See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/321736370

Attenuation of obesity and insulin resistance by fish oil supplementation is

associated with improved skeletal muscle mitochondrial function in mice fed
a high-fat diet

Article  in  The Journal of Nutritional Biochemistry · December 2017

DOI: 10.1016/j.jnutbio.2017.11.012

CITATIONS READS

6 224

19 authors, including:

Amanda Martins Amanda Rabello Crisma

AstraZeneca Brazil University of São Paulo
17 PUBLICATIONS   343 CITATIONS    39 PUBLICATIONS   290 CITATIONS   

SEE PROFILE SEE PROFILE

Laure Masi Catia Lira do Amaral

University of São Paulo Universidade Estadual de Goiás
19 PUBLICATIONS   109 CITATIONS    12 PUBLICATIONS   226 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Remission of type 1 diabetes in teenagers and adults View project

Vitamin D status and inflammation of Chronic Disease View project

All content following this page was uploaded by Gabriel Nasri Marzuca-Nassr on 16 April 2018.

The user has requested enhancement of the downloaded file.

 Available online at www.sciencedirect.com

ScienceDirect
Journal of Nutritional Biochemistry 55 (2018) 76 – 88

Attenuation of obesity and insulin resistance by fish oil supplementation is associated

with improved skeletal muscle mitochondrial function in mice fed a high-fat diet

Amanda R. Martins a , Amanda R. Crisma a , Laureane N. Masi b , Catia L. Amaral a, c , Gabriel N. Marzuca-Nassr a, d,
Lucas H.M. Bomfim e, Bruno G. Teodoro f , André L. Queiroz f , Tamires D.A. Serdan b , Rosangela P. Torres g,
Jorge Mancini-Filho g, Alice C. Rodrigues a , Tatiana C. Alba-Loureiro a , Tania C. Pithon-Curi b , Renata Gorjao b,
Leonardo R. Silveira e, Rui Curi a, b , Philip Newsholme h , Sandro M. Hirabara a, b,⁎
a
Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil
b
Institute of Physical Activity Sciences and Sports, Cruzeiro do Sul University, Sao Paulo, Brazil
c
Exact and Technological Sciences, State University of Goias, Anapolis, Goias
d
Department of Internal Medicine, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile
e
Institute of Biology, University of Campinas, Campinas, Brazil
f
Department of Biochemistry and Immunology, Faculty of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil
g
Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil
h
School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, Western Australia 6152

Received 26 July 2017; received in revised form 28 September 2017; accepted 14 November 2017

Abstract

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to improve insulin sensitivity and glucose homeostasis in animal models of insulin
resistance, but the involved mechanisms still remain unresolved. In this study, we evaluated the effects of fish oil (FO), a source of n-3 PUFAs, on obesity, insulin
resistance and muscle mitochondrial function in mice fed a high-fat diet (HFD). C57Bl/6 male mice, 8 weeks old, were divided into four groups: control diet (C),
high-fat diet (H), C+FO (CFO) and H+FO (HFO). FO was administered by oral gavage (2 g/kg b.w.), three times a week, starting 4 weeks before diet
administration until the end of the experimental protocol. HFD-induced obesity and insulin resistance associated with impaired skeletal muscle mitochondrial
function, as indicated by decreased oxygen consumption, tricarboxylic acid cycle intermediate (TCAi) contents (citrate, α-ketoglutarate, malate and
oxaloacetate), oxidative phosphorylation protein content and mitochondrial biogenesis. These effects were associated with elevated reactive oxygen species
production, decreased PGC1-a transcription and reduced Akt phosphorylation. The changes induced by the HFD were partially attenuated by FO, which
decreased obesity and insulin resistance and increased mitochondrial function. In the H group, FO supplementation also improved oxygen consumption;
increased TCAi content, and Akt and AMPK phosphorylation; and up-regulated mRNA expression of Gpat1, Pepck, catalase and mitochondrial proteins (Pgc1α,
Pparα, Cpt1 and Ucp3). These results suggest that dietary FO attenuates the deleterious effects of the HFD (obesity and insulin resistance) by improving skeletal
muscle mitochondrial function.
© 2017 Elsevier Inc. All rights reserved.

Keywords: Insulin resistance; Fish oil; Skeletal muscle; Mitochondrial function; Obesity

1. Introduction metabolic syndrome. Several factors are involved in the development

Insulin resistance is defined as a condition in which physiological
concentrations of insulin are not able to maintain normal concentra-
tions of blood glucose due to decreased glucose uptake (skeletal
muscle) and the impaired inhibition of gluconeogenesis [1] and
lipolysis (adipose tissue), which are regulated by the hormone [2].
This condition is associated with chronic diseases related to obesity
such as type 2 diabetes mellitus (T2DM), cardiovascular diseases, and
⁎ Corresponding author at: Institute of Physical Activity Sciences and Sports,
Cruzeiro do Sul University, Sao Paulo, Brazil. Tel.: +55 11 3385 3103.
E-mail address: sandromh@yahoo.com.br (S.M. Hirabara).
of insulin resistance, including genetic predisposition, physical
inactivity, high calorie diets, inflammation, oxidative stress, impaired
insulin signaling and mitochondrial function [3–5].
The ectopic accumulation of fatty acids in tissues with limited
capacity for lipid storage, such as skeletal muscle, is also strongly
associated with insulin resistance. Supporting this finding, several
researchers have reported that triacylglycerol accumulation and
decreased lipid oxidation in skeletal muscle are associated with
metabolic imbalance, leading to increased reactive oxygen species
(ROS) production and impaired insulin signaling. Oxidative stress,
therefore, contributes to the development of insulin resistance by
impairing mitochondrial function and decreasing β-fatty acid oxida-
tion and tricarboxylic acid cycle activity [3,6,7].

https://doi.org/10.1016/j.jnutbio.2017.11.012
0955-2863/© 2017 Elsevier Inc. All rights reserved.
 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88 77

Mitochondria are the main site for ROS production in skeletal muscle.
In conditions of muscle redox imbalance, mitochondrial DNA, proteins,
and lipids are exposed to high levels of these species, which may lead to
impaired mitochondrial function [6,8]. In addition, increased intramyo-
cellular lipid content has been associated with down-regulated expres-
sion of Pgc-1α and the genes encoding the mitochondrial respiratory
complexes (I, II, III, and IV) [9]. Researchers have also reported decreased
expression of carnitine palmitoyltransferase-1 (CPT-1) and other key
mitochondrial enzymes in skeletal muscle from obese and type 2 diabetic
individuals [10,11].
Evidence that impaired mitochondrial function is associated with
the initiation of insulin resistance has been described [12,13]. For
example, mitochondrial function and density are impaired during
obesity, T2DM, and aging and in well-characterized conditions of
reduced muscle oxidative capacity, as demonstrated by decreased
muscle oxidative phosphorylation, electron transport chain proteins,
and tricarboxylic acid cycle activity [14,15]. In addition, in animal and
human models exposed to elevated free fatty acid availability (by lipid
infusion or high-fat diet), several mitochondrial markers are affected,
including oxygen consumption and oxidative phosphorylation
[9,13,16]. In skeletal muscle cells, palmitic acid-induced ROS produc-
tion is closely associated with reduced FFA oxidation and PGC-1
expression [17]. Therefore, interventional strategies that prevent or
decrease mitochondrial dysfunction in skeletal muscle may be an
important tool for increasing insulin sensitivity in conditions of
elevated free fatty acid availability.
Dietary intervention is highly effective for reducing or prevent-
ing insulin resistance [18]. Several groups have investigated the
effects of n-3 PUFAs on insulin sensitivity and glucose homeostasis
in animal models of insulin resistance, but the precise mechanisms
involved are not known. The n-3 PUFAs reduce or abolish insulin
resistance by partially suppressing the inflammatory process
[19,20]. Other researchers have reported that the positive effects
of n-3 PUFAs on peripheral insulin sensitivity are mediated by
changes in mitochondrial function. N-3 PUFA supplementation
prevented insulin resistance in wild-type mice but failed to do so
in mice with either the α-2 catalytic subunit of AMP-activated
protein kinase (AMPK) [21] or PPARα [22] knocked out. Both AMPK
and PPARα are known to regulate the expression levels of genes
involved in mitochondrial biogenesis and fatty acid oxidation
[23,24]. These observations support the proposition that mitochon-
dria may play an important role in the reported effects of n-3 PUFAs
on insulin sensitivity. Herein, we describe a study to investigate
whether n-3 PUFAs attenuate diet-induced obesity and insulin
resistance by preventing the impairment of skeletal muscle
mitochondrial function.
2. Methods
2.1. Animals, diets, and fish oil supplementation protocol
All animal procedures were performed according to protocols
approved by the Animal Care and Use Committee of the Institute of
Biomedical Sciences, University of São Paulo. C57BL/6 male mice (8
weeks old) were housed in a room with a light–dark cycle of 12–12 h
and temperature of 23±2°C. Animals were randomly and equally
divided into four groups: a) control diet (C), b) high-fat diet (H), c)
control diet supplemented with fish oil (CFO), and d) high-fat diet
supplemented with fish oil (HFO). During the four-week period
preceding the onset of the high-fat diet (HFD) administration, all four
groups were fed ad libitum with a control diet (76% carbohydrates, 9%
fats, and 15% proteins). CFO and HFO were supplemented with fish oil
(n-3 PUFAs source) by oral gavage three times per week at 2 g per kg b.
w., whereas the C and H groups received water instead. After this four-
week period, animals from the H and HFO groups received a HFD (26%
carbohydrates, 59% fats, and 15% proteins) for the next eight weeks.
Supplementation with fish oil (CFO and HFO groups) or the
administration of water (C and H groups) was continued until the

