Animal handling and treatment plan:
After prior approval of Institutional animal ethical committee (IAEC), 5-6 weeks old female and male BALB/c mice (Mus musculus) were obtained from animal house facility of CSIR-IITR, Uttar Pradesh, India. After one week of acclimatization, female mice were randomly distributed into three groups; Untreated controls, low dose arsenic group (0.04 mg/kg) and mid dose arsenic group (0.4 mg/kg) (n=10 per group). The experimental plan is shown in Fig.1. The dose 0.04 mg/kg is equivalent to the human exposure through drinking water containing 0.2 ppm arsenic which is the median arsenic concentration in drinking water (Jadhav et al., 2007). Another dose is selected ten-fold higher. Inorganic arsenic was administered through oral gavage of sodium (meta)arsenite in drinking water while control group received purified water only. Arsenic doses were freshly prepared in RO purified water. However, there was no postnatal exposure in offspring. After weaning, male and female offspring were segregated and further experiments were carried out on males only. Some of the offspring were sacrificed at postnatal 6 weeks for isolation of AdSCs (n=5 per group) while remaining were monitored regularly and sacrificed at 20 weeks.
Analysis of serum biochemical parameters:
The serum was collected from isolated blood through centrifugation at 2000xg for 20 min and kept at -80⁰C deep freezer. The biochemical parameters including blood glucose, total cholesterol and serum triglycerides were analyzed in serum samples using clinical chemistry analyzer (Rx Daytona, Randox, U.K.).
Assessment of glucose tolerance:
To determine the effects of prenatal arsenic exposure on glucose homeostasis in F1 males, oral glucose tolerance test (OGTT) and Intraperitoneal insulin tolerance test (IPITT) were achieved at 20 weeks of age. Briefly, the animals (n=5 per group) were kept on fasting for 6 hours and administered with 2g/kg BW of D-Glucose through oral gavage after the determination of baseline glucose levels. For IPITT, the animals were fasted for 6 hours followed by intraperitoneal administration of Huminsulin (biphasic isophane insulin, 40 IU/kg solution, manufactured by Gland Pharma Limited, Hyderabad, India) according to 0.75 IU/kg BW after dilution in phosphate-buffered saline (PBS, pH 7.4). After glucose or insulin stimulation, blood glucose levels were measured using Accu-Check Active glucometer at different timepoints including 15, 30, 45, 60, 90, and 120 min of glucose or insulin administration.
Lipolysis Assay:
To determine in vivo basal lipolysis, the glycerol levels were measured in serum of 20 weeks males of control and arsenic exposed groups using glycerol assay kit (Sigma, MAK117) according to manufacturer’s instructions. For isoproterenol stimulated lipolysis, 10mg/kg of isoproterenol was administered through intraperitoneal injection and the serum was collected after 15 minutes for glycerol assay.
Glucose uptake assay:
To determine the ex vivo glucose uptake in EWAT at 20 weeks, a fluorescent glucose analogue, 2-NBDG (Invitrogen, N13195) was used as previously described with slight modifications (Alonso-Castro et al., 2008). Briefly, chopped tissue (20-50 mg pieces) was incubated with DMEM/F12 medium supplemented with 1% pen-strep for 24 hours. Next day, the cells were starved for 5-6 hours in serum and glucose free media. 100nM Insulin was administered to the starved cells to stimulate glucose uptake for 30 minutes. Afterwards, the starved adipocytes were treated with 100µM 2-NBDG for 30 minutes followed by cold 1X PBS washing twice to get rid of free NBDG. The fluorescence was measured using microplate reader (Thermo Scientific™ Varioskan™) at 467/542 nm. The results were represented as mean fluorescence intensities (MFI) compared to control after normalization with total protein measured by using protein estimation kit (Thermo, 23225).
