Maternal Nicotine Exposure Leads to Augmented Expression of the Antioxidant Adipose Tissue Triglyceride Lipase Long-Term in the White Adipose of Female Rat Offspring

Maternal Nicotine Exposure Leads to Augmented Expression of the Antioxidant Adipose Tissue... Abstract Globally, approximately 10%–25% of women smoke during pregnancy. Since nicotine is highly addictive, women may use nicotine-containing products like nicotine replacement therapies for smoking cessation, but the long-term consequences of early life exposure to nicotine remain poorly defined. Our laboratory has previously demonstrated that maternal nicotine exposed (MNE) rat offspring exhibit hypertriglyceridemia due to increased hepatic de novo lipogenesis. Hypertriglyceridemia may also be attributed to impaired white adipose tissue (WAT) lipid storage; however, the effects of MNE on WAT are not completely understood. We hypothesize that nicotine-induced alterations in adipose function (eg, lipid storage) underlie dyslipidemia in MNE adults. Female 6-month-old rats exposed to nicotine during gestation and lactation exhibited significantly decreased visceral adipocyte cell area by 40%, attributed, in part, to a 3-fold increase in adipose triglyceride lipase (ATGL) protein expression compared with vehicle. Given ATGL has antioxidant properties and in utero nicotine exposure promotes oxidative stress in various tissues, we next investigated if there was evidence of increased oxidative stress in MNE WAT. At both 3 weeks and 6 months, MNE offspring expressed 37%–48% higher protein levels of superoxide dismutase-1 and -2 in WAT. Since oxidative stress can induce inflammation, we examined the inflammatory profile of WAT and found increased expression of cytokines (interleukin-1β, tumor necrosis factor α, and interleukin-6) by 44%–61% at 6 months. Collectively, this suggests that the expression of WAT ATGL may be induced to counter MNE-induced oxidative stress and inflammation. However, higher levels of ATGL would further promote lipolysis in WAT, culminating in impaired lipid storage and long-term dyslipidemia. white adipose tissue, maternal nicotine exposure, oxidative stress, adipocytes Evidence now demonstrates that cigarette smoke plays a critical role in the development of dyslipidemia and obesity in children exposed during perinatal life (Wen et al., 2010; Weng et al., 2012). This is especially concerning given that approximately 10%–25% of women still smoke during pregnancy, with rates as high as 59% in certain Indigenous communities (Cui et al., 2014; Roman-Galvez et al., 2017; Tappin et al., 2010; Tong et al., 2013). To date, nicotine replacement therapies (NRTs) for smoking cessation (ie transdermal patches, gums, e-cigarettes) are thought to benefit pregnant women who are highly addicted and unable to quit smoking by other means (Oncken and Kranzler, 2003). However, the consequences of fetal and neonatal exposure to nicotine alone on the long-term metabolic health of the offspring have yet to be fully defined. Maternal nicotine use during gestation and lactation leads to significant exposure to the fetus and neonate since it is present in fetal blood and transferred through amniotic fluid, placental tissue, and breast milk (Luck and Nau, 1987; Luck et al., 1985). Numerous animal studies demonstrate that nicotine use during pregnancy leads to adverse neurobehavioral, pulmonary, cardiovascular, and metabolic outcomes in the offspring (Barra et al., 2017; Chou and Chen, 2014; Dasgupta et al., 2012; Ma et al., 2014; Pauly and Slotkin, 2008). Specifically, we and others have shown in rats that perinatal exposure to nicotine alone leads to increased blood pressure, adiposity, decreased glucose tolerance, and impaired pancreatic beta cell development (Fox et al., 2012; Gao et al., 2005, 2008; Holloway et al., 2008). Moreover, MNE during gestation and lactation leads to increased hepatic de novo lipogenesis and increased hepatic and circulating triglycerides (TGs) in postnatal life (Ma et al., 2014). This reciprocates human studies which have demonstrated that children exposed to maternal smoke in utero have higher TG levels later in life (Cupul-Uicab et al., 2012). Since increased circulating plasma TGs is strongly associated with cardiovascular disease (CVD) (Bansal et al., 2007), elucidating the underlying molecular mechanisms promoting dyslipidemia will undoubtedly uncover therapeutic targets to reduce unwarranted CVD risk to these nicotine-exposed individuals. Aside from the liver, the augmented circulating and hepatic TG accumulation observed in these MNE offspring could also be attributed to impairments in other metabolic tissues, such as white adipose tissue (WAT). The major role of WAT is to store excess circulating TGs to prevent glucose and lipid toxicity, and moreover, ectopic fat deposition (Abate, 2012). Excess circulating TGs promote adipocytes to undergo differentiation, hypertrophy, and/or hyperplasia to process and store lipids. Once the maximum TG storage capacity is reached, fatty acids can spillover in the plasma, increasing substrate availability for hepatic TG synthesis (Abate, 2012). Ultimately, hypertriglyceridemia contributes to various systemic abnormalities like dyslipidemia, insulin resistance, and CVD (Abate, 2012). Currently, the effects of MNE on WAT function are not completely understood. Studies by Somm et al. (2008) indicated that MNE during gestation resulted in adipocyte hypertrophy and increased expression of adipogenic transcription factors in 3-week male offspring, but the long-term effects were not examined. A more recent study by Fan et al. (2016) demonstrated that maternal exposure of nicotine (from gestational day 9 to weaning) led to decreased adipocyte size in 26-week-old male offspring, suggesting an impaired capacity for lipid storage. Furthermore, this study also found an increase in steady-state mRNA levels of adipocyte differentiation and lipogenic markers in MNE WAT compared with vehicle (Fan et al., 2016). However, given most women addicted to smoking would be exposed to nicotine (ie, NRT or cigarettes) in both prenatal and perinatal life, the goal of this study was to determine if this longer, more relevant, window of nicotine exposure impacts adipose function in both male and female offspring. Moreover, this window covers the entire period of rat adipose tissue differentiation (Greenwood and Hirsch, 1974). We hypothesize that in utero nicotine exposure will adversely impact WAT in the offspring, leading to dyslipidemia in adulthood. To examine this further, we will investigate if WAT function is affected due to altered adipocyte size, proliferation (ie, AKT-1), differentiation (ie, CCAAT/enhancer-binding protein [C/EBP]-α/β, sterol regulatory element-binding transcription factor [SREBP]-1c), lipogenesis (ie, lipoprotein lipase [LPL], acetyl-coA carboxlyase (ACCα), fatty acid synthase (FAS), Acsl1), fatty acid transport (ie, FATP1, FATP4), and/or lipolysis (ATGL) in our well-established rat model of maternal nicotine exposure (MNE). MATERIALS AND METHODS MNE rat model Nulliparous 200–250 g female Wistar rats (Harlan, Indianapolis, Indiana) were injected daily subcutaneously with either saline (vehicle) or nicotine bitartrate at 1 mg/kg/day (Sigma-Aldrich, St Louis, Missouri) 2 weeks prior to mating, during gestation, and until weaning (PND21). This nicotine dose results in maternal serum cotinine concentrations of 135.9 ± 7.86 ng/ml, which is comparable to “moderate” female smokers (80 ng/ml) or NRT users (169.9 ng/ml) (Holloway et al., 2006; Shahab et al., 2017). Litters were culled to 8 at birth and following weaning, rats were housed as sibling pairs until 6 weeks of age, then subsequently housed individually. Male and female offspring were sacrificed via carbon dioxide inhalation at 3 weeks and 6 months of age. Gonadal WATs were extracted, either fixed in formalin or frozen in liquid nitrogen and stored at −80°C for histological and molecular analyses, respectively. All rats were conventionally housed in polycarbonate microisolator cages with ad libitum access to water and standard chow diet (Teklad 22/5 rodent diet; Envigo) under controlled lighting (12:12 L:D), humidity (40%–50%), and temperature (22°C). In accordance with the Canadian Council for Animal Care guidelines, animal experiments were approved by the Animal Research Ethics Board at McMaster University. Gonadal adipocyte cell area A portion of gonadal WAT was fixed in 10% (v/v) neutral buffered formalin overnight, washed in water, and embedded in paraffin. Cross sections were stained with hematoxylin and eosin (H&E) and photographed using the Olympus BX50 microscope under a 10× objective. Cell areas were quantified from a minimum of 160 adipocytes from 2 cross sections/rat using Northern Eclipse software (Empix Imaging Inc.). RNA extraction and real time-polymerase chain reaction Total RNA was extracted from homogenized 3-week and 6-month WAT samples using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). Two micrograms of RNA were reversed-transcribed to cDNA (high-capacity cDNA Reverse Transcription Kit, Applied Biosystems). Forward and reverse primer sets used for real time-polymerase chain reaction (RT-PCR), listed in Table 1, were designed with the National Center for Biotechnology Information’s primer designing tool. Relative transcript abundance was determined using SensiFAST No-ROX SYBR Green Supermix (FroggaBio) and the Bio-Rad CFX384 Real Time System. Samples were assayed in triplicate and relative fold change was calculated using comparative cycle times (Ct) method normalized to β-actin. The relative abundance was calculated using the formula 2ΔΔCt, where ΔΔCt was the normalized value. Table 1. Forward and Reverse Primer Sequences Used for Quantitative Real-Time PCR. Gene  Forward  Reverse  GenBank/Reference  AKT-1  ATGTGTATGAGAAGAAGCTGAGCC  GTTCACTGTCCACACACTCCA  NM_033230.2  C/EBPα  GCCGGGAGAACTCTAACTCC  TCGATGTAGGCGCTGATGTC  NM_001287577.1  C/EBPβ  ACCACGACTTCCTTTCCGAC  TAACCGTAGTCGGACGGCTT  NM_024125.5  SREBP1c  CATGGACGAGCTACCCTTCG  TCTTCGATGTCGGTCAAGAGC  NM_001276707.1  LPL  GGATGCAACATTGGAGAACCC  GCTGGGGTTTTCTTCATTCAGC  NM_012598.2  ACCα  TCCGTATGTGACCAAAGACC  TACGTTGTTCCCAAGGACTG  NM_022193.1  FAS  GGACATGGTCACAGACGATGAC  CGTCGAACTTGGACAGATCCTT  NM_017332.1  ACSL1  CTACAGGCAACCCCAAAGGA  AATGCACTCTCCGTCGCTT  NM_012820.1  FATP1  CAGCCTCTGTGGCCTCATT  ACCCACGTACACACCGAAC  NM_053580.2  FATP4  CTTGGGCAACTTTGACAGCC  AGGACAGGATGCGGCTATTG  NM_001100706.1  ATGL  AACGCCACTCACATCTACGG  TACCAGGTTGAAGGAGGGGT  NM_001108509.2  EGR-1  CGAGCGAACAACCCTACGA  CGATGTCAGAAAAGGACTCTGTG  NM_012551.2  FOXO-1  TGCAGCAGACACCTTGCTAT  TTGGGGCTGGGGGAATTTAG  NM_001191846.2  IL-1β  CAGCTTTCGACAGTGAGGAGA  GTCGAGATGCTGCTGTGAGA  NM_031512.2  TNFα  CCGGGCAGGTCTACTTTGGA  AGGCCACTACTTCAGCGTCTCG  NM_012675.3  IL-6  CTTCCAGCCAGTTGCCTTCTTG  TGGTCTGTTGTGGGTGGTATCC  NM_012589.2  CD68  ACTGGGGCTCTTGGAAACTACAC  CCTTGGTTTTGTTCGGGTTCA  NM_001031638.1  SOD-1  ATTGGCCGTACTATGGTGGTC  GCAATCCCAATCACACCACA  NM_017050.1  SOD-2  ATTGCCGCCTGCTCTAATCA  TCCCACACATCAATCCCCAG  NM_017051.2  Gene  Forward  Reverse  GenBank/Reference  AKT-1  ATGTGTATGAGAAGAAGCTGAGCC  GTTCACTGTCCACACACTCCA  NM_033230.2  C/EBPα  GCCGGGAGAACTCTAACTCC  TCGATGTAGGCGCTGATGTC  NM_001287577.1  C/EBPβ  ACCACGACTTCCTTTCCGAC  TAACCGTAGTCGGACGGCTT  NM_024125.5  SREBP1c  CATGGACGAGCTACCCTTCG  TCTTCGATGTCGGTCAAGAGC  NM_001276707.1  LPL  GGATGCAACATTGGAGAACCC  GCTGGGGTTTTCTTCATTCAGC  NM_012598.2  ACCα  TCCGTATGTGACCAAAGACC  TACGTTGTTCCCAAGGACTG  NM_022193.1  FAS  GGACATGGTCACAGACGATGAC  CGTCGAACTTGGACAGATCCTT  NM_017332.1  ACSL1  CTACAGGCAACCCCAAAGGA  AATGCACTCTCCGTCGCTT  NM_012820.1  FATP1  CAGCCTCTGTGGCCTCATT  ACCCACGTACACACCGAAC  NM_053580.2  FATP4  CTTGGGCAACTTTGACAGCC  AGGACAGGATGCGGCTATTG  NM_001100706.1  ATGL  AACGCCACTCACATCTACGG  TACCAGGTTGAAGGAGGGGT  NM_001108509.2  EGR-1  CGAGCGAACAACCCTACGA  CGATGTCAGAAAAGGACTCTGTG  NM_012551.2  FOXO-1  TGCAGCAGACACCTTGCTAT  TTGGGGCTGGGGGAATTTAG  NM_001191846.2  IL-1β  CAGCTTTCGACAGTGAGGAGA  GTCGAGATGCTGCTGTGAGA  NM_031512.2  TNFα  CCGGGCAGGTCTACTTTGGA  