Am J Physiol Endocrinol Metab 310: E1003–E1015, 2016.
First published April 26, 2016; doi:10.1152/ajpendo.00521.2015.
CALL FOR PAPERS
Mitochondrial Dynamics and Oxidative Stress
Exposure to environmentally persistent free radicals during gestation lowers
energy expenditure and impairs skeletal muscle mitochondrial function in
adult mice
Erin J. Stephenson,1,2,3 Alyse Ragauskas,1,2,3 Sridhar Jaligama,2,3 JeAnna R. Redd,1,2,3
Jyothi Parvathareddy,2,3 Matthew J. Peloquin,1,2,3 Jordy Saravia,2,3 Joan C. Han,1,2,3
Stephania A. Cormier,1,2,3 and X Dave Bridges1,2,3
1
Submitted 18 December 2015; accepted in final form 19 April 2016
Stephenson EJ, Ragauskas A, Jaligama S, Redd JR, Parvathareddy J, Peloquin MJ, Saravia J, Han JC, Cormier SA,
Bridges D. Exposure to environmentally persistent free radicals
during gestation lowers energy expenditure and impairs skeletal
muscle mitochondrial function in adult mice. Am J Physiol Endocrinol
Metab 310: E1003–E1015, 2016. First published April 26, 2016;
doi:10.1152/ajpendo.00521.2015.—We have investigated the effects
of in utero exposure to environmentally persistent free radicals
(EPFRs) on growth, metabolism, energy utilization, and skeletal
muscle mitochondria in a mouse model of diet-induced obesity.
Pregnant mice were treated with laboratory-generated, combustionderived particular matter (MCP230). The adult offspring were placed
on a high-fat diet for 12 wk, after which we observed a 9.8% increase
in their body weight. The increase in body size observed in the
MCP230-exposed mice was not associated with increases in food
intake but was associated with a reduction in physical activity and
lower energy expenditure. The reduced energy expenditure in mice
indirectly exposed to MCP230 was associated with reductions in
skeletal muscle mitochondrial DNA copy number, lower mRNA
levels of electron transport genes, and reduced citrate synthase activity. Upregulation of key genes involved in ameliorating oxidative
stress was also observed in the muscle of MCP230-exposed mice.
These findings suggest that gestational exposure to MCP230 leads to
a reduction in energy expenditure at least in part through alterations to
mitochondrial metabolism in the skeletal muscle.
in utero exposure; environmentally persistent free radicals; oxidative
stress; skeletal muscle; mitochondria
OBESITY IS A MAJOR GLOBAL HEALTH CONCERN, and emerging data
support a role for environmental pollutants in the pathogenesis
of obesity and its comorbidities (2, 7, 9, 10, 12, 22, 24, 45).
Gestational and early-life exposure to combustion-derived particulate matter (PM) has been associated with an increased risk
of obesity in humans (9, 12, 18, 22, 24). This association is
supported by data obtained from animal studies, where the
offspring of pregnant mice, which have been exposed to diesel
exhaust in utero, are predisposed to weight gain as adults (6).
Furthermore, several studies have linked the exposure to combustion-derived PM to impaired metabolic health in humans (2,
Address for reprint requests and other correspondence: D. Bridges, Dept. of
Nutritional Sciences, Univ. of Michigan School of Public Health, 1415
Washington Heights, Ann Arbor, MI, 48109 (e-mail: davebrid@umich.edu).
http://www.ajpendo.org
7, 10, 45) and animals (29, 30, 43, 55, 56). Specifically,
cross-sectional studies of human subjects that are chronically
exposed to combustion-derived PM have shown associations
with type 2 diabetes and cardiovascular disease (2, 7, 45),
whereas murine models of chronic PM exposure indicate that
pollutants lead to elevated adipose tissue inflammation and
insulin resistance (30, 43, 55). Relatively stable radicals with
half-lives of ⬃21 days exist on the surface of airborne PM (13,
15, 31), and these are referred to as environmentally persistent
free radicals (EPFRs).
From a mechanistic stand point, exactly how environmental
pollutants result in obesity and other metabolic abnormalities is
currently unknown. However, mitochondrial deficiencies and
structural abnormalities have been observed in adipose tissue
(55, 56), vascular tissue (53), and cardiac muscle (28) following exposure to combustion-derived pollutants that should
contain EPFRs. Mitochondria are responsible for oxidative
cellular energy production and endogenous reactive oxygen
species production and are an essential component of the
antioxidant defense system (23). Thus, defects in mitochondrial metabolism, particularly in the context of obesity, are
likely to have profound effects on energy homeostasis and
other metabolic pathways. The importance of skeletal muscle
mitochondrial metabolism for maintaining metabolic health is
becoming well recognized (21, 37, 41) with deficits in muscle
quality and function, particularly during early development (8),
being closely linked to later life metabolic disturbances such as
impaired growth or insulin resistance (16, 37). However, the
effects of in utero exposure to EPFRs on skeletal muscle
mitochondrial quality remain to be determined. In this study,
we investigated the effects of in utero exposure to EPFRs on
growth, metabolism, energy utilization, and skeletal muscle
mitochondria in a mouse model of diet-induced obesity. We
hypothesized that gestational exposure to EPFRs reduces energy expenditure and results in mitochondrial impairments in
the skeletal muscle.
MATERIALS AND METHODS
MCP230 preparation. EPFR-containing particles [i.e., laboratorygenerated, combustion-derived particular matter (MCP230)] were
generated and characterized by our colleagues, as described previously (31). Suspensions of MCP230 and cabosil, a non EPFR-
Licensed under Creative Commons Attribution CC-BY 3.0: © the American Physiological Society. ISSN 0193-1849.
