of the
in skeletal muscle, associated with up-regulation of lumbar vertebrae (L2 to L5) and the region around the tail
protein phosphatase 2C, an enzyme leading to the head (Sanson et al. 1993).
dephosphorylation of AMPK at Thr 172 (Steinberg et al. Following a 12 h over-night fast on day 75 of gestation
2006). The pregnant sheep model has been extensively (term on day 150 of gestation), 14 pregnant sheep (7
studied to evaluate the regulation of fetal development and Con and 7 OB) were weighed. Sedation was induced
gain insight into problems of human pregnancy (Anderson by intravenous ketamine (10 mg kg−1 ) and anaesthesia
et al. 2001; Anthony et al. 2003; Elmes et al. 2004; Gentili was induced and maintained by isoflurane inhalation.
et al. 2006). The effect of maternal over-nutrition and A sample of maternal blood was collected via jugular
obesity on AMPK activity and fetal muscle development venipuncture into a chilled non-heparinized vacutainer
has not been examined in this model. We hypothesized that tube (no additives, Sigma, St Louis, MO, USA). Serum
maternal over-nutrition in the pregnant ewe would inhibit obtained was frozen at −80◦ C until assayed for insulin
the AMPK signalling pathways in fetal skeletal muscle as a and TNFα. Blood was also collected in a separate chilled


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J Physiol 586.10
tube (heparin plus sodium fluoride; 2.5 mg ml−1 ; Sigma),
and plasma was frozen at −80◦ C until assayed for glucose.
The abdomen and uterus were opened and fetal blood
was collected from the umbilical vein via a 20 gauge
needle and 3 ml syringe, and serum and plasma were
collected and stored as described for maternal blood.
After that, fetuses were quickly removed to obtain weight
and length and the semitendenosus (St) muscles on both
sides were collected and snap-frozen in liquid nitrogen
for biological analyses. Fetal muscle collected from five
ewes carrying twin pregnancy in each group was randomly
selected for analyses. No difference in body weight was
observed between twins and thus only one fetus of the twin
pregnancy was selected at random for analyses. Though
no difference in weight was observed among fetuses of
different sexes, the sex of fetuses in each group was
balanced.
Sheep were killed by administration of an overdose of
sodium pentobarbital (Abbott Laboratories, Abbott Park,
IL, USA) and exsanguinated. The St muscle from the left
side of each pregnant sheep was dissected immediately
and weighed. After trimming off all visible adipose and
connective tissues, a small piece of muscle (1 g) was
sampled in the anatomical centre of the muscle and
snap-frozen in liquid nitrogen for biological analyses.
Antibodies
Antibodies against AMPK, phospho-AMPK at Thr 172,
acetyl-CoA carboxylase (ACC), phospho-ACC at Ser 79,
IRS-1, phospho-IRS-1 at Ser 1101, Akt, phospho-Akt at
Ser 473, mTOR, phospho-mTOR at Ser 2448 l, phospho-
tuberous sclerosis 2 (TSC2) at Thr 1462, eukaryotic
translation initiation factor 4E-binding protein 1 (4E-BP1)
at Thr 37/46, peroxisome proliferator-activated receptor
γ (PPARγ ), and horseradish peroxidase linked secondary
antibody were purchased from Cell Signalling (Danvers,
MA, USA). Protein phosphatase 2C (PP2C) antibody
was purchased from Epigenomics (Berlin, Germany).
Cytochrome C antibody was purchased from EMD Chem.
Inc. (Gibbstown, NJ, USA). Anti-β-actin antibody was
obtained from Developmental Studies Hybridoma Bank
(DSHB, Iowa City, IA, USA).
Immunoblotting
St muscle (0.1 g) was powdered in liquid nitrogen and
homogenized in a polytron homogenizer (7 mm dia.
generator) with 400 μl of ice-cold buffer containing
137 mm NaCl, 50 mm Hepes, 2% SDS, 1% NP-40, 10%
glycerol, 2 mm PMSF, 10 mm sodium pyrophosphate,
10 μg ml−1 aprotinin, 10 μg ml−1 leupeptin, 2 mm
Na3 VO4 and 100 mm NaF, pH 7.4. The protein content of
lysates was determined by the Bradford method (Bio-Rad

