RESEARCH Open Access
Short-term, high-fat diet accelerates disuse
atrophy and protein degradation in a
muscle-specific manner in mice
Steven L. Roseno1,2, Patrick R. Davis1,2, Lance M. Bollinger3, Jonathan J. S. Powell1,2, Carol A. Witczak1,2,4,5
and Jeffrey J. Brault1,2,4,5*
Abstract
Background: A short-term high-fat diet impairs mitochondrial function and the ability of skeletal muscle to
respond to growth stimuli, but it is unknown whether such a diet alters the ability to respond to atrophy signals.
The purpose of this study was to determine whether rapid weigh gain induced by a high-fat (HF) diet accelerates
denervation-induced muscle atrophy.
Methods: Adult, male mice (C57BL/6) were fed a control or HF (60 % calories as fat) diet for 3 weeks (3wHF). Sciatic
nerve was sectioned unilaterally for the final 5 or 14 days of the diet. Soleus and extensor digitorum longus (EDL)
muscles were removed and incubated in vitro to determine rates of protein degradation and subsequently
homogenized for determination of protein levels of LC3, ubiquitination, myosin heavy chain (MHC) distribution, and
mitochondrial subunits.
Results: When mice were fed the 3wHF diet, whole-body fat mass more than doubled, but basal (innervated)
muscle weights, rates of protein degradation, LC3 content, mitochondrial protein content, and myosin isoform
distribution were not significantly different than with the control diet in either soleus or EDL. However in the
14 day denervated soleus, the 3wHF diet significantly augmented loss of mass, proteolysis rate, amount of the
autophagosome marker LC3 II, and the amount of overall ubiquitination as compared to the control fed mice. On
the contrary, the 3wHF diet had no significant effect in the EDL on amount of mass loss, proteolysis rate, LC3 levels,
or ubiquitination. Fourteen days denervation also induced a loss of mitochondrial proteins in the soleus but not the
EDL, regardless of the diet.
Conclusions: Taken together, a short-term, high-fat diet augments denervation muscle atrophy by induction of
protein degradation in the mitochondria-rich soleus but not in the glycolytic EDL. These findings suggest that the
denervation-induced loss of mitochondria and HF diet-induced impairment of mitochondrial function may combine
to promote skeletal muscle atrophy.
Keywords: Obesity, Skeletal muscle, Muscular atrophy
* Correspondence: braultj@ecu.edu
1East Carolina Diabetes and Obesity Institute, East Carolina University,
Greenville, NC, USA
2Human Performance Lab, Department of Kinesiology, College of Health and
Human Performance, East Carolina University, Greenville, NC, USA
Full list of author information is available at the end of the article
© 2015 Roseno et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Roseno et al. Nutrition & Metabolism  (2015) 12:39 
DOI 10.1186/s12986-015-0037-y
Background
Loss of muscle mass and strength increase disability [1]
and are independent predictors of mortality [2, 3]. The
deleterious effects of low muscle mass are compounded
during obesity, as obese individuals with less muscle are
at a greater risk for death due to certain cancers [4, 5],
cardiovascular disease [6, 7], and renal disease [8, 9].
Furthermore, severely obese individuals tend to be much
less active, and muscle disuse is a potent inducer of
muscle mass loss [10]. Despite the connection between
weight gain, low muscle mass, and increased mortality,
little is known about how obesity influences skeletal
muscle atrophy.
The duration of hypercaloric feeding seems to have an
effect on protein metabolism. Long-term (5 month)
high-fat feeding of C57BL/6 mice [11] or life-long gen-
etic mutation of the leptin receptor (db/db) [12] leads to
severe obesity, increased basal rate of muscle protein
degradation, and loss of skeletal muscle mass. In both
these models, blood glucose and insulin concentrations
are profoundly elevated demonstrating that the mice are
overtly diabetic, a condition known to induce muscle at-
rophy [13]. Conversely, mice fed a high-fat diet for a
shorter duration (2 months or less) have no apparent de-
fects in basal muscle protein metabolism [14], but dem-
onstrate whole-body insulin resistance and impaired
muscle mitochondria function [15, 16]. High-fat fed
mice also have an impaired ability to increase translation
of muscle proteins during load-induced hypertrophy
[17] and do not have the typical increased rate of muscle
protein synthesis in response to a meal [14] demonstrat-
ing that the ability to regulate protein synthesis in re-
sponse to external cues is impaired. However, whether a
short-term high fat diet affects the response of muscle to
atrophy signals is unknown.