AGGCCACTACTTCAGCGTCTCG  NM_012675.3  IL-6  CTTCCAGCCAGTTGCCTTCTTG  TGGTCTGTTGTGGGTGGTATCC  NM_012589.2  CD68  ACTGGGGCTCTTGGAAACTACAC  CCTTGGTTTTGTTCGGGTTCA  NM_001031638.1  SOD-1  ATTGGCCGTACTATGGTGGTC  GCAATCCCAATCACACCACA  NM_017050.1  SOD-2  ATTGCCGCCTGCTCTAATCA  TCCCACACATCAATCCCCAG  NM_017051.2  Table 1. Forward and Reverse Primer Sequences Used for Quantitative Real-Time PCR. Gene  Forward  Reverse  GenBank/Reference  AKT-1  ATGTGTATGAGAAGAAGCTGAGCC  GTTCACTGTCCACACACTCCA  NM_033230.2  C/EBPα  GCCGGGAGAACTCTAACTCC  TCGATGTAGGCGCTGATGTC  NM_001287577.1  C/EBPβ  ACCACGACTTCCTTTCCGAC  TAACCGTAGTCGGACGGCTT  NM_024125.5  SREBP1c  CATGGACGAGCTACCCTTCG  TCTTCGATGTCGGTCAAGAGC  NM_001276707.1  LPL  GGATGCAACATTGGAGAACCC  GCTGGGGTTTTCTTCATTCAGC  NM_012598.2  ACCα  TCCGTATGTGACCAAAGACC  TACGTTGTTCCCAAGGACTG  NM_022193.1  FAS  GGACATGGTCACAGACGATGAC  CGTCGAACTTGGACAGATCCTT  NM_017332.1  ACSL1  CTACAGGCAACCCCAAAGGA  AATGCACTCTCCGTCGCTT  NM_012820.1  FATP1  CAGCCTCTGTGGCCTCATT  ACCCACGTACACACCGAAC  NM_053580.2  FATP4  CTTGGGCAACTTTGACAGCC  AGGACAGGATGCGGCTATTG  NM_001100706.1  ATGL  AACGCCACTCACATCTACGG  TACCAGGTTGAAGGAGGGGT  NM_001108509.2  EGR-1  CGAGCGAACAACCCTACGA  CGATGTCAGAAAAGGACTCTGTG  NM_012551.2  FOXO-1  TGCAGCAGACACCTTGCTAT  TTGGGGCTGGGGGAATTTAG  NM_001191846.2  IL-1β  CAGCTTTCGACAGTGAGGAGA  GTCGAGATGCTGCTGTGAGA  NM_031512.2  TNFα  CCGGGCAGGTCTACTTTGGA  AGGCCACTACTTCAGCGTCTCG  NM_012675.3  IL-6  CTTCCAGCCAGTTGCCTTCTTG  TGGTCTGTTGTGGGTGGTATCC  NM_012589.2  CD68  ACTGGGGCTCTTGGAAACTACAC  CCTTGGTTTTGTTCGGGTTCA  NM_001031638.1  SOD-1  ATTGGCCGTACTATGGTGGTC  GCAATCCCAATCACACCACA  NM_017050.1  SOD-2  ATTGCCGCCTGCTCTAATCA  TCCCACACATCAATCCCCAG  NM_017051.2  Gene  Forward  Reverse  GenBank/Reference  AKT-1  ATGTGTATGAGAAGAAGCTGAGCC  GTTCACTGTCCACACACTCCA  NM_033230.2  C/EBPα  GCCGGGAGAACTCTAACTCC  TCGATGTAGGCGCTGATGTC  NM_001287577.1  C/EBPβ  ACCACGACTTCCTTTCCGAC  TAACCGTAGTCGGACGGCTT  NM_024125.5  SREBP1c  CATGGACGAGCTACCCTTCG  TCTTCGATGTCGGTCAAGAGC  NM_001276707.1  LPL  GGATGCAACATTGGAGAACCC  GCTGGGGTTTTCTTCATTCAGC  NM_012598.2  ACCα  TCCGTATGTGACCAAAGACC  TACGTTGTTCCCAAGGACTG  NM_022193.1  FAS  GGACATGGTCACAGACGATGAC  CGTCGAACTTGGACAGATCCTT  NM_017332.1  ACSL1  CTACAGGCAACCCCAAAGGA  AATGCACTCTCCGTCGCTT  NM_012820.1  FATP1  CAGCCTCTGTGGCCTCATT  ACCCACGTACACACCGAAC  NM_053580.2  FATP4  CTTGGGCAACTTTGACAGCC  AGGACAGGATGCGGCTATTG  NM_001100706.1  ATGL  AACGCCACTCACATCTACGG  TACCAGGTTGAAGGAGGGGT  NM_001108509.2  EGR-1  CGAGCGAACAACCCTACGA  CGATGTCAGAAAAGGACTCTGTG  NM_012551.2  FOXO-1  TGCAGCAGACACCTTGCTAT  TTGGGGCTGGGGGAATTTAG  NM_001191846.2  IL-1β  CAGCTTTCGACAGTGAGGAGA  GTCGAGATGCTGCTGTGAGA  NM_031512.2  TNFα  CCGGGCAGGTCTACTTTGGA  AGGCCACTACTTCAGCGTCTCG  NM_012675.3  IL-6  CTTCCAGCCAGTTGCCTTCTTG  TGGTCTGTTGTGGGTGGTATCC  NM_012589.2  CD68  ACTGGGGCTCTTGGAAACTACAC  CCTTGGTTTTGTTCGGGTTCA  NM_001031638.1  SOD-1  ATTGGCCGTACTATGGTGGTC  GCAATCCCAATCACACCACA  NM_017050.1  SOD-2  ATTGCCGCCTGCTCTAATCA  TCCCACACATCAATCCCCAG  NM_017051.2  Protein extraction and Western blot WAT was homogenized in RIPA buffer (50 mM Tris-HCL, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 0.25% C24H39NaO4) supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitors (20 mM NaF, 40 mM Na-pyrophosphate, 40 mM Na3VO4, 200 mM β-glycerophosphate disodium salt hydrate). The solution was sonicated, mixed in a rotator for 1 h at 4°C, and centrifuged at 300 × g for 15 min at 4°C. The total cellular protein extract in the collected supernatant was quantified by colorimetric DC protein assay (BioRad). Loading samples were heated at 50°C for 10 min to denature the proteins. Proteins (20 μg/well) were separated by size via gel electrophoresis and transferred onto polyvinylidene difluoride membrane (Millipore). Membranes were blocked in 1× Tris-buffered saline-Tween 20 buffer with 5% nonfat milk or 5% BSA (blocking solutions), and then probed using primary antibodies diluted in the blocking solution (Table 2). A mouse or rabbit secondary antibody was used to detect primary antibody diluted in the blocking solution at 1:5000 or 1:10 000 dilution, respectively (Table 2). Immuno-reactive bands were visualized, and relative band intensity was calculated using ImageLab software (BioRad) and normalized to β-actin, as previously performed (Ma et al., 2014). Table 2. Western Blot Antibodies, Dilutions Used in Experiments, and Company and Catalog Information Antibody Name  Source  Dilution  Company (No. Catalog)  4-HNE  Mouse monoclonal  1:500  R&D Systems Minneapolis, Minnesota (No. MAB3249)  AKT-1  Rabbit polyclonal  1:500  Abcam Inc., Cambridge, Massachusetts (No. abcam 5919)  ATGL (H-144)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-67355)  C/EBPα (14AA)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-61)  C/EBPβ (C-19)  Rabbit polyclonal  1:300  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-150)  SOD-1 (FL-154)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-11407)  SOD-2 (FL-222)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-30080)  SREBP 1c (H-160)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-8984)  Mouse IgG (H+L) Secondary  Sheep  1:5000  GE Healthcare UK, Pittsburgh, Pennsylvania (No. NA931)  Rabbit IgG (H+L) Secondary  Donkey  1:10 000  Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania (No. 711-001-003)  Antibody Name  Source  Dilution  Company (No. Catalog)  4-HNE  Mouse monoclonal  1:500  R&D Systems Minneapolis, Minnesota (No. MAB3249)  AKT-1  Rabbit polyclonal  1:500  Abcam Inc., Cambridge, Massachusetts (No. abcam 5919)  ATGL (H-144)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-67355)  C/EBPα (14AA)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-61)  C/EBPβ (C-19)  Rabbit polyclonal  1:300  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-150)  SOD-1 (FL-154)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-11407)  SOD-2 (FL-222)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-30080)  SREBP 1c (H-160)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-8984)  Mouse IgG (H+L) Secondary  Sheep  1:5000  GE Healthcare UK, Pittsburgh, Pennsylvania (No. NA931)  Rabbit IgG (H+L) Secondary  Donkey  1:10 000  Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania (No. 711-001-003)  Table 2. Western Blot Antibodies, Dilutions Used in Experiments, and Company and Catalog Information Antibody Name  Source  Dilution  Company (No. Catalog)  4-HNE  Mouse monoclonal  1:500  R&D Systems Minneapolis, Minnesota (No. MAB3249)  AKT-1  Rabbit polyclonal  1:500  Abcam Inc., Cambridge, Massachusetts (No. abcam 5919)  ATGL (H-144)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-67355)  C/EBPα (14AA)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-61)  C/EBPβ (C-19)  Rabbit polyclonal  1:300  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-150)  SOD-1 (FL-154)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-11407)  SOD-2 (FL-222)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-30080)  SREBP 1c (H-160)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-8984)  Mouse IgG (H+L) Secondary  Sheep  1:5000  GE Healthcare UK, Pittsburgh, Pennsylvania (No. NA931)  Rabbit IgG (H+L) Secondary  Donkey  1:10 000  Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania (No. 711-001-003)  Antibody Name  Source  Dilution  Company (No. Catalog)  4-HNE  Mouse monoclonal  1:500  R&D Systems Minneapolis, Minnesota (No. MAB3249)  AKT-1  Rabbit polyclonal  1:500  Abcam Inc., Cambridge, Massachusetts (No. abcam 5919)  ATGL (H-144)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-67355)  C/EBPα (14AA)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-61)  C/EBPβ (C-19)  Rabbit polyclonal  1:300  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-150)  SOD-1 (FL-154)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-11407)  SOD-2 (FL-222)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-30080)  SREBP 1c (H-160)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-8984)  Mouse IgG (H+L) Secondary  Sheep  1:5000  GE Healthcare UK, Pittsburgh, Pennsylvania (No. NA931)  Rabbit IgG (H+L) Secondary  Donkey  1:10 000  Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania (No. 711-001-003)  Statistical analysis Statistical analyses were performed using Graphpad Prism 6 software. Results were presented as the mean of arbitrary values ± SEM. Grubbs’ test was used to determine significant outliers and data were tested for normality and equal variance. Comparisons between vehicle and nicotine-exposed offspring were assessed using an unpaired Student’s t test, with a p-value of < .05 deemed as significant. RESULTS MNE Leads to Decreased Visceral Adipocyte Size in 6-Month-Old Offspring Using our well-established rat MNE model leading to dyslipidemia (Gao et al., 2005; Ma et al., 2014), we wanted to first confirm whether prenatal nicotine exposure has long term effects on adipocyte size in the offspring. Histological analyses revealed both MNE male and female offspring exhibited smaller gonadal adipocytes compared with vehicle controls at 6 months of age (Figs. 1A and 1B) . Given the more pronounced effects of perinatal nicotine exposure on female WAT area (13.2% male vs 39.7% female decrease in adipocyte size), we continued to examine only female offspring to explore the underlying mechanisms involved. Gondal adipose tissue weight did not differ between MNE and vehicle offspring (data not shown), suggesting a possible increase in adipocyte number due to prenatal nicotine exposure. With decreased adipocyte cell area and subsequent increase in cell number, transcript and protein levels of the proliferation marker AKT-1 were subsequently measured. Despite a significant increase in mRNA levels, there were no corresponding changes in AKT-1 protein levels in 6-month female MNE WAT (Figs. 1C and 1D). Figure 1. View largeDownload slide Maternal nicotine exposure leads to decreased visceral adipocyte cell area in 6-month-old offspring. Representative cross-sections of gonadal WAT excised from (A) vehicle and in utero nicotine exposed 6-month-old male and female offspring were stained with hematoxylin and eosin. B, Bar graph representing calculated cell areas analyzed using Northern Eclipse software (n = 4/group/sex). C, Transcript and (D) protein levels of AKT-1 in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 7/nicotine). **,*** Significant difference (**p < .01, ***p < .001). Figure 1. View largeDownload slide Maternal nicotine exposure leads to decreased visceral adipocyte cell area in 6-month-old offspring. Representative cross-sections of gonadal WAT excised from (A) vehicle and in utero nicotine exposed 6-month-old male and female offspring were stained with hematoxylin and eosin. B, Bar graph representing calculated cell areas analyzed using Northern Eclipse software (n = 4/group/sex). C, Transcript and (D) protein levels of AKT-1 in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 7/nicotine). **,*** Significant difference (**p < .01, ***p < .001). In Utero Nicotine Exposure Increases the Expression of the Differentiation Marker C/EBPα in WAT of 6-Month-Old Female Offspring Given impairments in adipocyte differentiation can also decrease adipocyte size, we next measured the expression of key targets involved in adipogenesis (Abate, 2012), including C/EBP-α/β and SREBP-1c. Protein analyses revealed a significant increase in the 42 kDa band of C/EBP-α in female MNE offspring compared with vehicle at 6 months independent of changes in transcript levels (Figs. 2A and 2B). Quantitative real time PCR analyses revealed that WAT from MNE exposed offspring had a significant increase in steady-state transcript levels of C/EBPβ and SREBP-1c compared with controls (Figs. 2C and 2E). However, immunoblot analyses revealed no significant differences at the protein level for either target (Figs. 2D and 2F). To examine if perinatal nicotine exposure had direct effects on C/EBPα, we measured the transcript and protein levels at 3 weeks age but found no difference in expression from vehicle (Figs. 2G and 2H). Figure 2. View largeDownload slide In utero nicotine