Fig. 1. Experimental design. C57BL/6 male mice (8 weeks old) were fed ad libitum with a control diet during 4 weeks, with fish oil or water supplementation by oral gavage (2 g per kg
b.w., three times per week). After this period, animals were divided in the following groups: a) control diet (C), b) high-fat diet (H), c) control diet supplemented with fish oil (CFO), and
d) H supplemented with fish oil (HFO). Control or high-fat diet was maintained during the next eight weeks. Supplementation with fish oil (CFO and HFO groups) or water (C and H
groups) was kept up to the end of the experimental protocol. Animals were euthanized at 20 weeks old.
78 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88
Fig. 2. Plasma levels of insulin (A) and glucose (B), HOMA-IR (C), and Kitt (D) from mice fed a control diet (C) or high-fat diet (H) and supplemented or not with fish oil (FO). Results are
means ± SE (n=8–10/group). Data were analyzed by ANOVA followed by Bonferroni post-test.
end of the experimental protocol (Fig. 1). Water was used in the
control group to promote the same handling stress (gavage proce-
dure) as experienced by the group supplemented with fish oil and to
avoid any side effects from different oils, which have frequently been
used as placebos, including soybean, coconut, and olive oils [25,26].
The fish oil dose was chosen based on our previous studies [27–29] and
was determined by body surface area normalization, as suggested by
Reagan-Shawet et al. [30]. According to this proposition, a fish oil dose
of 2 g per kg b. w. in mice corresponds to a dose of 162 mg per kg b.w. in
humans (i.e., 5.5 g per day for an individual weighing 70 kg). A similar
fish oil dosage (5–6 g per day) has been used in human studies [31,32].
(See Fig. 2.)
Body weight and food intake were recorded three times per week.
Energy efficiency was calculated by the following formula: energy
efficiency = body weight gain/energy intake (g/kcal). The energy
intake per mouse was determined using the following conversions:
standard chow=3.80 kcal/g and HFD=5.34 kcal/g. The visceral
adiposity index (in percentage) was calculated by the following
formula: (RP + EP + VI)/b.w. × 100. In this formula, RP is
retroperitoneal fat (g); EP is epididymal fat (g); VI is visceral fat (g);
and b.w. is body weight (g) [33]. During the last week of the
experimental protocol, glucose and insulin tolerance tests were
performed with an interval of three days between them. At the end
of the experimental protocol, mice were fasted for 6 h and euthanized
for tissue harvesting at 2:00 p. m. by cervical dislocation after being
anesthetized in a CO2 chamber.
2.2. Insulin tolerance test
Animals were fasted for 6 h before the insulin tolerance test. Blood
samples were collected for serum glucose determination before (time
0) the intraperitoneal (i.p.) insulin injection (0.75 U/kg b.w.) and 10,
20, 30, 40, and 50 min afterward. The constant for the glucose
disappearance rate during the test (Kitt) was calculated using the
following formula: Kitt=0.693/t1/2. In this formula, t1/2 was calculated
from the slope of the least-square analysis of the plasma glucose
concentrations of the linear decay phase [34].
2.3. Glucose metabolism in isolated soleus muscle
At the end of the experimental protocol, mice were fasted for 6 h
and euthanized by cervical dislocation after being anesthetized in a
CO2 chamber. Soleus muscles were rapidly and carefully isolated,
weighed, attached to stainless steel clips to maintain resting tension
and preincubated at 35°C in Krebs-Ringer bicarbonate buffer (KRBB)
containing 5.6 mM glucose and 1% BSA, pH 7.4, pregassed for 30 min
with 95% O2/5% CO2, followed by agitation at 100 oscillations per min
at 35°C. After this period, the muscles were transferred to other vials
containing the same buffer, but 0.3 μCi/ml [U-14C]-D-glucose and 0.2
μCi/ml 2-deoxy-[2,6-3H]-D-glucose were added. Phenylethylamine
(0.2 ml) diluted in methanol (1:1 v/v) was added into a separate
compartment for 14CO2 adsorption. Incubation was then performed
for 1 h under the same conditions and in the absence or presence of 10
mU/ml of insulin. During all preincubation and incubation periods, an
atmosphere of 95% O2/5% CO2 was maintained. After the incubation
period, the muscles were briefly washed in cold KRBB at 4°C, dried on
filter paper and frozen in liquid N2. Samples were processed for
measurements of 2-deoxy-[2,6-3H]D-glucose uptake, [U-14C]D-glu-
cose incorporation, [14C]-glycogen synthesis, and [U-14C]D-glucose
decarboxylation according to the methods described by Crettaz et al.
[35]. The same procedure was used in our previous studies [36–38].
2.4. Determination of ROS production
Soleus muscles were isolated and incubated, as described in
Section 2.3, in KRBB containing 50 μM Amplex® UltraRed and
 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88 79

peroxidase (0.1 UI/ml) for 1 h. At the end of this period, the medium
was kept at 4°C, and the fluorescent product was measured at 530 nm
excitation and 590 nm emission. The results were normalized by
muscle weight. Similar procedures have been used in previous studies
[39,40].
2.5. Muscle oxygen consumption
Soleus muscles were isolated as described in Section 2.3 and
were added to a cuvette containing 1 ml of Krebs-Ringer bicarbonate
buffer (KRBB), pH 7.4, at 35°C. Oligomycin (2 μg/ml) and p-
trifluoromethoxyphenylhydrazone (FCCP) (2 μM) were added as an
ATP synthase inhibitor and a mitochondrial uncoupling agent,
respectively (Wu et al., 1999). Muscle oxygen consumption was
monitored using a Clark-type oxygen electrode (Hansatech, Oxytherm
Electrode, UK) as previously used in our studies [41,42].
2.6. Serum measurements
The serum concentrations of glucose, triglycerides, total choles-
terol, and HDL and the activities of alanine (ALT) and aspartate (AST)
transaminases were determined using commercial kits (Labtest
Diagnostica, Santa Lagoa, Brazil). Serum NEFA concentration was
measured using the HR NEFA Series Kit (Wako Diagnostic, Richmond,
VA, USA) according to the manufacturer's instructions. The LDL
cholesterol concentration (in mg/dl) was determined using the
formula described by Friedewald et al. [43]: LDL-cholesterol = total
cholesterol – HDL-cholesterol – (triglycerides / 5). The homeostatic
model assessment – Insulin Resistance (HOMA-IR) index was
calculated by the following formula: HOMA-IR = [glycemia (mM) ×
insulin (mU/L)] / 22.5 [44].
2.9. Citrate synthase activity
Citrate synthase activity was determined by spectrophotometric
analysis using a kinetic assay based on the ability of 2,2′-dinitro-5,5′-
dithiobenzoic acid (DTNB) (Ellman reagent) to react with the CoA-SH
group (SH) generated from acetyl-CoA [48]. The results are presented
as nmol per min per mg protein. A similar procedure was used in our
previous studies [41,49].
2.10. Western blot analysis
Soleus muscles were isolated and preincubated as described in
Section 2.3. Subsequently, muscles were incubated in the absence or
presence of 10 mU insulin for 20 min. Soleus muscles were then
homogenized in lysis buffer containing protease and phosphatase
inhibitors and centrifuged (13,000×g, 10 min, 4°C). The protein
concentration of the homogenates was determined by the Bradford
method [2], and the supernatants were mixed with 4× SDS-PAGE
sample buffer. Equal amounts of proteins were separated by SDS-PAGE
and transferred to polyvinylidene difluoride membranes. Proteins
were detected using antibodies against phospho-Akt (Ser437),
phospho-GSK3-β, phospho-AMPK, phospho-JNK (T183/Y185), Akt,
GSK3- β and JNK from Cell Signaling Technology (Boston, MA); Mfn2
was from Santa Cruz Biotechnology (Dallas, TX), and MitoProfile®
Total OXPHOS Rodent WB Antibody Cocktail was from MitoSciences
(Eugene, Oregon). The results were normalized using β-actin or
Ponceau S staining and are presented as arbitrary units (AU). A similar
procedure was used in our previous studies [29,46].
2.11. Quantitative RT-PCR

2.7. Lipid extraction and determination of the composition of fatty acids
in the gastrocnemius muscle using gas chromatography
The AOAC 996.06 (AOAC, 2005) and AOCS Ce 1j-07 (AOCS, 2007)
methods were used with a C13:0 fatty acid as a standard in place of
C11:0. Fatty acid composition was determined in a GC 2010 Plus gas
chromatograph equipped with an automatic sample injector (AOC
20i), flame ionization detector, GC solution software (Shimadzu Co,
Kyoto, Japan), and a 100-m, fused silica SP-2560 capillary column with
0.25 mm film thickness (Supelco Park, Bellefonte, PA, USA). The
temperature conditions were as follows: 140°C for 5 min; 140°C to
240°C at a rate of 4°C/min; and 240°C for 20 min. The remaining
conditions were as follows: the injector temperature was 250°C and
the detector temperature was 260°C; helium was the carrier gas (at 1
ml/min); and a 1/50 split ratio was used. The reference fatty acid
methyl esters (FAMEs) were the 189–19 from Sigma-Aldrich (St.
Louis, MO, USA). The composition of the fatty acids in the fish oil was
Total RNA from the soleus muscles was extracted using TRIzol
reagent (Invitrogen Life Technologies, MA, USA) [5] and was reverse
transcribed to cDNA using the High-Capacity cDNA Kit (Applied
Biosystems, MA, USA). Gene expression was evaluated by real-time
PCR [17] using a Rotor Gene system (Qiagen, Hilden, Germany) and
SYBR Green as the fluorescent dye. Primer sequences are in Table 1.
The quantification of gene expression was performed using a
previously described method [27] with the Hprt1 gene as an internal
control. The same assay procedure was used in our previous studies
[50,51].
Table 1
Characteristics of mice fed with a control diet (C) or a high-fat diet (H) supplemented or
not with omega-3 PUFAs from fish oil (FO)
Parameter C H CFO HFO

