臨床環境医学（第29巻第2号）

45

Original article

The 7-days-exposure to gammahexachlorocyclohexane causes resistance to insulin
in differentiated mature 3T3-L1 adipocytes
Amire Alimu1), Junetsu Ogasawara1), Ken Shirato2), Takahiko Yoshida1)
1) Division in Hygiene and Health Science, Department of Social Medicine, Asahikawa Medical University, School of Medicine
2) Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine

Abstract
The object of this study is to elucidate the effect of gamma-hexachlorocyclohexane (c-HCH), an organochloride pesticide, on glucose uptake in fully differentiated mature 3T3-L1 adipocytes. Differentiatedmature adipocytes were treated with or without c-HCH for 7 days at 37°C in a 5% CO2 incubator. After
that, both cell viability and glucose uptake were measured by colorimetric assay, and generation of
intracellular reactive oxygen species (ROS) were measured by fluorescence assay. Levels of mRNA andprotein were analyzed using real-time polymerase chain reaction and immunoblotting, respectively.
Treatment with sub-toxic doses of c-HCH decreased insulin-stimulated glucose uptake, enhanced the
production of ROS, and upregulated glucose transporter 4 (GLUT4) while reducing its translocation to
the plasma membrane. Treatment with c-HCH also stimulated the expression of markers of ROS production and phosphorylation of c-Jun N-terminal kinase (JNK). Moreover, phosphorylation of serine307 (Ser307) in insulin receptor substrate-1 (IRS-1), which has an inhibitory effect on insulin-mediatedglucose uptake, was increased, whereas the phosphorylation of AKT2 and/or AKT substrate 160 (AS160)
proteins decreased. These findings indicate that c-HCH attenuated insulin-stimulated glucose uptake
in mature 3T3-L1 adipocytes by reducing the translocation of GLUT4 protein to the plasma membrane
via the phosphorylation of Ser307 in IRS-1 and ROS-stimulated activation of JNK.
(Jpn J Clin Ecol 29 : 45－59, 2020)《Key words》Gamma-hexachlorocyclohexane, mature 3T3-L1 adipocyte, insulin resistance, glucose
transporter 4

Introduction

countries.1,2) In addition to obesity, it has been

The prevalence of type 2 diabetes mellitus

reported that the exposure to persistent organic(T2DM) has recently increased in developing

compounds, most commonly pesticides, cause

受付：2021年２月18日 採用：2021年５月27日
責任著者：吉田貴彦
旭川医科大学医学部社会医学講座衛生学・健康科学分野〒078-8510 旭川市緑が丘東２条１丁目1-1

Jpn J Clin Ecol（Vol.29 No.2 2020）

46

T2DM.3-6) They also cause other metabolic diseases in humans

3-6)

this mechanism was impaired, it would result in

positively associated with

impaired glucose tolerance. The translocation of

T2DM and related metabolic conditions.5,6) It has

glucose transporter 4 (GLUT 4) protein from thebeen suggested a correlation between the devel-

cytosol to the plasma membrane plays a key role

opment of T2DM with chronic exposure to pesti-

in insulin-stimulated glucose uptake in adipo-

cides via direct oral intake and food chain.

cytes.13-15) Therefore, it is useful to determine theGamma-hexachlorocyclohexane (c-HCH) is a

modification of GLUT4, by determination of thecommon insecticide used in agriculture world-mRNA levels of GLUT4 and intracellular local-

7)wide. However, c-HCH has toxic effects on hu-

ization of GLUT4 protein for elucidation wheth-mans, such as in the development of diseases of

er c-HCH affects insulin-stimulated uptake via

the central nervous system, blindness, mental

GLUT4. It is known that excessive intracellular7)

confusion, convulsion, and dysarthria. There-

ROS suppresses the translocation of GLUT4

fore, c-HCH has been banned in several devel-

protein in 3T3-L1 adipocytes.16) Previous study

oped countries and it has been included as ademonstrated that chronic up-regulation of reac-

persistent organic pollutant in 2009 by thetive oxygen species (ROS) induced by c-HCH

Stockholm Convention. Nevertheless, c-HCH iscaused insulin resistance in 3T3-L1 preadipo-8)

popular in developing countries, although thiscytes.16) Also, c-HCH enhances intracellular ROS

chemical directly or indirectly damages the eco-production in Ehrlich ascites tumor cells17) andsystem. The increase in occurrence of T2DM in

human lymphocytes.18) To elucidate the mecha-

developing countries1,2) may be correlated withnism related to glucose uptake in adipose cells

chronic exposure to c-HCH. Indeed, it has been

resulting in insulin resistance in mature adipo-

reported that c-HCH is also associated with dys-cytes, it is necessary to examine the modification

lipidemia

9)and accumulates in adipose tis-

10,11)

sue.

of ROS production in adipocytes (intracellular
ROS production) by c-HCH.