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AMPK and fetal skeletal muscle 2653
Laboratories, Hercules, CA, USA) (Zhu et al. 2006) and
used for immunoblotting analyses as previously described
(Zhu et al. 2004).
IRS-1 associated PI3K activity analyses
The IRS-1-associated PI3K activity was analysed by
immunoprecipitation with anti-IRS-1 antibody (Kirwan
et al. 2000). Briefly, St muscles were lysed in a
buffer containing 50 mm Hepes pH 7.4, 1% NP-40 or
Triton-X 100, 10% glycerol, 2 mm Na3 VO4 , 100 mm NaF,
1% protease inhibitor cocktail, 1 mm MgCl2 , 1 mm CaCl2
and 2.5 mm EDTA. A 1 mg sample of total protein
was immunoprecipitated with 4 μg of IRS-1 antibody
and 40 μl protein A–Sepharose slurry. The pellet was
suspended in an assay buffer containing 0.1 mm ATP,
50 mm Hepes, pH 7.1, 1 mm EGTA, 20 mm MgCl2 ,
20 mm NaCl, 1 mm NaH2 PO4 , 0.2 m NaH2 CO3 and 5 μCi
of 32 P-γ ATP (GE Healthcare, Piscataway, NJ, USA).
l-Phosphatidylinositol (10 μg each−1 reaction) was used
as a substrate. The reaction mixture was incubated at 30◦ C
for 10 min. The reaction was stopped by adding 1 n HCl.
The phosphatidylinositol 3-phosphate was extracted with
chloroform : methanol (1 : 1). The lower phase containing
chloroform and extracted lipids was used for separation by
thin layer chromatography. The radioactivity incorporated
into phosphatydylinositol 3-phosphate was determined by
autoradiography (Kirwan et al. 2000).
Adenine nucleotides, creatine and phosphocreatine
analysis
St muscle homogenates (200 μl) obtained as described
above were added to 55% perchloric acid to a final
concentration of 5%, set for 30 min on ice, and then
neutralized to pH 6–8 with 2 m KOH. After centrifugation
at 13 000 g, 4◦ C for 10 min to remove KClO4 , the super-
natant was passed through a 0.2 μm filter prior to HPLC
analysis (Beckman Instruments, Inc., Fullerton, CA, USA).
HPLC conditions were the same as previously reported
(Shen et al. 2006). Lysate protein contents were determined
using a protein assay kit (Bio-Rad). Adenine nucleotides,
creatine and phosphocreatine contents are expressed as
micromoles per gram of muscle.
Glucose, insulin and TNFα analyses
Glucose was analysed using the Infinity. (ThermoTrace
Ltd, Cat. no. TR15498; Melbourne, Australia)
colourimetric assay modified in the following manner:
plasma was diluted 1 : 5 in distilled water, and 10 μl of
diluted plasma was added to 300 μl reagent mix. Insulin
was measured by radioimmuno assay in accordance with
the manufacturer’s recommendations (Coat-A-Count,
2654 M. J. Zhu and others J Physiol 586.10

Diagnostic Products Corporation, Los Angeles, CA, USA). Table 2. Body condition score, body weight, serum IGF-1, insulin
TNFα was analysed by using an ELISA kit in accordance and glucose, and semitendenosus (St) muscle weight of OB and
Con ewes and fetal sheep at 75 days gestation
with manufacturer’s recommendations (Endrogen, Cat.
no. ESS0011B; Rockford, IL, USA). All samples were run Category Con OB P value
in triplicate. Maternal
At the beginning of treatments
Body condition score 4.9 ± 0.4 5.0 ± 0.3
Glutathione : glutathione disulphide (GSH : GSSG) Body weight (kg) 68.3 ± 2.9 71.6 ± 3.2
ratio At the end of treatments
Body condition score 4.7 ± 0.4 8.0 ± 0.2 ∗∗
St muscle samples were homogenized in 4 volumes (w/v) ∗∗
Body weight (kg) 72.2 ± 3.3 102.2 ± 2.4
of 1% picric acid. Acid homogenates were centrifuged
Insulin (μIU ml−1 ) 4.8 ± 1.6 25.0 ± 4.0 ∗∗
at 16 000 g (30 min) and supernatant fractions collected.
Glucose (mg dl−1 ) 52.1 ± 3.4 70.2 ± 6.3 ∗
Supernatant fractions were assayed for total GSH and St muscle (g) 200.5 ± 22.4 267.2 ± 13.3 ∗
GSSG by the standard recycling method and GSH content
Fetal
was determined using a standard curve generated from ∗
Body weight (g) 268 ± 12 374 ± 10
known concentrations of GSH. The procedure consisted of Crown rump length (cm) 22.1 ± 0.2 24.6 ± 0.2 ∗
using one-half of each sample for GSSG determination and Glucose (mg dl−1 ) 25.35 ± 2.10 43.63 ± 5.58 ∗
the other half for GSH. Samples for GSSG determination IGF-1 (ng ml−1 ) 44.6 ± 0.9 53.3 ± 1.8 ∗

were incubated at room temperature with 2 μl of 4-vinyl Insulin (IU ml−1 ) 1.14 ± 0.35 7.29 ± 1.31 ∗∗

pyridine per 100 μl sample for 1 h after vigorous vortexing. St muscle (g) 0.49 ± 0.01 0.65 ± 0.05 ∗