expressed as a percentage of the total fatty acids from three analyses. Body weight gain (g) 2.87±0.37a 11.37±0.92b 3,65±0.52a 7.64±0.79c
The composition of the fatty acids in the gastrocnemius muscle was Food ingestion 3.35±0.07a 2.05±0.06b 3.68±0.03a 2.09±0.04b
(g/day/animal)
expressed as g/100 g tissue wet weight (AOAC, 2005; AOCS, 2007). The
Food efficiency (body 0.007±0.01a 0.084±0.01b 0.004±0.01a 0.07±0.02b
same procedure was used in our previous studies [45,46]. weight gain (g)/food
ingestion (g))
Visceral adiposity index⁎ 1.353±0.19a 3.789±0.16b 1.729±0.08a 2.807±0.23c
2.8. Determination of tricarboxylic acid cycle intermediates (TCAI) Triglycerides (mg/dl) 87.5±8.1 67.3±5.7 83.6±12.1 62.9±3.7
Total cholesterol (mg/dl) 155.6±5.8a 197.0±10.4b 103.3±11.0c 119.8±10.7a
Briefly, the soleus muscle contents were extracted in perchloric LDL-cholesterol (mg/dl) 127.4±5.15a 165.1±14.49 b
73.2±8.5c 99.34±4.25a,c
HDL-cholesterol (mg/dl) 14.73±0.83 15.52±1.86 17.58±2.23 16.22±1.50
acid (0.5 M) containing 1 mM EDTA and were neutralized with a 2.2 M
KHCO3 aqueous solution. The concentration of the TCAI was Oral supplementation with fish oil (2 g/kg body weight, three times/week, oral gavage)
determined using a standard curve containing different concentra- or water started four weeks before H and continued until the end of experiment (12
weeks). Animals were fed with H or C for eight weeks. Data are presented as means ± SE
tions of citrate, malate, oxaloacetate, and isocitrate. The fluorometric, (n=10). Groups with different superscript letters are significantly different (Pb.05,
enzymatic assays were performed as previously described [47]. The Bonferroni post-test). ⁎The index was calculated by the sum of retroperitoneal,
results are presented as μmol per g muscle protein. epididymal, and mesenteric adipose tissue.
80 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88

2.12. Transmission electron microscopy 3.3. Fatty acid composition and insulin responsiveness in skeletal muscle
were improved by fish oil supplementation
Sections of soleus muscles (1 mm × 1 mm) were fixed in 2.5%
glutaraldehyde buffer solution for 3 h at 4°C. Next, the fixation solution Fish oil supplementation increased the total n-3 PUFA content,
was replaced with cacodylate buffer containing 0.1 M glutaraldehyde mainly due to the increase in EPA and DHA concentration, and
and was maintained at 4°C until the samples were further processed. decreased the n-6:n-3 PUFA ratio in the gastrocnemius muscle
After being washed, dehydrated and embedded in resin, muscles were compared to that of the control and HFD groups (Table 2).
cut using an ultramicrotome (creates sections that are nanometers A HFD led to soleus muscle insulin resistance, whereas FO
thick). The cuts were observed under a Jeol JEM-100 CXII microscope supplementation restored glucose uptake and oxidation and total
(JEOL USA Inc., Peabody, USA) and images were obtained with glucose incorporation into the tissue (Fig. 3A, B, and D). In response to
increases in magnification of 8, 14, and 20 thousand times for the insulin, the phosphorylation of both Akt and GSK3-β (Fig. 3E and F)
further analysis of mitochondrial density, number, and area, was decreased by the HFD, and supplementation with fish oil
respectively. attenuated this reduction, confirming the insulin-sensitizing effect of
FO in skeletal muscle. No significant differences in Glut-4 and Irs-1
2.13. Statistical analysis mRNA expression levels were observed to be induced by FO, although
the expression of these genes was slightly higher in HFO mice than in
Digital image processing and analysis were performed using the H group (data not shown).
ImageJ software (NIH, MD, USA). The results are presented as the
means ± standard error of the mean (SEM), and comparisons between 3.4. Fish oil supplementation increased catalase expression and
groups were performed using Student's t test (for comparing two decreased H2O2 production in skeletal muscle
groups) or two-way ANOVA followed by Bonferroni's post-test (for
multiple analysis) in GraphPad Prism 7.0. The statistical test used for To investigate the contribution of oxidative stress to insulin
each analysis is noted in the figure and table legends. Pb.05 was resistance in our study, we measured H2O2 production and the
considered a statistically significant difference. expression levels of antioxidant enzymes (CAT, SOD2, and GPX1) in
soleus muscle. H2O2 production was significantly increased in the H
group compared with that of the C group, but this increase was
3. Results abolished in the HFO group (Fig. 4A). SOD2 and GPX1 mRNA
expression levels were decreased in the H group but were not
3.1. Fish oil supplementation reduced body weight gain and adiposity in significantly affected by FO supplementation (Fig. 4B). However, FO
high-fat-diet-fed mice supplementation increased CAT expression compared to that of the H
group.
At the end of the experimental protocol, both HFD groups gained
more body weight than the respective control diet group. However, 3.5. Fish oil supplementation improved skeletal muscle mitochondrial
body weight gain was attenuated by FO supplementation when bioenergetics
compared to the weight of the non-supplemented group (Table 1). A
similar effect was observed for the visceral adiposity index. FO not only Muscle O2 consumption was evaluated in isolated soleus muscle.
decreased the concentration of plasma total cholesterol and LDL in the Compared to animals receiving a balanced diet, mice that were fed a
control group but also abolished the increase induced by the HFD HFD had reduced basal oxygen consumption. Supplementation with
(Table 1). No differences were found for plasma triglyceride and HDL fish oil completely prevented this effect (Fig. 5A). The HFD decreased
levels. None of the hepatic enzyme activities, AST, ALT or γ-GT were the concentrations of citrate, α-ketoglutarate, malate, and oxaloace-
altered by the HFD and/or FO supplementation (data not shown). tate in the gastrocnemius muscle, although a statistically significant
effect was found only for α-ketoglutarate and oxaloacetate (Figs. 5C
3.2. Supplementation with fish oil attenuated high-fat-diet-induced and 4E). Supplementation with fish oil increased the concentrations of
insulin resistance citrate, α-ketoglutarate, and malate (Fig. 5B, C, and E).
The protein content of the electron transport chain complexes and
Fish oil supplementation reduced the insulin resistance induced by the expression levels of several genes that encode the proteins
the HFD, as demonstrated by an improved insulin response in the ITT composing these enzyme complexes were measured in the soleus
and a decreased HOMA-IR compared to that of the non-supplemented muscle. HFD and/or supplementation with fish oil had no clear effect
animals (Fig. 1). on the protein content or the mRNA expression levels of the analyzed
complexes, except for a slight decrease in complex II in the H group
(Fig. 5F).
Table 2
Fatty acid profile (mg 100/g of sample) in the gastrocnemius muscle from mice fed a
control diet (C), high-fat diet (H), C + fish oil supplementation (CFO), and H + fish oil 3.6. Skeletal muscle mitochondrial function-associated proteins
supplementation (HFO)
Fatty acid family C H CFO HFO We also analyzed the protein content and mRNA expression levels of
genes involved in the regulation of mitochondrial metabolism (Fig. 6).
SFA 0.80±0.29 0.86±0.24 0.85±0.26 0.99±0.34
MUFA 1.45±0.80 1.51±0.62 1.26±0.60 1.56±0.73 The HFD reduced Pgc1-α expression (mRNA and protein), a known co-
PUFA 0.68±0.22 0.96±0.22 0.76±0.22 1.07±0.40 activator of PPAR-α and NRF1, which promote mitochondrial biogen-
n-6 0.53±0.20 0.73±0.19 0.32±0.17 0.66±0.33 esis. The expression levels of both these transcriptional factors (PPAR-α
n-3 0.15±0.03 0.20±0.03 0.44±0.06* 0.39±0.06+
and NRF1) were reduced by the HFD. Fish oil supplementation
EPA + DHA 0.14±0.03 0.16±0.01 0.36±0.04* 0.32±0.03+
n-6/n-3 3.39±0.89 3.59±0.51 0.71±0.29* 1.63±0.57+ abolished the changes in the expression of PGC1-α and PPAR-α induced
by the HFD but did not alter the expression of NRF1 (Fig. 6A–C).
Results expressed as mean ± standard deviation of 5 animals per group. Abbreviations:
SFA, saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated
The expression levels of AMPK and CREB, two proteins that induce
fatty acids; n-3. *Pb.05 and +Pb.05 when compared to control group (C) and high-fat PGC1-α activation, were not modified by the HFD (Fig. 6D and E). FO
diet group (H), respectively. supplementation increased AMPK phosphorylation (Thr172) compared
 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88 81

A B
15 5
P<,01 P<,05 P<,01 P<,05

Glucose oxidation
( mol/g of tissue)
( mol/g of tissue)

4
Glucose uptake

10
3

2
5
1

0 0
C H CFO HFO C H CFO HFO

C D
4
15

Glucose incorporation
Glycogen synthesis

P<,05
( mol/g of tissue)

( mol/g of tissue)
3 P<,01 P<,05

10
2

5
1

0 0
C H CFO HFO C H CFO HFO

E F
2.0 P<,01
2.0
++ - insulin
P<,05
P<,01 +
+ insulin
P-GSK-3 /total (AU)