exposure increases the expression of the differentiation marker C/EBPα in WAT of 6-month-old female offspring. Transcript and protein levels of targets of interest in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month (A) C/EBPα mRNA and (B) protein levels. C, C/EBPβ mRNA and (D) protein levels. E, SREBP1-c mRNA and (F) protein levels. Three-week (G) C/EBPα mRNA and (H) protein levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 7/nicotine). *Significant difference (*p < .05). Figure 2. View largeDownload slide In utero nicotine exposure increases the expression of the differentiation marker C/EBPα in WAT of 6-month-old female offspring. Transcript and protein levels of targets of interest in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month (A) C/EBPα mRNA and (B) protein levels. C, C/EBPβ mRNA and (D) protein levels. E, SREBP1-c mRNA and (F) protein levels. Three-week (G) C/EBPα mRNA and (H) protein levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 7/nicotine). *Significant difference (*p < .05). MNE Does Not Increase the Expression of Markers Involved in Lipogenesis and Fatty Acid Transporters in 6-Month-Old Female Offspring Adipocyte size is influenced by its ability to breakdown TG molecules from circulation, transport its constitutive components intracellularly, and re-assemble TGs for storage (Kersten, 2014). Therefore, we wanted to determine whether perinatal nicotine exposure impaired TG transport and esterification by measuring the steady state transcript levels of key targets involved in lipogenesis and fatty acid transport. At 6 months of age we found no significant difference between groups in the expression of lipogenic markers including LPL, ACCα, and FAS, along with fatty acid transporters FATP1/4, and long-chain acyl CoA synthetase (ASCL)-1 in female MNE WAT (Figs. 3A–F). Figure 3. View largeDownload slide Maternal nicotine exposure does not increase the expression of markers involved in lipogenesis and fatty acid transporters in WAT of 6-month-old female offspring. Transcript levels of targets of interest in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR. Six-month (A) LPL, (B) ACCα, (C) FAS, (D) ASCL1, (E) FATP1, and (F) FATP4 mRNA levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Figure 3. View largeDownload slide Maternal nicotine exposure does not increase the expression of markers involved in lipogenesis and fatty acid transporters in WAT of 6-month-old female offspring. Transcript levels of targets of interest in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR. Six-month (A) LPL, (B) ACCα, (C) FAS, (D) ASCL1, (E) FATP1, and (F) FATP4 mRNA levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). In Utero Nicotine-Exposed Female Offspring Exhibit Increased Expression of ATGL at 6 Months, But Not at 3 Weeks Since enhanced lipolysis decreases adipocyte size (Zhang et al., 2017), we then examined whether MNE WAT had elevated expression of adipose triglyceride lipase (ATGL), the predominate enzyme involved in intracellular degradation (Zechner et al., 2009). At 6 months of age, we found a significant increase in both ATGL mRNA expression and protein levels in nicotine-exposed female WAT compared with vehicle controls (Figs. 4A and 4B), however this was not observed at 3 weeks (Figs. 4C and 4D). Altogether, these data suggest that augmented ATGL-induced lipolysis may lead to reduced adipocyte cell area in MNE WAT at 6 months. Figure 4. View largeDownload slide In utero nicotine-exposed female offspring exhibit increased expression of ATGL at 6 months, but not at 3 weeks. Transcript and protein levels of ATGL in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month ATGL (A) mRNA and (B) protein levels. Three-week ATGL (C) mRNA and (D) protein levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 8/nicotine). *,** Significant difference (*p < .05, **p < .01). Figure 4. View largeDownload slide In utero nicotine-exposed female offspring exhibit increased expression of ATGL at 6 months, but not at 3 weeks. Transcript and protein levels of ATGL in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month ATGL (A) mRNA and (B) protein levels. Three-week ATGL (C) mRNA and (D) protein levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 8/nicotine). *,** Significant difference (*p < .05, **p < .01). MNE Exhibit Enhanced Antioxidant Expression at 6 Months and 3 Weeks Evidence demonstrates that ATGL deficiency in mice is associated with accumulation of reactive oxygen species (ROS) and decreased superoxide dismutase (SOD)-2 expression, suggesting this enzyme may act as an antioxidant and suppress oxidative stress (Aquilano et al., 2016; Chen et al., 2017a,b). Moreover, we and others have previously shown that MNE promotes oxidative stress in the heart, pancreas, and placenta of adult offspring (Barra et al., 2017; Bruin et al., 2008; Sbrana et al., 2011). Therefore, we wanted to determine whether an imbalance between antioxidant defenses occurs in WAT of 6-month female MNE offspring by measuring the transcript and protein levels of the antioxidants SOD-1 and SOD-2. Both SOD 1 and 2 steady state mRNA and protein levels were significantly elevated in prenatal nicotine-exposed WAT in 6-month female offspring compared with vehicle (Figs. 5A–D). At 3 weeks of age, there was also a significant increase in SOD-1 protein levels, and a trending increase in SOD-2, in the nicotine-exposed WAT group compared with controls (Figs. 5E–H). To determine if there was oxidative stress, we measured protein levels of 4-hydroxynonenal (4-HNE) and found a nonsignificant upward trend in nicotine-exposed WAT at both 3-week and 6-month timepoints (Figs. 5I and 5J). Overall, these data suggest that prenatal nicotine exposure directly increases the expression of the antioxidant SOD-1 and SOD-2 in 6-month WAT which was enough to prevent lipid oxidative damage. Figure 5. View largeDownload slide In utero nicotine-exposed female offspring exhibit increased antioxidant expression in WAT at 3 weeks and 6 months. Transcript and protein levels of SOD-1 and SOD-2 in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month SOD-1 (A) mRNA and (B) protein levels and SOD-2 (C) mRNA and (D) protein levels. Three-week SOD-1 (E) mRNA and (F) protein levels and SOD-2 (G) mRNA and (H) protein levels. Protein levels of 4-HNE at (I) 6 months and (J) 3 weeks of age. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 8/nicotine). *,** Significant difference (*p < .05, **p < .01). Figure 5. View largeDownload slide In utero nicotine-exposed female offspring exhibit increased antioxidant expression in WAT at 3 weeks and 6 months. Transcript and protein levels of SOD-1 and SOD-2 in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month SOD-1 (A) mRNA and (B) protein levels and SOD-2 (C) mRNA and (D) protein levels. Three-week SOD-1 (E) mRNA and (F) protein levels and SOD-2 (G) mRNA and (H) protein levels. Protein levels of 4-HNE at (I) 6 months and (J) 3 weeks of age. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 8/nicotine). *,** Significant difference (*p < .05, **p < .01). In Utero Nicotine Exposure Exhibit Inflammation at 6 Months, But Not at 3 Weeks Enhanced oxidative stress can activate nuclear factor-κB (NF-κB), leading the expression of proinflammatory cytokines and inflammation (Li and Karin, 1999; Morgan and Liu, 2011). As well, activation of this pathway in differentiated 3T3-L1 cells leads to increased lipase expression and lipolysis (Chi et al., 2014). Since oxidative stress, inflammation, and lipolysis are integrated pathways, we wanted to determine whether inflammation was present in 6-month in utero nicotine-exposed WAT. At 6 months, we found a significant increase in the steady-state mRNA expression profile of proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and the macrophage marker CD68 (Figs. 6A–D). In contrast at 3 weeks of age, there was no significant difference in steady state mRNA levels of inflammatory markers between groups (Figs. 6E–H). Figure 6. View largeDownload slide Maternal nicotine-exposed female offspring exhibit increases in WAT inflammation at 6 months, but not at 3 weeks. Transcript levels of targets of interest in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR. Six-month (A) IL-1β, (B) TNFα, (C) IL-6, and (D) CD68 mRNA levels. Three-week (E) IL-1β, (F) TNFα, (G) IL-6, and (H) CD68 mRNA levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). *,**,*** Significant difference (*p < .05, **p < .01, **p < .001). Figure 6. View largeDownload slide Maternal nicotine-exposed female offspring exhibit increases in WAT inflammation at 6 months, but not at 3 weeks. Transcript levels of targets of interest in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR. Six-month (A) IL-1β, (B) TNFα, (C) IL-6, and (D) CD68 mRNA levels. Three-week (E) IL-1β, (F) TNFα, (G) IL-6, and (H) CD68 mRNA levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). *,**,*** Significant difference (*p < .05, **p < .01, **p < .001). DISCUSSION Under normal physiological conditions, WAT serves as the principle lipid storage site to prevent ectopic lipid deposition in peripheral organs. The balance between lipolysis and lipogenesis dictates the overall amount of TG molecules stored as a single large lipid droplet in the cytoplasm of adipocytes, ultimately influencing its size (Bolsoni-Lopes and Alonso-Vale, 2015). Adipocytes have a limited TG storage capacity. Once exceeded, excess lipids will spill-over into circulation resulting in dyslipidemia and increased lipid content at ectopic sites like the liver (Abate, 2012). In our model, we have previously shown that MNE offspring have dyslipidemia with increased levels of circulating TGs (Gao et al., 2005; Ma et al., 2014). Although we have demonstrated that hepatic de novo lipogenesis is increased in nicotine-exposed offspring, the effect of nicotine exposure on WAT function were not examined. Previous studies indicate MNE during gestation or from gestation until weaning altered adipocyte size and increased the expression of adipocyte differentiation markers, but this was only examined in male offspring (Fan et al., 2016; Somm et al., 2008). Moreover, this exposure to nicotine in these studies (gestation only or mid-gestation to weaning only) does not mimic women’s smoking habits and/or nicotine use prior to pregnancy. Therefore, our goal was to determine if WAT plays a role in promoting dyslipidemia in both male and female offspring exposed to nicotine in prenatal and perinatal life. In this study, we demonstrated that both male and female offspring exposed to nicotine during prenatal and perinatal life decreased adipocyte cell area compared with vehicle controls at 6 months of age. Somm et al. (2008) reported that rats exposed to nicotine during pregnancy alone (from gestational day 4 for 14 days at 3 mg/kg/day) had adipocyte hypertrophy compared with vehicle controls in 3-week-old male offspring, but the effect on WAT size into adulthood, after nicotine exposure, was not examined. Similar to our findings, Fan et al. (2016) demonstrated that in utero nicotine exposure from mid-pregnancy (eg, gestational day 9) until weaning decreased adipocyte size in male WAT at 26 weeks of age. The dose of nicotine (1 mg/kg) administered in this study is relevant as it corresponds to moderate female smokers or NRT users (Holloway et al., 2006; Shahab et al., 2017), while doses used in other studies (2–3 mg/kg/day) correspond to heavy smokers (Fan et al., 2016; Somm et al., 2008). Examining the effects of MNE on WAT for a longer duration is important given adipose development occurs both pre- and postnatally in rats. In rodent adipose tissue, growth and differentiation occurs from late gestation to 4 weeks in postnatal life, while in humans this occurs from 5 to 29 weeks gestation (Greenwood and Hirsch, 1974; Poissonnet et al., 1984). In both species, adipose tissue expansion happens throughout life (Greenwood and Hirsch, 1974; Spalding et al., 2008). Given the extensive differentiation of adipose tissue during perinatal