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Metabolic dysregulation and
decreased capillarization in skeletal
muscles of male adolescent
offspring rats exposed to
gestational intermittent hypoxia
Wirongrong Wongkitikamjorn
1
,
2†
, Eiji Wada
3†
, Jun Hosomichi
1
,
Hideyuki Maeda
4
, Sirichom Satrawaha
2
, Haixin Hong
1
,
5
,
Ken-ichi Yoshida
4
, Takashi Ono
1
and Yukiko K. Hayashi
3
*
1
Department of Orthodontic Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and
Dental University (TMDU), Tokyo, Japan,
2
Department of Orthodontics, Faculty of Dentistry, Chulalongkorn
University, Bangkok, Thailand,
3
Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan,
4
Department of Forensic Medicine, Tokyo Medical University, Tokyo, Japan,
5
Department of Stomatology,
Shenzhen University General Hospital, Shenzhen, China
Gestational intermittent hypoxia (IH) is a hallmark of obstructive sleep apnea that
occurs frequently during pregnancy, and effects caused by this environmental
change during pregnancy may be transmitted to the offspring. In this study, we
aimed to clarify the effects of IH in pregnant rats on the skeletal muscle of adolescent
offspring rats. Mother rats underwent IH from gestation day 7–21, and their 5-weeks-
old male offspring were analyzed. All male offspring rats were born and raised under
normoxia conditions. Although no general growth retardation was observed, we
found that exposure to gestational IH reduces endurance running capacity of
adolescent offspring rats. Both a respiratory muscle (diaphragm; DIA) and a limb
muscle (tibialis anterior; TA) showed no histological abnormalities, including fiber
size and fiber type distribution. To identify the possible mechanism underlying the
reduced running capacity, regulatory factors associated with energy metabolism
were analyzed in different parts of skeletal muscles. Compared with rats born under
conditions of gestational normoxia, gestational IH offspring rats showed significantly
lower expression of genes associated with glucose and lipid metabolism, and lower
protein levels of phosphorylated AMPK and AKT. Furthermore, gene expression of
adiponectin receptors one and two was significantly decreased in the DIA and TA
muscles. In addition, the DIA muscle from adolescent rats had significantly decreased
capillary density as a result of gestational IH. However, these changes were not
observed in a sucking muscle (geniohyoid) and a masticating muscle (masseter) of
these rats. These results suggest that respiratory and limb muscles are vulnerable to
gestational IH, which induces altered energy metabolism with decreased aerobic
motor function. These changes were partially owing to the decreased expression of
adiponectin receptors and decreased capillary density in adolescent offspring rats.
KEYWORDS
gestational intermittent hypoxia, skeletal muscle, developmental origins of health and
disease (DOHaD), energy metabolism, adiponectin receptors, capillarization
OPEN ACCESS
EDITED BY
Bruno Bastide,
Lille University of Science and Technology,
France
REVIEWED BY
Emiliana Giacomello,
University of Trieste, Italy
Alicia Mayeuf-Louchart,
Institut National de la Santé et de la
Recherche Médicale (INSERM), France
*CORRESPONDENCE
Yukiko K. Hayashi,
yhayashi@tokyo-med.ac.jp
†
These authors have contributed equally to
this work and share first authorship
SPECIALTY SECTION
This article was submitted to Striated
Muscle Physiology,
a section of the journal
Frontiers in Physiology
RECEIVED 12 October 2022
ACCEPTED 03 January 2023
PUBLISHED 12 January 2023
CITATION
Wongkitikamjorn W, Wada E, Hosomichi J,
Maeda H, Satrawaha S, Hong H,
Yoshida K-i, Ono T and Hayashi YK (2023),
Metabolic dysregulation and decreased
capillarization in skeletal muscles of male
adolescent offspring rats exposed to
gestational intermittent hypoxia.
Front. Physiol. 14:1067683.
doi: 10.3389/fphys.2023.1067683
COPYRIGHT
© 2023 Wongkitikamjorn, Wada,
Hosomichi, Maeda, Satrawaha, Hong,
Yoshida, Ono and Hayashi. This is an open-
access article distributed under the terms
of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that
the original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
Frontiers in Physiology frontiersin.org01
TYPE Original Research
PUBLISHED 12 January 2023
DOI 10.3389/fphys.2023.1067683
1 Introduction
Obstructive sleep apnea (OSA) is prolonged partial and/or
intermittent complete airway obstruction during sleep (ATS
American Thoracic Society, 1996), which consequently leads to
intermittent hypoxia (IH) (Dewan et al., 2015). The prevalence of
OSA in the third trimester of pregnancy is as high as 15.4% (Louis
et al., 2012), owing to reduced upper airway dimensions, partially
caused by pharyngeal edema (Izci et al., 2006) and pregnancy rhinitis
(Izci Balserak, 2015). The categorization of the origin of fetal hypoxia
by Kingdom and Kaufmann states that OSA during pregnancy causes
preplacental hypoxia, which leads to a lower fetal partial pressure of
oxygen (Nicolaides et al., 1987;Kingdom and Kaufmann, 1997).
