Biochemical and Biophysical Research Communications 531 (2020) 112e117

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Iron deficiency attenuates protein synthesis stimulated by branched-

chain amino acids and insulin in myotubes
Kazuhiko Higashida*, Sachika Inoue, Naoya Nakai
Laboratory of Exercise Nutrition, Department of Nutrition and Food Sciences, The University of Shiga Prefecture, 2500 Hassaka-cho, Hikone, Shiga, 522-
8533, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Iron deficiency anemia indicates poor nutrition and is a public health problem. Iron deficiency is also
Received 7 July 2020 associated with muscle weakness. However, the intracellular mechanisms by which iron deficiency in-
Accepted 8 July 2020 duces muscle weakness are obscure. The purpose of the present study was to evaluate the effect of iron
Available online 8 August 2020
deficiency on protein synthesis in basal and branched-amino acids (BCAA)- and insulin-stimulated state
in muscle cells. Differentiated C2C12 myotubes were incubated with an iron chelator, deferoxamine
Keywords:
mesylate, and then stimulated with BCAA or insulin to activate protein synthesis. This iron deprivation
p70 S6 kinase
resulted in a significant reduction in the abundance of iron-containing proteins, such as the mitochon-
Puromycin
HIF-1
drial complex 1 subunit protein, compared to control cells, but not of protein that does not contain iron,
Glucose transporter such as citrate synthase. Proteins involved in glucose utilization, such as glucose transpoter-1, hexokinase
AMP-activated protein kinase and AMP-activated protein kinase (AMPK), were upregulated under iron deficiency. Additionally, rates of
Mitochondria BCAA- and insulin-stimulated protein synthesis, measured by puromycin incorporation, were lower in
iron-deficient myotubes than in control cells. We suggest that low iron availability attenuates BCAA- and
insulin-stimulated protein synthesis, possibly via activation of AMPK in myotubes. The present findings
advance the understanding of the importance of iron to skeletal muscle protein synthesis and, thus, may
contribute to the prevention of sarcopenia and frailty.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction (mTORC1) plays an important role in controlling protein synthesis

Iron deficiency anemia, which is an indicator of poor nutrition, is
a public health problem that disproportionately affects developed
and developing countries. According to data collected from 1993 to
2005, the estimated global prevalence of anemia is about 25% [1].
Although it is well known that iron deficiency compromises
physical performance due to impairment of the supply and utili-
zation of oxygen in tissues [2], recent findings revealed that iron
deficiency is also associated with muscle weakness [3]. However,
the intracellular mechanisms by which iron deficiency induces
muscle weakness are yet to be elucidated.
Skeletal muscle mass is regulated by a balance between the
rates of protein synthesis and degradation. For example, sarcope-
nia, age-related reduction of muscle mass and strength, is thought
to be a result of an imbalance between protein synthesis and
degradation [4]. The mechanistic target of rapamycin complex 1
* Corresponding author.
E-mail address: higashida.k@shc.usp.ac.jp (K. Higashida).
in muscle tissues, acting as a nutrient- and growth factor-
responsive mediator of skeletal muscle protein synthesis [5].
Amino acids and insulin activate the mTORC1 pathway in a process
mediated via downstream targets of mTORC1, including p70 S6
kinase (p70S6K), eukaryotic translation initiation factor 4E-binding
protein 1 (4E-BP1), and eukaryotic elongation factor 2 (eEF2) [6,7].
Additionally, AMP-activated protein kinase (AMPK) is a known
energy sensor and regulates the rate of protein synthesis rate
phosphorylating mTORC1.
Several studies have proposed that AMPK acts antagonistically
against the mTORC1 signaling pathway and protein synthesis
[8e10], and it is activated in skeletal muscle of iron-deficient ani-
mals, probably due to compromised mitochondrial respiration
[11,12]. Although AMPK activation is not involved in muscle
contraction- or stretch-stimulated increases in protein synthesis
[13,14], the role of AMPK in nutrient- or hormone-stimulated pro-
tein synthesis is not known. It, therefore, could be possible that iron
deficiency attenuates the rate of protein synthesis in response to
amino acids or anabolic hormones via activation of AMPK in muscle

https://doi.org/10.1016/j.bbrc.2020.07.041
0006-291X/© 2020 Elsevier Inc. All rights reserved.
