| Research backgroundType 2 diabetes is a chronic non-communicable diseases,and its prevalence is increasing rapidly.The basic characteristic of type 2 diabetes is the disorder of carbohydrate metabolism caused by the relative or absolute deficiency of insulin.The excessive secretion of glucagon may be one of the important manifestations of diabetes.About 40 years ago,it was proposed to explain the pathophysiology of diabetes with the dual hormone theory;At present,GLP1 receptor agonist,which has attracted much clinical attention,can exert good hypoglycemic effect.One of its mechanisms is to inhibit the secretion of glucagon from a cells;Type 2 diabetes may be a complex diseases caused by abnormal insulin and glucagon secretion patterns.Recent studies have shown that short-term intensive insulin therapy can improveβ Cell function and the prognosis of newly diagnosed type 2 diabetes patients,reduce blood glucose variability,induce blood glucose remission,and maintain blood glucose homeostasis even after treatment is stopped.However,few studies have paid attention to α Cell function,α cell hyperfunction is a primary defect or is it due to β cell functional decline?These questions have not been answered.In most studies,the course of IIT is 2-4 weeks,and some studies are 4-8 weeks.The latter mainly studies the heterogeneity of IIT in different patients.The Chinese guidelines for the prevention and treatment of type 2 diabetes recommend that the course of IIT should be 2 weeks to 3 months.However,it is not clear whether extending the duration of IIT treatment will benefit patients.The predictive factors and mechanism of drug-free remission of IIT in newly diagnosed T2DM patients are also unclear.In view of this,this study is divided into three parts.The first part mainly discusses the effect of 12 week intensive insulin therapy on pancreatic islets α and β Cells and long-term metabolism in newly diagnosed T2DM patients.In the second part,we selected the patients who received intensive insulin therapy in the first part,and grouped them according to whether they were relieved in the long-term 12-month follow-up.The difference of blood glucose related indexes between the two groups was observed,and the influencing factors related to remission were analyzed.A prediction model of remission was constructed by receiver operating characteristic curve(ROC)and regression.The third part focuses on the molecular mechanism of IIT protection islet β Cells function.Part Ⅰ Effect of intensive insulin therapy on islet α and β cell Function and long-term metabolism of newly diagnosed T2DM patientsObjective1.To compare the effects of 12 weeks of intensive insulin therapy and 12 weeks of oral hypoglycemic therapy on hyperglycemia in newly diagnosed type 2 diabetic patients,and observe the function of α and β cells after intensive insulin therapy and the correlation between the two;2.To compare the long-term metabolic indexes of newly diagnosed type 2 diabetes patients after 12 weeks of intensive insulin therapy and 12 weeks of oral hypoglycemic drugs.Methods1.Research subjects and groupsThis section includes patients newly diagnosed with T2DM who were admitted to the First People’s Hospital of anqing City.These patients met the 1999 World Health Organization(World Health Organization,WHO)diagnostic criteria for diabetes.These patients were continuously included in this randomized controlled trial from January 2015 to 12 months 2015,and were randomly divided into two groups according to the random number table.A total of 108 patients were included in the trial,including 56 in the final Intensive Insulin Therapy(IIT)group and 52 in the Oral Hypoglycemic Agent(OHA)group.The IIT group was given four subcutaneous injections of insulin+metformin 0.5g 3 times a day,and the OHA group was given oral metformin 0.5g 3 times a day+gliclazide sustained-release tablets 60-120 mg once a day;when postprandial blood glucose is greater than 10 mmol/L,acarbose is given 50-100 mg 3 times a day;when the body mass index is greater than 25 kg/m2,pioglitazone is given 15-30 mg once a day.After 12 weeks,insulin was stopped in theIIT group,and metformin 0.5g was continued to be taken orally 3 times a day,and other hypoglycemic drugs were selected as needed;the OHA group maintained the original treatment and adjusted the hypoglycemic drugs as needed.Thirty health examiners in the health management center of our hospital during the same period were selected as controls.2.Research index detectionThe sex,age,height,weight