The purpose of this study was to determine whether
rapid weight gain induced by a high-fat diet accelerates
muscle atrophy due to disuse (loss of innervation). Given
that proper function of mitochondria are required to
maintain muscle mass [18, 19], we hypothesize that a
hypercaloric diet as characterized by rapid weight gain
will make muscles more susceptible to atrophy. To test
this, mice were fed a high fat diet for 3 weeks and atro-
phy induced by surgical sectioning of the sciatic nerve.
Methods
Animals and study design
Six-week old, male C57BL/6 mice were purchased from
Charles River Laboratories (Raleigh, NC). Mice were
housed at 22 °C in a 12:12 h light:dark cycle and pro-
vided free access to water and standard chow food. After
a two week acclimation period, mice were divided into
three groups; and each group (n = 18–20) was given free
access to a different diet for 12 weeks. The control group
(Control) was fed standard rodent chow (Prolab, RMH
3000) containing 14 % calories from fat. The 3-weeks high-
fat (3wHF) group was fed the control diet for 9 weeks and
then switched to a high-fat diet (Research Diets, D12492)
containing 60 % calories from fat for an additional 3 weeks.
The third group was fed only the high-fat diet (12wHF).
Mice were weighed weekly, and body composition (fat
mass and fat-free mass) was measured every third week
using an EchoMRI 700 Body Composition Analyzer. All
animal procedures were approved by the East Carolina
University Animal Care and Use Committee.
Muscle atrophy was induced by surgical, unilateral de-
nervation of the lower hindlimb as done previously [20]
at either 5 days or 14 days before the end of each diet
(n = 9–10 per group). Mice were anesthetized with in-
haled 2-3 % isoflurane with oxygen. Hair was removed
from the lateral surface of both hindlimbs, and the skin
disinfected with povidone-iodine. A small incision
(<5 mm) was made on the lateral surface of the hindlimb
at approximately mid-femur. The sciatic nerve was ex-
posed via blunt dissection, and a 2 mm section was cut
and removed. The skin was then closed with surgical glue
(3M Vetbond Tissue Adhesive). Sham surgery was per-
formed on the contralateral limb, leaving the sciatic nerve
intact. A subcutaneous dose of Buprenex (0.03 mg/kg)
was given as an analgesic.
Sample collection
Blood was collected from a small incision at the tip of
the tail and immediately tested for glucose. Mice were
anesthetized by an intraperitoneal injection of ketami-
ne:xylazine (100:10 mg/kg) and then euthanized by cer-
vical dislocation. Whole blood was collected by syringe
from the abdominal aorta and allowed to clot on ice.
The blood was centrifuged, and serum transferred to a
separate tube for storage at −80 °C. It is important to
note that samples were collected from fed mice, since fast-
ing induces a rapid increase in the rate of protein degrad-
ation and expression of atrophy-related genes [21].
Soleus and extensor digitorum longus (EDL) muscles
were removed, blotted dry, weighed and then mounted
for measures of protein degradation. These muscles rep-
resent extremes of metabolic fiber types in the mouse,
with the highly-oxidative soleus containing more than
80 % type I and type IIa fibers and the highly glycolytic
EDL having greater that 85 % type IIb fibers [22].
Blood glucose, serum insulin, and serum myostatin
Whole blood glucose levels were measured using a gluc-
ometer (OneTouch Ultra 2). Serum insulin was measured
in duplicate using an enzyme-linked immunosorbent assay
(ELISA) kit for rat/mouse insulin (EZRMI-13 K, Millipore)
as directed by the manufacturer. Serum myostatin was
measured in duplicate using an ELISA kit for mouse
Roseno et al. Nutrition & Metabolism  (2015) 12:39 Page 2 of 11
myostatin (E91653Mu, USCN Life Science Inc.) performed
as directed by the manufacturer. Importantly, the duration
of muscle denervation, which only effects a small propor-
tion of whole-body muscle mass, had no effect on measures
of blood glucose, insulin, or myostatin. Therefore, the data
of the two denervation duration groups were combined.
