Am J Physiol Endocrinol Metab 308: E990–E1000, 2015.
First published April 7, 2015; doi:10.1152/ajpendo.00602.2014.
Long-chain acylcarnitines activate cell stress and myokine release in C2C12
myotubes: calcium-dependent and -independent effects
Colin S. McCoin,1* Trina A. Knotts,2,3* Kikumi D. Ono-Moore,2 Pieter J. Oort,2 and Sean H. Adams1,3,4
1
Molecular, Cellular and Integrative Physiology Graduate Group, University of California, Davis, California; 2Obesity &
Metabolism Research Unit, United States Department of Agriculture-Agricultural Research Service Western Human Nutrition
Research Center, Davis, CA; 3Department of Nutrition, University of California, Davis, Davis, California; and 4Arkansas
Children’s Nutrition Center and Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Submitted 22 December 2014; accepted in final form 6 April 2015
cell death; interleukin-6; skeletal muscle
long been used as surrogate readouts reflecting tissue acyl-CoA pools (6). Thus, these indexes
are used in newborn screening as diagnostic biomarkers of
inherited disorders of metabolism involving enzymatic lesions
in lipid and amino acid metabolism, in which there are increases or decreases in specific acyl-CoA tissue pools (7, 37).
For example, long-chain fatty acid oxidation disorders (FAOD)
are characterized by defects in mitochondrial oxidative en-
PLASMA ACYLCARNITINES HAVE
* C. S. McCoin and T. A. Knotts contributed equally to this work.
Address for reprint requests and other correspondence: S. H. Adams,
Arkansas Children’s Nutrition Center, 15 Children’s Way, Little Rock, AR
72202 (e-mail: [email protected]).
E990
zymes, i.e., long-chain 3-hydroxyacyl-CoA dehydrogenase and
carnitine palmitoyltransferase 2 (CPT2) deficiencies, and these
lead to increased tissue, blood, and urine long-chain fatty
acylcarnitines. Under certain FAOD conditions, plasma longchain acylcarnitines can increase severalfold: L-C16 carnitine
(C16 carnitine), for instance, has been reported to increase
greater than five-fold, reaching levels between ⬃5 and 44 ␮M
in CPT2-deficient newborns (19, 23). Modest increases in
tissue or blood long-chain acylcarnitines have also been noted
following cardiac ischemia (17, 36, 38) and in type 2 diabetes/
insulin resistance (1, 18, 20, 24, 26).
In addition to their use as biomarkers, there is recent evidence supporting the hypothesis that acylcarnitines have bioactivities. Our previous work has shown that long-chain fatty
acylcarnitines activate proinflammatory signaling pathways in
RAW 264.7 murine macrophages (1, 29) and in HCT-116 cells
(29), and blunt the insulin signaling pathway in both murine
C2C12 and human primary skeletal muscle cells (2). These
activities were found to occur at initial extracellular concentrations of C14 carnitine and C16 carnitine as low as 5 ␮M.
Thus, whether or not these metabolites contribute to, or exacerbate, disease phenotypes or normal physiological processes
remains an open question. The mechanisms by which longchain fatty acylcarnitines impinge upon inflammatory systems
and insulin-associated cell signaling pathways in muscle have
yet to be fully elucidated. Evidence to date suggests that, at
least in macrophages, proinflammatory actions of acylcarnitines can occur in a MyD88-dependent manner (29), and
MyD88 is considered an important adaptor protein that can
serve as part of the membrane proximal signaling complexes,
i.e., for many pattern recognition receptors (14). However, we
found no evidence that specific Toll-like receptors or other
pattern recognition receptors are driving the proinflammatory
effects of C14- and C16-acylcarnitines (29).
In the course of studies examining the effect of long-chain
fatty acylcarnitines on insulin resistance and inflammation, we
noted changes in cell viability and function under conditions of
high acylcarnitines (2, 29). In our previous work, it was
observed that, at a higher concentration of C16 carnitine,
release of interleukin-6 (IL-6) in myocytes (2) and adenylate
kinase (AK, a cell permeability/death marker) in macrophages
(29) was increased. Furthermore, in cardiac ischemia, heart
muscle acylcarnitines accumulate (17, 36, 38) and long-chain
acylcarnitines have been associated with increased cardiac
cellular reactive oxygen species (ROS), apoptosis, and endoplasmic reticulum (ER) stress (32) and increases in intracellular calcium (39, 42). Considering that muscle is a site of robust
acylcarnitine generation, we have begun to consider if long-
0193-1849/15 Copyright © 2015 the American Physiological Society
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McCoin CS, Knotts TA, Ono-Moore KD, Oort PJ, Adams SH.
Long-chain acylcarnitines activate cell stress and myokine release in
C2C12 myotubes: calcium-dependent and -independent effects. Am J
Physiol Endocrinol Metab 308: E990 –E1000, 2015. First published
April 7, 2015; doi:10.1152/ajpendo.00602.2014.—Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites
that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular
inflammatory, stress, and death pathways in a skeletal muscle model.
