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Article
Di(2-ethylhexyl)phthalate exposureimpairs insulin receptor and glucosetransporter 4 gene expression in L6myotubes
P Rajesh1 and K Balasubramanian1
AbstractDi(2-ethyl hexyl)-phthalate (DEHP) is an endocrine disrupter and is the most abundantly used phthalate deri-vative, which is suspected to be an inevitable environmental exposure contributing to the increasing incidenceof type-2 diabetes in humans. Therefore, the present study was designed to address the dose-dependenteffects of DEHP on insulin signaling molecules in L6 myotubes. L6 myotubes were exposed to different con-centrations (25, 50, and 100 mM) of DEHP for 24 h. At the end of exposure, cells were utilized for assessingvarious parameters. Insulin receptor and glucose transporter4 (GLUT4) gene expression, insulin receptor pro-tein concentration, glucose uptake and oxidation, and enzymatic and nonenzymatic antioxidants were signifi-cantly reduced, but glutamine fructose-6-phosphate amidotransferase, nitric oxide, lipid peroxidation, andreactive oxygen species levels were elevated in a dose-dependent manner in L6 myotubes exposed to DEHP.The present study in turn shows the direct adverse effect of DEHP on the expression of insulin receptor andGLUT4 gene, glucose uptake, and oxidation in L6 myotubes suggesting that DEHP exposure may have a negativeinfluence on insulin signaling.
KeywordsL6 myotubes, di(2-ethylhexyl)phthalate, insulin signal transduction, reactive oxygen species, insulin receptor,glucose transporter4, glucose oxidation
Introduction
Polyvinyl chloride (PVC) plastics are used exten-
sively for a very wide range of purposes, such as inte-
rior surfaces, food wrappers, and covering of crops in
agriculture. Extensive use of PVC is related to its sta-
bility and flexibility, which is achieved by incorpora-
tion of plasticizers. More than 300 different types of
plasticizers have been identified, and between 50 and
100 are used commercially. Phthalates, diesters of
benzenedicarboxylic acid (phthalic acids), constitute
the most commonly used plasticizers. Phthalates are
multifunctional chemicals used to hold color and
scent in consumer and personal care products.1 The
probability of nonoccupational exposure to phthalates
is high given their use in a vast range of consumables,
including personal care products (e.g. perfumes,
lotions, and cosmetics), paints, industrial plastics, and
certain medical devices and pharmaceuticals. In
humans, phthalates are rapidly metabolized to their
monoesters, which can be further transformed to oxi-
dative products, conjugated, and eliminated.2 Di(2-
ethylhexyl)phthalate (DEHP), one of the most com-
monly used phthalates, leaches from blood storage
bags, intravenous and dialysate bags, and tubing made
with PVC.3 There is widespread exposure to DEHP in
the general population. Diet, particularly fatty food
(e.g. dairy, fish, and oils), is the main source of DEHP
1Department of Endocrinology, Dr A.L.M. Post Graduate Instituteof Basic Medical Sciences, University of Madras, Taramani, Chen-nai, India
Corresponding author:Karundevi Balasubramanian, Department of Endocrinology,Dr. A.L.M. Post Graduate Institute of Basic Medical Sciences, Uni-versity of Madras, Taramani, Chennai 600 113, India.Email: [email protected]
Human and Experimental Toxicology00(0) 1–16
ª The Author(s) 2013Reprints and permission:
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exposure in the general public.4 Since DEHP is not
chemically bound to the polymer, it leaches out of the
plastic matrix and therefore can be found to be nearly
ubiquitous in the environment; it has been detected in
air, water, soil, and food. As a consequence, DEHP is
incorporated into the human body and can be detected
in body fluids and tissues.5 Phthalates are known as
endocrine disrupters, an exogenous substance or mix-
ture that alters function(s) of the endocrine system,
and consequently causes adverse health effects in an
intact organism or its progeny.
Evidences have accumulated for association of
harmful health effects with exposure to phthalates,
particularly DEHP,6 raising public concerns and
debates. Although emphasis has been given to poten-
tial adverse reproductive and carcinogenic effects,
phthalates may have adverse effects on glucose
homeostasis, but the evidence has not been examined
systematically. This basic insight is supported by a
number of independent studies. For example, phtha-
late metabolite mono(ethylhexyl)phthalate (MEHP),
which is a breakdown product of the plasticizer
DEHP, was associated with diabetes in this study.7
In one study, levels of several phthalate metabolites
were associated with increased insulin resistance and
abdominal obesity in US men.8 In another study in
the US, people aged 6–80 yeras, various phthalate
metabolites were associated with higher body mass
index and waist circumference in men aged between
20 and 59 years.9
In a study of Mexican women, levels of three types
of DEHP metabolites were higher in adult women
with diabetes than those without diabetes, suggesting
that phthalate exposures may play a role in diabetes
development.10 While the type of diabetes was not
specified, it was presumably type-2, since the subjects
were older adults and many were overweight. Rats
administered with the phthalate DEHP developed
symptoms of diabetes, including higher blood sugar,
impaired glucose tolerance, altered insulin signaling
molecules, and lower insulin levels.11–13
Based on several studies, it has been suggested that
the onset of metabolic syndrome could be favored by
prolonged exposure to low concentrations of environ-
mental pollutants, including phthalates. However, the
mechanisms involved in these new aspects of meta-
bolic impact of phthalate remain poorly understood.