Primary culture of adipose derived stem cells:
AdSCs isolation protocol was adapted from previous work (Yu et al., 2011). Briefly, white epididymal fat deposits were excised following euthanasia (n = 5 per group). After washing in HBSS solution, fat tissue was minced and digested in a 1:1 (v/v) with ‘Collagenase/HBSS Solution” in a 37 °C shaking incubator for ∼45 min. After centrifugation at 300Xg for 7 min, the floating adipocytes were aspirated off. The remaining RBCs were lysed using RBC lysis buffer and after centrifugation, pellet was filtered through 100, 70 and 40 µm mesh filters to reduce cell heterogeneity. Remaining cell pellet was washed with ‘stromal media’ (DMEM:F12 (Sigma) with 10% FBS and 1× antibiotic-antimycotic (Gibco). Remaining cells were resuspended in stromal media and transferred to a flask, designated as passage 0. After 80-90% confluency, cells were trypsinized and subcultured.
Assessment of adipogenesis in adipose derived stem/stromal cells:
After two days of post-confluence, AdSCs were ready to differentiate. The stromal media was exchanged with differentiation media containing insulin (10µg/ml), dexamethasone (1µM), Indomethacin (100 µM) and 3-isobutyl-1-methylxanthine (0.5mM) for initiating adipogenesis (day 0). On day 6 post-induction, the differentiation media was replaced with the maintenance media containing only insulin (10µg/ml) and dexamethasone (1µM). Mature adipocytes were formed by day 8 post-induction. Assessment of lipid accumulation was done using Lipophilic dyes, Nile red (Himedia, RM9734) and Oil red O (Sigma, O0625). Nile red was administered to the cells through culture media (1µg/ml) for 15 minutes in CO2 incubator followed by fixation. After three times washing with 1X PBS, coverslips were mounted using fluoroshield mounting media containing DAPI (Sigma Aldrich, USA).
For Oil red O (ORO) staining, the fixed cells were washed with distilled water followed by 60% isopropanol washing for removal of extracellular lipids. 0.35% Oil red O stock solution was prepared in absolute propanol and freshly diluted in distilled water (3:2). After filtration, the cells were kept in ORO working solution for 15 minutes at room temperature. After excess washing with distilled water, the oil red O was eluted using absolute 2-propanol and absorbance was measured at 500nm using spectrophotometer (Thermo Scientific™ Varioskan™).
Multiplex based assessment of other metabolic markers, inflammatory cytokines and cell signaling proteins:
Bead based Multiplex Analysis of inflammatory cytokines, cell signaling molecules and metabolic biomarkers was done using MAGPIX Multiplex Reader (Bio-Rad Laboratories). The serum was recovered from the blood taken from the heart. Periodical profiling of biomarkers in blood was performed from the sample collected by retro-orbital bleeding. The Bio-PlexProTM Mouse cell signaling Assay Kit (Bio-Plex, customized panel for ERK1/2 (pT185/pY187), P38MAPK (pT180/pY182), Akt (pS473/pT308), TNFα mouse ProcartaPlex™ (Invitrogen, EPXS010-20607-901) and Bio-PlexProTM Mouse Diabetes 8-plex panel (Bio-Plex, 171F7001M) was used according to the manufacturer’s instructions. The fluorescence was read using MAGPIX Multiplex Reader (Bio-Rad Laboratories). Results were represented as net mean fluorescent intensity (MFI) of different groups.
Quantitative Real Time PCR:
Total RNA was extracted by using Trizol Reagent (Invitrogen, 15596026). Afterwards, cDNA was prepared using cDNA Reverse Transcription Kit (Thermo, 4368813). Then, the cDNA was subjected to quantitative real time PCR using 7500 Fast Real Time PCR System (ABI, USA) with Sybr Green containing master mix (Takara Bio, RR820A). Selected gene primers were purchased from Reliable-scientific. GAPDH and β-actin was used as endogenous control. The mean of threshold cycle number (Ct value) was calculated and relative gene expression compared to control was determined after normalization with endogenous control.