life, it is vulnerable to alterations by environmental cues (ie, drugs) during this developmental window. Furthermore, both mature adipocytes and adipocyte precursors like mesenchymal stem cells express nicotinic acetyl-choline receptor subunits, suggesting that nicotine may directly alter adipogenesis and adipocyte formation (Gochberg-Sarver et al., 2012; Hoogduijn et al., 2009). Since the effect on adipocyte size was more pronounced in female offspring, and previous studies focused on males (Fan et al., 2016; Somm et al., 2008), we decided to examine the effects of MNE on female adipose tissue function. One limitation of our study is that we did not evaluate the estrous cycle of the female rats. This is an important consideration since prenatal nicotine exposure increased progesterone levels in offspring compared with vehicle controls, which is reflective of estrous cycle perturbations (Holloway et al., 2006). Furthermore, others have reported that oscillations in metabolic related genes in the liver occur with estrous cycle (Villa et al., 2012). Since small adipocyte size results from aberrant adipogenesis, we examined whether MNE impaired adipocyte differentiation, lipid synthesis and transport, and/or lipolysis. We found that female MNE offspring exhibited increased mRNA and protein expression of the adipocyte differentiation factor C/EBPα and the lipolytic enzyme ATGL, without altering the expression of targets involved in lipogenesis and lipid transport. Similar to our results, Fan et al. (2016) found an increase in steady-state mRNA levels of adipocyte differentiation markers in MNE male offspring, including C/EBPα and SREBP-1c compared with control WAT in adulthood. Since these studies did not perform protein analyses, our study is the first to demonstrate that MNE induced C/EBPα protein levels in 6-month female WAT. Overall, the increased C/EBPα (42 kDa) expression in WAT suggests that MNE promote adipocyte differentiation and adipogenesis. C/EBPα contains 2 isoforms (30/42 kDa) alternatively translated from 1 mRNA transcript (Lin et al., 1993; Ossipow et al., 1993). Constitutive expression of the 42 kDa isoform can induce adipocyte differentiation in 3T3-L1 preadipocytes, suggesting that enhanced C/EBPα expression of this isoform can enhance adipocyte formation (Lin and Lane, 1994). Although the steady-state levels of AKT1, C/EBP-α, SREBP 1c mRNA were augmented in 6-month nicotine-exposed offspring, this did not translate to an alteration in protein levels. There are several cases in the literature whereby changes in the mRNA levels do not correlate to an alteration in protein, highlighting the importance of looking at both mRNA and protein (Vogel and Marcotte, 2012). Moreover, it is possible that the mRNA and protein levels of these key proliferation and differentiation markers may be altered earlier in development (ie, gestation and/or lactation) due to the direct effects of nicotine on their expression. For example, we have demonstrated that nicotine directly enhances the expression of the proliferation markers GADD45 and Rb1 in differentiating 3T3-L1 preadipocyte cells (data not shown). Therefore, in the absence of nicotine exposure after lactation, the levels of these proliferation and differentiation may have become normalized. This study was the first to demonstrate that both prenatal and perinatal nicotine exposure leads to increased ATGL mRNA and protein expression in adult female MNE offspring. This suggests that MNE may impair the lipid storage capacity of WAT via increased ATGL expression. Studies demonstrate that augmented ATGL leads to enhanced basal and stimulated WAT lipolysis and reduced adipocyte size (Ahmadian et al., 2009; Bezaire et al., 2009; Kershaw et al., 2006). The primary function of ATGL is to initiate lipolysis, by hydrolyzing the sn-2 ester bond of a TG molecule, resulting in the formation of diacylglycerol and fatty acid (Zechner et al., 2009). Therefore, decreased adipocyte cell size in gonadal WAT may be attributed to enhanced lipolysis in MNE offspring. Recently, evidence supports an additional role for ATGL—as an antioxidant and suppressor of oxidative stress. Oxidative stress is defined as an imbalance between antioxidant defenses and ROS production (free radicals) (Sena and Chandel, 2012). Prolonged oxidative stress can result in free radical damage, ultimately leading to mitochondrial-mediated apoptosis (Sena and Chandel, 2012). In vitro and in vivo analyses demonstrate that ATGL deficiency is associated with ROS accumulation and cellular apoptosis in renal podocytes and proximal tubules (Chen et al., 2017a,b). Similarly, ATGL inhibition in C2C12 myoblasts and in murine ATGL deficient skeletal muscle promotes oxidative damage and a defective cellular antioxidant response, characterized by a significant reduction in antioxidant SOD-2 expression and glutathione levels (Aquilano et al., 2016). Altogether, these findings suggest that ATGL may protect against oxidative stress. We and others have previously shown that MNE can cause oxidative stress in the offspring in various tissues including the heart, pancreas, and placenta (Barra et al., 2017; Bruin et al., 2008; Sbrana et al., 2011). Therefore, we examined the expression of antioxidants SOD-1/2 in WAT of 6-month female MNE offspring to assess whether an imbalance in antioxidant expression is present at the same time ATGL levels are elevated. Oxygen can oxidize other molecules to generate ROS, whereby majority of intracellular ROS are derived from superoxide (O2−). Superoxide is formed in the mitochondrial respiratory chain, emitted to the matrix and intermembrane space, and subsequently converted to hydrogen peroxide (H2O2) by SODs (Sena and Chandel, 2012). This study is the first to demonstrate that MNE WAT had increased transcript expression and protein levels of both SOD-1 and -2 at 6 months with evidence for increased SOD-1 at 3 weeks of age. Although nonsignificant, we also observed an upward trend for the oxidative marker 4-HNE in nicotine-exposed WAT compared with vehicle at both time points. This suggests that perinatal nicotine exposure may instigate WAT oxidative stress, triggering a rapid increase in antioxidant expression by weaning which persists into adulthood and may prevent long-term oxidative damage. Oxidative stress can contribute to altered adipocyte differentiation and lipolysis. Depending on environmental cues, mesenchymal stem cells can differentiate into various cell types derived from the mesodermal lineage including myocytes, adipocytes, osteocytes, and chondrocytes. Evidence suggests that elevated ROS levels, produced during oxidative stress, can stimulate adipogenesis while suppressing osteogenesis (Atashi et al., 2015). During conversion of stem cells to adipocytes, mitochondrial complexes I/III and the NADPH oxidase isoform NOX4 produce ROS to initiate adipocyte signaling cascades, leading to terminal differentiation (Atashi et al., 2015). Therefore, increased SOD-1 expression in fetal and neonatal nicotine-exposed WAT may suggest that augmented ROS production may directly promote the expression of adipogenic transcription factors like C/EBPα (Hu et al., 2005). In vitro analyses using both differentiated 3T3-L1 and human primary adipocyte cultures demonstrate that exposure to exogenous 4-HNE elevated lipolysis in basal and stimulated conditions characterized by a dose-dependent significant increase in glycerol and fatty acid release (Zhang et al., 2013). Altogether, these findings suggest that MNE may augment oxidative stress in WAT characterized by increased expression of antioxidants SOD-1/2 and ATGL, leading to altered adipocyte differentiation and lipolysis. Enhanced oxidative stress can promote pathways, like inflammation, that also influence adipose function. Exposure to hydrogen peroxide or ROS accumulation activates the transcription factor NF-κB, leading the expression of proinflammatory cytokines such as TNF-α or IL-1 (Lawrence, 2009; Li and Karin, 1999; Morgan and Liu, 2011). Intriguingly, treatment with either TNF-α or IL-6 in differentiated adipocytes can increase the expression of ATGL and enhance glycerol production (a marker of lipolysis). Moreover, in vitro studies reveal that TNF-α induces lipolysis in differentiated human and murine adipocytes through multiple distinct pathways (ie, mitogen-activated protein kinase kinase; extracellular signal-related kinase 1/2; and elevation of cyclic adenosine monophosphate; NH2-terminal kinase) (Ryden et al., 2002; Yang et al., 2011; Zhang et al., 2002). Similarly, treatment with IL-6 induces lipolysis through increased expression of ATGL in soleus muscles ex vivo (Macdonald et al., 2013) and enhanced glycerol production in 3T3-L1 cells (Ji et al., 2011). Our results reveal that fetal and neonatal nicotine exposure culminates in increased steady-state mRNA levels of proinflammatory mediators, including TNF-α and IL-6 by 6 months of age. These results coincide with previous findings demonstrating that in utero nicotine exposure resulted in increased circulating proinflammatory cytokines throughout development and in 11-week-old offspring (Mohsenzadeh et al., 2014; Orellana et al., 2014). Since inflammation was present in 6-month female WAT and not at 3 weeks, this suggests that nicotine does not have a direct effect on inflammation but that augmented oxidative stress may mediate the expression of proinflammatory cytokines. One research group demonstrated that CD-1 mice fed a nicotine-containing diet for 14 weeks postnatally decreased adipocyte cell size without affecting white adipose inflammation (Liu et al., 2018). In fact, treatment with 4-HNE in 3T3-L1 cells induced inflammation by activating p38, subsequently increasing the expression of cyclooxygenase-2 (Zarrouki et al., 2007). Further studies are warranted to determine whether inflammation may in part promote lipolysis in prenatal nicotine-exposed WAT long term. In summary, our findings demonstrate that MNE during pregnancy and lactation leads to decreased WAT adipocyte size and impaired lipid storage in postnatal life characterized by augmented lipolysis and inflammation triggered possibly by a pro-oxidative stress environment. This impairment in WAT lipid storage can contribute to increased circulating TGs, leading to enhanced hepatic de novo lipogenesis, high blood pressure, and increased CVD risk as we have previously shown (Gao et al. 2008; Ma et al., 2014). These findings provide insight into the consequence of prolonged oxidative stress driving WAT dysfunction long-term. Postnatal intervention strategies targeting oxidative stress may benefit children exposed to nicotine in utero through cigarette smoke and/or NRT. In fact, studies demonstrate that the use of antioxidants (ie, anthocyanin, quercetin, bottle gourd) can significantly reduce dyslipidemia (Li et al., 2015; Talirevic and Jelena, 2012). Overall, results from this study suggest that therapeutic strategies targeting oxidative stress may effectively protect nicotine-exposed offspring from WAT dysfunction and dyslipidemia. FUNDING Canadian Institutes of Health Research (MOP86474 to A.C.H., MOP111011 to D.B.H.); research grant from the Women’s Development Council to D.B.H. N.G.B. is a recipient of a Whaley Fellowship and a Molly Towell Perinatal Research Foundation Fellowship. ACKNOWLEDGMENT The authors would like to acknowledge Dr Lin Zhao for his technical support. REFERENCES Abate N. ( 2012). Adipocyte maturation arrest: A determinant of systemic insulin resistance to glucose disposal. J. Clin. Endocrinol. Metab.  97, 760– 763. Google Scholar CrossRef Search ADS PubMed  Ahmadian M., Duncan R. E., Varady K. A., Frasson D., Hellerstein M. 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Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

Maternal Nicotine Exposure Leads to Augmented Expression of the Antioxidant Adipose Tissue Triglyceride Lipase Long-Term in the White Adipose of Female Rat Offspring

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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1096-6080
