Animal maintenance and treatment

All procedures were performed in accordance with the guidelines of the Canadian Council of Animal Care. Nulliparous 200–250 g female Wistar rats (Envigo, Indianapolis, IN) were maintained under controlled lighting (12:12 L:D) and temperature (22°C) with ad libitum access to food and water. Two weeks prior to mating, the dams were randomly assigned to receive either saline (vehicle) or nicotine. Dams (N = 10 per group) were injected with saline or 1.0 mg/kg/day nicotine bitartrate (Sigma-Aldrich, St. Louis, MO) subcutaneously for 14 d prior to mating, and during pregnancy. Dams were allowed to deliver normally, and pups were euthanized at postnatal day 1 (PND1) for pancreas tissue collection. The dose of nicotine used in this animal model results in maternal serum cotinine concentrations of 136 ng/ml, ^{Reference Holloway, Kellenberger and Petrik13} which is within the range of cotinine levels reported in women who are considered “moderate smokers” (80–163 ng/ml), ^{Reference Eskenazi and Bergmann14} and serum cotinine concentrations of 26 ng/ml in the nicotine-exposed offspring at birth, ^{Reference Holloway, Kellenberger and Petrik13} which is also within the range (5–30 ng/ml) observed in infants nursed by smoking mothers. ^{Reference Luck and Nau15} Pancreas tissue was collected from two randomly selected pups per liter, one for RNA isolation and the other for histone extraction and quantification. Tissues were excised, flash frozen, and stored at −80°C. Total RNA was then isolated and extracted using TRIzol reagent (Invitrogen, Carlsbad, CA).

Cell culture maintenance and treatment

INS-1E rat insulinoma pancreatic beta cells (generously provided by Dr. Claes Wollheim, University of Geneva, Geneva, Switzerland) were cultured in RPMI-1640 media containing 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) and 2 mM L-glutamine (RPMI; Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco, Grand Island, NY), 50 μM 2-mercaptoethanol (EMD Millipore, Etobicoke, ON), 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37°C in a humidified atmosphere of 95% O₂ and 5% CO₂.

Cells were treated for 48 h with 0 (vehicle control) and 1 μM nicotine bitartrate (Sigma-Aldrich) diluted in supplemented RPMI as described above. This concentration was selected based on prior work showing impaired insulin secretion and mitochondrial dysfunction in INS-1E cells at this dose. ^{Reference Woynillowicz, Raha, Nicholson and Holloway16,Reference Yoshikawa, Hellström-Lindahl and Grill17} After 48 h, cells were washed with phosphate-buffered saline and total RNA was then isolated and extracted (N = 7–8 independent experiments) using TRIzol reagent (Invitrogen). Additional cells were collected and frozen at −80°C for histone extraction and quantification (N = 5–7 independent experiments).

RNA isolation and real-time quantitative polymerase chain reaction (qPCR)

