| Background:Diabetes mellitus is a chronic metabolic disease characterized by absolute or relative insulin deficiency,which is caused by genetic and environmental factors.In recent years,with the development of social economy and the change of lifestyle,the incidence and number of patients with diabetes are increasing rapidly worldwide.According to the latest statistics from the International Diabetes Federation,as of 2021,there are about 537 million diabetic individuals in the world.And this number is expected to reach 643 million by 2030 and 783 million by 2045,while the number of diabetic patients in our country is 141 million,ranking first in the world.Diabetes has become the third major disease threatening human health after cancer and cardiovascular disease.Therefore,it is of great significance to explore the mechanism of diabetes and find effective ways to treat diabetes.Diabetes mellitus is mainly divided into type 1 diabetes mellitus(T1DM)and type 2 diabetes mellitus(T2DM).In both T1 DM and T2 DM,the decrease of the functional pancreatic β cell mass and dysfunction of β cells are the main reasons for the development of diabetes mellitus.The current treatment of diabetes relies on diet control,increase exercise and the use of hypoglycemic drugs to control blood sugar levels.However,these methods can only alleviate the symptoms of diabetes to varying degrees and cannot achieve the purpose of long-term blood sugar control or reverse the process of diabetes,and most of them ultimately need to rely on insulin.Therefore,promoting the proliferation of pancreatic β cells and restoring the number and function of pancreatic β cells is the key to treating diabetes.However,what is the precise mechanism of beta cells proliferation? How to promote its proliferation remains unclear.Pancreatic β cells are differentiated mature cells and normally,adult individualshave a low rate of differentiated proliferation and inactive proliferation of β cells.However,in response to increased physiological and pathological metabolic demands in adults,such as insulin resistance,obesity,pregnancy or pancreatic injury,β cells may undergo compensatory proliferation that is critical for increasing insulin demands and maintaining glucose homeostasis.Studies have shown that in humans and rodents,a high-fat diet,a high-calorie diet and obesity can stimulate β-cell proliferation and increase β-cell mass.Thioredoxin-interacting protein(TXNIP)is a multifunctional protein which is ubiquitously expressed in multiple tissues and organs.TXNIP expression is closely associated with the development of many diseases such as diabetes,cardiovascular disease,central nervous system disease and cancer.Studies have shown that the expression of TXNIP is elevated in diabetic patients,which is involved in the apoptosis of β cells and has an important impact on glucose and lipid metabolism.Insulin levels were significantly higher in TXNIP-deficient Hc B-19(Hc B)mice compared to normal mice.Further studies revealed an increase in islet beta cell mass in Hc B-19 mice.The above results suggest that TXNIP may be involved in the regulation of pancreatic β-cell number and function.However,there is a lack of research on the role of TXNIP in β-cell proliferation,and whether TXNIP is involved in the compensatory proliferation of β cells under the above conditions has not been reported.It has also been shown that TXNIP is an important tumor suppressor,involved in cell growth regulation,inhibits cell proliferation and arrest cell cycle progression.Numerous studies have demonstrated that TXNIP expression is down-regulated in various cancers and human cancer cells,such as breast cancer,stomach cancer,lung cancer,colon cancer,cervical cancer,gastrointestinal cancer,prostate cancer,cutaneous T-cell lymphoma,and adult T-cell leukemia,and analysis of its mechanism suggests that TXNIP can inhibit the proliferation of tumor cells.However,it remains to be seen whether TXNIP is also involved in the regulation of pancreatic β cells proliferation,which has also not been reported.Therefore,in this study,we first used diabetic model mice,db/db mice,to observe the changes of TXNIP and the proliferation characteristics of pancreatic β-cells indiabetic model mice,and to preliminarily analyze the relationship between TXNIP and pancreatic β-cell proliferation.Then,a mouse model of TXNIP knockout was established,and an obesity model was prepared using TXNIP knockout transgenic animals in combination with high fat diet