Incubation with 4-vinyl pyridine conjugates any GSH
present in the sample so that only GSSG is recycled to
GSH without interference by GSH. The GSSG (as GSH
x2) was then subtracted from the total GSH to calculate
the ratio of GSH : GSSG (Li et al. 2005).
Real-time quantitative PCR (RT-PCR)
mRNA was extracted from the fetal St muscle using TRI
reagent (Sigma) and reverse transcribed into cDNA using
a kit (Qiagen, Valencia, CA, USA). Reversed transcribed
cDNAs were used for real-time PCR analyses by using
a SYBR Green RT-PCR kit from Bio-Rad (Hercules).
Primer sets used were: PPPARγ forward, 5 -CCGCA-
TCTTCCAGGGGTGTC-3 ; and reverse, 5 -CAAGGA-
GGCCAGCATCGTGAAAT-3 . The 18S RNA was used as
a control, forward, 5 -GTAACCCGTTGAACCCCATT-3 ;
and reverse, 5 -CCATCCAATCGGTAGTAGCG-3 . PCR
conditions were as follows: 20 s at 95◦ C, 20 s at 56◦ C,
and 20 s at 72◦ C for 35 cycles (Lomax et al. 2007). After
amplification, a melting curve (0.01◦ C s−1 ) was used to
confirm product purity. Data were expressed relative to
18S rRNA as previously described (Lomax et al. 2007).
Means ± S.E.M. n = 7 for maternal data and n = 5 for fetal data.
∗∗ P < 0.01, ∗ P < 0.05.
to primary myofibres was reported. Only those fasciculi
where primary and secondary myofibres were clearly
differentiated were counted (Zhu et al. 2004). In addition,
the relative density of muscle fibres was calculated by
counting all muscle fibres in each microscopic view.
Statistical analysis
Statistical analyses were conducted according to our
previous studies in sheep (Zhu et al. 2004, 2006). Briefly,
each animal was considered as an experimental unit.
Data were analysed as a complete randomized design
using GLM (General Linear Model of Statistical Analysis
System, SAS, 2000). The differences in the mean values
were compared by Tukey’s multiple comparison, and
mean ± standard errors of mean (s.e.m.) were reported.
Statistical significance was considered as P < 0.05.

Histochemical analyses
St muscle samples frozen in OCT compound (Tissue-Tek,
Sakura Finetek USA, Inc., Torrance, CA, USA) were
cut into 10 μm sections. Sections were stained with
haematoxylin–eosin for standard light microscopy. The
number of primary and secondary myofibres were counted
in 10 different microscopic fields at 200 × magnification
of each section and 10 sections (every fifth section)
were viewed for each sample. The ratio of secondary
Results
Fetal and maternal phenotypic changes, and serum
glucose and insulin levels
There was no difference in maternal body weight and
body condition score at the beginning of treatments
(Table 2). However, by the end of treatment, the body
weight and body condition score were much higher in OB
sheep compared to Con sheep (Table 2), showing that OB
sheep developed severe obesity. Maternal plasma glucose


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J Physiol 586.10 AMPK and fetal skeletal muscle 2655

and insulin concentrations were elevated (P < 0.05) in OB
versus Con ewes (Table 2).
Plasma glucose, IGF-1 and insulin concentrations were
elevated in fetuses of OB versus Con ewes. Body weight
and crown rump length of fetuses of OB ewes were ∼30%
greater than fetuses from Con sheep, as well as fetal muscle
weight (Table 2).
Akt and mTOR signalling pathway activity
in fetal St muscle
Phosphorylation of Akt at Ser 473, which is correlated with
its activation, was reduced by 41.2% and total Akt content
increased in the fetal muscle of OB sheep (Fig. 1A). TSC2
is a mediator between Akt and mTOR. Its phosphorylation
at Thr 1462 was reduced by 27.5% (Fig. 1B). mTOR
phosphorylation at Ser 2448 was reduced 29.1% in OB fetal
muscle without alteration in its total content (Fig. 1C).
mTOR signalling is a key signalling pathway controlling
protein synthesis. It controls protein synthesis through
phosphorylation of 4E-BP1 and S6K. The phosphorylation
of 4E-BP1 at Thr 37/46 was also reduced (Fig. 1D).
AMPK and ACC phosphorylation in fetal
St skeletal muscle
Phosphorylation of AMPK α subunit at Thr 172 and its
down-stream effector ACC at Ser 79 are frequently used to
assess AMPK activity. AMPK phosphorylation at Thr 172,
which is correlated with its activity, was down-regulated
in fetal St muscle of OB sheep compared to Con sheep
(14.9%, Fig. 2A). No change in the protein expression
of AMPK α subunits was observed. Phosphorylation of
ACC at Ser 79 is directly catalysed by AMPK and, thus,
its phosphorylation correlates with AMPK activity. ACC
phosphorylation was also reduced (18.9%) in fetal St