1.5 P<,05 ++ ++
P-Akt/total (AU)

+++ 1.5
+++

1.0 1.0
*

0.5 0.5

0.0 0.0
C H CFO HFO C H CFO HFO

Phospho
Total
B-actina

Fig. 3. Glucose uptake, metabolism, and insulin signaling in isolated soleus muscle. Glucose uptake (A), glucose oxidation (B), total glucose incorporation (C), and glycogen synthesis (D)
were evaluated in soleus muscle. Results are means ± SE (n=6–10/group). *Pb.05 vs. C; #Pb.05 vs. H (ANOVA with Bonferroni post-test). Immunoblot analysis of Akt phosphorylation
(Ser437) was performed in soleus muscle extracts. E: the relative levels of phosphorylated (p)-Akt/Akt and (p)-GSK3b/ GSK3b (F) ratio of muscle incubated or not with insulin (black
bars) are shown in the bar graph. G: representative Western blot showing Akt and GSK3b activation (n=5–6/group). Results are means ± SE. *Pb.05 vs. C; °Pb.05 vs. H; +Pb.05 vs. insulin
absence.

to that of the H group. A similar effect was observed for the transcription
of CPT-1 (Fig. 6C). There was no significant difference in the expression
of mitochondrial uncoupling protein-3 (UCP3) (Fig. 6C).
The expression of mitofusin-2 (Mfn-2), a protein involved in
mitochondrial fusion that has been associated with the metabolic
regulation in muscle cells, was not altered by the HFD, but it was
increased in the HFO group (Fig. 6F). We also examined the
transcription of the gene encoding the ATM protein, and it was
decreased in the H group but up-regulated in the HFO group (Fig. 6G).
There was no difference in the total number or average area of the
mitochondria among the groups, as indicated by electron microscopy.
Additionally, there was no apparent change in the shape or
distribution of the mitochondria. However, a significant difference
was found in the number of subsarcolemmal (SS) and intramyofi-
brillar (IMF) mitochondrial subpopulations. The IMF mitochondrial
number was increased by fish oil supplementation (Fig. 7).
4. Discussion
Some studies have suggested that insulin sensitivity is increased in
skeletal muscle after supplementation with polyunsaturated FAs
[29,52], but others have not found the same response [53,54]. Despite
82 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88
A
1500
fluoresc. un.xmg-1 x min-1
P<.05
1000
500
0
C H
B C
1.5
P<.05
mRNA (AU)
1.0
0.5
0.0
SOD1 SOD2
Fig. 4. Oxidative stress response in soleus muscle. H2O2 production in isolated soleus muscle using Amplex Red®. mRNA expression of antioxidant enzymes in soleus muscle from mice
fed a control diet (C) or high-fat diet (H) and supplemented or not with fish oil (FO). Results are means ± SE (n=6–10/group). ANOVA followed by Bonferroni post-test.
the conflicting findings, there is strong evidence that n-3 PUFAs,
especially EPA and DHA, contribute to improved insulin sensitivity in
skeletal muscle [55,56], although the mechanisms are still not well
understood.
This study was designed to test the hypothesis that the positive
effect of dietary n-3 PUFAs on HFD-induced insulin resistance is
associated with improved mitochondrial function in skeletal muscle.
After eight weeks of a HFD, body weight gain increased and insulin
sensitivity decreased in mice, and these alterations were partially
prevented by supplementation with fish oil. In addition, a HFD induced
an increase in H2O2 production and decreased the expression of Sod2
and Gpx-1 without altering catalase expression, leading to a redox
imbalance. Fish oil supplementation abolished the effect of a HFD on
superoxide production in the soleus muscle. Muscle mitochondrial
abundance and respiratory capacity, compared to those of the control
animals, were significantly increased in mice supplemented with fish
oil. In summary, the changes in the skeletal muscle respiratory
capacity and in ROS production associated with insulin resistance
induced by a HFD were partially attenuated by supplementation with
n-3 PUFAs.
After 8 weeks of a HFD, mice had a significant increase in plasma
insulin and glucose levels and impaired insulin sensitivity, as
demonstrated by the ITT and HOMA-IR index. Fish oil supplementa-
tion partially prevented the deleterious effects of the HFD on insulin
sensitivity. FO supplementation reduced the glucose and insulin
levels, improved blood glucose reduction kinetics, as assessed by ITT,
and improved the HOMA-IR index. These results are in agreement
with observations by others in different rodent models that n-3 PUFAs
have a preventive effect on insulin resistance.
The HFD led to an increase in plasma concentrations of total and
LDL cholesterol without any alteration in HDL-C and triglyceride
concentrations. These HFD-induced changes were prevented by FO
supplementation. Interestingly, fish oil also had a clear effect on these
parameters in animals fed the balanced diet. Similar changes in the
lipid plasma composition induced by obesity have been described [57],
P<.001
CFO HFO
H
P<.05
P<.001 CFO
HFO
GPX1 CAT
and an improvement in this profile by fish oil intervention has also
been reported [58,59]. Decreased plasma total cholesterol and
triglyceride concentrations after supplementation with n-3 PUFAs
were described by Hartweg et al. [58], and this effect has been
associated with increased beta-oxidation, which reduces triglyceride
accumulation and release by the liver [59].
Several studies, including the present study, have reported that
treating skeletal muscle cells with saturated FA impairs glucose
metabolism in the presence of insulin [60,61]. The insulin-stimulated
impairment of glycogen synthesis, lactate production, glucose uptake
and oxidation in skeletal muscle has been described. Decreased
insulin-stimulated glucose uptake, incorporation, and oxidation and
reduced glycogen synthesis were observed in the soleus muscles of
animals fed a HFD in this study. This feature of insulin resistance in
skeletal muscle was partially prevented by fish oil supplementation.
Saturated fatty acids do impair insulin intracellular signaling in
skeletal muscle, as reviewed by Schmitz-Peiffer [62]. The activation of
Akt and GSK-3β proteins was decreased in the soleus muscle from the
H group compared to that of the C mice. In a previous study, we also
reported that Akt phosphorylation is decreased in mice subjected to
the same obesity/insulin resistance model that was used here [63].
Tishinsky et al. [64] reported an increased activation of Akt in the
soleus muscle of rats that received fish oil in their diet compared to
that of animals fed only a HFD (55% of fat), similar to the findings in our
study. Some groups have reported that the phosphorylation of GSK-3β
is impaired in the skeletal muscles of rats fed a HFD [65,66] due to the
decreased activation of PI3 kinase. However, little was known about
the mechanism of action of n-3 PUFAs on the activation of this protein
in skeletal muscle. Evidence is presented herein that supplementation
with fish oil prevents the impaired insulin signaling induced by the
HFD.
The mechanisms contributing to the inhibition of insulin signaling
by the HFD include the activation of various kinases such as PKCs and
JNK. These proteins have been associated with decreased IRS-1
activation [67,68]. We found an increased activation of PKC-θ and
 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88 83

A
3

-1
P<,05
P<,05

nmol O2/min. x mg protein

2

0
C H CFO HFO

F
OXPHOS complexes (AU)

0.15
C
H
0.10 P<,05 CF
HF

0.05
CIII
CV
0.00
CI CII CIII CV CII

CI

Fig. 5. Mitochondrial function in skeletal muscle from mice fed a control (C) or high-fat diet (H) and supplemented or not with omega-3 from fish oil (FO). Basal oxygen consumption (A)
in isolated soleus muscle. Concentrations of trycarboxylic acid cycle intermediates (B-E) in gastrocnemius muscle homogenate. And content of Mfn2 (F) in soleus muscle analyzed by
Western blotting. Results are means ± SE (n=5/group). ANOVA followed by Bonferroni post-test.

JNK and decreased IRS-1 transcription (data not shown) after insulin sensitivity in healthy individuals receiving 3.6 g of n-3 PUFAs
consumption of the HFD, but fish oil had no significant effects. Thus, daily for 90 days. Lombardo et al. [56] used Wistar rats at 2 months of
the n-3 PUFA-mediated increase in the insulin sensitivity of skeletal age, fed them a diet rich in sucrose for 8 months and supplemented (or
muscle involves other mechanisms. not) with fish oil during the last 2 months of that period. The authors
Various studies involving supplementation with n-3 PUFAs have reported decreased levels of triglycerides in the liver and skeletal
described a subsequent reduction in insulin resistance, but none of muscle and improved skeletal muscle insulin sensitivity in animals
them have reported the prevention of insulin resistance. There is a receiving the fish oil supplementation. Rossmeisl et al. [69] used
huge variation in the provided doses, types of fish oil or sources of n-3 various derivatives of DHA to verify its preventive effect on obesity and
PUFAs, the periods of treatment, the experimental models, and the on the complications induced by a HFD in mice aged 3 months. The diet
design protocols. Giacco et al. [53] reported that there was no effect on was administered for 4 months, and the DHA derivatives either were
84 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88

A B
0.5 1.5 P<,01 P<,05
P<,05 P<,05

PGC1 protein (AU)

PGC1 mRNA (AU)

0.4
1.0
0.3

0.2
0.5
0.1

0.0 0.0
C H CFO HFO C H CFO HFO

C P<,001 P<,01 C
1.5
P<,01 P<,05 H
P<,05 CFO
mRNA (AU)

1.0 HFO

0.5

0.0
CPT1 PPARa UCP3 TFAM NRF1

D E
p-AMPK/AMPK total (UA)

p-CREB/CREB total (UA)

0.8 P<,05 1.0

0.6 0.8

0.6
0.4
0.4
0.2
0.2

0.0 0.0
C H CFO HFO C H CFO HFO

F G
3
Mfn2 - proteína (UA)

P<,01 1.5
P<,001 P<,05
ATM mRNA (UA)

2
1.0

1
0.5

0 0.0
C H CFO HFO C H CFO HFO

Fig. 6. Mitochondrial biogenesis and function. Effects of H and FO on PGC1a protein content (A) and mRNA expression (B). Expression of genes involved with mitochondrial activity in
soleus muscle (C). Phosphorylation of AMPK (D) and CREB (E) in soleus muscle (F). Electron microscopy mitochondrial images from soleus muscle. Results are means ± SE (n=5/
group). ANOVA followed by Bonferroni post-test.