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Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee; 2Department of Pediatrics,
University of Tennessee Health Science Center, Memphis, Tennessee; and 3Children’s Foundation Research Institute, Le
Bonheur Children’s Hospital, Memphis, Tennessee
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GESTATIONAL EPFR EXPOSURE AND ENERGY EXPENDITURE
CLAMS (Columbus Instruments). Mice were placed in the cages at
approximately ZT10 and monitored for 3– 4 days. Data from the first
6 h were discarded, as this was the amount of time determined to be
necessary for the mice to acclimate to their new environment. Oxymax software (Columbus Instruments) calculated the volumes of O2,
CO2, the respiratory exchange ratio, the ambulatory x- and y-phase
physical activity, and the food consumption. Heat production was
calculated using the Lusk equation (32) via the Oxymax software:
heat ⫽ (3.815 ⫹ 1.232 ⫻ RER) ⫻ V̇O2.
Tissue collection and nucleic acid preparation. After the 12-wk
HFD phase, mice were fasted overnight and anesthetized with ketamine-xylazine (180:10 mg/kg, respectively), which was delivered
intraperitoneally. Quadriceps muscles were carefully dissected out,
cleared of any visible adipose and connective tissue, and snap-frozen
in liquid N2. Nucleic acids were isolated from frozen quadriceps
samples via Trizol extraction using a Qiagen Tissue Lyser (30 Hz for
5 min). Following careful and complete removal of the RNA-containing aqueous phase and its subsequent column purification (PureLink
mRNA kit from Life Technologies), DNA extraction buffer [Tris base
(1 M), sodium citrate dibasic trihydrate (50 mM), and guanidine
thiocyanate (4 M)] was added to the tubes containing the remaining
Trizol-separated interphase and infranatant. Tubes were shaken vigorously and centrifuged at 12,000 g at room temperature for 30 min.
The aqueous phase was collected, and the genomic and mitochondrial
DNA were precipitated in isopropanol. Samples were respun at
12,000 g at 4°C to pellet the DNA. The DNA pellet was then washed
in 70% ethanol, respun, and, after careful ethanol removal, resuspended in TE buffer. cDNA was generated from purified RNA using
the Applied Biosystems cDNA Synthesis Kit.
Quantitative PCR analysis of mitochondrial DNA copy number and
mRNA transcripts. Primers designed for three mitochondrial-encoded
gene regions were used to assess mitochondrial DNA (mtDNA) copy
number in DNA, and primers designed for quantitative RT-PCR were
used to assess mRNA transcript levels (Table 1). Briefly, DNA or
cDNA from each sample extraction was added to the appropriate
working quantitative PCR master mix (containing SYBR Green and
the relevant primers at a final concentration of 100 nM each). PCR
conditions included an activation cycle of 95°C for 10 min followed
by 45 amplification cycles of 15 s at 95°C, 15 s at 60°C, and 10 s at
73°C. Cp values were quantified on a Light Cycler 480. Nucleic acid
Table 1. Mitochondrial DNA copy number and relative gene expression were determined using the following primer
sequences
Region/gene
d-Loop
mt-Nd1
mt-Cytb
mt-Nd4
Sdha
mt-Co2
Ppard
Ppargc1a
Ppargc1b
Nrf1
Nfe2l2
Tfam
Ucp2
Ucp3
Sod1
Sod2
Cat
Gpx1
Gclm
Tsc2
Rpl13a
Forward Primer
GGC
CGT
CTT
TAA
TCT
AAC
ACA
TGA
TTG
AGA
TGG
TCG
TGC
ACA
GGA
TTC
CAC
TTC
TGG
AAG
GGA
CCA
CCC
CAT
TCG
TCG
CGA
TGG
TGT
TAG
AAC
ACT
CAT
GGT
AAG
ACC
TGG
TGA
GGA
AGT
AAG
GTC
TTA
CAT
GTC
CAC
CTG
GTC
AAT
GAA
AGT
GGA
TGG
CCC
CCG
GAT
ATC
ACA
CGA
CAC
TCC
CCT
CGT
AAC
TCT
GGA
ATG
GTG
GTT
GTC
TGA
GCC
AAC
AGT
CTC
GAC
TTG
CAC
AAC
GAT
CAG
CAA
CTT
TGG
TTG
AAT
CGA
GCC
TGG
CTG
GGG
CTT
AGG
GGC
TGC
GTC
ACA
TGC
TTC
CTG
GGC
GAG
ATC
CTG
TCT
GGG
CGC
GGC
TCA
ATG
CCA
TGT
GGA
TGC
CTC
CAC
TAT
ATA
CCT
GAG
AGC
ACA
AAT
AGC
CTA
TGA
Reverse Primer
GT
CA
TT
CA
TC
AT
GC
TAC AGA CA
TG
AT
C
CA
G
CC
CA
CC
CT
GG
CC
CC
GG
TTC
ATG
CCT
GCT
CTT
CTA
CGG
GCT
GTG
GGC
TCT
AGT
GCC
TCA
CCC
GTC
TGT
TAA
CAA
CAG
GGC
TTC
GCG
CAT
GTG
CAG
GGG
AAG
CAT
TAT
TCT
TGC
TTT
TCC
AAA
ATG
ACG
GGA
AGA
CTC
CTC
CAA
ACC
TCT
GGA
GAT
CAC
AGG
AAG
TGT
CTG
GAG
CTC
GCA
AAG
CGG
CTG
CTT
GAA
GCG
CAA
CGA
GAT
Tsc2 and Rpl13a were used for normalization of genomic DNA and mRNA, respectively.
