Morphology And Function Study Of Conditionally Knockout Gene PDHA1 In Pancreatic Beta Cell

Background:Diabetes mellitus(DM)is a public health problem worldwide.Both its prevalence and the number of DM patients are increasing rapidly,posing a serious threat to human health.Current research suggests that diabetes is a metabolic disease primarily caused by a reduction in beta cell mass,decreased insulin secretion,and/or the impairment of insulin function.In recent years,however,the involvement of mitochondria in energy metabolism has become more apparent.Mitochondrial dysfunction can lead to impaired cell growth and function and the development of insulin resistance.The specific regulatory mechanisms underlying this process are complex and may be related to metabolic coupling,oxidative stress,calcium metabolism disorders,the decline of mitochondrial biosynthesis,and mitochondrial autophagy,among other factors.The specific pathogenesis of the disease is still being studied and explored.Pyruvate dehydrogenase complex(PDHc)is an important regulatory element in various mitochondrial processes,and it mediates the irreversible oxidation of pyruvate to acetyl coenzyme A and controls the secretion of insulin by cells through the circulation of tricarboxylic acid.PDHc is mainly composed of pyruvate dehydrogenase(E1),dihydrothiocinamide transferase(E2),dihydrothiocinamide dehydrogenase(E3),and several cofactors.Among these,the E1 subunit encoded by the PDHA1 gene is the most important component in the regulation of the PDHc.Catalytie enzymes can affect PDHc activity by regulating phosphorylation and dephosphorylation of three serine sites on the E1 subunit,thereby regulating pyruvate metabolism.Previous studies have found decreased PDH expression in both rodent models and human subjects with type 2 diabetes,suggesting that PDH plays a vital role in the development of the condition.However,the effect of PDHA1 on beta cells of the pancreas has not been thoroughly investigated.This study aimed to explore the correlation between PDHA1 and diabetes and further elucidate PDHA1 effect on the morphology and function of beta cells.Our findings will possibly provide a new theoretical basis for the mechanistic causes of diabetes and identify new molecular targets for treatment.Methods:We selected five mice with type 2 diabetes(db/db)and five wild-type C57BL/6 mice and measured PDHA1 expression in the islet cells of each group using immunohistochemistry.According to the principles of the Cre/loxP system,we used PDHAlflox/flox mice,whose beta cells exhibited Cre recombinant enzyme specificity.After 4 weeks of alkali decomposition,which allowed for the extraction of offspring tail DNA,PCR amplification was used to identify individual genes.After selecting individuals with the correct genotypes(PDHA1flox/-Cre+/-and PDHAflox/flox),PCR was used to identify genotypes and screenPDHA1flox/floxCre+/-mice.Five PDHA1flox/floxCre+/-mice and five Cre+/-mice of the same age(8 weeks)and body weight were selected as the experimental group and the control group,respectively,and tamoxifen(50mg/kg)was intraperitoneally injected into each individual at an age of 8 weeks to induce gene knockout.Western blot analysis and immunofluorescence staining of paraffin embedded pancreatic tissue sections were conducted to verify the knockout of PDHA1.qPCR was used to detect the expression of PDHA1 mRNA in the islets,liver,and adipose tissues of knockout mice.Immunohistochemical staining was performed to detect the localization and expression of PDHA1 in the pancreatic islets of the mice.Data concerning the hair,nutrition,activity,fasting blood glucose levels,and body weight of mice in both groups were continuously recorded.The glucose tolerance test(IPGTT)and insulin tolerance test(IPITT)were performed once the mice had reached 12 weeks of age.The serun insulin levels of mice after glucose stimulation were detected using the ELISA kit.HE scanning for pancreatic tissues of mice to observe the morphology of beta cells.Results:1.Immunofluorescence showed that PDHA1 was expressed in the islets of wild-type mice,while expression of PDHA1 was relatively lower in the diabetic(db/db)mice.This indicated that PDHA1 was associated with DM.2.A transgenic PDHA1 knockout mouse was bred using the Cer-loxP system,and PDHAlflox/floxCre+/-mice were screened for PCR amplification at an age of 4 weeks.At an age of 8 weeks,tamoxifen solution was injected to induce gene knockout.3.Fluorescenee microseopy was used to identify areas of heightened PDHA1 expression.PDHA1 was mainly expressed in the glandular duct and islets of the pancreas.The expression of PDHA1 was lower in the islets of mice than in control mice.When qPCR was performed using the islets,liver,and adipose tissues of knockout mice,the expression of PDHA1 was significantly decreased in only the pancreatic p cells(p<0.01).Immunohistochemical assays showed that the PDHA1 expression was significantly lower in the islets of knockout mice than in control mice.These results demonstrate the successful development of an islet-specific PDHA1 mouse.4.Observation of phenotypes of mice in the experimental group confirmed that there were no differences between the hair,development,activity,random blood glucose level,and body weight of mice in the control and experimental groups.However,the tolerance of mice to the intraperitoneal injection of glucose was reduced(p<0.01)in the experimental group,while the results of insulin tolerance tests showed no difference between the two groups(p>0.5).The ELISA results showed that following glucose injection,insulin secretion in knockout mice decreased 30min later relative to that of control mice(p<0.01).5.HE staining showed that the islet volume of mice in the knockout group was significantly increased,cell spacing was widened,nuclei were slightly stained,cells contained vacuoles,and the cytoplasm was loose.Conclusion:In this experiment,PDHA1 expression was lower in diabetic mice than in wild-type mice,confirming the correlation between PDHA1 expression and diabetes incidence.Furthermore,a transgenic PDHA1 knockout line was successfully developed.Although there was no difference in the hair,development,activity,random blood glucose level,and body weight between the control and experimental groups,glucose-stimulated insulin secretion was impaired and was not related to peripheral tissue insulin resistance.In addition,islet cells showed compensatory increases in various cell parameters and vacuolar expansion in the cytoplasm.