In view of this, the present study was designed to study
the impact of DEHP on insulin signal transduction in
L6 myotubes in vitro. Quantification of enzymatic,
nonenzymatic antioxidant, lipid peroxidation (LPO),
reactive oxygen species (ROS), nitric oxide (NO), glu-
tamine fructose-6-phosphate (F-6-P) amidotransferase
(GFAT), insulin receptor, and glucose transporter4
(GLUT4) would pave way for a better understanding
of the possible effect of DEHP on the induction of insu-
lin resistance. In addition, functional aspects like glu-
cose uptake, oxidation, and glycogen concentration
were assessed to delineate the impact of DEHP. L6
muscle cell line can be differentiated with high reliabil-
ity into a myotube muscle cell phenotype that naturally
expresses the GLUT4 glucose transporter protein and
has a significant insulin-stimulated glucose uptake,
biological response, thus providing an efficacious alter-
native to isolated skeletal muscle tissues or primary
skeletal muscle cell cultures.14 The L6 myotubes cell
line is the best characterized cellular model of skeletal
muscle origin to study glucose uptake and GLUT4
translocation.
Materials and methods
Reagents
All chemicals and reagents used in the present study
were of molecular and analytical grade and were pur-
chased from Sigma Chemical Company (St Louis,
Missouri, USA; Amersham Biosciences, UK; and
Sisco Research Laboratories, Mumbai, Maharashtra,
India. Dulbecco’s modified Eagle’s medium
(DMEM), DEHP, porcine insulin, dimethyl sulfoxide
(DMSO), penicillin sterptomycin solution, amphoter-
icin B, cytochalasin B, insulin receptor, GLUT4, �-
actin primers, and the �-actin monoclonal antibody
were purchased from Sigma Chemical Co. (St Louis,
Missouri, USA). Fetal bovine serum (FBS) and tryp-
sin–ethylenediaminetetraacetic acid (EDTA) solution
were purchased from HiMedia (Mumbai, Maharash-
tra, India). Total RNA isolation reagent and one-
step reverse transcriptase-polymerase chain reaction
(RT-PCR) kit were purchased from ABgene (Epsom,
UK) and Qiagen (Hilden, Germany), respectively.
Polyclonal insulin receptor �-subunit and GLUT4
antibodies were purchased from Santa Cruz Biotech-
nology Inc. (Dallas, Texas, USA). Iodine-125 ([125I]),14C-glucose, and 14C-2-deoxyglucose were procured
from the Board of Radiation and Isotope Technology
(Mumbai, Maharashtra, India).
Cell line and cell culture
L6 myotubes cell line was procured from National
Centre for Cell Science (Pune, Maharashtra, India).
2 Human and Experimental Toxicology 00(0)
The culture was maintained in DMEM containing
10% FBS and 1% antibiotic/antimycotic in a humidi-
fied atmosphere of 5% carbon dioxide (CO2) at 37�C.
Differentiation of myoblasts into myotubes was car-
ried out as described previously.15 L6 myoblast was
seeded in 10% FBS-DMEM until it reached 80–90%confluence; the FBS content was reduced to 2% for
a further 5–7 days to induce myotube formation. The
medium was changed every 2 days, and differentia-
tion was allowed to continue for up to 7 days before
the experimentation period. Reduction of serum
allowed cell-to-cell fusion and formation of myo-
tubes. The cells were examined each day to evaluate
the degree of differentiation, and it was determined
as the percentage of nuclei present in the multinu-
cleated myotubes under a phase contrast microscope
(Eclipse-80i, Nikon, Tokyo, Japan). Before all experi-
mental manipulations, L6 myotubes were deprived of
serum for 1 h to render the cells quiescent.
DEHP exposure
L6 myotubes were incubated with FBS-free DMEM
for 1 h and then exposed to different doses (0, 25,
50, 100, 200, 400, and 800 mM) of DEHP for 24,
48, and 72 h and a positive control Triton X-100
(0.1%) was used. The experiments lasted from 24 to
72 h, and test chemical DEHP was added to serum-
and antibiotic-free medium and renewed every 24 h.
DEHP was dissolved in DMSO, with a final concen-
tration, respectively, keeping DMSO end concentra-
tion not exceeding 0.01%. At the end of incubation,
cells were utilized for assessing various parameters.
Assessment of cell viability
Cell viability of L6 myotubes was assayed with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay as described previously.16
Briefly, cells were treated with different concentra-
tions of DEHP for 24, 48, and 72 h and then the
medium was aspirated from the wells of the culture
plates, and the cells were washed with phosphate-
buffered saline (PBS). Then, the cells were incubated
with 200 ml of DMEM containing 5 mg/ml MTT for a
further period of 4 h at 37�C. Medium was then
removed, and 200 ml of DMSO was added and agi-
tated for 10 min to dissolve the formazan crystal
formed. Then, 100 ml from each well was transferred
into an enzyme-linked immunosorbent assay reader
plate, and the absorbance of the converted dye was
read at 570 nm with background substitution at 630
nm. Data are expressed as a percentage of untreated
control.
Determination of cell and nuclear morphologicalchanges of cells
Analysis of cell morphology was performed using a
phase contrast microscope. To this, 3� 104 cells were
seeded in six- well plates and treated with DEHP for
24 h. Protocol was as mentioned for MTT assay. At
the end of the incubation period, the medium was
removed, and cells were washed once with PBS at
pH 7.4. The plates were observed under a phase con-
trast microscope (Eclipse-80i, Nikon, Tokyo, Japan).
