All animal procedures have undergone an extensive review process and were in accordance with the guidelines of Institutional Animal Care and Use Committee of the Oregon National Primate Research Center (ONPRC) and Oregon Health & Science University. Protocols involved in this study were developed to ameliorate suffering and have been approved under IACUC ID number: IS00000224 (0622 for internal purposes). The Animal Care and Use Program at the ONPRC abides by the Animal Welfare Act and Regulations (CFR 9, Ch 1, Subchapter A) enforced by the USDA, the Public Health Service Policy on Humane Care and Use of Laboratory Animals, in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the recommendations of the Weatherall report; The Use of Non-human Primates in Research.
Our model and materials and methods for fetal studies has previously been described in detail 
. Briefly, Japanese macaques matched for age (5–7 years at start) and weight (7–9 Kg) were randomly assigned to two dietary groups: 1: Control diet (CTR; 13% of calories from fat; Monkey Diet no. 5052, Lab Diet, Richmond, IN, USA) or 2: High-fat diet (HFD; 35.2% of calories from fat; Custom Diet 5A1F, Test Diet, Richmond, IN, USA). The HFD also included calorically dense treats made with peanut butter. Both diets are sufficient in vitamin, mineral, and protein content for normal growth. Prior to this study, all animals were maintained on standard monkey chow in large outdoor enclosures and were naive to any experimental protocols.
The animals were group housed and had ad libitum access to food and water. Each maternal group was housed with two males and the females were checked each successive year for pregnancies during the breeding season by ultrasound, which allows an estimate of gestational age ±5 days. Twice a year the animals underwent IV glucose tolerance tests (IVGTT), once during the late summer (nonpregnant state) and once during the early 3rd trimester of pregnancy. All of the above procedures were done under ketamine sedation (5–10 mg/kg).
For fetal studies, ONPRC veterinarians terminated singleton pregnancies from dams by cesarean section at gestational day 130 (G130), as determined by ultrasound. Normal full-term pregnancies for Japanese macaques is 175 days, thus G130 is in the early 3rd trimester. Pregnant dams were fasted overnight for approximately 16 hours prior to surgical procedure. Females were initially sedated with ketamine hydrochloride (100 mg/ml) at a dose of 10–15 mg/kg. Once animals were sedated they were delivered to the surgical area and placed on isoflurane gas; induced at 3%, then maintained at 1.0–1.5%. Cesarean sections were performed by trained ONPRC veterinarians and their staff. After cesarean section, fetuses were deeply anesthetized with sodium pentobarbital (>30 mg/kg i.v.) and exsanguinated. The liver was removed, weighed and the right lobe was stored for the subsequent RNA extractions and histological analyses used in this study. Pre and post-operative care was maintained by ONPRC veterinary staff. All surgical procedures used in this study, were performed each scheduled day in an identical manner, following a consistent routine in both technique and timing.
For our fetal studies, we are reporting differences in HFD fetuses whose mothers were exposed to the maternal diet for at least four consecutive years. In the fifth year of our studies, a diet-reversal protocol (REV) was initiated to assess dietary impact independent of maternal obesity. This protocol entailed switching a subgroup of adult females that had been exposed to a high-fat diet for four consecutive years, to a control diet 1–3 months before becoming pregnant and throughout the pregnancy. The CTR cohort contained 6–8 animals (2–5 females, 3–4 males), the HFD cohort contained 8 animals (4 females, 4 males) and the REV cohort contained 7 animals (5 females, 2 males).
For our juvenile studies, full-term offspring were maintained with their birth mothers on the same diet as consumed during pregnancy until weaning. Between 7 and 8 months, the offspring were weaned to create diet cohorts with the first dietary designation indicating the maternal diet in-utero and before weaning, and the second designation indicating the offspring’s diet after weaning. The two juvenile diet groups examined in this study were CTR/CTR and HFD/HFD. The CTR/CTR cohort contained 7 males and 5 females and the HFD/HFD cohort contained 6 males and 4 females.
The mean age of the juvenile offspring at time of necropsy was 12.9 months with a 95% confidence interval of between 12.7–13.1 months. Necropsy was carried out by an a-priori
protocol by the veterinary staff at ONPRC as previously described 
. The liver was removed, weighed and the right lobe was stored for the subsequent RNA extractions and histological analyses used in this study.