Together with anaerobic glycolysis, oxidative phosphorylation in
mitochondria is required for the increasing energy demand during
fetal development (Baker and Ebert, 2013). The concept that the
environmental factors of a fetus influence the organism’s adaptation to
conditions later in life is known as the Developmental Origins of
Health and Disease paradigm (DOHaD) (Heindel et al., 2015). Several
studies have demonstrated that gestational IH causes health concerns
in an organism later in life, such as diabetes mellitus, hypertension,
and cardiovascular diseases (Hutter et al., 2010;Giussani et al., 2012;
Svitok et al., 2016;Badran et al., 2019).
In recent years, animal models of prenatal hypoxia have been
widely used to understand the molecular mechanisms of adverse
outcomes in offspring. Prenatal hypoxia affects fetal growth and
elicits many disturbances after birth including the development of
the central nervous system and cardiovascular regulatory system
(Peyronnet et al., 2000;Wang et al., 2021;Sutovska et al., 2022).
Decreased oxygen supply and peripheral blood flow by gestational IH
to the fetal organs such as the heart and brain have critical impacts on
physiological functions of these organs (Baschat et al., 1997). In
addition, skeletal muscle is not fully developed in the fetus. Skeletal
muscle requires to increase the blood flow to meet the substantial
increase of oxygen demand from muscle contraction especially during
aerobic exercise; however, adverse outcomes of gestational IH on
skeletal muscles of the offspring remain unclear.
The effects of postnatal chronic IH exposure in the skeletal muscle
of rodents have been previously reported (Shortt et al., 2014),
including decreased muscle force and endurance associated with a
shift in fiber type from oxidative (slow) to glycolytic (fast) in the
diaphragm (DIA) muscle (Shortt et al., 2013), and changing muscle
endurance in sternohyoid muscles (upper airway muscles) (Dunleavy
et al., 2008). In contrast, gestational IH without postnatal IH exposure
did not cause ex vivo muscle dysfunction in the diaphragm and
sternohyoid muscles in both male and female adult offspring rats
(McDonald et al., 2016). Furthermore, we recently showed that
gestational IH without postnatal IH exposure induces
mitochondrial impairment in a sucking muscle (geniohyoid muscle;
GH), but not in a masticating muscle (masseter muscle; MAS) in male
adolescent offspring rats (Wongkitikamjorn et al., 2022). These results
indicate that skeletal muscles from offspring have site-specific
susceptibility to gestational IH. The aim of this study was to clarify
the effects of gestational IH on muscle morphology, function, and
metabolism in a respiratory muscle (DIA) and a limb muscle (tibialis
anterior; TA) in adolescent offspring rats. GH and MAS muscles were
also analyzed to compare site-specific differences.
2 Materials and methods
2.1 Experimental model of IH
IH causes hypoxemia and lower oxygen availability in animals.
Various protocols of gestational and postnatal IH exposure were used
in previous studies to analyze the effects of the severity of the
functional impairments, which were different in oxygen percentage,
number of cycles, duration of exposure, and timing (Navarrete-Opazo
and Mitchell, 2014). The IH protocol used in this study has been
described previously (Nagai et al., 2014;Hong et al., 2021). Briefly, 7-
weeks-olds rats were exposed to 4% oxygen every 3 min periods for
8 h/day for 14 days to evaluate autophagic regulation in cardiac muscle
(Maeda et al., 2013). In this study, Sprague-Dawley rats on the seventh
day of pregnancy were randomly divided into the normoxia (n=3)
and IH (n= 3) groups, and housed under normoxia and IH conditions,
respectively, for 14 days until the time of delivery. Decreased blood
oxygen saturation levels were observed using a pulse oximeter
(MouseOx; STARR Life Sciences Corp., United States) in pregnant
rats in the IH condition, as previously described (Wongkitikamjorn
et al., 2022). Offspring rats were born naturally and housed under
normoxia with their mothers until weaning at day 21 after birth. All
rats were maintained in a specific pathogen-free facility with 12-h/12-
h light/dark cycles, and food and water were given ad libitum.
Gestational normoxia and postnatal normoxia control group is
named as N/N, and gestational IH and postnatal normoxia group
is named as IH/N. Male offspring rats of each group were weighed
every week and euthanized at the age of 5 weeks for further analysis.
Food consumption was measured at 25 and 35 days after birth.
All experimental procedures were performed according to the
Guide for the Care and Use of Laboratory Animals published by the
United States National Institutes of Health (NIH publication 85-23).
The Animal Care and Use Committee of Tokyo Medical University
approved all experiments performed in this study (study approval
number: H31-0011).
2.2 Grip strength test
Forelimb grip strength was assessed using a grip strength meter
(CPM-101B, Melquest, Japan). Rats (5 weeks of age, n= 6 in each
group) were placed on a grid, and then was pulled backward by their
tails until they released the grid. The peak pull force was recorded on a
digital force transducer. The test was repeated 3 times for each rat, and
the interval of each test was 30 s. The averages of the repeated grip
strength measurements were normalized to body weights.
