Arch Toxicol DOI 10.1007/s00204-016-1889-2

MOLECULAR TOXICOLOGY

Polychlorinated biphenyls exposure‑induced insulin resistance is mediated by lipid droplet enlargement through Fsp27 Hye Young Kim1 · Woo Young Kwon1 · Yeon A. Kim1 · Yoo Jin Oh1 · Seung Hee Yoo1 · Mi Hwa Lee1 · Ju Yong Bae1 · Jong‑Min Kim1 · Young Hyun Yoo1 

Received: 10 May 2016 / Accepted: 7 November 2016 © Springer-Verlag Berlin Heidelberg 2016

Abstract  Although epidemiological and experimental studies demonstrated that polychlorinated biphenyls (PCBs) lead to insulin resistance, the mechanism underlying PCBs-induced insulin resistance has remained unsolved. In this study, we examined in vitro and in vivo effects of PCB-118 (dioxin-like PCB) and PCB-138 (nondioxin-like PCB) on adipocyte differentiation, lipid droplet growth, and insulin action. 3T3-L1 adipocytes were incubated with PCB-118 or PCB-138 during adipocyte differentiation. For in vivo studies, C57BL/6 mice were administered PCB-118 or PCB-138 (37.5 mg/kg) by intraperitoneal injection and we examined adiposity and whole-body insulin action. PCB-118 and PCB-138 significantly promoted adipocyte differentiation and increased the lipid droplet (LD) size in 3T3-L1 adipocytes. In mice, both PCBs increased adipose mass and adipocyte size. Furthermore, both PCBs induced insulin resistance in vitro and in vivo. Expression of fat-specific protein 27 (Fsp27), which is localized to LD contact sites, was increased in PCB-treated 3T3-L1 adipocytes and mice. Depletion of Fsp27 by siRNA resulted in the inhibition of LD enlargement and attenuation of insulin resistance in PCB-treated 3T3-L1 adipocytes. An anti-diabetic drug, metformin, attenuated insulin resistance in PCB-treated 3T3-L1 adipocytes through the

reduced expression of Fsp27 protein and LD size. This study suggests that PCB exposure-induced insulin resistance is mediated by LD enlargement through Fsp27. Keywords  Polychlorinated biphenyls · Insulin resistance · Lipid droplet enlargement · Fat-specific protein 27 Abbreviations ADRP Adipose differentiation-related protein aP2 Adipocyte protein 2 C/EBPα CCAAT/enhancer-binding protein alpha eWAT Epididymal white adipose tissue Fsp27 Fat-specific protein 27 GTT Glucose tolerance tests IRβ Insulin receptor beta IRS1 Insulin receptor substrate 1 ITT Insulin tolerance tests LD Lipid droplets PCBs Polychlorinated biphenyls PI3K Phosphoinositide 3-kinase PPARγ Peroxisome proliferator-activated receptor gamma

Introduction Electronic supplementary material  The online version of this article (doi:10.1007/s00204-016-1889-2) contains supplementary material, which is available to authorized users. * Young Hyun Yoo [email protected] 1



Department of Anatomy and Cell Biology, Dong-A University College of Medicine, Dongdaeshin‑dong 3‑1, Seo‑gu, Busan 602‑714, Republic of Korea

Persistent organic pollutants (POPs) such as organochlorine pesticides, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins PCDDs, and polychlorinated dibenzofurans PCDFs have become widespread environmental contaminants as a consequence of their extensive usage, long-range transport, and persistence. Because POPs are highly resistant to metabolic degradation, humans bioaccumulate these lipophilic and hydrophobic pollutants in fatty tissues for many years (Fisher 1999; Kiviranta et al. 2005).

13



Previous studies demonstrated that POPs including PCBs are involved in the development of diabetes. Numerous epidemiological data suggest an association between POPs burden, diabetes, and metabolic syndromes (Bertazzi et al. 1998; Fierens et al. 2003; Lee et al. 2006, 2007; Lee 2012; Ruzzin et al. 2010; Rylander et al. 2005). In addition, several experimental studies added clues supporting the association between POPs exposure and diabetes or insulin resistance (Gray et al. 2013; Ibrahim et al. 2011; Lv et al. 2013; Ruzzin et al. 2010). Ruzzin et al. (2010) demonstrated that chronic exposure to the low doses of POPs mixture commonly found in food chains induced severe impairment of whole-body insulin action and contributed to the development of abdominal obesity in rats and that the treatment in vitro of differentiated adipocytes with nanomolar concentrations of POP mixtures mimicking those found in crude salmon oil induced a significant inhibition of insulin-dependent glucose uptake. Subsequent studies demonstrated a causal relationship between POPs and insulin resistance. Ibrahim et al. (2011) showed that chronic consumption of farmed salmon containing POPs causes insulin resistance and obesity in mice. Gray et al. (2013) also presented data supporting that chronic exposure to PCBs (Aroclor 1254) exacerbates obesity-induced insulin resistance and hyperinsulinemia in mice. Lv et al. (2013) studying the consequence of gestational and lactational exposure to a POP perfluorooctane sulfonate (PFOS) on prediabetes effects in offspring indicated that glucose and lipid homeostasis in adult rats is impaired by earlylife exposure to PFOS. Although these epidemiological and experimental studies provide compelling evidence that exposure to POPs increases the risk of developing insulin resistance and metabolic disorders, the detailed molecular mechanism underlying POP-induced insulin resistance has remained unsolved. Millions of pounds of PCB compounds have been produced in multiple countries for industrial applications over the last several decades. PCB exposure induces various adverse health effects in animals and humans (Faroon and Ruiz 2015). Their high lipophilicity has resulted in bioaccumulation in various organisms through the food chain, and many PCB congeners have been detected in human blood, milk, and other tissues (Jursa et al. 2006; Shen et al. 2011). A common classification divides PCBs into dioxin like [which are agonists of the Ah receptor (AhR)] and non-dioxin like [which are agonists of the constitutive androstane receptor (CAR) or pregnane X receptor (PXR)] based on their structural and toxicological similarity with the dioxin molecule (Mesnier et al. 2015). The most frequently found congeners in white adipose tissue (WAT) are, according to IUPAC nomenclature, as follows—PCB No. 153, 138, 180, 170, 118, and 156 (Mullerova and Kopecky 2007). Irrespective of previous studies showing the

