- Academic Editor
†These authors contributed equally.
Background: Activation of the unfolded protein response (UPR) is
closely related to the pathogenesis of many metabolic disorders. Accumulating
evidence also shows that UPR and metabolic signaling pathways are interdependent.
The AMP-activated protein kinase (AMPK) signal pathway controls the energy
balance of eukaryotes. The aim of this study was therefore to investigate the
possible interaction between AMPK signaling and UPR in muscle cells exposed to
saturated fatty acids, as well as the potential mechanism. Methods: The
saturated fatty acid palmitate was used to induce UPR in C2C12 myotubes. Compound
C or knockdown of AMPK
Adult skeletal muscle shows considerable plasticity that allows it to respond rapidly under a variety of physiologic and pathologic conditions [1]. This is facilitated by the sarcoplasmic reticulum, a specialized form of the endoplasmic reticulum (ER) [2]. Environmental or cell-intrinsic stimuli such as nutrient or oxygen deprivation, exposure to toxic substances, and oxidative stress can disrupt cellular homeostasis and induce ER stress, thereby activating the unfolded protein response (UPR) [3, 4, 5]. The canonical UPR in mammals is initiated by activation of three major ER transmembrane sensors: PKR-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) [6, 7, 8]. These trigger the expression of downstream transcription factors including ATF4, ATF6c, C/EBP homologous protein (CHOP), and spliced X-box binding protein 1 (XBP1s). The main outcome of UPR signaling, particularly in the early phase, is the restoration of ER homeostasis through inhibition of protein synthesis or upregulation of ER chaperone proteins [9, 10]. However, prolonged UPR due to continuous stress can lead to the induction of apoptotic cell death [11, 12]. Thus, the UPR is a cellular mechanism that controls cell fate in response to stress.
ER stress and the UPR can also be activated in skeletal muscles that are exposed to metabolic stress, as occurs in diabetic patients [13], or by the consumption of a high-fat diet [14, 15]. The high concentration of free fatty acids and especially of saturated fatty acids (SFAs) in plasma under these conditions is one of the main triggers for UPR in skeletal muscle [16, 17, 18]. The UPR is closely associated with SFA-induced inflammation, insulin resistance, and apoptosis in skeletal muscle [18, 19, 20], indicating there is crosstalk between the UPR and signaling pathways that regulate metabolism [21, 22]. The AMP-activated protein kinase (AMPK) pathway is conserved across eukaryotes and integrates signals from multiple sources to control cellular energy balance [23]. Alterations to AMPK also contribute to the metabolic adaptations and progress of insulin resistance in muscles exposed to SFAs [24]. Given their critical influence on muscle health and metabolism, elucidating the interplay and synergisms (or antagomisms) between AMPK and UPR is prerequisite for the development of novel therapeutics or strategies to ameliorate muscle metabolic disorders. Although there is increasing evidence for interactions between AMPK signaling and the UPR [25, 26, 27, 28, 29, 30, 31, 32], the mechanistic basis for crosstalk between these two pathways has yet to be elucidated in different models of ER stress.
Therefore, in the present study we investigated whether there is crosstalk between AMPK signaling and the UPR following induction by palmitate in skeletal muscle cells, as well as the possible underlying mechanism. We found that AMPK was activated in myotubes in response to palmitate treatment. Moreover, we showed that AMPK signaling was associated with early activation of the UPR via a positive feedback mechanism. Additionally, pharmacologic activation of AMPK led to the induction of UPR. These findings provide novel insights into the interactions between metabolic signals and homeostatic mechanisms in skeletal muscle cells that may be perturbed in metabolic disorders.
