developmental stage: adult gender: Male diet: high fat/high sucrose (HFS) diet agent: Resveratrol tissue: Thoracic aorta
Treatment protocol
After baseline assessment, the twenty four male rhesus monkeys were quasi- randomized into one of three groups: a high fat/high sucrose (HFS) diet (n=10); a HFS diet supplemented with resveratrol (HFS+R) (n=10); or, remaining on the healthy standard diet (SD) (n=4). The standard diet was a purified biscuit consisting of 13% of kcal in fat and less than 5% sucrose by weight. The HFS diet was a specially formulated purified ingredient diet with 42% of kcal in fat and approximately 27% sucrose by weight (Harlan, Teklad, Madison, WI). The monkeys were gradually switched to the HFS diet over a 3-week period. All groups received 2 meals per day of the specified diet in allotments that represent ad libitum feeding, yet consumption was isocaloric across groups. Resveratrol was supplied by DSM Nutritional Products (Parsippany, NJ) and the dosing for monkeys was determined to be 40.7 mg. The resveratrol was used to formulate a flavored primate treat (Bi-Serv, Frenchtown, NJ) and given to the monkeys prior to each meal, thus, the monkeys received a total dose of 80 mg per day. All monkeys, regardless of weight, received the same amount of resveratrol. Monkeys in non-resveratrol groups receive a placebo treat. After completion of data collection at the one year time point, the resveratrol dose was increased to 240 mg per day. After baseline assessment, 10 male rhesus monkeys were begun on a high fat/high sucrose (HFS) diet (n=10) plus Resveratrol; the HFS diet was a specially formulated purified ingredient diet with 42% of kcal in fat and approximately 27% sucrose by weight (Harlan, Teklad, Madison, WI). The monkeys were gradually switched to the HFS diet over a 3-week period. All groups received 2 meals per day of the specified diet in allotments that represent ad libitum feeding, yet consumption was isocaloric across groups. Resveratrol treatment for the first year was 80mg per day. At the one year time point they were switched to 240mg per day until the end of 2 years of dietary intervention.
Growth protocol
Twenty-four adult (7-13 years old) male rhesus monkeys (Macaca mulatta) were housed individually in standard nonhuman primate caging on a 12h light/12h dark cycle, room temperature (78 +/- 2 degrees F), and humidity at 60 +/- 20%. One pairing was maintained throughout the study; all other monkeys had extensive visual, auditory, and olfactory but limited tactile contact with monkeys housed in the same room. Monkeys received 2 meals per day at estimated ad libitum levels throughout the study. Water was always available ad libitum. Monkeys were monitored minimally 3 times daily by trained animal care staff. During baseline assessments, all monkeys were maintained on a commercially available closed formula monkey chow (TestDiet #5038 Purina Mills, Richmond, IN).
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from rhesus monkey aorta at 24-months of dietary intervention using Trizol reagent (Invitrogen) and further purified using RNeasy mini columns (Qiagen). Quality and Quantity were tested with an Agilent Bio-Analyzer using RNA 6000-Nano Chips.
Label
biotin
Label protocol
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
Hybridization protocol
Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's SentrixHumanHT-12 v4 Expression BeadChips (Illumina, San Diego, CA). Each array on the HumanHT-12 v4 Expression BeadChip targets more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources and offers genome-wide transcriptional coverage of well-characterized genes, gene candidates, and splice variants, with approximately 15-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
Scan protocol
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
Data was extracted using the Illumina GenomeStudio software(v1.6.0). Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std.