13

Arch Toxicol

association of PCBs with the development of metabolic disorders, the mechanism by which PCBs interfere with metabolic regulation remains poorly understood. The lipid droplet (LD) is an important subcellular organelle responsible for lipid storage. Excess lipid storage in adipose tissue results in the development of obesity and other metabolic disorders such as insulin resistance or diabetes. Thus, LD size correlates with the susceptibility to insulin resistance and diabetes (Greenberg et al. 2011). Irrespective of the fact that adipose tissues are preferential sites for PCB accumulation (Mullerova and Kopecky 2007; Yu et al. 2011) to date the mechanism by which PCB accumulation affects the physiological role of adipose tissue and induce insulin resistance has not been delineated. We undertook this study to examine how PCBs induce insulin resistance through the modulation of LD size. As we demonstrate, exposure to PCBs induces insulin resistance in vivo and in vitro, and fat-specific protein 27 (Fsp27)-mediated lipid droplet enlargement in adipose tissues underlies PCB-induced insulin resistance.

Materials and methods Materials 3,3′,4,4′-Tetrachlorobiphenyl (PCB-77), 2,3′,4,4′,5-pentachlorobiphenyl (PCB-118), 2,2′,3,4,4′,5′-hexachlorobiphenyl (PCB-138) and 2,2′,4,4,5,5′-hexachlorobiphenyl (PCB153) were purchased from AccuStandard Inc. (New Haven, CT, USA). Mouse insulin ELISA kit was purchased from Shibayagi (Gunma, Japan). BODIPY 493/503 and Nile Red were purchased from Molecular Probes (Eugene, OR, USA). 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEXA), insulin, metformin, Oil Red O, corn oil, dimethyl sulfoxide (DMSO), and anti-beta-actin antibody were purchased from Sigma (St. Louis, MO, USA). The Lipofectamine 2000 and Lipofectamine® RNAiMAX transfection reagents were purchased from Invitrogen (Carlsbad, CA, USA). Anti-IRS1 (sc-559), C/EBPα, and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-aP2, perilipin, PPARγ, Akt1/2, phospho-Akt (Ser473), PI3K-p85, phospho-PI3Kp85 (Tyr458) antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-phospho-IRS1 (Ser307) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY, USA). Anti-Fsp27 antibodies were obtained from Abcam (Cambridge, MA, USA). Animals and PCBs exposure Adult male C57BL/6 mice (8 weeks old, 22–25 g) were purchased from SamTako Bio Korea (Osan, Korea). The

Arch Toxicol

animals were maintained in a temperature-controlled room (22 °C) on a 12-h:12-h light–dark cycle. All procedures were approved by the Committee on Animal Investigations at Dong-A University. Twelve-week-old mice were administered vehicle (corn oil), PCB-118 or PCB-138 (37.5 mg/kg) by intraperitoneal (ip) injection for a total of three injections (2, 3, and 5 weeks) during the 6-week study duration. Mice were randomly divided into three groups of ten animals each. Cell culture and treatment 3T3-L1 mouse embryo fibroblasts, purchased from American Type Culture Collection (Manassas, VA, USA), were maintained in standard Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS; Gibco-BRL, Gaithersburg, MD, USA) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 atmosphere. After confluence, 3T3-L1 cells were induced to differentiate using DMI induction medium (DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 0.5 μM dexamethasone, and 1  μg/ml insulin) for 2 days, followed by DMII (DMEM containing 10% FBS and 1 μg/ml insulin) for 2 days. The medium was subsequently replaced with fresh culture medium (DMEM with 10% FBS) every 2 days for 4 days. To define the effects of PCBs on adipocyte differentiation, we incubated preadipocytes with vehicle (DMSO) or PCBs (PCB-77, PCB-118, PCB-138, or PCB-153) at equivalent concentrations (20 μM) during DMI treatment. RNA interference and transfection For the siRNA-mediated downregulation of Fsp27, Fsp27specific siRNA and negative control siRNA were purchased from Bioneer (Daejeon, Korea) and used at a concentration of 20 nM. 3T3-L1 mouse embryo fibroblasts were transfected with either the siRNA molecule specific for Fsp27 or a negative control siRNA using Lipofectamine® RNAiMAX as per the manufacturer’s instructions.

