Studies On Molecular Structure Uniformity Of Liver Glycogen,Glycogen Structure Changes Over Diurnal Rhythm And Of Type 1 Diabetic Mice

Glycogen,a highly branched polymer of glucose which serves as a storage for glu-cose in animals,is found in many organisms and tissues.Liver glycogen plays an im-port role in maintaining blood glucose.In glycogen,glucose units join each other via?-(1?4)linkages to form chains.These chains are bonded together via ?-(1?6)link-ages as branch points to form ? particles(?20nm in diameter).? particles can further arranged into larger a particles(up to 300nm in diameter),which have a composite cauliflower-like appearance in transmission electron microscopy(TEM).The molecular structure of glycogen is important for various biological functions.For example,the highly but randomly branched structure enables glycogen to have good solubility in water;muscle glycogen consists largely of ? particles,because ? par-ticles can degrade more quickly,which is good for rapid supply of energy during exer-tion.Glycogen structure also enables informations to be contained about glycogen me-tabolism.Previous studies have found out that in an animal model for type 2 diabetes,db/db mice,a particles in liver glycogen were more fragile than in healthy mice liver glycogen,tending to degrade into smaller ? particles when treated with DMSO,a sol-vent which breaks hydrogen bonds but leaves chemical linkages intact.This fragility may have a close relationship with diabetes,as it has been shown that ? particles have a higher rate of enzymatic degradation to glucose than a particles because of a higher ratio of surface area to volume.Therefore,study on the molecular structure of glycogen has important meaning for understanding the property of glycogen and its metabolism as well as the mechanism of diabetes.This thesis expands the previous study of glycogen structure in three aspects,includ-ing exploring glycogen structure uniformity between liver lobes,studying the changes of liver glycogen structure in healthy and diabetic mice over the diurnal cycle and ex-ploring structure differences between glycogen from type 1 and healthy mice.The pur-pose of the study is to check whether the previous data are representive,provid infor-mation for choosing the way of obtaining glycogen from animal models with a bigger liver,study the glycogen structure and its relationship to glycogen metabolism as well as diabetes.The first is to explore any differences in glycogen molecular structure between liver lobes;if there were found to be significant differences,this would call into question the data previously obtained,which were for all portions of the liver treated together.It could provide some information for choosing method if bigger animals are used for study in the future,as it not convient to use the whole liver.This study use Wistar rats sacrificed at different times to extract glycogen from 6 different lobes.Size-exclusion chromatography(SEC),fluorophore-assisted carbohydrate electrophoresis(FACE)and transmission electron microscopy(TEM)were employed to examine the glycogen mo-lecular structure differences in weight distribution,chain length distribution(CLD)and appearance.It was found that glycogen structure from different lobes are similar,which means glycogen extracted from any part of liver can be representive,thus biopsy can be used for studying glycogen structure.In the next part of this study,the changes in molecular structure of healthy and dia-betic mice glycogen over a diurnal cycle were examined.This is because some data suggested that this structure could well undergo major changes over this cycle,and understanding these would contribute to understanding the molecular origin of the fra-gility.The previous study used a DMSO SEC method for molecular structural charac-terization which was subsequently greatly improved into an aqueous SEC method that have a better resolution of glycogen ? and ? particles.This study used the improved aqueous SEC method and the molecular fragility of glycogen were also checked.The result show that,all the mice liver glycogen from different time points were mainly composed of a particles,which is different from the previous result.Glycogen from healthy mice is fragile to DMSO during the glycogen synthesis phase but non-fragile during glycogenolysis and the non-fragile glycogen have a significant lower density than fragile one.Glycogen from diabetic mice(db/db mice)is always fragile.The re-sults are consistent with two alternative mechanisms.1.Healthy glycogen is fragile when first formed(optimizing rapid glucose storage)and becomes non-fragile during subsequent degradation,to optimize control of blood-sugar levels.The strengthening process were damaged in db/db mice.2.There are two kinds of glycogen:one is com-pact but fragile,the other is loose but non-fragile.The fragile glycogen is degraded first after the feeding cycle,and the non-fragile degrades more slowly to optimize blood-sugar control.The formation of non-fragile particles is less effective in db/db mice.These mechanisms have implication in understanding glycogen metabolism and sug-gest potential types of drug targets.The last part examined differences between the molecular structure of healthy and type 1 diabetic mice glycogen,db/db mice(type 2 diabetic mice)glycogen is found out to be more fragile than healthy glycogen,but it was unknown whether this fragility also exist in type 1 diabetic models.Type 1 diabetes have a different pathogenesis with type 2 diabetes.Type 1 diabetes is caused by immune-mediated pancreatic ? cells impair-ment while type 2 diabetes is accompanied with insulin resistant.Examining the glyco-gen molecular structure in type 1 diabetes will provide additional understanding in the relationship of glycogen structure and diabetes.The study used two types of diabetic animal model,non-obese diabetic mice(NOD mice)and streptozotocin(STZ)induced type 1 diabetic mice.The result show that glycogen from type 1 diabetic mice are fragile to DMSO,similar to glycogen from db/db mice,while glycogen from the control group were resistant to DMSO.This suggests that the fragility of glycogen may be related to the absolute or relative insufficient secretion of insulin.This thesis expands the previous study of glycogen structure in three aspects.Explor-ing differences in glycogen molecular structure between liver lobes helps to know how to get representative data of glycogen molecular structure.The results show that glyco-gen extracted any lobe of the liver is representive,which mean that biopsy can be used for glycogen structure study.Studying the changes of healthy and diabetic mice glyco-gen helps to understand the metabolism process of glycogen,and the differences on glycogen metabolism between healthy and diabetic mice.The results show that for the healthy mice,glycogen from degradation phase are more stable than glycogen from synthsis phase.While for the db/db mice,glycogen from all the time points are fragile.Further experiments such as exploring the expression of glycogen related proteins at different times could be done to find new potential drug targets.Examing the glycogen structure of two type 1 diabetic models can help to have a further understanding be-tween glycogen structure and its relationship to diabetes.The result show that glycogen from type 1 diabetic mice are fragile to DMSO treatment,while glycogen from control group are more stable.The instability of glycogen in type 1 diabetic mice may contrib-ute to hyperglycemia.Accroding to the results of expermints,it can be inferred that stability of glycogen structure may be related to the relative or absolute deficiency of insulin secretion.