Fructose ingestion impairs expression of genes involved in skeletal muscle’s adaptive response to aerobic exercise
Gonçalves et al. Genes & Nutrition
Fructose ingestion impairs expression of genes involved in skeletal muscle's adaptive response to aerobic exercise
Natalia Gomes Gonçalves 0 2
Stephanie Heffer Cavaletti 0 2
Carlos Augusto Pasqualucci 0 2
Milton Arruda Martins 1
Chin Jia Lin 0 2
0 Department of Pathology, School of Medicine, University of São Paulo , São Paulo , Brazil
1 Department of Internal Medicine, School of Medicine, University of São Paulo , São Paulo , Brazil
2 Department of Pathology, School of Medicine, University of São Paulo , São Paulo , Brazil
Background: The inverse relationship between exercise capacity and its variation over time and both cardiovascular and all-cause mortality suggests the existence of an etiological nexus between cardiometabolic diseases and the molecular regulators of exercise capacity. Coordinated adaptive responses elicited by physical training enhance exercise performance and metabolic efficiency and possibly mediate the health benefits of physical exercise. In contrast, impaired expression of genes involved in mitochondrial biogenesis or protein turnover in skeletal muscle-key biological processes involved in adaptation to physical training-leads to insulin resistance and obesity. Ingestion of fructose has been shown to suppress the exercise-induced GLUT4 response in rat skeletal muscle. To evaluate in greater detail how fructose ingestion might blunt the benefits of physical training, we investigated the effects of fructose ingestion on exercise induction of genes that participate in regulation of mitochondrial biogenesis and protein turnover in rat's skeletal muscle. Methods: Eight-week-old Wistar rats were randomly assigned to sedentary (C), exercise (treadmill running)-only (E), fructose-only (F), and fructose + exercise (FE) groups and treated accordingly for 8 weeks. Blood and quadriceps femoris were collected for biochemistry, serum insulin, and gene expression analysis. Expression of genes involved in regulation of mitochondrial biogenesis and autophagy, GLUT4, and ubiquitin E3 ligases MuRF-1, and MAFbx/Atrogin-1 were assayed with quantitative real-time polymerase chain reaction. Results: Aerobic training improved exercise capacity in both E and FE groups. A main effect of fructose ingestion on body weight and fasting serum triglyceride concentration was detected. Fructose ingestion impaired the expression of PGC-1α, FNDC5, NR4A3, GLUT4, Atg9, Lamp2, Ctsl, Murf-1, and MAFBx/Atrogin-1 in skeletal muscle of both sedentary and exercised animals while expression of Errα and Pparδ was impaired only in exercised rats. Conclusions: Our results show that fructose ingestion impairs the expression of genes involved in biological processes relevant to exercise-induced remodeling of skeletal muscle. This might provide novel insight on how a dietary factor contributes to the genesis of disorders of glucose metabolism.
Fructose; Exercise training; Skeletal muscle; Protein turnover; PGC-1α; Rats
The importance of physical activity as an essential
component of a healthy lifestyle cannot be overlooked.
Regular physical exercise enhances health span  while
lack of physical activity or decreased physical fitness
confers increased risk for premature death and increased
risk for several chronic, non-communicable diseases .