Protein degradation
Rates of protein degradation were determined by measur-
ing the release of the essential amino acid tyrosine from
isolated muscles, based on previous methods [23, 24]. Im-
mediately after weighing, muscles were secured with cus-
tom plastic clips [25] at approximately resting length.
Muscles were incubated for 30 min in Krebs-Henseleit buf-
fer containing 5 mM glucose and 0.15 mM pyruvate. All
buffers were maintained at 37 °C and gassed continuously
with 95 % O2/5 % CO2. The muscles were then blotted
and transferred to a new incubation well containing 3 ml
Krebs-Henseleit/glucose/pyruvate buffer and 0.5 mM cy-
cloheximide, to inhibit reincorporation of amino acids by
protein synthesis. Two hours later, muscles were removed,
blotted dry, and frozen in liquid nitrogen. The final incuba-
tion media was removed and treated with perchloric acid
(PCA) (0.2 N final concentration) to precipitate proteins
and small peptides. The PCA soluble tyrosine in the
buffer was measured by first derivatizing the samples with
Waters AccQTag technology [26] and then quantifying
the derivatized amino acids using a Waters Acquity Ultra
Performance Liquid Chromatograph H-class. Rates of
protein degradation are given as nmol tyrosine per mg
muscle per hour.
Protein analysis
Frozen soleus and EDL muscles were homogenized in
ice-cold RIPA buffer and diluted to a final protein con-
centration of 500–2500 ng/μl in sample buffer (2 % SDS,
80 mM Tris–HCl, 22 % glycerol, 50 mM DTT, bromo-
phenol blue). Protein concentration was determined by
bicinchoninic acid assay with bovine serum albumin as
the standard (ThermoScientific).
Myosin heavy chain (MHC) isoforms were separated
using a mini-gel electrophoresis system (BioRad) as we have
done previously in cultured myotubes [27]. Samples (5 μl
per well) were loaded and electrophoresis was performed
with a 35 % v/v glycerol, 8 % w/v acrylamide-N,N’-methyle-
nebisacrylamide (bis) (9:1) gel for 22 h at a constant 130
volts at 4 °C. The gels were then silver stained (Pierce Silver
Stain Kit), and images were captured using a BIO RAD
Molecular Imager ChemiDoc™ XRS+ Imaging System and
analyzed using Image Lab 3.0 software. Identity of specific
MHC isoforms was initially confirmed by immunoblotting.
For the autophagy related protein LC3, ubiquitin, and
mitochondrial proteins, equal amounts of total protein
were separated by 10 % SDS-PAGE and transferred to
polyvinylidene difluoride membranes. Membranes were
incubated overnight with a primary antibodies against:
mono and poly ubiquitinated conjugates (#BML-PW8810,
Enzo), LC3B (#3868, Cell Signaling), CoxIV (#4844, Cell
Signaling), or cocktail against proteins in electron trans-
port chain complexes II-V (Mitoprofile Total OXPHOS
Rodent, Mitosciences). Secondary antibodies were conju-
gated to horseradish peroxidase and detected using an en-
hanced chemiluminescent substrate (Millipore). Band
intensities were captured using a Bio-Rad Chemi Doc
XRS+ and analyzed using Image Lab 3.0 (BioRad) or
ImageJ (NIH) software [28].
Statistical analysis
All data are expressed as mean ± standard error of the
mean. Significant differences (P < 0.05) were assessed
using two-way ANOVA (for comparisons including both
diet and innervation status), one-way ANOVA (for com-
parisons of only diet groups). If significance was detected
by ANOVA, Tukey’s post hoc analysis was used to deter-
mine which groups were different. All analyses were per-
formed using GraphPad Prism for Mac, version 6.0 f.
