LRG1 Involved In Adipose Tissue Remodeling By Promoting Angiogenesis

ObjectiveTo identify the expression levels of LRG1 in normal and obese mice;To determine the effect of cold stimulation on LRG1 expression in adipose tissues of obese mice.To explore the expression level of LRG1 in mature 3T3-L1 adipocytes.To explore the effect of LRG1 on the function of human adipose microvascular endothelial cells.MethodsAnimalsA total of 60 C57BL/6J mice（male,8 weeks old）were obtained from Charles River Laboratories Animal Technology Company（Beijing,China）.All animals were raised in the same environment with constant temperature and humidity.Mice were randomly divided into two groups to receive different diets after one week of adaptive feeding.One group of mice were given normal diet with 5% fat（NC,n=10）,and the other group were fed with high-fat diet with 60 % fat（HFD,n=50）.The mice in the HFD group were confirmed as obesity when their weight was 20%heavier than the normal diet mice.All animal experiments were in accordance with the animal management regulations of the ministry of health of China.（Document no.55,2001）and the Guidelines from the national institutes of health conformed to the NIH guidelines（NIH Publication No.85-23,revised 1996）.Experiment in miceThe first part: Three normal and obese mice were randomly selected.And then the mature adipose cells and endothelial cells were separated from the adipose tissue by digestion and immunomagnetic beads for transcriptome sequencing.Blood was collected and stored in-80℃,and part of organs stored with4% paraformaldehyde,while the other part was stored in-80℃ for subsequent experiments.The second part: C57BL/6J male mice were randomly divided into 30℃,4℃,and-10℃ groups after the HFD mice model with obesity.The cold facilities were designed with a 12 h-light and 12 h-dark rhythm.Before cold exposure,all mice were adapted at 16 ℃for 1 week,then transferred to the environment with4℃ in daytime and 16℃ at night for three days.4℃ group remained at 4℃ for the entire four weeks of experimental duration.While-10℃ groups of mice were kept at 4℃ for three consecutive days.-10℃ mice were then exposed to-10 for 12 h ℃during daytime and 4 ℃for 12 h during nighttime for four days,and further maintained at-10 for 24 h for ℃ the rest 3 weeks.For-10℃-exposed animals,fresh drinking water was frequently changed.Extra snow and frost was put in the cage floor to make sure mice had free access to moisture.For thermoneutrality,30℃mice were directly transferred in 30 and maintained at this temperature for ℃ four weeks.After four-week cold exposure,mice were sacrificed.Subsequently,liver tissues,subcutaneous adipose tissues and epididymalvisceral adipose tissues were harvested and weighed.Blood was collected and stored in-80℃,and one part of organs stored with 4% paraformaldehyde,while the other part was stored in-80℃for subsequent experiments.Insulin tolerance test and glucose tolerance testsIPGTT and ITT were implemented at the fourth week after cold exposure.After fasting overnight,mice were treated with glucose at a dose of 2g/kg body weight and insulin at a dose of 1U/kg body weight.Then,the blood glucose contents were measured from the tail vein of mice at 0,15,30,60 and 120 min after the treatment.Serum Biochemical analysisBlood biochemical test was performed to detect blood lipids,including TG,TC,LDL-C and HDL-C.Immunohistochemistry and H&E staining analysisThe adipose tissues were fixed overnight in 4% paraformaldehyde,and then buried in paraffin.The tissue sections（5μm）made from embedded tissues were treated with immunohistochemistry staining of CD31 and LRG1.The positive shadow and cell diameter were analyzed by image-pro Plus 6.0 software.The liver tissues were buried in paraffin and optimal cutting temperature compound,then paraffin sections and frozen sections were prepared.H&E and Oil Red O stainings were performed according to standard protocol respectively.Cell Culture and TreatmentMouse 3T3-L1 preadipocytes were purchased from cell bank in the Shanghai Branch of Chinese Academy of Science.The cells were cultured in high glucose DMEM with 10% fetal bovine serum at 37°C incubator with saturated humidity and 5% CO2.The classic cocktail method was used to induce the 3T3-L1 preadipocytes.Briefly,two days after reaching confluency,cells treated with a mixture of 0.5 m M IBMX,1 μM dexamethasone,and 10 μg/m L insulin in DMEM containing 10% FBS for 4 days.Then the media were replaced with DMEM containing 10 μg/m L insulin and 10%FBS for another 2 days,and the media contain only DMEM with 10% FBS for any additional days.When more than 90%of the 3T3-L1 preadipocytes were induced into mature 3T3-L1 adipocytes,it can be used for further study.Human adipose microvascular endothelial cells（HAMECs）were purchased from cell bank in the Shanghai Branch of Chinese Academy of Science.The cells were grown in the ECM medium at 37°C incubator with saturated humidity and 5% CO2.Lipofectamin^TMRNAi MAX was used to transfect cells according to Lipofectamine^TMRNAi MAX specification.Oil Red O StainingMature adipocytes were washed with PBS three times and then fixed with 4%paraformaldehyde for 15 minutes at room temperature.Fixed cells were washed with distilled water and 60% isopropanol