Differentiated C2C12 myotubes treated with L-C14, C16, C18, and
C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death,
which was not observed for shorter chain lengths. L-C16 carnitine,
used as a representative long-chain acylcarnitine at initial extracellular
concentrations ⱖ25 ␮M, increased IL-6 production 4.1-, 14.9-, and
31.4-fold over vehicle at 25, 50, and 100 ␮M. Additionally, L-C16
carnitine activated c-Jun NH2-terminal kinase, extracellular signalregulated kinase, and p38 mitogen-activated protein kinase between
2.5- and 11-fold and induced cell injury and death within 6 h with
modest activation of the apoptotic caspase-3 protein. L-C16 carnitine rapidly increased intracellular calcium, most clearly by 10
␮M, implicating calcium as a potential mechanism for some
activities of long-chain acylcarnitines. The intracellular calcium
chelator BAPTA-AM blunted L-C16 carnitine-mediated IL-6 production by ⬎65%. However, BAPTA-AM did not attenuate cell
permeability and death responses, indicating that these outcomes
are calcium independent. The 16-carbon zwitterionic compound
amidosulfobetaine-16 qualitatively mimicked the L-C16 carnitineassociated cell stress outcomes, suggesting that the effects of high
experimental concentrations of long-chain acylcarnitines are
through membrane disruption. Herein, a model is proposed in
which acylcarnitine cell membrane interactions take place along a
spectrum of cellular concentrations encountered in physiologicalto-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology.
ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
chain acylcarnitines impact myocyte cell function. In the current cell culture experiments, it was hypothesized that the
naturally occurring zwitterionic metabolites, long-chain acylcarnitines, can elicit cell stress and cell death responses in a
model of skeletal muscle myotubes and that calcium-associated
pathways play a role.
MATERIALS AND METHODS
Cell Signaling Lysis Buffer ⫹ Pierce HALT phosphatase inhibitors
(Rockford, IL). Sample supernatants were sonicated and subject to
centrifugation at 12,000 g for 10 min at 4°C.
Immunoblotting. Lysates were resolved via 4 –12% Bis-Tris SDSPAGE (Life Technologies) and transferred to polyvinyl difluoride
membranes (Bio-Rad, Hercules, CA) using a Bio-Rad Trans-Blot
Turbo. Membranes were blocked in a 1X PBS and 0.1% (vol/vol)
Tween 20 (Fisher Scientific) (PBST) solution containing 2% wt/vol
dry milk. Membranes were probed for 1 h at room temperature or
overnight at 4°C with primary antibody in 1X PBST followed by
incubation with horseradish peroxidase-conjugated secondary antibody (Southern Biotech, Birmingham, AL) at a 1:10,000 dilution in
1X PBST ⫹ 2% milk for 1 h at room temperature. Bands were
visualized using Bio-Rad Clarity Western ECL reagent and imaged on
a Bio-Rad ChemiDoc XRS system.
ER stress analysis. Myotubes were grown in 96-well tissue culture
plates and differentiated for 4 days. Cells were serum starved (0.25%
FBS/DMEM) for 4 h and treated with C16 carnitine at 0, 5, 10, 25, 30,
40, 50, 75, and 100 ␮M and positive controls (staurosporine, thapsigargin, and tunicamycin) for 6 h in duplicate or triplicate. At 6 h,
media were harvested, cells were rinsed two times with cold HBSS
and lysed in 1X lysis buffer, and replicates were pooled. Media were
analyzed for AK, and concentrations and lysate protein concentrations
were measured and subjected to SDS-PAGE and Western blotting to
determine levels of ER stress markers [cleaved caspase 3, inositolrequiring protein-1␣ (IRE-1␣), binding immunoglobulin protein
(BiP), and CCAAT/enhancer-binding protein homologous protein
(CHOP)].
Live/dead assay. C2C12 myoblasts were seeded into 96-well clearbottom, black wall plates (BD Falcon) and differentiated as described
above. The cells were serum starved in 0.25% FBS phenol red-free
DMEM for 3– 4 h before treatment for 6 h with various compounds in
the same medium. Supernatants were removed, and 25 ␮l of HBSS
with Ca2⫹/Mg2⫹ were added to each well. Twenty-five microliters of
2X live/dead dye (catalog no. R37601; Life Technologies) was added
to each well 15 min before imaging. Imaging was done on a Nikon
Eclipse Ti microscope with an automated platform and Zyla Andon
camera, and the data acquired were analyzed using Nikon Elements
HCT software. Three to four images were captured per well. The total
GFP (green channel fluorescence) and RPE (red channel fluorescence)
intensities were calculated by the Nikon Element HCT software. The
ratio of GFP to RPE intensity was determined. A lower ratio indicates
increased cell death.
XTT viability assay. Myotubes were grown, starved, and treated as
done above for live/dead assay. After 6 h treatment, media were
removed, and fresh media with treatments were added, with 0.20
mg/ml XTT and 0.001 mM PMS activation reagent. Cells were
returned to 37°C for 4 h. Absorbance measurements were read on a
plate reader at 475 nm with a background correction of 690 nm.
Caspase assay. C2C12 myotubes were pretreated with caspase
inhibitors, caspase-3 inhibitor II (Z-DEVD-FMK) or caspase inhibitor
I (Pan inhibitor, Z-VAD-FMK) (EMD Millipore), for 1 h after a
serum starvation in 0.25% FBS for 2–3 h. C2C12 myotubes were
cotreated with the respective caspase inhibitor and various compounds
as indicated in the text for 6 or 20 h. Supernatants were collected and
stored at ⫺20°C. C2C12 were washed one time with cold HBSS and
lysed in 25 ␮l of reporter lysis buffer per well (catalog no. E3971;
Promega). The samples were stored at ⫺20°C. For assay, lysate
samples were thawed quickly at 50°C and mixed on an orbital shaker
for 15 min at room temperature, after which 20 ␮l were transferred to
white round-bottom 96-well plates, and 20 ␮l of the Caspase 3/7 Glo
working reagent were added to each sample (catalog no. G8091;
Promega). The plate was incubated (covered) at room temperature for
30 min on an orbital shaker. Luminescence was measured on a
Synergy 2 plate reader.
