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Transcriptomic and Functional Analyses on the Effects of Dioxin on Insulin Secretion of Pancreatic Islets and #-cells Keng Po Lai, Hin Ting Wan, Alice Hoi-Man Ng, Jing Woei Li, Ting-Fung Chan, and Chris Kong-Chu Wong Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02830 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 8, 2017

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Transcriptomic and Functional Analyses on the Effects of Dioxin on Insulin Secretion of Pancreatic Islets and β-cells.

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Short title: Transcriptomic and Functional Analysis of TCDD-treated Islets.

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8 Keng Po Lai2#, Hin Ting Wan1#, Alice Hoi-Man Ng1, Jing Woei Li 2,3, Ting Fung Chan3, Chris Kong-Chu Wong1*

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Croucher Institute for Environmental Sciences, Partner State Key Laboratory of Environmental and Biological Analysis, Department of Biology, Hong Kong Baptist University, 2Department of Chemistry, City University of Hong Kong, 3 School of Life Sciences, Hong Kong Bioinformatics Centre, The Chinese University of Hong Kong, Hong Kong SAR, China.

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Equal contribution *Corresponding author, Dr Chris KC Wong Croucher Institute for Environmental Sciences, Partner State Key Laboratory of Environmental and Biological Analysis, Department of Biology, Hong Kong Baptist University, Email address: [email protected]. Phone: 852-34117053

Keywords: Akt-mTOR, AMPK, ATP, GSIS, pyruvate dehydrogenase kinase.

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The authors declare they have no actual or potential competing financial interests.

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ABSTRACT

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In this study, transcriptomic and Ingenuity Pathway Analysis (IPA) underlined that an ex-vivo

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TCDD treatment (0.1 nM) stimulated insulin-release in mouse pancreatic islets via the effect on the

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Akt-mTOR-p70S6K, AMPK and ERK1/2 pathways. Functional studies using both ex-vivo islets

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and the mouse β-cell-line (Min-6) validated the stimulatory effects of TCDD (0.1 and 1 nM) on

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basal-insulin secretion. At 0.1 nM TCDD treatment on Min-6, western blot analysis showed

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activation of ERK1/2 and decreased expression of pyruvate dehydrogenase kinase (PDK). A

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reduction of PDK expression is associated with an increase of pyruvate dehydrogenase flux. This

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observation was supported by the detection of significantly higher cellular ATP levels, an

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increase of glucose-stimulated-insulin-secretion (GSIS), and an inhibition of the AMPK pathway.

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At 1 nM TCDD treatment on Min-6, significant inhibitions of the Akt-mTOR pathway, cellular ATP

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production, and GSIS were evident. The experimental studies in Min-6 supported the IPA of

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transcriptomic data in pancreatic islets. Collectively, TCDD treatment caused an elevated basal-

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insulin release in both islets and β-cell cultures. Moreover, our data revealed that the modulation of

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the Akt-mTOR-p70S6K, AMPK and ERK1/2 pathways might be an important component of the

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mechanism for the TCDD-perturbing effects on ATP production in β-cells in affecting insulin

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secretion.

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INTRODUCTION

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Type-2 diabetes (T2D) is a pandemic and constitutes 90% of people with diabetes. Genetic and

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nutritional factors are known to be major risk factors for this disease. However, these factors

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alone are still not able to account for the high prevalence of metabolic disease worldwide in

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recent years1,2. Therefore, additional efforts are required to reveal the pathogenesis of the disease.

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Recently, scientific researches have focused on characterizing other potential risk factors that

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may disrupt body energy homeostasis. The role of environmental chemical contaminants has

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drawn significant attention as potential contributors, independent of diet and physical activity3.

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Possible roles of chemical contaminants in metabolic diseases have been suggested, based on the

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observation of positive correlations between the increase in incidence of metabolic disorders and

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the use of industrial chemicals in the past 50 years4. Moreover, epidemiological studies in

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chemical-spill incidents and laboratory exposure studies hint at the possible link between

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pollutant exposure to disturbed glucose homeostasis and insulin resistance5.