C2C12 myoblast cells (cat. no. 1101MOU-PUMC000099) were purchased from National Infrastructure of Cell Line Resource (NICR) (Beijing, China). This cell line has been authenticated by NICR with flow cytometry. Venor
Mouse C2C12 myoblast cells were cultured in growth medium containing 89%
high-glucose Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY,
USA), 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 1%
Penicillin/Streptomycin at 37 °C in a 5% CO
Palmitate (Sigma-Aldrich; cat. no. P0500) was dissolved in ethanol and diluted
to 500 µmol/L in DMEM containing 2% AlbumiNZ bovine serum albumin (MP
Biomedicals, Solon, OH, USA; cat. no. 199896), 2% FBS (Atlanta Biologicals,
Flowery Branch, GA, USA), 2 mmol/L L-carnitine (Sigma-Aldrich; cat. no. C0283),
and 1% antibiotics [33]. Control C2C12 myotubes were incubated in the same
medium except that palmitate was substituted with an equal volume of ethanol. For
some treatment conditions, 10 µmol/L compound C (prepared in
dimethylsulfoxide [DMSO]; Sigma-Aldrich) was coincubated with palmitate for 12 h.
DMSO was also used as a vehicle control for the treatments. To inhibit ER stress,
C2C12 myotubes were pretreated for 1 h with 1 mM taurourdodeoxycholic acid
(TUDCA) (Millipore, Billerica, MA, USA; cat. no. 580549) before the addition of
palmitate for another 12 h. To activate AMPK signaling, the AMPK agonists
5-amino-1-
Adenoviral constructs containing short hairpin RNA (shRNA) against
AMPK
Total RNA was extracted from C2C12 myotubes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA; cat. no. 15596-026) and then 500 ng of total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Bio, Otsu, Japan; cat. no. RR037A) according to manufacturer’s instructions. RT-PCR was performed using a StepOnePlus RT-PCR system (Invitrogen) with fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA; cat. no. 4385612). Each RT-PCR mixture contained 21 µL sterile water, 25 µL SYBR Green, 2 µL cDNA (500 ng/µL), and 1 µL each of forward and primers (10 pmol/µL). The reaction was performed by an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at the melting temperature of the specific primer set for 10 s, elongation at 72 °C for 15 s, and concluded with a melting curve step. Target gene expression levels were normalized to those of the 18S rRNA gene. The sequences of the primers used are listed in Supplementary Table 1.
C2C12 myotubes were lysed in RIPA buffer containing 20 mM Tris
Data are presented as mean
We investigated the AMPK phosphorylation status and the expression of UPR
markers in C2C12 myotubes treated with palmitate (a major component of dietary
saturated fats) for different times. While the total AMPK
AMPK signaling is activated within 12 h of palmitate treatment.
(A) C2C12 myotubes were incubated with 0.5 mM palmitate for 0, 3, 6, 12, and 24
h. The proteins levels of AMPK
To further investigate the interaction between the AMPK pathway and UPR, C2C12 myotubes were treated with palmitate for 12 h with or without compound C, a widely used specific inhibitor of AMPK. As expected, palmitate caused activation of AMPK, as well as increased expression of the ATF4 and CHOP proteins (Fig. 2A). Compound C completely abolished the AMPK activation induced by palmitate (Fig. 2A) and reduced the palmitate-induced upregulation of ATF4 and CHOP protein levels (Fig. 2A). In agreement with the above findings, palmitate induced the upregulation of gene expression for multiple components of UPR, including ATF4, CHOP, GADD34, chaperone BIP, XBP1u and XBP1s (Fig. 2B). This upregulation was also attenuated by treatment with compound C (Fig. 2B).