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10.1093/toxsci/kfy083
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Abstract

Abstract Globally, approximately 10%–25% of women smoke during pregnancy. Since nicotine is highly addictive, women may use nicotine-containing products like nicotine replacement therapies for smoking cessation, but the long-term consequences of early life exposure to nicotine remain poorly defined. Our laboratory has previously demonstrated that maternal nicotine exposed (MNE) rat offspring exhibit hypertriglyceridemia due to increased hepatic de novo lipogenesis. Hypertriglyceridemia may also be attributed to impaired white adipose tissue (WAT) lipid storage; however, the effects of MNE on WAT are not completely understood. We hypothesize that nicotine-induced alterations in adipose function (eg, lipid storage) underlie dyslipidemia in MNE adults. Female 6-month-old rats exposed to nicotine during gestation and lactation exhibited significantly decreased visceral adipocyte cell area by 40%, attributed, in part, to a 3-fold increase in adipose triglyceride lipase (ATGL) protein expression compared with vehicle. Given ATGL has antioxidant properties and in utero nicotine exposure promotes oxidative stress in various tissues, we next investigated if there was evidence of increased oxidative stress in MNE WAT. At both 3 weeks and 6 months, MNE offspring expressed 37%–48% higher protein levels of superoxide dismutase-1 and -2 in WAT. Since oxidative stress can induce inflammation, we examined the inflammatory profile of WAT and found increased expression of cytokines (interleukin-1β, tumor necrosis factor α, and interleukin-6) by 44%–61% at 6 months. Collectively, this suggests that the expression of WAT ATGL may be induced to counter MNE-induced oxidative stress and inflammation. However, higher levels of ATGL would further promote lipolysis in WAT, culminating in impaired lipid storage and long-term dyslipidemia. white adipose tissue, maternal nicotine exposure, oxidative stress, adipocytes Evidence now demonstrates that cigarette smoke plays a critical role in the development of dyslipidemia and obesity in children exposed during perinatal life (Wen et al., 2010; Weng et al., 2012). This is especially concerning given that approximately 10%–25% of women still smoke during pregnancy, with rates as high as 59% in certain Indigenous communities (Cui et al., 2014; Roman-Galvez et al., 2017; Tappin et al., 2010; Tong et al., 2013). To date, nicotine replacement therapies (NRTs) for smoking cessation (ie transdermal patches, gums, e-cigarettes) are thought to benefit pregnant women who are highly addicted and unable to quit smoking by other means (Oncken and Kranzler, 2003). However, the consequences of fetal and neonatal exposure to nicotine alone on the long-term metabolic health of the offspring have yet to be fully defined. Maternal nicotine use during gestation and lactation leads to significant exposure to the fetus and neonate since it is present in fetal blood and transferred through amniotic fluid, placental tissue, and breast milk (Luck and Nau, 1987; Luck et al., 1985). Numerous animal studies demonstrate that nicotine use during pregnancy leads to adverse neurobehavioral, pulmonary, cardiovascular, and metabolic outcomes in the offspring (Barra et al., 2017; Chou and Chen, 2014; Dasgupta et al., 2012; Ma et al., 2014; Pauly and Slotkin, 2008). Specifically, we and others have shown in rats that perinatal exposure to nicotine alone leads to increased blood pressure, adiposity, decreased glucose tolerance, and impaired pancreatic beta cell development (Fox et al., 2012; Gao et al., 2005, 2008; Holloway et al., 2008). Moreover, MNE during gestation and lactation leads to increased hepatic de novo lipogenesis and increased hepatic and circulating triglycerides (TGs) in postnatal life (Ma et al., 2014). This reciprocates human studies which have demonstrated that children exposed to maternal smoke in utero have higher TG levels later in life (Cupul-Uicab et al., 2012). Since increased circulating plasma TGs is strongly associated with cardiovascular disease (CVD) (Bansal et al., 2007), elucidating the underlying molecular mechanisms promoting dyslipidemia will undoubtedly uncover therapeutic targets to reduce unwarranted CVD risk to these nicotine-exposed individuals. Aside from the liver, the augmented circulating and hepatic TG accumulation observed in these MNE offspring could also be attributed to impairments in other metabolic tissues, such as white adipose tissue (WAT). The major role of WAT is to store excess circulating TGs to prevent glucose and lipid toxicity, and moreover, ectopic fat deposition (Abate, 2012). Excess circulating TGs promote adipocytes to undergo differentiation, hypertrophy, and/or hyperplasia to process and store lipids. Once the maximum TG storage capacity is reached, fatty acids can spillover in the plasma, increasing substrate availability for hepatic TG synthesis (Abate, 2012). Ultimately, hypertriglyceridemia contributes to various systemic abnormalities like dyslipidemia, insulin resistance, and CVD (Abate, 2012). Currently, the effects of MNE on WAT function are not completely understood. Studies by Somm et al. (2008) indicated that MNE during gestation resulted in adipocyte hypertrophy and increased expression of adipogenic transcription factors in 3-week male offspring, but the long-term effects were not examined. A more recent study by Fan et al. (2016) demonstrated that maternal exposure of nicotine (from gestational day 9 to weaning) led to decreased adipocyte size in 26-week-old male offspring, suggesting an impaired capacity for lipid storage. Furthermore, this study also found an increase in steady-state mRNA levels of adipocyte differentiation and lipogenic markers in MNE WAT compared with vehicle (Fan et al., 2016). However, given most women addicted to smoking would be exposed to nicotine (ie, NRT or cigarettes) in both prenatal and perinatal life, the goal of this study was to determine if this longer, more relevant, window of nicotine exposure impacts adipose function in both male and female offspring. Moreover, this window covers the entire period of rat adipose tissue differentiation (Greenwood and Hirsch, 1974). We hypothesize that in utero nicotine exposure will adversely impact WAT in the offspring, leading to dyslipidemia in adulthood. To examine this further, we will investigate if WAT function is affected due to altered adipocyte size, proliferation (ie, AKT-1), differentiation (ie, CCAAT/enhancer-binding protein [C/EBP]-α/β, sterol regulatory element-binding transcription factor [SREBP]-1c), lipogenesis (ie, lipoprotein lipase [LPL], acetyl-coA carboxlyase (ACCα), fatty acid synthase (FAS), Acsl1), fatty acid transport (ie, FATP1, FATP4), and/or lipolysis (ATGL) in our well-established rat model of maternal nicotine exposure (MNE). MATERIALS AND METHODS MNE rat model Nulliparous 200–250 g female Wistar rats (Harlan, Indianapolis, Indiana) were injected daily subcutaneously with either saline (vehicle) or nicotine bitartrate at 1 mg/kg/day (Sigma-Aldrich, St Louis, Missouri) 2 weeks prior to mating, during gestation, and until weaning (PND21). This nicotine dose results in maternal serum cotinine concentrations of 135.9 ± 7.86 ng/ml, which is comparable to “moderate” female smokers (80 ng/ml) or NRT users (169.9 ng/ml) (Holloway et al., 2006; Shahab et al., 2017). Litters were culled to 8 at birth and following weaning, rats were housed as sibling pairs until 6 weeks of age, then subsequently housed individually. Male and female offspring were sacrificed via carbon dioxide inhalation at 3 weeks and 6 months of age. Gonadal WATs were extracted, either fixed in formalin or frozen in liquid nitrogen and stored at −80°C for histological and molecular analyses, respectively. All rats were conventionally housed in polycarbonate microisolator cages with ad libitum access to water and standard chow diet (Teklad 22/5 rodent diet; Envigo) under controlled lighting (12:12 L:D), humidity (40%–50%), and temperature (22°C). In accordance with the Canadian Council for Animal Care guidelines, animal experiments were approved by the Animal Research Ethics Board at McMaster University. Gonadal adipocyte cell area A portion of gonadal WAT was fixed in 10% (v/v) neutral buffered formalin overnight, washed in water, and embedded in paraffin. Cross sections were stained with hematoxylin and eosin (H&E) and photographed using the Olympus BX50 microscope under a 10× objective. Cell areas were quantified from a minimum of 160 adipocytes from 2 cross sections/rat using Northern Eclipse software (Empix Imaging Inc.). RNA extraction and real time-polymerase chain reaction Total RNA was extracted from homogenized 3-week and 6-month WAT samples using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). Two micrograms of RNA were reversed-transcribed to cDNA (high-capacity cDNA Reverse Transcription Kit, Applied Biosystems). Forward and reverse primer sets used for real time-polymerase chain reaction (RT-PCR), listed in Table 1, were designed with the National Center for Biotechnology Information’s primer designing tool. Relative transcript abundance was determined using SensiFAST No-ROX SYBR Green Supermix (FroggaBio) and the Bio-Rad CFX384 Real Time System. Samples were assayed in triplicate and relative fold change was calculated using comparative cycle times (Ct) method normalized to β-actin. The relative abundance was calculated using the formula 2ΔΔCt, where ΔΔCt was the normalized value. Table 1. Forward and Reverse Primer Sequences Used for Quantitative Real-Time PCR. Gene  Forward  Reverse  GenBank/Reference  AKT-1  ATGTGTATGAGAAGAAGCTGAGCC  GTTCACTGTCCACACACTCCA  NM_033230.2  C/EBPα  GCCGGGAGAACTCTAACTCC  TCGATGTAGGCGCTGATGTC  NM_001287577.1  C/EBPβ  ACCACGACTTCCTTTCCGAC  TAACCGTAGTCGGACGGCTT  NM_024125.5  SREBP1c  CATGGACGAGCTACCCTTCG  TCTTCGATGTCGGTCAAGAGC  NM_001276707.1  LPL  GGATGCAACATTGGAGAACCC  GCTGGGGTTTTCTTCATTCAGC  NM_012598.2  ACCα  TCCGTATGTGACCAAAGACC  TACGTTGTTCCCAAGGACTG  NM_022193.1  FAS  GGACATGGTCACAGACGATGAC  CGTCGAACTTGGACAGATCCTT  NM_017332.1  ACSL1  CTACAGGCAACCCCAAAGGA  AATGCACTCTCCGTCGCTT  NM_012820.1  FATP1  CAGCCTCTGTGGCCTCATT  ACCCACGTACACACCGAAC  NM_053580.2  FATP4  CTTGGGCAACTTTGACAGCC  AGGACAGGATGCGGCTATTG  NM_001100706.1  ATGL  AACGCCACTCACATCTACGG  TACCAGGTTGAAGGAGGGGT  NM_001108509.2  EGR-1  CGAGCGAACAACCCTACGA  CGATGTCAGAAAAGGACTCTGTG  NM_012551.2  FOXO-1  TGCAGCAGACACCTTGCTAT  TTGGGGCTGGGGGAATTTAG  NM_001191846.2  IL-1β  CAGCTTTCGACAGTGAGGAGA  GTCGAGATGCTGCTGTGAGA  NM_031512.2  TNFα  CCGGGCAGGTCTACTTTGGA  AGGCCACTACTTCAGCGTCTCG  NM_012675.3  IL-6  CTTCCAGCCAGTTGCCTTCTTG  TGGTCTGTTGTGGGTGGTATCC  NM_012589.2  CD68  ACTGGGGCTCTTGGAAACTACAC  CCTTGGTTTTGTTCGGGTTCA  NM_001031638.1  SOD-1  ATTGGCCGTACTATGGTGGTC  GCAATCCCAATCACACCACA  NM_017050.1  SOD-2  ATTGCCGCCTGCTCTAATCA  TCCCACACATCAATCCCCAG  NM_017051.2  Gene  Forward  Reverse  GenBank/Reference  AKT-1  ATGTGTATGAGAAGAAGCTGAGCC  GTTCACTGTCCACACACTCCA  NM_033230.2  C/EBPα  GCCGGGAGAACTCTAACTCC  TCGATGTAGGCGCTGATGTC  NM_001287577.1  C/EBPβ  ACCACGACTTCCTTTCCGAC  TAACCGTAGTCGGACGGCTT  NM_024125.5  SREBP1c  CATGGACGAGCTACCCTTCG  TCTTCGATGTCGGTCAAGAGC  NM_001276707.1  LPL  GGATGCAACATTGGAGAACCC  GCTGGGGTTTTCTTCATTCAGC  NM_012598.2  ACCα  TCCGTATGTGACCAAAGACC  TACGTTGTTCCCAAGGACTG  NM_022193.1  FAS  GGACATGGTCACAGACGATGAC  CGTCGAACTTGGACAGATCCTT  NM_017332.1  ACSL1  CTACAGGCAACCCCAAAGGA  AATGCACTCTCCGTCGCTT  NM_012820.1  FATP1  CAGCCTCTGTGGCCTCATT  ACCCACGTACACACCGAAC  NM_053580.2  FATP4  CTTGGGCAACTTTGACAGCC  AGGACAGGATGCGGCTATTG  NM_001100706.1  ATGL  AACGCCACTCACATCTACGG  TACCAGGTTGAAGGAGGGGT  NM_001108509.2  EGR-1  CGAGCGAACAACCCTACGA  CGATGTCAGAAAAGGACTCTGTG  NM_012551.2  FOXO-1  TGCAGCAGACACCTTGCTAT  TTGGGGCTGGGGGAATTTAG  NM_001191846.2  IL-1β  CAGCTTTCGACAGTGAGGAGA  GTCGAGATGCTGCTGTGAGA  NM_031512.2  TNFα  CCGGGCAGGTCTACTTTGGA  AGGCCACTACTTCAGCGTCTCG  NM_012675.3  IL-6  CTTCCAGCCAGTTGCCTTCTTG  TGGTCTGTTGTGGGTGGTATCC  