for 1 h with 10% (w/v) formaldehyde in PBS. After two washes in 60% isopropyl alcohol, the cells were stained for 30 min in freshly diluted Oil Red O solution. Then, the stain was removed, and the cells were washed four times in water. After adding 100% 2-propanol at 500 nm, the absorbance of the eluted Oil Red O was measured in a spectrophotometer. Plasma glucose concentrations and tolerance tests for glucose and insulin As previously described (Kim et al. 2015), intraperitoneal glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed after the mice were fasted for 16 h. Plasma glucose concentrations were measured in tail blood using a GlucoDr Blood Glucose Test Strip (Hasuco, Seoul, South Korea) prior to and 30, 60, 90, and 120 min after intraperitoneally injecting a bolus of glucose (1 mg/g) for the GTT and at the same time points after intraperitoneally injecting 0.75 U/kg body weight insulin for the ITT. RNA isolation and RT‑PCR Total RNA was prepared from cell lines or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Then, 5 μg of total RNA was converted into single-stranded cDNA using MMLV reverse transcriptase (Promega, Madison, Wisconsin, USA) with random hexamer primers. A one-tenth aliquot of cDNA was subjected to PCR amplification using gene-specific primers (Online Resource 1). Western blot analysis

Epididymal white adipose tissues (eWAT) were fixed in 10% neutral buffered formalin and embedded in paraffin. Four-micrometer sections were prepared and stained with hematoxylin and eosin. The morphology of the liver tissue was photographed using an Aperio ScanScope (Aperio Technologies, Vista, CA, USA).

Cells and tissues were washed with ice-cold PBS, resuspended in 100 μl ice-cold RIPA buffer, and incubated at 4 °C for 30 min. Lysates were centrifuged at 13,000 rpm for 30 min at 4 °C. Equal amounts of proteins were subjected to 7.5–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane and reacted with each antibody. Immunostaining with antibodies was performed using the SuperSignal West Pico (Thermo Scientific, Hudson, NH, USA) enhanced chemiluminescence substrate and detected with LAS-3000 Plus (Fuji Photo Film, Tokyo, Japan). Quantification and normalization to actin or GAPDH control bands were performed using ImageJ 1.48q (NIH imaging software, Bethesda, Maryland, USA).

Oil Red O staining

Statistical analysis

As previously described (Kim et al. 2015), cells were washed twice in phosphate-buffered saline (PBS) and fixed

At least three independent experiments were conducted. The results are expressed as the means ± standard

Histology staining

13



Arch Toxicol

Corn oil PCB-118 PCB-138 0

1

2

3

weeks

4

5

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

C

1.6 1.4

*

**

D

1.2

Body Fat (%) g fat/g BW

B

Total Body Fat (g)

35 34 33 32 31 30 29 28 27 26 25

Liver Weight (g)

Body Weight (g)

A

1 0.8 0.6

0.4 0.2 0

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

**

**

6

E

F G 2000

none

corn oil

none

Adipocyte size (µm2)

1800

corn oil

**

**

1600 1400 1200 1000 800 600 400 200 0

PCB-118

PCB-138

PCB-118

PCB-138

100µM

Fig. 1  PCB-118 and PCB-138 increase adipose mass and adipocyte size in C57BL/6 mice. Body weight (a), liver weight (b), body fat (c), and percent body fat (d) of mice administered either vehicle (corn oil) or 37.5 mg/kg PCBs (PCB-118 or PCB-138) for 6 weeks. e Exposed ventral view of representative mice from each group. f Representative

H&E-stained sections of eWAT. Bar 100 µm. g Average adipocyte size of eWAT was measured from H&E images using ImageJ 1.48q. n  = 5–10 per group. *p < 0.05 and **p < 0.01 compared with the experimental controls

deviations (±SD). The statistical significance of the differences was determined using a Mann–Whitney U test. p < 0.05 indicated statistical significance.

differentiation. These results indicate that PCB-118 and PCB-138 promote adipogenesis in vitro. PCB‑118 and PCB‑138 increase adipose mass and adipocyte size in C57BL/6 mice

Results PCBs promote adipocyte differentiation in 3T3‑L1 adipocytes Using four types of PCBs (PCB-77, PCB-118, PCB138, and PCB-153), we examined the effects of PCBs at equivalent concentrations (20 μM) on the differentiation of 3T3-L1 adipocytes. Among these four types, PCB-118 and PCB-138 significantly increased Oil Red O staining (Online Resource 2a and b). Thus, PCB-118 and PCB-138 were utilized in further in vitro and in vivo studies. In vitro, PCB-118 and PCB-138 increased both mRNA (Online Resource 2c) and protein levels (Online Resource 2d) of aP2, PPARγ, and C/EBPα, which are markers of adipocyte

13

To determine whether PCB (PCB-118 or PCB-138) exposure affects adiposity, adult male C57BL/6 mice were administered PCB-118 or PCB-138 (37.5 mg/kg) by intraperitoneal injection. Both PCB-118 and PCB-138 increased adipose mass (Fig. 1c–e) and adipocyte size (Fig. 1f, g) without affecting body weight (Fig. 1a) or liver weight (Fig. 1b). PCBs promote large LD formation Phase-contrast microscopy, Oil Red O stain, and confocal microscopy demonstrated that numerous small LDs appear in control adipocytes. Importantly, fewer and larger LDs were formed in adipocytes treated with PCBs (Fig. 2a–c).