AMPK inhibition with compound C attenuates palmitate-induced UPR
in C2C12 myotubes. (A) Western blot analysis of AMPK
To more specifically inhibit the AMPK signaling pathway, C2C12 myotubes were
infected with adenovirus that expressed AMPK
AMPK
We further investigated whether inhibition of UPR alters AMPK activation in
C2C12 myotubes. Myotubes were pretreated with the UPR inhibitor TUDCA for 1 h
before the addition of palmitate for 12 h. TUDCA significantly attenuated the
palmitate-induced upregulation of ATF4 and CHOP (Fig. 4), thereby demonstrating
pharmacologic inhibition of the UPR. Interestingly, TUDCA also abolished
palmitate-induced AMPK
TUDCA attenuates palmitate-induced AMPK activation in C2C12
myotubes. C2C12 myotubes were pretreated for 1 h with 1 mM TUDCA or left
untreated before the addition of palmitate for another 12 h. Protein levels for
AMPK
Given the finding that AMPK activation contributes to palmitate-induced UPR, we
speculated that pharmacologic activation of AMPK would be sufficient to induce
the UPR in C2C12 myotubes. To test this hypothesis, C2C12 myotubes were treated
with the AMPK agonist AICAR at concentrations ranging from 0.125–2 mM for 12 h
to activate AMPK signaling. AMPK phosphorylation increased with increasing AICAR
concentration, and was highest at 1 mM AICAR (Fig. 5A). Moreover, ATF4 and CHOP
protein levels were upregulated by AICAR in a dose-dependent manner at
concentrations
Pharmacologic activation of AMPK induces UPR. (A) C2C12
myotubes were treated with different concentrations of AICAR (125–2000 µM)
for 12 h. The proteins levels for AMPK
The results of this study provide novel evidence of the interaction between the
AMPK pathway and UPR in muscle cells exposed to palmitate, a major component of
dietary saturated fats [35]. Specifically, we first observed the unexpected
activation of AMPK signaling within 12 h of palmitate treatment, which was
accompanied by acute induction of the UPR. In support of these findings, a
previous report showed that peroxisome proliferator-activated receptor gamma
coactivator (PGC)-1
The association between the UPR and AMPK has been investigated previously [27, 37, 38, 39, 40]. Several studies on palmitate-induced ER stress have demonstrated an
inhibitory effect of AMPK signaling on the UPR in different tissues and cells
[27, 37, 40]. For example, pharmacologic activation of AMPK with AICAR was shown
to suppress palmitate-induced ER stress in rat vascular endothelial cells [29].
In C2C12 myotubes, both GW501516 (a peroxisome proliferator-activated receptor
[PPAR]
Interestingly, we also found that pharmacologic activation of AMPK with AICAR or
ex229 was sufficient to induce upregulation of UPR components in myotubes. In
line with this finding, PGC-1
In summary, we have provided evidence of bidirectional crosstalk between AMPK signaling and early activation of the UPR in muscle cells exposed to SFAs (Fig. 6). We also showed that pharmacologic activation of AMPK was sufficient to induce mild UPR in skeletal muscle cells. These findings demonstrate an essential role for the AMPK pathway in restoring ER homeostasis via activation of the UPR in response to metabolic stress. Furthermore, they may guide the development of new strategies for the treatment of diseases such as obesity and diabetes through improvements in skeletal muscle metabolism.
Bidirectional crosstalk between AMPK signaling and unfolded protein response (UPR) in muscle cells exposed to saturated fatty acids (SFAs). During the early stages after palmitate treatment of muscle cells, AMPK signaling and UPR are activated and crosstalk through a positive feedback mechanism, thereby facilitating restoration of ER homeostasis. Physiological (e.g., exercise) or pharmacological (e.g., phenformin) interventions can accelerate the restoration of ER homeostasis and muscle health by promoting bidirectional crosstalk between AMPK signaling and UPR during the early stages of regular high-fat diet. ER, endoplasmic reticulum.
AICAR, 5-amino-1-
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
PZ and XC designed the research study. JG, LW, WT, ZL, XP, and WL performed the research. PZ, JG, WL, and SL analyzed the data. PZ, JG, LW, and XC wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
This work was supported by grants from the National Natural Science Foundation of China (81871522, 32171173) and the State Key Laboratory Grant of Space Medicine Fundamentals and Application (SMFA18B01, SMFA20A02).
The authors declare no conflict of interest.
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