NM_012589.2  CD68  ACTGGGGCTCTTGGAAACTACAC  CCTTGGTTTTGTTCGGGTTCA  NM_001031638.1  SOD-1  ATTGGCCGTACTATGGTGGTC  GCAATCCCAATCACACCACA  NM_017050.1  SOD-2  ATTGCCGCCTGCTCTAATCA  TCCCACACATCAATCCCCAG  NM_017051.2  Table 1. Forward and Reverse Primer Sequences Used for Quantitative Real-Time PCR. Gene  Forward  Reverse  GenBank/Reference  AKT-1  ATGTGTATGAGAAGAAGCTGAGCC  GTTCACTGTCCACACACTCCA  NM_033230.2  C/EBPα  GCCGGGAGAACTCTAACTCC  TCGATGTAGGCGCTGATGTC  NM_001287577.1  C/EBPβ  ACCACGACTTCCTTTCCGAC  TAACCGTAGTCGGACGGCTT  NM_024125.5  SREBP1c  CATGGACGAGCTACCCTTCG  TCTTCGATGTCGGTCAAGAGC  NM_001276707.1  LPL  GGATGCAACATTGGAGAACCC  GCTGGGGTTTTCTTCATTCAGC  NM_012598.2  ACCα  TCCGTATGTGACCAAAGACC  TACGTTGTTCCCAAGGACTG  NM_022193.1  FAS  GGACATGGTCACAGACGATGAC  CGTCGAACTTGGACAGATCCTT  NM_017332.1  ACSL1  CTACAGGCAACCCCAAAGGA  AATGCACTCTCCGTCGCTT  NM_012820.1  FATP1  CAGCCTCTGTGGCCTCATT  ACCCACGTACACACCGAAC  NM_053580.2  FATP4  CTTGGGCAACTTTGACAGCC  AGGACAGGATGCGGCTATTG  NM_001100706.1  ATGL  AACGCCACTCACATCTACGG  TACCAGGTTGAAGGAGGGGT  NM_001108509.2  EGR-1  CGAGCGAACAACCCTACGA  CGATGTCAGAAAAGGACTCTGTG  NM_012551.2  FOXO-1  TGCAGCAGACACCTTGCTAT  TTGGGGCTGGGGGAATTTAG  NM_001191846.2  IL-1β  CAGCTTTCGACAGTGAGGAGA  GTCGAGATGCTGCTGTGAGA  NM_031512.2  TNFα  CCGGGCAGGTCTACTTTGGA  AGGCCACTACTTCAGCGTCTCG  NM_012675.3  IL-6  CTTCCAGCCAGTTGCCTTCTTG  TGGTCTGTTGTGGGTGGTATCC  NM_012589.2  CD68  ACTGGGGCTCTTGGAAACTACAC  CCTTGGTTTTGTTCGGGTTCA  NM_001031638.1  SOD-1  ATTGGCCGTACTATGGTGGTC  GCAATCCCAATCACACCACA  NM_017050.1  SOD-2  ATTGCCGCCTGCTCTAATCA  TCCCACACATCAATCCCCAG  NM_017051.2  Gene  Forward  Reverse  GenBank/Reference  AKT-1  ATGTGTATGAGAAGAAGCTGAGCC  GTTCACTGTCCACACACTCCA  NM_033230.2  C/EBPα  GCCGGGAGAACTCTAACTCC  TCGATGTAGGCGCTGATGTC  NM_001287577.1  C/EBPβ  ACCACGACTTCCTTTCCGAC  TAACCGTAGTCGGACGGCTT  NM_024125.5  SREBP1c  CATGGACGAGCTACCCTTCG  TCTTCGATGTCGGTCAAGAGC  NM_001276707.1  LPL  GGATGCAACATTGGAGAACCC  GCTGGGGTTTTCTTCATTCAGC  NM_012598.2  ACCα  TCCGTATGTGACCAAAGACC  TACGTTGTTCCCAAGGACTG  NM_022193.1  FAS  GGACATGGTCACAGACGATGAC  CGTCGAACTTGGACAGATCCTT  NM_017332.1  ACSL1  CTACAGGCAACCCCAAAGGA  AATGCACTCTCCGTCGCTT  NM_012820.1  FATP1  CAGCCTCTGTGGCCTCATT  ACCCACGTACACACCGAAC  NM_053580.2  FATP4  CTTGGGCAACTTTGACAGCC  AGGACAGGATGCGGCTATTG  NM_001100706.1  ATGL  AACGCCACTCACATCTACGG  TACCAGGTTGAAGGAGGGGT  NM_001108509.2  EGR-1  CGAGCGAACAACCCTACGA  CGATGTCAGAAAAGGACTCTGTG  NM_012551.2  FOXO-1  TGCAGCAGACACCTTGCTAT  TTGGGGCTGGGGGAATTTAG  NM_001191846.2  IL-1β  CAGCTTTCGACAGTGAGGAGA  GTCGAGATGCTGCTGTGAGA  NM_031512.2  TNFα  CCGGGCAGGTCTACTTTGGA  AGGCCACTACTTCAGCGTCTCG  NM_012675.3  IL-6  CTTCCAGCCAGTTGCCTTCTTG  TGGTCTGTTGTGGGTGGTATCC  NM_012589.2  CD68  ACTGGGGCTCTTGGAAACTACAC  CCTTGGTTTTGTTCGGGTTCA  NM_001031638.1  SOD-1  ATTGGCCGTACTATGGTGGTC  GCAATCCCAATCACACCACA  NM_017050.1  SOD-2  ATTGCCGCCTGCTCTAATCA  TCCCACACATCAATCCCCAG  NM_017051.2  Protein extraction and Western blot WAT was homogenized in RIPA buffer (50 mM Tris-HCL, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 0.25% C24H39NaO4) supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitors (20 mM NaF, 40 mM Na-pyrophosphate, 40 mM Na3VO4, 200 mM β-glycerophosphate disodium salt hydrate). The solution was sonicated, mixed in a rotator for 1 h at 4°C, and centrifuged at 300 × g for 15 min at 4°C. The total cellular protein extract in the collected supernatant was quantified by colorimetric DC protein assay (BioRad). Loading samples were heated at 50°C for 10 min to denature the proteins. Proteins (20 μg/well) were separated by size via gel electrophoresis and transferred onto polyvinylidene difluoride membrane (Millipore). Membranes were blocked in 1× Tris-buffered saline-Tween 20 buffer with 5% nonfat milk or 5% BSA (blocking solutions), and then probed using primary antibodies diluted in the blocking solution (Table 2). A mouse or rabbit secondary antibody was used to detect primary antibody diluted in the blocking solution at 1:5000 or 1:10 000 dilution, respectively (Table 2). Immuno-reactive bands were visualized, and relative band intensity was calculated using ImageLab software (BioRad) and normalized to β-actin, as previously performed (Ma et al., 2014). Table 2. Western Blot Antibodies, Dilutions Used in Experiments, and Company and Catalog Information Antibody Name  Source  Dilution  Company (No. Catalog)  4-HNE  Mouse monoclonal  1:500  R&D Systems Minneapolis, Minnesota (No. MAB3249)  AKT-1  Rabbit polyclonal  1:500  Abcam Inc., Cambridge, Massachusetts (No. abcam 5919)  ATGL (H-144)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-67355)  C/EBPα (14AA)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-61)  C/EBPβ (C-19)  Rabbit polyclonal  1:300  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-150)  SOD-1 (FL-154)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-11407)  SOD-2 (FL-222)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-30080)  SREBP 1c (H-160)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-8984)  Mouse IgG (H+L) Secondary  Sheep  1:5000  GE Healthcare UK, Pittsburgh, Pennsylvania (No. NA931)  Rabbit IgG (H+L) Secondary  Donkey  1:10 000  Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania (No. 711-001-003)  Antibody Name  Source  Dilution  Company (No. Catalog)  4-HNE  Mouse monoclonal  1:500  R&D Systems Minneapolis, Minnesota (No. MAB3249)  AKT-1  Rabbit polyclonal  1:500  Abcam Inc., Cambridge, Massachusetts (No. abcam 5919)  ATGL (H-144)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-67355)  C/EBPα (14AA)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-61)  C/EBPβ (C-19)  Rabbit polyclonal  1:300  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-150)  SOD-1 (FL-154)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-11407)  SOD-2 (FL-222)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-30080)  SREBP 1c (H-160)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-8984)  Mouse IgG (H+L) Secondary  Sheep  1:5000  GE Healthcare UK, Pittsburgh, Pennsylvania (No. NA931)  Rabbit IgG (H+L) Secondary  Donkey  1:10 000  Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania (No. 711-001-003)  Table 2. Western Blot Antibodies, Dilutions Used in Experiments, and Company and Catalog Information Antibody Name  Source  Dilution  Company (No. Catalog)  4-HNE  Mouse monoclonal  1:500  R&D Systems Minneapolis, Minnesota (No. MAB3249)  AKT-1  Rabbit polyclonal  1:500  Abcam Inc., Cambridge, Massachusetts (No. abcam 5919)  ATGL (H-144)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-67355)  C/EBPα (14AA)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-61)  C/EBPβ (C-19)  Rabbit polyclonal  1:300  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-150)  SOD-1 (FL-154)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-11407)  SOD-2 (FL-222)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-30080)  SREBP 1c (H-160)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-8984)  Mouse IgG (H+L) Secondary  Sheep  1:5000  GE Healthcare UK, Pittsburgh, Pennsylvania (No. NA931)  Rabbit IgG (H+L) Secondary  Donkey  1:10 000  Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania (No. 711-001-003)  Antibody Name  Source  Dilution  Company (No. Catalog)  4-HNE  Mouse monoclonal  1:500  R&D Systems Minneapolis, Minnesota (No. MAB3249)  AKT-1  Rabbit polyclonal  1:500  Abcam Inc., Cambridge, Massachusetts (No. abcam 5919)  ATGL (H-144)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-67355)  C/EBPα (14AA)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-61)  C/EBPβ (C-19)  Rabbit polyclonal  1:300  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-150)  SOD-1 (FL-154)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-11407)  SOD-2 (FL-222)  Rabbit polyclonal  1:1000  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-30080)  SREBP 1c (H-160)  Rabbit polyclonal  1:500  Santa Cruz Biotechnology Inc., Santa Cruz, California (No. sc-8984)  Mouse IgG (H+L) Secondary  Sheep  1:5000  GE Healthcare UK, Pittsburgh, Pennsylvania (No. NA931)  Rabbit IgG (H+L) Secondary  Donkey  1:10 000  Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania (No. 711-001-003)  Statistical analysis Statistical analyses were performed using Graphpad Prism 6 software. Results were presented as the mean of arbitrary values ± SEM. Grubbs’ test was used to determine significant outliers and data were tested for normality and equal variance. Comparisons between vehicle and nicotine-exposed offspring were assessed using an unpaired Student’s t test, with a p-value of < .05 deemed as significant. RESULTS MNE Leads to Decreased Visceral Adipocyte Size in 6-Month-Old Offspring Using our well-established rat MNE model leading to dyslipidemia (Gao et al., 2005; Ma et al., 2014), we wanted to first confirm whether prenatal nicotine exposure has long term effects on adipocyte size in the offspring. Histological analyses revealed both MNE male and female offspring exhibited smaller gonadal adipocytes compared with vehicle controls at 6 months of age (Figs. 1A and 1B) . Given the more pronounced effects of perinatal nicotine exposure on female WAT area (13.2% male vs 39.7% female decrease in adipocyte size), we continued to examine only female offspring to explore the underlying mechanisms involved. Gondal adipose tissue weight did not differ between MNE and vehicle offspring (data not shown), suggesting a possible increase in adipocyte number due to prenatal nicotine exposure. With decreased adipocyte cell area and subsequent increase in cell number, transcript and protein levels of the proliferation marker AKT-1 were subsequently measured. Despite a significant increase in mRNA levels, there were no corresponding changes in AKT-1 protein levels in 6-month female MNE WAT (Figs. 1C and 1D). Figure 1. View largeDownload slide Maternal nicotine exposure leads to decreased visceral adipocyte cell area in 6-month-old offspring. Representative cross-sections of gonadal WAT excised from (A) vehicle and in utero nicotine exposed 6-month-old male and female offspring were stained with hematoxylin and eosin. B, Bar graph representing calculated cell areas analyzed using Northern Eclipse software (n = 4/group/sex). C, Transcript and (D) protein levels of AKT-1 in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 7/nicotine). **,*** Significant difference (**p < .01, ***p < .001). Figure 1. View largeDownload slide Maternal nicotine exposure leads to decreased visceral adipocyte cell area in 6-month-old offspring. Representative cross-sections of gonadal WAT excised from (A) vehicle and in utero nicotine exposed 6-month-old male and female offspring were stained with hematoxylin and eosin. B, Bar graph representing calculated cell areas analyzed using Northern Eclipse software (n = 4/group/sex). C, Transcript and (D) protein levels of AKT-1 in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 7/nicotine). **,*** Significant difference (**p < .01, ***p < .001). In Utero Nicotine Exposure Increases the Expression of the Differentiation Marker C/EBPα in WAT of 6-Month-Old Female Offspring Given impairments in adipocyte differentiation can also decrease adipocyte size, we next measured the expression of key targets involved in adipogenesis (Abate, 2012), including C/EBP-α/β and SREBP-1c. Protein analyses revealed a significant increase in the 42 kDa band of C/EBP-α in female MNE offspring compared with vehicle at 6 months independent of changes in transcript levels (Figs. 2A and 2B). Quantitative real time PCR analyses revealed that WAT from MNE exposed offspring had a significant increase in steady-state transcript levels of C/EBPβ and SREBP-1c compared with controls (Figs. 2C and 2E). However, immunoblot analyses revealed no significant differences at the protein level for either target (Figs. 2D and 2F). To examine if perinatal nicotine exposure had direct effects on C/EBPα, we measured the transcript and protein levels at 3 weeks age but found no difference in expression from vehicle (Figs. 2G and 2H). Figure 2. View largeDownload slide In utero nicotine exposure increases the expression