and blood pressure of all subjects were collected,and the body mass index(BMI)was calculated.T2DM patients need to record the course of disease.All three groups of subjects received the steamed bread meal test,using 100g standard steamed bread.During the test,glucagon,C-peptide,blood glucose,fasting proinsulin,HbAlc,TC and TG were measured at 0,30 and 120 minutes respectively.AUCg was calculated according to glucagon,the ratio of fasting proinsulin to CP was calculated,and ΔCP30/ΔG30,ΔCP120/ΔG120,HOMA-β,HOMA-IR was calculated based on blood glucose and C peptide.Finally,glucagon and AUCg were used to evaluate the function of islet α cells,the ratio of fasting proinsulin to CP,ΔCP30/ΔG30,ΔCP120/ΔG120 and HOMA-β were used to evaluate the function of islet β cells,HOMA-IR was used to evaluate insulin resistance,and TC,TG and HbAlc were used for evaluation of long-term metabolism.The remission rate during the 12-month follow-up and the occurrence of nausea,abdominal distension,and hypoglycemia during the trial were recorded.Result1 Comparisons of paremeters between two groups at baseline1.At baseline,there was no significant difference in diastolic blood pressure,systolic blood pressure,age,sex ratio,BMI,TC,TG and ACR among the control group,IIT group and OHA group(all P>0.05).The baseline characteristics of the three groups were balanced and comparable.The blood glucose related indexes in group IIT and group OHA were statistically different from those in the control group.The blood glucose and glucagon levels of diabetic patients in group IIT and group OHA were higher than those in control group at 0,30 and 120 minutes in OGTT test(P<0.05),HbA lc,fasting proinsulin,fasting proinsulin/CP,AUCg and HOMA-IR in the IIT and OHA groups were also higher than those in the control group(P<0.05),while the CP of the 0,30 and 120 minutes and ΔCP30/ΔG30,ΔCP120/ΔG120 and HOMA-β were lower than those of the control group,in the IIT and OHA groups.There was no statistically significant difference in any analyzed blood glucose parameters at baseline between OHA and IIT group(P>0.05).2 Comparisons of paremeters after insuin intensive treatment among the groups(1)For glucagon,the inter-group,time,and time-to-group interactions between the IIT group and the OHA group are statistically significant(P<0.001).Glucagon levels at 12 week and 12 month in the IIT group were lower than those at baseline(P<0.05),and the difference between 12 week and 12 month was not statistically significant;while there was a difference in glucagon levels between 12 week and baseline in the OHA group.There was no statistically significant difference in glucagon levels in the OHA group at 12 week and at baseline,while the glucagon levels in the OHA group at 12 month were higher than at 12 week and at baseline(P<0.05).The differences in glucagon levels between the two groups at 12 week and 12 month were statistically significant(P<0.016).The glucagon levels in the OHA group at 12 week and at 12 month were higher than those in the IIT group.For AUCg,the interaction between the IIT group and the OHA group,time,and time and group interactions were statistically significant(P<0.001).For different time points in the same group,the 12-week and 12-month AUCg levels of the IIT group were lower than the baseline(P<0.05),and the difference between 12 month and 12 week was not statistically significant;while the 12-month AUCg level of the OHA group was higher than12 week and baseline(P<0.05),there was no statistically significant difference between the latter two.For the comparison between the two groups at the same time point,the difference in AUCg levels between the two groups at 12 week and 12 month was statistically significant(P<0.016).The AUCg levels at 12 week and 12 month in the OHA group were higher than those in the IIT group.(2)For fasting proinsulin/CP,after 12 weeks of intensive treatment in IIT group,fasting proinsulin/CP decreased significantly compared with baseline,and the effect lasted until 12 months;In OHA group,the ratio of fasting pro insulin/CP at 12 week was lower than that at baseline(P<0.05),and the ratio at 12 month was higher than that at 12 week(P<0.05);The ratio of fasting pro insulin/CP at 12 week and 12 month in IIT group was lower than that in OHA group(P<0.016).(3)For Δ CP30/Δ G30 and Δ CP120/Δ G120:After 12 week of treatment in IIT group and OHA group,Δ CP30/Δ G30 and Δ CP120/ΔG120 increased significantly(all<0.01),especially in IIT group,especially in early phase,and the effect lasted until 12 months.