Figure 1. Immunoblots and combined data for Akt, mTOR and their phosphorylation in fetal St muscle
of Con (open bars) and OB (filled bars) sheep
A, Akt and phospho-Akt (p-Akt) immunoblots of fetal muscle; B, TSC2 and phospho-TSC2 (p-TSC2) immunoblots
of fetal muscle; C, mTOR and phospho-mTOR (p-mTOR) immunoblots of fetal muscle; D, 4E-BP1 immunoblots of
fetal muscle. ∗ P < 0.05. Mean ± S.E.M.; n = 5.


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2656 M. J. Zhu and others J Physiol 586.10

Figure 2. Immunoblots and combined data for AMPK, ACC and their phosphorylation in fetal St muscle
of Con (open bars) and OB (filled bars) sheep
A, AMPK and phospho-AMPK (p-AMPK) immunoblots of fetal muscle; B, acetyl-CoA carboxylase (ACC) and
phospho-ACC (p-ACC) immunoblots of fetal muscle. ∗ P < 0.05; ∗∗ P < 0.01. Mean ± S.E.M.; n = 5.

muscle of OB sheep, in the absence of any change in total
ACC concentration (Fig. 2B).
IRS-1 associated PI3K activity in fetal skeletal muscle
IRS-1 is a key mediator of IGF-1/insulin signalling. IRS-1
regulates IGF-1/insulin signalling through altering its
ability to recruit PI3K. The content of IRS-1 was not
altered but the phosphorylation of IRS-1 at Ser 1101
(this phosphorylation included both IRS-1 and IRS-2
since this antibody cross-reacts with IRS-2) was increased
(93.6%) in fetal St muscle of OB sheep compared to Con
sheep (Fig. 3A). Consistent with this increase in serine
phosphorylation of IRS-1, the PI3K activity associated
with IRS-1 was lower in fetal St muscle of OB sheep
compared to Con sheep (Fig. 3B).
Adenosine nucleotides, creatine and phosphocreatine
contents in fetal St skeletal muscle
There were no differences in ATP, ADP, AMP, creatine
and phosphocreatine contents between Con and OB sheep
for fetal muscle, indicating that AMPK inhibition was not
due to alteration in energy status (Table 3). No differences

Figure 3. Immunoblots and combined data for IRS-1 and its phosphorylation, and IRS-1-associated PI3K
activity in fetal St muscle of Con (open bars) and OB (filled bars) sheep
A, IRS-1 and phospho-IRS-1 immunoblots of fetal muscle; B, PI3K activity associated with IRS-1 in fetal muscle
measured by immunoprecipitation. ∗ P < 0.05. Mean ± S.E.M.; n = 5.


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Table 3. ATP, AMP, ADP, creatine and phosphocreatine

concentrations in fetal semitendinosus muscle of OB and
4

GSH/GSSG ratio
Con sheep at 75 days gestation
3
Con OB

ATP (μmol (g muscle)−1 ) 0.36 ± 0.09 0.38 ± 0.08 2 *

ADP (μmol (g muscle)−1 ) 0.42 ± 0.06 0.51 ± 0.03
AMP (μmol (g muscle)−1 ) 0.48 ± 0.04 0.53 ± 0.04 1
AMP/ATP ratio 1.87 ± 0.39 1.63 ± 0.50
Creatine phosphate 7.38 ± 0.28 7.37 ± 0.36 0
(μmol (g muscle)−1 )
Creatine (μmol (g muscle)−1 ) 33.46 ± 1.26 35.28 ± 1.10
Con OB
Creatine/creatine phosphate ratio 4.55 ± 0.13 4.83 ± 0.14 Figure 5. Glutathione : glutathione disulphide (GSH : GSSG)
Data are presented as means ± S.E.M. No differences were ratio in fetal muscle of Con (open bars) and OB (filled bars) fetal
St muscle
observed. n = 5. ∗ P < 0.05. Mean ± S.E.M.; n = 5.