Mature adipocytes constitute the principal

Many ROS markers have been known such aspart of adipose tissue. Therefore, it is hypothe-

cytochrome P450 1b1, haptoglobin, ceruloplas-

sized that c-HCH affects adipocytes resulting in

min, and xanthine dehydrogenase. ROS markers

the occurrence of T2DM. However, little isare overexpressed upon the generation of intra-

known about the toxic effects of c-HCH on thecellular ROS.19) In addition, the levels of NADPH

physiological responses in mature adipocytes.

oxidase-4 (Nox-4) mRNA and nuclear factor ery-

The NOEL (dose, no observable effect level) of c-throid 2-related factor 2 (Nrf2) have shown to be

12)

increased by intracellular ROS generation inThis concentration corresponds to approximately

3T3-L1 adipocytes.20,21) Therefore, it is useful to

2 mM (MW: 290.83). Therefore, to determine theexamine levels of these markers in adipose tis-

cytotoxic and sub-toxic dose of c-HCH in maturesues exposed to c-HCH by determination of

3T3-L1 adipocytes, cell viability should be exam-

mRNA expressions of them.

HCH is 600 mg/L in L6-derived myotubes.

ined at various concentrations of c-HCH fromlow levels to 1000 nM.

Excessive ROS stimulates the phosphorylation of c-Jun N-terminal kinase (JNK), thereby

It is also hypothesized that c-HCH affects theactivating JNK in 3T3-L1 adipocytes.22) The

many mechanisms related to the T2DM. Adipo-

phosphorylation of IRS-1 at Ser307 is a target ofcyte uptakes glucose by insulin stimulation. If

JNK. Other targets of JNK are known, which

臨床環境医学（第29巻第2号）

are related to glucose uptake in adipocytes, such

47

fluence in 3T3-L1 preadipocyte medium (Zen-Bio,as phosphorylation of AS160, which participates

Inc., NC). The cells were maintained in the same

in insulin-stimulated GLUT4 translocation

medium for 2 days post-confluence at 37°C in adownstream of AKT2,23) and phosphorylation of

5% CO2 incubator. Cellular differentiation was

AKT2, a key molecule in the activation of insu-

induced by treatment with 3T3-L1 differentia-

24)

lin-stimulated glucose uptake.

Previous study

tion medium (Zen-Bio, Inc., NC) for 3 days. Cells

reported that the inactivation of IRS-1 upon

were cultured for 4 more days in 3T3-L1 adipo-

phosphorylation at Ser307 and subsequent inhi-

cyte medium (Zen-Bio, Inc., NC) to be differenti-

25)

bition of insulin-related signal transduction.

Itated mature adipocytes.

is useful to examine the expressions of these

Subsequently, differentiated mature 3T3-L1

genes to elucidate the possible impaired mecha-

adipocytes were cultured in collagen 1-coated 24-

nism of glucose uptake by c-HCH whether or not

well plates (Corning, Inc., ME) with (c-HCH

c-HCH affect the phosphorylation. It has showngroup) or without c-HCH (control group) in 1 mlthat TNF-a triggers the activation of JNK,

of medium per well for 7 days in incubator. The

thereby impairing insulin action.26) It is also use-

medium was changed every 2-3 days.

ful to examine determine whether c-HCH stimulates selection of TNF-a, or increases the mRNA

Cell viability of the mature adipocytes exposed

expression of TNF-a.

to c-HCH

The objective of this study is to elucidate theDifferentiated mature adipocytes were treatedeffects of c-HCH on insulin resistance in mature

with 0 nM, 25 nM, 50 nM, 100 nM, and 1000 nM

3T3-L1 adipocytes and the molecular mecha-c-HCH for 7 days. At the end of cultivation, cell

nisms underlying possible c-HCH-induced modi-

culture media were collected and cell viability

fication in signal transduction cascade of glucose

was determined using the LDH cytotoxicity de-uptake by insulin stimulation. For this objective,

tection kit (Takara Bio, Inc., Shiga, Japan) ac-

the effects on c-HCH on the followings in thecording to the manufacturer’s instructions. The

mature adipocytes were examined; the cytotoxic

subtoxic level was determined based on the re-level of c-HCH, insulin-stimulated glucose up-

sults of cell viability. The subtoxic level would

take, translocation event of GLUT 4 protein on

be the level in which there was no significant

the plasma membrane, ROS production, the ac-

difference in the cell viability compared to thetivation of AKT2 and the phosphorylation ofcontrol and there were no big variances in the