(4)For HOMA-IR:HOMA-IR levels at 12 week and 12 month in both groups were lower than those at baseline(P<0.05),there was no significant difference in HOMA-IR level between the two groups at different time points(P>0.05).(5)For HOMA-β index:The HOMA-β index of the two groups at 12 week and 12 month was higher than the baseline level(P<0.05),and the HOMA-β index at 12 month was lower than that at 12 week(P<0.05);the HOMA-β index in the IIT group at 12 week and 12 month were higher than that in the OHA group,and the difference was statistically significant(P<0.016);Spearman correlation analysis did not find that the correlation between the changes in AUCg at 12 week and the changes in β-cell function-related indicators at 12 week was statistically significant(P>0.05).(6)Spearman correlation analysis did not find that the correlation between the changes in AUCg at 12 week and the changes in β-cell function-related indicators at 12 week was statistically significant(P>0.05).(7)The changes of blood lipids:The blood lipid levels of the two groups decreased first and then increased.The levels of TC and TG in IIT group and OHA group decreased significantly compared with the baseline at 12 week(P<0.05);the levels of TC and TG at 12 month were higher than those at 12 week(P<0.05);there was no significant difference in TC and TG levels between OHA group and baseline at 12 month(P>0.05),and there was no significant difference in TC levels between IIT group and baseline at 12 month(P>0.05),while TG levels at 12 month were lower than baseline(P<0.05).(8)The target rates of HbAlc and remission rate of diabetes:The level of HbA1c inIIT group at 12 month was lower than that in OHA group(P<0.016).The level of HbA1c in the two groups at 12 week and 12 month was lower than that at baseline(P<0.05),and the level of HbAlc at 12 month was higher than that at 12 week(P<0.05).The level of HbAlc in IIT group at the 12th month was lower than that in OHA group,the difference was statistically significant(P<0.016);At 12 week and 12 month of IIT,98.0%and 82.2%of patients reached the target blood glucose target(defined as HbAlc<7%);only 62.5%and 35.7%of OHA group reached the blood glucose target(P<0.05 compared with IIT group at the same time point).Compared with OHA group,patients in IIT group used significantly fewer OHAs at 12 week and 12 month.At the final follow-up,the remission rate was 31.1%in IIT group and 7.1%in OHA group.The remission rate in IIT group was higher than that in OHA group,and the difference was statistically significant(χ2=7.938,P=0.005).(9)There were no significant differences in the proportion of nausea,abdominal distention,asymptomatic hypoglycemia and symptomatic hypoglycemia between IIT group and OHA group(P>0.05).Conclusion1.The 12-week intensive insulin therapy is associated with the improvement of β and αcell function in newly diagnosed T2DM patients,allowing better long-term metabolic control with minimal OHAs or only life style intervention.2.Decreased insulin secretion by β cells and accompanying a cell dysfunction(weakened paracrine inhibition+primary α cell hyperfunction)may jointly participate in the pathogenesis of T2DM.Part Ⅱ Predictors of diabetes remission after intensive insulin therapy among newly diagnosed type 2 diabetes patientsObjectiveTo analyze the predictive factors of no drug remission in patients with newly diagnosed type 2 diabetes who received 12 weeks of short-term intensive insulin therapy.Methods1.Research subjects and groupsThe original data of 56 patients with type 2 diabetes who received short-term intensive insulin treatment in group IIT were re analyzed.All patients were newly diagnosed with T2DM from anqing first people’s Hospital and were continuously included in the study from January 2015 to December 2015.The study group was divided into remission group and non remission group.2.Indexes detectionThe patients’ gender,age,duration of diabetes,blood pressure,height and weight were recorded and BMI was calculated.All subjects received the steamed bread meal test of 100g standard steamed bread at baseline and 12 weeks after treatment.Glucagon,C-peptide and blood glucose were measured at 0,30 and 120 minutes,and fasting proinsulin,HbA1c,TC and TG were detected.AUCg was calculated according to glucagon,the ratio of fasting proinsulin to CP was calculated,and the ratio ofΔCP30/ΔG30、ΔCP120/ΔG120、HOMA-β、HOMA-IR was calculated according to blood glucose and C-peptide.The remission rate at 12-month follow-up was