| Diabetes is a chronic metabolic disease that occurs when the body is unable to produce insulin or when the body is unable to use insulin effectively.Insulin,which directly lowers blood glucose,has always been one of the most important drugs used in the clinical treatment of diabetes.However,insulin is susceptible to degradation by proteases and has a short halflife in the body,resulting in poor efficacy.Therefore,the study of recombinant human insulin that can resist protease degradation,and thus improve the long-lasting efficacy of insulin drugs,is of profound significance for the treatment of type Ⅱ diabetes.1.Design,synthesis and validation of long-acting recombinant human insulin analogueBased on the design of insulin icodec,a long-acting recombinant human insulin derivative 13-acy containing three amino acid mutations and a 20C fatty acid side chain at lysine B29 was designed.13-acy is obtained by E.coli expression system,protein denaturation,anion exchange chromatography,recombinant trypsin digestion,cation chromatography,acylation reaction and reverse phase chromatography.The molecular weight of 13-acy was determined and the acylation position was verified using V8 enzyme and high performance liquid chromatography,and the results showed that the molecular weight of 13-acy and the position of the acylation-linked side chain were correct.The results indicated that the longacting recombinant human insulin analogue 13-acy has been successfully obtained in this study.2.Hypoglycemic effects of long-acting recombinant human insulin analogueIn the study,blood glucose monitoring experiments were performed in BABL/c mice,SD rats,BKS-db/db mice,and HFD+STZ-induced diabetic mice after a single subcutaneous administration.The insulin drugs Tresiba and insulin icodec(named 0-acy),were used as controls.Before the experiment,the mice were free to eat and drink freely,and after the administration,the normoglycemic mice were still free to eat and drink freely,while the BKSdb/db mice and HFD+STZ induced diabetic mice were restricted in food intake and time.Each drug group received subcutaneous injections of normal saline(10 ml/kg),Tresiba(0.75 mg/kg),0-acy(3 mg/kg),or 13-acy(3 mg/kg).The duration of action of 13-acy was significantly longer than that of Tresiba,and 13-acy had the same glucose-lowering effect as insulin icodec(48 h in BABL/c mice and 72 h in SD rats).In order to explore the ability of recombinant human insulin analogues to resist enzymatic degradation and further explore the mechanism of their long-term hypoglycemic effect,the anti-enzyme digestion experiment was carried out in vitro,and the remaining protein was detected by high performance liquid chromatography.The results showed that the 50%degradation time of 13-acy was 90 min,which was the same as that of 0-acy and 6-fold longer than that of native human insulin.In order to explore the role of combined mutations in 13-acy in promoting long-term glucose-lowering effect,we performed glucose-lowering experiments and anti-enzyme digestion experiments on 41-acy with B17 mutation,42-acy with B17 and B25 mutation,and 13-acy with B17,B25 and A14 mutation.The results showed that 41-acy,with only B17 mutation,had significantly prolonged glucose-lowering effect(24 h in vivo)and enzyme resistance(2-fold longer than that of native human insulin)in BABL/c mice.When the amino acids at B25 and A14 sites were further changed,the long-term effect and anti-digestion ability were further increased.In conclusion,13-acy is comparable to insulin icodec in glucose-lowering ability and resistance to enzyme digestion,and the B17 amino acid mutation plays a key role in the longterm glucose-lowering effect of 13-acy.3.The antidiabetic effects of long-acting recombinant human insulin analogue and the underlying mechanismsIn this study,treatment experiments were performed in BKS-db/db mice for up to 6 w in vivo.BKS-db/db mice were divided into blank control group(saline),control group A(Tresiba),control group B(0-acy)and experimental group(13-acy).Six BKS-wt/wt mice were set as WT group(normal saline).The mice were free to drink and eat throughout the experiment.Body weight and fasting blood glucose were measured weekly after administration,and oral glucose tolerance test was performed at the 3rd and 6th weeks.Three mice from each group were randomly selected for sampling after the experiment,and the serum levels of GHbAlc,TG,T-CHO,BUN and CRE were detected,and the liver and kidney were observed histopathologically.At the same time,mRNA was extracted from liver and muscle tissue to quantitatively detect the expression of glucose metabolism-related genes.The results showed that 13-acy could reduce blood glucose by stimulating the expression of glucose metabolism related genes Insr,Irs-2 and Glut4.The expression of Irs-2 and Glut4 in the experimental group was significantly higher than that in the control group A and control group B.The results of body weight,FBG and GHbAlc showed that 13-acy had a good blood glucose control effect.The weight gain and loss of the experimental mice were relatively stable,and the FBG decreased by 37.2%compared with the initial level,and the total GHbA1c decreased by 33.6%compared with the blank control group.OGTT test showed that 13-acy could improve the glucose tolerance of BKS-db/db mice,and AUC glucose(0-120 min)value decreased by 25.2%compared with blank control group.The results of TG and T-CHO and oil red O staining showed that 13-acy had no effect on lipid metabolism.The results of BUN and CRE and renal PAS staining showed that compared with the blank control group,the experimental group had a significant reduction in BUN and CRE by 60.5%and 53.9%,and the thickness of glomerular mesangial matrix,capillary loop and basement membrane was reduced.13-acy could slow down the progression of diabetic kidney disease.In summary,in this study,based on the design of insulin icodec,a recombinant human insulin analogue 13-acy with significant resistance to enzymatic degradation was prepared by mutating the amino acid at position B17 of insulin.It can regulate glucose metabolism,improve glucose tolerance,and slow down the development of diabetic nephropathy.It is expected to become a diabetes drug injected once a week. |
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