feeding to induce compensatory proliferation of pancreatic islet β-cells.To explore the relationship between TXNIP and pancreatic β cell proliferation and its role in the compensatory proliferation of pancreatic β cells.To further elucidate the mechanism of TXNIP in the regulation of islet β-cell proliferation and to provide new ideas and targets for intervention in the prevention and treatment of diabetes.Part I The expression of islet TXNIP and proliferation changes of pancreatic β cells in diabetic model miceObjective:To clarify the changes of TXNIP in the pancreatic islets of db/db mice,observe the proliferation characteristics of pancreatic β-cells,and preliminarily analyze the relationship between TXNIP and pancreatic β-cell proliferation in diabetic model mice.Methods:(1)Experimental animals and cellsIn this study,8-week-old male diabetic model mice db/db mice and control db/m mice were used.Both mice were purchased from Gem Pharmatech Co.,Ltd(Jiangsu,China).The Min6 mouse islet β cell line was obtained from Beijing Beina Chuanglian Biotechnology Research Institute.Lentiviral transfection constructed TXNIP overexpression stable cell line(Ad-TXNIP-GFP)and empty viral vector(Ad-GFP),which were packaged by Guangzhou Saiye Biotechnology Co.,Ltd.(2)Measurement of blood glucose and insulin levels in mice.(3)Glucose tolerance test(OGTT)and insulin tolerance test(ITT)were performed to observe changes in glucose tolerance and insulin sensitivity in mice.(4)Primary mouse islets were isolated and the expression level of TXNIP protein in mouse islets was detected by Western blot.(5)Weigh the mice,isolate the mouse pancreas tissue and calculate the mouse pancreas/body weight ratio.(6)Screening for changes in cytokines associated with proliferation of diabetic pancreatic β cells using suspension microarray technology.(7)HE staining to observe morphological changes in the islets of mice.(8)Western blot to detect changes in protein expression of the proliferationassociated protein PCNA.(9)Flow cytometry was used to detect changes in the proliferation cycle progression of Min6 β-cells.Statistical methodsAll data were statistically analyzed using SPSS 17.0 software and plotted using Prism Graph Pad 7.0 software.Data comparison between two groups was performed by independent sample t-test.Statistical results were expressed as mean ± standard error(Mean±SEM)and P?0.05 was considered statistically significant.Results:(1)The blood glucose level of db/db mice was significantly increased(19.30±0.62 mmol/L vs 6.23±0.20 mmol/L,P<0.01);At the same time,the serum insulin level was significantly increased(19724±2085 pg/m L vs 2335±222 pg/m L,P<0.01).(2)The results of glucose tolerance test showed that the area under the OGTT curve(AUCOGTT)was significantly increased in db/db mice(P<0.01),indicating that Glucose tolerance was impaired in mice;similarly,the results of insulin tolerance test showed that the area under the curve(AUCITT)of db/db mice was also significantly increased(P <0.01),indicating decreased insulin sensitivity in mice.(3)Western blot results showed that,compared with the control group db/m mice,the expression of TXNIP protein in the pancreatic islets of the diabetic model mice db/db mice was significantly increased(P<0.01).(4)Body weight was significantly increased in db/db mice(37.90±1.74 g vs 20.97±0.38 g,P<0.01)and the pancreas/body weight ratio decreased(0.0041±0.0007 vs 0.0115±0.0003,P<0.01).(5)The results of the suspension microarray showed that compared with the db/m mice in the control group,the GLP-1 content in the serum of the mice in the db/db group was significantly increased(P<0.01);Similarly,Leptin(P<0.01),GIP(P<0.05)and PAI-1(P<0.05)increased;Ghrelin(P<0.01)decreased,while the expression of Resistin(P>0.05)did not change significantly in the two groups of mice and was not statistically significant.(6)HE staining showed that the islets of the control db/m mice were morphologically intact,round or oval in shape,with clear boundaries between the islets and the surrounding tissues and uniform distribution of islet cells,whereas the islets of the db/db group were morphologically and structurally destroyed,with irregular shape,hyperplasia and hypertrophy of the islets,and some of the islets infiltrated into the surrounding tissues and had unclear boundaries with the surrounding tissues.(7)The expression of PCNA,a proliferation-related protein,was significantly higher in the pancreatic islets of the db/db group compared to the control db/m mice(P<0.01).