Physical fitness or exercise capacity is a better predictor
of mortality than traditional cardiovascular risk factors
, and changes in exercise capacity over time are
strong and inversely associated with all-cause mortality
in men . Interestingly, rats selectively bred for low
exercise capacity exhibited defects characteristic of
metabolic syndrome such as elevated blood pressure,
impaired glucose tolerance, visceral adiposity, and
elevated circulating levels of triglycerides . These
observations suggest that cardiometabolic diseases and
the molecular determinants of exercise capacity are
Physical exercise produces mechanical, metabolic,
nutritional, and oxidative stresses in engaged skeletal
muscles. These stimuli trigger a set of coordinated
adaptive responses which result in modification of volume,
protein content, mechanical properties, and metabolic
capacities . These responses restore homeostasis and
improve the performance of challenged muscle groups
[6, 7]. Contracting skeletal muscle can also modulate the
function of metabolically relevant tissues with
production and release of myokines . The enhancement of
muscle metabolic efficiency and crosstalk of muscle with
other tissues constitute the fundamental ingredients by
which physical exercise improves the health of whole
organism . One of the most relevant exercise-induced
muscle remodeling responses, from the perspective of
metabolic efficiency, is the increase in mitochondrial
density and enzyme activity, termed mitochondrial
biogenesis . Mitochondrial biogenesis is a complex
process that requires co-expression of genes from two
distinct genomes (nuclear and mitochondrial) and is
regulated by transcription factors and transcription
Peroxisome proliferator receptor-γ co-activator-1α
(PGC-1α) is an inducible transcription co-activator that
interacts with many different transcription factors to
activate distinct biological programs in a multitude of
tissues. In skeletal muscle, PGC-1α is readily induced by
endurance exercise and regulates the coordinated
expression of mitochondrial proteins encoded in both
nuclear and mitochondrial genomes. Induction of
PGC1α in skeletal muscle leads to activation of genetic
programs characteristic of slow-twitch (type I,
predominantly oxidative) muscle fibers and phenotypical
changes such as increase in functional mitochondria,
improvement in whole-body VO2max, shift of fuel usage
from carbohydrate to fat during submaximal exercise,
and improved endurance performance . Moreover,
PGC-1α mediates the exercise-dependent up-regulation
of fibronectin type III domain-containing protein 5
(FNDC5) which is proteolytically cleaved to generate
irisin—a myokine that enhances thermogenesis and
promotes conversion of white adipose cell to brown
adipose cell . Therefore, exercise induction of
PGC1α seems to be an event that orchestrates adaptive
responses of skeletal muscle to physical exercise
although the results from loss-of-function studies suggest
that PGC-1α is probably not mandatory for some of the
training-induced adaptive responses [11–13].
Protein turnover—proteolysis coupled with de novo
protein synthesis—is another cellular process involved
with exercise-induced muscle remodeling. Damaged
proteins and organelles need to be removed by proteasome
and autophagy proteolysis and replaced by newly
synthesized ones in exercised muscles during the recovery .
Autophagy also plays an essential role in maintaining
the mass of skeletal muscle and provides skeletal muscle
cells with an alternative energy source during energy
stress caused by physical training [14, 15]. Interestingly,
loss-of-function studies have shown that autophagy is
required for exercise-dependent mitochondrial
biogenesis and improvement of endurance capacity [14, 16].
Metabolic syndrome (MetS) and obesity have a
longknown relationship with decreased muscle mass and
strength. Morphological and functional alterations have
been observed in skeletal muscle of obese or MetS
subjects [17–19], and mice exposed to high-fat diet have
decreased total muscle mass of hind limbs, muscle fiber
diameter, muscle protein content, and grip strength .
Moreover, skeletal muscle myotubes from severely obese
individuals are shown to have altered proteasome and
autophagic proteolytic flux . These findings suggest
that MetS (or obesity) is associated with morphological
and functional abnormalities of skeletal muscle which
might be a consequence of MetS (or obesity) per se or
caused, at least in part, by altered proteolytic pathways
or other cellular processes due to dietary habits or
Increased consumption of high-fructose corn syrup
(HFCS) or sucrose via ingestion of ultra-processed food
and sugar-sweetened beverages (SSB) has been linked to
the obesity and diabetes epidemics in the USA .
Fructose is a major monosaccharide component of both HFCS
and sucrose and has been considered as responsible for
the metabolic effects of these sweeteners [22, 23]. The
liver is the major site of fructose metabolism which breaks
fructose down into metabolic intermediates that enter
promptly the triose pool in a process that bypasses the
rate-limiting phosphofructokinase step. The expansion of
triose phosphate pool is responsible for metabolic
adaptations to acute fructose load while the responses to
long-term load will depend on enzymatic adaptation .
In the liver of fed animals, the increase in the flux through
the glycolytic pathway leads to lactate production,
activation of pyruvate dehyderogenase, and enhancement of
oxidative pathway with carbon dioxide and ketone body
production . This metabolic milieu also favors
esterification of non-esterified fatty acids (NEFAs) augmenting
the liver production and secretion of very low density
lipoprotein (VLDL) . In starved animals, activation of
gluconeogenesis enzymes leads to formation of glucose
from fructose . Long-term load of fructose causes the
liver to form more glucose and glycogen from fructose
and respond more intensely to the actions of fructose in
promoting VLDL output. In adipose tissue, fructose
impairs both glucose utilization and esterification of fatty
acids. This raises NEFAs concentration and increases
VLDL production. Increased concentration of triglyceride
and NEFAs impairs glucose utilization in skeletal muscle
. The consequence is increased insulin resistance,
hyperinsulinemia, and formation of a vicious cycle in
which insulin resistance will stimulate the already
increased VLDL production by the liver. Thus, chronic
fructose feeding will produce metabolic derangement similar
to those found in the MetS.