Cytotoxicity assay
Lactate dehydrogenase (LDH) is a stable cytosolic
enzyme that is released in the culture medium upon cell
lysis and the released LDH is measured colorimetrically
with maximum absorbance read at 440 nm.17 LDH cat-
alyzes the readily reversible reaction involving the oxi-
dation of lactate to pyruvate, forming oxidized
nicotinamide adenine dinucleotide (NADþ) from
NADH, and the determination of LDH is based on the
detection of NADH in the reaction. The treatment pro-
tocol was as mentioned for MTT assay, and the condi-
tioned medium alone was taken for LDH leakage
assay. To 1 ml of buffered substrate, 0.1 ml of condi-
tioned media was added and kept in water bath at
37�C. Then, 0.2 ml of NADþ solution was added,
mixed gently, and incubated at 37�C for 15 min. To this,
1 ml of 2,4-dinitrophenylhydrazine reagent was added
and incubated for further 15 min. Finally, 10 ml of
sodium hydroxide (NaOH; 0.4 N) was added, and after
1–5 min, the absorbance was read at 440 nm. Standards
were also run simultaneously and treated for assays with
sodium pyruvate to prepare the standard graph. The
amount of color formed is proportional to the number
of lysed cells. LDH activity ¼ OD of unknown/OD
of known � standard concentration ¼ microgram of
Lactate liberated/milliliter of conditioned media.
DNA fragmentation assays
L6 myotubes treatment protocol was as mentioned for
MTT assay. The medium of serum-deprived cells was
collected and added to the scraped cells to allow cen-
trifugation of both adhering and floating cells. Cell
pellets were resuspended in lysis buffer (10 mM
Tris-hydrochloric acid (HCl), pH 8.0, 25 mM EDTA,
100 mM sodium chloride (NaCl), 0.5% sodium
Rajesh and Balasubramanian 3
dodecyl sulfate (SDS), and 0.5 mg/ml proteinase K)
and incubated overnight at 56�C. Afterward, samples
were treated with 50 mg/ml RNase A for
2 h followed by a phenol/chloroform extraction.
The DNA was precipitated using 0.3 M final concen-
tration of sodium acetate, pH 5.2, and isopropyl alco-
hol. The DNA pellet was then rinsed with 75% cold
ethanol and resuspended in water. After being spun
for 20 min in a microcentrifuge, samples were loaded
onto a 1.8% agarose and Tris-boric acid-EDTA buffer
gel containing ethidium bromide to allow electro-
phoretic separation of the fragmented DNA. The
separated fragments were visualized on the agarose
gel using an ultraviolet transluminator scanning, and
profiles of oligonucleotides were taken using a digital
camera (Bio Rad, Hercules, California, USA).
Determination of ROS and enzymatic andnonenzymatic antioxidants
The medium was removed by suction from the dishes,
and the cells were washed three times at 1–2�C with
Hanks’ balanced salt solution. Glycyl-glycine buffer
(1200 ml) was added to each dish, and the cells were
scraped loose with a rubber policeman and homoge-
nized with the dish on ice by ultrasonication at 50
W for 5 s. The homogenates were kept frozen at
�80�C until analyzed. LPO was measured by the
method described by Devasagayam and Terachand.18
The malondialdehyde (MDA) concentration of the
sample is expressed in nanomoles of formed per min-
ute per milligram protein. Hydrogen peroxide (H2O2)
generation was assessed by spectrophotometric
method.19 The H2O2 concentration is expressed in
micromoles per minute per milligram protein. Hydro-
xyl radical (OH*) production was quantified by the
method of Puntarulo and Cederbaum20 and expressed
in micromoles per minute per milligram protein.
Superoxide dismutase (SOD) was assayed by the
method of Marklund and Marklund.21 The enzyme
activity is expressed in units per milligram protein.
One enzyme unit corresponds to the amount of
enzyme required to bring about 50% inhibition of pyr-
ogallol auto-oxidation. Catalase activity was assayed
by the method followed by Sinha.22 Catalase activity
is expressed in micromoles of H2O2 consumed per
minute per milligram protein. Glutathione peroxidase
(GPx) was assayed by the method described by
Rotruck et al.23 GPx activity is expressed in micro-
gram of glutathione (GSH) utilized per minute per
milligram protein. Reduced GSH was determined by
the method described by Moron et al.24 The amount
of GSH is expressed in microgram per milligram pro-
tein. Vitamin C was estimated by the method followed
by Omaye et al.25 Vitamin C level is expressed in milli-
gram per 5� 105 cells. Vitamin E was estimated by the
methoddescribed by Quaife and Dju26 and expressed in
milligram per 5 � 105 cells.
Nitrite determination using Griess reaction as anassay of NO production (iNOS activity)
Nitrite, a stable oxidation product of NO, was used as a
measure of NOS activity. Nitrite present in the condi-
tioned culture media was determined by a spectropho-
tometric method based on the Griess reaction.27 For
determination of nitrite concentration, 24-well plates
were used, each well containing 1.5 � 105 cells cul-
tured in 0.5 ml DMEM free of phenol red. Then, 100
ml of the conditioned medium was incubated with the
same volume of Griess reagent (0.1% N-(1-naphtyl)-
ethylenediamine and 1% sulfanilamide in 5% ortho-
phosphoric acid) at room temperature for 10 min. The
absorbance at 540 nm was measured using a microplate
reader, and the nitrite concentration was determined
from a standard curve generated with dilutions of
sodium nitrite in DMEM free of phenol red.
Insulin receptor assay
For insulin receptor assay, insulin was radioiodinated
with [125I] by lactoperoxidase method, according to the
procedure described by Thorell and Johansson.28 Cell-
surface insulin receptors were quantified following the
method of Habberfield et al.29 Briefly, the cells were
exposed to different doses (0, 25, 50, and 100 mM) of
DEHP using FBS-free DMEM for 24 h. After the expo-
sure period, medium was aspirated, and the cells were
incubated with saturating concentration of [125I]-
insulin-containing medium in the presence or absence
of unlabeled insulin at 4�C for 16 h to determine the
total and nonspecific binding. After the incubation
period, the medium was aspirated from all the wells,
and the cells were washed twice with ice-cold fresh
medium to remove any unbound radioactivity. The
cells were then solubilized with 500 ml of ice-cold 1
N NaOH, pipetted out into radioimmuno assay vials
and the radioactivity was counted in a gamma counter
for 1 min. Specific binding of [125I]-insulin was calcu-
lated by subtracting nonspecific binding from the total
cell-surface-bound radioactivity, and the results are
expressed in femtomole per 5 � 105 cells.