in creatine and phosphocreatine contents were observed

(Table 3).
TNFα content in the fetal circulation and oxidative
stress in fetal muscle
Obesity leads to low-grade inflammation. As a marker
of inflammation, TNFα leads to AMPK inhibition. To
identify whether TNFα was a reason for AMPK inhibition,
its concentration was measured in the fetal circulation.
The TNFα content was higher in OB fetuses compared to
Con (Fig. 4A). In agreement with this increase in TNFα
content, the expression of PP2C was enhanced in OB fetal
St muscle compared to Con muscle (Fig. 4B).
The ratio of GSH : GSSG was lower in OB fetal muscle,
indicating that OB fetal skeletal muscle experienced
oxidative stress compared to Con fetal muscle (Fig. 5).
Fetal skeletal muscle development
To test whether the down-regulation of AMPK signalling
in the presence of increased plasma levels of fetal IGF-1 and
insulin affected fetal muscle development, sections of fetal
St muscle were stained with haematoxylin and eosin. Both
primary fibres with centrally located nuclei and secondary
muscle fibres with peripherally located nuclei were clearly
visible forming primary muscle bundles (Fig. 6A and B).
No difference in the ratio of primary to secondary muscle
fibres was observed (Fig. 6C). However, the density of
muscle fibres per area was significantly lower in OB fetal
muscle compared to Con fetal muscle (Fig. 6D).
The content and mRNA expression of PPARγ were
enhanced in OB fetal muscle compared to Con fetal muscle
(Fig. 7), indicating enhanced adipogenesis.
Discussion
Value of pregnant sheep as a model for studying
fetal development as affected by maternal obesity
and over-nutrition
Obesity and over-nutrition pose an ever increasing health
threat to pregnant women and their fetuses (Catalano &

Figure 4. TNFα concentration in fetal plasma and expression of PP2C in fetal St muscle of Con (open
bars) and OB (filled bars) sheep
A, TNFα concentration in fetal plasma; B, PP2C immunoblot of fetal St muscle. ∗ P < 0.05. Mean ± S.E.M.; n = 5.


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2658 M. J. Zhu and others J Physiol 586.10

Figure 6. Hematoxylin and eosin staining of fetal St muscle

A, Con fetal muscle; B, OB fetal muscle; C, secondary to primary muscle fibre ratio; D, density of muscle fibres.
∗ P < 0.05. Mean ± S.E.M.; n = 5.

Ehrenberg, 2006; Siega-Riz et al. 2006) and are associated
with several obstetric problems as well as an increased
risk of obesity and diabetes in offspring (Warram et al.
1990; Petersen et al. 2004; Petersen et al. 2005). However,
the associated mechanisms are unclear. Most of the recent
studies on the effects of maternal over-nutrition on fetal
development have been conducted in rodents (Armitage
et al. 2005; Reusens et al. 2007). Rodent studies provide
data of great value; however, it must always be born in
mind that rodents are polytocous animals with a large

Figure 7. PPARγ content and mRNA expression in fetal St muscle of Con (open bars) and OB (filled bars)
sheep
A, PPARγ immunoblot and statistical data; B, PPARγ mRNA expression measured by RT-PCR. ∗ P < 0.05.
Mean ± S.E.M.; n = 5.