of the differentiation marker C/EBPα in WAT of 6-month-old female offspring. Transcript and protein levels of targets of interest in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month (A) C/EBPα mRNA and (B) protein levels. C, C/EBPβ mRNA and (D) protein levels. E, SREBP1-c mRNA and (F) protein levels. Three-week (G) C/EBPα mRNA and (H) protein levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 7/nicotine). *Significant difference (*p < .05). Figure 2. View largeDownload slide In utero nicotine exposure increases the expression of the differentiation marker C/EBPα in WAT of 6-month-old female offspring. Transcript and protein levels of targets of interest in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month (A) C/EBPα mRNA and (B) protein levels. C, C/EBPβ mRNA and (D) protein levels. E, SREBP1-c mRNA and (F) protein levels. Three-week (G) C/EBPα mRNA and (H) protein levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 7/nicotine). *Significant difference (*p < .05). MNE Does Not Increase the Expression of Markers Involved in Lipogenesis and Fatty Acid Transporters in 6-Month-Old Female Offspring Adipocyte size is influenced by its ability to breakdown TG molecules from circulation, transport its constitutive components intracellularly, and re-assemble TGs for storage (Kersten, 2014). Therefore, we wanted to determine whether perinatal nicotine exposure impaired TG transport and esterification by measuring the steady state transcript levels of key targets involved in lipogenesis and fatty acid transport. At 6 months of age we found no significant difference between groups in the expression of lipogenic markers including LPL, ACCα, and FAS, along with fatty acid transporters FATP1/4, and long-chain acyl CoA synthetase (ASCL)-1 in female MNE WAT (Figs. 3A–F). Figure 3. View largeDownload slide Maternal nicotine exposure does not increase the expression of markers involved in lipogenesis and fatty acid transporters in WAT of 6-month-old female offspring. Transcript levels of targets of interest in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR. Six-month (A) LPL, (B) ACCα, (C) FAS, (D) ASCL1, (E) FATP1, and (F) FATP4 mRNA levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Figure 3. View largeDownload slide Maternal nicotine exposure does not increase the expression of markers involved in lipogenesis and fatty acid transporters in WAT of 6-month-old female offspring. Transcript levels of targets of interest in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR. Six-month (A) LPL, (B) ACCα, (C) FAS, (D) ASCL1, (E) FATP1, and (F) FATP4 mRNA levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). In Utero Nicotine-Exposed Female Offspring Exhibit Increased Expression of ATGL at 6 Months, But Not at 3 Weeks Since enhanced lipolysis decreases adipocyte size (Zhang et al., 2017), we then examined whether MNE WAT had elevated expression of adipose triglyceride lipase (ATGL), the predominate enzyme involved in intracellular degradation (Zechner et al., 2009). At 6 months of age, we found a significant increase in both ATGL mRNA expression and protein levels in nicotine-exposed female WAT compared with vehicle controls (Figs. 4A and 4B), however this was not observed at 3 weeks (Figs. 4C and 4D). Altogether, these data suggest that augmented ATGL-induced lipolysis may lead to reduced adipocyte cell area in MNE WAT at 6 months. Figure 4. View largeDownload slide In utero nicotine-exposed female offspring exhibit increased expression of ATGL at 6 months, but not at 3 weeks. Transcript and protein levels of ATGL in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month ATGL (A) mRNA and (B) protein levels. Three-week ATGL (C) mRNA and (D) protein levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 8/nicotine). *,** Significant difference (*p < .05, **p < .01). Figure 4. View largeDownload slide In utero nicotine-exposed female offspring exhibit increased expression of ATGL at 6 months, but not at 3 weeks. Transcript and protein levels of ATGL in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month ATGL (A) mRNA and (B) protein levels. Three-week ATGL (C) mRNA and (D) protein levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 8/nicotine). *,** Significant difference (*p < .05, **p < .01). MNE Exhibit Enhanced Antioxidant Expression at 6 Months and 3 Weeks Evidence demonstrates that ATGL deficiency in mice is associated with accumulation of reactive oxygen species (ROS) and decreased superoxide dismutase (SOD)-2 expression, suggesting this enzyme may act as an antioxidant and suppress oxidative stress (Aquilano et al., 2016; Chen et al., 2017a,b). Moreover, we and others have previously shown that MNE promotes oxidative stress in the heart, pancreas, and placenta of adult offspring (Barra et al., 2017; Bruin et al., 2008; Sbrana et al., 2011). Therefore, we wanted to determine whether an imbalance between antioxidant defenses occurs in WAT of 6-month female MNE offspring by measuring the transcript and protein levels of the antioxidants SOD-1 and SOD-2. Both SOD 1 and 2 steady state mRNA and protein levels were significantly elevated in prenatal nicotine-exposed WAT in 6-month female offspring compared with vehicle (Figs. 5A–D). At 3 weeks of age, there was also a significant increase in SOD-1 protein levels, and a trending increase in SOD-2, in the nicotine-exposed WAT group compared with controls (Figs. 5E–H). To determine if there was oxidative stress, we measured protein levels of 4-hydroxynonenal (4-HNE) and found a nonsignificant upward trend in nicotine-exposed WAT at both 3-week and 6-month timepoints (Figs. 5I and 5J). Overall, these data suggest that prenatal nicotine exposure directly increases the expression of the antioxidant SOD-1 and SOD-2 in 6-month WAT which was enough to prevent lipid oxidative damage. Figure 5. View largeDownload slide In utero nicotine-exposed female offspring exhibit increased antioxidant expression in WAT at 3 weeks and 6 months. Transcript and protein levels of SOD-1 and SOD-2 in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month SOD-1 (A) mRNA and (B) protein levels and SOD-2 (C) mRNA and (D) protein levels. Three-week SOD-1 (E) mRNA and (F) protein levels and SOD-2 (G) mRNA and (H) protein levels. Protein levels of 4-HNE at (I) 6 months and (J) 3 weeks of age. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 8/nicotine). *,** Significant difference (*p < .05, **p < .01). Figure 5. View largeDownload slide In utero nicotine-exposed female offspring exhibit increased antioxidant expression in WAT at 3 weeks and 6 months. Transcript and protein levels of SOD-1 and SOD-2 in 3-week and 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR and Western blot, respectively. Six-month SOD-1 (A) mRNA and (B) protein levels and SOD-2 (C) mRNA and (D) protein levels. Three-week SOD-1 (E) mRNA and (F) protein levels and SOD-2 (G) mRNA and (H) protein levels. Protein levels of 4-HNE at (I) 6 months and (J) 3 weeks of age. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). Protein levels were expressed as means normalized to β-actin ± SEM (n = 8/vehicle; n = 8/nicotine). *,** Significant difference (*p < .05, **p < .01). In Utero Nicotine Exposure Exhibit Inflammation at 6 Months, But Not at 3 Weeks Enhanced oxidative stress can activate nuclear factor-κB (NF-κB), leading the expression of proinflammatory cytokines and inflammation (Li and Karin, 1999; Morgan and Liu, 2011). As well, activation of this pathway in differentiated 3T3-L1 cells leads to increased lipase expression and lipolysis (Chi et al., 2014). Since oxidative stress, inflammation, and lipolysis are integrated pathways, we wanted to determine whether inflammation was present in 6-month in utero nicotine-exposed WAT. At 6 months, we found a significant increase in the steady-state mRNA expression profile of proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and the macrophage marker CD68 (Figs. 6A–D). In contrast at 3 weeks of age, there was no significant difference in steady state mRNA levels of inflammatory markers between groups (Figs. 6E–H). Figure 6. View largeDownload slide Maternal nicotine-exposed female offspring exhibit increases in WAT inflammation at 6 months, but not at 3 weeks. Transcript levels of targets of interest in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR. Six-month (A) IL-1β, (B) TNFα, (C) IL-6, and (D) CD68 mRNA levels. Three-week (E) IL-1β, (F) TNFα, (G) IL-6, and (H) CD68 mRNA levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). *,**,*** Significant difference (*p < .05, **p < .01, **p < .001). Figure 6. View largeDownload slide Maternal nicotine-exposed female offspring exhibit increases in WAT inflammation at 6 months, but not at 3 weeks. Transcript levels of targets of interest in 6-month vehicle and perinatal nicotine-exposed WAT were determined via real time PCR. Six-month (A) IL-1β, (B) TNFα, (C) IL-6, and (D) CD68 mRNA levels. Three-week (E) IL-1β, (F) TNFα, (G) IL-6, and (H) CD68 mRNA levels. mRNA levels were expressed as means normalized to β-actin ± SEM (n = 15/vehicle; n = 11/nicotine). *,**,*** Significant difference (*p < .05, **p < .01, **p < .001). DISCUSSION Under normal physiological conditions, WAT serves as the principle lipid storage site to prevent ectopic lipid deposition in peripheral organs. The balance between lipolysis and lipogenesis dictates the overall amount of TG molecules stored as a single large lipid droplet in the cytoplasm of adipocytes, ultimately influencing its size (Bolsoni-Lopes and Alonso-Vale, 2015). Adipocytes have a limited TG storage capacity. Once exceeded, excess lipids will spill-over into circulation resulting in dyslipidemia and increased lipid content at ectopic sites like the liver (Abate, 2012). In our model, we have previously shown that MNE offspring have dyslipidemia with increased levels of circulating TGs (Gao et al., 2005; Ma et al., 2014). Although we have demonstrated that hepatic de novo lipogenesis is increased in nicotine-exposed offspring, the effect of nicotine exposure on WAT function were not examined. Previous studies indicate MNE during gestation or from gestation until weaning altered adipocyte size and increased the expression of adipocyte differentiation markers, but this was only examined in male offspring (Fan et al., 2016; Somm et al., 2008). Moreover, this exposure to nicotine in these studies (gestation only or mid-gestation to weaning only) does not mimic women’s smoking habits and/or nicotine use prior to pregnancy. Therefore, our goal was to determine if WAT plays a role in promoting dyslipidemia in both male and female offspring exposed to nicotine in prenatal and perinatal life. In this study, we demonstrated that both male and female offspring exposed to nicotine during prenatal and perinatal life decreased adipocyte cell area compared with vehicle controls at 6 months of age. Somm et al. (2008) reported that rats exposed to nicotine during pregnancy alone (from gestational day 4 for 14 days at 3 mg/kg/day) had adipocyte hypertrophy compared with vehicle controls in 3-week-old male offspring, but the effect on WAT size into adulthood, after nicotine exposure, was not examined. Similar to our findings, Fan et al. (2016) demonstrated that in utero nicotine exposure from mid-pregnancy (eg, gestational day 9) until weaning decreased adipocyte size in male WAT at 26 weeks of age. The dose of nicotine (1 mg/kg) administered in this study is relevant as it corresponds to moderate female smokers or NRT users (Holloway et al., 2006; Shahab et al., 2017), while doses used in other studies (2–3 mg/kg/day) correspond to heavy smokers (Fan et al., 2016; Somm et al., 2008). Examining the effects of MNE on WAT for a longer duration is important given adipose development occurs both pre- and postnatally in rats. In rodent adipose tissue, growth and differentiation occurs from late gestation to 4 weeks in postnatal life, while in humans this occurs from 5 to 29 weeks gestation (Greenwood and Hirsch, 1974; Poissonnet et al., 1984). In