Ser307 in insulin receptor substrate-1 (IRS-1),

cell viability in the group. The concentration 50

and the mRNA expression and secretion of TNF-

nM of c-HCH in the following experiments werea.

the subtoxic level determined here.
To adjust the data determined by LDH

Materials and Methods

amount, the remaining cells were harvested us-

Culturing 3T3-L1 preadipocyte to mature adipo-

ing a cell scraper and resuspended in a solution

cytes and treatment of c-HCH

of protease inhibitor cocktail (Nacalai Tesuque,

3T3-L1preadipocytes were purchased from

Kyoto, Japan) and phosphatase inhibitor (Naca-

KAC Co. Ltd. (Kyoto, Japan). 3T3-L1 preadipo-

lai Tesuque, Kyoto, Japan) including M-PER

2

cytes were seeded in a 25 cm collagen 1-coatedMammalian protein extraction reagent (Thermo

flask (Thermo Scientific, NY) and grown till con-

Scientific, NY). Data were calculated by dividingJpn J Clin Ecol（Vol.29 No.2 2020）48

the total protein concentration of each well.

Intracellular ROS levels were measured using
the ROS-Glo TM H2O2 Assay kit (Promega, WI)

Insulin-stimulated glucose uptake

and fluorescence microplate reader. After theAfter 7 days of cultivation with or without c-

measurement, cell numbers were counted in

HCH, the culture medium was aspirated andeach well using the Cell Count Normalization

cells were cultured in serum-free culture medi-Kit (Dojindo Laboratories, Kumamoto, Japan)

um for 6 h in CO2 incubator. After incubation,according to the manufacturer’s instructions.

cells were washed three times with buffer A(Krebs Ringer Phosphate Hepes buffered with

Quantitative real-time polymerase chain reac-

1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCl2,

tion (PCR)

118 mM NaCl, 5 mM KCl, 30 mM Hepes, pH 7.5).

After 7 days treatment of 3T3-L1 cells with or

Buffer A containing 2% BSA (essential fatty acidwithout c-HCH, total RNA in the cytoplasm was

free, Sigma Ca. No. A0281, Sigma Aldrich, MO).

extracted for following experiment. The total

Ten nl of 100 nM insulin was added to each well

RNA was extracted using TRI Reagent, Direct-

at the final concentration as 1 nM and the plates

zol TM RNA Miniprep kit (Zymo Research, CA).

were incubated for 18 min in CO2 incubator.

Complementary DNA was synthesized using the

Next, 2-deoxyglucose (Nacalai Tesuque, Kyoto,

Super Script® IV First-Strand cDNA synthesis

Japan) was added at the final concentration as 1

reaction system using the Oligodt primer (Invit-

mM and incubated for 20 min in CO2 incubator.

rogen) and RNase inhibitor (Toyobo, Tokyo, Ja-

The culture medium was removed and cells werepan). For real-time PCR, RNA was reverse tran-

washed three times with phosphate-buffered sa-

scribed using the PowerUp TMSYBER Greenline containing 200 nM phloretin (Sigma-Aldrich,

Master Mix (Thermo Fisher Scientific, CA) andMO), a glucose uptake inhibitor. The cells were

amplif ied using the Applied Biosystems

harvested using a cell scraper in 10 mM Tris-

StepOne®Real-Time PCR system (Waltham,HCl (pH 8.0). The cellular suspension was ho-MA). The amplification protocol included a holdmogenized by 30 passages using a 5/8-inch

step for 2 min at 50°C and pre-denaturation for

27-gauge needle attached to a syringe. The ho-

2 min at 95°C followed by 40 cycles of the follow-

mogenate was heated at 80°C for 15 min anding: denaturation for 15 s at 95°C and annealing

centrifuged at 15,000×g at 4°C for 20 min. The

for 1 min at 60°C. Relative gene expression was

obtained supernatant was used as the sample

normalized to b-actin expression using the 2-DDCt

for the analysis of glucose uptake. The amountmethod. Amplification of specific transcripts wasof 2-deoxyglucose was determined using theconfirmed based on the melting curves between

commercially available 2-Deoxyglucose uptake

60°C and 95°C following PCR. Primers havemeasurement kit (Cosmo Bio Co., Ltd., Tokyo,

been listed in the Table 1.