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fetal and placental biomass relative to maternal biomass
with important nutritional consequences. In addition,
offspring are born at a very altricial level without having
completed many stages of development that are completed
in utero in precocial mammals such as sheep or primates. In
addition, rodents tend to decrease food consumption when
fed high-energy-containing diets. To avoid this problem,
obese rat and mouse strains (carrying a mutation in leptin
or leptin receptor which impairs satiety control), such as
Zucker rat carrying a leptin receptor missense mutation
(Phillips et al. 1996) and ob/ob mice with leptin deficiency
(Pelleymounter et al. 1995), are frequently used in obesity
studies. However, leptin is a known crucial mediator of
placental growth (Szczepankiewicz et al. 2006; Lepercq
et al. 2007; O’Connor et al. 2007) and extensively involved
in regulation of metabolism in pregnancy.
The pregnant sheep has been extensively studied to
evaluate the effects of decreased or increased nutritional
supply to the fetus and the effects on fetal growth
and development and pregnancy outcomes (Battaglia
& Meschia, 1986; DiGiacomo & Hay, 1990; Hay, 1995;
Anderson et al. 2001, 2005; Anthony et al. 2003; Elmes
et al. 2004; Manikkam et al. 2004; Gentili et al. 2006; Zhu
et al. 2007; Muhlhausler et al. 2007; Steckler et al. 2007;
Taylor & Poston, 2007). However, no fetal studies have
been reported in ewes made obese prior to pregnancy.
We present here data from sheep made obese prior to
pregnancy and maintained at an elevated body weight
during pregnancy.
The body condition score and body weight of OB
sheep were much higher than those in Con sheep. Plasma
insulin and glucose levels in both maternal and fetal
circulations were increased in OB sheep. Furthermore, we
recently determined that OB sheep on a dietary protocol
identical to that utilized in this study exhibited marked
insulin resistance and glucose intolerance to a bolus i.v.
injection of glucose on day 75 of gestation (SP Ford, MM
Miller, MJ Zhu, L Zhang, BW Hess, GE Moss & PW
Nathanielsz, unpublished observations). Fetal weight and
crown rump length were increased in OB ewes, showing
enhanced fetal growth. These general data show that this
model has potential for studying the negative physiological
consequences of maternal obesity and over-nutrition on
the fetus.
Effect of maternal obesity on the AMPK and m-TOR
signalling pathways
The phosphoinositide-3 kinase/protein kinase B
(PI3K/Akt) and AMPK signalling pathways interact to
control the activation of mTOR signalling and play a
central role in cell energetics and protein synthesis. The
down-stream targets of mTOR signalling are proteins
involved in mRNA translation, including 4E-BP1 and
S6K.