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00521.2015 • www.ajpendo.org
GTA
GCA
AGG
CCG
CTG
GGA
CCC
TGT
GGC
TTT
CAA
TCT
GTC
AGA
GCC
GAT
TCG
GGT
GGA
CCA
GCA
GGT
AAT
ACG
TTC
TCC
CTG
TTG
ACT
CAA
CCG
AGG
GGG
AAG
TTC
TTC
AGC
AAC
GAG
CGG
TGA
CTA
GCG
GGT
TAG
GTA
CTT
CTC
CAC
GGT
CGG
AAG
ATG
TGT
CTT
CCG
AGT
CTC
GGC
CCT
AGC
AGT
TCG
TC
TG
CC
GT
GT
AT
C
TGG ATA TG
AA
CA
TCA
TTA GC
CT
CA
TA
CA
AA
TC
AT
G
GA
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containing amorphous silica particle control (1 mg/ml), were prepared
in irrigation saline containing 0.02% Tween 80, and the resulting
particle suspension was monodispersed by probe sonication.
Animals, particulate exposure, and high-fat diet. C57BL/6NHsd
mice were purchased from Harlan (Indianapolis, IN). Mice were
maintained in a 12-h light-dark cycle room at constant temperature
and humidity and allowed unrestricted access to food and water.
Breeder mice (6 wk of age) were mated and pregnant dams administered 50 ␮l of MCP230 particle suspension via oropharyngeal aspiration on days 10 and 17 of gestation, as described earlier (51).
Control mice received 50 ␮l of saline or cabosil. Pregnant mice were
anesthetized by inhalant anesthetic isoflurane (5%) and placed in a
holder and physically supported in an upright position. The suspension was instilled just above the vocal cords while the tongue was held
with forceps to prevent swallowing. Offspring were weaned at 4 wk
of age. Male mice were selected for the study and were fed standard
rodent chow until 10 wk of age. At 10 wk of age, mice were switched
from chow to a high-fat diet (HFD) consisting of 45% of calories from
fat (catalog D12451; Research Diets). Mice were maintained on HFD
for 12 wk. One mouse, an MCP230-treated animal, had malocclusion
and was removed from all data analyses. The University of Tennessee
Health Science Center Institutional Animal Care and Use Committee
approved all mouse procedures.
Metabolite assays. Blood was collected in both the fed and 16-hfasted state. Blood glucose was determined using an AccuCheck
glucometer. Serum hormone levels were determined using a Bio-Plex
pro mouse diabetes multiplex immunoassay (no. 171-F7001M; BioRad), following the manufacturer’s instructions using a MAGPIX
LMX200 system. Homeostatic model assessment of insulin resistance
(HOMA-IR) was calculated from 16-h-fasting glucose and insulin
values.
Body composition and metabolic cages. Mice were weighed
weekly from approximately Zeitgerber time (ZT)10. Body composition was determined noninvasively using an echo-MRI 100. Food
intake during the HFD phase was determined on a per-cage level by
weighing the food on a weekly basis. For food intake pre-HFD, this
was determined by scaled feeders within the Comprehensive Laboratory Animal Monitoring System (CLAMS) system.
V̇O2, energy expenditure, ambulatory locomotor activity, and respiratory exchange ratios were determined in a home cage style
GESTATIONAL EPFR EXPOSURE AND ENERGY EXPENDITURE
Citrate synthase activity. Muscle homogenates were prepared in
KCl-EDTA buffer (pH 7.4) from ⬃10 to 40 mg of frozen quadriceps.
Following three freeze-thaw cycles, samples were centrifuged at 4°C
for 10 min at 1,000 g to settle cellular debris. Supernatants were
analyzed for citrate synthase activity using a modified method (40).
Briefly, aliquots of supernatant were added to the appropriate wells of
a 96-well microplate containing an assay solution comprised of Tris
(72.5 mM), acetyl-CoA (0.45 mM), and 5,5=-dithiobis-2-nitrobenzoate (DTNB; 0.1 mM) at a pH of 8.3. After the plate was monitored for
possible background activity, activity reactions were initiated by the
addition of oxaloacetic acid (0.5 mM) to each well. Changes in
absorbance at 405 nm were recorded for each well every 9 –11 s over
3 min at room temperature. Citrate synthase activity was calculated
using the extinction coefficient for DTNB (which is reduced by the
CoA-SH released during the cleavage of acetyl-CoA by citrate synthase).
Statistics. All raw data, and analysis scripts are available at http://
bridgeslab.github.io/ObesityParticulateTreatment (42). Statistics and
calculations were performed using R version 3.1.1 (34). For longitudinal data, mixed linear models were used and ␹2 tests performed to
determine the significance of the MCP230 treatment. Mixed linear
models used the R package lme4 (version 1.1–7; Ref. 4). In all cases,
normality of the data and models were determined via Shapiro-Wilk
Test, and equal variance was tested using Levene’s test from the car
package (version 2.0 –21; Ref. 19). Pairwise Student’s t-test, Welch’s
t-test, or Wilcoxon rank sum tests were performed as indicated in
RESULTS and the figure legends and were dependent on normality and
homoscedasticity. In cases where cabosil and saline treatment were
not significantly different, these data were combined and designated
as a single control group. For energy expenditure calculations, we
performed an ANCOVA analysis with lean body mass and the
treatment group as noninteracting covariates and the averaged light or
dark V̇O2 as the responding variable as described previously (48).