2.3 Exhaustion treadmill running test
Aerobic motor function was evaluated using a treadmill. Rats from
both groups (5 weeks of age, n= 6 in each group) were familiarized
with a motorized treadmill containing shocker plates for 2 days (at
5 m/min for 30 min per day). The protocol used for the exhaustion test
was as previously described (Wada et al., 2019). Briefly, the test was
started at 5 m/min for 5 min. The speed was gradually increased by
1 m/min every min until the rat could no longer run continuously.
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2.4 Sample preparation and histological
staining
Five-week-old male rats that did not undergo muscle function
tests were anesthetized with isoflurane and euthanized. Soon after
being sacrificed, blood samples were collected via the caudal vena cava,
and serum samples were separated by incubation on ice for 2 h,
followed by centrifugation at 8,000 rpm for 15 min. Serum total
protein, total cholesterol, triglyceride, high-density lipoprotein
cholesterol, glucose, and lactate levels were measured using a
biochemistry automatic analyzer (model 7180; Hitachi High-Tech,
Japan). Serum adiponectin levels were measured using an ELISA kit
(Mouse/Rat Adiponectin ELISA-OY kit; Oriental Yeast Co., Japan), in
accordance with the manufacturer’s instructions. Muscle samples were
collected and frozen immediately with isopentane in liquid nitrogen
then stored at −80°C until use. All frozen samples were cut into
transverse 10-µm-thick sections using a Leica CM 3050S cryostat, and
collected on micro cover glasses (Matsunami, Japan). Each sample was
stained with H&E, modified Gomori Trichrome, nicotinamide
adenine dinucleotide reductase (NADH), and periodic acid schiff
(PAS). For the NADH staining, cryosections were stained with the
NADH solution (1.0% nitro blue tetrazolium and .8% beta-NADH in
.05 M Tris-HCl buffer) for 30 min at 37°C. Semiquantitative
measurements of the intensity of the NADH and PAS staining in
each fiber type was analyzed based on previous reports (Scribbans
et al., 2014;Cong et al., 2016;White et al., 2016;Giacomello et al.,
2020;Zheng et al., 2020;Corona et al., 2022). For fiber type
classification, each section was stained with primary antibodies
against myosin heavy chains (MHC) as listed in Supplementary
Table S1. Alexa Fluor 350 and 488 anti-mouse and 598 anti-rabbit
secondary antibodies (1:1,000; Thermo Fisher Scientific) were used for
detection. The percentage area of NADH-positive or PAS-stained
from each muscle fiber was calculated from serial sections using NIH
ImageJ software, and around 40 fibers of each muscle fiber type were
counted from the DIA (type I, IIA, IIX/D) and TA (type I, IIA, IIX/D,
IIB) samples (total around 240 fibers of each muscle fiber type in six
samples). Each fiber was classified as strong-, medium- or weak-
stained by the staining intensity.
2.5 Analysis of muscle fiber size and fiber type
distribution
Transverse 8-µm-thick muscle cryosections were prepared. After
blocking with 2% bovine serum albumin in phosphate-buffered saline,
each section was stained with primary antibodies against MHCs and
laminin to detect muscle cell membranes at 37°C for 80 min as
previously described (Bloemberg and Quadrilatero, 2012). Primary
antibodies used for immunofluorescence staining are listed in
Supplementary Table S1. For fiber size analysis, Alexa Fluor
488 anti-mouse and 568 anti-rabbit secondary antibodies (1:1,000;
Thermo Fisher Scientific, United States) were used for detection. For
fiber type distribution analysis, Alexa Fluor 350 and 488 anti-mouse
and 598 anti-rabbit secondary antibodies (1:1,000; Thermo Fisher
Scientific) were used for detection. All staining images were acquired
using a fluorescence microscope (Zeiss, Germany). The whole image
of each section was captured by the IN Cell Analyzer 2200 imaging
system for calculating muscle fiber size (diameters in the minor axis)
with IN Cell Developer Toolbox software (GE Healthcare,
United States). Basal membranes were detected by laminin staining
to calculated fiber size, and each MHC-positive fiber was automatically
selected by staining intensity. Muscle fiber size was assessed by
quantifying the short diameters on the cross-sectional images.
More than 3,000 fibers (3,000–10,000) from each sample were used
for the quantification (n= 5 per group). The fiber size distribution was
compared between the N/N and IH/N groups. Fiber type
compositions were counted using NIH ImageJ software.
2.6 Protein extraction and western blotting
Muscle samples were homogenized in a sample buffer solution
(Fujifilm, Japan) comprised of RIPA buffer containing protease
inhibitors and phosphatase inhibitors (Roche, Switzerland), then
centrifuged at 15,000 rpm at 4°C for 5 min. Supernatants were
collected, and total protein concentrations were measured using
BioPhotometer (Eppendorf, Germany). Equal amounts of protein
for each sample were loaded onto 10%–20% or 15% SDS-PAGE
gels (Fujifilm) and blotted onto PVDF membranes by the semi-dry
technique using Trans-Blot Turbo system (Bio-Rad, United States).
The PVDF membranes were incubated with primary antibodies,
followed by incubation with horseradish peroxidase-conjugated
secondary antibodies (Thermo Fisher Scientific). The primary
antibodies used for Western blotting are listed in Supplementary
Table S1. All bands were detected with Clarity Western ECL
Substrate (Bio-Rad) and visualized using Image Lab 5.0 system
(Bio-Rad). All data were normalized using expression levels of
GAPDH and analyzed as relative band intensities using Image Lab 5.0.