 K. Higashida et al. / Biochemical and Biophysical Research Communications 531 (2020) 112e117
cells. Indeed, treatment with the iron chelator deferoxamine (DFO)
reduces phosphorylation of p70S6K in cardiomyocytes [15].
In this context, the present study was undertaken to determine
whether iron deprivation causes activation of AMPK and subse-
quent decreases mTORC1 signaling in myotubes, and, if so, to
determine the effect of iron deficiency on mTORC1 signaling and
protein synthesis in response to BCAA and insulin stimulation in
myotubes. The present findings contribute to the understanding of
the importance of iron to skeletal muscle protein synthesis and
provide the informative knowledge for the prevention of sarcope-
nia and frailty.
2. Materials and methods
2.1. Cell culture
All reagents for cell culture were obtained from Nacalai Tesque
(Kyoto, Japan), unless otherwise indicated. A mouse myoblast cell
line, C2C12 (ATCC, VA) cells were maintained in growth medium
(GM) containing Dulbecco’s modified Eagle’s medium (DMEM), 10%
fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml strep-
tomycin in a 5% CO2-humidified incubator at 37
C. Cells were
grown approximately 80% confluence, and the culture medium was
changed to differentiation medium (DM). DM comprised DMEM
supplemented with 2% horse serum, 100 units/ml penicillin and
100 mg/ml streptomycin. Cells were cultured in DM for 5 days to
form multinucleated myotubes; DM was changed every 48 h. After
formation, myotubes were treated with DFO (Sigma, MO) at the
indicated concentrations for 2 days, after which (day 7) the cells in
all groups were analyzed. In some experiments, 100 mM FeCl3 was
added into DM. DFO and FeCl3 were dissolved in distilled H2O. For
nutritional and hormonal stimulation, serum and BCAA were
excluded from the medium for 90 min to starve myotubes, then
BCAA (Leucine: Isoleucine: Valine ¼ 2:1:1 in mM) or insulin (2 mU
humalin, Eli Lilly Japan, Hyogo, Japan) was added for 45 min before
harvesting cells. To measure protein synthesis rate, 1 mM puromycin
was added 30 min before harvesting the cells.
2.2. Glucose and lactate concentration measurement
Differentiated C2C12 myotubes were washed twice with PBS
and incubated with phenol-red free DMEM containing 2% horse
serum, 100 units/ml penicillin and 100 mg/ml streptomycin at 37
C
for 48 h with or without DFO. After 48-h incubation, aliquots of the
medium were collected and glucose and lactate concentrations
were assayed using a Glucose II-test (Wako, Osaka, Japan) and N-
assay L LAC (Nittobo Medical, Tokyo, Japan), respectively.
2.3. Western blotting
Myotubes were washed with PBS twice and harvested in ice-
cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA, and a protease/
phosphatase inhibitor cocktail (Nacalai Tesque). Cell lysates were
centrifuged at 15,000  g for 5 min. The supernatants were
collected and protein concentrations were measured using a
bicinchoninic acid assay kit. Samples were prepared in 5 Laemmli
sample buffer. Proteins (10e20 mg) were separated by 7.5% or 15%
SDS-PAGE, transferred to a PVDF membrane, and incubated over-
night at 4
C with primary antibodies. Primary antibodies used in
this study are listed in Supplement Fig. 1. Bands were visualized by
enhanced chemiluminescence (Merck, Darmstadt, Germany) and
quantified by densitometry. Equal loading was confirmed by
staining the blot with Coomassie Brilliant Blue (CBB) [16].
113
2.4. RNA extraction and real time PCR
Myotubes were washed with PBS twice and harvested in TRIzol
(Invitrogen, CA), and total RNA was extracted and precipitated us-
ing chloroform and isopropanol. The DNase-treated total RNA
(1 mg) was reverse-transcribed into cDNA using random primers
with the High-Capacity cDNA Reverse Transcription Kit (Applied
Biosystems, CA). Real-time PCR was performed on a QuantStudio
real-time PCR system (Applied Biosystems, CA). Gene expression
was determined using commercially available Taqman primers and
probes for Atrogin-1 (Fbox32, Mm00499523_m1), MuRF-1
(Trim63, Mm01185221_m1), b-actin (ActB, Mm00637939_s1) and
the Taqman Universal PCR Master Mix (Applied Biosystems, CA).