Results
Body composition and insulin resistance
To establish a model of weight gain without overt dia-
betes, mice were separated into three dietary groups.
Mice were fed for 12 weeks one of the following diets:
standard rodent chow (control), high-fat chow (12wHF),
or standard chow for 9 weeks followed by a high-fat
chow for 3 weeks (3wHF). At the completion of the di-
ets, mice on the 3wHF diet were 22 % heavier and mice
on the 12wHF diet were 42 % heavier than the control
group (Fig. 1a). The majority of this increase in body
weight was due to an increase in the amount of fat. As
compared to mice on the control diet, the 3wkHF mice
had 220 % more fat, while the 12wHF mice had 280 %
more fat (Fig. 1b). Only the 12wHF group gained signifi-
cantly more fat-free mass (8 %) (Fig. 1c).
Serum insulin and blood glucose were analyzed to esti-
mate insulin resistance, a known complication of obesity
[13, 29]. Serum insulin was increased 9.1-fold (P < 0.05)
by the 12wkHF diet but was not significantly altered
with the 3wHF diet (Fig. 1d). However, blood glucose
concentration was not significantly different among the
diet groups (Fig. 1e).
Myostatin is a muscle secreted protein that negatively
regulates muscle mass [30] and increases in severely obese
rodents [31] and humans [32]. Serum levels of myostatin
of the 3wkHF group were not different than controls, but
myostatin concentrations were more than doubled (2.2-
fold greater, p ≤ 0.05) in the 12wkHF group (Fig. 1f). Taken
together, the 3wHF diet, with continuous weigh gain even
through the final week of the diet and modestly, but non-
Roseno et al. Nutrition & Metabolism  (2015) 12:39 Page 3 of 11
significantly, increased insulin and myostatin levels, estab-
lishes a model to test the effects of rapid weight gain in the
absence of overt diabetes. Therefore, all subsequent studies
were conducted with muscles of the 3wHF mice only.
Loss of mass of denervated muscles
Since skeletal muscle is a major component of fat-free
mass, differences in fat-free mass may be reflected by
differences in muscle mass. To determine whether a
high-fat diet increased the weight of specific muscles, in-
dividual innervated muscles of the lower hind limbs
were removed and weighed. In agreement with fat-free
mass (Fig. 1c), the 3wHF diet had no significant effect
on the mass of innervated soleus or EDL muscles
(Fig. 2a). To induce muscle atrophy, hindlimb muscles
were surgically denervated with the contralateral limb
Fig. 1 Body composition and blood metabolites of mice on control or high-fat diets. Starting at week 0, mice were kept on the low-fat diet for
12 weeks (control), fed the high-fat for 12 weeks (12wHF), or fed the control diet for 9 weeks followed by the high-fat diet for 3 weeks (3wHF). Body
weight (a) was measured weekly. Fat mass (b) and fat-free mass (c) were measured by EchoMRI every 3 weeks. At the completion of the diet, serum
insulin (d), whole blood glucose (e), and serum myostatin (f) were measured. n = 18–20 per diet; *P < 0.05; #P < 0.05 vs control and 3wHF
Roseno et al. Nutrition & Metabolism  (2015) 12:39 Page 4 of 11
serving as the innervated control. Five days later, little
atrophy had occurred; the denervated muscles weighed
within 5 % of the contralateral innervated muscles re-
gardless of the diet (data not shown). However, in the
control-fed mice, fourteen days denervation induced
16 % loss of mass in the soleus and 18 % loss of mass of
the EDL (Fig. 2b). Interestingly, the 3wkHF diet substan-
tially increased the amount of atrophy only in the soleus.
The soleus muscles of the 3wkHF group lost 26 % of
their mass (Fig. 2b).