used together with the diet to investigate the preventive action or activity of mitochondrial enzymes, biogenesis and function, suggest-
were used two months after the induction of obesity to examine the ing that this organelle is not involved in the pathogenesis of obesity-
possible reversal effect. These authors found beneficial effects of n-3 associated insulin resistance [72]. In fact, the known effects of n-3
PUFA supplementation on obesity and on related metabolic disorders PUFAs on mitochondrial function in skeletal muscle are still scarce
in both protocols. and controversial.
Ectopic fat accumulation is one of the main mechanisms associated Several mechanisms have been proposed to explain the positive
with HFD-induced obesity that is involved in the development of effects of n-3 PUFAs on insulin sensitivity. The modulation of
insulin resistance [70]. The lipid accumulation in skeletal muscle has inflammation is one of the leading mechanistic explanations
been postulated to decrease mitochondrial function [15], as demon- [20,73]. According to previous studies, mitochondria are directly
strated by the decreased mitochondrial content and impaired associated with insulin sensitivity, thus our aim was to evaluate a
transcription of genes involved in mitochondrial biogenesis [9,71]. possible direct link between n-3 PUFAs and mitochondrial function.
However, others have reported that HFD consumption increases the Long-chain fatty acyl-CoAs can be used for mitochondrial oxidation,
 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88
C H
B C
60 P<,05
number of mitoch./
field 14.000x
40
20
0 0.0
C H CFO HFO
D
P<,001
Mitochondrial density (n/8kx)
90
60 P<,05
*
30
0
C H
Fig. 7. Mitochondrial abundance is increased with fish oil by electron microscopy. (A) Representative transmission electron micrographs (27.000 times of magnification) of skeletal
muscle from C, H, CFO and HFO mice. (B, C, and D) Quantification of the mitochondrial number, area, and density, respectively. Results are means ± SE (n=5/group). ANOVA followed by
Bonferroni post-test.
phospholipid synthesis, ceramide production, and the generation of
other fatty acid-derived metabolites. In insulin resistance, several of
these lipid metabolites are accumulated into the cell and are
responsible for impairing insulin signaling pathways [74,75]. Thus,
the improved mitochondrial function induced by n-3 PUFAs may
decrease the accumulation of lipid metabolites, preferentially
diverting LCA-CoA to mitochondrial beta-oxidation rather than the
generation of fatty acid-derived metabolites associated with insulin
resistance. This proposition is based on preceding literature
suggesting a potential role for mitochondria in the insulin-
sensitizing effects of n-3 PUFAs. In our study, we did not find any
alterations in the TG or NEFA content of skeletal muscle (data not
shown).
The n-3 PUFAs prevent insulin resistance in wild-type mice but not
in mice lacking the α-2 catalytic subunit of AMPK [21] or PPAR-α [22].
AMPK and PPAR-α are transcriptional factors known to up-regulate
genes involved in mitochondrial biogenesis and fatty acid oxidation
[24,76]. Additionally, n-3 PUFAs are strong PPAR ligands [77]. The
treatment of glioma cells with EPA increased PGC-1α, TFAM, and
cytochrome c oxidase expression and mitochondrial function, as
demonstrated by increased mitochondrial membrane potential and
ATP levels [78]. By activating several proteins through the regulation
of transcriptional factor activity (PPAR, AMPK, and PGC-1α), n-3
85
CFO HFO
2.0
Area mitoch./
1.5
20.000x ( m)
1.0
0.5
C H CFO HFO
*** SS
P<,001 IMF
**
CFO HFO
PUFAs can stimulate mitochondrial biogenesis, increase mitochondrial
oxidative capacity and lipid oxidation, and decrease the abundance of
intracellular lipid metabolites that would otherwise interfere with
insulin signaling. Herein, fish oil attenuated the lipid accumulation
and oxidative damage in skeletal muscle induced by the HFD.
According to previous reports [77,78], fish oil increased the gene
transcript expression level for PPAR-α and PGC1-α, which regulate
mitochondrial biogenesis. FO supplementation also increased the
abundance of mitochondria in skeletal muscle, as observed by electron
microscopy. In fact, mitochondrial abundance was significantly higher
in skeletal muscle from the HFO group than in that of the C and H
groups. Significant differences were observed in the subsarcolemmal
(SS) and intramyofibrillar (IMF) mitochondrial subpopulations; the
IMF population was increased after FO supplementation. This
alteration is important because this subpopulation of mitochondria
may have unique metabolic influences on lipid oxidation and may
respond differently to stimuli. Therefore, a better understanding of
how specific mitochondrial subpopulations are related to insulin
sensitivity and the patterns of fuel use may help in elucidating the
etiology of insulin resistance under the conditions described.
Mitochondrial function was impaired by the HFD, and the
consumption of fish oil attenuated these alterations. Soleus muscle
isolated from the H group exhibited decreased O2 consumption
86 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88
compared to that of the C group, and this impairment was completely
prevented by fish oil administration. Similarly, Yokota et al. [79]
reported a decreased respiration capacity in mitochondria isolated
from the skeletal muscle of HFD-fed mice, and Holmström et al. [80]
described a lower oxygen consumption rate in the soleus muscle of db/
db obese insulin-resistant mice compared to that of mice fed a control
diet. On the other hand, Lanza et al. [81] reported an improved O2
respiration rate in mitochondria isolated from the soleus muscle of
mice fed a HFD and no significant effect of FO supplementation.
Although important difference in the fiber composition between
soleus and gastrocnemius muscles, in accordance with impaired
mitochondrial activity observed in soleus muscle, the HFD decreased
the activity of tricarboxylic acid cycle, as shown by the reduced muscle
concentrations of the Krebs cycle intermediates in gastrocnemius
muscle. Similar effects from a HFD were reported by Barbosa et al. [41].
FO supplementation increased the concentrations of the Krebs
cycle intermediates, indicating an improvement in mitochondrial
function.
Sparks et al. [9] described the impaired transcription of various
OXPHOS complex genes in the skeletal muscle of diabetic patients. We
also analyzed OXPHOS protein content and found that only the
expression level of CII was decreased in the H group compared with
that of the C group. FO supplementation prevented this HFD effect.
There was no alteration observed in the expression of several other
genes of the OXPHOS complexes: NDUFB3, NDUFB5, SDHB, SLC25A12,
CYC1, and SURF (data not shown).
Supplementation with FO increased the expression of PGC1-α,
AMPK, and Mfn2, which are associated with mitochondrial biogenesis
and function. Similarly, it has been demonstrated that n-3 PUFAs
increase energy expenditure in mice fed a HFD by increasing that
expression of genes (PGC-1α, AMPK, and SIRT-1) in brown adipose
tissue and skeletal muscle related to mitochondrial biogenesis and
function [82–84]. AMPK is a key protein involved in energy
homeostasis and is activated in several conditions, including muscle
contraction, hypoxia, and oxidative stress (for review, see [85]). In
regards to muscle glucose metabolism, AMPK stimulates insulin-
independent glucose uptake, and this glucose is utilization through an
increased expression and translocation of GLUT-4 to the plasma
membrane and through the increased activities of two key glycolytic
enzymes: 6-phosphofructo-2-kinase (PFK-2) and hexokinase-2 (HK-
2). AMPK activity may have contributed, at least in part, to the effects
of fish oil observed in our study since we found that improved glucose
homeostasis was associated with an increase in AMPK expression. The
increase in Mfn2 expression by FO may explain the low incidence of
mitochondrial morphological alterations, such as mitochondrial
fusion, that have been associated with improved mitochondrial
function [86,87]. Increased Mfn2 expression in skeletal muscle is not
only associated with mitochondrial remodeling [88] but also with
increased O2 consumption, mitochondrial membrane potential and
glucose oxidation [89]. PGC1-α also regulates the mitochondrial
dynamics that stimulate the fusion process [86]. Thus, Mfn2 is not only
another nuclear co-activator target of PGC1-α but also an intracellular
signaling component that regulates skeletal muscle metabolism. This
is the first evidence that n-3 PUFAs regulate Mfn2 expression in
skeletal muscle.
The expression of PGC-1α, a transcription factor that plays a key
role in mitochondrial biogenesis, is stimulated by oxidative stress [90]
and AMPK and CREB (cAMP response element-binding) activity [91].
CREB can also be activated by pathways sensitive to calcium and cyclic
AMP (cAMP), such as CaMKII (calcium/calmodulin-dependent protein