The levels of gene expression were determined using the DDCT
method using b-actin as the control gene.
2.5. Statistical analysis
Data are expressed as means ± standard deviations of the mean.
Statistical analyses were carried out using BellCurve for Excel (So-
cial Survey Research Information, Tokyo, Japan). Differences be-
tween groups were assessed by one- or two-way analysis of
variance (ANOVA) followed by Tukey’s post-hoc test, or unpaired t-
tests. A value of p < 0.05 was considered statistically significant.
3. Results
3.1. Effects of iron deficiency on protein synthesis signaling
pathway and mitochondrial proteins
We first examined whether iron deficiency caused by the iron
chelator DFO affected protein synthesis pathways. Phosphorylation
of p70S6K, a well-established downstream target of mTORC1 [6,7],
was not affected by 24-h DFO treatment. However, 48-h DFO
treatment reduced the phosphorylation of p70S6K level compared
to control cells. (Fig. 1 B, D). The protein content of NDUFB8, an iron-
containing subunit of mitochondrial complex I, did not differ be-
tween the treatment groups in the 24-h DFO-treated cells, but did
decrease in the 48-h treated cells compared to control cells. The
non-iron-containing mitochondrial protein, such as citrate syn-
thase (CS), was unaffected by either 24- or 48- h DFO treatment.
3.2. Effects of iron deficiency on glucose metabolism and hypoxia
signaling
Because iron deficiency reduced the content of protein that is
involved in mitochondrial respiration, we investigated glucose
metabolism proteins and hypoxia signaling in DFO-treated cells.
The reduction of iron-containing proteins by 48-h treatment with
DFO caused concomitant increases in hypoxia inducible factor (HIF)
-1a, glucose transporter (GLUT) 1, and hexokinase protein content
(Fig. 2A and B). Furthermore, DFO treatment at 100 mM significantly
increased phosphorylation of Thr172 in the AMPKa subunit. In line
with these changes, the glucose concentration in the culture me-
dium was lower by DFO treatment compared to that in the control
group after 48-h incubation, but lactate concentration was higher
(Fig. 2C and D). The increased levels of HIF-1a and decreased levels
of phospho-p70S6K resulting from DFO treatment were restored to
control levels by the addition of FeCl3 to the culture medium
(Fig. 2E and F).
3.3. Effect of iron deficiency on BCAA-stimulated protein synthesis
in myotubes
Activation of AMPK is thought to inhibit protein synthesis [17].
114 K. Higashida et al. / Biochemical and Biophysical Research Communications 531 (2020) 112e117

Fig. 1. Effects of iron deficiency on protein synthesis and mitochondrial proteins. (A) Representative blots for phosphorylated (P) - and total (T)-p70S6K, NDUFB8 and citrate
synthase (CS) after 24-h treatment with DFO. (B) Quantitation of P-p70S6K, NDUFB8 and CS after 24-h treatment with DFO. (C) Representative blots for P- and T-p70S6K, NDUFB8
and CS after 48-h treatment with DFO. (D) Quantitation of P-p70S6K, NDUFB8 and CS after 48-h treatment with DFO. In all experiments, n ¼ 3e6 and one-way ANOVA with Tukey’s
post hoc test was used to determine statistical significance; *p < 0.05 vs. control (CON) group.

Fig. 2. Effect of iron deficiency on HIF-1a and glucose metabolism-related proteins. (A) Representative blots for and (B) quantitation of HIF-1a, GLUT1, HK, phosphorylated (P)-and
total (T)-AMPK. (C) Glucose and (D) lactate concentration in the medium after 48-h treatment with DFO. (E) Represented blots and (F) quantitation of P-p70S6K, GLUT1, NDUFB8 and
P-AMPK in the presence and absence of DFO or FeCl3. In all experiments, n ¼ 5e6 and unpaired t-test or one-way ANOVA with Tukey’s post hoc test was used to determine statistical
significance; *p < 0.05 vs. control (CON) group.