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The well-known anthropogenic pollutant dioxin (i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin,

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TCDD) is proposed to be a putative diabetogenic factor6, based on the analysis of numerous

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human epidemiological and laboratory animal data. During the Vietnam War in the early 60s,

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dioxin exposure (via Agent Orange) was reported to be associated with an increase in the

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prevalence of diabetes mellitus, with 111,726 identified cases in Korean Vietnam veterans7.

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From the residential exposure to dioxins at the chemical explosion in Seveso in 1976, an inverse

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association between maternal serum dioxins with birth weight was recognized in 1,211 cases of

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post-explosion pregnancies8. A connection between size at birth to the risk of developing insulin

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resistance and cardiovascular diseases is known9. Moreover, a positive association between

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serum dioxin concentrations and metabolic syndrome was found in women who were ≤ 12 years

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of age at the time of exposure in Seveso10. An increased risk for diabetes was reported in other

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incidences of dioxin exposure, like in phenoxy-herbicide and chlorophenol production workers11,

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and cohorts in the Yucheng accident12. Recent studies showed positive associations between

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serum dioxin concentrations and insulin resistance13,14, hypertension and glucose intolerance15,16.

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However, a prospective association analysis in elderly Swedish adults showed no direct

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association between serum dioxin levels and T2D17. These observations suggest that the risk of

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dioxins may be greater when exposed at younger age. Nevertheless, these studies do not establish

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causality, because the cross-sectional study design makes it difficult to assess the exposure-

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disease association. On the other hand, laboratory data provided some mechanistic data to

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support the notion that dioxin exposure is associated with dysglycemia. An earlier study of

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dioxin exposure in rats reported a pathological outcome of wasting syndrome, where diminishing

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availability of glucose for metabolism was noted18,19. The hallmark of wasting syndrome, a

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decrease of hepatic gluconeogenesis, was found to be mediated by the activation of the cytosolic

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receptor, aryl hydrocarbon receptor (AhR), which stimulates the expression of TCDD-inducible

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poly(ADP-ribose) polymerase to suppress hepatic glucose synthesis20,21. In addition, inhibitory

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effects of dioxins on glucose uptake in the brain, adipose and pancreas were demonstrated in

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guinea pigs20,22, via modulation of glucose transporting activities23.

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In fact, establishing an association between TCDD exposure and the development of diabetes is

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inherently difficult. A comparison of the gene expression pattern of pancreatic islets between

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normal and T2D people revealed a significant reduction in the levels of AhR nuclear translocator

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(ARNT) in patients with the disease24. ARNT is a member of the bHLH/PAS family of

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transcriptional factors, and is a heterodimer partner of AhR or hypoxia-inducible factor-1α25.

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Depletion of ARNT expression in pancreatic β-cells of ARNT-knockout mice caused an 4 ACS Paragon Plus Environment

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impairment of glucose-stimulated insulin release (GSIS), with no significant effects on insulin

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content and the total number of isolated islets24. The actions of ARNT were found to regulate

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changes in islet gene expression. Since the dysfunction of pancreatic β-cells is known to be a key

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component in the pathogenesis of T2D, and the AhR/ARNT heterodimer regulates many

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pancreatic β-cell genes24, it warrants further analysis of the underlying mechanistic effects of

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dioxin on pancreatic islet functions.

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MATERIALS AND METHODS

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Experimental Animals and Chemicals

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CD-1 mice were kept and maintained according to the “Guidelines and Regulations” of the

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Department of Health, Hong Kong Special Administrative Region. The animal handling

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procedure was approved by the Animal Committee of the Hong Kong Baptist University (Permit

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no. 261812). Male CD-1 mice (6-8 weeks old) were received from the “Animal Unit” of the

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University of Hong Kong. The animals, housed in polypropylene cages with sterilized bedding,

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were kept under controlled temperature (22 °C) and 12-h/12-h light-dark cycle. TCDD was

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purchased from Sigma Aldrich (CA, USA). A stock solution of TCDD (1 µM in dimethyl

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sulfoxide, DMSO) was made. Further dilutions were prepared in DMSO and phenol red-free

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DMEM-high glucose medium with charcoal-stripped serum. The final DMSO concentration was

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less than 0.1%. The corresponding percentage of DMSO was added as the vehicle control.

