Evaluation Of Irisin On Browning Of White Adipose Tissue And Activiation Of Brown Adipose Tissue By MRI

Part 1 Irisin Induces White Adipose Tissue Browning in Mice as Assessed by Magnetic Resonance ImagingPurpose:To non-invasively evaluate the effect of Irisin promoting the browning of white adipose tissue(WAT)in vivo and related mechanism using magnetic resonance imaging(MRI).Methods:Subcutaneous white adipose tissue(s WAT)was isolated from the inguinal region of the mice.The primary white adipocytes extracted from mice were isolated,cultured,induced and differentiated into mature primary white adipocytes,and identified by Oil red O staining.Immunohistochemistry(IHC)and western blotting were used to determine the expression levels of uncoupling protein 1(UCP1)and deiodiase type II(DIO2)(a specific protein in brown fat cells)to assess the effect of different concentrations of Irisin(0,20 and 40 nmol/ml)on primary white adipocytes after 6 days of treatment.In vivo,MRI was used to assess the intervention effect of 14days after intraperitoneal injection of irisin on normal diet mice(NCD).The volume of viscera,subcutaneous and total WAT was measured by T1 weighted imaging(T1WI),and the fat fraction(FF)in WAT was measured by chemical shift selective imaging(CSSI).In order to further evaluate whether Irisin has the function of improving metabolism,the changes of serum indexes and hepatic steatosis were monitored in mice fed with high-fat diet(HFD).Results:The primary mouse primordial mature white adipocytes were successfully cultured and identified in vitro.The expression level of UCP1 and DIO2 protein in the primary mouse white adipocytes was significantly increased after Irisin intervention.The best intervention dose of Irisin was 20 nmol/ml and the intracellular red stained lipid drops were significantly reduced.After Irisin intervention,the weight of NCD mice significantly lost weight(28.23±1.58g vs 27.54±1.60g).MRI measured volumes of subcutaneous(0.86±0.42 cm~3),visceral(0.81±0.34 cm~3)and total(1.61±0.74cm~3)WAT were significantly lower after treatment comparing to the controls(s WAT:1.52±0.22 cm~3,v WAT:1.24±0.27 cm~3,t WAT:2.76±0.34 cm~3).Gross pathology also confirmed the decrease of WAT.CSSI quantitative data showed fat fraction(FF)in WAT reduced(77.82±1.53%)compared to the controls(81.42±1.64%).In vitro histopathology showed significant reduction in the area of white adipocytes,and immunohistochemistry(UCP1:208.75 vs 1928.42;DIO2:282.08 vs 820.36)and WB confirmed that after Irisin intervention,the UCP1 and the DIO2 expression significantly increased.After Irisin treatment of HFD mice,blood glucose(7.72±1.13 mmol/L)and cholesterol(2.55±0.40mmol/L)levels were significantly reduced than controls(blood glucose:12.88±2.01mmol/L;cholesterol:3.78±0.76 mmol/L),and the content of lipid droplets in liver(2.39±1.92%)was significantly reduced to control groups(12.03±7.30%).Conclusion:Irisin can promote browning of primary white adipocytes and white adipocytes in vivo.At the animal experiments,the effect of Irisin in promoting the browning of WAT can be accurately detected by MRI noninvasively in vivo.In addition,Irisin improved diet-induced obesity and related metabolic disorders in mice.Part 2 Irisin Activated the Brown Adipose Tissue in Mice as Assessed by Magnetic Resonance ImagingObjective: To explore the effect of a hormone Irisin on brown adipose tissue(BAT)and related mechanism by magnetic resonance imaging(MRI)and near infrared spectroscopy(NIRS).Methods: Brown adipose tissue(BAT)was isolated from the scapular area of mice.The primary progenitor brown adipocytes were isolated,cultured,induced and differentiated into mature adipocytes and identified by Oil red O staining.Immunohistochemistry(IHC)and western blotting were used to determine the expression of uncoupling protein 1(UCP1),a specific protein of brown adipocytes,and to assess the effects of Irisin at different concentrations(0,20 and 40 nmol/m L)on cultured brown adipocytes after 6 days of treatment.Fourteen days after intraperitoneal injection of Irisin,magnetic resonance chemical shift selective imaging(CSSI)of fat in normal food(NCD)mice was performed to measure fat fraction(FF)in BAT.Meanwhile,the activity of BAT was analyzed by near infrared spectroscopy(NIRS).To further evaluate the activity of Irisin in obese mice induced by high fat diet(HFD).Results: The primary mouse brown adipocytes were successfully cultured successfully,and Oil red O staining showed a large number of small multicellular red stained lipid droplets in the cells.After Irisin intervention,the expression of UCP1 protein in primary brown adipocytes was significantly increased.The optimal intervention dose of Irisin was 20 nmol/m L.After Irisin intervention,NCD mice significantly lost weight(control: 28.23± 1.58g;test: 27.54± 1.60g).In vivo MR imaging showed that the FF value in BAT(43.82±4.31%)by CSSI was significantly lower than that in the control group(60.80±6.59%);NIRS showed that the fluorescence signal intensity(SI)of BAT in Irisin treated group was significantly enhanced,and the size of brown adipocytes in vitro was significantly increased by histopathology(experimental group: 0.94±0.19 μm).The control group was 0.42±0.12 μm),which was in the activated state.Meanwhile,the expression level of browned protein UCP1 after Irisin intervention was significantly higher than that of the PBS group.After Irisin treatment of HFD mice,the lipid droplet area in brown fat cells was significantly reduced to normal(experimental group: 0.05±0.04 μm2;control group: 0.20±0.08 μm2).The expression level of UCP1 protein in brown adipocytes increased significantly with Irisin treatment.Conclusion: Irisin can activate the activity of primary brown adipocytes and BAT.The effect of Irisin on BAT can be non-invasively verified by MRI and near infared spectroscopy(NIRS)in vivo.In addition,Irisin can effectively activate the BAT of diet induced obese mice.