recorded.Results1.At baseline,the difference in age and BMI between remission group and non remission group was statistically significant.The age of remission group was lower than that of non remission group(P=0.029),and the BMI of remission group was higher than that of non remission group(P=0.001).Other differences were not statistically significant(P>0.05).2.At 12 week,the 30 minute C peptide and Δ CP30/Δ G30 was higher than that in the non remission group.HbAlc,30 minute glucagon,120 minute glucagon and AUCg in the remission group were lower than those in the non remission group.3.After treatment,the blood lipids TC and TG of the remission group were decreased.FBG,P30BG,P120BG,HbAlc,fasting proinsulin,glucagon,fasting proinsulin/CP,HOMA-IR,AUCg were all lower than before treatment,while fasting C Peptide,30 minutes,120 minutes C peptide,ΔCP30/ΔG30,ΔCP120/ΔG120,HOMA-β index were all significantly higher than before treatment.4.After treatment,the blood lipids TC and TG of the non-remission group were decreased,FBG,P30BG,P120BG,HbAlc(%),fasting proinsulin,glucagon,fasting proinsulin/CP(%),HOMA-IR index,AUCg were all decreased.The indexes of fasting C-peptide,120-minute C-peptide,ΔCP30/ΔG30,ΔCP120/ΔG120,and HOMA-β were all significantly higher than those before treatment.5.Spearman correlation analysis did not find that the correlation between the changes in AUCg at 12 week and the changes in β-cell function-related indicators at 12 week was statistically significant(P>0.05)in either the remission group and the non-remission group.6.Multivariate logistic regression analysis showed that the impact of baseline BMI,12-week AUCg and 12-week ΔCP30/ΔG30 on remission was statistically significant.For every unit increase in baseline BMI,the OR of remission increased to 1.538 times the original,;for every unit increase in AUCg at 12 week,the OR of remission decreased to the original 0.998;for every unit increase in ΔCP30/ΔG30 at 12 week,the OR of remission increases to 3.027 times.7.ROC analysis showed that baseline BMI,12-week AUCg,12-week ΔCP30/ΔG30,and the AUC values of the three combined indicators were 0.809(95%CI:0.664-0.911),0.838(95%CI:0.697-0.930),0.795(95%CI:0.648-0.901),and 0.945(95%CI:0.833-0.991).According to the AUC value,the combined index>12-week AUCg>baseline BMI>12-weekΔCP30/ΔG30;in terms of sensitivity,12-week AUCg>combined index>12-weekΔCP30/ΔG30>baseline BMI;in terms of specificity,the combined index>baseline BMI>12 week ΔCP30/ΔG30>12 week AUCg.ConclusionBaseline BMI,12-week AUCg,12-week ΔCP30/ΔG30,and the combined indicators of the three have a certain predictive value for the remission of diabetes in intensive insulin therapy.Part Ⅲ Molecular mechanism of the effect of intensive insulin therapy on the function of a and β cells in DM RatsObjectiveTo establish a DM rat model,compare the therapeutic effects of insulin intensive therapy(IIT)and oral hypoglycemic drug therapy(OHA)in rats,and analyze the repair of insulin intensive therapy β Possible molecular mechanisms of cell function.MethodsIn order to observe the effect and possible mechanism of IIT treatment,we took 24 male SD rats and randomly selected 6 as the normal control group(NC group).The remaining 18 rats were fed with high-fat diet for 4 weeks and then injected with STZ intraperitoneally.After 1 week of adaptive feeding,six rats were randomly selected as the normal control group(NC group)after fasting for 12h.The remaining 18 rats were fed with high-fat diet for 4 weeks and then injected with STZ intraperitoneally.One week later,the random blood glucose≥16.7mmol/l was determined as a successful DM rat model.T2DM rats were then divided into three groups on average:DM model group(no treatment),-insulin intensive therapy(IIT)group(3U/kg/D),oral hypoglycemic drug(OHA)treatment group(glibenclamide 10mg/kg/D),NC group was given an equal amount of normal saline intraperitoneal injection,and was gavaged with normal saline once a day for 8 weeks.During the experiment,NC group was fed with conventional diet,and T2DM rats continued to be fed with high sugar and high fat diet.After the experiment,the mice in each group were anesthetized by intraperitoneal injection of 10%chloral hydrate.Blood and pancreas tissues were taken,and some pancreas tissues were fixed with 4%neutral formaldehyde to prepare paraffin sections for pathological staining and indirect immunofluorescence analysis of isletsα and β Distribution and number of cells.qRT-PCR and Western blot were used to detect the expression levels of PDX1 and