4 Human and Experimental Toxicology 00(0)
Reverse transcriptase-polymerase chain reaction
For reverse transcriptase (RT)-polymerase chain reac-
tion (PCR), 5 � 105 cells were seeded in 8 �8 cm2
cell culture plate after differentiation and treatment
protocol was followed. Total RNA was isolated from
control and experimental samples, concentration and
purity of RNA were determined spectrophotometri-
cally at an absorbance of A260/280. The purity of RNA
obtained was 1.8–1.9. The yield of RNA is expressed
in microgram. Total RNA (2 mg) extracted from L6
myotubes of control and experimental samples were
reverse transcribed in a reaction volume of 20 ml using
1 mM oligo dT primer, 0.5 mM deoxynucleotide tri-
phosphates, 10 U ribonuclease inhibitor, and 4 U
omniscript reverse transcriptase (Qiagen, Hilden,
Germany). The reaction was carried out in an autori-
sierter (Eppendorf, Hamburg, Germany) thermocycler
(37�C for 60 min). The resulting complementary
DNAs were stored at �20�C until used for PCR. The
rat-specific primer sequences used in this study for
RT-PCR are listed in Table 1. Each RT-PCR prod-
ucts (10 ml) was analyzed by gel electrophoresis on
a 2% agarose gel. The molecular size of the amplified
products (GLUT4, insulin receptor (IR), and �-actin)
was determined by comparison with molecular weight
marker (100 bp DNA ladder) run in parallel with RT-
PCR products. Then, the gels were subjected to den-
sitometric scanning (Bio Rad, Hercules, California,
USA) to find out the optical density units of each
band and then normalized against that of internal
control (�-actin).
Myotubes subcellular fractionation
Treated cells were washed with ice-cold PBS and
scraped in homogenization buffer containing 20
mM Tris-HCl (pH 7.4), 2 mm ethylene glycol tetra-
acetic acid, 2 mM EDTA, 1 mm phenylmethylsulfo-
nyl fluoride, 10 mM �-mercaptoethanol, 10 mg/ml
aprotinin, and 10 mg/ml leupeptin. After 10 min of
incubation, cells were homogenized with 30 strokes
of a Dounce homogenizer using a tight fitting pestle.
Nuclei were collected by centrifugation at 500g for 5
min, and the low-speed supernatant was centrifuged
at 100,000g for 30 min. The high-speed supernatant
constituted the cytosolic fraction. The pellet was
washed three times and extracted in ice-cold homo-
genization buffer containing 1% Triton X-100 for
60 min. The Triton-soluble component (plasma
membrane fraction) was separated from the Triton-
insoluble material (cytoskeletal fraction) by centri-
fugation at 100,000g for 15 min.30 Plasma mem-
brane and cytosolic fractions were kept at �80�Cbefore protein quantification and Western blotting.
Western blot analysis
Cell lysates were separated via SDS-polyacrylamide
gel electrophoresis and the separated proteins were
blotted onto polyvinylidene fluoride (PVDF) mem-
branes, which were subsequently blocked using
Tris-buffered saline (TBS) containing 0.1% (v/v)
Tween 20 (TBS-T) and 5% (w/v) milk. Membranes
were probed with anti-�-actin/anti-GLUT4/anti-IR�primary antibody and were diluted 1:1000 in TBS-
T. The membranes were washed three times in TBS,
TBS-T for 15 min prior to incubation with horseradish
peroxidase (HRP)—conjugated mouse/rabbit second-
ary antibody—which was diluted 1:7500 with TBS-T
as deemed appropriate. Protein bands on PVDF were
visualized using enhanced-chemiluminescence detec-
tion kit.
Determination of 14C-2-deoxy glucose uptake
Briefly, the rat L6 myotubes were exposed to different
doses (0, 25, 50, and 100mM) of DEHP using FBS-
free DMEM for 24 h. After the exposure period,
medium was aspirated and L6 myotubes were washed
with DMEM supplemented with 0.5% bovine serum
Table 1. List of primer sequences used in the study.
Genes
Gene sequence (50!30)
Ampliconsize (bp)
Senseprimer
Anti-senseprimer
Rat IR 50-GCC ATCCCG AAAGCG AAGATC-30
50-TCT GGGTCC TGATTG CAT-30
224
RatGLUT4
50-GGG CTGTGA GTGAGT GCTTTC-30
50-CAG CGAGGC AAGGCT AGA-30
150
Rat �-actin
50-AAG TCCCTC ACCCTC CCAAAA-30
50-AAG CAATGC TGTCAC CTTCCC-30
96
IR: insulin receptor; GLUT4: glucose transporter 4.
Rajesh and Balasubramanian 5
albumin. After washing, insulin (100 nM) was added
to the serum-free medium and further incubated for 30
min. The above procedure was carried out without
insulin also. Myotubes were washed with HEPES-
buffered saline (HBS; 140 nM NaCl, 20 mM HEPES
pH 7.4, 5.0 mM potassium chloride (KCl), 2.5 nM
magnesium sulfate, and 1.0 mM calcium chloride)
and deoxy glucose uptake was measured as described
by Blair et al[Q: Please provide expansion for
‘‘HEPES,’’ if applicable.].31 Briefly, after insulin sti-
mulation, myotubes were incubated with 14C-2-deoxy
glucose (0.5 mCi/ml) in HBS for 10 min at 37�C.