Japan) according to the manufacturer’s instructions.

Protein extraction from the plasma membrane
and cytosol

Intracellular ROS levels

After 7 days treatment of 3T3-L1 cells with or

The cells were seeded and grown in collagen

without c-HCH, the protein of both plasma

1-coated 96-well plates and differentiated/treat-membrane and cytosol were extracted by modi-

ed with or without c-HCH as described above.

fying a previously described protocol.27) Cells

臨床環境医学（第29巻第2号）

49

Table
1 Primer sequences for RT-qPCR
Table 1 Primer sequencesfor RT-qPCR
Primer name-actinPrimerLength(mer)
21
20

Amplaic
Sequenceon Size(bp)
237 bp Sense: 5’-TCGTACCACAGGCATTGTGAT-3’
Antisense: 5’-TGCTCGAAGTCTAGAGCAAC-3’

GLUT4

20
20

99 bp

Sense: 5’- GCCTGCCCGAAAGAGTCTAA-3’
Antisense: 5’-CGCTCTCTCTCCAACTTCCG-3’

Cytochrome
P450,1b1

22
20

234 bpSense: 5’-TATTACGGACATCTTCGGAGCC-3’
Antisense: 5’-TGTGGAATGGTGACAGGCAA-3’

Haptoglobin

20
21

196 bpSense: 5’-GTGGAGCACTTGGTTCGCTA-3’
Antisense: 5’-CCATAGAGCCACCGATGATGC-3’

Ceruloplasmin

20
24

226 bpSense: 5’-CCTGGAGTCTGGATGCTCAG-3’
Antisense:5’-CTGAATCACTTCCAGAGGCTGTTA-3’

Xanthine
Dehydrogenase20
20

120 bpSense:5’-GGATGCTAATCGCAGAATAC-3’
Antisense: 5’-GCTTCTGGTTGAAGTGAGTC-3’

Nox-4

20
20

196 bpSense: 5’-GTGTCTGCATGGTGGTGGTA-3’
Antisense: 5’-AGACTAATGCAGCCAGGAGG-3’

Nrf2

20
20

178 bpSense: 5’-AGCTGGCTGATACTACCGCT-3’
Antisense: 5’-CTGGCACATCAGTGGAGAGG-3’

Abbreviations:
GLUT4:
glucosetransporter
4; Nox-4:NADPH
oxidase-4;
Nrf2:
NuclearfactorAbbreviations:
GLUT4:
glucosetransporter
NADPH
oxidase-4;
Nrf2:
Nuclearfactorerythroid
4; Nox-4;
erythroid
2-relatedfactor2
factor2-related2

were homogenized in ice-cold buffer A, i.e., M-v) NP-40 and incubated on ice for overnight with

PER mammalian protein extraction reagent

gentle mixing followed by centrifugation at(Thermo Scientific, IL) containing 0.1% (v/v) NP-

16,000×g for 30 min at 4°C. The supernatant

40 (FUJIFILM Wako Pure Chemical Corpora-

was collected as the plasma membrane fraction.

tion, Osaka, Japan), protease inhibitor cocktail

In this fraction, we confirmed that expression of(Nacalai Tesque, Kyoto, Japan), and phospha-

ATPase protein, a marker of plasma membranetase inhibitor cocktail (Nacalai Tesque, Kyoto,

fraction, by immunoblotting. Finally, cytosolic

Japan) by passing ten times through a 5/8-inch

fractions 1 and 2 were centrifuged at 16,000×g

27-gauge needle attached to syringe at 4°C. The

for 30 min at 4°C. The supernatants were col-homogenate was centrifuged at 1,000×g for 10

lected as the final cytosolic fractions. All samples

min at 4°C and the supernatant was collected

were stored at -80°C until further use.

and comprised the cytosolic fraction 1. The obtained precipitate was resuspended in NP-40-

Immunoblotting

free buffer A and incubated on ice for 10 min

The protein samples, which were described aswith gentle mixing. Subsequently, the resus-

above, were mixed with Laemmli’s sample buf-

pended participates was centrifuged at 1,000×g

fer and incubated in a heat block at 100°C for 3

for 10 min at 4°C and the supernatant was col-min (cytosol fraction) and 37°C for 10 min (plas-

lected as cytosolic fraction 2. This precipitate

ma membrane fraction). Cooled samples at room

was resuspended in buffer A containing 1.0% (v/temperature were loaded onto a 9%–12% sodiumJpn J Clin Ecol（Vol.29 No.2 2020）