(8)The results of the proliferation cycle of Min6 mouse pancreatic β-cells using flow cytometry showed that the percentage of cells in the G1 phase was significantly higher in the TXNIP overexpression group compared to the empty viral vector(AdGFP)group,indicating that the cell proliferation cycle was blocked in the G1 phase.Conclusion:The db/db mice showed typical T2 DM features,and the islet β cells may have compensatory proliferation.Cytological studies have shown that TXNIP affected the proliferation of islet β cells.Part II Proliferative changes in pancreatic β cells in TXNIP knockout mouse modelObjective:To observe the phenotypic characteristics of TXNIP knockout mouse model.The TXNIP knockout mouse animal model was used to observe the proliferation changes of pancreatic β cells in the TXNIP knockout mouse model.Methods:(1)Experimental animalTXNIP knockout mice(KO),with C57BL/6J background mice,were entrusted to Gem Pharmatech Co.,Ltd(Jiangsu,China)to prepare them using CRISPR/Cas9 technology.Mice were divided into two groups: control group(WT)and TXNIP knockout group(KO).(2)Mouse genotype was identified by PCR.Primary mouse islets were isolated,and Western blot detected the protein expression level of TXNIP in mouse islets.Realtime PCR detects m RNA expression levels of TXNIP in mouse islets.(3)Observe the behavioral characteristics of TXNIP knockout mice,and record the phenotype characteristics of the mice.(4)Calculation of pancreas/body weight ratio in TXNIP knockout mice.(5)Blood glucose and insulin levels of mice were measured.(6)Suspension chip technology was used to detect the changes of cytokines related to the proliferation of islet β cells.(7)HE staining was used to observe changes in the shape,volume,and size of mouse islets;Immunohistochemical staining was used to observe changes in mouse insulin-secreting cells.(8)Insulin/Ki67 and Insulin/PCNA immunofluorescence double-labeled stainingwas performed to observe the changes in the number of Ki67 and PCNA positive cells in TXNIP knockout mice.Statistical methodsAll data were statistically analyzed using SPSS 17.0 software and plotted using Prism Graph Pad 7.0 software.Data comparison between two groups was performed by independent sample t-test.Statistical results were expressed as mean ± standard error(Mean±SEM)and P?0.05 was considered statistically significant.Results:(1)The results of PCR gene identification showed that the TXNIP knockout(KO)mice showed a target band at 262 bp,while the wild-type(WT)mice showed a target band at 407 bp.According to the gene identification scheme,the TXNIP knockout mice were determined to be homozygous mice by band alignment.Western blot results showed that there was no significant TXNIP protein expression in the KO group mice compared to the WT group mice.Similarly,the results of Real-time PCR showed that compared with WT mice,KO mice had no expression of TXNIP m RNA.(2)TXNIP knockout mice were in good mental condition,with normal activity level,shiny hair,normal diet,water intake and urine output,and responsive to external stimuli.There was no significant change in phenotypic characteristics compared to the normal group of mice.Anatomy revealed that TXNIP knockout mice had increased epididymal fat compared to the control group.(3)Compared with the control group,the body weight of TXNIP knockout mice did not increase significantly(24.93±0.38 g vs 24.10±0.21 g,P>0.05).The mouse pancreas/body weight ratio did not change significantly(0.0096±0.0003 g vs 0.0104±0.0003 g,P>0.05).(4)Compared with the control group,the fasting blood glucose level in TXNIP knockout mice was decreased(3.55±0.24 mmol/L vs 5.28±0.24 mmol/L,P<0.05),and the serum insulin level was significantly increased(5315.7±302.5 pg/m L vs 4139.8±271.5 pg/m L,P<0.05).(5)The GLP-1 content in serum of TXNIP knockout mice was not significantly increased(P>0.05);Leptin(P<0.01),Resistin(P<0.05),GIP(P<0.05)and PAI-1(P<0.01)content in serum was decreased(P<0.05),while Ghrelin content in serum was increased(P<0.05).(6)The results of Real-time PCR showed that,compared with the WT mice in the control group,the m RNA expressions of transcription factors such as Pdx1,Mafa,Mafb,and Foxo1 in the mice in the TXNIP knockout group were not significantly changed(P> 0.05).