Intracellular calcium readouts. C2C12 myotubes in a black 96-well
tissue culture plate were serum starved for 2–3 h in 0.25% FBS plus
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Reagents. Lipopolysaccharide (LPS) was purchased from List Biologicals (Campbell, CA). Lot-tested (lot no. K0109) premium-select
fetal bovine serum (FBS) and horse serum were purchased from
Atlanta Biologicals (Lawrenceville, GA) and Hyclone (Logan, UT),
respectively. Dulbecco’s Modified Eagle’s Medium (DMEM), phenol
red-free Hank’s Balanced Salt Solution (HBSS), penicillin/streptomycin, sodium pyruvate, GlutaMax, and calcium green-1 AM, were all
purchased from Life Technologies (Grand Island, NY). Ionomycin,
BAPTA-AM, caspase-3 inhibitor II (Z-DEVD-FMK), and caspase
inhibitor I (pan inhibitor, Z-VAD-FMK) were purchased from EMD
Millipore (Billerica, MA), and cyclosporine A was purchased from
Cell Signaling Technologies (Danvers, MA). Acyl-L-carnitines of
varying chain lengths and L-carnitine were purchased from Advent
Bio (Downers Grove, IL), amidosulfobetaine-16 (ASB-16) {3-[N,Ndimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate} (catalog no. DG062, lot no. 092605) was purchased from G-Biosciences
(St. Louis, MO), and Toxilight and IL-6 ELISA Assays were purchased from Lonza (Basel, Switzerland) and R&D Systems (Minneapolis, MN), respectively. 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt (XTT) sodium
salt was purchased from Biotium (Hayward, CA) and phenazine
methosulfate 98% (PMS) from Acros Organics (Geel, Belgium).
Antibodies against phospho-p44/42 mitogen-activated protein kinase
(MAPK) [extracellular signal-regulated kinase (ERK1/2)] (Thr202/
Tyr204) (catalog no. 4370), total p44/42 MAPK (ERK) (catalog no.
9102), p-c-Jun NH2-terminal kinase (JNK) (catalog no. 4668), JNK
(catalog no. 9252), p-p38 (catalog no. 4511), p38 (catalog no. 8690),
lamin A/C (catalog no. 2032), and ER stress antibody sampler kit
(catalog no. 9956) were all purchased from Cell Signaling Technology
(Danvers, MA). The ␤-tubulin (Clone Tub 2.1, catalog no. ab11308)
was purchased from Sigma-Aldrich (St. Louis, MO).
Cells and cell culture. C2C12 (catalog no. CRL-1772 murine
myoblast cell line) were purchased from ATCC (Manassas, VA).
Cells were cultured in DMEM containing 10% FBS (premium select
FBS, catalog no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, 100 ␮g/ml streptomycin, 2 mM
GlutaMAX-I (Life Technologies, Grand Island, NY), and 100 ␮M
L-carnitine (Advent Bio). Because penicillin and streptomycin were
included in all treatment conditions, any potential effects on mitochondrial function or metabolism would not influence treatmentassociated differences. C2C12 myoblasts were maintained at 37°C in a
5% CO2 atmosphere until ⬎90% confluent at which time FBS was
replaced with 10% horse serum (HyClone, Logan, UT) for 4 –5 days
to induce myotube differentiation.
Long-term acylcarnitine treatment. Myotubes (4- to 5-day differentiated) were grown in 0.25% FBS starvation media for 2– 4 h and
then treated at various doses with different-chain-length acylcarnitines
for 18 h, as indicated. Myotubes were pretreated for 1 h during
starvation and overnight with sample treatment with BAPTA-AM, a
calcium chelator, at the concentrations indicated. Conditioned media
were harvested and frozen at ⫺20°C and analyzed for IL-6. Media were
further analyzed by Toxilight assay for presence of AK, a surrogate
marker of cytotoxicity.
Short-term acylcarnitine treatment. Fully differentiated myotubes
were serum starved for 2–3 h in 0.25% FBS media and then treated
with corresponding L-acylcarnitines at times and doses indicated.
Plates were then placed on ice, media were removed and discarded,
and cells were rinsed two times in ice-cold HBSS and lysed in 1⫻
E991
E992
ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
RESULTS
Acylcarnitine chain-length effects on IL-6 production. Because our previous studies showed that C14- and C16-acylcarnitine can elicit proinflammatory gene and cytokine expression
in RAW 264.7 murine monocyte/macrophages and (at higher
concentrations) in C2C12 murine myotube models (2, 29), we
sought to fully characterize a panel of short-, medium-, and
long-chain L-acylcarnitine treatments on media IL-6 cytokine
and AK concentrations in the C2C12 myotube model. These
markers were chosen as factors reflecting activation of inflammatory cascades (IL-6) or cytotoxicity typically tracking cell
death (AK). L-Acylcarnitines of acyl chain lengths from C2through C12- did not alter IL-6 cytokine production or AK
release into the medium after 18 h of treatment (Fig. 1).