50

A

B
120

120
100
80
60
40

*

20

Glucose uptake(mg / mg protein)

Cell viability(Control=100)

140

100
80
60
20
0

0

*

40

Controlg-HCH

Figure 1. Effect of c-HCH on cell viability and glucose uptake in mature 3T3L1 adipocytes(A) The cell viability. Bars and vertical lines indicate mean ± standard deviation (SD).
Figure1 uptake. *P<0.05.
(B) Glucose

dodecyl sulfate-polyacrylamide gel. After electro-

detection reagent:HRP (Bio-Rad Laboratories

phoresis, the proteins were transferred onto aInc., CA). The membranes were washed and im-

polyvinylidene fluoride (PVDF) membrane (Mil-munoreactive bands were detected using Chemi-

lipore Corporation, Billerica, MA). The PVDF

Lumi One Super (Nacalai Tesque, Kyoto, Japan)

membrane was first incubated for 5 min in Bul-

with the ChemiDoc imaging system (Bio-Rad

let Blocking One (Nacalai Tesque, Kyoto, Japan)

Laboratories Inc., CA).

at room temperature. After blocking, the PVDF
membrane was incubated overnight with specific

Enzyme-Linked Immuno Sorbent Assay of TNF-aprimary antibodies in Tris-buffered saline sup-The protein levels of tumor necrosis factor-al-

plemented with Tween 20 at 4°C. We used thepha (TNF-a) in the medium and cell lysate, were

following primary antibodies at a 1:1,000 dilu-

determined by commercially available kit in ac-tion: b-actin, ATPase (Abcam, Cambridge, UK),cordance with the manufacturer’s instructions

GLUT4 (Cell Signaling Technology, Inc., Dan-(R and D systems, MN).

vers, MA), JNK, JNK phosphorylated at tyrosine-183/185 (Cell Signaling Technology), IRS-1

Statistical analysis(Abcam), IRS-1 phosphorylated at serine-307

Parametric data were represented as mean ±(Ser307) (Merck KGaA, Darmstadt, Germany),

standard deviation. The mean values were com-

AKT-2, AKT-2 phosphorylated at serine-474

pared by the Dunnett’s test and Student’s t-test.

(Ser474), AS160, AS160 phosphorylated at ser-

Non-parametric data was shown with boxplot.

ine-588 (Ser588) (Abcam). After washing, theThe values were compared by Mann-Whitney U

membranes were incubated for 60 min with an-

test. P < 0.05 was considered statistically signif-ti-rabbit or anti-mouse immunoglobulin G

icant.

(1:2,000 or 5,000 dilution) antibodies conjugatedwith horseradish peroxidase (DakoCytomation,
Glostrup, Denmark) and TidyBlot western blot

Results
As shown in Fig. 1A, cell viabilities were sig-

臨床環境医学（第29巻第2号）

A

51

B

Cytosol fraction

GLUT4 mRNAb-actin*300

Control

250

200

GLUT4/ b-actin
Relative density(Control = 100)

Relative density(Control = 100)

400

GLUT4

100
0

Control

g-HCH

*

200
150
100
50
0

Plasma Membrane
GLUT4
120

Relative density(Control = 100)Controlg-HCH

DControl

g-HCH

*

100
80
60
40
20
0

Controlg-HCH

Arbitrary unit
(Average luminescence / Cell number)

C

g-HCH

ROS production

*

100

50

0

Controlg-HCH

Figure 2. Effect of c-HCH on the mRNA and protein levels of GLUT4 and ROS
Figure2 generation in mature 3T3-L1 adipocytes(A) The mRNA and (B) protein levels of GLUT4 in the cytosol fraction, (C) protein levels of
GLUT4 in the plasma membrane, (D) intracellular reactive oxygen species (ROS) levels.
Results were representative of three independent experiment (n = 5 for each group). Barsand vertical lines indicate mean ± SD. *P<0.05.

nificantly reduced at 1,000 nM of c-HCH com-stimulated glucose uptake as compared to thatpared with that in non-treated control cells (0in control cells (Fig. 1B).

nM). No significant changes were observed at 25

As shown in Fig. 2A, GLUT4 mRNA levels

nM, 50 nM and 100 nM of c-HCH. In 100 nM ofwere significantly higher in c-HCH-treated cells

c-HCH, however, the cell viability had large

than that in control cells. The protein levels ofvariation compared with 50 nM of c-HCH. We