kinase II) and PKA (protein kinase A), which are activated in insulin
deficiency [92]. Roberts-Wilson et al. [93] reported that the reduction
in PGC-1α content in the gastrocnemius muscle of type 1 diabetic rats,
despite the activation of other pathways that can induce PGC-1α, is
due to a reduction in calcineurin signaling, a calcium-dependent
phosphatase associated with the stimulation of PGC-1α expression.
This same mechanism may apply to the model used in our study.
Similar to the results of PGC-1α, PPARα mRNA expression was
decreased in the soleus muscle of animals fed a HFD, and this effect
was reversed by fish oil supplementation. PPARα and β are abundant
in skeletal muscle, and their activation results in the induction of genes
that participate in the regulation of fatty acid oxidation [94,95].
Impaired energy metabolism was described in the diaphragm of
PPARα knockout mice but did not alter fiber typing or myosin heavy
chains [96]. The oxidative stress in HFD-induced insulin-resistant
skeletal muscle, therefore, appears to reduce PPARα expression in the
soleus muscle, contributing to the impairment of mitochondrial
energy metabolism. FO supplementation also increased the
expression of CPT1, a key enzyme for FA oxidation that is regulated
by PPARα.
The increased production of ROS has been directly related to the
development of diet-induced insulin resistance. Animals fed a HFD
had elevated ROS production in the mitochondria isolated from the
liver [97], skeletal muscle [81], and adipose tissue [98]. Skeletal muscle
cells treated with palmitate also have an enhanced production of these
species [17,41]. Herein, a decrease in O2 consumption was associated
with an increase in hydrogen peroxide production in the soleus
muscle of animals fed a HFD. Supplementation with FO abolished
these effects, which is consistent with previous studies showing the
antioxidant and anti-inflammatory effects of n-3 PUFAs in adipose
tissue [99–101], macrophages [102,103], and skeletal muscle
[104,105].
An increased expression of the protein kinase ATM (ataxia
telangiectasia mutated) by FO supplementation was also described.
This protein is a redox sensor and an important regulator of
metabolism, as reviewed by Ditch and Paul [106]. ATM is expressed
in most tissues and is a member of the phosphatidylinositol 3-kinase
(PI3-K)-kinase (PI3KK) family and the insulin-signaling pathway
[107]. A strong association between ATM deficiency, oxidative stress
and favorable conditions for the development of neurodegenerative,
oncogenic and metabolic diseases has been reported. Compared to
controls with a high content of ATM, the consumption of high-fat diets
by ATM−/− knockout mice caused insulin resistance, atherosclerosis
and glucose intolerance. The reduced activity of ATM promotes insulin
resistance in peripheral tissues, including skeletal muscle, and this is
induced by ROS production.
In summary, fish oil supplementation partially prevented the HFD-
induced obesity, insulin resistance, and mitochondrial dysfunction by
improving glucose metabolism, insulin signaling, and mitochondrial
function in skeletal muscle. The improvement in mitochondrial
function by FO supplementation was associated with reduced ROS
production and increased ATM expression, which activates antioxi-
dant systems and AMPK directly, leading to the stimulation of PGC1-α
and, consequently, the increased expression of genes involved in
mitochondrial biogenesis and oxidative respiration. Thus, our results
suggest that n-3 PUFAs are a potential intervention for obesity-
induced mitochondrial dysfunction and insulin resistance in skeletal
muscle and, consequently, may be a potential strategy for preventing
or attenuating obesity-associated diseases, including type 2 diabetes
mellitus, dyslipidemias, cardiovascular diseases, and the metabolic
syndrome.
Acknowledgements
FAPESP, CNPq, CAPES, and Guggenheim Foundation supported this
study. PN received FAPESP travel support and is grateful to the Curtin
University School of Biomedical Sciences for research support. Authors
are grateful for the technical assistance from Dr. Gilson Masahiro
Murata and Mr. Jose Roberto Mendonca.
 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88
References
[1] Lionetti L, Mollica MP, Crescenzo R, D'Andrea E, Ferraro M, Bianco F, et al. Skeletal
muscle subsarcolemmal mitochondrial dysfunction in high-fat fed rats exhibit-
ing impaired glucose homeostasis. Int J Obes (Lond) 2007;31:1596–604.
[2] Schenk S, Saberi M, Olefsky JM. Insulin sensitivity: modulation by nutrients and
inflammation. J Clin Invest 2008;118:2992–3002.
[3] Di Meo S, Iossa S, Venditti P. Skeletal muscle insulin resistance: role of
mitochondria and other ROS sources. J Endocrinol 2017;233:R15–42.
[4] Karaderi T, Drong AW, Lindgren CM. Insights into the Genetic Susceptibility to
Type 2 Diabetes from Genome-Wide Association Studies of Obesity-Related
Traits. Curr Diab Rep 2015;15:1–12.
[5] Ruiz-Nunez B, Dijck-Brouwer DA, Muskiet FA. The relation of saturated fatty
acids with low-grade inflammation and cardiovascular disease. J Nutr Biochem
2016;36:1–20.
[6] Martins AR, Nachbar RT, Gorjao R, Vinolo MA, Festuccia WT, Lambertucci RH, et al.
Mechanisms underlying skeletal muscle insulin resistance induced by fatty acids:
importance of the mitochondrial function. Lipids Health Dis 2012;11:1–11.
[7] Savage DB, Petersen KF, Shulman GI. Disordered lipid metabolism and the
pathogenesis of insulin resistance. Physiol Rev 2007;87:507–20.
[8] Schrauwen P, Hesselink MK. Oxidative capacity, lipotoxicity, and mitochondrial
damage in type 2 diabetes. Diabetes 2004;53:1412–7.
[9] Sparks LM, Xie H, Koza RA, Mynatt R, Hulver MW, Bray GA, et al. A high-fat diet
coordinately downregulates genes required for mitochondrial oxidative phos-
phorylation in skeletal muscle. Diabetes 2005;54:1926–33.
[10] Hulver MW, Berggren JR, Cortright RN, Dudek RW, Thompson RP, Pories WJ, et al.
Skeletal muscle lipid metabolism with obesity. Am J Physiol Endocrinol Metab
2003;284:E741–7.
[11] Kim JY, Nolte LA, Hansen PA, Han DH, Ferguson K, Thompson PA, et al. High-fat
diet-induced muscle insulin resistance: relationship to visceral fat mass. Am J
Physiol Regul Integr Comp Physiol 2000;279:R2057–65.
[12] Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science
2005;307:384–7.
[13] Szendroedi J, Schmid AI, Meyerspeer M, Cervin C, Kacerovsky M, Smekal G, et al.
Impaired mitochondrial function and insulin resistance of skeletal muscle in
mitochondrial diabetes. Diabetes Care 2009;32:677–9.
[14] Kelley DE, He J, Menshikova EV, Ritov VB. Dysfunction of mitochondria in human
skeletal muscle in type 2 diabetes. Diabetes 2002;51:2944–50.
[15] Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI. Impaired mitochondrial
activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J
Med 2004;350:664–71.
[16] Brehm A, Krssak M, Schmid AI, Nowotny P, Waldhausl W, Roden M. Increased
lipid availability impairs insulin-stimulated ATP synthesis in human skeletal
muscle. Diabetes 2006;55:136–40.
[17] Lambertucci RH, Hirabara SM, Silveira Ldos R, Levada-Pires AC, Curi R, Pithon-
Curi TC. Palmitate increases superoxide production through mitochondrial
electron transport chain and NADPH oxidase activity in skeletal muscle cells. J
Cell Physiol 2008;216:796–804.
[18] Hamdy O, Goodyear LJ, Horton ES. Diet and exercise in type 2 diabetes mellitus.
Endocrinol Metab Clin North Am 2001;30:883–907.
[19] Bhaswant M, Poudyal H, Brown L. Mechanisms of enhanced insulin secretion and
sensitivity with n-3 unsaturated fatty acids. J Nutr Biochem 2015;26:571–84.
[20] Oh DY, Talukdar S, Bae EJ, Imamura T, Morinaga H, Fan W, et al. GPR120 is an
omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-
sensitizing effects. Cell 2010;142:687–98.
[21] Jelenik T, Rossmeisl M, Kuda O, Jilkova ZM, Medrikova D, Kus V, et al. AMP-
activated protein kinase alpha2 subunit is required for the preservation of
hepatic insulin sensitivity by n-3 polyunsaturated fatty acids. Diabetes 2010;59:
2737–46.
[22] Neschen S, Morino K, Dong J, Wang-Fischer Y, Cline GW, Romanelli AJ, et al. n-3
Fatty acids preserve insulin sensitivity in vivo in a peroxisome proliferator-
activated receptor-alpha-dependent manner. Diabetes 2007;56:1034–41.
[23] Biswas D, Ghosh M, Kumar S, Chakrabarti P. PPARalpha-ATGL pathway improves
muscle mitochondrial metabolism: implication in aging. FASEB J 2016;30:
3822–34.
[24] Garcia-Roves PM, Osler ME, Holmstrom MH, Zierath JR. Gain-of-function R225Q
mutation in AMP-activated protein kinase gamma3 subunit increases mito-
chondrial biogenesis in glycolytic skeletal muscle. J Biol Chem 2008;283:
35724–34.
[25] Deol P, Evans JR, Dhahbi J, Chellappa K, Han DS, Spindler S, et al. Soybean Oil Is
More Obesogenic and Diabetogenic than Coconut Oil and Fructose in Mouse:
Potential Role for the Liver. PLoS One 2015;10:e0132672.
[26] Schwingshackl L, Christoph M, Hoffmann G. Effects of Olive Oil on Markers of