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Pancreatic islets isolation

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We followed the method of pancreatic islet isolation described previously by Nunemaker and

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coworkers26. Briefly, male mice were euthanized by cervical dislocation. For each mouse, the

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animal was dissected and the common bile duct attached to the duodenum at the papilla was

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identified. A 30-gauge 0.5-inch needle secured to a 5 ml syringe was cannulated into the bile

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duct and tied with a suture. A 3 ml aliquot of collagenase solution [1.4 mg/ml Collagenase-P

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(Roche) in HBSS/1% BSA] was injected slowly to completely inflate the pancreas. The inflated

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pancreas was then dissected and placed into a 50 ml conical tube containing 3 ml of collagenase

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solution, and then incubated at 37 oC for 11 min, without agitation. The tissue suspension was

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then shaken vigorously by hand for approximately 5 s. The enzymatic digestion was then slowed

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down by adding 15 ml of ice-cold HBSS/1% BSA. The suspension was centrifuged for 2 min at

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1200 rpm. The pellet was washed with 10 ml of HBSS/1% BSA twice and the final suspension

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was filtered through a 420 µm sized sieve into a new 50 ml conical tube. The filtered suspension

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was filled up to 20 ml with HBSS/1% BSA, followed by centrifugation for 2 min at 1200 rpm.

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The pellet was resuspended in 10 ml of Histopaque 1100 solution (Sigma), and then centrifuged

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for 20 min at 1200 rpm. The supernatant was collected and diluted with 25 ml of HBSS/1% BSA

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in a new 50 ml conical tube, and then centrifuged for 4 min at 1500 rpm. The pellet was washed

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in 10 ml of HBSS/1% BSA and finally resuspended in RPMI 1640 (10% FBS, 1% Pen/Strep).

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After overnight incubation, islets from the preparation of the individual pancreas were picked

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under a light microscope. Islets from two mice were pooled as one biological sample (~150 islets

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per replicate), and three replicates (6 animals per group) were used for the control and TCDD

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treatment. The islets were then treated with either DMSO (solvent control) or 0.1 nM TCDD for

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24 h.

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Transcriptomic Analysis of Ex-vivo Islets

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Total RNA was isolated from the control and TCDD-treated islets using the mirVanaTM RNA

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isolation kit (Applied Biosystems). The RNA quality was determined (Agilent 2100 Bioanalyzer

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system), RNA with a RNA Integrity Number > 8 was used for library construction and

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sequencing (pair-end reads, 150 bp read-length) using the Illumina MiSeq sequencer. Sequence-

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reads were trimmed according to BWA's – q algorithm as described previously27. Quality-

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trimmed sequence reads were mapped onto the mouse genome reference GRCm38 using STAR

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v2.5.2b. Read-count data were used for differential expression analysis using the edgeR

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package28. Genes with a FDR < 0.05 were considered as differentially expressed genes (DEGs).

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The DEGs were analysed using the Database for Annotation, Visualization and Integrated

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Discovery (DAVID) v6.829,30 for functional annotation and to determine the biological processes.

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Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to identify the

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alteration of pathways caused by TCDD treatment. Moreover, Ingenuity Pathway Analysis

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(IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity) was applied to underline the

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molecular interaction networks. Canonical pathways with p < 0.05 were set as statistically

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significant.

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Real-time PCR analysis

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Total RNA with an A260/A280 > 1.8 were used for quantitative real-time PCR analyses. For each

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sample, complementary DNA (cDNA) was prepared from 150 ng of total RNA using the VILO

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cDNA synthesis Kit (Invitrogen, ThermoFisher). Gene-specific primers were designed according

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to the published sequences (Supplementary Table 1). Real-time PCR was implemented with a

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program of 10 min at 95 oC, followed by 40 cycles of 98 °C for 20 s, 60 °C for 1 s and 72 °C for

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60 s. The copy numbers of standards and cDNAs of the samples were measured using the

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StepOne Real-time PCR system with the KAPA HiFi Hotstart ReadyMix PCR kit (KAPA

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Biosystems). The data were normalized with the transcript levels of the housekeeping gene,

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mouse actin. Control amplifications were performed either without the step of reverse

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transcription or RNA.