Radioactive medium was aspirated rapidly, and the
cells were washed four times with ice-cold isotonic
saline (0.9% NaCl). The cells were lysed in 0.05 M
NaOH, and transferred to vials with scintillation cock-
tail. The radioactivity was determined by liquid scin-
tillation counting. Nonspecific glucose uptake was
determined in the presence of cytochalasin-B (50
mM), an inhibitor of facilitative glucose transport and
was subtracted from total uptake. Results are
expressed in counts per minute of 14C-2-deoxy glu-
cose uptake per 5 � 105 cells.
Determination of 14C-glucose oxidation14C-glucose oxidation was estimated as per the
method followed by Kraft and Johnson.32 The treat-
ment protocol was as mentioned above for deoxy glu-
cose uptake (with/without 100 nM insulin). Briefly,
20 ml of cell suspension containing 5 � 105 cells were
pipetted and placed in a 2-ml ampule containing 170
ml DMEM (pH 7.4), 10 IU penicillin in 10 ml of
DMEM and 0.5 mCi of 14C-glucose. After aeration
with gas mixture (5% CO2 and 95% air) for 30 s, the
ampule was tightly closed with a rubber cork contain-
ing CO2 trap and incubated at 37�C. The CO2 traps
were replaced every 2 h. Upon removal of the second
trap, 0.01 ml of 1 N sulfuric acid was added to the
ampule to halt further metabolism. The system was
again closed for 1 h before the third and final trap was
removed and all the CO2 traps were placed in the scin-
tillation vials containing 10 ml of scintillation fluid,
and the samples were counted in a beta counter for
1 min. Results are expressed in counts per minute of14CO2 released per 5 � 105 cells.
Estimation of glycogen
The medium was removed from the dishes by suction
(5� 105 cells), and 1000 ml of 0.4 N potassium hydro-
xide was added to each dish. After 5 min at room
temperature, aliquots of the homogenates were stored
frozen at �20�C until analyzed. Glycogen was deter-
mined by previously described method.33
GFAT assay by glutamate dehydrogenase method
For GFAT assay,34 5� 105 cells were seeded in 8 cm2
cell culture plate after differentiation and treatment
protocol was followed. The myotubes were washed
twice with ice-cold PBS and scraped with 180 ml
ice-cold GFAT buffer. Then, the cells were sonicated.
The samples were centrifuged at 5000g for 5 min, and
the supernatants were centrifuged again at 60,000g for
30 min. All these procedures were carried out at 4�C.
The supernatants were collected, and the protein con-
centrations were determined. In the typical assay, the
total volume was 200 ml, and the reaction was held in
96-well plates. The reaction system included 100 ml
reactive buffer (containing 0.8 mM fructose-6-
phosphate; 6.0 mM glutamine; 0.3 mM acetylpyridine
adenine dinucleotide (APAD); 50 mM KCl,; 0.1 mM
monopotassium phosphate; and 6 U with pH 7.8 glu-
tamate dehydrogenase,) and 100 ml GFAT buffer
(containing 50 mM Tris; 5 mM EDTA; 5 mM GSH;
5 mM glucose-6-phophate disodium salt; 50 mM with
pH 7.8 KCl). The plates were shaken at 37�C for 90
min; the changes in absorbance caused by reduction
of APAD were measured at 370 nm by microplate
spectrometers. The absorbance values of the reaction
mixtures without F-6-P were considered as ‘‘blank
values’’. A unit of the enzyme activity is defined as
one nanomole of glutamate formed per milligram pro-
tein per minute at 37�C.
Statistical analysis
Experiments were repeated three times, each time in
triplicate. Values are expressed as mean + SE.
One-way analysis of variance followed by Turkey’s
test was used to determine significant differences
between groups. The values of p < 0.05 were consid-
ered as statistically significant.
Results
DEHP inhibits cell growth in L6 myotubes
The effect of DEHP on cell viability in a time- and
dose-dependent manner was determined using MTT
assay (Figure 1(a)). Percentage of cell viability was
significantly decreased with increase in concentra-
tion of DEHP. In L6 myotubes, 50% inhibition was
found at 200 mM at 24 h, and hence for further
6 Human and Experimental Toxicology 00(0)
analyses 24 h was considered for treatment and also
25, 50, and 100 mM were considered to identify the
effect of DEHP on the parameters studied. Morpho-
logical examinations of the L6 myotubes were
observed and photographed using phase contrast
microscope. The morphology of L6 myotubes
treated with DEHP (25, 50, 100, 200, 400, and 800
mM) for 24 h compared to untreated cells and cells
treated with 0.01% DMSO showed characteristic of
apoptotic cells, such as cell shrinkage and reduced
cell density (Figure 1(b)). Cells undergoing apopto-
sis also displayed other types of morphological
changes such as rounded up cells that shrink and lose
contact with neighboring cells. Some sensitive cells
were even detached from the surface of plates.
Cytotoxic effects of DEHP
To evaluate cell membrane permeability and integrity,
LDH activity was assayed. As shown in Figure 2(a),
groups exposed to DEHP alone presented a significant
increase in LDH release when compared with the
control. As shown in Figure 2(b), apoptosis-inducible
ability of DEHP was confirmed by DNA fragmentation
in L6 myotubes. At lower doses (25 and 50 mM)
of DEHP exposure, intact DNA was observed, whereas
Figure 1. Cytotoxicity of DEHP in L6 myotubes. Viability of the cells was assessed by MTT method (a) and morphology ofthe cells was analyzed using phase contrast microscope (�10 magnification; (b)). Each value represents the mean + SEMof three experiments. *p < 0.05: compared with their respective control. DEHP: di(2-ethylhexyl)phthalate; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Rajesh and Balasubramanian 7
fragments of DNA were seen at higher dose (100 mM)
of DEHP. The fragments of DNA have interval
molecular weight of approximately180 bp suggesting
an apoptotic event.