Ingestion of fructose or maltodextrin has been shown
to suppress the exercise-induced glucose transporter
type 4 (GLUT4) adaptive response in rat skeletal muscle
. Motivated by this work, we conducted the present
study to investigate if ingestion of fructose can impair
the expression of genes involved in post-exercise muscle
remodeling which is our primary aim in this study. The
secondary aim of this study is to assess the effects of
fructose ingestion and physical training on expression of
selected genes involved in protein degradation in skeletal
Animals and experimental protocol
Eight-week-old male Wistar rats were provided by the
University of São Paulo School of Medicine’s Animal
Facility which keeps the animals in cages with four to
five animals and feeds them with standard chow from
weaning to the moment they started the protocol. The
animals were randomly allocated into the following
groups: sedentary control (C, n = 6), exercise-only (E, n
= 7), sedentary fructose (F, n = 8), and fructose + exercise
(FE, n = 8) and treated accordingly for 8 weeks. The
pretreatment weight of the rats ranged from 194.64 to
342.0 g, and there was no inter-group difference (F(
) = 2.23, p = 0.110). The animals were kept in cages
with four to five animals under a 12-h light/dark cycle
and were given ad libitum access to food and water.
Standard chow (2990 kcal/kg) was given as a solid diet.
The rats assigned to fructose treatment (F and FE
groups) were given a 15% fructose solution as drinking
solution. The fructose treatment began on the same day
as the exercise training (see the next section). The
quantity and volume of unconsumed food and fluid for each
cage were verified each morning. The daily consumption
of food and fluid was calculated as a difference between
what was provided on previous day and what was left
unconsumed. Due to limited quantity of tissue and
blood samples, biochemical and molecular analyses were
not performed in all the animals.
This study was approved by the Ethics Committee of
University of São Paulo School of Medicine under the
number 073/13, and all animal experiments were
performed according to the procedures approved at our
Treadmill exercise protocol
The rats in exercise training groups were initially
acclimatized to the treadmill (KT 400, Imbramed, RS, Brazil)
for 3 days (10 min/day, 0.3 km/h). Afterwards, a
maximal exercise capacity test was performed with an initial
velocity of 0.3 km/h for 5 min followed by an increase of
0.1 km/h every 1.5 min until animal exhaustion which
was defined as the moment when an animal sat at the
lower end of the treadmill and was unresponsive to 10
gentle taps to continue running. Total test time, velocity,
and distance were recorded for each rat. The rats were
trained at moderate intensity (60% of maximal velocity
achieved in exercise capacity test) for 60 min/day, 5 days
a week for 8 weeks. After 8 weeks, the maximal exercise
capacity test was repeated. One of us (NGG) oversaw
personally all treadmill trainings and, whenever
necessary, provided with stimulation to any animal that was
running slower than the speed established by the
treadmill. No electrical shock was applied to the animals
throughout the training period.
Tissue collection and biochemical analysis
The rats were euthanized 1 day after the last training
session. After an overnight fast, the animals were
anesthetized with intraperitoneal injection of 75 mg/kg
ketamine and 10 mg/kg xilazine. Blood was collected by
cardiac puncture. Following blood collection, the rats
were euthanized by decapitation, and the quadriceps
femoris was dissected and preserved in RNAlater
(Ambion) while blood samples were centrifuged at
5000 rpm at 4 °C and the resulting serum samples
transferred to a fresh microcentrifuge tube. Both muscle and
serum specimens were stored at −80 °C until use.
Serum insulin levels were measured with an ELISA kit
(Millipore) as per the manufacturer’s instructions. Serum
triglyceride and glucose levels were measured by
enzymatic colorimetric assay in the Cobas c111 analyzer
We used the HOMA2 model [25, 26] to evaluate
insulin resistance (HOMA2-IR), pancreatic beta cell reserve
(HOMA2-%B), and insulin sensitivity (HOMA2-%S).
The indexes were calculated with the Oxford HOMA
RNA extraction and gene expression analysis
Total RNA from quadriceps muscle was isolated with
TRI Reagent (Sigma-Aldrich) as per the manufacturer’s
instructions. Genomic DNA was removed by treating
the RNA samples with DNase I for 20–30 min at 37 °C.
RNA was reversely transcribed into complementary
DNA with a commercial kit (High Capacity cDNA
Reverse Transcription Kit, ABI) as per the manufacturer’s
instructions. Gene expression analysis was performed
using quantitative real-time polymerase chain reaction
in assay buffer which contains EvaGreen fluorescent dye
(5× HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX),
Solis BioDyne, Tartu, Estonia) using the primers listed in
Table 1. Relative gene expression was calculated using
procedures reported previously , and cyclophilin
A (CypA) was adopted as internal normalization
control. A sample collected from an untreated control
was used as a calibrator in all real-time PCR
All data are presented as mean ± SEM. Normality of
samples was assessed with Shapiro-Wilk test.