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Cell culture of mouse pancreatic β-cell-line

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Min-6 cells were maintained in a humidified atmosphere with 5% CO2 in a complete DMEM-

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high glucose medium (Gibco, ThermoFisher) supplemented with 10% heat-inactivated fetal

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bovine serum, 50 µM 2-mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin

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(Invitrogen). Cell cytotoxicity was assessed by the MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-

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Diphenyltetrazolium Bromide, Invitrogen] following the manufacturer’s instructions. In brief,

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cells were incubated in MTT solution at 37 °C for 4 h, followed by the addition of 100 µl of

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DMSO and mixed thoroughly. After incubating at 37 °C for 10 min, the absorbance at 540 nm

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was read using a microplate reader.

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Basal and Glucose-stimulated Insulin Secretion

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Min-6 cells were seeded at a density of 5 x 104 cells/cm2 in a 24-well plate for 48 h before drug

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treatment. Isolated pancreatic islets were evenly distributed in a 24-well-plate (60 islets per well).

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Each culture was treated with either DMSO, 0.1 nM or 1 nM TCDD for 24 h. The Min-6 cells

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and islets were then washed twice with Krebs-Ringer bicarbonate buffer (KRBB, containing 2.4

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mmol/L CaCl2, 5.4 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 120 mmol/L NaCl,

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20 mmol/L HEPES, 0.1% BSA, pH 7.0) to remove leftover glucose in the medium. Cells were

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then pre-incubated in KRBB containing 1.67 mmol/L glucose for 1 h in the incubator, and

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afterwards incubated with 1.67 mmol/L, followed by 16.7 mmol/L glucose, 1 h for each. The

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supernatants were collected as basal and stimulated insulin secretions respectively. The levels of

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insulin were measured by using the Insulin AlphaLISA detection kit and the fluorescent signals

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were measured by the EnSight Multimode plate reader (PerkinElmer). Data were normalized by

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measuring protein concentrations using the Bio-Rad DC protein assay.

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ATP Determination

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Min-6 cells were seeded at a density of 5 x 104 cells/cm2 in a 24-well plate for 48 h before drug

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treatment. After 24 h of TCDD (0.1 and 1 nM) incubation, the cells were lysed in 1X passive

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lysis buffer (Promega) followed by centrifugation at 10,000 x g for 2 min. Aliquots of 10 µl of

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samples were subjected to ATP determination using the ATP determination kit (Invitrogen) and

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read by the VICTOR X4 multilabel plate reader (PerkinElmer). Data were normalized by

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measuring the protein concentrations.

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Western Blotting and Immunofluorescence Analysis (IF)

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Min-6 cells were seeded at a density of 2 x 105 cells/cm2 in a 6-well plate for 48 h before TCDD

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treatment. The protein samples were lysed in the radioimmunoprecipitation assay buffer (RIPA:

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150 mM sodium chloride, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 50 mM Tris-

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Cl, pH 8.0) mixed with a cocktail of protease inhibitors (Sigma-Aldrich) at 1:100 dilution.

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Concentrations of protein were measured and 15-20 µg of samples were subjected to

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electrophoresis in 7.5% or 10% polyacrylamide gels. The gels were then blotted onto a PVDF

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membrane (Bio-Rad) and blocked in 5% non-fat dry milk (wt/vol) in PBS-0.1% Tween-20 buffer.

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Primary and secondary antibodies (Supplementary Table 2) were prepared using 0.1% BSA

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(wt/vol) or 3% non-fat dry milk in PBS-Tris buffer (10 mM Tris, pH 7.4). The protein blots

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were incubated with the primary antibody overnight at 4oC, then secondary antibody for 1 h at

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room temperature. The blots were incubated with WESTSAVE Up (AbFrontier) and protein

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bands were analyzed using ImageJ software.

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For immunofluorescent staining, the cells were seeded at a density of 1 x 105 cells/cm2 on a

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cover glass in each well of a 12-well plate for 48 h before TCDD treatments. After 24 h of the

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treatment, cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) at room temperature for

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10 min. The cells were permeabilized using 0.1% Triton X-100 in PBS for 10 min. The cells

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were then incubated with 1% (wt/vol) BSA in a PBS-Tris buffer, followed by the primary

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antibody of guinea pig anti-insulin (DAKO) or rabbit anti-pdx-1 (Millipore) overnight, and a 1 h

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incubation with Alexa-Fluor-conjugated secondary antibodies (Invitrogen, Thermofisher). Nuclei

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were visualized with Vectashield antifade mounting medium with DAPI (4’,6’-diamidino-2-

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phenylindole, Vector laboratories). Images in TIFF format were examined using Adobe

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PhotoShop in Creative Suite 6. The imaging data were the representative results from at least

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four independent experiments.

