This research conforms to the Institutional and National Guide for the Care and Use of Laboratory Animals. The present study was conducted in accordance with the ARRIVE guidelines and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Twenty female C57BL/6 mice (21 ± 1.5 g) were housed according to the standard protocol for maintenance of laboratory animals (21–23 °C; 50 ± 5 % humidity; and 12 h artificial light cycle). After two weeks of adaptation with AIN 93M diet, female mice were mated overnight. The vaginal plug was confirmed, and mothers were randomly divided to the LF-HC and HF-LC diets, which contain 16% and 48% of calories as fat, respectively. LF-HC diet supplies 64% of calorie as carbohydrate compared with 32% in the HF-LC group. Both diets had 3.97 kcal/g. (Table 1) Mothers received LF-HC and HF-LC diets during gestation and lactation periods in a pair-fed model. The number of offspring was equaled in all cages (n=4), nursed, and lactated with their mothers for three weeks. Mice were separated according to the sex, and all offspring received LF-HC diet up to 6 weeks. In the end, one male and one female offspring were randomly selected and euthanized by ketamine and xylazine. Liver tissues were removed, snap froze and kept at -80 °C for final gene and protein expression, as well as gas chromatography- mass spectroscopy (GC/MS) analysis at six weeks of age. The study protocol illustrated at figure 1.
SIRT1 gene expression
Frozen liver tissues were powdered in liquid nitrogen (N2), and total RNA was extracted using TRIzol Lysis Reagent (QIAGEN Inc., Valencia, CA 91355, USA). After tissue washing by PBS, 300 𝜇l of TRIzol was added to prepare the lysates. Then, chloroform was added, and samples were centrifuged (10000 rpm) for 15 min at 4 °C. To purify the RNA supernatants were collected, isopropanol was added, and samples were stored for 30-60 min at -20 °C. Finally, the samples were centrifuged (10000 rpm) for 15 min at 4 °C. To omit the possible contaminants such as lipids, 700 𝜇l of alcohol (70-80%) was added and centrifuged (7500 rpm) for 10 min at 4 °C. RNA sediments were dissolved in 20 𝜇l of distilled water for 5 min at 45-55 °C. The ND-1000 spectrophotometer (DPI-1, QIAGEN Inc., USA) was used to assess RNA quantity at 260 nm. Quality (integrity) of the extracted RNA was checked by agarose gel electrophoresis. The cDNA was synthesized from one microgram of total RNA using Fermentas protocols (Fermentas Co. USA).
ABI StepOne sequence detection system (Applied Biosystems, California, USA) was used for the real-time polymerase chain reaction (RT-PCR) with 10 pmol of the forward and reverse primers for SIRT1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, housekeeping gene), 1 μl of the synthesized cDNA and SYBR Green I Master Mix (Roche) in duplicate runs. Both primers were designed by the Primer Express software 2.0.0 as bellows: SIRT1 (Forward: GTGTCATAGGATAGGTGGTG; Reverse TATGAAGAGGTGTTGGTGG) and GAPDH gene (Forward: CTATGTTTGTGATGGGTGTGA; Reverse AGTGGATGCAGGGATGATGT).
The copy threshold (Ct) values are calculated and normalized against GAPDH mRNA as an appropriate control . The amplification profile included 40 three-step cycles: 94°C for 20, 58-60 °C for 30 s and 72 °C for 30 s. The results were generated and analyzed using the 2^-∆∆Ct method in which was computed as follows:
Liver tissues were washed by PBS, powdered on ice, and transferred to a micro tube. Then, 200 𝜇l of Rippa buffer, as a protease inhibitor, was added and stored for 90 min at 4 °C. The lysates were centrifuged by a refrigerator centrifuge. 50 mg of protein (from each sample) was added to the same amounts of loading buffer for 5 min at 95 °C. Then, denaturized proteins were loaded on wells, inserted in the western pons and running buffer was added at 90-100 mV for 220-230 min. Proteins were separated by 10% SDS-PAGE (PH=6.8) and transferred to nitrocellulose filter membrane at 80 mV for 70-80 min at 4 °C. After overnight incubation in blocking buffer (skim dried milk), diluted SIRT1 antibodies were diluted by TBS-Tween buffer (1:200), added to the membrane and shaken for 120 min. Samples were washed three times with TBS-Tween buffer and shaken for 10 min in each step. Anti-SIRT1 antibody (1:3000) was added and stored on the shaker for 90 min. Then, samples were washed by TBS-Tween for three times and shaken for 10 min. GAPDH antibody was used as the housekeeping protein. Chemiluminescence detection system and ImageJ software were used for detection of protein bands and analyzing the data, respectively.
Gas chromatography-mass spectrometry (GC/MS)
Frozen samples were powdered in N2, and a mixture of chloroform-methanol (1 mL, 2:1; v/v) was added to the 0.5 g of each sample and shaken for 10 min. The chloroform phase containing the lipids was separated, and the aqueous phase was extracted again, similarly. Then, samples were centrifuged (4500 rpm) for 5 min and supernatants collected for the derivatization step. The 2% H2SO4-methanol, as a methylation/transesterification solution, was added to the extracted samples and refluxed for 45 min at 80 °C. After neutralization with NaOH (1 M; PH=7), n-hexane was added to each sample, and supernatant was collected for GC/MS analyses.
Analyses were performed on a 5977A MS and 7890B GC (Aligent Co., USA) equipped with the split/splitless column as injector system and HP5-MS (60 m × 0.25 m × 0.25 𝜇m) column for fatty acid profile analysis. The oven temperature was kept at 70 ◦C for 5 min, then programmed at 15 °C /min to 150 °C and kept for 2 min. Finally, the system programmed at 20 °C /min to 290 °C and kept for 10 min. Electron impact ionization (EI+, 70 eV) was used for all samples. The split ratio was settled on 1:20.
By considering a power of 80% and α=0.05, sample size was computed according to differences in gene and protein expression of SIRT-1, weight of offspring and lipid profile in the liver through a power analysis using the Mann-Whitney-Wilcoxon test and one-way ANOVA in IBM SPSS statistics software (version 18; IBM Corp). The required sample size was six (for gene and protein expression of SIRT-1), ten (for weight) and five (for lipid profile of liver) offspring in each group to determine the variations. Data were tested for normal distribution using the Kolmogorov-Smirnov test and did not have normal distributions in gene expression even after all of the transformation methods. Then, the differences were measured by the Mann-Whitney-Wilcoxon test. One-way ANOVA was used to compare the lipid profile of liver among the groups. All data are expressed as the means ± SE. The level of significance was set at P<0.05.