2.7 Quantitative-PCR analysis
Total RNA was extracted from frozen muscle samples using
RNeasy Plus Universal Mini kit (QIAGEN, Germany) in
accordance with the instructions provided by the manufacturer.
Complementary DNA (cDNA) was synthesized from 1,000 ng of
total RNA with Oligo (dT) primers using SuperScript IV VILO
Master Mix (Thermo Fisher Scientific) in accordance with the
manufacturer’s instructions. Real-time PCR was performed using
10 ng of cDNA template for each gene using an Applied
Biosystems QuantStudio3 real-time PCR system (Thermo Fisher
Scientific). Primers were chosen for real-time PCR as listed in
Supplementary Table S2. All results were normalized using Actb
(beta-actin), and gene expression levels were calculated by the
ΔΔCT method of relative quantification. Data were analyzed as
relative messenger RNA expression levels.
2.8 Quantification of capillary numbers per
muscle area and per myofiber
Capillaries, skeletal muscle area and fiber numbers were counted
from muscle sections stained with anti-CD31 (an endothelial cell
marker) and anti-laminin antibodies, respectively. Alexa Fluor
488 anti-rabbit and 568 anti-goat secondary antibodies (1:1,000;
Thermo Fisher Scientific) with DAPI solution were used for
detection. Staining sections were observed using a fluorescence
microscope Axio Scope A1 (Zeiss), and four random fields of 200-
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times magnification from each sample (n= 6 per group) were used for
the measurements. The skeletal muscle area, number of fibers and
capillaries in the field were counted using NIH ImageJ software. Data
were analyzed as the capillaries per muscle area and per myofiber
number.
2.9 Statistical analysis
Data are shown as the mean ± standard deviation (SD), and
analyzed using the independent t-test. A p-value of less than .05 was
considered to indicate a statistically significant difference between
groups. All statistical analyses were performed using SPSS statistics
28 software (IBM, United States).
3 Results
3.1 Gestational IH reduces endurance running
capacity in offspring rats
Both N/N and IH/N offspring rats were born naturally under
normoxia, and their body weights gradually increased weekly with no
significant difference between the groups (Figure 1A). No general
growth retardation was observed in IH/N rats. Food intake was similar
in IH/N rats and N/N rats at 25 and 35 days after birth (Figure 1B).
Serum biochemical analysis demonstrated that no notable
abnormalities were observed, including total cholesterol and
triglyceride levels in the IH/N group (Table 1). Serum adiponectin
levels were also similar between N/N and IH/N rats.
For functional performance tests, the scores of forelimb grip
strength (normalized to body weight) showed no significant
difference between N/N and IH/N rats (Figure 1C). One day after
the grip strength test, maximal exercise performance was evaluated in
the rats by treadmill running until exhaustion. When the running
speed was gradually increased, IH/N rats stopped running at a slower
speed than N/N rats (N/N rats: 43.0 ± 1.4 m/min; IH/N rats: 37.8 ±
2.8 m/min, p<.01) (Figure 1D).
3.2 Muscle morphology and mitochondria
contents are preserved in the DIA and TA from
IH/N rats
No pathological changes, such as muscle fiber degeneration,
internal nuclei, or inflammatory cellular infiltration were identified
by H&E and modified Gomori Trichrome staining in both DIA and
TA muscles from N/N and IH/N rats (Figures 2A, B). In addition,
FIGURE 1
Changes in body weight, food intake, grip strength, and running to exhaustion treadmill test of offspring adolescent rats (N/N and IH/N groups). (A)
Weekly average body weights of N/N and IH/N rats from day 14 to day 35 after birth were shown. (B) Food consumption of N/N and IH/N rats, measured on day
25 and day 35 after birth, was similar. (C) Grip strength normalized to body weight showed no significant difference between N/N and IH/N rats on day 35. (D)
IH/N rats had significantly lower scores on the treadmill running test compared with N/N rats (n= 6 in each group) **p<.01.
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there is no notable change in the intensity (classification) of NADH
and PAS-stained area of each fiber type in the DIA and TA muscles
between N/N and IH/N rats (Figures 2A, B;Supplementary Figures
S1A–E). Western blot analysis of mitochondrial biogenesis and fusion
proteins indicated that no specific change was observed in the DIA and
TA muscles in the adolescent rats exposed to gestational IH
(Supplementary Figures S2A–D). In our initial study, gestational
IH induced a significant downregulation of mitochondrial proteins
in the GH muscle, but not in the MAS muscle (Wongkitikamjorn et al.,
2022). The proportion and size distribution of skeletal muscle fiber
TABLE 1 Serum biochemistry data of N/N and IH/N rats.