Homoscedasticity (homogeneity of variances) was assessed with
Fligner-Killeen test due to robustness of this test .
Since there was no violation of normality or
homogeneity of variances, no transformation of original data was
necessary. Differences among groups of weight,
metabolic profile, and exercise capacity were assessed by
analysis of variance (ANOVA). The status of fructose
ingestion and exercise training were used as factors and
factorial ANOVA was used to assess the effect of each
treatment on gene expression. This study has a power of
0.34 when an effect size of 0.4 (a large conventional
effect size according to Cohen ) is used in the
calculation. All statistical analyses were performed in R
version 3.3.1. Study power was calculated using R
packages pwr and pwr2 [31, 32]. A value of p < 0.05 was
considered statistically significant.
Effect of fructose and exercise on food, water, and calorie intake
Food and water intake were measured daily. The animals
were kept in cages with four or five rats; therefore, it
was not possible to perform statistical analysis of food,
water, and calorie intake, only the means were
compared. The animals assigned to groups F and FE ingested
less food than groups C and E. On the other hand, F and
FE consumed more water than C and E, resulting in
higher calorie intake in the former (data not shown).
These data agree with previous study .
Effect of fructose and exercise in body weight
The animals were weighted before the diet/exercise
protocols started (week 0) and again after the end of the
diet/exercise protocols (week 8). There was no difference
between groups in pre-treatment weight (F(
) = 2.23,
p = 0.110). At the end of the 8th week, the rats in E
group presented with the lowest while the rats in FE
group with the highest body weight (Table 2). A main
effect of fructose ingestion on body weight (F(
6.885, p = 0.01354) as well as an interaction between
fructose and exercise (F(
) = 7.791, p = 0.00905) were
Effect of fructose in metabolic profile of the animals
To assess the metabolic profile of the animals, after the
end of the diet/exercise protocols, serum glucose,
insulin, and triglyceride levels were measured. HOMA2
model was used to evaluate insulin resistance
(HOMA2IR), pancreatic beta cell reserve (HOMA2-%B), and
insulin sensitivity (HOMA2-%S). No significant effect of
fructose ingestion or exercise training was detected for
serum insulin (respectively, F(
) = 0.397, p = 0.534
) = 0.121, p = 0.731), glucose levels
) = 1.226 p = 0.279 and F(
) = 1.401, p =
0.248), HOMA2-IR (respectively, F(
) = 1.042, p =
0.317 and F(
) = 0.627, p = 0.436), HOMA2-%S
) = 0.488, p = 0.491 and F(
0.560, p = 0.461), or HOMA2-%B (respectively, F(
= 0.002, p = 0.963 and F(
) = 0.002, p = 0.966). In
contrast, there is a main effect of fructose ingestion on
serum triglyceride levels (F(
) = 4.601, p = 0.0418),
Effect of exercise in the physical conditioning
To evaluate their physical conditioning, the animals
underwent a maximal exercise capacity test before the
diet/exercise protocols started and after the end of the
protocols. In the initial maximal exercise capacity test,
there was no statistical difference between the groups
) = 1.08, p = 0.379). After 8 weeks of treadmill
training, groups E and FE were both able to run
significantly faster than the non-trained groups C and F (F(
) = 37.24, p < 0.001) and to reach higher speeds than
they did during the initial test (E: p = 0.047; FE: p =
0.001). Interestingly, non-trained animals performed
poorer in the final test relative to the initial test (C: p =
0.001; F: p = 0.035, Fig. 1a). The same trend is seen both
in duration and distance. There were no between group
differences regarding the duration of running (F(
0.59, p = 0.660) and traveled distance (F(
) = 2.28, p
= 0.090) at the initial assessment. After 8 weeks of
training, both E and FE improved the duration (E: p < 0.001;
FE: p = 0.001) and distance (E: p < 0.001; FE: p < 0.001).
Both groups E and FE also ran for longer time (F(
= 58.66, p < 0.001) and a greater distance (F(
42.34, p < 0.001) than their littermates assigned to
sedentary groups (C and F). The non-trained animals (groups
C and F) also performed poorer relative to their own
initial test in both duration (C: p = 0.003; F: p = 0.008, Fig. 1b)
and traveled distance (C: p = 0.005; F: p = 0.009, Fig. 1c).