GLUT4 were significantly increased and de-determined, therefore, 50 nM of c-HCH as a sub-creased in the cytosolic (Fig. 2B) and plasma

toxic dose of c-HCH in this study. Under thesemembrane fractions (Fig 2C), respectively of c-conditions, c-HCH significantly reduced insulin-

HCH-treated cells as compared to the levels in

52

Jpn J Clin Ecol（Vol.29 No.2 2020）

A

B

C

0

Control

100

0

g-HCH

D

*

0

Control

g-HCH

g-HCH

Nrf2

Nox-4

*

400

Relative density(Control = 100)

Relative density(Control = 100)

100

ControlF

Xanthine Dehydrogenase

*

100

0

g-HCH

Control

E
200

Relative density(Control = 100)

100

*

200

200

300
200
100
0

Control

g-HCH

200

Relative density(Control = 100)*

Relative density(Control = 100)

Relative density(Control = 100)

200

Ceruloplasmin

Haptoglobin

Cytochrome P450 1b1

*

100

0

Controlg-HCH

Figure 3. Effect of c-HCH on the mRNA levels of ROS markers

Figure3P450 1b1, (B) haptoglobin, (C) ceruloplasmin 350, (D) xanthine dehydrogenase. (E) NADPH oxi(A) Cytochromedase-4 (Nox-4), and(F) nuclear factor erythroid 2-related factor 2 (Nrf2). Bars and vertical lines indicate mean ± SD.
*P<0.05.

control cells. Intracellular ROS production was

4A). Under this condition, c-HCH also enhanced

enhanced in c-HCH-treated cells as compared tophosphorylation of IRS-1 at Ser307, a target ofthat in control cells (Fig. 2D). The exposure to c-

JNK (Fig. 4B). The phosphorylation of AKT2

HCH enhanced the levels of ROS markers, such

was suppressed upon treatment with c-HCH

as cytochrome P450 1b1 (Fig. 3A), haptoglobin(Fig. 4C). Phosphorylation of AS160 was also

(Fig. 3B), ceruloplasmin (Fig. 3C), and xanthine

downregulated by c-HCH (Fig. 4D).

dehydrogenase (Fig. 3D), as compared to thoseIn the incubation medium, there were noin the control cells. The levels of NADPH oxi-change in the protein levels of TNF-a with ordase-4 (Nox-4) mRNA (Fig. 3E) and nuclear fac-without g-HCH (Fig. 5A), while significant de-

tor erythroid 2-related factor 2 (Nrf2) (Fig. 3F)

crease was observed at the levels of both TNF-a

were also significantly augmented in c-HCH-

mRNA (Fig. 5B) and protein (Fig. 5C) in the ly-treated cells.

sate of the cells exposed to c-HCH compared toTreatment with c-HCH enhanced intracellular ROS levels and phosphorylation of JNK (Fig.

non-treated control cells.

臨床環境医学（第29巻第2号）

B

Cytosol fraction
p54-phospho
p46-phospho
p54
p46

JNK-phospho

JNK-phospho/ JNK-pan
Relative density(Control = 100)JNKControl
400

g-HCH

*

300
200
100
0

Controlg-HCH

IRS1-phospho

Cytosol fraction(Ser307)

IRS1
IRS1-phospho/ IRS1-pan
Relative density(Control = 100)

A

53

200

Control

g-HCH

*

100

0

Control

g-HCH

D

C

Cytosol fraction

Cytosol fraction
AKT2-phospho

AS160-phospho

AKT2

AS160

(Ser474)(Ser588)

g-HCH

*

80
60
40
20
0

Controlg-HCH

AS160- phospho/AS160- pan
Relative density(Control = 100)

AKT2- phospho/AKT2-pan
Relative density(Control = 100)Control
120
100

160
140
120
100
80
60
40
20
0

Controlg-HCH*
Controlg-HCH

FigureFigure4.4 Effect of c-HCH on the expression and phosphorylation of JNK, IRS1, AKT2, and AS₁₆₀ in mature 3T3-L1 adipocytes
Phosphorylation of (A) JNK and (B) IRS-1, (C) AKT2 and (D) AS160. Results were representative of three independent experiment (n = 5 for each group). Bars and vertical lines indicate mean ± SD. *P<0.05.