Arch Toxicol

Our analysis on the size distribution of the LDs corroborates that PCBs promote large LD formation (Fig. 2d). Western blot assay showed that PCB-118 and PCB-138 increased protein expression levels of Fsp27 and perilipin, which are the proteins associated with the surface of the intracellular LD in vitro (Fig. 2e) and in vivo (Fig. 2f). These results indicate that PCB-118 and PCB-138 promote large LD formation. Fsp27 mediates PCB‑induced LD enlargement We next examined whether PCB-induced large LD formation is mediated by Fsp27. Noticeably, more numerous and smaller LDs were observed in Fsp27-depleted adipocytes compared to experimental controls (Fig. 3a, b). Total lipid content is decreased by siFsp27 in PCBtreated 3T3-L1 adipocytes (Fig. 3c). These data suggest that siFsp27 prevented the enlargement of lipid droplets in adipocytes treated with PCB-118 and

A

PCBs impair insulin action Next, we assessed the impacts of PCBs (PCB-118 or PCB138) on insulin action in vivo and in vitro. Both PCBs increased blood glucose level (Fig. 4a) and plasma insulin level (Fig. 4b). Furthermore, the amount of hyperglycemia in GTT (Fig. 4c) was increased and the efficiency of insulin in ITT (Fig. 4d) was reduced in PCB-administered mice. We further determined the signaling pathway by which PCBs induce the development of insulin resistance. Both PCBs impaired the insulin-induced upregulation of p-Akt(Ser473) and p-PI3K p85(Tyr458) in 3T3-L1 adipocytes (Fig. 4e). We further examined whether PCB-77 and PCB-153, which showed the meager effect on the adipocyte differentiation compared to PCB-118 and PCB-138, also affect the insulin action in adipocytes (Online Resource 3).

B

C DMSO

+ PCB118

+ PCB118

DMSO

+ PCB138

+ PCB138

10 µM

Fig. 2  PCB-118 and PCB-138 promote large LD formation. LD morphologies determined using inverted phase-contrast microscopy (a) Oil Red O staining (b), confocal microscopy (c) (upper, immunofluorescence staining with anti-perilipin antibody; lower, Nile Red staining) demonstrated that PCB-118 and PCB-138 increased LD size in 3T3-L1 adipocytes. d LD size distribution demonstrated that the

70 60

43

8 10

45

18

32

26

28

50 40

29

30 20

41

10

28

37 21

>6 (µM) 4~6 2~4 0~2

Fsp27 perilipin actin

F

18

0 + PCB138

10 µ M

80

7 9

+ PCB118

+ PCB138

90

+ PCB138

100

PCB138

E

+ DMSO

+ PCB118

10 µ M

PCB118

x100

+ DMSO

x100

corn oil

10 µ M

+ Mock

x40

none

x40

+ Mock

+ PCB118

+ PCB138

+ DMSO

Mock

+ PCB118

LD size distribution (%)

DMSO

D

PCB-138, indicating that Fsp27 mediates PCB-induced LD enlargement.

Fsp27 perilipin GAPDH

population of LDs with a diameter 4–6 and >6 µm increased in PCBtreated 3T3-L1 adipocytes. Data were collected from 30 cells stained with BODIPY 493/503 in each group. Bar 10 µm. e, f Western blot demonstrated that PCB-118 and PCB-138 increased the expression level of LD-associated proteins in PCBs-treated 3T3-L1 adipocytes (e) and mice (f)

13



Arch Toxicol

A

sc DMSO

+ PCB118

siFsp27 + PCB138

+ PCB118

+ PCB138

10 µM

10 µ M

sc

4 3 2.5 2 1.5 1

siF27

sc

+ PCB138

+ PCB118

0

+ PCB138

0.5 + DMSO

21 18

41 35

**

3.5

+ PCB118

28

**

4.5

+ Mock

0

41 37

>6 (µM) 4~6 2~4 0~2

Total lipid content (OD)

+ PCB138

20

29

+ PCB138

+ PCB118

40

+ PCB118

DMSO

60

+ PCB138

siFsp27

80

+ PCB118

+ PCB138

C 7 8 12 14 9 10 18 26 20 21 32 43 45 28 27 30

+ Mock

+ PCB118

100

LD size distribution (%)

Mock

sc

+ DMSO

B

siF27

Fig. 3  Fsp27 mediates PCB-induced LD enlargement. Inverted phase-contrast microscopy (a) and confocal microscopy (b) demonstrated that siFsp27 markedly reversed PCB-induced LD enlargement. The data of LD size distribution were collected from 30 cells

stained with BODIPY 493/503 in each group. Bar 10 µm. c ORO staining demonstrated that siFsp27 significantly decreased total lipid content in PCB-treated 3T3-L1 adipocytes. **p < 0.01 compared with the experimental controls

Fsp27 mediates PCB‑induced insulin resistance via IRS1 downregulation

Metformin reduces LD size and increases IRS1 protein level in PCB‑treated 3T3‑L1 adipocytes through downregulation of Fsp27 protein

We further examined the molecular mechanism underlying PCB-induced large LD formation that mediates insulin resistance in vivo and in vitro. The protein level of IRS1, which is a critical element in insulin-signaling pathways, was markedly reduced in both PCB-administered mice (Fig. 5a) and 3T3-L1 adipocytes (Fig. 5b). However, PCBs did not alter mRNA levels of IRS1 (Fig. 5a, b). We further determined whether Fsp27 plays a role in PCB-induced reduction of IRS1 protein. Importantly, siFsp27 reversed PCB-induced IRS1 reduction (Fig. 5c). These results indicate that Fsp27 mediates PCB-induced insulin resistance through IRS1 reduction.