This degradation of exercise capacity of C and F is
probably a result of physical deconditioning that the untrained
animals underwent after 8 weeks of sedentarism.
Impact of fructose ingestion and exercise training on
expression of PGC-1α and FNDC5
Ingestion of fructose negatively affected expression of
both PGC-1α and FNDC5 in rat skeletal muscle regardless
of their exercise status. Fructose-ingesting sedentary rats
exhibited a less intense expression of PGC-1α and FNDC5
than littermates that did not ingest fructose (Fig. 2a, b).
Furthermore, expression of these two genes after exercise
is also decreased in the fructose-fed animals when
compared to the exercised animals that did not ingest
fructose (Fig. 2a, b). Indeed, fructose was the only
treatment that affected the expression of both PGC-1α and
) = 11.720, p = 0.00759 and F(
) = 11.310,
p = 0.00835, respectively, PGC-1α and FNDC5).
To gain further insight on the effects of fructose
ingestion on molecular mediators of beneficial effects of
exercise training, we studied the expression of transcription
factors nuclear receptor subfamily 4 group A member 3
(NR4A3/Nor-1), estrogen-related receptor alpha (Errα),
Results are presented as mean ± SEM. The numbers in parenthesis represent the number of animals included in the experiment. A main effect of fructose (p = 0.01354)
and an interaction between fructose and exercise (p = 0.00905) on animals’ weight were observed. There is also an effect of fructose on serum triglycerides (p = 0.0418).
See the text for more details
and peroxisome proliferator activated receptor δ (Pparδ)
which are induced in skeletal muscle by endurance
exercise. Although average expression of NR4A3/Nor-1 was
higher in rats undergoing physical training, no
statistically significant effect of treadmill running was observed
among rats that did not ingest fructose. In contrast,
fructose-treated (F and FE) rats exhibited a 80% decrease
in expression of NR4A3/Nor-1 when compared to the
littermates that did not ingest fructose (C and E groups,
p = 0.027, Fig. 2c). In fact, an effect of fructose on
expression of NR4A3/Nor-1 was noted (F(
) = 7.651,
p = 0.0244). Expression of Errα and Pparδ exhibited a
very similar pattern. Both were strongly induced by
treadmill running in skeletal muscle (79 and 66%,
respectively, Errα and Pparδ, Fig. 2c). There are main
effects of fructose ingestion, exercise training, and
interaction between fructose and exercise on expression of
both Errα (respectively, F(
) = 17.61, p = 0.001494,
) = 47.38, p = 2.64 × 10−05, and F(
) = 29.61, p
= 0.000203) and Pparδ (respectively, F(
) = 20.43, p =
) = 14.54, p = 0.00341, and F(
19.26, p = 0.00136). We also assessed how fructose
ingestion affects expression of calcium/calmodulin-dependent
protein kinase type IV (CAMK IV) in skeletal muscle as
this kinase is reported to transduce muscle contraction
into a regulatory signal for the expression of PGC-1α
. No effect of fructose ingestion or treadmill training
on expression of CAMK IV was observed (F(
0.712, p = 0.417 and F(
) = 0.001, p = 0.982; Fig. 2c).
We also assayed the expression of GLUT4 which is
induced by exercise training and is responsible for the
enhanced muscle glucose uptake caused by chronic exercise.
Fructose ingestion attenuated expression of GLUT4 by
78% in skeletal muscle of either sedentary or exercised rats
(p = 0.0156, Fig. 2c), and a significant main effect of
fructose ingestion on GLUT4 expression was detected (F(
) = 5.848, p = 0.0324). Therefore, ingestion of fructose
globally attenuates expression of key genes involved in
metabolic adaptation of skeletal muscle to physical
Expression of forkhead box O3 (FoxO3A)—a
transcriptional factor reported to interact with PGC-1α1 to
regulate expression of oxidative stress genes —was
also assessed in the skeletal muscle. While fructose
showed no effect (F(
) = 2.055, p = 0.1710; Fig. 2c) a
main effect of aerobic training on the expression of
FoxO3A was detected (F(
) = 5.711, p = 0.0295).
Expression of genes involved in protein degradation
The results on the expression of PGC-1α and FNDC5
and their transcriptional regulators led us to seek
whether fructose ingestion might affect other molecular
pathways that also mediate adaptive metabolic response
of skeletal muscle to physical exercise. Should this be
the case attenuation of exercise-induced remodeling of
skeletal muscle might be, in addition to excessive caloric
accumulation, a relevant mechanism underlying
metabolic derangement associated with fructose ingestion.