High-fat diet increases protein degradation rate in the
soleus
In lean mice, muscle atrophy due to denervation is me-
diated mainly by an increase in the rate of protein deg-
radation [7, 33]. To determine whether the increased
muscle atrophy in the soleus of the 3wHF was a result of
increased proteolysis, we measured protein degradation
by the release of the essential amino acid tyrosine. In so-
leus muscles, the rates of protein degradation increased
rapidly in the 5 day denervated muscles to 70 % greater
than innervated muscles regardless of the diet. By 14 days
post-denervation, the degradation rates of the control
fed mice decreased to rates not different from inner-
vated, while rates in the 3wHF mice remained signifi-
cantly increased (Fig. 3a). In EDL muscles, the rates of
protein degradation increased approximately 33 % in
5 day denervated muscles and further increased (68 % or
more) in 14 day denervated muscles (Fig. 3b). The high-
fat diet had no effect on rates of protein degradation in
the EDL.
The increased protein degradation of muscle atrophy
is mediated largely by activation of the autophagic/lyso-
somal and ubiquitin/proteasomal pathways [9, 34]. One
indicator of the activation of autophagy/lysosomal path-
way is the abundance of LC3 [35]. In mammalian cells
LC3 is predominantly in two forms: LC3-I, which is
cytosolic, and LC3-II, which is associated with autopha-
gosomes (Fig. 4a). In the soleus and EDL muscles, both
LC3-I (Fig. 4b) and LC3-II (Fig. 4c) were more abundant
in the denervated than in the innervated muscles. Import-
antly, the 3wHF diet induced LC3-II further (P < 0.05) in
the denervated soleus muscle leading to an increase in the
LC3-I/LC3-II ratio (Fig. 4d). However, in the EDL, the
3wHF diet had no significant effect on LC3-I abundance
Fig. 2 Mass and atrophy of hindlimb muscles. a Weights of innervated
soleus and extensor digitorum longus (EDL) muscles. b Percent change
of muscle mass of 14 day denervated muscles = (weight of denervated
muscle - weight of contralateral innervated muscle)/wet weight of
innervated muscle x 100. n= 9–10 per group; *P< 0.05 vs control
Fig. 3 Muscle protein degradation rates of mice fed a high-fat diet for three or twelve weeks. Soleus (a) and EDL (b) muscles were incubated in Krebs-
Henseleit buffer containing cycloheximide, and tyrosine release was measured over time. n= 9–10 per group; *P< 0.05, vs innerv. †P< 0.05 vs control
Roseno et al. Nutrition & Metabolism  (2015) 12:39 Page 5 of 11
(Fig. 4b), LC3-II abundance (Fig. 4c), or the ratio of LC3-
I/LC3-II (Fig. 4d).
Proteins are targeted for degradation by the proteasome
by being labeled by ubiquitin chains. Therefore, as an indi-
cator of upregulation of the ubiquitin/proteasome pathway,
we next examined the amount of ubiquitin-protein conju-
gates. Denervation increased the amount of ubiquitination
in both the soleus and EDL (Fig. 5b). Furthermore, 3wHF
diet had a main effect to increase ubiquitination in the so-
leus but had no effect in the EDL (Fig. 5b)
Fig. 4 LC3 content is increased by 14 days denervation. Mice were fed a low-fat diet (control) or a high-fat diet for 3 weeks (3wHF). The muscles
of one hindlimb were denervated (Denv) for the final 14 days, and the other hindlimb served as the innervated control (Inn). a Representative
LC3 immunoblot. b Relative quantification of LC3 I, c LC3 II, and d the ratio of the band intensities of LC3 II/LC3 I. n = 9–10 per group; *P < 0.05
main effect vs innervated. †P < 0.05 vs control
Roseno et al. Nutrition & Metabolism  (2015) 12:39 Page 6 of 11
Myosin heavy chain and mitochondrial proteins
Muscle atrophy varies among skeletal muscles with dif-
ferent fiber types [36], and fiber type may change in re-
sponse to a high-fat diet [37] or denervation [38].
Therefore, differences in muscle mass loss may be ex-
plained by changes in fiber type. As an indicator of
muscle type, the percent of myosin heavy chain (MHC)
isoforms was determined by high-resolution electrophor-
esis and silver staining of homogenates of the soleus
(Fig. 6a) and EDL (Fig. 6b). The soleus muscles
expressed an abundance (>75 %) of type I and type IIa
MHC (Table 1). Fourteen days of denervation had a
main effect to increase the percent of type I MHC pro-
tein and decrease type IIa. EDL muscles expressed pre-
dominantly (>85 %) fast-twitch type IIb MHC (Table 2).
