The Effect Of Roux-en-Y Gastric Bypass On Browning Of Adipose Tissue Through Downregulating The Pro-inflammatory Effects Of Lipopolysaccharide

Background and objective:Diabetes has become a major public health problem globally.About 450 million people worldwide have diabetes.The incidence of people over 18 years old in China is10.8%,of which more than 90%are Type 2 diabetes mellitus?T2DM?.The incidence rate of T2DM is high,complications are numerous,long-term hazards are great,and the economic burden of treatment is heavy.It is a major chronic disease that needs further study.Surgical treatment is an effective method to treat obesity combined with T2DM,and its exact curative effect shows the hope of curing T2DM.Roux-en-Y gastric bypass?RYGB?is one of the mainstream standard surgical procedures for metabolic surgery.Its anti-T2DM effect is longer than other surgical procedures and it is not easy to relapse.However,it is difficult to carry out RYGB in T2DM patients with normal weight at present due to the more common complications such as trauma,postoperative diarrhea,severe malnutrition and other adverse factors such as changing the normal intestinal anatomy of human body is not conducive to the management of postoperative gastrointestinal diseases.However,the immediate T2DM relieving effect after RYGB confirmed that there is an anti-T2DM mechanism independent of weight loss caused by limiting energy intake.To clarify these mechanisms will contribute to the drug transformation of RYGB in the treatment of T2DM.However,RYGB's mechanism to alleviate T2DM is very complicated,which has not been fully defined.Adipose tissue is the main tissue for insulin resistance and plays an important role in the pathogenesis of T2DM.Adipose tissue can be divided into white adipose tissue?WAT?with energy storage and brown adipose tissue?BAT?with high energy consumption,and phenotypic transformation can take place under certain conditions.Among them,the transformation of WAT to BAT is called browning of adipose tissue.In recent years,it has been found that browning of adipose tissue can effectively increase glucose uptake and consumption,improve the body's insulin sensitivity,and is expected to become a new target for the treatment of obesity and T2DM.Some studies have found that RYGB can promote the browning of adipose tissue.The browning of adipose tissue may be one of the important mechanisms for RYGB to alleviate T2DM,but the specific mechanism has not been fully clarified.The browning of adipose tissue is related to the inflammatory state of adipose tissue.The change of intestinal microflora after operation can reduce the blood inflow of LPS and the level of LPS induced inflammation.But whether RYGB can mediate browning of adipose tissue by down-regulating LPS is not clear.In summary,the purpose of this study is to clarify the relationship between LPS changes and inflammation induced by RYGB and fatty tissue browning in obese T2DM rats,and to further explore the specific mechanism through cell experiments,so as to provide a new theoretical basis for RYGB to alleviate T2DM and the browning of adipose tissue in the treatment of obesity and T2DM.Part 1:Effect of RYGB on inflammation and browning of adipose tissue in obese T2DM ratsObjective:To explorer the effect of RYGB on the serum LPS,serum inflammatory factors,and the infiltration macrophages and browning of adipose tissue of obese T2DM rats.Methods:According to the experimental purpose,normal SD rats and streptozocin?STZ?induced obese T2DM rats were divided into normal control group?Control,CON,n=6?,RYGB surgery group?RYGB,n=6?,sham surgery group?Sham surgery,Sham,n=6?and weight matched group?WM,n=6?.The levels of fasting blood glucose,serum insulin,LPS,TNF-?,IL-1?and IL-10 in each group were measured at 4 weeks postoperatively.The infiltration of macrophages in adipose tissue of each group was assessed by immunohistochemical staining.The expression of uncoupling protein 1?UCP1?,a browning marker in adipose tissue,was measured by Real-time quantitative PCR?q PCR?and Western blot?WB?to compare the browning differences of adipose tissue of each group.Results:The levels of fasting blood glucose,serum insulin,HOMA-IR,LPS and TNF-?in RYGB group were significantly lower than those in Sham group?P<0.01?and WM group?P<0.05?.The level of IL-10 in RYGB group was significantly higher than that in Sham group?P=0.042?and WM group?P=0.034?.Compared with Sham group and WM group,RYGB significantly reduced the number of CD68⁺macrophages infiltration polarized M1 macrophages was decreased?BAT:P<0.01;WAT:P<0.01?and the number of CD206⁺polarized M2 macrophages was increased?BAT:P<0.01;WAT:P<0.05?.In group RYGB,the expression of UCP1 m RNA and protein in BAT and WAT were significantly higher than those in group Sham?m RNA:BAT,P=0.002?WAT,P=0.004;protein:BAT,P<0.001?WAT,P=0.001?and group WM?m RNA:BAT,P=0.002?WAT,P=0.007;protein:BAT,P=0.013?WAT,P=0.001?.The expression of UCP1 was negatively correlated with the level of serum LPS?m RNA:BAT,r=-0.561,P<0.001?WAT,r=-0.761,P=0.004;protein:BAT,r=-0.709,P<0.001?WAT,r=-0.614,P=0.001?.Conclusions:RYGB can alleviate the inflammatory level of obese T2DM rats,downregulate