Inflammation and Endothelial Function-A Systematic Review and Meta-Analysis.
Forum Nutr 2015;7:7651–75.
[27] de Sa RD, Crisma AR, Cruz MM, Martins AR, Masi LN, do Amaral CL, et al. Fish oil
prevents changes induced by a high-fat diet on metabolism and adipokine
secretion in mice subcutaneous and visceral adipocytes. J Physiol 2016;594:
6301–17.
[28] Folador A, de Lima-Salgado TM, Hirabara SM, Aikawa J, Yamazaki RK, Martins EF,
et al. Effect of fish oil supplementation for two generations on changes of
lymphocyte function induced by Walker 256 cancer cachexia in rats. Nutr Cancer
2009;61:670–9.
87
[29] Hirabara SM, Folador A, Fiamoncini J, Lambertucci RH, Rodrigues Jr CF, Rocha MS,
et al. Fish oil supplementation for two generations increases insulin sensitivity in
rats. J Nutr Biochem 2013;24:1136–45.
[30] Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human
studies revisited. FASEB J 2008;22:659–61.
[31] Couet C, Delarue J, Ritz P, Antoine JM, Lamisse F. Effect of dietary fish oil on body
fat mass and basal fat oxidation in healthy adults. Int J Obes Relat Metab Disord
1997;21:637–43.
[32] McGlory C, Wardle SL, Macnaughton LS, Witard OC, Scott F, Dick J, et al. Fish oil
supplementation suppresses resistance exercise and feeding-induced increases
in anabolic signaling without affecting myofibrillar protein synthesis in young
men. Physiol Rep 2016;4:1–11.
[33] Jeyakumar SM, Lopamudra P, Padmini S, Balakrishna N, Giridharan NV,
Vajreswari A. Fatty acid desaturation index correlates with body mass and
adiposity indices of obesity in Wistar NIN obese mutant rat strains WNIN/Ob and
WNIN/GR-Ob. Nutr Metab (Lond) 2009;6:1–8.
[34] Bonora E, Moghetti P, Zancanaro C, Cigolini M, Querena M, Cacciatori V, et al.
Estimates of in vivo insulin action in man: comparison of insulin tolerance tests
with euglycemic and hyperglycemic glucose clamp studies. J Clin Endocrinol
Metab 1989;68:374–8.
[35] Crettaz M, Prentki M, Zaninetti D, Jeanrenaud B. Insulin resistance in soleus
muscle from obese Zucker rats. Involvement of several defective sites. Biochem J
1980;186:525–34.
[36] Farina AC, Hirabara S, Sain J, Latorre ME, Gonzalez M, Curi R, et al. Conjugated
linoleic acid improves glucose utilization in the soleus muscle of rats fed linoleic
acid-enriched and linoleic acid-deprived diets. Nutr Res 2014;34:1092–100.
[37] Massao Hirabara S, de Oliveira Carvalho CR, Mendonca JR, Piltcher Haber E,
Fernandes LC, Curi R. Palmitate acutely raises glycogen synthesis in rat soleus
muscle by a mechanism that requires its metabolization (Randle cycle). FEBS
Lett 2003;541:109–14.
[38] Reis FC, Haro AS, Bacurau AV, Hirabara SM, Wasinski F, Ormanji MS, et al.
Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise
Performance. PLoS One 2015;10:e0134844.
[39] Guimaraes-Ferreira L, Pinheiro CH, Gerlinger-Romero F, Vitzel KF, Nachbar RT,
Curi R, et al. Short-term creatine supplementation decreases reactive oxygen
species content with no changes in expression and activity of antioxidant
enzymes in skeletal muscle. Eur J Appl Physiol 2012;112:3905–11.
[40] Pinheiro CH, Gerlinger-Romero F, Guimaraes-Ferreira L, de Souza-Jr AL, Vitzel KF,
Nachbar RT, et al. Metabolic and functional effects of beta-hydroxy-beta-
methylbutyrate (HMB) supplementation in skeletal muscle. Eur J Appl Physiol
2012;112:2531–7.
[41] Barbosa MR, Sampaio IH, Teodoro BG, Sousa TA, Zoppi CC, Queiroz AL, et al.
Hydrogen peroxide production regulates the mitochondrial function in insulin
resistant muscle cells: effect of catalase overexpression. Biochim Biophys Acta
1832;2013:1591–604.
[42] Hirabara SM, Silveira LR, Alberici LC, Leandro CV, Lambertucci RH, Polimeno GC,
et al. Acute effect of fatty acids on metabolism and mitochondrial coupling in
skeletal muscle. Biochim Biophys Acta 1757;2006:57–66.
[43] Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-
density lipoprotein cholesterol in plasma, without use of the preparative
ultracentrifuge. Clin Chem 1972;18:499–502.
[44] Turner RC, Holman RR, Matthews D, Hockaday TD, Peto J. Insulin deficiency and
insulin resistance interaction in diabetes: estimation of their relative contribu-
tion by feedback analysis from basal plasma insulin and glucose concentrations.
Metabolism 1979;28:1086–96.
[45] Abreu P, Pinheiro CH, Vitzel KF, Vasconcelos DA, Torres RP, Fortes MS, et al.
Contractile function recovery in severely injured gastrocnemius muscle of rats
treated with either oleic or linoleic acid. Exp Physiol 2016;101:1392–405.
[46] Marzuca-Nassr GN, Vitzel KF, De Sousa LG, Murata GM, Crisma AR, Rodrigues
Junior CF, et al. Effects of high EPA and high DHA fish oils on changes in signaling
associated with protein metabolism induced by hindlimb suspension in rats.
Physiol Rep 2016;4:1–16.
[47] Bergmeyer HU. Methods of enzymatic analysis. New York: Academic Press;
1974; 1562–615.
[48] Srere PA. Citryl-Coa. An Substrate for the Citrate-Cleavage Enzyme. Biochim
Biophys Acta 1963;73:523–5.
[49] Alba-Loureiro TC, Hirabara SM, Mendonca JR, Curi R, Pithon-Curi TC. Diabetes
causes marked changes in function and metabolism of rat neutrophils. J
Endocrinol 2006;188:295–303.
[50] da Silva-Santi LG, Antunes MM, Caparroz-Assef SM, Carbonera F, Masi LN, Curi R,
et al. Liver Fatty Acid Composition and Inflammation in Mice Fed with High-
Carbohydrate Diet or High-Fat Diet. Nutrients 2016;8:1–14.
[51] Kuwabara WM, Curi R, Alba-Loureiro TC. Autophagy Is Impaired in Neutrophils
from Streptozotocin-Induced Diabetic Rats. Front Immunol 2017;8:1–10.
[52] Gao H, Geng T, Huang T, Zhao Q. Fish oil supplementation and insulin sensitivity:
a systematic review and meta-analysis. Lipids Health Dis 2017;16:1–9.
[53] Giacco R, Cuomo V, Vessby B, Uusitupa M, Hermansen K, Meyer BJ, et al. Fish oil,
insulin sensitivity, insulin secretion and glucose tolerance in healthy people: is
there any effect of fish oil supplementation in relation to the type of background
diet and habitual dietary intake of n-6 and n-3 fatty acids? Nutr Metab
Cardiovasc Dis 2007;17:572–80.
[54] Mostad IL, Bjerve KS, Basu S, Sutton P, Frayn KN, Grill V. Addition of n-3 fatty
acids to a 4-hour lipid infusion does not affect insulin sensitivity, insulin
secretion, or markers of oxidative stress in subjects with type 2 diabetes mellitus.
Metabolism 2009;58:1753–61.
88 A.R. Martins et al. / Journal of Nutritional Biochemistry 55 (2018) 76–88
[55] Jucker BM, Cline GW, Barucci N, Shulman GI. Differential effects of safflower oil
versus fish oil feeding on insulin-stimulated glycogen synthesis, glycolysis, and
pyruvate dehydrogenase flux in skeletal muscle: a 13C nuclear magnetic
resonance study. Diabetes 1999;48:134–40.
[56] Lombardo YB, Hein G, Chicco A. Metabolic syndrome: effects of n-3 PUFAs on a
model of dyslipidemia, insulin resistance and adiposity. Lipids 2007;42:427–37.
[57] Lu J, Borthwick F, Hassanali Z, Wang Y, Mangat R, Ruth M, et al. Chronic dietary n-
3 PUFA intervention improves dyslipidaemia and subsequent cardiovascular
complications in the JCR:LA- cp rat model of the metabolic syndrome. Br J Nutr
2011;105:1572–82.
[58] Hartweg J, Farmer AJ, Perera R, Holman RR, Neil HA. Meta-analysis of the effects
of n-3 polyunsaturated fatty acids on lipoproteins and other emerging lipid
cardiovascular risk markers in patients with type 2 diabetes. Diabetologia 2007;
50:1593–602.
[59] Lombardo YB, Chicco AG. Effects of dietary polyunsaturated n-3 fatty acids on
dyslipidemia and insulin resistance in rodents and humans. A review. J Nutr
Biochem 2006;17:1–13.
[60] Lipina C, Macrae K, Suhm T, Weigert C, Blachnio-Zabielska A, Baranowski M, et al.
Mitochondrial substrate availability and its role in lipid-induced insulin
resistance and proinflammatory signaling in skeletal muscle. Diabetes 2013;
62:3426–36.
[61] Mazibuko SE, Muller CJ, Joubert E, de Beer D, Johnson R, Opoku AR, et al.
Amelioration of palmitate-induced insulin resistance in C(2)C(1)(2) muscle cells
by rooibos (Aspalathus linearis). Phytomedicine 2013;20:813–9.
[62] Schmitz-Peiffer C. Signalling aspects of insulin resistance in skeletal muscle:
mechanisms induced by lipid oversupply. Cell Signal 2000;12:583–94.
[63] Vinolo MA, Rodrigues HG, Festuccia WT, Crisma AR, Alves VS, Martins AR, et al.
Tributyrin attenuates obesity-associated inflammation and insulin resistance in
high-fat-fed mice. Am J Physiol Endocrinol Metab 2012;303:E272–82.
[64] Tishinsky JM, Gulli RA, Mullen KL, Dyck DJ, Robinson LE. Fish oil prevents high-
saturated fat diet-induced impairments in adiponectin and insulin response in
rodent soleus muscle. Am J Physiol Regul Integr Comp Physiol 2012;302:R598–605.
[65] Cleasby ME, Lau Q, Polkinghorne E, Patel SA, Leslie SJ, Turner N, et al. The adaptor
protein APPL1 increases glycogen accumulation in rat skeletal muscle
through activation of the PI3-kinase signalling pathway. J Endocrinol 2011;210:81–92.
[66] Tremblay F, Lavigne C, Jacques H, Marette A. Dietary cod protein restores insulin-
induced activation of phosphatidylinositol 3-kinase/Akt and GLUT4 transloca-
tion to the T-tubules in skeletal muscle of high-fat-fed obese rats. Diabetes 2003;