N/N IH/N p-value
TP (g/dL) 5.88 ± .08 5.82 ± .13 .415
T-CHO (mg/dL) 120.2 ± 13.0 123.0 ± 5.6 .677
TG (mg/dL) 85.4 ± 20.5 102.8 ± 28.2 .301
HDL-C (mg/dL) 44.6 ± 3.65 46.4 ± 2.07 .374
GLU (mg/dL) 430.6 ± 109.9 326.2 ± 67.9 .114
LA (mg/dL) 112.1 ± 21.3 98.3 ± 24.5 .369
Adiponectin (ng/μl) 8138.4 ± 1713.7 10367.9 ± 2085.0 .100
Values are shown as the mean ± SD (n= 5 in each group). TP, total protein; T-CHO, total cholesterol; TG, triglyceride; HDL-C, high density lipoprotein cholesterol; GLU, glucose; LA, lactate.
FIGURE 2
Histological images from the DIA and TA muscles of offspring rats. Histological images of H&E, modified Gomori Trichrome, NADH, and PAS staining
showed no pathological features in the (A) DIA, and (B) TA muscles from N/N and IH/N rats.
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types showed no significant difference in both DIA and TA muscles
from IH/N rats compared with those from N/N rats (Figures 3A, B,
respectively). These results indicate that gestational IH affects partly
and functionally different skeletal muscles within a body.
3.3 Downregulation of genes associated with
glucose and lipid metabolism in skeletal
muscles from IH/N rats
Metabolic changes in skeletal muscles in response to gestational
IH were assessed by analyzing their relative gene expression changes.
The expression of several genes involved in glucose and fatty acid
metabolism were downregulated, particularly in the DIA and TA
muscles. Among the genes associated with glucose metabolism, the
expression of Slc2a4, which encodes muscle-enriched glucose
transporter 4 (GLUT4), was significantly reduced in the DIA and
TA muscles but not in the GH and MAS muscles from IH/N rats
(Figures 4A–D). Gene expression levels of Slc2a1 for GLUT1 and its
positive regulator Hif1a were also decreased only in the DIA muscle.
The expression of glucose metabolic enzymes (Hk2,Pkfm,Pkm, and
Gys1) were substantially decreased only in the DIA and TA muscles
from IH/N rats, and Pygm and Chrebp, a transcriptional regulator of
Pygm, were similarly decreased in the DIA, TA, and MAS muscles, but
not in the GH muscle from IH/N rats (Figures 4A–D). The expression
of several genes involved in fatty acid metabolism was also
FIGURE 3
Muscle fiber type distribution and muscle fiber size in the DIA and TA muscles of offspring rats. Fiber type-specific immunohistochemical staining for type
I, type IIA, type IIX/D, and type IIB fibers with skeletal muscle membrane protein, laminin (red). Each panel shows the cross-sectional image of the (A) DIA, and
(B) TA muscles. Green areas indicate immuno-positive muscle fibers. Muscle fiber type distribution and size frequency in the DIA and TA showed no significant
difference between N/N and IH/N rats. Bars represent (A) 100 µm for DIA sections, and (B) 200 µm for TA sections.
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downregulated particularly in the DIA and TA muscles. Gestational
IH reduced the gene expression of triglyceride metabolism (Lpl) and
fatty acid metabolism (Ppara,Ppard, and Ucp3) in the DIA and TA
muscles. In the DIA muscle, a gene associated with beta-oxidation
(Cpt1) was significantly decreased by gestational IH. Additionally,
genes associated with sterol metabolism (Srebf) and lipid metabolism
(Lxra and Ppargc1b) were significantly downregulated in the TA
muscle from IH/N rats. The expression level of Srebf was also
commonly decreased in the GH and MAS muscles, whereas Ppard
and Ucp3 were significantly decreased in the MAS muscle but not in
the GH muscle from IH/N rats (Figures 5A–D). Therefore, alterations
in glucose and fatty acid metabolism were substantial in the DIA and
TA muscles, but minimal in the GH and MAS muscles.
3.4 Suppression of AMPK and AKT activation
in the DIA and TA muscles by gestational IH
Glucose and/or lipid metabolism is partially regulated by the
AMP-activated protein kinase (AMPK) and phosphatidylinositol-3
kinase (PI3K)/protein kinase B (AKT) signaling pathways. The
phosphorylation of AMPK, PI3K, AKT, and mammalian target of
rapamycin (mTOR), and the expression of associated proteins were
analyzed by Western blotting. In the DIA muscle, a significant
decrease in AMPK and AKT phosphorylation and increase in total
AKT levels were detected (Figures 6A, C). Reduced levels of
phosphorylated AMPK and AKT in the TA muscle were also
observed (Figures 6B, D). Phosphatase and tensine homolog
(PTEN) and total PI3K levels were significantly decreased only in
the TA muscle. The expression levels of these proteins were
comparable in both the GH and MAS muscles from IH/N rats
(Figures 7A–D). Although gene expression levels of Slc2a4 and
Hif1a were decreased, the protein levels of GLUT4 and HIF1α
were unchanged by gestational IH in all the analyzed skeletal
muscles from adolescent rats.