Fourteen days of denervation had a main effect to
increase the percent type IIa fibers and decrease the per-
cent IIb fibers, i.e. a shift away from the fastest fiber-
type. The 3wHF diet had no significant effect on MHC
isoform expression either in the soleus or EDL.
Aside from myosin heavy chain, different muscles are
characterized by different metabolic properties [39].
Therefore, we also measured the amount of several mito-
chondrial electron transport chain proteins as indicators
Fig. 5 Ubiquitinated conjugates accumulate by 14 days denervation. Mice were fed a low-fat diet (control) or a high-fat diet for 3 weeks (3wHF). The
muscles of one hindlimb were denervated (Denv) for the final 14 days, and the other hindlimb served as the innervated control (Inn). a Representative
ubiquitin immunoblot. b Relative quantification band intensities. n = 9–10 per group; *P < 0.05 main effect vs innervated. †P < 0.05 vs control
Fig. 6 Representative images of myosin heavy chain isoform separation by high-resolution electrophoresis. Soleus (a) and EDL (b) muscles that
were denervated for 14 days (Denrv) or innervated (Inn) were homogenized, and equal amounts of total protein were separated by high-
resolution electrophoresis. Proteins were silver stained for visualization
Roseno et al. Nutrition & Metabolism  (2015) 12:39 Page 7 of 11
of mitochondrial content. In the soleus, 14 days denerv-
ation decreased the protein amount of all mitochondrial
proteins measured: ATP5A, SDHB, UQRC2, MTCO1,
and CoxIV (Fig. 7a-e). The 3wHF diet had no effect on
these proteins in the innervated or denervated muscles.
In EDL muscles, the abundance of mitochondrial pro-
teins was not significantly different between the dener-
vated and innervated muscle (Fig. 7f-j). The 3wHF diet
had a significant effect on only one protein, UQRC2,
which is part of the electron transport chain complex
III. UQRC2 increased in both the innervated and dener-
vated EDL muscle in response to the 3wHF. Taken to-
gether, the pattern of response differs substantially
between the soleus and EDL muscles, with 14 days de-
nervation inducing loss of mitochondrial proteins only
in the soleus.
Discussion
The major finding of this study is that a short-term
high-fat diet accelerates skeletal muscle atrophy in the
primarily oxidative soleus muscle. A high-fat diet for
three weeks, which has little effect on basal muscle size
and rates of protein degradation, augments denervation-
induced loss of mass, induction of rates of protein deg-
radation, accumulation of the autophagosome marker
LC3, and accumulation of ubiquitinated proteins in the
soleus. The high-fat diet had no effect on these parame-
ters in the EDL muscle. Therefore, a high-fat diet en-
hances the activation of protein degradation in the
predominantly oxidative fibers of the soleus muscle.
Muscle type-specific differences in protein degradation
and atrophy demonstrated here due to a hypercaloric
diet are consistent with muscle type-specific responses
to various atrophy signals. For instance, Duchenne mus-
cular dystrophy [40], fasting [41], and glucocorticoid ad-
ministration [42] preferentially affect fast skeletal muscle
fibers. On the other hand, muscles composed of a sub-
stantial proportion of slow fibers (e.g. rodent soleus) are
often, but not always, more susceptible to inactivity or
spinal cord injury [43]. The soleus is phenotypically
unique among mouse lower hindlimb muscles in that it
has a substantial proportion of slow twitch, oxidative
type I fibers and few to no fast-twitch glycolytic type IIb
fibers [22, 44]. The other muscle examined herein, the
EDL, has little to no MHC type I fibers and a substantial
amount of type IIb fibers [22, 44]. The mouse soleus
muscle also has greater oxidative capacity as evidence by
having greater activity of several mitochondrial enzymes
[45] and greater coupled and uncoupled respiration [46]
as compared to the EDL. Therefore, our findings suggest
that high-fat feeding preferentially induces the susceptibil-
ity to enhanced rates of protein degradation and loss of
mass in the highly oxidative, type I skeletal muscle fibers.