52:29–37.
[67] Evans JL, Maddux BA, Goldfine ID. The molecular basis for oxidative stress-
induced insulin resistance. Antioxid Redox Signal 2005;7:1040–52.
[68] Paz K, Hemi R, LeRoith D, Karasik A, Elhanany E, Kanety H, et al. A molecular basis
for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and
IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor
and impairs their ability to undergo insulin-induced tyrosine phosphorylation. J
Biol Chem 1997;272:29911–8.
[69] Rossmeisl M, Jelenik T, Jilkova Z, Slamova K, Kus V, Hensler M, et al. Prevention
and reversal of obesity and glucose intolerance in mice by DHA derivatives.
Obesity (Silver Spring) 2009;17:1023–31.
[70] Samuel VT, Shulman GI. Mechanisms for insulin resistance: common threads and
missing links. Cell 2012;148:852–71.
[71] Crunkhorn S, Dearie F, Mantzoros C, Gami H, da Silva WS, Espinoza D, et al.
Peroxisome proliferator activator receptor gamma coactivator-1 expression is
reduced in obesity: potential pathogenic role of saturated fatty acids and p38
mitogen-activated protein kinase activation. J Biol Chem 2007;282:15439–50.
[72] Turner N, Bruce CR, Beale SM, Hoehn KL, So T, Rolph MS, et al. Excess lipid
availability increases mitochondrial fatty acid oxidative capacity in muscle:
evidence against a role for reduced fatty acid oxidation in lipid-induced insulin
resistance in rodents. Diabetes 2007;56:2085–92.
[73] Serhan CN, Chiang N. Endogenous pro-resolving and anti-inflammatory lipid
mediators: a new pharmacologic genus. Br J Pharmacol 2008;153(Suppl. 1):
S200–15.
[74] Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, et al. Free fatty acid-
induced insulin resistance is associated with activation of protein kinase C theta
and alterations in the insulin signaling cascade. Diabetes 1999;48:1270–4.
[75] Schmitz-Peiffer C, Craig DL, Biden TJ. Ceramide generation is sufficient to account
for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal
muscle cells pretreated with palmitate. J Biol Chem 1999;274:24202–10.
[76] Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory
and pro-resolution lipid mediators. Nat Rev Immunol 2008;8:349–61.
[77] Larter CZ, Yeh MM, Cheng J, Williams J, Brown S, dela Pena A, et al. Activation of
peroxisome proliferator-activated receptor alpha by dietary fish oil attenuates
steatosis, but does not prevent experimental steatohepatitis because of hepatic
lipoperoxide accumulation. J Gastroenterol Hepatol 2008;23:267–75.
[78] Jeng JY, Lee WH, Tsai YH, Chen CY, Chao SY, Hsieh RH. Functional modulation of
mitochondria by eicosapentaenoic acid provides protection against ceramide
toxicity to C6 glioma cells. J Agric Food Chem 2009;57:11455–62.
[79] Yokota T, Kinugawa S, Hirabayashi K, Matsushima S, Inoue N, Ohta Y, et al.
Oxidative stress in skeletal muscle impairs mitochondrial respiration and limits
exercise capacity in type 2 diabetic mice. Am J Physiol Heart Circ Physiol 2009;
297:H1069–77.
[80] Holmstrom MH, Iglesias-Gutierrez E, Zierath JR, Garcia-Roves PM. Tissue-specific
control of mitochondrial respiration in obesity-related insulin resistance and
diabetes. Am J Physiol Endocrinol Metab 2012;302:E731–9.
View publication stats
[81] Lanza IR, Blachnio-Zabielska A, Johnson ML, Schimke JM, Jakaitis DR, Lebrasseur NK,
et al. Influence of fish oil on skeletal muscle mitochondrial energetics and lipid
metabolites during high-fat diet. Am J Physiol Endocrinol Metab 2013;304:E1391–403.
[82] Pahlavani M, Razafimanjato F, Ramalingam L, Kalupahana NS, Moussa H, Scoggin S,
et al. Eicosapentaenoic acid regulates brown adipose tissue metabolism in high-fat-
fed mice and in clonal brown adipocytes. J Nutr Biochem 2017;39:101–9.
[83] Cavaliere G, Trinchese G, Bergamo P, De Filippo C, Mattace Raso G, Gifuni G, et al.
Polyunsaturated Fatty Acids Attenuate Diet Induced Obesity and Insulin
Resistance, Modulating Mitochondrial Respiratory Uncoupling in Rat Skeletal
Muscle. PLoS One 2016;11:e0149033.
[84] Philp LK, Heilbronn LK, Janovska A, Wittert GA. Dietary enrichment with fish oil
prevents high fat-induced metabolic dysfunction in skeletal muscle in mice. PLoS
One 2015;10:e0117494.
[85] Sanchez AM, Candau RB, Csibi A, Pagano AF, Raibon A, Bernardi H. The role of
AMP-activated protein kinase in the coordination of skeletal muscle turnover
and energy homeostasis. Am J Physiol Cell Physiol 2012;303:C475–85.
[86] Soriano FX, Liesa M, Bach D, Chan DC, Palacin M, Zorzano A. Evidence for a
mitochondrial regulatory pathway defined by peroxisome proliferator-activated
receptor-gamma coactivator-1 alpha, estrogen-related receptor-alpha, and
mitofusin 2. Diabetes 2006;55:1783–91.
[87] Lionetti L, Mollica MP, Donizzetti I, Gifuni G, Sica R, Pignalosa A, et al. High-lard
and high-fish-oil diets differ in their effects on function and dynamic behaviour
of rat hepatic mitochondria. PLoS One 2014;9:e92753.
[88] Munoz JP, Zorzano A. Analysis of mitochondrial morphology and function under
conditions of mitofusin 2 deficiency. Methods Mol Biol 2015;1265:307–20.
[89] Pich S, Bach D, Briones P, Liesa M, Camps M, Testar X, et al. The Charcot-Marie-
Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through
expression of OXPHOS system. Hum Mol Genet 2005;14:1405–15.
[90] Uldry M, Yang W, St-Pierre J, Lin J, Seale P, Spiegelman BM. Complementary
action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat
differentiation. Cell Metab 2006;3:333–41.
[91] Handschin C, Spiegelman BM. Peroxisome proliferator-activated receptor
gamma coactivator 1 coactivators, energy homeostasis, and metabolism. Endocr
Rev 2006;27:728–35.
[92] Mayr B, Montminy M. Transcriptional regulation by the phosphorylation-
dependent factor CREB. Nat Rev Mol Cell Biol 2001;2:599–609.
[93] Roberts-Wilson TK, Reddy RN, Bailey JL, Zheng B, Ordas R, Gooch JL, et al.
Calcineurin signaling and PGC-1alpha expression are suppressed during muscle
atrophy due to diabetes. Biochim Biophys Acta 1803;2010:960–7.
[94] Gilde AJ, Van Bilsen M. Peroxisome proliferator-activated receptors (PPARS):
regulators of gene expression in heart and skeletal muscle. Acta Physiol Scand
2003;178:425–34.
[95] Muoio DM, MacLean PS, Lang DB, Li S, Houmard JA, Way JM, et al. Fatty acid
homeostasis and induction of lipid regulatory genes in skeletal muscles of
peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice.
Evidence for compensatory regulation by PPAR delta. J Biol Chem 2002;277:
26089–97.
[96] Lecarpentier Y, Krokidis X, Martin P, Pineau T, Hebert JL, Quillard J, et al.
Increased entropy production in diaphragm muscle of PPAR alpha knockout
mice. J Theor Biol 2008;250:92–102.
[97] Zacarias AC, Barbosa MA, Guerra-Sa R, De Castro UGM, Bezerra FS, de Lima WG,
et al. Swimming training induces liver adaptations to oxidative stress and insulin
sensitivity in rats submitted to high-fat diet. Redox Rep 2017;22:515–23.
[98] Manna P, Achari AE, Jain SK. Vitamin D supplementation inhibits oxidative stress
and upregulate SIRT1/AMPK/GLUT4 cascade in high glucose-treated 3T3L1
adipocytes and in adipose tissue of high fat diet-fed diabetic mice. Arch Biochem
Biophys 2017;615:22–34.
[99] Bhaskaran S, Unnikrishnan A, Ranjit R, Qaisar R, Pharaoh G, Matyi S, et al. A fish
oil diet induces mitochondrial uncoupling and mitochondrial unfolded protein
response in epididymal white adipose tissue of mice. Free Radic Biol Med 2017;
108:704–14.
[100] Fan C, Zirpoli H, Qi K. n-3 fatty acids modulate adipose tissue inflammation and
oxidative stress. Curr Opin Clin Nutr Metab Care 2013;16:124–32.
[101] Bargut TC, Mandarim-de-Lacerda CA, Aguila MB. A high-fish-oil diet prevents
adiposity and modulates white adipose tissue inflammation pathways in mice. J
Nutr Biochem 2015;26:960–9.
[102] Bashir S, Sharma Y, Elahi A, Khan F. Amelioration of obesity-associated
inflammation and insulin resistance in c57bl/6 mice via macrophage polariza-
tion by fish oil supplementation. J Nutr Biochem 2016;33:82–90.
[103] Wang HH, Hung TM, Wei J, Chiang AN. Fish oil increases antioxidant enzyme
activities in macrophages and reduces atherosclerotic lesions in apoE-knockout
mice. Cardiovasc Res 2004;61:169–76.
[104] Dannenberger D, Nuernberg G, Renne U, Nuernberg K, Langhammer M, Huber K,
et al. High-fat diets rich in omega-3 or omega-6 polyunsaturated fatty acids have
distinct effects on lipid profiles and lipid peroxidation in mice selected for either
high body weight or leanness. Nutrition 2013;29:765–71.
[105] Capel F, Acquaviva C, Pitois E, Laillet B, Rigaudiere JP, Jouve C, et al. DHA at
nutritional doses restores insulin sensitivity in skeletal muscle by preventing
lipotoxicity and inflammation. J Nutr Biochem 2015;26:949–59.
[106] Ditch S, Paull TT. The ATM protein kinase and cellular redox signaling: beyond
the DNA damage response. Trends Biochem Sci 2012;37:15–22.
[107] Matsuoka S, Ballif BA, Smogorzewska A, McDonald III ER, Hurov KE, Luo J, et al.
ATM and ATR substrate analysis reveals extensive protein networks responsive
to DNA damage. Science 2007;316:1160–6.