Beginning with C14 chain-length acylcarnitine and continuing
with the higher chain lengths tested (C16, C18, and C18:1
carnitines), L-acylcarnitine induced both IL-6 production and
AK release in parallel. The minimum concentration tested that
elicited an increase in IL-6 cytokine production was 25 ␮M
C16- and C18- carnitine, with the caveat that other concentrations between 10 and 25 ␮M were not evaluated. These effects
increased in a dose-dependent manner up to 100 ␮M, the
highest concentration tested. AK tracked IL-6 production under all conditions except for C18:1 carnitine, where AK release
increased at 50 ␮M without a corresponding increase in media
IL-6.
Because C16 carnitine had the most robust effects on the
C2C12 myotubes, all further experiments were performed using
C16 carnitine as a representative long-chain acylcarnitine.
Previously, the lots of FBS and C16 carnitine were subjected to
endotoxin testing and found to be negative (29). These results
indicate that the inflammatory IL-6 and AK responses are a
direct result of C16 carnitine and not endotoxin contamination.
Furthermore, in experiments not shown, LPS treatment of cells
failed to elicit an AK response despite the expected robust IL-6
response.
Acylcarnitine activation of MAPK pathway. Our previous
work demonstrated that C14 carnitine increases phosphorylation of the JNK and ERK MAPKs in RAW 264.7 murine
macrophages. Furthermore, IL-6 cytokine production is known
to be mediated in part by the MAPK cell stress signaling
pathway in the C2C12 model (13). As shown in Fig. 2, C16
carnitine elicited an increase in phosphorylation of p38, JNK,
Fig. 1. L-Acylcarnitine chain length-dependent induction of the inflammatory
cytokine interleukin-6 (IL-6) and secretion of adenylate kinase (AK) in a
C2C12 murine myotube skeletal muscle cell model. Myotubes were serum
starved for 3– 4 h (0.25% FBS/DMEM) and then treated for 18 h with varying
concentrations (0 –100 ␮M) and chain lengths (C2 to C18:1) of L-acylcarnitines. Media concentrations of IL-6 (A) and AK (B) were measured by
ELISA (R&D Systems Quantikine) and luminescence (Lonza Toxilight) assays, respectively; n ⫽ 6 or more over 3 experiments. Two-way ANOVA with
Dunnett’s test (post hoc) for dose response: *P ⬍ 0.05, ***P ⬍ 0.001, and
****P ⬍ 0.0001 vs. vehicle-treated control; mean ⫾ SE. Data are expressed
as fold of vehicle control. Mean vehicle IL-6 ⫽ 27.4 pg/ml.
and ERK in C2C12 myotubes. The pathway activation occurred
in both time- and concentration-dependent manners that were
consistent for all MAPK pathways; the effects appeared to be
triggered by concentrations between 10 and 25 ␮M. This effect
suggests that C16 carnitine can elicit global activation of
MAPK pathways in the C2C12 model in a dose-dependent
manner.
C16 carnitine at higher concentrations elicits cell death
within 6 h. Because it was observed that C16 carnitine at
concentrations greater than 25 ␮M cause significant increases
in AK after 18 h (Fig. 1B), a time course of treatment was
designed to determine when and at what concentration this
effect occurs. By 6 h, media AK had increased at concentrations of 40 ␮M and higher (Fig. 3A), indicating that cells had
permeabilized and begun to release intracellular contents into
the media. As a secondary readout for cell damage, lactate
dehydrogenase (LDH) was also assessed in the media. While
variable, LDH increased in a dose-dependent manner with C16
carnitine treatment, similar to AK (data not shown).
When cells are stressed and begin to die, mitochondrial
function is compromised (21, 22). To further characterize the
C2C12 response to C16 carnitine on cell stress and viability,
XTT was used to assess mitochondrial redox potential at 6 h of
treatment (Fig. 3B). The ability of mitochondria to convert
XTT to formazan was significantly reduced starting at 40 ␮M
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10 ␮M calcium green-1 AM. Cells were rinsed two times in warm
HBSS (phenol red free), and 100 ␮l phenol red-free 0.25% DMEM
were added back. The plate was incubated at room temperature on a
Synergy 2 plate reader, and 100 ␮l of a 2X treatment solution
(containing acylcarnitines or other factors indicated in the text) were
added to each well using the Synergy 2 injectors. Fluorescence
readings were taken immediately following injection and continuing
every 2 s for a 1-min duration. Baseline to maximum values was
calculated by subtracting the initial fluorescent reading value from the
highest value recorded.
Data analysis. Each experiment was performed a minimum of three
independent times, as indicated in RESULTS; typical experiments each
included at least three replicates per treatment. Depending on the
nature of the experiment, results were analyzed by one- or two-way
ANOVA with Dunnett’s (comparing against control) or Tukey’s
(comparing all treatments with one another) post hoc tests, and data
are presented as means ⫾ SE. Statistical analyses were performed
using PrismGraph 6.0 (GraphPad Software, San Diego, CA).
ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
Max Fold of Vehicle
A
E993
C16-carnitine
Time (min)
0
5 15 30 60 120 0 5 15 30 60 120 0 5 15 30 60 120
p-JNK
Total JNK
Max Fold of Vehicle
Time (min)
0 5 15 30 60 120 0 5 15 30 60 120 0 5 15 30 60 120
p-ERK
Total ERK
Max Fold of Vehicle
C
Time (min)
0 5 15 30 60 120 0 5 15 30 60 120 0 5 15 30 60 120
p-p38
Total p38
Fig. 2. C16 carnitine increases phosphorylation of the c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated
protein kinase (p38-MAPK) in both a dose- and time-dependent manner. C2C12 myotubes were serum starved for 3– 4 h (0.25% FBS/DMEM) before treatment
with varying doses of C16 carnitine (0 –50 ␮M) for the indicated times. Cell lysates were assessed via electrochemiluminescent ELISA (Meso Scale Discovery)
and Western blot (representative blot from 4 separate experiments). Maximum phosphorylation of p-JNK (A), p-ERK (B), and p-p38 (C) over basal was
quantitated by ELISA (p-JNK, p-ERK) and densitometry (p-p38). Maximum was calculated from n ⫽ 5/treatment over 4 experiments. One-way ANOVA with
Dunnett’s test: **P ⬍ 0.01 and ***P ⬍ 0.001 vs. basal; mean ⫾ SE. Data are expressed as fold over basal.
C16 carnitine after a 6-h treatment, suggesting mitochondrial
dysfunction. The results for AK, LDH, and XTT support
significant cell damage and are suggestive of cell death. A
live/dead imaging assay (Fig. 3, C and D) coupled to AK
measurement (Fig. 3E) confirmed that higher concentrations of
C16 carnitine cause C2C12 myotube cell death.
Long-chain acylcarnitines do not activate ER stress pathways but weakly activate caspases. After observing that C16
carnitine can elicit myotube cell death, the mechanism by
which death occurs was examined. Cellular death can occur
through a number of well-described pathways, including activation of ER stress/unfolded protein response pathways that
can occur through a disruption in lipid homeostasis (4). After
6 h of acylcarnitine treatment, none of the ER stress markers,
IRE-1␣, BiP, nor CHOP, were increased, in contrast to positive
control treatments (Fig. 4A). Interestingly, C16 carnitine modestly increased the apoptotic marker cleaved caspase-3, beginning at 10 –25 ␮M.
Staurosporine is a well-known activator of caspase-associated cell death and as expected triggered a large upregulation
of cleaved caspase-3 in the cells (showing ⬃10-fold increase
vs. control; Fig. 4B, inset). Addition of either a caspase-3/7 or
pan-caspase inhibitor was capable of preventing the cell from
eliciting a staurosporine-mediated increase in caspase-3/7 (Fig.
4B, inset). However, neither of the caspase inhibitors was
capable of protecting the cells from AK release following
acylcarnitine treatment (Fig. 4B). Together, these results indicate that acylcarnitines are capable of activating cell stress
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B
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ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
A
B
1.25
6
1.00
XTT (Fold of Vehicle)
5
AK (Fold of Vehicle)
40 μM
50 μM
75 μM
100 μM
0 μM
5 μM
10 μM
25 μM
30 μM
4
*
3
2
***
***
0.75
***
***
0.50
0
10
75
50
0.00
24
40
8
30
6
25
4
Time (hours)
10
3
5
2
0
1
C16-carnitine (µM)
C
C16-carnitine (μM)
25
0
40
D
E
4
*
***
1.00
***
***
0.75
***
***
0.50
AK (Fold of Vehicle)
3
***
*** ***
2
1
0.25
0
10
75
50
40
30
25
10
0
10
75
50
40
30
25
10
5
0
C16-carnitine (µM)
5
0
0.00
0
Live/Dead (Fold of Vehicle)
1.25
100
50
C16-carnitine (µM)
Fig. 3. Dose-dependent cell death occurs within 6 h of C16 carnitine treatment. C2C12 myotubes were serum starved for 4 h (0.25% FBS/DMEM) and then treated with varying
concentrations of C16 carnitine (0–100 ␮M). Concentrations ⬎40 ␮M resulted in increased AK release after 6 h (A). At 6 h, conversion of 2,3-Bis-(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt (XTT) to its formazan product (B), a live/dead imaging assay (live: green, dead: red) (C and D), and release of AK
(E) confirmed concentrations of C16 carnitine 40 ␮M and greater result in dose-dependent injury/death of C2C12 myotubes. C: representative images of at least 3 experiments.
One-way ANOVA with Dunnett’s test: *P ⬍ 0.05 and ***P ⬍ 0.001; mean ⫾ SE. Data are expressed as fold over vehicle.
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0.25
1
0
5
10 25 30 40 50 75 100
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Tg
C16-carnitine (µM)
Tm
A
STS
ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
20kDa
Cleaved
Caspase 3
100kDa
IRE1-α
BiP
75kDa
CHOP
25kDa
Tubulin
14
12
10
8
6
4
P 10
0
C3 10
2
DMSO
Caspase 3/7 Glo
(Fold of Vehicle)
16
STS
pathways but that the observed cell death and membrane
permeability to AK was not likely mediated through classical
caspase-dependent apoptosis.
Acylcarnitine treatment rapidly increases intracellular calcium and promotes IL-6 production. Increased intracellular
calcium is required for caspase-3 activation (34) and has been
associated with increases in acylcarnitines during cardiac ischemia (39). Additionally, increases in cytosolic calcium are well
established to induce IL-6 transcription (3), which can be
blunted by a calcineurin inhibitor (12). Thus, we assessed
whether addition of exogenous C16 carnitine could elicit an
increase in intracellular calcium in C2C12 myotubes. C16
carnitine rapidly elicited a dose-dependent increase in intracellular calcium when measured by a fluorescent calcium indicator using a microplate reader (Fig. 5, A and B). Whereas the
rate of the calcium increase was slower than that of the positive
control ionophore, ionomycin (Fig. 5A), the average maximum
fluorescence from baseline was comparable between 50 ␮M
C16 carnitine and 1 ␮M ionomycin (Fig. 5B). Increased calcium above vehicle control was detectable by 10 ␮M C16
carnitine.