 K. Higashida et al. / Biochemical and Biophysical Research Communications 531 (2020) 112e117 115

Because AMPK is activated in DFO treated cells, iron deficiency
might attenuate both basal protein synthesis and nutrition-
stimulated protein synthesis pathways in myotubes. As shown in
Fig. 3, the phosphorylation levels of p70S6K were increased by
BCAA treatment in both control and DFO treated cells, though DFO
reduced the degree of increase in phospho-p70S6K protein content
in response to BCAA treatment. 4E-BP1 is also a known down-
stream target of mTORC1 [6], but DFO treatment did not affect the
BCAA-stimulated increase in phosphorylation 4E-BP1. In addition
to stimulating translation initiation, BCAA also stimulates trans-
lation elongation via eEF2 activation. Accordingly, the phosphory-
lation of eEF2 Thr56 was increased by DFO treatment, but the
degree of increase was lowered by BCAA stimulation. In addition,
DFO treatment decreased BCAA-stimulated protein synthesis, as
estimated by puromycin incorporation (Fig. 3B).
3.4. Effect of iron deficiency on insulin-stimulated protein synthesis
in myotubes
Insulin is a well-known activator of the mTORC1 pathway [18].
Therefore, we next examined the mTORC1 signaling pathway and
protein synthesis in response to insulin in control and iron-
deficient muscle cells. Unlike BCAA stimulation, insulin stimula-
tion increased phosphorylation of p70S6K to the same degree in
both the control and DFO-treated cells (Fig. 4A and B). Similar to
p70S6K, the increased phosphorylation of 4E-BP1 stimulated by
insulin was not affected by DFO treatment. However, the insulin-
induced decrease in phosphorylation of eEF2 and the rate of pro-
tein synthesis were significantly lower in DFO-treated cells than in
control cells (Fig. 4D and E).
3.5. Effect of iron deficiency on ubiquitin lipase mRNA expression
Furthermore, we investigated the expression of ubiquitin ligase
mRNA in response to DFO treatment. The atrogin-1 mRNA
expression was elevated by DFO treatment, and was normalized by
the addition of FeCl3 in culture medium (Control, 1.00 ± 0.07
arbitrary unit; DFO, 1.58 ± 0.09; DFO þ FeCl3, 1.14 ± 0.12; Control vs
DFO, p < 0.05). In contrast, the mRNA expression of MuRF-1 was
slightly decreased by DFO treatment and was not affected by the
addition of FeCl3 (Control, 1.00 ± 0.07 arbitrary unit; DFO,
0.75 ± 0.05; DFO þ FeCl3, 0.84 ± 0.13; Control vs DFO, p < 0.05).
4. Discussion
In this study, we examined the effect of iron deficiency on
mTORC1 and protein synthesis in C2C12 myotubes. We showed
that iron deficiency reduced the abundance of iron-containing
protein and activated glucose utilization and AMPK signaling.
Moreover, both mTORC1 signaling and protein synthesis were
decreased under basal as well as nutrition- and hormone-
stimulated conditions in iron-deficient cells. These results suggest
that iron plays an important regulatory role in protein synthesis in
muscle cells.
Iron homeostasis is important for cellular respiration and ATP
production since iron is an essential component for the electron
transport chain. Therefore, oxidative metabolism, such as fatty
acids and pyruvate oxidation, is compromised during iron defi-
ciency [11,12,19]. On the other hand, iron deficiency upregulates
transport and utilization of glucose in skeletal muscle to compen-
sate for the impairment of oxidative metabolism [11,12]. There is
abundant literature describing the relationship between iron and

Fig. 3. Effect of iron deficiency on BCAA-stimulated protein synthesis and puromycin incorporation. (A) Representative blots for and quantitation of (B) phosphorylated (P)-p70S6K,
(C) P-4E-BP1, (D) P-eEF2 and (E) protein synthesis. # Significantly different from PBS-treated cells in the same treatment group (p < 0.05), * Significantly different from control cells
in the same treatment group (p < 0.05). In all experiments, n ¼ 3e6 and two-way ANOVA with Tukey’s post hoc test was used to determine statistical significance.
