The Expression Of Proteins Which Ralative To Metabolism In Hypothalamus And Peripheral Organs Of DIO/DR Rats

Obesity has been increased rapidly all over the world for the reason of changing lifestyle and increasing energy intake of people. The chronic disease which caused by obesity have serious risk of developing complications. High energy intake may result in imbalance between energy intake and consume. Then the body fat mass increased and obesity occurred. As we know, some proteins and peptides play crucial roles in regulating the energy homeostasis of the body. By the development of protein research, some new proteins which play key roles in obesity have been found.Objective:Our study aims at established diet induced obesity (DIO) and diet resistant (DR) SD rat model by high fat diet feeding. Then we selected differential expressed proteins of hypothalamus in DIO-DR rats by proteomic analysis. We use techniques of immunofluorescence, western blotting and RT-PCR to test some differential expressed proteins expressed in peripheral organs which relative to glucose and fat metabolism. Our purpose of research is to illuminate the effect of proteins and peptides during the progress of obesity and provide new target of therapy in the future.Methods:1. We established DIO-DR rat models.2. We used two-dimensional differential in-gel eletrophoresis and MALDI-TOF-MS/MS to separate and identify differential expressed proteins of hypothalamus in DIO-DR rat.3. We used techniques of immunofluorescence, western blotting and RT-PCR to test some differential expressed proteins which expressed in pancreas, liver and skeletal muscle. These proteins include molecular chaperon (PDIA3), synapse associated proteins (SNAP-25), cytoskeleton proteins (β3-Tubulin) and glucose transport protein (GLUT4).Results:1. Phenotype differentiations between DIO and DR rats.1) DIO rats did not grow faster than DR rats during the high fat fed period in initial period (4weeks) because they have similar body weights. DIO rats grew faster and took in more energy per day than DR rats did in middle period (4-8weeks).2) At the end of14weeks, weight of main organs which including heart, gastrocnemius muscle and testes of DIO rats were similar to that of DR rats. The weight of visceral fat in DIO rats including epididymal fat and perinephrit fat were significantly higher than that of DR rats. Plasma triglycerides and free fatty acid (FFA) level of DIO rats were higher than of DR rats.3) Serum level of insulin and leptin were measured by Elisa in different period. We found no significant difference between DIO and DR rats in early stage (4weeks) and middle stage (8weeks). But at the end of14weeks, the serum levels of insulin and leptin were significantly increased in DIO rats, and no significant difference between DR and control rats. This result suggests that DIO rats are insulin-resistant and leptin-resistant after long time high energy diet intake.2. Separate and identify differential proteins of hypothalamus by2D-DIGE integrated with MALDI-TOF-MS/MS.42differential protein spots were separated by2D-DIGE and then33proteins were identified by MALDI-TOF-MS/MS. These proteins were classified into molecular chaperon, metabolism enzymes, synapse associated proteins, cytoskeleton proteins and antioxidant proteins.3. Partial differential proteins in peripheral organs were validated by molecular biology techniques. The results of our research demonstrated PDIA3, and β3-Tubulin were significantly over expressed of DIO rats as well as PDIA3, β3-Tubulin were down regulated of DR rats in pancreas, liver and skeletal muscle. On the contrary, SNAP-25and GLUT4were over expressed of DR rats and down regulated of DIO rats in pancreas, liver and skeletal muscle.Conclusions:1. DIO and DR rat models were built up successfully which have many characteristics similar to that of human beings. The obesity induced by diet could result in insulin-resistant and leptin-resistant.2. Differential proteins were separated and identified successfully by2D-DIGE integrated with MALDI-TOF-MS/MS. The results of2D-DIGE provide foundations for obesity research and metabolic disease.3. Chronic high fat diet may result in dysfunction of glucose transport and destruction of cell skeletal structure of DIO rats. Oxidative stress and endoplasmic reticulum stress of DIO rats may be happened too. All the disorder mentioned above could induce disorder of metabolism in DIO rats.