(7)The results of HE staining of pancreatic tissues showed that the islets in the pancreatic tissues of WT and KO mice had regular morphology and clear boundaries with the surrounding alveolar cells,and the islet cells were more numerous,densely and uniformly distributed.Islets size were larger in TXNIP knockout mice compared to WT group mice.Immunohistochemical staining showed that insulin secretion was increased in TXNIP knockout mice compared with normal wild-type mice.(8)Immunofluorescence results showed that compared with the control group,the number of Ki67 or PCNA positive cells in TXNIP knockout mice did not increase significantly.The results suggested that the proliferation of islet β cells in TXNIP knockout mice was not obvious.Conclusion:The phenotype characteristics of TXNIP knockout mouse model are stable.The proliferation of islet β cells in TXNIP knockout mouse model was not obvious.Part III The role and mechanism of TXNIP on compensatory proliferation of islet β cells in high-fat diet-induced obese miceObjective:The obese mouse model was prepared by high-fat feeding to induce the compensatory proliferation of pancreatic β-cells,and the relationship between TXNIP and the proliferation of pancreatic β-cells and its role in the compensatory proliferation of pancreatic β-cells were explored.Further elucidate the mechanism of TXNIP in the regulation of islet β cell proliferation,and provide new ideas and intervention targets for the prevention and treatment of diabetes.Methods:(1)Experimental animals and groupsMale TXNIP knockout mice(KO),which were genetically identified as homozygous,and wild-type mice(WT)from the same litter were used as experimental animals in this study.The experimental mice were fed with normal diet and high-fat diet.Normal chow feeding groups: WT and KO groups;high-fat chow feeding groups: WT-HFD and KO-HFD groups.The mice were fed high-fat diet from 4 weeks of age for 12 weeks,weighed weekly and the samples were collected after 16 weeks of age for relevant measurement and analysis.(2)Effect of TXNIP knockout on mouse islet β cell mass(β cell mass)The pancreatic/body weight ratios of the mice were calculated.The protein expression levels of TXNIP in the islets of each group were detected by Western blot;The m RNA expression levels of TXNIP in the islets of each group were detected by Real-time PCR.Immunohistochemical staining was performed to observe the morphology,number,size and insulin content of the islets.The β cell mass(β cell mass)of the islets was calculated using the serial sectioning method.(3)Effect of TXNIP knockout on glucose metabolism and glucose tolerance and insulin sensitivity in miceThe fasting blood glucose values of the mice in each group were measured,and the serum insulin and glucagon levels of the mice were measured.Insulin/Glucagon immunofluorescence double-labeled staining was performed to observe the changes in islet cell classification.Glucose tolerance test(OGTT)and insulin tolerance test(ITT)were performed to observe the changes of glucose tolerance and insulin sensitivity in mice.(4)Effect of TXNIP knockout on the number of proliferating pancreatic islet β cellsImmunofluorescence double-labeling of Insulin/Ki67 and Insulin/PCNA was performed to observe the changes in the number of Ki67 and PCNA positive cells in each group of mice;The percentage of Ki67+Ins+ and PCNA+Ins+ double-positive cells was calculated;Real-time PCR was performed to detect the m RNA expression levels of Ki67 and PCNA in each group of mice.(5)Effect of TXNIP knockout on proliferation-associated transcription factors in pancreatic islet β cellsReal-time PCR was used to detect the m RNA expression changes of Pdx1,Mafa,Mafb,Foxo1,Foxm1,Nkx6.1,Ins2 and Glut2 and other related transcription factors.(6)Effect of TXNIP knockout on cell proliferation cycle regulators in β cellsWestern blot was used to detect the protein expression changes of key cell cycle regulators Cyclin D2,CDK4 and p27;Real-time PCR was used to detect the m RNA expression changes of Ccnd2,Cdk4 and Cdkn1b;Real-time PCR was used to detect the m RNA expression changes of Rb,Ccna2,Ccnb1,Ccnb2 Ccnd1,Ccne1,Cdk2,Cdkn1 a,Cdkn2a and Cdkn1 c,and the m RNA expression changes of other cell cycle regulators were detected by Real-time PCR.To analyze the effect of TXNIP knockout on the proliferation cycle of pancreatic β cells.