3.5 Decreased gene expression levels of
adiponectin receptors and capillaries per
muscle area and per myofiber
Adiponectin, which is an adipocyte-derived circulating hormone,
is known to activate AMPK via adiponectin receptors, and to improve
the utilization of glucose and fatty acids in skeletal muscles (Yamauchi
et al., 2002). In skeletal muscle, two adiponectin receptors
(AdipoR1 and AdipoR2) are expressed. These two receptors have
distinct roles; i.e., adiponectin receptor 1 controls metabolic activity,
and adiponectin receptor 2 is associated with vascular homeostasis
(Parker-Duffen et al., 2014). Expression levels of both Adipor1 and
Adipor2 genes were significantly decreased in the DIA and TA
muscles, but not in the GH and MAS muscles by gestational IH
(Figure 8A). As energy metabolism and blood flow to the skeletal
muscle are closely associated with each other, the capillary density in
each muscle was calculated as capillaries per muscle area and as a ratio
of capillaries-to-myofiber. IH/N rats were found to have decreased
capillary per muscle area in the DIA (p<.01) and TA (p= .07);
FIGURE 4
Quantitative PCR analysis of genes associated with glucose metabolism. Relative gene expression of Pkfm,Pkm,Pygm,Slc2a1,Slc2a4,Hk1,Hk2, and
Gys1 in the (A) DIA, (B) TA, (C) GH, and (D) MAS muscles were normalized by Actb and shown as fold increase of the N/N group *p<.05, **p<.01, ***p<.001.
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however, capillarization was not altered in the GH and MAS muscles
(Figures 8B, C). These data were consistent with the quantification of
capillary numbers per myofiber ratio (Supplementary Figure S3).
These results suggested that there is an interaction between
reduced Adipor2 levels and capillary density in the DIA and TA
muscles from adolescent offspring rats exposed to gestational IH.
FIGURE 5
Quantitative PCR analysis of genes associated with lipid metabolism. Relative gene expression of Chrebp,Srebf,Cpt1,Lpl,Lxra,Ppargc1b,Ppara,Ppard,
Pparg,Ucp3, and Hif1a in the (A) DIA, (B) TA, (C) GH, and (D) MAS muscles were normalized by Actb and shown as fold increase of the N/N group *p<.05, **p<
.01, ***p<.001.
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FIGURE 6
Western blot analysis of proteins involved in glucose and lipid metabolism in the DIA and TA muscles. Western blots were performed on six individual
samples to quantify the levels of mTOR, PI3K, AKT, AMPK, PTEN, and HIF1αin the (A) DIA, and (B) TA muscles. Graphs represent the ratio between the
phosphorylated forms of mTOR, PI3K, AKT, and AMPK, and the total amount of each target protein. Relative expression levels of each protein were normalized
to the level of GAPDH expression. Expression levels are shown with those for (C) DIA, and (D) TA muscles from the N/N group set to 1 *p<.05, **p<.01,
***p<.001.
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FIGURE 7
Western blot analysis of proteins involved in glucose and lipid metabolism in the GH and MAS muscles. Western blots were performed on six individual
samples to quantify the levels of mTOR, PI3K, AKT, AMPK, PTEN, and HIF1αin the (A) GH, and (B) MAS muscles. Graphs represent the ratio between the
phosphorylated forms of mTOR, PI3K, AKT, and AMPK and the total amount of each target protein. Expression levels of each protein are shown relative to the
level of GAPDH expression, for the (C) GH, and (D) MAS muscles from the N/N group set to 1.
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4 Discussion
To date, only a limited number of studies have reported the
effects of gestational IH on the offspring’s skeletal muscle. In this
study, we clearly demonstrated that gestational IH results in a
significant reduction in endurance motor function in male
adolescent offspring rats, associated with potential metabolic
alterations and reduced capillary density in the DIA and TA
muscles. Previous studies reported that gestational IH leads to
reduced body weight of the offspring at birth, but promotes catch-
up growth after birth. Subsequently, adult offspring rats exposed to
gestational IH gradually increase their food consumption and gain
body weight (Gozal et al., 2003;Camm et al., 2011). Likewise,
increases in food intake and body weight, together with metabolic
abnormalities are observed in an age-dependent manner in adult
offspring mice exposed to gestational IH (Khalyfa et al., 2017;
Badran et al., 2019). Interestingly, these postnatal changes are sex-
dependent. Only male offspring mice are affected, whereas adult
female offspring have no metabolic dysfunctions (Badran et al.,
2019). Males can be more vulnerable to maternal insults (Dearden
and Balthasar, 2014;Sundrani et al., 2017), and therefore, only male
offspring rats were analyzed in this study.
FIGURE 8
Quantitative PCR analysis of genes encoding adiponectin receptors, and quantification of capillary numbers per muscle area (mm
2
). (A) Relative gene
expression of Adipor1 and Apipor2 in the DIA, TA, GH, and MAS muscles, normalized by Actb and shown as fold increase of the N/N group. (B)
Immunohistochemical staining for CD31 (red) and a merged image with laminin (green) and DAPI (blue) from the TA muscle. Bar represents 100 µm. (C)
Number of capillaries per muscle area (mm
2
). Results are presented as mean ± SD (n=6)**p<.01, ***p<.001.