13

We examined whether metformin, a representative insulin resistance-improving drug, alleviates PCB-induced insulin resistance through Fsp27. Metformin not only reduced the expression level of Fsp27, but reversed PCB-induced upregulation of Fsp27 expression (Fig. 6a). Phase-contrast microscopy showed that metformin reversed PCB-induced LD enlargement (Fig. 6b). The reversal by metformin of Fsp27 upregulation was correlated with the reversal by metformin of IRS1 downregulation in adipocytes exposed to PCBs (Fig. 6c). Remarkably, metformin reversed the impairment by PCBs of the insulin-induced upregulation

Arch Toxicol

**

4 3 2

PCB-138

PCB-118

corn oil

1 0

0

30

*

60

90

Time (min)

E

**

120

Mock

insulin (100 nM) + PCB138

**

**

+ PCB118

**

+ DMSO

**

+ PCB138

*

p-Akt (Ser473) Akt 1/2

D

**

Blood glucose (mg/dl)

5

PCB-138

0

PCB-118

50

corn oil PCB-118 PCB-138

*

+ PCB118

100

800 700 600 500 400 300 200 100 0

+ DMSO

150

B Plasma insulin (ng/ml)

C

**

Blood glucose (mg/dl)

**

200

corn oil

Blood glucose (mg/dl)

A

p-PI3K p85 (tyr458) 200

corn oil PCB-118 PCB-138

150

**

100

*

50 0

0

30

PI3K p85 actin

** *

60

Time (min)

90

120

Fig. 4  PCB-118 and PCB-138 impair insulin action. Blood glucose level (a) and plasma insulin level (b) were significantly higher in PCB-administered mice. n = 7–10 per group. The blood glucose levels over the entire time course of the GTT (c) and ITT (d) were significantly higher in PCB-administered (PCB-118 or PCB-138) mice.

n  = 7–10 per group. *p < 0.05 and **p < 0.01 compared with the experimental controls. e Western blot demonstrated that PCB-118 and PCB-138 impaired the insulin-induced upregulation of p-Akt(S473) and p-PI3K p85(Y458) in 3T3-L1 adipocytes

of p-Akt(Ser473) and p-PI3K p85(Tyr458) (Fig. 6d). These findings indicate that metformin may improve PCB-induced insulin resistance through inhibition of LD enlargement via downregulation of Fsp27 protein.

by which PCBs affect adipocyte physiological function have not been defined until the present study. In the present study, we revealed that PCBs induce insulin resistance through Fsp27-mediated LD enlargement. We also demonstrated that Fsp27-specific siRNA and metformin alleviated PCBs-induced insulin resistance through the reduced LD enlargement, which is correlated with the reduced accumulation of PCBs. Dysfunction of WAT causes serious metabolic disruption in energy/nutrient homeostasis and may result in several pathological conditions. The excess of adipose tissue can be caused by both hypertrophy (increased lipid accumulation as LDs) and hyperplasia (increased proliferation and/ or differentiation) of adipocytes (Gesta et al. 2007). Particularly, hypertrophied adipocytes not only lead to local inflammation and ischemia of WAT but also inhibit the production of adipokines, such as adiponectin, resulting in systemic insulin resistance (Jiang et al. 2011; Lumeng and Saltiel 2011). Recent reports indicate that alterations of the regulation of LD physiology influence the risk of developing metabolic diseases (Greenberg et al. 2011). LDs are subcellular organelles found in most cells, which regulate the storage

Both Fsp27‑specific siRNA and metformin reverts the accumulation of PCBs To this end, we examined whether Fsp27-specific siRNA and metformin revert accumulation of PCBs. Both Fsp27specific siRNA and metformin reverted the accumulation of PCB-118 and PCB-138 in adipocytes (Online resource 4).

Discussion White adipose tissue represents a reservoir of POPs. Previous studies showed that POPs accumulated in WAT may modulate the activity of key transcriptional factors engaged in the control of differentiation, metabolism, and the secretory function of adipocytes (Mullerova and Kopecky 2007). Despite the potential for adipocytes to be frequently exposed to PCBs, the cellular and molecular mechanisms

13



IRβ

IRβ

GAPDH

actin

IRS1

IRS1

GAPDH

actin

13

+ PCB138

+ PCB118

+ DMSO

0.4 0.2

+ PCB138

+ PCB118

+ DMSO

+ Mock

0

sc

and hydrolysis of neutral lipids. The core of LDs in most cells includes triacylglycerol (TAG) and/or sterol esters (SEs). The core of an LD is surrounded by a monolayer of polar lipids with some attached or embedded proteins, such as adipose differentiation-related protein (ADRP), perilipin 1, and Fsp27, to regulate their formation and mobilization (Greenberg et al. 2011; Miura et al. 2002; Wolins et al. 2006). Adipocytes are characterized by their large LDs. The increased rate of adipocyte lipolysis results in elevated levels of circulating fatty acids, which are stored as TAG in LDs within skeletal muscle and hepatocytes. Local and circulating free acids are principal etiological agents in the development of insulin resistance (Boden et al. 2005; Dresner et al. 1999; Zhai et al. 2010). Numerous cross-sectional studies have indicated that enlarged LD size is associated with hyperinsulinemia, insulin resistance, and glucose tolerance (Brook and Lloyd 1973; Kissebah et al. 1982; Salans et al. 1968). Additional reports indicate that the number and size of the LDs is often correlated with obesity and other pathologies linked with fat accumulation (Grahn et al. 2013; Rizzatti et al. 2013). Because excess lipid storage in adipose tissue results in insulin resistance and diabetes, LD size is correlated with the susceptibility to insulin resistance and diabetes

+ PCB138

0.6

+ PCB138

+ PCB138

+ PCB118

0.8

+ PCB118

actin

**

1

+ DMSO

Fsp27

**

1.2

+ Mock

IRS1

+ DMSO

+ Mock

+ PCB138

+ PCB118

+ DMSO

+ Mock

siFsp27

IRS1 protein/actin (fold)

IRS1

sc

+ PCB118

+ DMSO

PCB138

PCB118

corn oil

IRS1

C

insulin (100 nM)