Discussion

insulin-related signal transduction and resulted

We firstly confirmed sub-toxic dose of c-HCH

in insulin resistance. This was supported by thein mature 3T3-L1 adipocytes as 50 nM, and ap-

following experimental data. GLUT4 protein

plied the concentration for whole experiments inlevels in the plasma membrane decreased in c-this study. Thereby the results of this study are

HCH-treated cells as compared with that in un-

thought to be occurred under the normal situa-

treated control cells, although the mRNA andtion without cell damage.

protein levels of GLUT4 in the cytosol fraction

This study demonstrated that the addition ofwere higher in c-HCH-treated cells than thosec-HCH significantly reduced glucose uptake byin the untreated control cells. In addition, c-

fully differentiated mature 3T3-L1 adipocytes

HCH augmented intracellular ROS generation

via the increased phosphorylation of IRS-1 atwith elevation in the mRNA levels of ROS mark-Ser307 following JNK activation that impaired

ers as compared to those in the control cells.

Jpn J Clin Ecol（Vol.29 No.2 2020）

54

A

B

40

30
20
10
0

ControlC
TNF-a production (pg/ ml)

TNF-a mRNA
150

50

Relative density(Control = 100)

TNF-a production (pg/ ml)

TNF-a in medium

g-HCH

*

100
50
0

Controlg-HCH

TNF-a in Lysate
50
40
30

*

20
10
0

Controlg-HCH

Figure 5. Effect of c-HCH on secretion of TNF-a into the cell culture medium
and TNF-a mRNA and its protein in the lysate of mature 3T3-L1 adipocytes.

(A) secretion
Figure5 levels of TNF-a into the cell culture medium, (B) TNF-a mRNA and (C) its
protein in the lysate. Bars and vertical lines indicate mean ± SD. * P < 0.05 vs control values.

Moreover, c-HCH hindered the phosphorylation

tion in differentiated 3T3-L1 mature adipocytes;

of AKT2 and AS160, downstream molecules ofthis is in accordance with the enhanced genera-

phosphorylated IRS-1 at Ser307, suggesting ation of ROS in other cells, such as renal distal

negative role of c-HCH on glucose uptake via

tubule cells,28) dopaminergic neurons,29) and Eh-the inhibition of GLUT4 translocation from therlich ascites tumor cells.30) NADPH oxidase-1

cytosol to the plasma membrane, thereby caus-(Nox-1), a key enzyme involved in the production

ing insulin resistance in mature 3T3-L1 adipo-of cellular ROS, has a prominent role in c-HCH-

cytes.

induced ROS generation in dopaminergic neu-

c-HCH enhanced intracellular ROS produc-rons.31) Interestingly, Nox-4, a member of the臨床環境医学（第29巻第2号）

InsulinPreadipocytes

Insulinreceptor

7 days Differentiation

Ser307 P

Mature adipocytes

JNK

HCH
(50mM)

7 daysP

glucoseGlucose uptake

IRS-1
Plasamamembrane

AKT-2

ROS

55

Translocation

P

Cytosol

GLUT4:IncreaseP:Decrease

AS160

P :Phosphorylation

Figure 6. The schematic model of the current study
Figure6
Intracellular ROS generation is increased by treatment of c-HCH, in turn, JNK is activated
via its phosphorylation. Activated-JNK provokes phosphorylation of IRS-1 at Ser307. Consequently, translocation of GLUT4 from the cytosol to the plasma membrane is impaired, thereby attenuating the glucose uptake into mature 3T3-L1 adipocytes.

NADPH oxidase family of proteins, is only ex32)

ed that the c-HCH-stimulated phosphorylation

A dramatic in-of Ser307 in IRS-1 inactivated AKT2 and AS160,

crease in the ROS content of adipocytes has

resulting in the reduced translocation of GLUT4

shown to be related with elevation of Nox-4

from the cytosol to the plasma membrane.

pressed in 3T3-L1 adipocytes.

20)

Moreover, Nrf2 mRNA

Therefore, c-HCH-induced phosphorylation oflevels are associated with the increased ROS

Ser307 in IRS-1 might play a critical role in themRNA and protein.

21)

Conse-

attenuation of insulin-stimulated glucose up-

quently, the c-HCH-induced increase in ROS

take, which is mediated by the translocation ofgeneration might be regulated by Nox-4 along

GLUT4 protein. These results indicate thatgeneration in rat white adipocytes.

with elevated levels of Nrf2 mRNA in 3T3-L1

ROS-mediated activation of JNK upon treat-

adipocytes. This is in accordance with the find-ment with c-HCH inhibits the insulin-stimulat-

ings of this study: c-HCH induced the overex-

ed translocation of GLUT4 protein via an in-pression of Nox-4 and Nrf2 mRNAs.

crease in the phosphorylation of IRS-1 at Ser307,

JNK activation is enhanced by oxidative
33,34)

stress in 3T3-L1 adipose cells

and ROS-medi-

ated activation of JNK promotes insulin resis35,36)

thereby reducing insulin-stimulated glucose uptake in 3T3-L1 adipocytes.
On the other hand, it has been widely accept-

Previous studies haveed that TNF-a also attenuates insulin-induced

demonstrated that the mechanism underlying

glucose uptake via impaired insulin signal-

JNK-mediated insulin resistance is closely asso-

ing. 40-42) One of the mechanisms underlying

ciated with the phosphorylation of Ser307 in

TNF-a-induced insulin resistance has shown totance in adipose cells.