Frontiers in Physiology frontiersin.org11
Wongkitikamjorn et al. 10.3389/fphys.2023.1067683
In our gestational IH model (IH/N), the growth curves and food
intake of these rats were similar to those of control (N/N) rats between
2 and 5 weeks after birth. Serum metabolic parameters, including
glucose, total cholesterol, and triglyceride showed no differences
between N/N and IH/N rats at 5 weeks of age. These results
indicate that gestational IH does not cause overt growth or
metabolic abnormalities in adolescent offspring rats.
Importantly, the 5-weeks-old offspring male IH/N rats showed
normal grip strength, which is an indicator of forelimb strength,
but showed significantly reduced aerobic motor performance as
assessed by the forced exhaustion running test using the treadmill,
which is a reliable method to assess whole-body muscle function
(Wada et al., 2019). Skeletal muscle fiber type composition may be
changed by physical activity, pathological or stress conditions, and
nutritional conditions. Endurance training, low energy availability,
and higher body metabolic rates induce skeletal muscle fiber
conversion from glycolytic to oxidative fibers (Purohit and
Dhawan, 2019). A previous study reported that conversion from
fast to slow fiber type was observed in a CoCl
2
-simulated hypoxic
environment of muscle cells (Lixin et al., 2019). Similarly,
gestational IH causes hypoxemia and lower O
2
availability in the
fetus (Carter, 2015), which is required for energy demand in
aerobic metabolism (Baker and Ebert, 2013). Interestingly, as
showninthisstudy,nohistological changes were observed in
the DIA and TA muscles in rats exposed to IH, and muscle fiber size
and fiber-type composition were comparable between N/N and IH/
N rats. Mitochondrial impairment was also not detected in the DIA
and TA muscles from IH/N rats. Our recent study demonstrated
that gestational IH induces smaller type IIA fibers and
mitochondrial impairment in a sucking muscle (GH), but not in
a masticating muscle (MAS) in the adolescent offspring rats that
had been exposed to gestational IH (Wongkitikamjorn et al., 2022).
Even though the effects of mitochondrial impairment on muscle
function of GH was not determined, these results indicate that
gestational IH may exert different effects on different parts of
muscles.
To elucidate the pathomechanism of the reduced aerobic
performance observed in IH/N rats, we analyzed the expression of
genes associated with energy metabolism. We demonstrated
significantly reduced expression of several genes associated with
glucose and fatty acid metabolism in the DIA and TA muscles,
whereas these changes were minimal in the GH and MAS muscles
from the same rats. The downregulation of genes associated with
glucose and fatty acid metabolism can be explained by a decrease in
phosphorylated AMPK and AKT protein levels (Long and Zierath,
2006). Previous studies reported that offspring rodent pups that were
exposed to gestational IH can develop alterations in energy
metabolism and have an increased risk of insulin resistance in later
life (Camm et al., 2011;Rueda-Clausen et al., 2011;Khalyfa et al., 2014;
Badran et al., 2019). Badran et al. reported that offspring mice exposed
to gestational IH had increased insulin resistance and decreased
phosphorylation of AKT in the gastrocnemius muscle (Badran
et al., 2019). On the other hand, Camm et al. demonstrated
decreased total AKT2 levels but not phosphorylated AKT levels in
skeletal muscle (the specific muscle part was not stated) from adult rat
offspring exposed to gestational IH (Camm et al., 2011). These results
support our data that the effects of gestational IH on activation of the
AKT pathway is different among skeletal muscles of the offspring.
AMPK is another key regulator of energy homeostasis, which
increases glucose uptake and fatty acid oxidation in skeletal muscle
(Merrill et al., 1997). Furthermore, AMPK can facilitate mitochondrial
biogenesis in skeletal muscle (Zong et al., 2002). AMPK is activated by
ATP depletion upon rapid muscle contraction, hypoxia, or glucose
deprivation. It is also activated by adipokines, such as adiponectin and
leptin. Khalyfa et al. (2017) reported decreased serum adiponectin
levels and increased serum leptin levels in adult male offspring mice
exposed to gestational IH. Interestingly, these mice have less
locomotor activity and reduced daily energy expenditure compared
with controls (Khalyfa et al., 2017). Low adiponectin levels in serum
and perivascular adipose tissue associated with hypermethylation of
the adiponectin gene promoter were reported in male adult offspring
rats exposed to gestational IH (Badran et al., 2019). Adiponectin, an
adipocyte-derived circulating hormone, is known to improve the
utilization of glucose and fatty acids in skeletal muscles (Yamauchi
et al., 2002). Recent studies demonstrated that adiponectin receptors
are also expressed in skeletal muscle. Adiponectin mediates specific
effects in organs via binding to its receptors, AdipoR1 and AdipoR2
(Bjursell et al., 2007). These two receptors have distinct roles in that
AdipoR1, which is mainly expressed in skeletal muscle, regulates
energy metabolism, where AdipoR2 controls vascular homeostasis
(Parker-Duffen et al., 2014). In our study, reduced expression of the
gene encoding AdipoR1 in the DIA and TA muscles but not the GH
and MAS muscles was observed by exposure to gestational IH.