Statistical significance was designated as P ⬍ 0.05.
Fig. 1. In utero exposure to laboratory-generated, combustion-derived particular matter (MCP230) results in
increased body size. A: schematic of the experimental
design. B: body weight throughout the high-fat diet
phase of the intervention. C–E: absolute body fat (C),
fat-free mass (D), and %body fat (E) after 12 wk of
high-fat diet Zeitgerber time (ZT)12. Data shown are
group means ⫾ SE. §P ⬍ 0.05 via mixed linear model,
compared by ␹2 test (B); *P ⬍ 0.05 via Student’s t-test
(C and D). Black bars, saline-exposed mice; gray bars,
MCP230-exposed mice.
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levels were calculated using the ⌬⌬CT method, with data for mtDNA
copy number being normalized to values obtained for a nuclearencoded genomic locus (Tsc2) and mRNA levels being normalized to
Rpl13a, which was determined to be unaffected by MCP230 treatment
compared with other commonly used normalization controls, including Rplp0 and Gapdh.
Preparation of protein lysates and Western blotting. Skeletal muscle homogenates were prepared from ⬃30 to 50 mg of frozen
quadriceps in RIPA buffer [Tris basic (50 mM), sodium deoxycholate
(0.25%), NP-40 (1%), NaCl (150 mM), EDTA (1 mM), Na3VO4 (100
␮M), NaF (5 mM), sodium pyrophosphate (10 mM), protease inhibitor cocktail] using stainless-steel beads and a Qiagen Tissue Lyser
(30 Hz for 5 min). Homogenates were centrifuged at 4°C for 10 min
at 14,000 g, after which the protein concentration of supernatants was
determined by Bradford assay. Lysates of equal protein concentration
were prepared in 2⫻ Laemmli buffer containing 2-mercaptoethanol
and heated at 37°C (for mitochondrial proteins) or 95°C (for all
nonmitochondrial proteins) for 5 min. Proteins were separated by
SDS-PAGE and transferred to nitrocellulose membranes for Western
blotting. After ponceau staining to ensure equal protein loading,
membranes were blocked in BSA for 1 h and incubated overnight in
total OXPHOS rodent WB antibody cocktail (no. ab110413; Abcam),
anti-PGC-1␣ (no. SAB4200209; Sigma), anti-phospho AMPK Thr172
(Cell Signaling Technology no. 2535S), anti-AMPK (no. 2793S; Cell
Signaling Technology), anti-phospho S6K Thr389 (no. 9206; Cell
Signaling Technology), anti-S6K (no. 2708; Cell Signaling Technology), anti-phospho Akt Ser473 (no. 4060; Cell Signaling Technology),
anti-Akt (no. 9272; Cell Signaling Technology), anti-LC3 (no. 12741;
Cell Signaling Technology), or anti-␤-actin (no. 60008-1-Ig; Proteintech) at 4°C. Blots were visualized after a 1-h incubation with infrared
anti-mouse or anti-rabbit secondary antibody, using a LI-COR Odyssey fluorescent Western blotting system. Protein expression was
quantified using densitometry (Image Studio Lite; LI-COR) and
normalized to Akt, which was unchanged by the treatments. LC3 was
presented as the ratio of light chain 3 (LC3)II/LC3I as prescribed (25).
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GESTATIONAL EPFR EXPOSURE AND ENERGY EXPENDITURE
RESULTS
Fig. 2. Gestational exposure to MCP230 causes a reduction in food intake and alters “hunger hormone”
concentrations on a high-fat diet. A and B: food intake
per mouse was calculated on a weekly (A) and cumulative (B) basis throughout the high-fat diet phase of the
intervention. C–E: MCP230-exposed mice had elevated
serum concentrations of leptin (C), ghrelin (D), and
glucagon-like peptide-1 (GLP-1; E) after access to the
high-fat diet. F: serum glucose-dependent insulinotropic
polypeptide (GIP) tended to be elevated during the
fasted state, although this did not attain statistical significance. Fed serum was collected at ZT12. Fasting
serum was collected following an overnight fast
(⬃16 h) at ZT4. Data shown are group means ⫾ SE; n
⫽ 8 –14/group. §P ⬍ 0.05 by mixed linear model,
compared by ␹2 test (B); †main effect for feeding state
(C–F); ‡main effect for MCP230 exposure by 2-way
ANOVA (C–E); *P ⬍ 0.05 via a Wilcoxon rank sum
test (D and E). Black bars, saline-exposed mice; gray
bars, MCP230-exposed mice.
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Gestational exposure to MCP230 leads to increased body
size on a high-fat diet. To test the metabolic effects of gestational exposure to EPFRs, pregnant females were exposed to
MCP230 on days 10 and 17 of gestation. As controls, mice
were exposed to either cabosil (the nonconjugated particulate
without the EPFR group) or saline. After birth, these mice were
left with their dams until weaning onto standard rodent chow at
28 days of age. At 10 wk of age, male mice were placed on a
high-fat diet (HFD) consisting of 45% of calories from lard to
induce obesity (Fig. 1A).