successively,andincubated with oil red O for 1h.Rinse the surface dyeing of stained 3T3-L1 adipocyte with distilled water.Cells were observed under a microscope.Western Blot and q PCR AnalysisThe total protein of 3T3-L1 adipocytes,human adipose microvascular endothelial cells and mouse adipose tissue were extracted by total protein extraction kit.The protein expression levels of LRG1,samd1/5/8 and PPARγ were detected.The proteins were separated by 10% SDS-PAGE gel electrophoresis and then transferred to 0.22μm PVDF membranes（Bio-Rad）.Membranes were blocked in confining liquid,then incubated overnight at 4°C with corresponding primary antibodies.Secondary antibody was goat anti-rabbit immunoglobulin G antibody.Total RNA was extracted from 3T3-L1 adipocytes and mouse adipose tissue according to the manufacturer’s instruction.q PCR was used to assess relative m RNA expression levels of LRG1,PPARγ and CEBPα.Statistical AnalysisAll data were expressed as the means ± SEM.Data analysis involved unpaired Student’s t-test and multiple group comparisons were analyzed by ANOVA method.The level of statistical significance was defined as p < 0.05Results1.Compared with the body weight of the normal diet mice,the HFD mice were 1 to 1.5 times heavier.The mice models for obesity were successfully induced.2.Differential gene analysis of transcriptome sequencing results showed that,compared with normal mice,the expression of LRG1 gene in subcutaneous inguinal adipose tissue of obese mice was down-regulated（p<0.05）,and the change was more significant in mature adipocytes than endothelial cells.3.Differential gene analysis of transcriptome sequencing results showed that,compared with normal mice,the expression of LRG1 gene in visceral epididymal adipose tissue of obese mice was down-regulated（p<0.05）,and the changes in endothelial cells were more significant than mature adipose cells.4.Compared with normal mice,RNA and protein expression levels of LRG1 in subcutaneous inguinal and visceral epididymis were all decreased in obese mice（p<0.05）.5.Compared with the 30℃ control group,the obese mice in the 4℃ and-10℃ cold stimulation groups had significant weight and adipose tissue loss and increased food intake（p<0.001）,and the weight loss was increased with the decreasing of temperature（p<0.001）.6.Compared with the 30℃ control group,The expression of UCP1 in adipose tissue of obese mice in the 4℃/-10℃ group increased gradually with the decrease of temperature（p<0.001）.The change is more obvious in i WAT than in e WAT.7.IPGTT experiment was conducted to observe the glucose tolerance of obese mice.Compared with the 30℃ group,the blood glucose regulatory ability of obese mice in the 4℃ and-10℃ cold stimulation groups was significantly enhanced（p<0.001）.And with the decreasing of temperature,the change was more significant（p<0.01）.8.ITT experiment was treated to observe the insulin tolerance of obese mice.Compared with the 30℃ group,the insulin sensitivity of obese mice in the 4℃and-10℃ cold stimulation groups was significantly enhanced（p<0.001）,especially in the-10℃ group（p<0.001）.9.Compared with the control group at 30℃,TG,TC,LDL-C and HDL-C levels in the serum of obese mice in the 4℃ and-10℃ cold stimulation groups decreased（p<0.001）.With the decreasing of temperature,TG levels decreased more significantly（p<0.01）,and there was no significant difference in the other three levels.10.Compared with the 30℃ group,the severity of fatty liver inthe 4℃ and-10℃ cold stimulation groups was reduced（p<0.001）.11.The result of immunohistochemistry staining showed that the expression levels of CD31 and LRG1 in subcutaneous inguinal fat and visceral epididymal fat of mice in the 4℃/-10℃ cold stimulation groups were increased（p<0.001）.It also increased with the decreasing of temperature（p<0.001）.It confirmed that cold stimulation caused angiogenesis in adipose tissue and LRG1 participated in this process.12.Compared with the 30℃ group,protein expression level of LRG1 in subcutaneous inguinal and visceral epididymal adipose tissues increased in the 4℃and-10℃ cold stimulation groups（p<0.05）.13.It showed that the expression levels of PPARγ and LRG1 gradually increased over time during the differentiation of 3T3-L1 cells in vitro,and it reached the highest in mature fat cells（p<0.001）.The expression level of CEBPαincreased first and then decreased.14.LRG1 promoted tube formation of HAMECs in vitro.High levels of LRG1 and phosphorylated Smad1/5/8 in HAMECs.The phosphorylation of Smad1/5/8 was inhibited in HAMECs by interfere with the expression of LRG1（p<0.001）.Conclusions1.LRG1 expression in adipose tissue of obese mice was lower than that of normal mice.2.LRG1 is involved in cold stimulation induced adipose tissue remodeling.3.LRG1 is highly expressed in mature adipose cells.4.LRG1 recombinant protein can promote the tubular-forming ability of HAMECs.HAMECs has high expression of LRG1 and Smad1/5/8,and phosphorylation of Smad1/5/8 is affected by LRG1.5.The above results indicate that LRG1 is a potent endogenous pro-angiogenic factor and is involved in adipose tissue angiogenesis by affecting Smad1/5/8 phosphorylation.This conclusion provides a new target for angiogenic drugs in the treatment of obesity and its related complications.