Proteomics Research. People With Type 2 Diabetes Red Cell Membrane

Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose) level, which results from defect in insulin secretion, or action, or both. Over time, diabetes can lead to blindness, kidney failure, and nerve damage. This disease is also an important factor in accelerating the hardening and narrowing of the arteries (atherosclerosis), leading to strokes, coronary heart disease, and other blood vessel diseases. And type 2 diabetes mellitus accounts for 90—95% of all diabetes. Current therapeutic approaches are largely developed in the absence of defined molecular target or even solid understanding of type 2 diabetes pathogenesis. The blood should be regarded as the primary target organ in diabetes mellitus. It is reasonable to presume that the properties of RBC(red blood cell), especially plasma membrane, must suffer variation during disease development. It has observed that the rate of glucose entry into RBC was decreased significantly in type 2 diabetic patients compared with healthy controls and this is due to the structural change of GLUT1(glucose transporter 1). There is substantial interest in the identification of more membrane proteins related to the disease development using proteomics.Proteomics is the study of the dynamic expression pattern, activities, modifications and localization of all proteins encoded by the genome, and protein-protein interactions in a given cell, tissue or organism. So there is intense interest in use of proteomic approach to foster a better understanding of disease processes, develop new biomarkers for diagnosis, and accelerate drug development. 2-DE(two-dimensional electrophoresis) is first, important step in the traditional proteomics strategy, which separates proteins on the gel in two dimensions on the basis of independent charge and molecular weight. We have employed it in this study to permit the simultaneous investigation of the largest possible range of RBC membrane proteins. Because hydrophobic membrane proteins simply do not dissolve in traditional solvents used for IEF(isoelectric focusing), the first step of 2-DE, soclassical techniques used in 2-DE are of poor efficiency for their analysis. In this study, several types of surfactants have been used for optimizing the condition of solubilization for RBC membrane proteins during IEF. By comparing the complex 2-DE protein patterns, among about total of 600 protein spots in 2-DE gel, analysis by PD-Quest software, 42 proteins displayed change, 27 proteins increased in expression and 15 proteins decreased in diabetes versus normal controls. Among them Flotillin-1, Syntaxin 1C, and Arginase appear to be affected in the diseaseThe present study shows, for the first time, that Flotillin-1 protein is affected in type 2 diabetes RBC membranes. Flotillin-1 protein is first identified in a screen of mouse adipose tissue for novel marker of lipid rafts/caveolae. It is enriched in the Triton X-100 insoluble buoyant fraction after sucrose density centrifugation, which is indicative of their association with lipid rafts microdomains in the membranes. Recently, it is reported that Flotillins are the most abundant raft proteins in human RBC. It is now clear that these lipid rafts microdomains act as platforms for conducting a variety of cellular functions, such as vesicular trafficking and signal transduction. Our result shows that Flotillin-1 in RBC membranes appears to be affected in the type 2 diabetes. Although we do not know yet the relationship between Flotillin-1 and the disease of type 2 diabetes, but it is quite clear now about the function of Flotillin-1 in translocation of glucose transporter GLUT4. In muscle and adipose tissue, Flotillin-1 function as an adaptor protein that recruits a signaling complex of proto-oncoprotein c-Cbl/c-Cbl-associated protein to lipid rafts leading to Glut 4 translocation to the plasma membranes in response to insulin. Syntaxin 1 on the target membrane binds to SNAP-25 and vesicular synaptobrevin/VAMP to assemble the core complex of the membrane fusion machine. Decreased expression of Syntaxin 1C in patient 2D-gel infers that translocation of GLUT4 to the plasma membranes would be impaired, even if the expression of Flotillin 1 compensatory increased. On the other hand, impaired membrane fusion machine must affect exocytosis of insulin secretory granule in the pancreatic b-cell. Arginase is an enzyme that breaks down arginine. It shares a common substrate, L-arginine, with NOS, thus increased expression of Arginase in diabetic human cavernosal tissue maydownregulate NO production by competition with NOS. Our result demonstrated that the expression of Arginase1 in RBC membranes is also high in patients, which shows that NO and Arginase1 pathways are involved in the development of human type 2 diabetes.For further research of molecular mechanism of Flotillin-1 and Arginase1 involving in diabetes mellitus, we have constructed recombination gene to express Flotillin-1, Flotillin-2, Arginase1 and other membrane proteins, such as Cdb3. The interaction methods of Far-Western and Pull-down assay were used to verify their interaction in vitro. The result showed that there is direct interaction between Flotillin-1 and Arginase 1. In combination with the previous result, it is deduced that Flotillin-1 and Arginase1 in RBC membranes would be directly interacted, and perhaps they are regulateing signal pathway by their interaction.Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipid. These regions concentrate certain signaling molecules. This concentration of signaling molecules suggests that lipid rafts might function as a site for compartmentalization of signaling events. We observed that Flotillin-1 and Arginase1 translocation to Lipid rafts membrane fraction. For investigation of mechanism of Lipid rafts proteins in diabetes. We alsostudy proteomics analysis of Lipid rafts with 54 proteins displayed change, 18 proteins increased, 15 proteins decreased, 10 proteins expressed only in diabetes and 11 proteins expressed only in normal controls.In conclusion, the present investigation has provide initiate 2-D pattern of normal human and diabetes RBC membrane proteins; has make primary efforts on elucidating molecular mechanism of diabetic RBC membrane proteins; has discovered several cellular activation involving in diabetic pathological; the possible role of several disregulated protein are disccused. All above has established groundwork for further investigation to clarify the molecular mechanism of diabetic pathway.