As shown in Fig. 1B, at 10 wk of age, mice that were
exposed to MCP230 had a 7.6% higher body weight than the
saline-exposed mice and remained heavier, gaining more
weight throughout the HFD phase (P ⫽ 3.5 ⫻ 10⫺5). After 12
wk of HFD, the MCP230-exposed mice were 4.5 g heavier
than saline-exposed mice (9.8%, P ⬍ 0.001; Fig. 1B). We
assessed body composition after 12 wk of HFD and observed
significant elevations in both fat mass (10.1% increase, P ⫽
0.011) and fat-free mass (10.2% increase, P ⫽ 2.2 ⫻ 10⫺4) in
the MCP230-exposed mice (Fig. 1, C and D). The relative
adiposity of these mice, as determined by the percent fat mass,
was not different between groups (Fig. 1E).
MCP230-exposed mice have reduced caloric intake and
increased serum concentrations of leptin, ghrelin, and GLP-1.
To determine how energy balance was affected in MCP230exposed mice, we examined their food intake throughout the
HFD feeding period. As shown in Fig. 2A, all mice tended to
eat less food each week, although this did not reach statistical
significance. Cumulatively, the MCP230-exposed mice ate less
food throughout the diet (⫺6.3 ⫾ 1.8 kcal·wk⫺1·mouse⫺1, P ⫽
8.0 ⫻ 10⫺4; Fig. 2B). Throughout the 12-wk HFD treatment,
this corresponds to a 19.2% reduction in total caloric intake.
During the metabolic cage experiments, which occurred prior
to HFD feeding, the MCP230-exposed mice tended to eat less
food per feeding bout, whereas each feeding bout also tended
to be shorter in duration; however, neither of these parameters
were significantly different (data not shown). There were no
differences between groups for the frequency of feeding bouts.
Leptin concentrations were modestly elevated in serum from
MCP230-exposed mice (main effects feeding state, P ⫽ 0.002,
and treatment, P ⫽ 0.011, by 2-way ANOVA, with post hoc
t-test P values of 0.058 under fasting and P ⫽ 0.097 under fed
conditions; Fig. 2C). Elevations in circulating leptin levels are
consistent with the increase in fat mass observed in MCP230exposed mice (Fig. 1C). We observed significant serum elevations in both the fasting and fed states for ghrelin (for main
effects of feeding state P ⫽ 0.001, and MCP230 treatment P ⫽
6.5 ⫻ 10⫺6, with post hoc t-test P values of 0.024 in the fasting
and P ⫽ 0.0002 in the fed state; Fig. 2D), which is consistent
with a reduction in food intake (Fig. 2, A and B) and reduced
energy expenditure (Fig. 4, A–E) observed in the MCP230exposed mice (11, 46, 47, 54). Similarly, GLP-1 was elevated
in serum from MCP230-exposed mice in both the fasted and
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fed states (main effects for feeding state P ⫽ 0.002, and
treatment P ⫽ 3.6 ⫻ 10⫺5, with post hoc t-test P values of
0.024 in the fasted and P ⫽ 0.001 in the fed state; Fig. 2D),
which is also consistent with the MCP230-exposed mice eating
less (Fig. 2, A and B) (3, 49). There was an effect of the feeding
state with respect to glucose-dependent insulinotropic polypeptide (GIP) concentrations (P ⫽ 6.0 ⫻ 10⫺9 by 2-way ANOVA;
Fig. 2F), and GIP was elevated in serum from MCP230exposed mice in the fasted state, although these values did not
quite attain statistical significance (P ⫽ 0.069 by Wilcoxon
rank sum test). Although there were main effects of the feeding
state for PAI-1 and resistin levels, these were not different
between the two treatment groups (data not shown).
We next evaluated the extent of obesity-related comorbidities in these mice after 12 wk of high-fat feeding. We observed
no differences in fasting blood glucose as a result of MCP230
exposure (Fig. 3A). As shown in Fig. 3B, there was a main
effect of feeding state on serum insulin concentrations (P ⫽
3.3 ⫻ 10⫺6); however, MCP230 exposure had no effect.
Calculation of the HOMA-IR revealed that both the saline and
MCP230-exposed groups had similar HOMA-IR scores
(12.77 ⫾ 1.29 vs. 12.14 ⫾ 0.96 for saline and MCP230,
respectively, P ⫽ 0.74; Fig. 3C). Taken together, these data
indicate that although HFD did impair insulin sensitivity, there
was no difference between these two groups. Consistent with
this, we observed no changes in the levels of fasted Akt
phosphorylation in muscle tissue (data not shown). With respect to glucagon levels, both feeding state (P ⫽ 7.3 ⫻ 10⫺5)
and MCP230 treatment (P ⫽ 4.0 ⫻ 10⫺3) increased serum
glucagon concentrations. MCP230-exposed mice had elevated
glucagon concentrations in the fasted and fed state, although
fed state levels did not quite attain statistical significance
(32.65%, P ⫽ 0.009 for fasting and 28.46%, P ⫽ 0.059 for fed
by post hoc Wilcoxon rank sum tests; Fig. 3D).
MCP230 mice have reduced energy expenditure. Since the
MCP230 mice did not appear to be larger due to excessive
caloric intake, we next examined their energy utilization. To
evaluate energy expenditure, we individually housed 9-wk-old
mice (prior to HFD) in metabolic cages for indirect calorimetry, physical activity monitoring, and evaluation of gas exchange rates. As shown in Fig. 4, A and B, the MCP230exposed mice had lower oxygen consumption (V̇O2) in both the
light and dark phases (⫺19.1%, P ⫽ 0.031, and ⫺16.8%, P ⫽
0.019, respectively). This reduction in V̇O2 translated to a
similar reduction in energy expenditure in both the light and
dark phases (⫺18.4%, P ⫽ 0.032, and ⫺16.4%, P ⫽ 0.021,
respectively; Fig. 4, C and D). In Fig. 4, B and D, each circle
represents the average value for each individual mouse plotted
against fat-free mass. Accounting for change in lean mass is
necessary due to known associations between this covariate
and rates of oxygen consumption (48). To determine whether
these decreases in energy expenditure were associated with
changes in physical activity, we monitored the ambulatory
movements of these mice while they were housed in the
metabolic cages. As shown in Fig. 4E, compared with the
control group, we observed 21.4 (P ⫽ 0.040) and 26.2% (P ⫽
0.0099) reductions in physical activity for the MCP230-exposed mice in the dark and light phases, respectively.