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Statistical analysis

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Statistical evaluations were implemented using SigmaStat version 3.5. Data were analyzed by the

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Student’s t-test or one-way analysis of variance (ANOVA) followed by Duncan’s multiple range

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test. All data are presented as statistical mean ± SD. A p value < 0.05 was used as the cutoff for

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statistical significance.

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RESULTS AND DISCUSSION

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Comparative transcriptomic analysis revealed the activation of the insulin signalling pathway

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upon TCDD treatment.

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In an attempt to understand the molecular mechanisms underlying the effects of TCDD on pancreatic

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islets, comparative transcriptomic analysis on 0.1 nM TCDD-treated and DMSO (solvent control)-

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treated islets were conducted. Quality-trimmed Illumina reads of 20.65 M and 23.14 M were

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acquired from the TCDD- and DMSO -treated islets, respectively. A total of 3.3 Gb of clean bases

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was retrieved from the RNA sequencing (Supplementary Table S3). A total of 5484 differentially

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expressed genes (DEGs), including 5179 up- and 305 down-regulated genes, were identified from

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comparing the data between TCDD- and DMSO-treated islets (Supplementary Table S4). The DEGs

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were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID)

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v6.7 and Ingenuity Pathway Analysis (IPA) to identify the altered gene networks and canonical

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pathways. The result of KEGG from DAVID analysis showed that TCDD treatment perturbed

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molecular and biochemical pathways that affected islet functions (Fig. 1A and Supplementary Table

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S5). The pathways directly related to the islet function and insulin secretion include the (i)

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phosphatidylinositol signaling system, (ii) calcium signaling pathway, (iii) mammalian Target of

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Rapamycin (mTOR) signaling pathway, (iv) Wnt signaling pathway, (v) insulin signaling pathway

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and (vi) Type II diabetes mellitus. Analysis of the Ingenuity Canonical Pathway (IPA) revealed that

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the TCDD treatment significantly modulated 275 pathways (p < 0.05), in which 154 pathways were

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stimulated and 13 pathways were inhibited. The canonical pathways directly associated with islet

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function and insulin secretion are (i) PI3K/AKT, (ii) mTOR signaling, (iii) p70S6K signaling, (iv)

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AMP-activated protein kinase (AMPK) signaling, (v) calcium signaling, (vi) insulin receptor

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signaling, (vii) Type I diabetes mellitus signaling, and (viii) Type II diabetes mellitus signaling

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(Supplementary Table S6). The canonical pathways associated with TCDD binding include (i) aryl

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hydrocarbon receptor signaling and (ii) estrogen receptor signaling. Based on the pathway analyses,

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several candidate genes were selected for validation by real-time PCR assays using an independent

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cohort of isolated pancreatic islets (Fig. 1B). A highlighted IPA canonical pathway – insulin receptor

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signaling was schemed (Fig. 1C). Perturbation of the Akt-mTOR-p70S6K and AMPK pathways

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was observed, which predicted a stimulation of insulin release upon exposure to 0.1 nM TCDD.

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Intriguingly, the mTOR and AMPK-pathways are known to be important in nutrient and energy

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sensing in pancreatic β-cells31. This observation prompted us to conduct experiments using ex vivo

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pancreatic islet culture and the mouse islet β-cells, Min-6, to elucidate the effects of TCDD on insulin

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secretion.

