The Study Of The Effects And Mechanism Of Beinaglutide On Weight And Adipocyte Differentiation

Background:Nowadays,obesity has been the most serious pandemic diseases in the World,thereby leading to a decline in both life expectancy and the quality of life.It is essential to find out effective and safe treatment strategies for obesity.The pathological characteristics of obesity was hyperplasia and hypertrophy of adipocytes,and the inhibition of hyperplasia and hypertrophy can alleviate the expansion of adipose tissue and thus reduce body weight.In addition,gut microbiota was involved in the occurrence and development of obesity by regulating the energy metabolism of host.Glucagon like peptide-1 receptor agonists（GLP-1 RAs）can effectively reduce blood glucose and body weight and has been widely approved for the treatment of overweight/obese patients with or without type 2 diabetes.According to the molecular structure,GLP-1 RAs can be divided into two types,and the first type was artificially synthesized based on the structure of exodin-4,and the others were locally modified and processed based on the structure of natural GLP-1 in human such as liraglutide and beinaglutide.Beinaglutide is a recombinant GLP-1 which was 100%homologous to human GLP-1 and the effect on hypoglycemic and weight loss effect in clinical practice has been proved in the clinical practice.However,the effect on intestinal flora,inflammation,adipocyte differentiation and lipid accumulation and underlying mechanisms remains unclear yet.Based on the above background,this study first evaluated the effects of beinaglutide on body weight,body composition,laboratory parameters,inflammatory cytokines and intestinal microflora in enrolled non-diabetic obese patients.Then,we explored the effect and potential mechanism of beinaglutide on adipocyte differentiation and lipid accumulation by3T3-L1 adipocytes in vitro.PartⅠ:Effects of Beinaglutide on body weight,intestinal flora andinflammatory factors in non-diabetic obese patients.Objective:To observe the effect of beinaglutide on body weight and intestinal microflora and inflammatory factors.Methods:According to inclusion criteria and exclusion criteria,25patients who were newly diagnosed with non-diabetic obesity was recruited in the Department of Endocrinology of Xiangya Hospital of Central South University from May 2019 to May 2022.Based on the lifestyle intervention,the subjects received the subcutaneous injection of beinaglutide three times a day for 3 months,which was initiated at 0.06mg per time in the first week,then 0.1mg in the second week,0.14 mg in the third week and reaching to 0.2mg per time in the fourth week.The final dose of 0.2mg per time was maintaining in the following treatment.The body composition evaluated by dual-energy X-ray（DXA）and laboratory parameters were monitored at baseline and the end of the study,including serum glucose,glycated hemoglobin A1c（Hb A1c）,insulin levels at 0,30,60 and 120 minutes during the 75g oral glucose tolerance test（OGTT）,hepatic and renal function,lipid profiles and pro-inflammatory cytokines.Various indices of islet beta cell function and insulin sensitivity derived from OGTT results were calculated,including homeostasis model assessment index of insulin resistance（HOMA-IR）,homeostasis model assessment index of B cell function（HOMA-B）,delta I₃₀/delta G_30,CIR₃₀,delta I₁₂₀/delta G₁₂₀,CIR₁₂₀.Fresh stool samples were collected for metagenomic sequencing of intestinal flora.Statistical Product Service Solutions 25.0（SPSS 25.0）software was performed for analysis.The Shapiro-Wilk test was first used to verify whether the delta value between baseline and the post 3months matched the normal distribution.If the delta value matched the normal distribution,the paired T-test was preformed;otherwise,Wilcoxon rank sum test was used.The results were expressed as mean±standard deviation.p<0.05indicated that the difference was statistically significant.Results:1.Anthropometric indicators improved and body weight was reduced significantly.Compared with baseline,body weight decreased significantly at the end of the study（94.93±14.89 kg vs 87.7±12.87 kg）（p<0.01）,the proportion of weight loss greater than 5%reached 80%（20/25）,and 28%（7/25）lost more than 10 percent of their weight.Also,the waist circumference,hip circumference,waist to hip ratio and total body fat rate decreased significantly（p<0.05）.2.Various laboratory parameters and insulin resistance improved.Compared with baseline,the Hb A1c,1h PG,2h PG decreased significantly（p<0.05）.The secretion of insulin decreased at every time point（p<0.05）.And the HOMA-IR and CIR₁₂₀improved significantly（p<0.05）while the change of HOMA-B,delta I₃₀/delta G₃₀,CIR₃₀,delta I₁₂₀/delta G₁₂₀was not significant（p>0.05）.3.The chronic inflammatory state improved.Compared with baseline,the concentration of serum lipopolysaccharide,pro-inflammatory cytokines TNF-αand IL-6 levels were significantly decreased at the end of the study（p<0.01）.4.Intestinal flora improved.Details are as follows:（1）The composition of intestinal bacteria changed:compared with baseline,at the phylum level,the relative abundance of Firmicutes decreased while the relative abundance of.Bacteroidetes increased,and the Firmicutes/Bacteroidetes（F/B）ratio decreased;at the genus level,the relative abundance of Bacteroides increased obviously while Faecaliba-cterium decreased obviously;at species level,the relative abundance of conditional pathogenic bacteria decreased,such as Faecalibacterium prausnitzii at the end of study.