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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: kbala82@rediffmail.com

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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