Mock

B

A none

Fig. 5  Fsp27 mediates PCBinduced (PCB-118 or PCB-138) IRS1 downregulation. a, b Western blot (upper) demonstrated that the protein level but not mRNA level (lower) of IRS1 was reduced in PCBadministered mice (a) and PCBtreated 3T3-L1 adipocytes (b). c Western blot demonstrated that siFsp27 reversed PCB-induced IRS1 downregulation. n = 4. Western blot bands were quantified and normalized to anti-actin control bands using ImageJ 1.48q. **p < 0.01 compared with the experimental controls

Arch Toxicol

siFsp27

(Greenberg et al. 2011). LD size varies depending on the cell type and metabolic conditions. Maintaining small LDs provides more surface area for efficient lipolysis. Under conditions of lipid overload, cells respond to lipid storage pressure by increasing the number and/or volume of LDs. There have been at least three models explaining how LDs may grow. LDs can grow by acquiring lipids through targeted delivery. LDs also can grow by local lipid synthesis. Finally, LDs can grow through fusion with existing LDs (Yang et al. 2012). Insulin acts by binding to its cell surface receptor, thus activating the receptor’s intrinsic tyrosine kinase activity, resulting in receptor autophosphorylation and phosphorylation of several substrates (Kang et al. 2016; Pirola et al. 2004). Understanding how insulin action is modulated by these factors provides suitable targets for pharmacological agents, to enable the control of altered glucose and lipid metabolism and diabetes. Metformin is the most widely used drug for the treatment of type 2 diabetes. Previous studies provide direct/ indirect evidence of the mechanisms through which metformin reduces LD size. This insulin-sensitizing agent has known beneficial effects on control of abnormal lipid accumulation by not only inhibiting adipogenesis (Alexandre

Arch Toxicol DMSO

B

A

+ PCB138

+ PCB138

+ PCB118

+ PCB138

+ PCB118

+ PCB138

+ DMSO

+ PCB118

+ PCB118

Metformin (0.1mM)

DMSO + DMSO

D8

Metformin (0.1mM)

Fsp27 actin 10 µ M

Fsp27 actin

+

+

+

+

+

PCB138

Insulin (100nM)

PCB118

** **

DMSO

**

Metformin (0.1mM)

DMSO PCB138

*

D

PCB118

1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

DMSO + DMSO PCB138 + DMSO DMSO + Metformin PCB138 + Metformin

DMSO

+ PCB138

+ PCB118

+ DMSO

Metformin (0.1mM) + PCB138

+ PCB118

Mock + DMSO PCB118 + DMSO Mock + Metformin PCB118 + Metformin

Protein/actin

IRS1

+ DMSO

+ Mock

DMSO

+ Mock

C

+

p-Akt (Ser473) Akt 1/2 p-PI3K p85 (tyr458) PI3K p85 IRS1

Fsp27

actin

Fig. 6  Metformin reduces LD size and increases IRS1 protein level in PCB-treated 3T3-L1 adipocytes through downregulation of Fsp27 expression. a Western blot demonstrated that metformin not only reduced Fsp27 protein level, but reversed PCB-induced upregulation of Fsp27 protein. b Phase-contrast microscopy demonstrated that metformin markedly reduced PCB-induced LD enlargement. Bar 10 µm. c Western blot demonstrated that the reversal by metformin of

Fsp27 upregulation was correlated with the reversal by metformin of IRS1 downregulation. n = 3. Western blot bands were quantified and normalized to anti-actin control bands using ImageJ 1.48q. *p < 0.05 and **p < 0.01 compared with the experimental controls. d Western blot demonstrated that metformin reversed the impairment by PCBs of the insulin-induced upregulation of p-Akt(Ser473) and p-PI3K p85(Tyr458)

et al. 2008) but also ameliorating NAFLD (Liu et al. 2014). Particularly, Liu et al. (2014) has reported that metformin prevents hepatic steatosis by regulating the expression of ADRP, which is a major LD-associated protein expressed in almost all cells. The efficiency and capacity of adipocytes to esterify fatty acids into triglyceride and protect triglyceride stores within the cells are controlled within LDs surrounded by a phospholipid layer and LD proteins. These factors are involved in regulating LD size. The “PAT” domain-containing proteins perilipin, TIP47, and ADRP are targeted to LDs and regulated the size and biogenesis of these organelles (Wolins et al. 2006). Cidec/Fsp27 was recently reported to be a LD-binding protein and to promote lipid accumulation in adipocytes (Keller et al. 2008; Nishino et al. 2008; Puri et al. 2008; Toh et al. 2008). Fsp27 belongs to the CIDE family of proteins which has three members in mice (Cidea, Cideb, and Fsp27) and humans (CIDEA, CIDEB, and CIDEC, similar to Fsp27) (Liang et al. 2003). Despite previous studies on Fsp27, the

physiological role of Fsp27 in regulating adipocyte function and whole-body energy homeostasis is largely unknown. Only several reports deal with this subject. However, they appear to be opposing and mutually contradictory. Nishino et al. (2008) show that Fsp27 deficiency protects mice from obesity, hepatosteatosis, and insulin resistance induced by high-fat diet (HFD), which was likely associated with increased energy expenditure. Toh et al. (2008) show that Fsp27 deficiency causes reduced white fat pads, decreased adiposity, increased whole-body metabolism, enhanced insulin sensitivity, and a lean phenotype in Fsp27−/−mice. We also observed that LD enlargement through Fsp27 mediates PCB-induced insulin resistance. On the other hand, it has been reported that adipocyte-specific disruption of Fsp27 causes insulin resistance in HFD-fed mice (Tanaka et al. 2015). Further future study on this issue is a challenging task. For the present study, we adopted the single POPs treatment for a short duration. However, humans are exposed to very low dose, complex mixtures of POPs. Thus, it is not