25,37)The knockdown of JNK rescues insu-

involve the phosphorylation of Ser307 in IRS-

lin resistance in 3T3-L1 adipocytes38) and the in-

1.43,44) Therefore, it was hypothesized that c-HCH

activation of JNK attenuates Ser307 phosphory-

increases the secretion of TNF-a from adipo-

lation of IRS-1 and confers insulin resistance incytes, thereby stimulating the phosphorylation

IRS-1.

39)

3T3-L1 adipocytes.

Our data also demonstrat-of Ser307 in IRS-1 in adipose cells via autocrine

Jpn J Clin Ecol（Vol.29 No.2 2020）56

and paracrine signaling. However, c-HCH had

porter 4; JNK: c-Jun N-terminal kinase; IRS-1:

no effect on the secretion of TNF-a from matureinsulin receptor substrate-1; AS160: Akt Sub-

3T3-L1 adipocytes into the culture medium, al-strate of 160 kDa; TNF-a: tumor necrosis factor

though the levels of both TNF-a mRNA and pro-alphatein was significantly reduced in c-HCH-treatedcells as compared to those in control cells. Thus,
the ability of c-HCH at phosphorylating Ser307
in IRS-1 through enhanced JNK activation via

Acknowledgements
Funding: This study was supported in part by
Asahikawa Medical University Fund.

increase in ROS generation might be independent on TNF-a-induced phosphorylation event of
IRS-1. These results suggest that c-HCH-induced reduction of insulin-stimulated glucose

Competing interests
The authors declare that they have no competing interests.

uptake is independent of secreted TNF-a fromcell themselves.
In conclusion, c-HCH exhibited inhibitory effects on the insulin-stimulated uptake of glucose
by mature 3T3-L1 adipocytes via ROS generation, JNK-induced phosphorylation of Ser307 of
IRS-1, impaired translocation of GLUT4 protein

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臨床環境医学（第29巻第2号）

59

抄録

本研究の目的は、有機塩素系農薬であるガンマ型ヘキサクロロシクロヘキサン（c-HCH）が成熟型脂肪細胞へおよぼす影響について検討することである。成熟型に分化した3T3-L1脂肪細胞を c-HCH の存在下と非存在下で37℃、５％濃度の二酸化炭素の環境において７日間培養した。培養終了後に細胞の生存率、インスリン添加によるグルコースの取り込み能、細胞内活性酸素の発現量を蛍光アッセイ法にて測定した。
mRNA とタンパク質の発現は、それぞれ定量 PCR 法とイミュノブロッティング法にて測定した。c-HCHの添加は、非添加と比較してインスリン刺激による細胞へのグルコースの取り込みを有意に低下させ、細胞内の活性酸素量を有意に増加させた。この状況下では、細胞膜上における GLUT4タンパク質の発現が有意に低下した。c-HCH の添加によって、活性酸素の発現マーカーである分子群の mRNA 発現と JNK タ
ンパク質のリン酸化も有意に増加した。さらに、c-HCH の添加はインスリン刺激による糖取り込みを抑制する IRS-1タンパク質の307番目にあるセリン残基のリン酸化を有意に増加させ、その下流に存在する
AKT2タンパク質と AS160タンパク質のリン酸化を有意に抑制した。一連の結果より、c-HCH の添加は細胞内の活性酸素量を増加させ、それに伴い活性化された JNK タンパク質が IRS-1の307にあるセリン残基のリン酸化を増加させることによって AKT2タンパク質と AS160タンパク質の活性が有意に低下し、これに伴い GLUT4タンパク質の細胞膜への移行が阻害されることによってインスリン刺激によるグルコースの取り込みが低下する、インスリン抵抗性が生じることが明らかとなった。
（臨床環境 29：45－59，2020）《キーワード》ガンマ型ヘキサクロロシクロヘキサン、成熟型白色脂肪細胞、インスリン抵抗性、グルコー
ス輸送単体４