116 K. Higashida et al. / Biochemical and Biophysical Research Communications 531 (2020) 112e117
Fig. 4. Effect of iron deficiency on insulin-stimulated protein synthesis and puromycin incorporation. (A) Representative blots for and quantitation of (B) phosphorylated (P)-
p70S6K, (C) P-4-BP1, (D) P-eEF2 and (E) protein synthesis. # Significantly different from PBS-treated cells in the same treatment group (p < 0.05), * Significantly different from
control cells in the same treatment group (p < 0.05). In all experiments, n ¼ 3e6 and two-way ANOVA with Tukey’s post hoc test was used to determine statistical significance.
glucose-fat metabolism, whereas few studies have investigated the
relationship between iron availability and protein metabolism. In
this study, we found that iron deficiency increases the phosphor-
ylation of AMPK and concomitantly stimulated transcription of
genes encoding proteins involved in glucose utilization. In addition,
DFO treatment reduced phosphorylation of p70S6K in muscle cells,
suggesting that the basal rate of protein synthesis was decreased.
Similarly, it has been reported that the rate of protein synthesis is
decreased in iron-deficient conditions in many organs, including
the liver, spleen, and thymus [20,21]. Taken together, these results
suggest that iron is a key microelement for regulation of protein
synthesis in various organs, including skeletal muscle.
To clarify the cellular responses to anabolic stimulation in iron-
deficient states, we investigated the effect of BCAA and insulin
stimulation on protein synthesis in myotubes. Protein synthesis,
measured by puromycin incorporation, decreased in response to
BCAA or insulin (Figs. 3E and 4E). These results suggested that iron
deficiency reduces not only basal protein synthesis, but also BCAA-
and insulin-stimulated protein synthesis in muscle cells. In protein
synthesis signaling pathways, 4E-BP1 phosphorylation levels
responded similarly to BCAA and insulin, whereas p70S6K and eEF2
showed different responses to these two stimuli (Figs. 3 and 4). For
example, the increase in p70S6K phosphorylation in response to
BCAA was attenuated by DFO treatment (Fig. 3B), but its response to
insulin was comparable to the control treatment group (Fig. 4B). It
has been proposed that insulin activates both mTORC1 and
mTORC2 but that amino acids only activate mTORC1 [22,23]. The
precise mechanisms underlying the differential response to BCAA
and insulin stimulation in DFO treated cells are not clear from the
current study, though the upstream signaling pathway may be
involved.
Our finding that iron deficiency reduces protein synthesis in
muscle cells seem to contradict recent work demonstrating iron-
induced muscle atrophy via activation of ubiquitin ligase expres-
sion in iron-overloaded rat skeletal muscle [24]. In that paper, it
was reported that muscle atrophy induced by iron overload was
mediated by oxidative stress, and treatment with Tempol, a radical
scavenger reagent, reversed the iron overload-induced gene
expression of ubiquitin ligases. Contrarily, we found that iron
deficiency-induced activation of AMPK and reduction of iron con-
taining proteins in mitochondria (Fig. 1), both of which could be
reversed by addition of FeCl3 to the culture medium (Fig. 2). In
addition, DFO treatment induced the expression of Atrogin-1
mRNA, which could also be reversed by FeCl3. These results indi-
cate that either deficiency or excess iron compromises cellular
homeostasis and causes concomitant decreases in muscle protein
synthesis. In the future, more extensive studies to investigate the
effect of iron deficiency on muscle protein synthesis in response to
nutritional and anabolic hormone in vivo should be performed.
Our results indicate the importance of micronutrients for
maintenance of skeletal muscle mass. Although the regulatory roles
of this micronutrient in muscle protein synthesis warrant further
investigation, the present findings advance the understanding of
the importance of iron to skeletal muscle protein synthesis and,
thus, may contribute to the prevention of sarcopenia and frailty.
5. Conclusion
Iron deprivation reduced the abundance of iron containing
protein and activated AMPK in skeletal muscle cells. AMPK acti-
vation caused by low iron availability may contribute, at least in
part, to attenuation of amino acids- or insulin-stimulated protein
 K. Higashida et al. / Biochemical and Biophysical Research Communications 531 (2020) 112e117
synthesis in myotubes.
Author contribution
KH, SI and NN conceived and designed the study. KH and SI
performed the experiments. KH, SI and NN analyzed and discussed
the data and wrote the manuscript. KH, SI and NN discussed the
results, commented on the manuscript, and approved the manu-
script submission.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
We thank for Riki Ogasawara, Satoru Ato and Eri Imai for an
intellectual discussion for this study. This work was supported by
Grant-in-Aid for Scientific Research (C) (20K11364 to HK) and
Grant-in-Aid for Scientific Research (C) (19K11553 to NN) from the
Japan Society for the Promotion of Science (JSPS).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2020.07.041.
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