(7)Mechanism of TXNIP knockout on compensatory proliferation of islet β cellsWestern blot was used to detect the changes of PI3 K,phosphorylated PI3 K,AKT,phosphorylated AKT,m TOR,phosphorylated m TOR,GSK3β and phosphorylatedGSK3β protein levels in the PI3K/AKT signaling pathway;Changes of ERK/1/2,phosphorylated ERK1/2,MEK1/2 and phosphorylated MEK1/2 protein levels in the ERK signaling pathway;Changes of β-catenin and phosphorylated β-catenin protein levels in the Wnt/β-catenin signaling pathway.Analyze the signaling pathways that TXNIP participates in regulating the proliferation of islet β cells,and preliminarily clarify the mechanism of TXNIP on the proliferation of islet β cells.Statistical methodsAll data were statistically analyzed using SPSS 17.0 software and plotted using Prism Graph Pad 7.0 software.Data comparison between two groups was performed by independent sample t-test,and comparison between multiple groups was performed by one-way ANOVA.Statistical results were expressed as mean ± standard error(Mean ± SEM)and P?0.05 was considered statistically significant.Results:1.TXNIP knockout can further increase islet β cell mass in high-fat fed obese mice(1)TXNIP protein expression level and m RNA expression level in each group of mice.The results of Western blot showed that compared with the control group of WT mice,there was no obvious expression of TXNIP protein in the knockout KO mice.Similarly,in high-fat fed mice,compared with control WT-HFD mice,there was no obvious expression of TXNIP protein in knockout KO-HFD mice.The m RNA assay revealed that no m RNA in TXNIP was detected in the pancreatic islets of TXNIP knockout mice.(2)Changes in mouse body weight and pancreas/body weight ratioCompared with the mice fed with normal diet,in the high-fat fed group,the body weight of the mice in the WT-HFD group increased significantly with the prolongation of the feeding time,while the body weight of the mice in the KO-HFD group did not increase significantly.The results of the pancreas/body weight ratio of mice in each group showed that there was no significant change in the pancreas/body weight ratio ofmice in the KO and WT groups(0.0093±0.0004 vs 0.0089±0.0002,P>0.05),while the pancreas/body weight ratio of the mice in the KO-HFD group was significantly higher than that of the mice in the WT-HFD group in the high-fat feeding(0.0108±0.0003 vs 0.0089±0.0005,P<0.01).This result suggested that the pancreas mass of TXNIP knockout mice fed with high fat increased.(3)TXNIP knockout increased pancreatic β cell mass in high-fat-fed miceThe results of immunohistochemical staining showed that compared with normal feeding,the number and size of islets in WT-HFD group and KO-HFD group were increased,and the β cell mass was significantly increased in high-fat-fed mice;Further observation found that there was no significant difference in islet number,size and β cell mass between KO group and WT group(1.2919±0.0593 mg vs 1.1208±0.0465 mg,P>0.05);While the number and size of islets increased,and the β cell mass in the KOHFD group was significantly higher than that in the WT-HFD group(1.9639±0.1180 mg vs 1.4626±0.1453 mg,P<0.01).The above results suggested that high-fat feeding can induce compensatory proliferation of pancreatic islet β-cells and that TXNIP knockout can increase the pancreatic β cell mass in high-fat fed mice.2.TXNIP knockout improved glucose tolerance and increased insulin sensitivity in high-fat-fed mice(1)Changes of insulin levels and fasting blood glucose in mice in each groupCompared with WT mice,the insulin level of TXNIP knockout mice was increased,and the insulin level of high-fat-fed mice was higher than that of normal-fed mice,and the insulin level of mice in KO-HFD group was significantly higher than that in WTHFD group mice(9118.5±200.6 pg/m L vs 6025.6±151.6 pg/m L,P<0.01).Fasting blood glucose levels were significantly higher in the high-fat fed mice than in the normal fed mice,while mice in the KO and KO-HFD groups were significantly lower compared to their controls(3.55±0.24 mmol/L vs 5.28±0.24 mmol/L,P<0.05 and 7.57±0.76 mmol/L vs 13.91±0.71 mmol/L,P<0.01),respectively.The results showed that TXNIP was involved in the regulation of glucose metabolism in mice and affected the function of islet β cells.Knockout TXNIP couldreduce fasting blood glucose and increase insulin secretion.(2)TXNIP knockout reduced serum glucagon levels in high-fat-fed miceInsulin/Glucagon immunofluorescence double-labeling staining showed that islet cells were evenly distributed around the islets in the WT and KO groups,while islet cells were significantly increased in the WT-HFD and KO-HFD groups,and a small number of cells infiltrated into the interior of the islets.The serum glucagon levels of the mice in each group showed no significant difference between the KO and WT groups(1938.3±82.1 pg/m L vs 2368.4±98.0 pg/m L,P>0.05),while the serum glucagon levels of the mice in the KO-HFD group were significantly lower than those of the mice in the WT-HFD group(2356.6±77.4 pg/m L vs 3143.9±225.1 pg/m L,P<0.05).