DEHP increases in thiobarbituric acid reactivesubstance, OH*, H2O2, and NO levels in L6myotubes
Oxidative stress plays a major role in the development
of insulin resistance. LPO, OH*, H2O2, and nitrite sta-
tus in the L6 myotubes are shown in Figure 3(a) to (d).
Due to DEHP treatment, a significant increase in thio-
barbituric acid reactive substance (irrespective of indu-
cers such as H2O2, ascorbic acid, and ferrous sulfate)
was seen in 50 and 100 mM doses (Figure 3(a)). A
dose-dependent increase was observed in OH* and
H2O2 levels in DEHP-treated L6 myotubes (Figure
3(b) and (c)). Nitrite present in the conditioned culture
media was determined by a spectrophotometric method
(Figure 3(d)). Dose-dependent significant increase in
nitrite level was seen at 50 and 100 mM DEHP doses,
whereas 25 mM dose had no effect on nitrite level.
DEHP suppresses activities of enzymaticantioxidants
Three groups of enzymes play significant roles in pro-
tecting cells from oxidant stress; SODs, catalase, and
GPx. In the present study, the enzymatic antioxidants
registered significant a dose-dependent decrease in
DEHP-treated L6 myotubes (Figure 4).
DEHP reduces the level of nonenzymaticantioxidants
Nonenzymatic antioxidants of particular importance
are GSH and vitamin C and E. DEHP treatment signif-
icantly decreased the GSH level at 50 and 100 mM
doses, but it had no effect at 25 mM dose compared
with the control. Vitamin C and E showed a significant
decline in a dose-dependent manner (Figure 5).
Effect of DEHP on IR and GLUT4 geneexpression and IR concentration
IR and its downstream molecules play a vital role in
insulin signal transduction in L6 myotubes. L6 myo-
tubes exposed to DEHP showed a decline in IR con-
centration in a dose-dependent manner (Figure 6(a)).
IR messenger RNA (mRNA; Figure 6(b)) registered
a significant decrease at 25 and 100 mM doses of
DEHP treatment, whereas no change was found at
50 mM dose compared with the control group. Plasma
membrane IR protein in L6 myotubes was reduced
significantly upon DEHP treatment (Figure 6(c)).
Among the isoforms of glucose transporter pro-
teins, GLUT4 is the one which is insulin sensitive /
Figure 2. Dose-dependent effects of DEHP on cytotoxic (a) and DNA fragmentation (b) of L6 myotubes. Cytotoxic effectwas assessed by LDH leakage method in conditioned media. Apoptosis was evaluated by assessing the presence of DNAladdering by gel electrophoresis. Each bar represents mean + SEM of three experiments. p < 0.05: (a0) compared withcontrol; (b0) compared with 25 mM; and (c) compared with 50 mM. M: Marker; C: Control; DEHP: di(2-ethylhexyl)phtha-late; LDH: lactate dehydrogenaseAQ 1 .
8 Human and Experimental Toxicology 00(0)
Figure 3. Dose-dependent effects of DEHP on LPO(a), H2O2 (b), hydroxyl radical (c), and NO level (d) in L6 myotubes.LPO, H2O2, hydroxyl radical, and NO levels were assessed by spectrophotometric method. Each bar represents mean +SEM of three observations. Significance at p < 0.05: (a0) compared with control; (b0) compared with 25 mM; (c0) comparedwith 50 mM. LPO: lipid peroxidation; DEHP: di(2-ethylhexyl)phthalate; H2O2: hydrogen peroxide; NO: nitric oxide; SEM:standard error mean.
Figure 4. Dose-dependent effects of DEHP on enzymatic antioxidants in L6 myotubes. SOD, catalase and GPx levelswere assessed by spectrophotometric method. Each bar represents mean + SEM of three observations. p < 0.05: (a)compared with control; (b) compared with 25 mM; and (c)compared with 50 mM. DEHP: di(2-ethylhexyl)phthalate; SOD:superoxide dismutase; GPx: glutathione peroxidase; SEM: standard error mean.
Rajesh and Balasubramanian 9
insulin responsive transporter. The dose-dependent
effect of DEHP on GLUT4 mRNA in L6 myotubes
is represented in Figure 7(a). DEHP treatment (50 and
100 mM dose) significantly decreased the GLUT4
mRNA but 25 mM dose of DEHP did not have any
effect on GLUT4 mRNA. Figure 7(b) and (c) depicts
the effect of DEHP on GLUT4 protein in the cytosolic
and plasma membrane fractions in L6 myotubes.
DEHP treatment caused a significant decrease in
cytosolic and plasma membrane GLUT4 concentra-
tion in a dose-reliant manner compared with the coe-
val control.
Glucose uptake, oxidation, glycogenconcentration, and GFAT
Figure 8(a) and (b) represent the dose-dependent
effect of DEHP on 14C-2-deoxyglucose uptake and14C-glucose oxidation in L6 myotubes. Also, 25 mM
DEHP treatment caused a significant decrease in glu-
cose uptake and oxidation in L6 myotubes, and it was
further decreased in 50 and 100 mM DEHP-treated
groups. Further, preincubation with 100 nM insulin
significantly increased the glucose uptake and oxida-
tion follows the same trend of basal uptake and
oxidation.
DEHP treatment caused a significant decrease in
glycogen level at 25 and 50 mM doses, and it was fur-
ther diminished at 100 mM dose in L6 myotubes (Fig-
ure 8(c)). Figure 8(d) shows the dose-dependent effect
of DEHP on cellular GFAT activity in L6 myotubes.