Consistently, decreased expression of genes associated with energy
metabolism together with decreased levels of phosphorylated AMPK
and AKT were observed in the DIA and TA, but not in the GH and
MAS. Moreover, gene expression of AdipoR2 was similarly reduced in
the DIA and TA muscles in male adolescent offspring exposed to
gestational IH. In a model of hind limb ischemia, AdipoR2 knockout
mice showed delayed recovery of blood flow, impaired perfusion and
reduced capillary density in the gastrocnemius muscle (Parker-Duffen
et al., 2014). Capillary density is strictly regulated in skeletal muscles in
human and rodents, and the ratio depends on the muscle part
(O’Reilly et al., 2021;Høeg et al., 2009). Consistent with reduced
Adipor2 expression in the DIA and TA muscles, the number of
capillaries per muscle area and per myofiber were significantly
decreased in the DIA and slightly decreased in the TA muscles of
offspring rats exposed to gestational IH. Collectively, these data
indicate that gestational IH impairs energy homeostasis and
reduces the number of capillaries per muscle area in the DIA and
TA muscles associated with reduced aerobic performance from a
younger age. Further analyses are needed to clarify the differences to
the responses to gestational IH among the muscles from different body
parts.
The present study provides clear evidence regarding the
possible adverse effects of gestational IH in the regulation of
energy homeostasis and vasculature, at least in part, owing to
decreased adiponectin receptor expression in the DIA and TA
but not in the GH and MAS muscles of male adolescent offspring
rats. The results of the comparison of various skeletal muscle parts
are particularly interesting because alterations in energy
metabolism by gestational IH depends on the body region. Our
findings indicate that a further comprehensive approach to
understand the effects of gestational IH on the skeletal muscles
of offspring, with consideration of their developmental origins and
functions after birth is required.
Frontiers in Physiology frontiersin.org12
Wongkitikamjorn et al. 10.3389/fphys.2023.1067683
Data availability statement
The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be directed
to the corresponding author.
Ethics statement
The animal study was reviewed and approved by the Animal Care
and Use Committee of Tokyo Medical University (study approval
number: H31-0011).
Author contributions
All authors contributed to design the study. WW, EW, JH, and
HM prepared the animal model, conducted skeletal muscle
performance tests, and collected the samples. WW and EW
performed histological analysis, Western blot analysis, and
real-time PCR analysis of skeletal muscle tissues. KY, TO, and YH
interpreted the data. WW and EW prepared the manuscript and
figures. JH, HM, HH, SS, KY, TO, and YH drafted and edited the
manuscript. All authors approved the final version of the manuscript.
Funding
This work was financially supported in part by Grants-in-Aid for
Scientific Research (16K11778, 18K15052, 20H03895, 20H03594)
from the Japanese Ministry of Education, Culture, Sports, Science
and Technology (KAKENHI), and an Intramural Research Grant for
Neurological and Psychiatric Disorders of NCNP (2-5 and 29-4), and a
Follow-up Grant from Tokyo Medical University (2022).
Acknowledgments
We thank Dr. Helena Popiel (Tokyo Medical University) for
editing the manuscript, Ms. Nao Naruse (Tokyo Medical
University) for performing histological staining and the Animal
Research Center, Tokyo Medical University and the Research Core
Center, Tokyo Medical and Dental University for their technical
support.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fphys.2023.1067683/
full#supplementary-material
SUPPLEMENTARY FIGURE S1
Quantification of histological properties of fiber type-dependent NADH activity
from NADH-stained sections and glycogen content from PAS-stained sections.
(A) Representative images of serial sections from the DIA and TA muscles,
stained for MHC type I (blue), type IIA (green), type IIX/D (not stained, black) and
type IIB (red), NADH-TR, and PAS staining. Bar represents 50 µm. Qualitative
analysis of fiber-type-dependent NADH activity in the (B) DIA, and (C)TA
muscles, and of glycogen content from PAS staining in the (D) DIA, and (E) TA
muscles fromN/N and IH/N groups. Results are presented as mean ± SD (n=6).
SUPPLEMENTARY FIGURE S2
Western blot images for PPAR-gamma coactivator 1-alpha (PGC1α),
mitochondrial transcription factor A (TFAM), NADH:Ubiquinone
oxidoreductase complex assembly factor 1 (NDUFAF1), ATP5A1, optic atrophy
1 (OPA1), mitofusin (MFN)1, MFN2, and mitochondrial fission 1 (FIS1) and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous
control marker in the (A) DIA and (B) TA muscles. Quantification of Western
blot analysis results of the mitochondrial metabolic markers from the (C) DIA
and (D) TA muscles are shown. The expression level of each protein was
normalized to the level of GAPDH expression, and relative expression levels
are shown with those for the N/N group set to 1.
SUPPLEMENTARY FIGURE S3
Ratios of CD31-positive capillaries-to-myofiber. The ratios in the DIA (counted
around 2,100 capillaries and 1,200 myofibers from each sample), TA (counted
around 2,200 capillaries and 1,300 myofibers from each sample), GH
(counted around 2,000 capillaries and 1,800 myofibers from each sample), and
MAS (counted around 1,900 capillaries and 2,300 myofibers from each
sample) muscles were measured from double-stained immunohistochemical
images. Results are presented as mean ± SD (n= 6) ***p<0.001.
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