We next investigated whether C16 carnitine-mediated increases in intracellular calcium were responsible for the increases in media IL-6. The intracellular calcium chelator
BAPTA-AM was used to decrease free intracellular calcium.
BAPTA-AM completely blunted the stimulation of IL-6 by 25
␮M C16 carnitine and caused an ⬃78% drop at 50 ␮M (Fig.
5C). Notably, intracellular calcium chelation had no effect on
Toll-like receptor-mediated IL-6 production from LPS treatment (data not shown). Whereas BAPTA-AM strongly blunted
the production of IL-6 with acylcarnitine treatment, it did not
reduce the AK marker of cell death (Fig. 5D). The data indicate
that changes in intracellular calcium are important for some,
but not all, of the effects of C16 carnitine in myotubes.
Mechanism of acylcarnitine-mediated cell death. Observations from the current experiment and our previous studies of
inflammation (29) and insulin sensitivity (2) in response to
acylcarnitines have illustrated that types and magnitude of
bioactivities differ dramatically when considering lower (i.e.,
5–10 ␮M) and higher (i.e., ⬎25 ␮M) concentrations of these
metabolites. The latter consistently has led to cell permeability
in both RAW cells and C2C12 myotubes, whereas this effect is
not observed at lower long-chain acylcarnitine concentrations,
despite proinflammatory or insulin-resistance phenotypes at the
lower levels of the metabolites. It is possible that acylcarnitines
elicit some or all of their effects through interaction with
cellular membranes by virtue of their zwitterionic character,
perhaps even at the lower concentrations. At very high con-
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B
Fig. 4. C16 carnitine weakly activates cleavage of
caspase-3 but not endoplasmic reticulum (ER)
stress and cannot be rescued from death by caspase
inhibitors in C2C12 myotubes. C16 carnitine weakly
elicits the cleavage of caspase-3 but does not appear to induce markers of ER stress [inositol-requiring protein-1␣ (IRE-1␣), binding immunoglobulin protein (BiP), and CCAAT/enhancer-binding
protein homologous protein (CHOP)] in myotubes.
Positive controls: staurosporine (STS), Tunicamycin (Tm), and thapsigargin (Tg) (A). Inhibition of
caspases does not affect release of AK (i.e., cell
death) (B). Caspase inhibitors effectively inhibit
staurosporine (STS)-induced caspase 3/7 activity
(B, inset). Three independent experiments done in
duplicate (n ⫽ 6/bar); mean ⫾ SE. Data are expressed as fold over vehicle.
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ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
centrations, membrane integrity may be compromised, and cell
stress responses ensue. To begin to evaluate this concept, we
employed the 16-carbon zwitterionic compound ASB-16 that
displays similar chemical characteristics with C16 carnitine
(Fig. 6). In dose-response studies under identical conditions to
those used for C16 carnitine, ASB-16 elicited comparable
patterns for increasing intracellular calcium (Fig. 6A). Additionally, IL-6 and AK release were evident at similar concentrations to those seen for acylcarnitine (Fig. 6B). Although the
studies were not designed to statistically compare ASB-16 and
L-C16 carnitine, overall the molecules displayed qualitatively
similar activities on the parameters tested. These results support the idea that at least some of the effects of acylcarnitine
involve cell components sensitive to zwitterion interaction with
cellular membranes.
DISCUSSION
A growing body of research related to inefficient or impaired
long-chain fatty acid oxidation has implicated some lipid
intermediates as being associated with insulin resistance, inflammation, and cell stress responses (1, 20). Recent studies by
our laboratory have focused on the bioactive properties of
acylcarnitines in eliciting an inflammatory response in murine
macrophages (29) and in blunting insulin signaling in murine
and human myotubes (2). These effects were qualitatively
equivalent with either C14- or C16-acylcarnitines, suggesting a
similar mechanism. One particularly striking finding was that
higher concentrations of long-chain acylcarnitines (i.e., C14or C16-acylcarnitines) elicited apparent cell death in macro-
phages; however, the pathophysiological relevance of these
observations, if any, remains to be determined. Because skeletal muscle is a major source of whole body acylcarnitine
production, we sought to identify the inflammatory and cell
stress effects of long-chain acylcarnitines on murine C2C12
myotubes and explored the mechanism by which these effects
might occur.
Herein, we provide evidence that C16 carnitine, a representative long-chain acylcarnitine elevated under certain disease
conditions such as FAOD (23), cardiac ischemia (17, 36, 38),
or more modestly in insulin resistance/type 2 diabetes (1, 18,
20, 24, 26), elicits the activation of cell death and stress
pathways in a concentration-dependent manner in murine myotubes. C16 carnitine rapidly activated the JNK/ERK/p38
MAPK stress pathways (in concert with AK release), increased
intracellular calcium, and elicited markers of cell death within
6 h. C16 carnitine also modestly activated the proapoptotic
caspase-3 catalytic protein but did not increase markers of ER
stress. These results are in line with a study by Mutomba and
colleagues who reported activation of recombinant caspase-3
enzyme activity by palmitoylcarnitine (25). In the latter study,
the idea that long-chain acylcarnitines can activate caspases
was also supported by the observation that staurosporinemediated apoptosis was blunted in cells lacking CPT1 (25).
