Tissue-Specific Expression and the Regulation of Porcine FTO
|School||Nanjing Agricultural College|
|Course||Basic Veterinary Science|
|Keywords||Pig FTO Tissue distribution Maternal protein restricted feeding Hippocampus Neurons|
FTO (Fat mass and obesity associated gene) is obesity-related gene which belongs to the demethy super family relying on Fe (II) and 2-oxoglutarate. Researches on FTO gene have been mostly focused on the effect of different single nucleotide polymorphism (SNP) alleles on body mass index (BMI) and its demethy function. Studies on porcine FTO are limited, no information is available, up to now, regarding the tissue distribution of FTO protein and mRNA, and the regulation of FTO expression in the pig. Therefore, the present study was aimed to describe the tissue distribution of FTO protein in Meishan pigs, to demonstrate the localization of FTO mRNA in hippocampus and the effect of maternal protein restriction on hippocampal expression of FTO in pig offspring at different ages. Furthermore, primary porcine hippocampal neurons were used to investigate the effect of glucocorticoids on FTO protein expression in vitro.1 Tissue specific expression of porcine FTO protein and the effect of maternal protein restrictionMeishan sows were divided into control and protein restriction groups fed with either standard protein level diet containing 12% protein during pregnancy,14% protein during breast feeding or protein restriction diet containing 6% protein during pregnancy,7% protein during lactation, respectively. The piglets were fed with standard protein level diet after weaning. At 8 months of age, male offspring pigs were sacrificed and hippocampus, adipose tissue, liver and muscle were collected. FTO protein content was determined by using Western Blot. The results showed that FTO protein was highly expressed in hippocampus, moderately in adipose tissue, liver and rarely in muscle. No significant effect of maternal protein restriction was observed on the expression of FTO protein in all the tissues investigated.2 Localization of FTO mRNA in porcine hippocampus and the effect of maternal protein restriction on hippocampal FTO expressionAt 70 days of pregnancy (E70),1 day (D1) and 35 days (D35) after birth, fetuses or piglets were sacrificed and hippocampus was sampled. FTO mRNA localization in hippocampus was demonstrated by using fluorescent in situ hybridization (FISH); FTO mRNA expression in the hippocampus was quantitated with Real-time PCR and protein expression was detected by using Western Blot. FTO mRNA was widely expressed in the pyramidal cell layer of hippocampus but not in the glial cells. FTO mRNA expression in hippocampus was greatly increased in protein restriction group at E70 and D1 (P< 0.05), but no change was found in D35 piglets; FTO protein expression in hippocampus did not match the mRNA content, maternal protein restriction did not affect hippocampal FTO protein content in E70 fetuses, D1 or D35 piglets.3 Effect of glucocorticoid treatment on protein expression of FTO in porcine primary cultured hippocampal neuronsMale piglets were sacrificed within 24 hours after birth and the hippocampus was dissected for isolating neurons. After 7 days of incubation. neurons were treated with cortisol (1μM,10μM) and dexamethasone (10μM) for 4 h before harvest for cell viability test using MTT and FTO expression quantification using Western Blot. Cortisol and dexamethasone treatment significantly decreased the cell viability (P< 0.05).10μM cortisol and dexamethasone treatment significiantly increased FTO protein expression in hippocampal neurons in vitro (P<0.05).