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AMPK and fetal skeletal muscle 2659
IRS-1 plays a key regulatory role at the entrance to the
mTOR pathway. We observed that PI3K activity associated
with IRS-1 was reduced in OB fetal skeletal muscle.
To determine potential mechanisms for the functional
impairment of IRS-1, we analysed AMPK activity as
assessed by AMPK phosphorylation at Thr 172 and ACC
phosphorylation at Ser 79, a method widely used by
other researchers (Hutchinson & Bengtsson, 2006; Han
et al. 2007; Tanaka et al. 2007). In a previous study, we
have shown that phosphorylation of AMPK and ACC is
highly correlated with AMPK activity (Shen et al. 2007),
though there are reports that phosphorylation status and
measured activity were not well correlated (Richter et al.
2004).
AMPK activation sensitizes insulin/IGF-1 signalling
through several possible mechanisms. Activation of
AMPK phosphorylates IRS-1 at Ser 789 which enhances
IGF/insulin signalling (Jakobsen et al. 2001), though
this claim has been disputed (Qiao et al. 2002). In
addition, AMPK promotes lipid oxidation and inhibits
lipid synthesis, thereby reducing cellular fatty acyl-CoA
content and protein kinase C (PKC) activity, as a result of
which IRS-1 function and insulin sensitivity are improved
(Steinberg et al. 2006). On the other hand, AMPK
inhibition led to the accumulation of lipids in cells,
impairing IRS-1 function. Indeed, the phosphorylation
of IRS-1 at Ser 1101, a site phosphorylated by PKC,
was enhanced in OB fetal muscle compared to Con
muscle. Furthermore, it has been reported that AMPK
inhibits mTOR by phosphorylation of TSC2 at Thr 1227
and Ser 1345 (Inoki et al. 2003). Since activated mTOR
phosphorylates IRS-1 at Ser 636/639, which negatively
affects IRS-1 function and down-regulates insulin
signalling (Um et al. 2004; Khamzina et al. 2005), the
down-regulation of AMPK should activate mTOR, which
de-sensitizes insulin signalling. However, in this study,
both mTOR and AMPK phosphorylation were lower in
OB compared to Con fetal muscle. This down-regulation
of mTOR in OB fetal muscle may well be due to the
down-regulation of Akt, its main upstream kinase in OB
fetal muscle. The resulting down-regulation of mTOR
may be only partially compensated by the inhibition
of AMPK, resulting in an overall inhibition of mTOR
signalling in OB fetal muscle. In support of the above
notions, activation of AMPK pharmacologically reverses
insulin resistance (Fisher et al. 2002; Inoki et al. 2003;
Bandyopadhyay et al. 2006). Knockout of the AMPKα2
catalytic subunit in mice leads to insulin resistance (Viollet
et al. 2003). Therefore, the reduction of AMPK activity
we observed in fetal muscle in OB sheep should provide
the mechanism leading to IRS-1 function impairment
and the down-regulation of IGF-1/insulin down-stream
signalling. Inhibition of AMPK in mature skeletal muscle
has been observed in high-fat-fed rats (Liu et al. 2006;
Ye et al. 2006). However, this is the first demonstration
2660 M. J. Zhu and others
that AMPK activity is inhibited in fetal skeletal muscle
at mid-gestation in the presence of maternal obesity and
over-nutrition.
Mechanisms leading to AMPK inhibition in fetal
skeletal muscle of OB sheep
Low cellular energy status as indicated by low
ATP/AMP and phosphocreatine/creatine ratios activates
AMPK. By providing excessive nutrients to cells,
over-nutrition should enhance cellular energy level.
However, no difference was observed in ATP/AMP and
phosphocreatine/creatine ratios between OB and Con
sheep. These data show that the AMPK inhibition in the
OB fetal skeletal muscle was probably due to changes in
other regulatory systems than a decreased energy level in
the fetal muscle cells.
Recently, hormones/cytokines have been shown to act as
mediators of AMPK activity. Leptin, a hormone secreted by
adipocytes, increases AMPK activity through alteration of
the ATP/AMP ratio in skeletal muscle (Minokoshi et al.
2002). Since no significant alteration in the ATP/AMP
ratio was observed, leptin seems unlikely to be responsible
for the inhibition of AMPK activity in fetal and maternal
muscle of OB compared to Con sheep. In addition, we have
shown that fetal leptin levels are below the sensitivity of
available assays in both the CON and OB fetuses (MJ Zhu,
M Du, SP Ford, PW Nathanielsz, unpublished data).
Obesity commonly leads to a low-grade inflammation
response (Steinberg, 2007), which is associated with the
increased production of TNFα. TNFα reduces AMPK
activity in skeletal muscle through up-regulation of PP2C,
an enzyme dephosphorylating Thr 172 at the AMPK
α subunit (Steinberg et al. 2006; Shen et al. 2007). Our data
show that maternal obesity and over-nutrition increase
TNFα levels in the fetal circulation and the expression
of PP2C was increased in OB fetal muscle compared to
Con muscle. A high level of TNFα is known to induce
IGF-1/insulin resistance in skeletal muscle (Peraldi &
Spiegelman, 1998), which is linked to AMPK inhibition
(Steinberg et al. 2006; Li et al. 2007), consistent with our
observations. Therefore, the high level TNFα we have
demonstrated in the fetal circulation may play a role in
the decreased signalling of the AMPK pathway we have
shown and would also tend to increase insulin resistance
in fetal skeletal muscle. In addition, we observed increased
oxidative stress in OB fetal muscle, which is consistent with
an inflammation response indicated by high TNFα in fetal
circulation.
Mitochondrial function is associated with insulin
sensitivity and AMPK is known to increase mitochondrial
protein expression in skeletal muscle. To assess whether the
inhibition of AMPK led to the alteration in mitochondrial
protein expression, we analysed the expression of
cytochrome C, a protein in mitochondria. However, no
J Physiol 586.10
significant difference was detected between treatments
(Con versus OB, 1.00 ± 0.10 versus 0.91 ± 0.09 arbitrary
units). The possible reason could be due to the low
mitochondrial content in fetal muscle compared to adult
muscle (Lee et al. 2005). It will be interesting to observe
long-term effects of such changes on the mitochondrial
function of adult muscle.
Fetal muscle development due to obesity
During prenatal muscle development, primary myofibres
are first formed, followed by the formation of secondary
myofibres (Beermann et al. 1978). Primary myofibres
have centrally located nuclei and larger diameters while
secondary muscle fibres have peripherally located nuclei
and small sizes (Swatland, 1973; Beermann, 1978). The
secondary myofibres are derived from muscle precursor
cells which are initially maintained in a proliferating,
undifferentiated state (Swatland, 1973). Those precursor
cells differentiate into myoblasts and fuse to form
secondary myofibres parallel to primary myofibres
(Beermann, 1978). The number of secondary fibres
present in prenatal muscles is susceptible to maternal
nutrients (Zhu et al. 2004). In this study, maternal obesity
and over-nutrition did not affect the ratio of secondary to
primary muscle fibres halfway through gestation. Maternal
nutrient deficiency reduces muscle fibre numbers and
secondary to primary ratio (Ward & Stickland, 1991;
Dwyer et al. 1994; Zhu et al. 2004). To our knowledge,
the effect of maternal obesity and over-nutrition on the
ratio of secondary to primary muscle fibres has not
been examined previously. In this study, we observed
that the density of muscle fibres was reduced in OB
fetuses compared to Con fetuses. The reason for the
reduced muscle fibre density is unclear, but may be
associated with an increase in adipogenesis (diversion from
myogenesis to adipogenesis) and lipid accumulation. The
expression of PPARγ , a marker of adipogenesis, was
higher in OB fetal muscle compared to Con fetal muscle.
Though PPARγ is also expressed in skeletal muscle, its
level is very low (Vidal-Puig et al. 1996; Verma et al.
2004). Therefore, the elevated PPARγ expression we have
observed probably relates to enhanced adipogenesis in
adipocytes between the muscle cells. Another possible
reason is due to inflammation. Inflammation in fetal
muscle due to maternal obesity might contribute to the
accumulation of fluid and other extracellular substrates,
resulting in the reduction in muscle fibre density. These
data showed that maternal obesity and over-nutrition
affected fetal muscle development.
Adipocytes existing inside muscle bundles may secrete
adipokines, including TNFα, that would have paracrine
effects on muscle insulin signalling leading to insulin
resistance (Sell et al. 2006). In vivo mechanisms
controlling adipogenesis from pluripotent cells within