While speculative, results from the current study and the
literature are consistent with the hypothesis that, under certain
disease conditions or events, such as FAOD or severe lipotoxicity, increases in long-chain acylcarnitines could elicit muscle
cell inflammation and stress.
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Fig. 5. Intracellular calcium is rapidly increased with C16 carnitine treatment, and its
chelation blunts C16 carnitine-mediated IL-6
production but does not protect from cell
death. Differentiated C2C12 myotubes were
serum starved in 0.25% FBS/DMEM for 4 h
and preloaded with calcium green-1 AM (10
␮M) (A and B) or BAPTA-AM (7.5 ␮M) (C
and D) for the last hour of starvation. Cells
were treated with C16 carnitine (0, 5, 10, or
25 ␮M) or positive control ionomycin (1
␮M) for 1 min, and maximum fluorescence
over basal levels (B) was determined by
plate reader as described in MATERIALS AND
METHODS. Fluorescence trace is representative of 5 experiments (A). Myotubes were
treated for 18 h with C16 carnitine (0, 25,
and 50 ␮M) with and without BAPTA-AM
(7.5 ␮M) (C and D). Media IL-6 and AK were
analyzed. One-way ANOVA with Tukey’s
test: *P ⬍ 0.05, **P ⬍ 0.01 and ****P ⬍
0.0001 vs. basal; mean ⫾ SE. Data are
expressed as fold over vehicle.
ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
E997
Lipotoxicity is a well-described phenomenon that occurs
when lipids and lipid intermediates accumulate abnormally
throughout the body, as seen in insulin resistance and more
severely in poorly controlled type 2 diabetes (30, 35). There are
a host of cellular consequences to lipotoxic conditions, including increases in cellular ROS, ER stress, inflammation, and, in
its most severe manifestation, cell death (5, 30). Increases in
cardiac tissue acylcarnitines have been noted during ischemiareperfusion and associated with a range of cellular complications, including derangements in ionic flux that control cardiac
electrophysiology (9, 32, 40 – 42) and stress and death pathway
activation (32). Although the skeletal muscle metabolic effects
of lipotoxicity associated with type 2 diabetes or insulin resistance are well documented, the specific metabolites that trigger
cell stress are less conclusive. There have been reports of
increased caspase-3/apoptosis (33), ROS, and mitochondrial
damage (31) in type 2 diabetes mellitus and insulin resistance.
This current study, as well as a previous study (29), raise the
possibility that, under certain conditions of inefficient ␤-oxidation, an abnormal increase in long-chain acylcarnitines contributes to lipid-associated cell stress.
The mechanisms-of-action of long-chain acylcarnitines on
cell inflammation (29), insulin sensitivity (2), and cell stress
responses has been elusive. Based on the aggregate of results,
it is speculated that these effects involve associations with cell
membranes (28) and fall along a spectrum from modest (i.e.,
impacting insulin signaling or inflammation) to severe (i.e.,
triggering cell death and loss of membrane integrity). At higher
concentrations of long-chain acylcarnitines, disruption of
membrane integrity may be associated with a host of negative
consequences, including arrhythmias, myopathies, and necrosis, since loss of proper membrane function overrides the cells’
ability to maintain homeostasis (10, 11). Long-chain acylcarnitines at high concentrations were also shown to trigger red
blood cell lysis (8). Consistent with our perspective, it was
observed that the zwitterionic compound ASB-16, often used
to solubilize membranes and membrane proteins (16), displayed effects qualitatively similar to high-concentration longchain acylcarnitines with respect to IL-6 release, cell death, and
permeabilization in C2C12 myotubes. C16 carnitine and
ASB-16 are structurally similar compounds. Furthermore, the
C16 carnitine effects on cell death were seen at concentrations
within the same order of magnitude as its 75- to 100-␮M
estimated critical micelle concentration (CMC) (27). As depicted in a working model (Fig. 7), it is proposed that the
long-chain acylcarnitine interaction with cell membranes also
manifests at lower, nonpermeabilizing concentrations that impact cell signaling outcomes. In other words, long-chain acylcarnitines at concentrations that are normally seen in vivo may
hypothetically interact with cell membranes to impact cellular
function and receptor signaling. In contrast, at pathophysiologically high concentrations, the membrane effects could
also lead to cellular damage.
While this concept provides a provocative perspective on
acylcarnitine biology, a major limitation is that it remains to be
determined if effects seen using the initial extracellular concentrations in cell culture studies mimic acylcarnitine actions at
concentrations circa or within cells in vivo. It is notable that, in
studies by Mutomba et al. (25), the lowest concentration of
palmitoylcarnitine tested, 1 ␮M, activated recombinant puri-
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Fig. 6. Amidosulfobetaine-16 (ASB-16)
elicits similar effects on IL-6 cytokine production and AK secretion in C2C12 myotubes as C16 carnitine. C2C12 cells were
starved and preloaded as done previously
and then treated with ASB-16 (0, 5, 10, or 25
␮M) or ionomycin (1 ␮M). As seen with
C16 carnitine, ASB-16 increased intracellular calcium fluorescence in a dose-dependent