Histology and immunostaining:
For histological analysis of epididymal white adipose tissue (EWAT), paraffin-embedded blocks were prepared after fixation in 10% neutral buffered formalin (NBF) and serial dehydration in ethanol. 5μm thick histological sections were stained with hematoxylin and eosin (H&E). Images were acquired using bright-field microscope (Nikon Eclipse Ni-U microscope) and cell diameter was measured using NIS-Elements Microscope Imaging Software from five different regions per slide after analyzing three slides (n=3 animal per group).
For immunostaining, 5μm thick sections of adipose tissue were deparaffinized, rehydrated and kept for antigen retrieval at 60˚C for 45 minutes. After washing with TBST (pH 7.6), blocking was done using 3% BSA in TBST. Anti-F4/80 (sc-26643), anti-Glut4 (Abcam, ab654), and anti-Perilipin (Abcam, ab35026) antibodies were used at 1:100 dilution and incubated overnight at 4 ˚C. After overnight incubation, the sections were washed with TBST followed by incubation with Alexa Fluor 488 (Invitrogen A-11008) and Cy3 (Invitrogen A-10520) tagged anti-rabbit IgG antibody for 2-3 hours at room temperature. The sections are mounted using DAPI (Sigma, F6057) after washing with TBST.
Chromatin immunoprecipitation (ChIP) analysis of AdSCs:
ChIP was performed as previously described (Abcam). Briefly, 5X107 cells were cross-linked using 1% HCHO for 10 minutes at room temperature, which was quenched by glycine at final concentration of 0.125M. Further, the cells were sonicated using Ultravibra sonicator for 10 cycles (30 sec on/ 30 sec off) at 35% amplitude to acquire the fragments ranging between 500-1000bp. 25µg of sonicated chromatin was incubated with 5µg of CTCF antibody (CST, 3418S) overnight, while 10% chromatin was kept aside for use as input. The antibody bound chromatin was precipitated using protein A/G beads after multiple washing with low salt and high salt buffer to avoid nonspecific binding. Further, DNA was isolated from precipitated chromatin using 10% Chelex for rt-PCR. Results were calculated as % input.
Methylated DNA immunoprecipitation (medIP) analysis of AdSCs:
meDIP was performed as previously described (Thu et al., 2009). Briefly, the DNA was isolated using standard PCI method. Further, 1µg of isolated DNA was sonicated using Ultravibra sonicator for 20 sec at 35% amplitude to acquire DNA fragments ranging between 300-1000bp. 80% of sonicated DNA was denatured at 95⁰C for 10 min while 20% was kept aside as input. After immediate cooling, denatured DNA was incubated with 5µg of 5-mC antibody (Abcam, ab214727) overnight at 4⁰C. The antibody bound DNA was precipitated using protein A/G beads after multiple washing with low salt and high salt buffer to avoid nonspecific binding. Further, DNA was isolated from the complex using 10% Chelex for rt-PCR. Results were calculated as % input.
Western blotting:
For western blot analysis, the tissue/cells were homogenized in RIPA buffer and centrifuged for removal of insoluble cell debris. The cell lysate containing 40µg of protein was separated using 10% SDS-PAGE and transferred on PVDF membrane for further analysis. After blocking of nonspecific sites for 60-120 minutes at room temperature, blots were incubated with primary antibody as anti-PPARγ (Abcam, ab209350), anti-Perilipin (Abcam, ab35026), anti-SCD1 (CST, C12H5), anti-p-IGF1R (Abcam, ab205332), and anti-IGF2 (Abcam, ab262713) at 1:1000 dilution for overnight in a refrigerated shaker. After incubation with HRP-tagged secondary antibody (CST, 7074S) at 1:2000 dilution, the blots were analyzed using Imagelab software (Bio-rad). β-Actin (sc-47778) was used as an endogenous control to quantify the relative expression.
Statistics:
All data are represented as mean±SD. One tailed t-test, one-way analysis of variance (ANOVA) and two-way ANOVA was used to determine the statistical significance of data between the groups using Microsoft office Excel and GraphPad Prism 8 software. The p-value<0.05 was considered statistically significant in all the experiments.