Next, we evaluated energy substrate preference by analyzing
the respiratory exchange ratio of the three groups. When this
ratio nears 1, that indicates preference for predominately car-
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Fig. 3. Gestational exposure to MCP230 causes an increase in serum glucagon
but does not differentially alter glucose or insulin concentrations following
exposure to a high-fat diet. Fasting blood glucose (A), serum insulin (B),
homeostatic model assessment of insulin resistnace (HOMA-IR; C), and serum
glucagon (D) concentrations were determined after a 16-h fast at ⬃ZT4. Fed
serum was collected at ZT12 and analyzed for insulin (B) and glucagon (D).
Data shown are group means ⫾ SE; n ⫽ 8 –14/group. †Main effect for feeding
state (B and D); ‡main effect for MCP230 exposure by 2-way ANOVA (D);
*P ⬍ 0.05 via a Wilcoxon rank sum test (D). Black bars, saline-exposed mice;
gray bars, MCP230-exposed mice.
bohydrate as fuel, and as it nears 0.7 it indicates utilization of
mainly lipids (5). Although there was no difference in the
respiratory exchange ratio between MCP230- and cabosilexposed mice, we did observe a significant elevation (carbohydrate preference) in the saline-exposed mice during both the
light and dark phases relative to mice exposed to either the
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vehicle control (cabosil) or MCP230 (the EPFR) (Fig. 4F).
These data indicate that particle exposure alone (cabosil) altered substrate preference but that exposure to the EPFR did
not alter substrate preference.
Skeletal muscle from MCP230-treated mice have reduced
mtDNA copy number and a lower citrate synthase activity.
Because of the observed reductions in whole body oxygen
consumption and total energy expenditure, we next explored
the hypothesis that MCP230-exposed mice have skeletal muscle mitochondrial deficits, as muscle is the major organ responsible for variations in resting energy expenditure (57). To test
this, we first determined mitochondrial DNA (mtDNA) copy
number in quadriceps muscle after the 12-wk HFD phase.
Figure 5A demonstrates that MCP230-exposed mice have a
marked reduction in mtDNA copy number relative to the
saline-exposed mice, as determined using primers designed for
three distinct mtDNA-encoded gene regions. Decreases of
61.2, 68.0, and 51.9% were observed for the mitochondrial
D-loop, mt-Cytb, and mt-Nd1, respectively (P ⫽ 0.039, P ⫽
0.031, and P ⫽ 0.032, respectively), suggesting that MCP230exposed mice may have reduced skeletal muscle mitochondrial
content. Since citrate synthase activity is better associated with
skeletal muscle mitochondrial content than mtDNA copy number (and is also a good indicator of tricarboxylic acid cycle
activity; see Ref. 27), we measured citrate synthase activity to
further evaluate mitochondrial content and function in the
skeletal muscle from MCP230-exposed mice. As shown in Fig.
5B, maximal citrate synthase activity was reduced 24.1% in the
quadriceps from MCP230-exposed mice (P ⫽ 0.03). Together,
reduced mtDNA copy number and lower citrate synthase
activity suggest that mice exposed to MCP230 may have
reduced mitochondrial oxidative enzyme content and, as a
result, reduced skeletal muscle oxidative capacity, which,
along with the reduction in physical activity, would likely
contribute to the reduced V̇O2 seen in these mice. Consistent
with this hypothesis, mRNA transcript levels for the mitochondrial- and nuclear-encoded electron transport genes mt-Nd4
(25.2%), Sdha (35.9%), mt-Cytb (35.4%), and mt-Co2 (35.1%)
were reduced in the quadriceps from MCP230-exposed mice,
although not all of these reductions attained statistical significance (P ⫽ 0.12, P ⫽ 0.08, P ⫽ 0.04, and P ⫽ 0.10,
respectively; see Fig. 5C). To determine whether differences in
skeletal muscle mitochondrial electron transport enzymes were
present at the protein level, we measured the relative expression of several electron transport chain proteins via Western
blotting (Fig. 5D). We did not observe differences in levels of
any of the five oxidative phosphorylation proteins measured in
skeletal muscle, nor did we see changes in PGC-1␣ protein
expression (Fig. 5, D and E) with MCP230 exposure. To test
whether the lack of change in mitochondrial protein expression
was due to suppression of autophagy, we blotted for processing
of LC3 and observed no evidence of decreased autophagy (Fig.
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Fig. 4. In utero exposure to MCP230 reduces
energy expenditure and lowers physical activity. A and B: O2 consumption rates (V̇O2;
A) and V̇O2 analysis (B) normalized to fatfree mass during both the light and dark
phases. Each circle represents the average
O2 consumption of each mouse. C and D:
time course of energy expenditure (C) and
energy expenditure normalized to fat-free
mass (D) during both the light and dark
phases. Each circle represents the average
energy expenditure of each mouse. E: quantification of ambulatory movement during
the light and dark phases. F: respiratory
exchange ratio of each group. Saline and
cabosil groups were not combined for this
analysis because there was a significant reduction in the respiratory exchange ratio for
both the cabosil- and MCP230-exposed
groups. Data shown are either individual (B
and D) or group means (A, C, E, and F) ⫾
SE (E and F); n ⫽ 18, 6, or 14 for MCP230,
saline, and cabosil groups, respectively.