13



completely clear that the data obtained from our experimental system are relevant human exposure. Moreover, we did not test all types of PCBs. Despite these limitations, through presenting data supporting that Fsp27-mediated LD enlargement underlies PCB exposure-induced insulin resistance, we not only provide important insights into the mechanisms governing PCB-mediated insulin resistance but also could offer a suitable target for interventions targeting PCB-induced insulin resistance. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MISP) (Nos. 2015R1A2A1A10051603, 2016R1C1B2011721, and 2016R1A5A2007009). Compliance with ethical standards  Conflict of interest  The authors declare that they have no conflict of interest. Ethical standards  The manuscript does not contain clinical studies or participant data.

References Alexandre KB, Smit AM, Gray IP, Crowther NJ (2008) Metformin inhibits intracellular lipid accumulation in the murine pre-adipocyte cell line, 3T3-L1. Diabetes Obes Metab 10:688–690 Bertazzi PA, Bernucci I, Brambilla G, Consonni D, Pesatori AC (1998) The Seveso studies on early and long-term effects of dioxin exposure: a review. Environ Health Perspect 106(Suppl 2):625–633 Boden G, She P, Mozzoli M, Cheung P, Gumireddy K, Reddy P, Xiang X, Luo Z, Ruderman N (2005) Free fatty acids produce insulin resistance and activate the proinflammatory nuclear factor-kappaB pathway in rat liver. Diabetes 54:3458–3465 Brook CG, Lloyd JK (1973) Adipose cell size and glucose tolerance in obese children and effects of diet. Arch Dis Child 48:301–304 Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI (1999) Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity. J Clin Invest 103:253–259 Faroon O, Ruiz P (2015) Polychlorinated biphenyls: new evidence from the last decade. Toxicol Ind Health. doi: 10.1177/0748233715587849 Fierens S, Mairesse H, Heilier JF, De Burbure C, Focant JF, Eppe G, De Pauw E, Bernard A (2003) Dioxin/polychlorinated biphenyl body burden, diabetes and endometriosis: findings in a population-based study in Belgium. Biomarkers 8:529–534 Fisher BE (1999) Most unwanted. Environ Health Perspect 107:A18–A23 Gesta S, Tseng Y, Kahn CR (2007) Developmental origin of fat: tracking obesity to its source. Cell 131:242–256 Grahn TH, Zhang Y, Lee MJ, Sommer AG, Mostoslavsky G, Fried SK, Greenberg AS, Puri V (2013) FSP27 and PLIN1 interaction promotes the formation of large lipid droplets in human adipocytes. Biochem Biophys Res Commun 432:296–301 Gray SL, Shaw AC, Gagne AX, Chan HM (2013) Chronic exposure to PCBs (Aroclor 1254) exacerbates obesity-induced insulin resistance and hyperinsulinemia in mice. J Toxicol Environ Health A 76:701–715

13

Arch Toxicol Greenberg AS, Coleman RA, Kraemer FB, McManaman JL, Obin MS, Puri V, Yan QW, Miyoshi H, Mashek DG (2011) The role of lipid droplets in metabolic disease in rodents and humans. J Clin Invest 121:2102–2110 Ibrahim MM, Fjaere E, Lock EJ, Naville D, Amlund H, Meugnier E, Le Magueresse BB, Froyland L, Madsen L, Jessen N, Lund S, Vidal H, Ruzzin J (2011) Chronic consumption of farmed salmon containing persistent organic pollutants causes insulin resistance and obesity in mice. PLoS ONE 6:e25170 Jiang C, Qu A, Matsubara T, Chanturiya T, Jou W, Gavrilova O, Shah YM, Gonzalez FJ (2011) Disruption of hypoxia-inducible factor 1 in adipocytes improves insulin sensitivity and decreases adiposity in high-fat diet-fed mice. Diabetes 60:2484–2495 Jursa S, Chovancová J, Petrík J, Lokša J (2006) Dioxin-like and nondioxin-like PCBs in human serum of Slovak population. Chemosphere 64:686–691 Kang S, Tsai LT, Rosen ED (2016) Nuclear mechanisms of insulin resistance. Trends Cell Biol 26:341–351 Keller P, Petrie JT, De Rose P, Gerin I, Wright WS, Chiang SH, Nielsen AR, Fischer CP, Pedersen BK, MacDougald OA (2008) Fat-specific protein 27 regulates storage of triacylglycerol. J Biol Chem 283:14355–14365 Kim HY, Park SY, Lee MH, Rho JH, Oh YJ, Jung HU, Yoo SH, Jeong NY, Lee HJ, Suh S, Seo SY, Cheong J, Jeong JS, Yoo YH (2015) Hepatic STAMP2 alleviates high fat diet-induced hepatic steatosis and insulin resistance. J Hepatol 63:477–485 Kissebah AH, Vydelingum N, Murray R, Evans DJ, Kalkhoff RK, Adams PW (1982) Relation of body fat distribution to metabolic complications of obesity*. J Clin Endocrinol Metab 54:254–260 Kiviranta H, Tuomisto JT, Tuomisto J, Tukiainen E, Vartiainen T (2005) Polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls in the general population in Finland. Chemosphere 60:854–869 Lee DH (2012) Persistent organic pollutants and obesity-related metabolic dysfunction: focusing on type 2 diabetes. Epidemiol Health 34:e2012002 Lee DH, Lee IK, Song K, Steffes M, Toscano W, Baker BA, Jacobs DR Jr (2006) A strong dose-response relation between serum concentrations of persistent organic pollutants and diabetes: results from the National Health and Examination Survey 1999– 2002. Diabetes Care 29:1638–1644 Lee DH, Lee IK, Porta M, Steffes M, Jacobs DR Jr (2007) Relationship between serum concentrations of persistent organic pollutants and the prevalence of metabolic syndrome among nondiabetic adults: results from the National Health and Nutrition Examination Survey 1999–2002. Diabetologia 50:1841–1851 Liang L, Zhao M, Xu Z, Yokoyama KK, Li T (2003) Molecular cloning and characterization of CIDE-3, a novel member of the celldeath-inducing DNA-fragmentation-factor (DFF45)-like effector family. Biochem J 370:195–203 Liu F, Wang C, Zhang L, Xu Y, Jang L, Gu Y, Cao X, Zhao X, Ye J, Li Q (2014) Metformin prevents hepatic steatosis by regulating the expression of adipose differentiation-related protein. Int J Mol Med 33:51–58 Lumeng CN, Saltiel AR (2011) Inflammatory links between obesity and metabolic disease. J Clin Invest 121:2111–2117 Lv Z, Li G, Li Y, Ying C, Chen J, Chen T, Wei J, Lin Y, Jiang Y, Wang Y, Shu B, Xu B, Xu S (2013) Glucose and lipid homeostasis in adult rat is impaired by early-life exposure to perfluorooctane sulfonate. Environ Toxicol 28:532–542 Mesnier A, Champion S, Louis L, Sauzet C, May P, Portugal H, Benbrahim K, Abraldes J, Alessi M, Amiot-Carlin M (2015) The transcriptional effects of PCB118 and PCB153 on the liver, adipose tissue, muscle and colon of mice: highlighting of Glut4 and Lipin1 as main target genes for PCB induced metabolic disorders. PLoS ONE 10:e0128847