Autophagy and ubiquitin-proteasome pathways are
major protein degradation pathways in the skeletal
muscle. In addition to regulating the net amount and
the quality of muscle protein, autophagy (basal and
acute, exercise-induced) has been shown to play a
critical role in exercise-induced muscle remodeling and
improvement of insulin sensitivity [16, 36].
We observed a statistically significant main effect of
exercise training on expression of autophagy-related
protein 6 (Atg6/beclin 1) (F(
) = 23.856, p = 0.000484),
autophagy-related protein 7 (Atg7) (F(
) = 27.609, p =
0.000156), and autophagy-related protein 12 (Atg12) (F(
) = 8.157, p = 0.012), and they all showed significant
induction in skeletal muscle after treadmill running (Fig. 3c).
No significant effect for fructose ingestion or interaction
between exercise and fructose ingestion was observed on
expression of these genes. In contrast, autophagy-related
protein 9 (Atg9) expression in both sedentary and
exercised rats was impaired by fructose ingestion (Fig. 3c), and
there was a significant main effect of fructose (F(
28.972, p = 9.66 × 10−05) and interaction between exercise
and fructose (F(
) = 4.653, p = 0.0489) on expression of
Atg9. Expression of microtubule-associated protein 1 light
chain 3 isoform B (LC3B)—a marker of autophagosome
accumulation was not affected by physical training or
fructose (respectively, F(
) = 0.951, p = 0.341 and F(
) = 0.811, p = 0.378; Fig. 3a). However, ingestion of
fructose impaired expression of lysosome-associated
membrane protein 2 (Lamp-2, F(
) = 7.750, p = 0.0127;
Fig. 3a). Fructose ingestion also attenuated expression of
lysosomal cathepsin L (Ctsl) in skeletal muscle (F(
6.768, p = 0.0209, Fig. 3b). No statistically significant main
effect for exercise or fructose on expression of BCL2/
adenovirus E1B interacting protein B (Bnip3)—a marker
of mitochondrial autophagy—was detected except for a
trend for interaction between exercise and fructose (F(
) = 3.401, p = 0.0923, Fig. 4a).
Effects of exercise and fructose ingestion on
ubiquitinproteasome pathway were also evaluated by studying the
expression of E3 ubiquitin ligases muscle RING-finger
protein-1 (Murf-1) and muscle atrophy F-box (MAFBx,
also known as atrogin-1) (Fig. 4b). There was a main
effect of fructose on expression of both Murf-1 (F(
= 12.181, p = 0.00245) and MAFBx (F(
) = 4.897, p =
0.0409) and a marginally significant interaction between
exercise and fructose on expression of Murf-1 (F(
= 4.000, p = 0.05999).
The main finding of this study is that fructose ingestion
impairs the expression of genes involved in
transcriptional regulation of both oxidative metabolism and
mitochondrial biogenesis and of genes of proteolytical
pathways in the skeletal muscle. This negative effect of
fructose ingestion was seen in both sedentary and
exercised animals for most of these genes, but a few of these
genes showed blunted expression only in
treadmilltrained animals. Our results not only confirm the finding
of a previous work which reported that fructose
consumption impairs adaptive response of GLUT4 
but also suggest that ingestion of fructose might impair
other responses of skeletal muscle to exercise.
Our results are similar to a recent study with human
volunteers in which failure to upregulate mitochondrial
fuel oxidation genes was shown as the mechanism
behind the inability of human subjects to improve their
insulin sensitivity upon aerobic training . Like our
study, the skeletal muscle of those who were unable to
respond to aerobic training displayed deficient
exerciseinduced expression of PGC-1α, ERRα, and of
5′-AMPactivated protein kinase catalytic subunit alpha-2
(AMPKα2) . This study and ours highlighted the
importance of oxidative muscle fibers in the genesis of
insulin resistance and related metabolic diseases.
Decreased oxidative phosphorylation in skeletal muscle
has been reported as the earliest defect leading to insulin
resistance and glucose intolerance in elderly subjects
and non-diabetic offspring of type 2 diabetes patients
[38, 39]. In fact, the latter group also displayed a reduced
ratio of inorganic phosphate to phosphocreatine in
soleus muscle which is compatible with a diminished
content of type I (oxidative) fibers relative to type II
fibers . Content of type I fibers has also been shown
to correlate inversely with fat body mass and positively
with the response to weight loss intervention .
Therefore, our results open the possibility that a dietary
factor might lead to disorders associated to insulin
resistance via reduction of number or function of
mitochondria in skeletal muscle.