Significant dose-dependent increase in GFAT activity
was observed.
Discussion
Insulin resistance condition is associated with the
development of several syndromes, such as obesity,
type 2 diabetes mellitus, and metabolic syndrome. It
can be defined as a failure of target tissues to increase
whole body glucose disposal in response to insulin.
The tissues responsible, especially skeletal muscle
and fat, exhibit reduced insulin-stimulated glucose
uptake and metabolism. The insulin resistance seen
in myotubes/muscle is of particular importance
because this is the major site of insulin-stimulated
glucose uptake.35 Although factors linking insulin
resistance to these syndromes are not precisely
defined yet, evidence suggests that the exposure to
certain environmental endocrine disruptors like
phthalate and bisphenol-A plays an important role in
the development of insulin resistance.36
Figure 5. Dose-dependent effects of DEHP on nonenzymatic antioxidants in L6 myotubes. GSH, vitamin C, and vitamin Elevels were assessed by spectrophotometric method. Each bar represents mean + SEM of three observations. p < 0.05:(a) compared with control; (b) compared with 25 mM; and (c)compared with 50 mM. DEHP: di(2-ethylhexyl)phthalate;GSH: glutathione.
10 Human and Experimental Toxicology 00(0)
The present study was undertaken to examine the
effects of DEHP on insulin signaling in L6 myotubes.
Initially, we assessed cytotoxic effect of DEHP in cul-
tured L6 myotubes. When the differentiated myotubes
were treated with DEHP for 24, 48, and 72 h, induction
of cytotoxicity were observed in a concentration-
dependent manner. However, DEHP elicited a toxic
effect at higher concentration above 200 mM and
increased time points such as 48 and 72 h. The half
maximal inhibitory concentration values of DEHP for
cytotoxicity (measured by MTT and LDH leakage
assay) was 200mM DEHP at 24 h time point in L6 myo-
tubes. So, further experiments were carried out at the
doses of 25, 50, and 100 mM DEHP for 24 h period.
At this juncture, it is worth to recall the other reports
shown that phthalates have the capacity to induce cyto-
toxity in vitro.37–40
Previous evidence suggested that phthalate induces
free radical production in vivo by activating NADPH
oxidase complex that generates superoxide anion.
Superoxide anion is rapidly transformed to H2O2 and
then to hydroxyl radical.41 An increased production of
H2O2 would lead to formation of highly ROS. ROS
production is shown to totally dependent on DEHP-
induced calcium (Ca2þ) ion influx, plausibly through
the Ca2þ-mediated activation of NADPH complex.42
Similarly, in the present study also there is an increase
in the lipid peroxidation, OH*, H2O2, and NO levels
in DEHP-treated L6 myotubes suggesting that it may
inhibit the cell survival.
Figure 6. Effects of DEHP on insulin receptor concentration (a), insulin receptor mRNA (b), and insulin receptor protein(c) in L6 myotubes. The mRNA expression was analyzed by isolating total RNA using TRIR and converting into cDNA usingRT-PCR, identified on AGE and quantified by densitometric scanning. IR protein was analyzed using Western blot. Totalprotein concentration was determined prior to Western blot analysis. Each bar represents the mean+ SEM of three obser-vations. p < 0.05: (a) compared with control; (b) compared with 25 mM; and (c)compared with 50 mM. DEHP: di(2-ethylhex-yl)phthalate; TRIR: total RNA isolation reagent; cDNA: complementary DNA; RT-PCR: reverse transcriptase polymerasechain reaction; AGE: agarose gel electrophoresis; IR: insulin receptor; mRNA: messenger RNA; SEM: standard error mean.
Rajesh and Balasubramanian 11
Further, intracellular LDH leakage, a well-known
indicator of cell membrane integrity and cell viability
was increased in the conditioned media in a propor-
tionate manner with DEHP doses. In this context, it
has been shown that DEHP induces DNA fragmenta-
tion, which is a key feature of apoptosis, a type of pro-
grammed cell death. Apoptosis is characterized by the
activation of endogenous endonucleases with subse-
quent cleavage of chromatin DNA into internucleoso-
mal fragments of roughly 180 bp.43
A major characteristic of type-2 diabetes mellitus
(T2DM) is insulin resistance in skeletal muscle. A
growing body of evidence indicates that oxidative
stress that results from increased production of ROS
and/or reactive nitrogen species leads to insulin resis-
tance, tissue damage, and other complications
observed in T2DM.44–46 The balance between oxida-
tion and antioxidation (redox balance) is critical in
maintaining a healthy biological system.47 In cellular
redox state, the double-edged effect does not only
concern ROS but also antioxidants. Cells have an ela-
borate defense system against ROS, consisting of
antioxidant enzymes and low-molecular weight sub-
stances capable of scavenging many different ROS.48
In this system, SOD convert superoxide radical (O2�)into H2O2, whereas GPx and catalase convert H2O2
into water.49 Therefore, two toxic species, O2� and
H2O2, are converted into the harmless product water.
Figure 7. Effects of DEHP on GLUT4 mRNA (a), GLUT4 protein cytosol (b), and GLUT4 protein plasma membrane (c) inL6 myotubes. The mRNA expression was analyzed by isolating total RNA using TRIR and converting into cDNA using RT-PCR, identified on AGE and quantified by densitometric scanning. GLUT4 protein was analyzed using Western blot. Totalprotein concentration was determined prior to Western blot analysis. Each bar represents the mean + SEM of three obser-vations. p < 0.05: (a) compared with control; (b) compared with 25 mM; and (c)compared with 50 mM. DEHP: di(2-ethylhex-yl)phthalate; TRIR: total RNA isolation reagent; cDNA: complementary DNA; RT-PCR: reverse transcriptase polymerasechain reaction; AGE: agarose gel electrophoresis; GLUT4: glucose transporter 4; SEM: standard error mean.