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TCDD stimulated the basal insulin secretion

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To verify the predicted effects of TCDD on islet functions, mouse pancreatic islets were isolated to

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measure insulin release upon TCDD treatment (Fig. 2A). Ex-vivo islet culture incubated with 0.1 nM

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or 1 nM TCDD for 24 h showed increases of basal insulin secretion (Fig. 2B). No significant

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differences were observed in the glucose-stimulated-insulin-release (GSIS) in both the control and

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TCDD treated groups. However, the ratio of GSIS/basal insulin secretion was significantly lower in

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the TCDD-treated islets (Fig. 2C). To validate the effects and elucidate the mechanistic action of

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TCDD, the mouse β-cell line, Min-6 was used in the subsequent experiments. No noticeable effect

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on cell viability was observed from increasing the concentrations of TCDD treatment (1 pM – 4 nM)

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(Fig. 3A). Effects of low (1.67 mM) and high glucose (16.7 mM) levels on cellular ATP levels of the

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control and TCDD treated cells were measured (Fig. 3B). TCDD treatments (0.1 and 1 nM) caused

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significant increases of ATP levels in cells incubated at the low glucose level. Only 0.1 nM TCDD

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treatment increased ATP production in cells incubated at the high glucose medium. The increases of

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cellular ATP levels were positively correlated with the levels of insulin release at both low and high

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glucose incubation (Fig. 3C). At the low glucose level, the basal insulin release in TCDD treatment

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was significantly elevated. At the high glucose incubation, the level of insulin release (GSIS) was

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significantly induced in 0.1 nM TCDD treatment as compared with the control. Similar to the results

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of the ex-vivo pancreatic culture, the ratios of GSIS/basal insulin secretion were significantly lower

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in the TCDD-treated cells (Fig. 3D). We then tested if the TCDD treatment affected the expression

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levels of AhR-target genes (i.e. CYP1A1) and the transcriptional factors for β-cell differentiation

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and maturation [i.e. paired Box 4 (Pax4), pancreatic and duodenal homeobox 1 (Pdx1)]. A dose-

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dependent induction of CYP1A1 protein was observed (Fig. 3E). These results implied that the

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AhR-ARNT pathway responded to TCDD treatment in the β-cells. No significant changes in the

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expression levels of Pax4 and Pdx1 were observed among the control and treatment groups (Fig.

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3E). No noticeable changes in the cellular levels of Pdx1 and insulin were observed in the

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immunofluorescent staining (Fig. 4). Taken together, both ex vivo and cell-line culture models

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showed elevations of basal insulin secretion upon TCDD treatment. The functional analysis

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supported the results of the transcriptomic and bioinformatics analyses.

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TCDD altered cell signaling pathways in β-cells

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With reference to the canonical pathways identified in the transcriptomic data, western blot

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analyses of the AKT-mTOR-p70S6K and AMPK signaling pathways were implemented to

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elucidate the modulations of these signaling pathways upon TCDD treatment in Min-6. At 0.1 nM

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TCDD treatment of Min-6, the western blot analysis showed activation of ERK1/2 but decreased

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levels of pyruvate dehydrogenase kinase (PDK) (Fig. 5A). Functionally, the stimulation of

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ERK1/2 pathway could positively regulate pyruvate dehydrogenase (PDH) flux via the reduction

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of PDK expression32, resulting in an increase of mitochondria-derived ATP. Consistently,

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significant higher cellular ATP levels were detected in the cells treated with 0.1 nM TCDD at

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both low and high glucose conditions. In addition, the TCDD treatment significantly reduced the

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levels of pAMPKα (Fig. 5A). Since AMPK is a key regulator of cellular energy status in

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response to changes in cellular ATP levels33, these results supported the data of greater cellular

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ATP contents in the 0.1 nM TCDD treated cells. The treatment however showed no noticeable

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effect on the phosphorylation of the protein kinases Akt, mTOR and p70S6K as compared with the

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control. In fact, multiple factors control basal insulin secretion and GSIS. The conventional model

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establishes that an increase of cellular ATP leads to the closure of ATP-sensitive K+-channels,

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followed by the opening of voltage Ca2+-channels, which triggers insulin exocytosis34. Intriguingly, a

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previous study using the insulin-secreting β-cell line (INS-1E) demonstrated that TCDD treatment

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at high doses (50-100 nM) could increase the cellular Ca2+-levels to trigger insulin exocytosis,

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independent of glucose stimulation35. Taken together, our data and the previous study supported

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our observation that 0.1 nM TCDD treatment elicited greater levels of basal insulin release and GSIS

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in the culture.