(3)TXNIP knockout improved glucose tolerance and increased insulin sensitivity in high-fat-fed miceThe results of glucose tolerance test showed that compared with normal-fed mice,the area under the curve of high-fat-fed mice increased,and the difference was significant,indicating that high-fat feeding caused impaired glucose tolerance.Compared with the WT group,the area under the curve of the KO group was not significantly decreased,and there was no statistical significance(P>0.05).In high-fat fed mice,the area under the curve was significantly lower in the KO-HFD group compared to the WT-HFD group(P<0.01),indicating that TXNIP knockout improved high-fat feeding-induced impairment of glucose tolerance.Similarly,the results of insulin tolerance test showed that the area under the curve of high-fat-fed mice was increased compared with that of normal-fed mice,indicating that their insulin tolerance was impaired and their insulin sensitivity was reduced;The difference in the area under the curve between the WT and KO groups was not significant and not statistically different(P>0.05).In high-fat fed mice,the area under the curve was significantly lower in the KO-HFD group compared to the WT-HFD group(P<0.01).The above results suggested that high-fat feeding reduced insulin sensitivity,and TXNIP knockout can improve the decrease in insulin sensitivity caused by high-fat feeding.3.TXNIP knockout can further promote high-fat feeding-induced compensatory proliferation of pancreatic β cells(1)TXNIP knockout increased the number of Ki67 or PCNA positive cells in islets of high-fat fed miceImmunofluorescence double-labeling staining and statistical results showed that in normal fed mice,no Ki67 or PCNA positive cells were seen in the islets of the WT group,and a small number of Ki67 or PCNA positive cells were found in the islets of the KO group,but the increased was not significant and not statistically significant(Percentage of Ki67+Ins+ double positive cells,0.7668±0.0363 vs 0.5536±0.0369,P>0.05;Percentage of PCNA+Ins+ double positive cells,0.8546±0.0628 vs 0.6903±0.0239,P>0.05).In contrast,the number of Ki67 or PCNA-positive cells in the islets of high-fat fed mice was significantly increased,and the number of Ki67 or PCNA-positive cells in the KO-HFD group was further increased compared to the WTHFD group,with the percentage of Ki67+Ins+ double-positive cells being approximately 1.8 times higher than that of mice in the WT-HFD group(1.7773±0.1441 vs.0.9626± 0.0493,P<0.01).The percentage of PCNA+Ins+ double-positive cells was about 1.9 times that of WT-HFD mice(2.2218±0.1467 vs 1.1787±0.0560,P<0.01).(2)Knockout of TXNIP further increased m RNA expressions of Ki67 and PCNA in islets of high-fat-fed miceReal-time PCR results showed that there was no significant difference in the m RNA expression of Ki67 between the KO and WT groups of normal fed mice(P>0.05);Compared with normal fed mice,the m RNA expression levels of Ki67 in KO-HFD and WT-HFD groups were significantly higher in high-fat fed mice,,and the m RNA expression level of KO-HFD group was significantly higher than that of WTHFD group(P<0.01).The changes of PCNA m RNA expression in each group of mice were roughly consistent with the expression of Ki67 m RNA.The above results suggested that TXNIP knockout further promoted proliferation of pancreatic β-cells in high-fat fed mice.4.Effect of TXNIP knockout on the expression of proliferation-related transcription factors in pancreatic islet β-cellsReal-time PCR of mRNA levels of transcription factors such as Pdx1,Mafa,Mafb,Foxm1 and Ins2,which are related to the proliferation of pancreatic β-cells orcharacteristic of β-cells,showed no significant difference in the m RNA expression levels of the above factors between the WT and KO groups of mice(P>0.05).The m RNA levels of all factors were elevated in the high-fat-fed group compared to the corresponding normal-fed control;In the high-fat-fed group,the m RNA expression levels of the above factors were significantly upregulated in the KO-HFD group of mice compared to the WT-HFD group(P<0.01).In contrast,Foxo1 m RNA expression levels were reduced in the high-fat fed group and were significantly lower in mice in the KOHFD group compared to the WT-HFD group(P<0.01).The changes in