Reactions were run on an Applied Biosystems 7300 as relative quantification plates using macaque specific primers as previously described 
. Additional primers were designed to quantify autonomic targets of interest (Definition of abbreviations: F, forward primer; R, reverse primer):
Alpha 1A Adrenergic Receptor (ADRA1A; F: CGACACCTGCACTCAGTCACA,
R: CCTCGAAGATGGCGGAGAA, Genbank Accesion No.: NW_001122890),
Alpha 1B Adrenergic Receptor (ADRA1B; F: CAGCTAAGACGTTGGGCATTG,
R: GGCTTCAGGGTGGAGAACAA, Genbank Accesion No.: NW_001120992),
Alpha 2A Adrenergic Receptor (ADRA2A; F: CTGGTGGCCACGCTTGTC,
R: CGTCGAGCGCCAGGTAGAT, Genbank Accesion No.: NW_001124223),
Type-1 Cannabinoid Receptor (CB1R; F: ACGCTTTCCGGAGCATGTT,
R: GCGTTGTTTGCGTGTTTGTG, Genbank Accesion No.: NM_001032825),
Type-2 Cannabinoid Receptor (CB2R; F: GGGCATGTTCTCTGGAAAGC,
R: ACCTCACGTCCAGCCTCATT, Genbank Accesion No.: NW_001111036),
Cholinergic Receptor, Nicotinic, Alpha 7 (CHRNA7; F: TGGTGGTGACGGTGATCGT,
R: CACGCGCACCAGTTCAGA, Genbank Accesion No.: NM_001032883),
Neuropeptide-Y Y1 Receptor (NPY1R; F: ATTTCCGGTCTCGGGATGAT,
R: AATGCGACTGGGCTTGCTT, Genbank Accesion No.: NM_001032866),
Glucose Transporter 2 (GLUT2; F: GACCACGTCCTGCTGCTTTAG,
R: GGTCCACAGAAGTCCGCAAT, Genbank Accesion No.: NW_001112558).
Real-Time PCR reaction products were run on a 2% agarose gel to verify amplicon singularity and size. Bands of the expected size were excised, gel purified (Qiaquick gel extraction kit, Qiagen #28706) and sequenced. Target specificity was confirmed by BLAST and comparing amplicon sequence with the NCBI macaque database.
NPY immunoreactivity was detected with a sheep polyclonal NPY antibody (Cat. #AB1583, Lot # JC1676120, Millipore, Temecula, CA, 1
4000). Tyrosine hydroxylase immunoreactivity was detected with mouse monoclonal ascites (Cat. # MAB318 - clone LNC1, Millipore, Temecula, CA, 1
200). Both antibodies have been previously characterized for specificity in the macaque and antibody control reactions were also employed in this study 
Liver tissue postfixed in 10% zinc formalin was embedded in paraffin and sectioned at 16 µm. Sections were deparaffinized with xylene and rehydrated in a descending alcohol series (100%, 70%, 50%). Heat induced epitope retrieval (HIER) was performed with a citrate based antigen unmasking solution (100X, Vector Laboratories, cat. # H-3300) for approximately 15 minutes in a commercial autoclave. Following HIER, sections were washed in 1X PBST (1X PBS with 0.05% Tween 20) and blocked for 1 hour at room temperature in 5% normal serum (NPY; donkey, TH; goat). Primary antibodies were incubated for 48 hrs at 4°C in a humidified chamber. Sections were then washed in 1X PBST and the appropriate secondary antibody (Alexa-fluor donkey anti-sheep 555, Alexa-fluor goat anti-mouse 488 or 555, Molecular Probes, Invitrogen), diluted 1
500 in PBST was applied for 1 hr at room temperature. For doubled labeled immunohistochemistry, a cocktail of each antibody was used. Sections were washed in 1X PBST and coverslips were applied using elvanol mounting medium.
Liver sections were de-identified and imaged with a C-APO 40X 1.2 W Corr M27 objective on an LSM710 laser-scanning confocal microscope using Zen software (Carl Zeiss, Thornwood, NY). To minimize bias between samples, we selected portal and parenchymal regions according to specific criteria. Utilizing epifluorescence from the GFP channel, which contained no observable signal of nerve staining, we visually identified portal triads that: 1) contained a distinct portal vein, hepatic artery and bile canniculi and 2) were of a similar size that would fit cleanly into our field of view. For parenchymal regions, we chose regions that had no discernable structural elements other than sinusoids and that were located roughly midway within the hepatic lobule.
Image acquisition of nerve structures was performed with a 561 nm laser and acquisition parameters were optimized to maximize the signal-to noise ratio of the 555 fluorophore without saturating individual pixel signal intensity. Z- stacks, with a step-size of 1µm/section, were acquired for each target region using as many optical sections as necessary to image through the entire thickness of each liver section. For both fetal and juvenile studies, we acquired at least 8–10 fields for each region (portal triad, parenchyma) conforming to our selection criteria for each animal. One liver section from each animal was examined (Fetal study: 6 CTR, 8 HFD, 7 REV; Juvenile study: 12 CTR/CTR, 9 HFD/HFD). All images for the fetal and juvenile studies were acquired in an identical manner.