（2）The richness and diversity of intestinal flora decreased significantly:The Simpson,Shannon and ACE indices representing alpha diversity were significantly decreased significantly（p<0.05）.The results showed a decrease in both the uniformity of intestinal flora distribution and the total number of intestinal flora species decreased.After three months’treatment,βdiversity index indicated that the intestinal microflora changed significantly（p<0.05）.（3）The top 30 most important bacteria at the species level classification were screened by the random forest model.The area under the Receiver Operating characteristic curve（ROC）based on the test set was 0.933.This showed that the model could effectively distinguished the population between baseline and post treatment of beinaglutide.Conclusion:1.Beinaglutide significantly reduced the body weight and body fat in simple obese indiviuals,and improved anthropometric indicators,including waist circumference,hip circumference and waist to hip ratio as well as glycometabolism and insulin resistance,lipid profiles,and chronic low-grade inflammation.2.Beinaglutide can improve the composition of intestinal flora,increase the relative abundance of probiotics,and reduce the relative abundance of harmful intestinal bacteria.PartⅡ:Effects of beinaglutide on differentiation and lipidaccumulation in 3T3-L1 preadipocytes.Objective:To screen the effect of beinaglutide on differentiation and lipid accumulation in 3T3-L1 preadipocytes and to elucidate the underlying mechanisms.Methods:The differentiation medium（MDI）was performed to induce adipocyte differentiation.Preadipocytes were treated with various concentrations（0-10μM）of Beinaglutide for 8 days.The cell viability of adipocytes treated with Beinaglutide was measured by the Cell Count kit-8（CCK-8）.Quantitative real-time polymerase chain reaction（q RT-PCR）assessed the expression of adipogenesis,lipogenesis related genes,toll-like receptor（TLR4）and pro-inflammatory cytokines.Oil red O staining and isopropanol extraction assay were performed to evaluate the differentiation and lipid accumulation in adipocyte.Western blot was performed to measure the nuclear factor kappa-B（NF-κB）pathway.Results:1.The maximum concentration of beinaglutide determined by CCK-8 was 10μmol/L.Compared with the MDI group,beinaglutide increased the expression of adipogenic genes:peroxisome proliferator-activated receptor gama（PPARγ）,sterol regulatory element binding protein 1（SREBP1）,CCATT enhancer binding protein alpha（C/EBPα）,fatty acid synthase（FAS）,acetyl-Co A carboxylase 1（ACC1）,meanwhile decreasing the expression of lipolytic genes:adipose triglyceride lipase（ATGL）and hormone-sensitive lipase（HSL）and inflammatory cytokines:tumor necrosis factor–alpha（TNF-α）and interleukin–6（IL-6）showed by q RT-PCR（p<0.05）.Oil red O staining and isopropanol extraction assay revealed that beinaglutide reduced the number of mature adipocytes and lipid droplet size.2.QRT-PCR showed that beinaglutide downregulated the expression of TLR4.Western blot revealed that beinaglutide inhibited the TLR4/NF-κB signaling pathway.3.The maximum concentration of the specific agonist trifluoro-acetate salt（RS 09）of TLR4 determined by CCK-8 was 10μmol/L.RS09 was used to activate TLR4 on adipocyte membranes.QRT-PCR showed that compared with only beinaglutide group,beinaglutide+RS 09group increased the expression of TLR4,pro-inflammatory cytokines（TNF-αand IL-6）,adipogenic differentiation genes（PPARγ,SREBP1,C/EBPα,FAS and ACC1）and lipogenic genes（ATGL and HSL）.However,oil red O staining and isopropanol extraction quantitative experiment revealed that there was no significant difference in the number of mature adipocytes and the size of lipid droplets between beinaglutide+RS 09 group and only beinaglutide group（p>0.05）.Conclusion:1.Beinaglutide inhibited the differentiation and intracellular lipid accumulation of 3T3-L1 preadipocytes in a concentration-dependent manner.2.Beinaglutide inhibited the activation of TLR4/NF-κB signaling pathway in a concentration-dependent manner,thereby contributing to a decrease of the inflammatory cytokines.TLR4/NF-κB signaling pathway did not affect adipocyte differentiation and lipid accumulation,which may be attributed to the increase of lipolytic enzymes ATGL and HSL.