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J Physiol 586.10
fetal muscle fibres are poorly defined, though there
are numerous in vitro cell culture studies (Rosen
& MacDougald, 2006) that demonstrate that PPARγ
and CCAAT-enhancer-binding proteins (C/EBPs) are
crucial intracellular factors controlling adipogenesis. Their
expression leads to adipogenesis from pluripotent cells. We
observed that the expression of PPARγ was enhanced at
both the protein and message level in fetal muscle due to
maternal obesity and over-nutrition. Activation of AMPK
inhibits adipogenesis in 3T3L1 cells and also in obese
mice (Giri et al. 2006). The enhancement of adipogenesis
may also relate to the inflammatory response. An altered
cytokine environment has been shown to direct cells to an
adipogenic phenotype during muscle regeneration in adult
mice (Contreras-Shannon et al. 2007). A similar shift from
myogenesis to adipogenesis may have potential impacts
on the functionality of muscle in offspring by increased
adipokine effects on muscle function.
The effect of maternal obesity and over-nutrition on
the insulin/IGF-1 signalling in ovine fetal muscle has
not been examined previously. Here, we observed that
the down-stream signalling of insulin/IGF-1 signalling
pathways were attenuated in OB fetal muscle, which
impacts on fetal muscle development.
In order to identify mechanisms leading to
down-regulation of the down-stream insulin/IGF-1
signalling, we examined the function of IRS-1, which is
a key site mediating insulin/IGF-1 signalling through its
ability to recruit PI3K (Shao et al. 2002). In agreement
with the down-regulation of insulin/IGF-1 down-stream
signalling pathways observed in OB sheep, the PI3K
activity associated with IRS-1 was reduced in fetal St
muscle. Therefore, this attenuation of insulin/IGF-1
down-stream signalling in OB sheep at least partially
occurs due to the functional impairment of IRS-1.
The observation that both IGF-I and insulin were
elevated in the fetal circulation of the OB fetuses in the
presence of decreased phosphorylation of key components
of the AMPK and mTOR signalling pathways indicates the
existence of resistance to the action of these two potent
growth factors. Rats feeding on a high-fat diet developed
insulin/IGF-1 resistance in skeletal muscle (Guo & Zhou,
2004). The proximate causes of the observed changes in
fetal skeletal muscle remain to be determined.
In conclusion, our data indicate that pregnant sheep
feeding on 150% of nutrient requirements provide a good
model for studying the physiological consequences on
fetal skeletal muscle development of obese pregnancy.
Signalling in the AMPK and mTOR pathways was
down-regulated in fetal St muscle of OB sheep despite
high plasma IGF-1/insulin levels. This down-regulation
of AMPK activity in OB fetal skeletal muscle was not
due to an alteration in energy status (ATP/AMP) in
muscle. Increased TNFα, an indicator of inflammation
associated with obesity, may be a major factor responsible
for the down-regulation of AMPK. We hypothesize that the

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AMPK and fetal skeletal muscle 2661
changes described may play a role in the insulin resistant
phenotype that has been demonstrated in the offspring of
obese mothers.
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Acknowledgements
The authors would like to thank Dr Myrna Miller and Mr
Ryan Gustafson for assistance with animal care and tissue
collection. The monoclonal antibody of actin developed by
Jim Jung-Ching Lin was obtained from the Developmental
Studies Hybridoma Bank developed under the auspices of the
NICHD and maintained by the University of Iowa, Department
of Biological Sciences, Iowa City, IA 52242, USA. This work
was supported by NIH INBRE P20 RR016474-04 and Research
Initiative Grant 2006-55618-16914 and 2007-35203-18065 from
the USDA Cooperative State Research, Education and Extension
Service.

C 2008 The Authors. Journal compilation 
C 2008 The Physiological Society