§P ⬍ 0.05 by ANCOVA (B); *P ⬍ 0.05 by
Student’s t-test (E) or Wilcoxon rank sum
test (F). Black bars, saline-exposed mice;
open bars, cabosil-exposed mice; gray bars,
MCP230-exposed mice; black and white
striped bars, combined saline- and cabosilexposed groups.
GESTATIONAL EPFR EXPOSURE AND ENERGY EXPENDITURE
E1009
5, D and F). We did not see alterations in any of the other
measured markers of skeletal muscle metabolism and growth
(phospho-Akt Ser473, phospho-AMPK Thr172, or phospho-S6K
Thr389; data not shown).
To test whether reductions in mtDNA copy number and
citrate synthase activity were due to lowered mitochondrial
biogenesis, we evaluated the expression level of several known
mitochondrial biogenesis genes. Although we observed increases in the mRNA of Ppard and Ppargc1b (Fig. 6, A and C),
there were no differences in the expression levels of Ppargc1a,
Nrf1, Nfe2l2, or Tfam (Fig. 6, B and D–F). In the absence of
reduced mitochondrial biogenesis markers or changes in the
levels of oxidative phosoporylation enzymes, the mitochondrial deficits we observe in the skeletal muscle of mice exposed
to MCP230 may be a response to oxidative stress rather than
transcriptional downregulation of mitochondrial biogenesis per
se. In support of this notion, we found robust increases in the
mRNA for Ucp2, Sod1, Sod2, Cat, Gpx1, and Gclm, enzymes
activated in response to oxidative stress (Fig. 7).
DISCUSSION
Although epidemiological studies have linked exposure to
particulate matter (PM) with obesity and its comorbidities, few
have evaluated energy expenditure changes in response to
acute gestational exposure. In this study, we tested some of the
metabolic effects of a limited gestational exposure to a recently
realized environmental pollutant that is present in most combustion-derived PM: EPFRs. Each exposure of MCP230 that
the mothers received was the equivalent to a human breathing
200 ␮g/m3 of EPFR, which is similar to what would be inhaled
on a typical day in one of the major US cities (38). We noted
that pups born from mothers that were acutely exposed to PM
grew larger, despite reductions in food intake, and that this was
associated with reduced energy expenditure and mitochondrial
impairments in skeletal muscle.
One potential explanation for reductions in energy expenditure and skeletal muscle mitochondrial function is the observed
reduction in physical activity for MCP230-exposed mice. It is
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Fig. 5. Exposure to MCP230 in utero results
in skeletal muscle mitochondrial abnormalities following high-fat diet consumption as
adults. mtDNA copy number (A), citrate
synthase activity (B), and mRNA levels of
oxidative phosphorylation genes (C) were
reduced in the quadriceps muscles of mice
that were indirectly exposed to MCP230 in
utero and subjected to 12 wk of high-fat diet
as adults. Quadriceps PPAR␥ coactivator-1
␣ (PGC-1␣), light chain 3 (LC3), and select
electron transport chain protein expression
was unchanged in the MCP230-exposed
mice (D, representative blots, and E and F,
relative quantification). Data shown are
group means ⫾ SE. *P ⬍ 0.05 via Student’s
t-test; n ⫽ 7–12/group. Black bars, salineexposed mice; gray bars, MCP230-exposed
mice. ATP5A, ATP synthase H⫹ transporting ␣-subunit; UQCRC2, ubiquinol-cytochrome c reductase core protein II; MtCO1,
mitochondrially encoded cytochrome c oxidase I; SDHB, succinate dehydrogenase
complex, subunit B; NDUFB8, NADH,
ubiquinone oxidoreductase subunit B8.
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GESTATIONAL EPFR EXPOSURE AND ENERGY EXPENDITURE
also possible that muscle weakness (due to reduced skeletal
muscle oxidative capacity; see Refs. 44 and 57) could contribute to the reduced physical activity of MCP230-exposed animals. Both of these hypotheses are consistent with crosssectional studies showing negative associations between ambient air pollutant exposure and leisure time physical activity
(35) and exercise performance (14, 33, 36). Our current data
are unable to distinguish whether reduced mitochondrial function is the primary cause of these reductions in energy expenditure or whether this observation is secondary to a reduced
propensity for physical activity or some other mechanism.
However, our observations of reductions in mtDNA, citrate
synthase activity, and mRNA transcripts support the hypothesis that gestational exposure to EPFRs can affect skeletal
muscle mitochondrial oxidative function, which would contribute to the overall changes we observe in energy expenditure.