Along with myosin heavy chain and mitochondrial
proteins, a large number of other proteins are differen-
tially expressed among different muscle fiber types of
the mouse [39, 47], any of which may be critical for the
differential response shown here to the high-fat diet. A
promising explanation for a soleus-specific response is
the transcriptional coactivator PGC-1α, which is a po-
tent stimulator of mitochondrial synthesis. PGC-1α is
more highly-expressed in the soleus than in other hind-
limb muscles [48], and its mRNA and protein levels de-
crease in most, if not all, atrophy conditions [21, 49]. On
the other hand, PGC-1α protein, but not mRNA, levels
increase following 4 weeks or more of a high-fat diet
[50]. Importantly, PGC-1α inhibits muscle protein deg-
radation and disuse atrophy, and this inhibition can be
disassociated from mitochondrial content [20, 51].
Therefore, a pronounced decrease in PGC-1α expression
or intracellular sequestration to keep PGC-1α out of the
nucleus would accelerate protein degradation and en-
hance atrophy. To determine the role of PGC-1α in
high-fat diet accelerated atrophy, future studies would
be required that determine the muscle fiber-type specific
PGC-1α expression pattern, intracellular localization,
Table 1 Percent MHC isoform protein of innervated (Inn) or 14 day denervated (Denv) soleus muscles
Type I Type IIa Type IIx Type IIb
Inn Denv Inn Denv Inn Denv Inn Denv
Control 29.6 ± 1.3 41.7 ± 2.4* 50.3 ± 2.2 40.1 ± 2.3* 15.7 ± 1.9 12.4 ± 1.6 4.5 ± 1.6 5.9 ± 3.5
3wHF 30.7 ± 1.4 40.9 ± 2.2* 47.9 ± 1.8 41.3 ± 1.6* 15.3 ± 1.5 10.0 ± 1.2 6.1 ± 1.9 7.8 ± 2.7
*P < 0.05 vs Inn
Table 2 Percent MHC isoform protein of innervated (Inn) or 2 week denervated (Denv) EDL muscles
Type I Type IIa Type IIx Type IIb
Inn Denv Inn Denv Inn Denv Inn Denv
Control n.d. n.d. 13.4 ± 0.9 17.1 ± 1.4 n.d. 3.6 ± 1.6 86.6 ± 0.9 79.3 ± 1.0*
3wHF n.d. n.d. 12.4 ± 1.0 22.5 ± 2.3* n.d. n.d. 87.6 ± 1.0 77.5 ± 2.3*
n.d. = none detected. *P < 0.05 vs Inn
Roseno et al. Nutrition & Metabolism  (2015) 12:39 Page 8 of 11
and manipulation of PGC-1α expression in a muscle
specific manner during different diet conditions, perhaps
by electroporation of overexpression constructs, as done
previously [20].
Alternatively, another possible explanation for the in-
creased protein degradation in the soleus is that the
3wHF diet may impart a metabolic defect, perhaps by
lipid induced mitochondrial stress [52], that is com-
pounded by atrophy. Mitochondrial defects sufficient to
impair energetics would activate the low-energy sensing
molecule AMP activated protein kinase, which has been
shown to activate protein degradation and lead to atro-
phy [53], perhaps by activating FoxO [54]. Even modest
mitochondrial mutations, insufficient to change oxida-
tive capacity, induce profound mRNA transcript changes
[55]. In support of a metabolic defect induced by a
short-term high-fat diet, a 5 week but not a 10 week
high-fat diet impairs state 3 respiration and ATP synthe-
sis in permeabilized soleus fibers of C57BL/6 mice [15],
while a 6 week HF diet impairs state 3 respiration and
ATP synthesis in permeabilized fibers of rat soleus but
not TA muscles [16]. Importantly, any mitochondrial de-
fect in the soleus imparted by the high-fat diet would be
exacerbated on the whole tissue level by denervation at-
rophy, because the soleus, but not the EDL, have re-
duced content of mitochondrial proteins.