Ngn3,the transcription factors related to islet precursor cell generation,α Expression levels of cell differentiation related transcription factors ARX,Brn-4 and MafB β-The expression levels of Pax4,MafA and Nkx6.1,the key transcription factors of cell differentiation.Blood glucose,blood lipid level,C-peptide,insulin,Proinsulin and glucagon were detected in serum.Results1.Compared with the NC group,the BG level in the DM group was significantly increased(P<0.0001),and the BG level in the IIT group and the OHA group was significantly decreased(P<0.0001),and there was no significant difference between the IT group and the OHA group.The TG level of DM group was significantly higher than that of NC group(P<0.0001),and the TG level of IIT group and OHA group was significantly lower than that of DM group(P<0.0001),but the TG level of IIT group was closer to that of NC group,and there was no significant difference between IIT group and OHA group.Compared with the NC group,the TC and LDL-C level of DM group increased significantly(P<0.001),and the TC and LDL-C level of IIT group and OHA group decreased significantly compared with the DM group,but the TC level of rats in IIT group was closer to the NC group,and there was a significant difference compared with OHA group(P<0.05).Compared with the NC group,the HDL-C level of DM group was significantly lower(P<0.0001),and the HDL-C level ofIIT group and OHA group was significantly higher than that of DM group,but the HDL-C level of IIT group was closer to that of NC group,and there was no significant difference compared with OHA group.2.Compared with the NC group,the glucagon and proinsulin level of DM group was significantly increased(P<0.0001),and the glucagon and proinsulin level of IIT group and OHA group was significantly lower than that of DM group(P<0.0001),but the glucagon and proinsulin level of IIT group was closer to that of NC group,and there was a significant difference compared with OHA group(P<0.05).The level of C-peptide in DM group was significantly lower than that in NC group(P<0.0001),and the level of C-peptide in IIT group and OHA group was significantly higher than that in DM group(P<0.05),but the level of C-peptide in IIT group was closer to that in NC group,and there was a significant difference compared with OHA group(P=0.0229).3.In the DM group,the islets of the rats were atrophied,the cells were shrunk,and became narrow in a strip shape.The cell morphology was small,the nucleus was dense,and the cytoplasm was few.Compared with DM group,the injury of islets in IIT group and OHA group was reduced,and the effect of IIT group was better than that of OHA group.Indirect immunofluorescence was used to observe the secretion of glucagon in islets of Langerhans αCell number and distribution,found in NC group rats α.The cells were evenly distributed at the edge of the islet and showed a ring shape,α Cells are concentrated in the whole islet.Compared with DM group,glucagon positive cells in islets of rats in IIT group and OHA group gradually distributed at the edge of islets,and the effect of IIT group was better than that of OHA group.NC group rats β Cells were evenly distributed in the islets,and the number was large,while the islets of rats in DM group β cells decreased sharply,and most of them lost the ability to secrete insulin.Compared with DM group,IIT group and OHA group rat islets.The number of cells increased,and the effect of IIT group was better than OHA group.4.The results of real-time PCR and Western blot showed that compared with the NC group,the expression levels of Pdx1,Ngn3,Pax4,MaFa and Nkx6.1 mRNA in the pancreas of the DM group were significantly decreased(P<0.001),and compared with the DM group,the expression levels of Pdxl,Ngn3,Pax4,MaFa and Nkx6.1 mRNA in the islets of the IIT group and the OHA group were significantly increased(P<0.001),and the effect of the IIT group was better than that of the OHA group.The results of real time PCR and Western blot showed that the expression levels of ARX,Brn-4 and MafB mRNA in pancreas of rats in DM group were significantly higher than those in NC group(P<0.001),and the expression levels of ARX,Brn-4 and MafB mRNA in pancreas of rats in IIT group and OHA group were significantly lower than those in DM group(P<0.001).ConclusionIIT may maintain the islets of DM rats by affecting the expression of transcription factors related to islet cell differentiation β Cell number,control blood glucose level. |
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