Arch Toxicol Miura S, Gan J, Brzostowski J, Parisi MJ, Schultz CJ, Londos C, Oliver B, Kimmel AR (2002) Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium. J Biol Chem 277:32253–32257 Mullerova D, Kopecky J (2007) White adipose tissue: storage and effector site for environmental pollutants. Physiol Res 56:375–381 Nishino N, Tamori Y, Tateya S, Kawaguchi T, Shibakusa T, Mizunoya W, Inoue K, Kitazawa R, Kitazawa S, Matsuki Y, Hiramatsu R, Masubuchi S, Omachi A, Kimura K, Saito M, Amo T, Ohta S, Yamaguchi T, Osumi T, Cheng J, Fujimoto T, Nakao H, Nakao K, Aiba A, Okamura H, Fushiki T, Kasuga M (2008) FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets. J Clin Invest 118:2808–2821 Pirola L, Johnston AM, Van Obberghen E (2004) Modulation of insulin action. Diabetologia 47:170–184 Puri V, Virbasius J, Guilherme A, Czech M (2008) RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27. Acta Physiol 192:103–115 Rizzatti V, Boschi F, Pedrotti M, Zoico E, Sbarbati A, Zamboni M (2013) Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution. Eur J Histochem 57:e24 Ruzzin J, Petersen R, Meugnier E, Madsen L, Lock EJ, Lillefosse H, Ma T, Pesenti S, Sonne SB, Marstrand TT, Malde MK, Du ZY, Chavey C, Fajas L, Lundebye AK, Brand CL, Vidal H, Kristiansen K, Froyland L (2010) Persistent organic pollutant exposure leads to insulin resistance syndrome. Environ Health Perspect 118:465–471

Rylander L, Rignell-Hydbom A, Hagmar L (2005) A cross-sectional study of the association between persistent organochlorine pollutants and diabetes. Environ Health 4:28 Salans LB, Knittle JL, Hirsch J (1968) The role of adipose cell size and adipose tissue insulin sensitivity in the carbohydrate intolerance of human obesity. J Clin Invest 47:153–165 Shen K, Shen C, Yu J, Yu C, Chen L, Shi D, Chen Y (2011) PCB congeners induced mitochondrial dysfunction in Vero cells. J Hazard Mater 185:24–28 Tanaka N, Takahashi S, Matsubara T, Jiang C, Sakamoto W, Chanturiya T, Teng R, Gavrilova O, Gonzalez FJ (2015) Adipocytespecific disruption of fat-specific protein 27 causes hepatosteatosis and insulin resistance in high-fat diet-fed mice. J Biol Chem 290:3092–3105 Toh SY, Gong J, Du G, Li JZ, Yang S, Ye J, Yao H, Zhang Y, Xue B, Li Q, Yang H, Wen Z, Li P (2008) Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice. PLoS ONE 3:e2890 Wolins NE, Brasaemle DL, Bickel PE (2006) A proposed model of fat packaging by exchangeable lipid droplet proteins. FEBS Lett 580:5484–5491 Yang H, Galea A, Sytnyk V, Crossley M (2012) Controlling the size of lipid droplets: lipid and protein factors. Curr Opin Cell Biol 24:509–516 Yu GW, Laseter J, Mylander C (2011) Persistent organic pollutants in serum and several different fat compartments in humans. J Environ Public Health 2011:417980 Zhai W, Xu C, Ling Y, Liu S, Deng J, Qi Y, Londos C, Xu G (2010) Increased lipolysis in adipose tissues is associated with elevation of systemic free fatty acids and insulin resistance in perilipin null mice. Horm Metab Res 42:247–253

13