Interestingly, in our study, fructose feeding also
prevented exercise induction of selected autophagy
genes and muscle-specific E3 ubiquitin-protein ligases
Murf-1 and MAFBx. A functioning autophagy pathway
seems to be required for muscle mass maintenance,
muscle regeneration, and exercise-induced muscle
remodeling [14, 16, 36] while both expression of E3
ligases and proteasome activity in skeletal muscle have
been reported to increase with either acute or chronic
endurance exercise . Such activation of proteasomal
proteolysis might be an adaptive response as it allows
for removal of damaged proteins and facilitates
myofilament restructuring . Therefore, fructose ingestion
seems to affect multiple cellular functions that are
related to skeletal muscle remodeling and metabolic
adaptation to endurance training.
Intriguingly, in the present study, the fructose-loaded
rats that underwent treadmill training (FE group)
improved their exercise capacity to a similar extent as did
their exercise-only counterparts (E group). Such finding is
not what one might predict considering the altered gene
expression exhibited by FE rats and evidences from
overexpression experiments regarding the effects of PGC-1α
on mitochondrial biology and energy metabolism. Possible
explanations include the existence of other transcriptional
co-activators that might provide redundancy for PGC-1α
signaling or that PGC-1α might not be mandatory for
some of training-induced adaptations. In fact, PGC-1α is a
prototypical member of a family of transcriptional
coactivators that regulates mitochondrial biogenesis and energy
production, and there seems to be a redundancy between
members of this family [41, 42]. Also, loss-of-function
studies have shown that PGC-1α might not be mandatory
for some of training-induced responses in skeletal muscle
[11, 13, 43]. It is noteworthy that in the study by Bohm
et al. , no group of volunteers showed significant
training-related improvement of VO2max regardless of
their ability (or inability) to improve insulin sensitivity
with aerobic exercise or to induce expression of PGC-1α,
ERRα, and AMPKα2.
One might speculate the mechanism underlying the
defective induction of genes related to skeletal muscle
response to aerobic training in fructose-fed animals.
These observed effects of fructose ingestion are probably
mediated by transcriptional mechanism as the affected
genes encompass multiple cellular processes.
Cyclooxygenase 2-mediated inflammation have been reported to
be the underlying mechanism of fructose-induced
insulin resistance in rats [44, 45]. A persistent inflammation
caused by fructose ingestion might lead to defective
activation of PGC-1α and other transcriptional regulators of
skeletal muscle adaptation via a TGFβ-dependent
mechanism like the one underlying the defective activation of
PGC-1α and AMPKα2 in individuals who failed to
improve insulin sensitivity upon aerobic training .
This hypothesis, however, contradicts the existing
evidence of anti-inflammatory properties of chronic
aerobic exercise in rodent models of diabetes and
tobacco smoking [46–48]. Alternatively, fructose might
impair exercise-induced skeletal muscle remodeling by
interfering with post-exercise glycogen accumulation in
skeletal muscle. Exercise-induced activation of Pparδ—a
known activator of PGC-1α transcription —varies
inversely with the glycogen content of muscle fiber .
Also, exercise-trained rats that ingest fructose exhibit
higher content of both liver and muscle glycogen
content than their exercise-trained, control diet-fed
littermates . Therefore, ingestion of fructose might impair
the activation of Pparδ and its downstream transcription
targets including PGC-1α by enhancing the
accumulation of glycogen in skeletal muscle. Whether Pparδ
functions as an upstream transcriptional regulator of
proteolytic pathways remains to be determined. Fructose
ingestion might also affect expression PGC-1α and
training-induced adaptive genes responses by promoting
the accumulation of lactate or lipids. In the liver, where
most of absorbed fructose is metabolized, fructose is first
phosphorylated by fructokinase to form
fructose-1phosphate then broken down to glyceraldehyde and
dihydroxyacetone phosphate by aldolase B . The
glyceraldehyde thus generated is phosphorylated to
glyceraldehyde-3-phosphate by triokinase after which it
can follow any triose phosphate metabolic pathway
including conversion to lactate [23, 52]. Conversion to
lactate is a means to release fructose-derived carbon
from liver for extrahepatic utilization, and about a
quarter of ingested fructose is converted to lactate .