The ability of a short-term high-fat diet to foster high
rates of protein degradation during disuse atrophy may
have important clinical implications. For instance,
muscle disuse during bed rest, which occurs during hos-
pital stays, is a potent stimulus for atrophy, which then
leads to greater weakness and fatigue and extends hos-
pital stays further. Humans that gain weight (i.e. are in
positive energy balance) during 5 weeks of bed rest lose
more muscle mass than those subjects that are more
weight stable even though both groups consumed the
same macronutrient composed diet [56]. It is unclear
whether the increased atrophy is due to greater lipid ac-
cumulation or whether it is the mismatch in energy sup-
ply and demand. Interestingly, our body weight data
demonstrate that the 3wHF mice are in positive energy
balance (cf. Fig. 1), and overnutrition, i.e. providing fuel in
excess of what mitochondria require for ATP resynthesis,
is proposed to lead to mitochondrial dysfunction [52].
Another noteworthy finding is that the time course re-
sponse to denervation varies markedly between the so-
leus and EDL. In control fed mice, basal rates of protein
degradation are faster in the soleus versus the EDL (cf.
Fig. 3), which agrees well with studies in young rats
demonstrating protein turnover is faster in the slow-
twitch soleus muscles [41, 57, 58]. Denervation induces
a rapid increase in proteolysis in the soleus, with a peak
at 5 days after denervation (increased ~85 % versus
innervated). In the EDL, the increase in proteolysis is
delayed, with the peak occurring at 14 days post-
denervation (increased ~90 % versus innervated). The
rapid induction of proteolysis in the soleus may explain
the apparent greater sensitivity of the soleus muscle to
inactivity or denervation atrophy [43, 59]. Furthermore,
our findings emphasize that fiber-type differences in
Fig. 7 Mitochondrial protein expression decreases with 14 day
denervation in the soleus but not in the EDL. Immunoblot analysis
for several mitochondrial proteins was performed on the soleus (a-e)
and EDL (f-j) to determine the response to denervation and a high-fat
diet. Data normalized to control diet, innervated muscle. *P < 0.05 main
effect vs Innerv
Roseno et al. Nutrition & Metabolism  (2015) 12:39 Page 9 of 11
atrophy may not simply be differences in the magnitude
of response, but must also account for differences in the
timing of responses.
Conclusions
In summary our results indicate that a high-fat diet for
three weeks induces the rate of protein degradation and
increases the amount of disuse atrophy in soleus muscles
of mice, but this high-fat diet has no effect on basal pro-
tein degradation. This implies that a mismatch between
energy supply and energy demand, as evidenced by
weight gain during the entire diet, exacerbates the loss
of muscle mass when atrophy is activated. Importantly,
this increased loss of muscle with the high-fat diet does
not appear to be related to circulating glucose or ele-
vated myostatin levels, both known complications of
prolonged obesity.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SR and JB conceived and designed the study, participated in all data
collection, performed statistical analysis, and drafted the manuscript. PD and
LB participated in collection and analysis of the protein degradation studies.
JP carried out the myosin heavy chain data collection and analysis. CW
participated in study design and western blotting. All authors read and
approved of the final manuscript.
Acknowledgements
This study was supported by Start-up funds from East Carolina University to
JJB, and Start-up funds from East Carolina University and NIH R00-AR056298
to CAW.
Author details
1East Carolina Diabetes and Obesity Institute, East Carolina University,
Greenville, NC, USA. 2Human Performance Lab, Department of Kinesiology,
College of Health and Human Performance, East Carolina University,
Greenville, NC, USA. 3Department of Kinesiology and Health Promotion,
College of Education, University of Kentucky, Lexington, KY, USA.
4Department of Physiology, Brody School of Medicine, East Carolina
University, Greenville 27834NC, USA. 5Department of Biochemistry and
Molecular Biology, Brody School of Medicine, East Carolina University,
Greenville, NC, USA.
Received: 26 August 2015 Accepted: 25 October 2015
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