The Effects Of Insulin Like Growth Factor 2 On Cell Proliferation,Differentiation And Glucose Uptake In Pre-adipose 3T3-L1 Cell Line

BackgroundInsulin-like growth factor-2(IGF2)is a widely expressed peptide hormone in cell division.Although it is abundant in serum,understanding of its physiological and pathological role is limited compared with that of IGF1.IGF2 regulates foetal development and differentiation,but its role in adults is still poorly understood.Evidence suggests roles in these tissues including skeletal muscle,adipose tissue,bone and ovary.Altered IGF2 expression has been observed in metabolic conditions,obesity,diabetes and the polycystic ovary syndrome.Here we observed effects of IGF2 on cell proliferation,differentiation and glucose uptake in 3T3-L1 cells and explored its mechanism.MethodsCell culture and differentiationMouse embryo 3T3-L1 fibroblasts were grown at 37? under a humidified 5%C02 atmosphere in medium containing 10%fetal bovine serum(FBS).Two days after confluence,pre-adipocytes of 3T3-L1(designated as day 0)were cultured in differentiation medium(DM)containing 10%FBS,10 pg/mL insulin,0.5 mM isobutylmethylxanthine,and 1 ?M dexamethasone.After 2 days,DM was switched to 10%FBS and 10 ?g/mL insulin for two days,and then changed to 10%FBS medium and the medium was replaced every 2 days.MTS assayCell viability was determined by an MTS assay in 96-well plates.3T3-L1 cells were seeded at a density of 5 × 103 cells per well.After 24 h incubation,cells were treated with different concentrations of IGF2(10ng/ml-100ng/ml)for 48 h.After 48 h,cells were then treated with 5 mg/ml MTS at 37? for 4 hours.Absorbance at 490 nm was considered to represent the viability of the cells.ELISADuring the period of 3T3-L1 differentiation,the concentrations of IGF2 in the supernatant were determined by enzyme linked immunosorbent assay(ELISA).Glucose uptakeGlucose uptake activity was measured through detection of 2NBDG uptake,which was accomplished by performing fluorescence microscope and flow cytometer.Oil Red O stainingTo evaluate the effects of IGF2 on 3T3-L1 differentiation,the cells were cultured with DM in the presence of various concentrations of IGF2(10ng/ml-100ng/ml).Oil Red-O staining was performed after 48 hours.3T3-L1 adipocyte cells were washed with phosphate buffered saline(PBS)and fixed with 4%paraformaldehyde.After Oil Red-O stain,cells were photographed using a phase-contrast microscope in combination with a digital camera at 100 × magnification.RNA extraction and real-time quantitative RT-PCRTotal RNA was isolated from 3T3-L1 adipocytes using the RNase kit and used to synthesize cDNA for analysis by real-time reverse transcription-polymerase chain reaction(RT-PCR).Quantitative real-time PCR was performed in a 20 ?l reaction mixture.The cycle conditions were as follows:95°C for 5 min,followed by 45 cycles involving denaturing at 95 ? for 10 s,annealing at 60? for 30 s,and extension at 72?for 10 s.Results1.We performed an MTT assay to analyze the viability of 3T3-L1 pre-adipocyte cells treated with IGF2.The results revealed that treatment of IGF2 exhibited a dose-dependent increase in the absorption at 490nm.2.During the 3T3-L1 differentiation,IGF2 expression did not change according to the results of ELISA.However,in the last phase of differentiation(day 12),the expression reached the peak.3.High dose of IGF2 promoted 3T3-L1 differentiation by Oil Red-O staining.Treatment with 100ng/ml IGF2 could enlarge the lipid droplets as well as enhance the lipid accumulation in 3T3-L1 adipocytes.4.Both fluorescence microscope and flow cytometer revealed that IGF2 induced an increase in glucose uptake.The fluorescence intensity was increased 11.6%?22%and 24.8%after exposing to IGF2 of lOng/ml?50ng/ml and 100ng/ml,compared with the control group.5.We performed qPCR to analyze the mRNA expression levels of PPARy?CEBP??LPL?FAS?leptin and adiponectin.The mRNA expression of PPAR??PGC1?and LPL was increased with the treatment of IGF2.On the contrary,the expression of leptin was reduced.ConclusionsOur results uncovered that IGF2 could promote the proliferation and differentiation of 3T3-L1 cells.Furthermore,glucose uptake was also induced with different concentrations of IGF2 in a dose-dependent manner,during which period the effect of IGF2 on the expression of glucolipid metabolism and adipose tissue differentiation related genes might play an indispensable role.