Thus, lactate might be a fructose-derived metabolic
intermediate that causes the muscle to impair
exerciseinduced gene response. The caveat for this hypothesis is
the fact that exposure to lactate has been reported to
promote expression of PGC-1α and genes involved in
mitochondrial biogenesis in both cultured L6 cells 
and C57BL/6J mice . Finally, excessive exposure of
skeletal muscle to lipids results in muscle
insulinresistance and accumulation within muscle fiber of fatty
acid metabolites . Since, by both augmenting lipid
synthesis and decreasing lipid clearance, fructose loading
increases plasma triglyceride and NEFAs [23, 53], metabolic
overload of skeletal muscle mitochondria might impair
the training-induced gene expression in skeletal muscle.
This study presents a number of limitations that
should be mentioned. Firstly, we did not include
isocaloric controls of other sugar preparations. For this reason,
we could not test whether the observed effects on gene
expression is specific to fructose ingestion or is a general
phenomenon associated to excessive carbohydrate (or
caloric) consumption. Second, animal’s insulin sensitivity
status was assessed only after the treatment/training
period, and this hinders inferences that can be made
regarding the effect of training or fructose on insulin
sensitivity. Third, we used maximal exercise capacity on
treadmill running to evaluate the effect of training
instead of VO2max. Since exercise capacity is determined
by a combination of factors which include VO2max ,
we might not have captured adequately the impact of
altered gene response on the physiology of skeletal muscle.
Fourth, we did not allow the animals in this study to
perform voluntary physical activity. For this reason, it is
possible that our test for exercise capacity was
comparing physical conditioning with physical deconditioning.
The latter two deficiencies of our study might also be
the reason why no apparent difference in exercise
capacity between E and FE animals was detected. Finally, in
view of the limited power of this study, we might have
failed to detect an effect of fructose or exercise. To
assess how the design of this study would affect our ability
to detect an effect of treatment factors on gene
expression, we calculated the power using as parameters the
effect sizes obtained from our PGC-1α and FNDC5
expression data (Table 3). Post hoc power analysis showed
that the power of this study to detect an effect of
fructose on PGC-1α or FNDC5 is 0.8 but only 0.05 for effect
of exercise on either gene. Importantly, we were able to
detect interaction between fructose and exercise on
expression of a few genes despite of small effect size
attributable to exercise (Table 3). In our opinion, the
limitations mentioned here do not invalidate the main
conclusion of our study regarding the possibility of
excessive ingestion of a macronutrient impairing beneficial
adaptive responses in skeletal muscle.
Our results suggest that fructose might impair exercise
induction of genes involved in regulation of metabolic
adaptation of the skeletal muscle. This finding indicates the
need for a more detailed examination of the role of
dietexercise interaction in the pathophysiology of
cardiometabolic diseases. Further studies are needed to elucidate the
mechanisms underlying the impairment of skeletal muscle
metabolic adaptation induced by fructose consumption.
AMPKα2: 5′-AMP-activated protein kinase catalytic subunit alpha-2;
Atg12: Autophagy-related protein 12; Atg6/beclin 1: Autophagy-related
protein 6; Atg7: Autophagy-related protein 7; Atg9: Autophagy-related
protein 9; Bnip3: BCL2/adenovirus E1B interacting protein B; CAMK
IV: Calcium/calmodulin dependent protein kinase type IV; Ctsl: Lysosomal
cathepsin L; CypA: Cyclophilin A; Errα: Estrogen-related receptor alpha;
FNDC5: Fibronectin type III domain-containing protein 5; FoxO3A: Forkhead
box O3A; GLUT4: Glucose transporter type 4; Lamp-2: Lysosome-associated
membrane protein 2; LC3B: Microtubule-associated protein 1 Light Chain 3
Isoform B; MAFBx/atrogin-1: Muscle atrophy F-box; Murf-1: Muscle
RINGfinger protein-1; NR4A3/Nor-1: nuclear receptor subfamily 4 group A
member 3; PGC-1α: Peroxisome proliferator receptor-γ co-activator-1α;
Pparδ: Peroxisome proliferator activated receptor δ
This is study was supported by FAPESP – Fundação de Amparo à Pesquisa
do Estado de São Paulo (Grant Number 2013/06720–5).
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
NGG designed the study, performed the experiments, performed the
statistical analysis, interpreted the results, and wrote the manuscript. SHC
performed the gene expression experiments and drafted the manuscript.
CAP participated in the interpretation of the results and contributed to the
writing of the definitive version of the manuscript. MAM designed the
exercise protocol, interpreted the results, and contributed to the writing of
the definitive version of the manuscript. CJL designed the study, performed
the statistical analysis, interpreted the results, and wrote the manuscript. All
authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Ethics Committee of University of São Paulo
School of Medicine under the number 073/13, and all animal experiments
were performed according to procedures approved at our institution.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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