Total RNA concentration from PND1 pancreas tissue and INS-1E cells was determined using the Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA). Complementary DNA (cDNA) was synthesized from 2 μg of RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) as per the manufacturer’s instructions. The resulting cDNA was used as a template for qPCR, which was performed using PerfeCTa® SYBR® Green FastMix® (Quanta Biosciences, Gaithersburg, MD) on the LightCycler® 480 (Roche, Mississauga, ON). To explore expression of p66shc and methylation drivers in histone H3 lysine 9 (H3K9) methylation, we quantified p66shc and markers of both histone demethylation and histone methylation. For genes involved in H3K9 demethylation, we selected lysine demethylase 4A (Kdm4a) and 4C (Kdm4c). ^{Reference Marmorstein and Trievel18,Reference Su, Wang and Lee19} For genes involved in H3K9 methylation, we selected suppressor of variegation 3–9 homolog 1 (Suv39h1) ^{Reference Costantino, Paneni and Virdis20} and 2 (Suv39h2) ^{Reference Lehnertz, Ueda and Derijck21} and ASH1-like histone lysine methyltransferase (Ash1l). ^{Reference Gregory, Vakoc and Rozovskaia22} The primers were (listed as gene, forward and reverse sequence): p66shc, 5′-GTCCGACTACCCTGTGTTCCTT-3′ and 5′-CAGCAGGATTGGCCAGCTT-3′; Kdm4a, 5′-CTTTAGGCTCCAGGTTGTTCCT-3′ and 5′-TCATCATAGGAGGTTCGAGGC-3′; Kdm4c, 5′-GGTGGAGAGTCCCCTAAATCC-3′ and 5′-GAACTCCCGAAA-CTCCTCCAT-3′; Suv39h1, 5′-TTTCTGTGTACCCTCCTCAGC-3′ and 5′-GAGCAACCCCAGTTAACCTCT-3′; Suv39h2, 5′-AACCTTGATACTCGGCTTCCC-3′ and 5′-TGGCAGGGCTGTAGTCAATAG-3′; and Ash1l, 5′-ACAGACCCAAAGAGCCGATT-3′ and 5′-CAACGTAGACATCAACATGAATGG-3′. The cycling conditions included polymerase activation (95°C for 10 min), followed by 40 cycles of denaturing (95°C for 15 s) and annealing/elongation (60°C for 1 min). Levels of gene expression were calculated using the ΔΔCt method ^{Reference Livak and Schmittgen23} and normalized using the geometric means of two reference genes: hypoxanthine phosphoribosyltransferase 1 (Hprt1) (5′-GCAGTACAGCCCCAAAATGG-3′ and 5′-GGTCCTTTTCACCAGCAAGCT-3′) and peptidylprolyl isomerase A (Ppia) (5′-CCGCTGTCTCTTTTCGCC-3′ and 5′-GCTGTCTTTGGAACTTTGTCTGC-3′).

Acid extraction of histones

Pancreas tissue and INS-1E cell pellets were homogenized with lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, and protease inhibitor tablets; 1:5 ratio tissue pellet:lysis buffer) and HCl added to a final concentration of 0.2 M. Samples were placed on a rocker at 4°C for 1 h and then centrifuged at 11,000g for 10 min in an Eppendorf 5417R centrifuge at 4°C. The acid-insoluble pellet was discarded and the supernatant (approximately 500 µl) was dialyzed in 200 ml of dialysis buffer using the Pierce Slide-A-Lyzer® MINI dialysis units (3500 MW cut-off) in the following manner at 4°C on a rocker: twice in 0.1 M acetic acid for 1 h, once in water for 1 h, once in water for 3 h, and in water overnight. The samples were then aliquoted into separate tubes and stored at −20°C.

Histone quantification and immunoblotting

Protein loading samples were prepared utilizing NuPAGE lithium dodecyl sulfate Sample Buffer NuPAGE Sample Reducing Agent (Invitrogen). Loading amounts were 10–15 µg for histone extracts. Samples were run on NuPage 4%–11.5% Bis-Tris gels (Invitrogen) in 2-(N-morpholino)ethanesulfonic acid (MES) - sodium dodecyl sulphate (SDS) running buffer at 150 V for 1–2 h. After transfer to nitrocellulose membranes, membranes were blocked for 2 h in 5% non-fat dried milk in 0.1% tris buffered saline with Tween 20 (TBST). Membranes were incubated with antibodies targeted against total nuclear histone H3 (EMD Millipore, 05-499), and trimethylated histone H3 (lysine 9, EMD Millipore, 07-422). After washing in 0.1% TBST, membranes were incubated for 2 h with the appropriate secondary antibody (1:10,000 dilution). Bands were normalized to appropriate reference proteins. Beta-actin was probed with antibody (Sigma-Aldrich) for a 2-h room temperature incubation. All bands were detected with a SuperSignal® West Dura Extended Duration Substrate (Thermo Scientific) and imaged with a VersaDoc Imaging System (Bio-Rad). Densitometric analysis on blots was performed with Quantity One Software (Bio-Rad). Data were normalized to reference proteins (beta-actin for cytoplasmic proteins, or total histone H3 for nuclear proteins) by applying stripping buffer (200 mM glycine, 0.1% sodium dodecyl sulfate, and 1% Tween 20) for 10 min, and then the detection protocol was implemented as above.

Statistical analysis

All statistical analyses were performed using SigmaPlot (v.11.2, Systat Software, San Jose, CA). Data were tested for outliers (Grubbs’ test), normality, and equal variance. Means were compared between control and nicotine groups using Student’s t-test. Data that failed normality and/or equal variance were analyzed by using the Mann–Whitney rank sum test. Values are presented as mean ± standard error of the mean (SEM).