the level of serum LPS and TNF-?,upregulate the concentration of IL-10,inhibit the inflammatory background of adipose tissue mainly infiltrated by polarized M1macrophages,and promote the browning of adipose tissue.The browning level of adipose tissue was negatively correlated with serum LPS concentration.Part 2:The role of LPS in RYGB promoting browning of adipose tissue in obese T2DM ratsObjective:To verify the role of LPS downregulation in alleviating inflammation and promoting browning of adipose tissue after RYGB.Methods:The normal SD rats and the STZ induced obese T2DM rats after RYGB were respectively given intraperitoneal continuous injection of LPS?150ug/kg/day×8weeks?and the same amount of normal saline,which were divided into four groups:normal rats control group?NC,n=5?,normal rats+LPS intervention group?normal+LPS,NL,n=5?,surgical rats+saline control group?Surgery control,SC,n=5?and surgical rats+LPS intervention group?Surgery+LPS,SL,n=5?.After 8 weeks,fasting blood glucose,serum insulin,inflammatory factor concentration,macrophage infiltration and UCP1 expression in adipose tissue were measured between NC and NL,SC and SL groups.Results:NL group received LPS intervention for 8 weeks,except for 7^th week of fasting plasma glucose was lower than NC group?P=0.048?,there was no significant difference in other time points;Fasting blood glucose was gradually increased in SL group after LPS intervention,which was significantly higher in the 5^th,7^th,and 8^th week after LPS injection than in the SC group(5^th week:P=0.025;7^th week:P=0.017;8^th week:P=0.044).At the 8^th week,HOMA-IR,serum insulin,IL-1?,and TNF-?in NL group were significantly higher than those in NC group?P<0.05?;HOMA-IR,serum insulin,IL-1?and TNF-?in SL group were significantly higher than those in SC group?P<0.05?;While IL-10 in SL group was significantly lower than that in SC group?P=0.019?.LPS significantly increased the number of CD68⁺macrophages in adipose tissue of NL group and SL group?NL vs.NC:P<0.01;SL vs.SC:P<0.01?,in which the number of i NOS⁺polarized M1 macrophages was increased significantly(NL vs.NC:P<0.05;SL vs.SC:P decreased significantly?BAT:SL vs.SC,P=0.019;WAT:SL vs.SC,P=0.015?.LPS significantly downregulated UCP1 m RNA and protein expression in NL group?m RNA:BAT,P=0.048?WAT,P=0.025;protein:BAT,P=0.006?WAT,P=0.036?,and SL group?m RNA:BAT,P=0.041?WAT,P=0.037;protein:BAT,P=0.003?WAT,P<0.001?.Conclusions:LPS can increase the inflammation level of obese T2DM rats after RYGB,promote the inflammatory infiltration of macrophages in adipose tissue and inhibit browning,which significantly inhibit the effects of RYGB on inflammation and browning of adipose tissue.LPS may be a key target molecule for RYGB to alleviate inflammation and promote the browning of adipose tissue.Part 3:The effect and mechanisms of LPS,and M1?M2 polarized macrophages on the browning of adipocytesObjective:To investigate the effect of LPS,and polarized M1?M2 macrophages on the browning of adipocytes and its possible mechanism.Methods:The rat primary vascular stromal cells from subcutaneous adipose tissue of inguinal region were isolated,and induced to adipocytes.Norepinephrine and triiodothyronine induced brown like adipocytes were stimulated by LPS.The differential expression of UCP1 and Toll-like receptors?TLR?inflammation related molecules of LPS stimulated group?LPS,n=4?and PBS control group?CON,n=4?were detected by q PCR and WB.Rat bone marrow macrophages were isolated and cultured to induce polarized M1and M2 macrophages.The expression of UCP1 and VCAM1 in the control group?CON,n=4?,polarized M1 macrophage co-culture group?M1,n=4?and polarized M2 macrophage co-culture group?M2,n=4?were detected.Results:The m RNA expression of TLR2,TLR4,and TNF-?in LPS group were significantly upregulated compared to CON group?TLR2:12.19±2.43 folds,P=0.019;TLR4:8.07±1.72 folds,P=0.026;TNF-?:31.78±6.05 folds,P=0.015?,but the differential expression of UCP1 m RNA and protein were not found?m RNA:P=0.702;protein:P=0.514?.There was no significant difference in UCP1 m RNA and protein expression among CON group,M1 group and M2 group in the mode of non-contact co-culture of macrophages and adipocytes?m RNA:P=0.222;protein:P=0.741?.The expression of UCP1 m RNA in M1 group was 0.18±0.01 folds downregulated compared to CON group?P<0.001?,and significantly downregulated compared to M2 group?P=0.020?.The expression of UCP1 protein in M1 group was 0.50±0.06 folds downregulated compared to CON group?P=0.004?,and significantly downregulated compared to M2 group?P=0.006?.The expression of VCAM1 m RNA in M1 group was 8.83±0.94 folds upregulated compared to CON group?P=0.004?,and significantly upregulated compared to M2 group?P=0.007?.The expression of VCAM1 m RNA was negatively correlated with that of UCP1 m RNA?r=-0.909,P=0.002?.Conclution:LPS can significantly activate TLR-TNF-?pathway of adipocytes,but has no significant effect on the browning of adipocytes.Polarized M1 macrophages can inhibit the browning of adipocytes by direct contact mode,and the upregulation of VCAM1 may be one of the important mechanisms of polarized M1 macrophages inhibiting the browning of adipocytes.