12 Human and Experimental Toxicology 00(0)
The removal of H2O2 or other hydroperoxides by GPx
requires reduced GSH as cofactor. Antioxidants sca-
venge ROS before they cause damage to the various
biological molecules, or prevent oxidative damage
from spreading, for example, by interrupting the rad-
ical chain reaction of LPO. In the current investiga-
tion, enzymatic and nonenzymatic antioxidants were
declined suggesting that this effect of DEHP might
be exerted through oxidative stress.
Increased oxidative stress has been suggested to
play a role in many pathophysiological conditions.
Altered transcriptional regulation of various genes,
postulated to be mediated by transcriptional activation
factors such as activator protein 1 and nuclear factor-
�B, is a well-described cellular reaction to oxidative
stress.50–52 IR is the master switch of the signaling
pathway of insulin and therefore, the effect of DEHP
was assessed at the level of IR gene expression and its
concentration. DEHP alters the IR mRNA as well as IR
protein levels and insulin binding, which may be the
result of defective IR gene transcription and translation
of mRNA. In this regard, it is worth to recall our
previous article which reported that DEHP reduces IRs
and glucose oxidation in cultured Chang liver cells.53
Glucose transport is a rate-limiting step in the metabo-
lism of many cell types and therefore in energy produc-
tion.54 This process is mediated by a family of
transmembrane glycoproteins differing in their kinetics
and tissue distribution.55 Among the 13 known glucose
transporters, GLUT4 is the insulin-sensitive glucose
transporter. Its gene expression is regulated in different
insulin sensitive cell types by various stimuli. In the
current investigation, GLUT4 mRNA level was signif-
icantly reduced due to DEHP exposure. It has been
reported that peroxisome proliferator-activated recep-
tor-g(PPARg) represses transcriptional activity of the
GLUT4 promoter via direct and specific binding of the
(PPARg) /retinoid X receptor, heterodimer to a �66/
þ163 bp of GLUT4-promoter region.56 DEHP and its
metabolites, particularly MEHP induces PPARg in a
time- and dose-dependent manner in HRP 1 tropho-
blast cells.57 Probably, the reduction in GLUT4 mRNA
may be the result of impaired GLUT4 gene transcrip-
tional activity mediated through enhanced PPARg due
Figure 8. Effects of DEHP on glucose uptake (a), glucose oxidation (b), glycogen concentration (c), and GFAT (d) in L6myotubes. Glucose uptake in L6 myotubes was estimated by the 14C-2-deoxy glucose uptake assay. Glucose oxidationwas assessed using 14C-glucose. Glycogen was estimated by anthrone reagent method. GFAT activity was assessed byspectrophotometric method. Each bar represents mean + SEM of three observations. p < 0.05: (a) compared with con-trol; (b) compared with 25 mM; and (c)compared with 50 mM. DEHP: di(2-ethylhexyl)phthalate; GFAT: glutamine fructose-6-phosphate amidotransferase; SEM: standard error mean.
Rajesh and Balasubramanian 13
to DEHP exposure. Cytosolic and plasma membrane
GLUT4 protein level was significantly decreased in a
dose-dependent manner in DEHP-exposed L6 myo-
tubes. The reduced membrane GLUT4 may partly be
due to impaired GLUT4 translocation from cytosol to
plasma membrane. It was shown that GLUT4 is a
membrane-bound protein, which is likely to get
affected once the membrane integrity is lost.58
Additionally, we investigated the functional aspect
of GLUT4 such as glucose uptake and oxidation by
the L6 myotubes which shows reduced glucose uptake
and oxidation even after insulin stimulated condition.
In the present study, DEHP reduces the capacity of
insulin to elicit an increase in glucose uptake and
metabolism in myotubes, which is clearly evident
from the decreased membrane-bound GLUT4 reflect-
ing a hallmark of insulin resistance. Glycogen con-
centration in the myotubes was decreased due to
DEHP treatment. In this respect, it is worth to recall
the in vivo study that DEHP treatment reduced the
hepatic glycogen concentration via enhanced glyco-
genolysis in male Wistar rats.59 GFAT is the rate-
limiting enzyme of the hexosamine biosynthetic path-
way (HBP) pathway that catalyzes the amidation of
F-6-P to glucosamine-6-phosphate in the presence
of glutamine. 60 Overproduction of superoxide through
the mitochondrial electron transport system is consid-
ered as a unifying mechanism responsible for aberra-
tions in several biochemical pathways61 of which the
HBP is the key pathway where glucose flux through
this pathway is considered as a form of nutrient sen-
sing, and HBP is one of the mechanisms that mediate
peripheral insulin resistance. In the current investiga-
tion, we found increased GFAT activity which may
be attributed to the elevated ROS.
Conclusion
The most striking finding of the present study is that
exposure to DEHP disrupts cell viability via oxidative
stress and causes a dose-dependent decline in IR,
glucose transporter 4, and antioxidant levels, resulting
in decreased glucose uptake and oxidation. Taken
together, our study supports the hypothesis that certain
environmental chemicals such as DEHP can contribute
to the development of diabetes/insulin resistance even
at relatively low levels.
Conflict of interest
The authors declared no conflicts of interest.
Funding
The author RP was financially supported by a fellowship in
the form of Junior Research Fellowship from the Depart-
ment of Science and Technology, Government of India,
New Delhi, India (Award letter no: DST/ INSPIRE Fellow-
ship (119) /2010, dated 6 May 2010)also from the Depart-
ment of Endocrinology by UGC-SAP-DRS, UGC-ASIST,
DST-FIST program.
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