both species, adipose tissue expansion happens throughout life (Greenwood and Hirsch, 1974; Spalding et al., 2008). Given the extensive differentiation of adipose tissue during perinatal life, it is vulnerable to alterations by environmental cues (ie, drugs) during this developmental window. Furthermore, both mature adipocytes and adipocyte precursors like mesenchymal stem cells express nicotinic acetyl-choline receptor subunits, suggesting that nicotine may directly alter adipogenesis and adipocyte formation (Gochberg-Sarver et al., 2012; Hoogduijn et al., 2009). Since the effect on adipocyte size was more pronounced in female offspring, and previous studies focused on males (Fan et al., 2016; Somm et al., 2008), we decided to examine the effects of MNE on female adipose tissue function. One limitation of our study is that we did not evaluate the estrous cycle of the female rats. This is an important consideration since prenatal nicotine exposure increased progesterone levels in offspring compared with vehicle controls, which is reflective of estrous cycle perturbations (Holloway et al., 2006). Furthermore, others have reported that oscillations in metabolic related genes in the liver occur with estrous cycle (Villa et al., 2012). Since small adipocyte size results from aberrant adipogenesis, we examined whether MNE impaired adipocyte differentiation, lipid synthesis and transport, and/or lipolysis. We found that female MNE offspring exhibited increased mRNA and protein expression of the adipocyte differentiation factor C/EBPα and the lipolytic enzyme ATGL, without altering the expression of targets involved in lipogenesis and lipid transport. Similar to our results, Fan et al. (2016) found an increase in steady-state mRNA levels of adipocyte differentiation markers in MNE male offspring, including C/EBPα and SREBP-1c compared with control WAT in adulthood. Since these studies did not perform protein analyses, our study is the first to demonstrate that MNE induced C/EBPα protein levels in 6-month female WAT. Overall, the increased C/EBPα (42 kDa) expression in WAT suggests that MNE promote adipocyte differentiation and adipogenesis. C/EBPα contains 2 isoforms (30/42 kDa) alternatively translated from 1 mRNA transcript (Lin et al., 1993; Ossipow et al., 1993). Constitutive expression of the 42 kDa isoform can induce adipocyte differentiation in 3T3-L1 preadipocytes, suggesting that enhanced C/EBPα expression of this isoform can enhance adipocyte formation (Lin and Lane, 1994). Although the steady-state levels of AKT1, C/EBP-α, SREBP 1c mRNA were augmented in 6-month nicotine-exposed offspring, this did not translate to an alteration in protein levels. There are several cases in the literature whereby changes in the mRNA levels do not correlate to an alteration in protein, highlighting the importance of looking at both mRNA and protein (Vogel and Marcotte, 2012). Moreover, it is possible that the mRNA and protein levels of these key proliferation and differentiation markers may be altered earlier in development (ie, gestation and/or lactation) due to the direct effects of nicotine on their expression. For example, we have demonstrated that nicotine directly enhances the expression of the proliferation markers GADD45 and Rb1 in differentiating 3T3-L1 preadipocyte cells (data not shown). Therefore, in the absence of nicotine exposure after lactation, the levels of these proliferation and differentiation may have become normalized. This study was the first to demonstrate that both prenatal and perinatal nicotine exposure leads to increased ATGL mRNA and protein expression in adult female MNE offspring. This suggests that MNE may impair the lipid storage capacity of WAT via increased ATGL expression. Studies demonstrate that augmented ATGL leads to enhanced basal and stimulated WAT lipolysis and reduced adipocyte size (Ahmadian et al., 2009; Bezaire et al., 2009; Kershaw et al., 2006). The primary function of ATGL is to initiate lipolysis, by hydrolyzing the sn-2 ester bond of a TG molecule, resulting in the formation of diacylglycerol and fatty acid (Zechner et al., 2009). Therefore, decreased adipocyte cell size in gonadal WAT may be attributed to enhanced lipolysis in MNE offspring. Recently, evidence supports an additional role for ATGL—as an antioxidant and suppressor of oxidative stress. Oxidative stress is defined as an imbalance between antioxidant defenses and ROS production (free radicals) (Sena and Chandel, 2012). Prolonged oxidative stress can result in free radical damage, ultimately leading to mitochondrial-mediated apoptosis (Sena and Chandel, 2012). In vitro and in vivo analyses demonstrate that ATGL deficiency is associated with ROS accumulation and cellular apoptosis in renal podocytes and proximal tubules (Chen et al., 2017a,b). Similarly, ATGL inhibition in C2C12 myoblasts and in murine ATGL deficient skeletal muscle promotes oxidative damage and a defective cellular antioxidant response, characterized by a significant reduction in antioxidant SOD-2 expression and glutathione levels (Aquilano et al., 2016). Altogether, these findings suggest that ATGL may protect against oxidative stress. We and others have previously shown that MNE can cause oxidative stress in the offspring in various tissues including the heart, pancreas, and placenta (Barra et al., 2017; Bruin et al., 2008; Sbrana et al., 2011). Therefore, we examined the expression of antioxidants SOD-1/2 in WAT of 6-month female MNE offspring to assess whether an imbalance in antioxidant expression is present at the same time ATGL levels are elevated. Oxygen can oxidize other molecules to generate ROS, whereby majority of intracellular ROS are derived from superoxide (O2−). Superoxide is formed in the mitochondrial respiratory chain, emitted to the matrix and intermembrane space, and subsequently converted to hydrogen peroxide (H2O2) by SODs (Sena and Chandel, 2012). This study is the first to demonstrate that MNE WAT had increased transcript expression and protein levels of both SOD-1 and -2 at 6 months with evidence for increased SOD-1 at 3 weeks of age. Although nonsignificant, we also observed an upward trend for the oxidative marker 4-HNE in nicotine-exposed WAT compared with vehicle at both time points. This suggests that perinatal nicotine exposure may instigate WAT oxidative stress, triggering a rapid increase in antioxidant expression by weaning which persists into adulthood and may prevent long-term oxidative damage. Oxidative stress can contribute to altered adipocyte differentiation and lipolysis. Depending on environmental cues, mesenchymal stem cells can differentiate into various cell types derived from the mesodermal lineage including myocytes, adipocytes, osteocytes, and chondrocytes. Evidence suggests that elevated ROS levels, produced during oxidative stress, can stimulate adipogenesis while suppressing osteogenesis (Atashi et al., 2015). During conversion of stem cells to adipocytes, mitochondrial complexes I/III and the NADPH oxidase isoform NOX4 produce ROS to initiate adipocyte signaling cascades, leading to terminal differentiation (Atashi et al., 2015). Therefore, increased SOD-1 expression in fetal and neonatal nicotine-exposed WAT may suggest that augmented ROS production may directly promote the expression of adipogenic transcription factors like C/EBPα (Hu et al., 2005). In vitro analyses using both differentiated 3T3-L1 and human primary adipocyte cultures demonstrate that exposure to exogenous 4-HNE elevated lipolysis in basal and stimulated conditions characterized by a dose-dependent significant increase in glycerol and fatty acid release (Zhang et al., 2013). Altogether, these findings suggest that MNE may augment oxidative stress in WAT characterized by increased expression of antioxidants SOD-1/2 and ATGL, leading to altered adipocyte differentiation and lipolysis. Enhanced oxidative stress can promote pathways, like inflammation, that also influence adipose function. Exposure to hydrogen peroxide or ROS accumulation activates the transcription factor NF-κB, leading the expression of proinflammatory cytokines such as TNF-α or IL-1 (Lawrence, 2009; Li and Karin, 1999; Morgan and Liu, 2011). Intriguingly, treatment with either TNF-α or IL-6 in differentiated adipocytes can increase the expression of ATGL and enhance glycerol production (a marker of lipolysis). Moreover, in vitro studies reveal that TNF-α induces lipolysis in differentiated human and murine adipocytes through multiple distinct pathways (ie, mitogen-activated protein kinase kinase; extracellular signal-related kinase 1/2; and elevation of cyclic adenosine monophosphate; NH2-terminal kinase) (Ryden et al., 2002; Yang et al., 2011; Zhang et al., 2002). Similarly, treatment with IL-6 induces lipolysis through increased expression of ATGL in soleus muscles ex vivo (Macdonald et al., 2013) and enhanced glycerol production in 3T3-L1 cells (Ji et al., 2011). Our results reveal that fetal and neonatal nicotine exposure culminates in increased steady-state mRNA levels of proinflammatory mediators, including TNF-α and IL-6 by 6 months of age. These results coincide with previous findings demonstrating that in utero nicotine exposure resulted in increased circulating proinflammatory cytokines throughout development and in 11-week-old offspring (Mohsenzadeh et al., 2014; Orellana et al., 2014). Since inflammation was present in 6-month female WAT and not at 3 weeks, this suggests that nicotine does not have a direct effect on inflammation but that augmented oxidative stress may mediate the expression of proinflammatory cytokines. One research group demonstrated that CD-1 mice fed a nicotine-containing diet for 14 weeks postnatally decreased adipocyte cell size without affecting white adipose inflammation (Liu et al., 2018). In fact, treatment with 4-HNE in 3T3-L1 cells induced inflammation by activating p38, subsequently increasing the expression of cyclooxygenase-2 (Zarrouki et al., 2007). Further studies are warranted to determine whether inflammation may in part promote lipolysis in prenatal nicotine-exposed WAT long term. In summary, our findings demonstrate that MNE during pregnancy and lactation leads to decreased WAT adipocyte size and impaired lipid storage in postnatal life characterized by augmented lipolysis and inflammation triggered possibly by a pro-oxidative stress environment. This impairment in WAT lipid storage can contribute to increased circulating TGs, leading to enhanced hepatic de novo lipogenesis, high blood pressure, and increased CVD risk as we have previously shown (Gao et al. 2008; Ma et al., 2014). These findings provide insight into the consequence of prolonged oxidative stress driving WAT dysfunction long-term. Postnatal intervention strategies targeting oxidative stress may benefit children exposed to nicotine in utero through cigarette smoke and/or NRT. In fact, studies demonstrate that the use of antioxidants (ie, anthocyanin, quercetin, bottle gourd) can significantly reduce dyslipidemia (Li et al., 2015; Talirevic and Jelena, 2012). Overall, results from this study suggest that therapeutic strategies targeting oxidative stress may effectively protect nicotine-exposed offspring from WAT dysfunction and dyslipidemia. FUNDING Canadian Institutes of Health Research (MOP86474 to A.C.H., MOP111011 to D.B.H.); research grant from the Women’s Development Council to D.B.H. N.G.B. is a recipient of a Whaley Fellowship and a Molly Towell Perinatal Research Foundation Fellowship. ACKNOWLEDGMENT The authors would like to acknowledge Dr Lin Zhao for his technical support. REFERENCES Abate N. ( 2012). Adipocyte maturation arrest: A determinant of systemic insulin resistance to glucose disposal. J. Clin. Endocrinol. Metab.  97, 760– 763. Google Scholar CrossRef Search ADS PubMed  Ahmadian M., Duncan R. E., Varady K. A., Frasson D., Hellerstein M. 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Toxicological SciencesOxford University Press

Published: Mar 30, 2018

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