The mechanisms by which gestational exposure to EPFRs
result in reduced mitochondrial function are not yet clear. Our
data are consistent with chronic models of PM2.5 exposure,
which show reduced mitochondrial numbers in white adipose
tissue (55, 56). Analyses of placental tissues from mothers
showed a strong correlation between late-gestational PM10
exposure and placental mtDNA content (22). Given the elevated sensitivity of mitochondria to free radicals and oxidative
stress, it is reasonable to hypothesize that, during development,
EPFR-mediated mitochondrial damage may result in chronic
decreases in mitochondrial oxidative function either directly
via reactive oxygen species (ROS) or indirectly via inflammatory processes. Indeed, the bioenergetics proteins in skeletal
muscle are highly susceptible to ROS-induced posttranslational
modifications, changes thought to be important for reducing
endogenous ROS production and protect against irreversible
oxidative damage during periods of cellular stress (26). In line
with this concept, Siegel et al. (39) have shown that mild
oxidative stress in vivo impairs skeletal muscle oxidative
efficiency and reduces oxidative phosphorylation coupling
without altering the expression of key electron transport chain
proteins or their respiratory activities ex vivo. This suggests
that reduced skeletal muscle oxidative capacity in response to
oxidative stress is probably not due to downregulation of the
Fig. 7. The antioxidant defense system is upregulated in the
quadriceps of MCP230-exposed mice. Data shown are
group means ⫾ SE. *P ⬍ 0.05 via Student’s t-test (Cat),
Welch’s t-test (Sod1, Sod2, Gpx1), or Wilcoxon rank sum
test (Ucp2, Gclm); n ⫽ 7–12/group. Black bars, salineexposed mice; gray bars, MCP230-exposed mice.
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Fig. 6. Indirect exposure to MCP230 in utero is not
associated with reductions in the mRNA of upstream
regulators of mitochondrial biogenesis Ppard (A) and
Ppargc1b (C). mRNA was elevated in the MCP230exposed mice, whereas Ppargc1a (B), Nrf1 (D), Nfe2l2
(E), and Tfam mRNA (F) were not different. Data
shown are group means ⫾ SE. *P ⬍ 0.05 via Student’s
t-test (A) or Wilcoxon rank sum test (C); n ⫽ 7–12/
group. Black bars, saline-exposed mice; gray bars,
MCP230-exposed mice.
GESTATIONAL EPFR EXPOSURE AND ENERGY EXPENDITURE
utero exposure to EPFRs can affect energy metabolism later in
life highlights a need for further research in this area.
ACKNOWLEDGMENTS
We thank the other members of the Bridges, Cormier, O’Connell, and Han
laboratories for helpful discussions and insights. We also thank the University
of Tennessee Health Science Center Molecular Resource Center, including
William Taylor and Felicia Waller, for assistance with quantitative RT-PCR.
GRANTS
We acknowledge funding from the Memphis Research Consortium (to J. C.
Han and D. Bridges), National Institute of Diabetes and Digestive and Kidney
Diseases Grant R01-DK-107535, Le Bonheur Grant no. 650700 (to D.
Bridges), and National Institutes of Health Grants (R01-AI-090059, R01-ES015050, and P42ES013648) to S. A. Cormier.
DISCLOSURES
The authors have no conflicts of interest, financial or otherwise, to disclose.
AUTHOR CONTRIBUTIONS
E.J.S., A.R., S.J., J.P., M.J.P., and D.B. performed experiments; E.J.S.,
A.R., S.J., J.S., S.A.C., and D.B. analyzed data; E.J.S., A.R., J.C.H., S.A.C.,
and D.B. interpreted results of experiments; E.J.S. prepared figures; E.J.S. and
S.A.C. drafted manuscript; E.J.S., A.R., S.J., J.C.H., S.A.C., and D.B. edited
and revised manuscript; E.J.S., A.R., S.J., J.R.R., J.P., M.J.P., J.S., J.C.H.,
S.A.C., and D.B. approved final version of manuscript; S.A.C. and D.B.
conception and design of research.
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mitochondrial biogenesis pathways or irreversible oxidative
damage to bioenergetic proteins. Similarly to previous reports
on oxidative stress-induced mitochondrial dysfunction (39), we
did not observe decreases in upstream regulators of mitochondrial biogenesis, increases in autophagy, or changes in mitochondrial protein expression as part of the chronic effects of
acute in utero MCP230 exposure. However, we did observe
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ROS-induced posttranslational changes and chronic oxidative
stress. Future studies with more direct measurements of mitochondrial function and the oxidative stress response will provide more mechanistic insight into this process.
In contrast to previous studies that use chronic pollution
models (1, 5, 9, 33, 35), we did not observe any indications that
glycemic control was impaired to a greater extent in MCP230exposed mice compared with the control groups following the
HFD despite differences in fat mass. It should be noted that all
of the mice in this study received the HFD to induce obesity
and its metabolic comorbidities, and although we did not
measure fasting glucose or insulin concentrations prior to the
change in diet, the fasting glucose and insulin concentrations of
all mice post-HFD were elevated compared with chow-fed
mice of a similar age, regardless of exposures. There were no
differences in fasting glucose, insulin, HOMA-IR score (Fig. 3,
A–C), or Akt phosphorylation in muscle tissue (data not
shown). We did not measure insulin sensitivity directly, which
limits our ability to make strong conclusions about the effects
of acute in utero PM exposure on insulin sensitivity. That said,
our data suggest that the effects of acute gestational particulate
exposure may not mimic the effects of chronic exposure, and
the risk profiles and mechanisms associated with these exposures may differ.
In conclusion, we have investigated the effects of limited
gestational exposure to combustion-derived pollutants in a
mouse model of diet-induced obesity. Our findings show that
even brief gestational exposure to environmental pollutants
such as EPFRs can result in chronic changes in growth,
metabolism, and energy balance. These changes are associated
with skeletal muscle mitochondrial deficits and reductions in
physical activity, which likely contribute to reduced energy
expenditure and a predisposition to elevated body weight when
exposed to a HFD. Although the mechanisms behind these
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