Quantification of Nerve Fiber Density
Fetal and juvenile sympathetic nerve fiber density was quantified in an identical and blinded manner. Sympathetic nerve fiber density was normalized to hepatic volume using Imaris 7.1 image analysis software (Bitplane, Zurich Switzerland). Background autofluorescence was utilized to calculate the volume of hepatic tissue in each image by constructing a surface object with the following parameters: Smoothing 5 µm, background 9 µm, threshold.250, number of voxels above 10.
An additional surface was constructed to determine the volume of sympathetic immunoreactivity present in each image by the following parameters: Smoothing 0.2 µm, background 0.5 µm, threshold 5, maximum intensity 35, number of voxels above 10. Each image was visually inspected for the integrity of the surface calculations and the volume statistics were exported into Excel. The nerve fiber volume was then normalized to total hepatic volume for each image.
Data is expressed as the median normalized nerve density for each respective diet group. All graphs were made with Prism software (GraphPad Software, Inc., La Jolla, CA.). Representative images were created using Imaris 7.1 as maximum intensity projections and linear adjustments to brightness and contrast were made using Adobe Photoshop CS (Adobe Inc., Los Altos, CA.).
TUNEL Assay and Apoptosis Quantification
All tissue sections were treated according to the manufacturer instructions using the fluorescent staining of paraffin-embedded tissue protocol from the Apo Tag Fluorescein In Situ Apoptosis Detection Kit (#S7110 Millipore, Temecula, CA) without modification. Sections were counter stained with DAPI, mounted and viewed by fluorescence microscopy using a standard GFP filter at 20x with a Leica DM4000B microscope. Five random, non-overlapping digital images were obtained from each animal (11 CTR/CTR, 10 HFD/HFD) using the Leica Application Suite V3.6 and apoptotic events were counted by a blinded observer using Image J and normalized to the total surface area.
For reverse transcriptase-PCR (RT-PCR) of CHRNA7, 1.3 ug of total RNA prepared from two randomly selected juvenile livers (1 CTR/CTR, 1 HFD/HFD) was reverse transcribed in a 75 ul reaction using a Taqman Reverse Transcription kit (Applied Biosystems N808-0234) according to manufacturer’s protocol. 50 ng of cDNA was amplified using Taq platinum polymerase in a 50 µl reaction. 200 pmol of the 5′ and 3′ primers were used in a Touchdown protocol to amplify a single 500 bp band (95°C for 2 min., [(95°C for 30 s, 64°C for 30 s, 72°C for 1 min) x 20 cycles], 95°C for 30 s, [(54°C for 30 s, 72°C for 1 min) x 20 cycles], 72°C for 2 min, 4°C hold). The primers used for this reaction were: (Forward: CGCTCACCGTCTACTTCTCC, Reverse: GTAGGGCTCTTTGCAGCACT, Genbank Accesion No.: NM_001032883). PCR reaction products were run on a 1.5% agarose gel to verify amplicon singularity and size. Bands of the expected size were excised, gel purified (Qiaquick gel extraction kit, Qiagen #28706) and sequenced. Target specificity was confirmed by BLAST and comparing amplicon sequence with the NCBI macaque database.
Fresh frozen right lobe of juvenile liver was sectioned at 10 µm, fixed in 10% zinc formalin at 4°C for 10 minutes and stained with Oil-Red-O and hematoxylin according to manufacturers instructions (American Master Tech Scientific Inc.). Sections were coded and read in a blinded manner by a board certified pathologist.
The right lobe of the juvenile liver was used for this procedure. Briefly, 100 mg of hepatic tissue was pulverized and digested in 2 ml of 30% KOH at 95°C for 30 min. The homogenate (150 µl) was placed on No. 1 Whatman filter paper and washed in 66% ethanol with constant stirring for 30 min. The filter paper was removed, dried, and cut into small pieces. Glycogen was converted to glucose with 31.1 U amyloglucosidase (Sigma Chemical) in 0.2 M acetate buffer (pH 4.8, 0.5% glacial acetic acid, 0.12 M sodium acetate) at 37°C for 60 min. Glucose concentration of this solution was determined in triplicate on a microplate using glucose oxidase method (Sigma) and compared with concurrently run standards of glycogen (Sigma Chemical). Results are expressed as milligrams glycogen per grams tissue (wet weight). This expanded juvenile cohort consisted of 17 CTR/CTR animals (8 females, 9 males) and 14 HFD/HFD animals (7 females, 7 males) and included all the animals used in the nerve fiber assay, TUNEL assay and Real-Time PCR experiments.
Data for all analyses were first compiled and tested for normality by Shapiro-Wilk. Data were then tested for overall significance by Kruskal-Wallis rank sum, followed by a Wilcoxon rank sum test with a Bonferroni adjusted alpha, when needed, to determine significance between multiple diet groups. Data analysis was performed with STATA (College Station, Texas) statistical software and graphs were made with Prism software (GraphPad Software, Inc., La Jolla, CA).