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On the other hand, inhibition of the Akt/mTOR signaling pathway at 1 nM TCDD treatment was

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observed. The treatment showed significant reductions in the levels of p-Akt (ser 773) and p-mTOR

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(ser 2448) (Fig. 5B). No significant changes in the levels of pAMPKα, pERK1/2 and PDK were

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found. The inhibition of the Akt-mTOR pathway supported the observation of significantly lower

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cellular ATP levels and GSIS in the cells at the high glucose condition, as compared to the 0.1 nM

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TCDD treatment. Even though the 1 nM TCDD treatment did not cause cytotoxic effects on the

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cells as demonstrated by the MTT assay, suppression on the mTOR (the sensor for cellular

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nutrient levels)36 for the coordination of energy metabolism was evident. Indeed, another study

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showed the cytotoxic effects of high concentrations of TCDD treatment (12.5 and 25 nM) on

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INS-1E, mediated by increased rates of Ca2+ influx and mitochondria depolarization37. While the

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treatment of INS-1E with 1 nM TCDD reduced GSIS. Similar findings on TCDD-impaired GSIS

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were also reported in other animal studies. In TCDD-treated rats (1 µg/kg b.w. i.p.), a decrease

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of glucose uptake in isolated pancreatic islets was found to be associated with an inhibitory

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effect on GSIS38. Moreover, an impairment of GSIS was shown using TCDD pre-treated mice

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(10 µg/kg b.w. i.p.) and the mechanistic action was found to be AhR-dependent39. Although our

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data of using 1 nM TCDD treatment on Min-6 did not demonstrate an inhibition of GSIS, treatment

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at this concentration showed a significant decrease of GSIS when compared with the cells incubated

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at 0.1 nM TCDD. The dissimilar observation in GSIS at the two different doses of TCDD treatments

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might be due to the differential effects on the energy and nutrient sensors, AMPK-signaling (0.1 nM

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TCDD) and AKT-mTOR-signaling (1 nM TCDD), resulting in the perturbation of cellular ATP

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levels and insulin secretion.

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Collectively, the present study characterized the transcriptional and functional effects of TCDD on

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mouse pancreatic islets and the β-cells (Min-6). The transcriptome and IPA of the pancreatic islets

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underlined the effects of TCDD on the perturbations of the Akt-mTOR-p70S6K, AMPK and

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ERK1/2 pathways, predicting the outcome of stimulating insulin release. Subsequent experimental

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studies confirmed the stimulatory effects of TCDD on basal insulin secretion in the two culture

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systems. With the benefit of hindsight and the data of the present study, the stimulation of insulin

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release was suggested to be mediated by an elevation of cytosolic Ca2+ and ATP levels. Moreover,

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western blot analysis of the mouse β-cell-line supported the data of IPA and identified the effects of

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TCDD on AMPK and mTOR signaling, the energy and nutrient sensors in the β-cells31. The

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modulation of these signaling pathways might be an important component of the mechanism for

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TCDD-perturbing effects on ATP production in β-cells in affecting basal and glucose-stimulated

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insulin release.

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ACKNOWLEDGEMENTS

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This work was supported by the Partner State Key Laboratory of Environmental and Biological

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Analysis (SKLP-16-17-P01, HKBU) and Department of Biology (Strategy Development Fund,

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40-49-038, HKBU) to Dr Chris KC Wong (Hong Kong Baptist University).

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FIGURE LEGENDS

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Figure 1. Comparative transcriptomic analysis of mouse pancreatic islets upon TCDD

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treatment. Pancreatic islets were prepared separately from individual mouse. Islets from two

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mice were pooled as one biological sample (~150 islets per replicate), and three replicates (6

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animals per group) were used for the control and TCDD treatment. (A) KEGG pathway analysis

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using DAVID on TCDD-dysregulated genes. (B) Validation of transcriptomic results on

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independent cohorts of islet-culture using qPCR analysis (experimental replicates n = 4). (C)

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Canonical pathway analysis of IPA shows the activation of insulin receptor signaling. Grey shade

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represents overexpression; dot represents suppression. The intensity of color shade or dot represents

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the degree of overexpression or suppression, respectively.

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Figure 2. Effects of TCDD exposure for 24 h on ex-vivo pancreatic islets. (A) A

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photomicrograph of isolated mouse islets. Effects of TCDD on (B) the levels of the basal insulin and

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glucose-stimulated insulin release (n = 4, experimental replicates). The levels of basal insulin

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secretion were significant greater in the TCDD-treated islets as compared with the control group (p