m RNA expression levels of transcription factors Nkx6.1 and Glut2 were not statistically significant in the mice in each group(P>0.05).The above results suggested that TXNIP can affect pancreatic β-cell proliferation and insulin secretion by influencing the expression of various transcription factors that regulate pancreatic β-cell proliferation and insulin synthesis.5.TXNIP knockout promoted islet β-cell proliferation by regulating cell cycle factors and accelerating cell cycle progression(1)TXNIP knockout upregulated Cyclin D2 and CDK4 and downregulated p27 protein and m RNA expression levels in high-fat fed miceThe results of Western blot showed that in normal-fed mice,there was no significant difference in the expression levels of Cyclin D2 and CDK4 between KO and WT groups(P>0.05);In high-fat fed mice,the protein expression levels of Cyclin D2 and CDK4 were significantly increased,and the KO-HFD group was higher than that of the WT-HFD group(P<0.01).As an inhibitory protein of the cell cycle,in normal feeding mice,compared with the WT group,the expression of p27 protein in the KO group was not significantly changed,and there was no statistical significance(P>0.05).The p27 protein expression was reduced in the high-fat fed mice and was significantly lower in the KO-HFD group than in the WT-HFD group(P<0.01).Real-time PCR results were generally consistent with the Western Blot results.(2)Effect of TXNIP knockout on mRNA expression of islet cell cycle-related regulators in high-fat-fed miceReal-time PCR showed that the differences in mRNA expression of cell cycleregulators such as Rb,Cdkn2 a,Cdkn1c,Ccna2,Ccnb1,Ccnb2 and Cdk2 between the KO and WT groups of mice fed with normal chow were not significant and not statistically significant(P>0.05).In high-fat fed mice,compared with WT-HFD group,m RNA expression of the negative cell cycle regulators Rb,Cdkn2 a and Cdkn1 c in KOHFD group were significantly down-regulated(P<0.05),while m RNA expression of the pro-proliferative factors Ccna2,Ccnb1,Ccnb2 and Cdk2 were significantly upregulated(P<0.01).The m RNA expressions of Ccnd1,Ccne1 and Cdkn1 a were not significantly changed in both high-fat fed and normal fed mice(P>0.05).The above results suggested that TXNIP knockout can accelerate the cell cycle process by affecting the expression of the above cell cycle regulators in the pancreatic islets of high-fat-fed mice,thus promoting the proliferation of pancreatic β-cells.6.TXNIP involved in proliferation regulation of pancreatic β-cells through PI3K/AKT and ERK signaling pathways(1)TXNIP knockout activated PI3K/AKT signaling pathway in pancreatic β-cells of high-fat-fed miceWestern bot results showed that the expression of phosphorylated PI3 K,phosphorylated AKT,phosphorylated m TOR and phosphorylated GSK3β proteins were significantly higher in the KO-HFD group of mice compared with the WT-HFD group(P<0.05).The results suggest that TXNIP knockout can promote the activation of PI3K/AKT signaling pathway in pancreatic β-cells induced by high-fat feeding.(2)TXNIP knockout activated ERK signaling pathway in pancreatic β-cells of high-fat-fed miceWestern bot results showed that the expression of phosphorylated ERK/1/2 and phosphorylated MEK1/2 proteins were significantly higher in the KO-HFD group compared to the WT-HFD group(P<0.01).The results showed that TXNIP knockout could promote the activation of ERK signaling pathway in pancreatic β-cells induced by high-fat feeding.(3)TXNIP knockout had no effect on Wnt/β-catenin signaling pathway in pancreatic β-cells of high-fat-fed miceWestern bot results showed that there was no significant change in the expressionof both phosphorylated and non-phosphorylated β-catenin protein in the KO-HFD group compared to the WT-HFD group(P>0.05).It was suggested that TXNIP knockout had no effect on the Wnt/β-catenin signaling pathway in pancreatic β-cells of high-fat fed mice.The above results indicated that TXNIP knockout may promote the compensatory proliferation of islet β cells by further promoting the activation of PI3K/AKT and ERK signaling pathways in islets of high-fat-fed mice.Conclusion:1.TXNIP knockout can promote the compensatory proliferation of pancreatic β cells induced by high-fat feeding.2.TXNIP can affect the expression of cell cycle-related regulatory factors by activating PI3K/AKT and ERK signaling pathways,and participate in the regulation of compensatory proliferation of pancreatic β cells. |
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