manner (A and B). ASB-16 also increased
secretion of both IL-6 (C) and AK (D) in a
dose-dependent manner. BAPTA-AM was
also able to reduce IL-6 secretion but, as
seen with C16 carnitine, could not prevent
the myotubes from increased permeabilization to AK. *P ⱕ 0.05, ***P ⱕ 0.001, and
****P ⱕ 0.0001.
E998
ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
Pathological, high acylcarnitines
compromise cellular function
Modestly-increased acylcarnitines impact
activity of membrane-associated systems
LDH
CK
AK
IR
PRR
[Ca2+]
P
COX-2
MAPK
Akt
Cleaved
caspase-3
Glucose Uptake
Pro-Inflammatory genes and cytokines
(macrophages)
JNK
ERK
P
p38
IL-6
Cell Stress
= Long-Chain Acylcarnitine
Fig. 7. Proposed hypothesis and model for acylcarnitine action on cells and cell membranes. At modest concentrations, zwitterionic long-chain acylcarnitines
interact with cellular membranes, which influences membrane-associated systems (i.e., certain receptors and receptor complexes) (left). This could be a natural
physiological phenomenon that serves to link metabolism to cellular function. At least under certain conditions, the acylcarnitine-membrane associations can
increase intracellular calcium, activate inflammatory pathways, or decrease insulin-stimulated phosphorylation of protein kinase B (Akt) and glucose uptake. At
higher concentrations of long-chain acylcarnitines that elicit cell membrane disruption, cell permeabilization ensues (right). Under this condition, excessive tissue
long-chain acylcarnitine accumulation, singly or in concert with other stressors, may contribute to pathology outcomes [i.e., episodic myopathies in fatty acid
oxidation disorders (FAOD) or cardiomyocyte death following cardiac ischemia]. IR, insulin receptor; MyD88, myeloid differentiation primary response gene
88; CK, creatine kinase; P, phosphorylated; COX-2, cyclooxygenase-2.
fied caspase-3, supporting our assertion that long-chain acylcarnitine accumulation could trigger some cell stress mechanisms well below the CMC. We have also observed inflammatory phenotypes in immune cells treated with as low as 5
␮M long-chain acylcarnitines (29). The working model of
membrane associations would apply most clearly to long-chain
acylcarnitines and would be less likely to explain bioactivities,
if any, of shorter-chain acylcarnitines. Indeed, we observed a
sharp increase of cell stress outcomes with increasing acyl
chain length, consistent with the relative affinities for acylcarnitines for membranes, as indicated by a decrease in CMCs.
For instance, Haeyaert et al. (15) reported CMCs of C12
carnitine (700 ␮M), C14 carnitine (100 ␮M), and C16 carnitine
(40 ␮M). The absolute CMCs are highly dependent on the
system being tested and the pH, yet, despite variability of these
values in the literature, the chain-length associations would
still hold. As a final consideration, it is likely that the free
acylcarnitine concentrations in situ, and hence their cellular
activities, are modified through binding to other lipids and
proteins. Thus, future studies to examine the validity of the
model components could include, for instance, determination,
albeit challenging, of the in situ cellular concentrations of
acylcarnitines (i.e., immune and myocyte cells), monitoring
cellular effects of manipulation of intracellular generation of
acylcarnitines (i.e., genetic modifications such as CPT2 knockdown, specific enzyme inhibitors), protein binding, and experiments that determine if acylcarnitine association with membrane preparations corresponds with receptor- or membrane
protein-based outcomes.
In summary, we have shown that long-chain acylcarnitines
have the potential to rapidly increase intracellular calcium and
to activate skeletal muscle cell stress pathways, which under
certain conditions elicit cell death. Our studies employed
acylcarnitine administration externally to cells, which were in
line with the high concentrations of long-chain acylcarnitines
within plasma and muscle under conditions of FAOD (23, 43).
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MyD88
ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH
However, it is acknowledged that the amounts used herein may
differ from the concentrations found at the sarcolemma or
within muscle cells. Thus, it remains speculative as to the
potential role of long-chain acylcarnitine accumulation in the
episodic myopathy typical of certain FAOD conditions.
Whether acylcarnitines trigger cellular responses through specific receptors, membrane-associated proteins, nonspecific amphipathic interactions, or a combination of these remains to be
fully elucidated.
ACKNOWLEDGMENTS
We thank Dr. Michael Paddy of the University of California Davis MCB
LM Imaging Facility for technical support and training.
GRANTS
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
The content is solely the responsibility of the authors and does not necessarily
represent the official views of the NIH. The USDA is an equal opportunity
provider and employer.
AUTHOR CONTRIBUTIONS
Author contributions: C.S.M., T.A.K., K.D.O.-M., P.J.O., and S.H.A. conception and design of research; C.S.M., T.A.K., and K.D.O.-M. performed
experiments; C.S.M., T.A.K., and K.D.O.-M. analyzed data; C.S.M., T.A.K.,
K.D.O.-M., P.J.O., and S.H.A. interpreted results of experiments; C.S.M.,
T.A.K., K.D.O.-M., and S.H.A. prepared figures; C.S.M. and S.H.A. drafted
manuscript; C.S.M., T.A.K., K.D.O.-M., and S.H.A. edited and revised manuscript; C.S.M., T.A.K., K.D.O.-M., P.J.O., and S.H.A. approved final version
of manuscript.
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This work was supported by the American Diabetes Association (1-12-BS02), the National Institutes of Health (NIH) (R01-DK-078328 and R01-DK078328-02S1), and United States Department of Agriculture-Agricultural Research Service intramural Project 5306-51530-019-00. This project was also
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ACYLCARNITINES CAN ELICIT MUSCLE CELL STRESS AND DEATH