| The number of obese people has increased rapidly all over the world for the reason of an increase in food availability. Obese subjects have an increased risk of developing comorbidities and lie heavy upon society. Obesity is a complex disease mainly caused by an increase in body fat mass resulting from an imbalance between energy intake and expenditure. Hypothalamus plays an crucial role in regulating the energy homeostasis of the body. Previous research has found that hypothalamus could release orexia peptides and anorexia peptides such as neuropeptide Y and a-MSH to maintain the balance of energy by regulating food intake. By the development of proteomic approaches, more and more new proteins related to obesity have been found which play key roles in obesity.Objective:Our study aims at selecting and identifying differential expressed proteins of hypothalamus in DIO-DR rats by proteomic analysis, in order to illuminate the effect of proteins and neuropeptides during the progress of obesity and provide new target of therapy in the future.Methods:1. Build DIO-DR model.2. The two-dimensional gel electrophoresis was performed to separate differential expressed proteins of hypothalamus in DIO-DR rat. The differential proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry.3. Use two-dimensional differential in-gel eletrophoresis and MALDI-TOF-MS/MS to separate and identify differential expressed proteins of hypothalamus in DIO-DR rat.4. The partial proteins including PDI, GSTM1 and SNAP-25 were validated by immunohistochemistry and western blotting.Results:1. Phenotype differentiations between DIO and DR rats.1) Although initial body weights were similar, DIO rats grew faster than DR rats did during the high fat fed period. DIO rats also took in more energy per day than DR rats did.2) Weight of main organs, including heart, gastrocnemius muscle and testes were similar between DIO and DR rats, but weight of visceral fats in DIO rats including fats of epididymis and around kidneys were significantly higher than that in DR rats.3) Plasma triglycerides and free fatty acid (FFA) level were higher in DIO rats than in DR rats.4) 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 (4 weeks) and middle stage (8 weeks). But in later stage (14 weeks), insulin and leptin were significantly increased in DIO rats, and no significant difference between DR and control rats. This result suggests that DIO rats have insulin-resistant and leptin-resistant in the later stage.2. Separate and identify differential proteins of hypothalamus by 2DE integrated with MALDI-TOF-MS.1) 22 differential proteins were identified,7 of them were over expressed and 7 were down expressed in DIO rats; 6 of them were over expressed and 6 were down expressed in DR rats.2) These identified proteins were classified into antioxidant proteins (PRDX1, GSTM1), metabolism enzymes (UCHL-1), modulins of cell cycle, cytoskeleton proteins and unknown proteins.3. Separate and identify differential proteins of hypothalamus by 2D-DIGE integrated with MALDI-TOF-MS/MS.1) 42 differential protein spots were separated by 2D-DIGE and then 33 proteins were identified by MALDI-TOF-MS/MS.9 of them were up-regulated and 2 were down-regulated in DIO rats compared to control; 8 were up-regulated and 9 were down-regulated in DIO compared to DR; 8 were up-regulated and 4 were down-regulated in DR compared to control.2) 2D-DIGE is more sensible and specific than 2DE and can separate more protein spots. MALDI-TOF-MS/MS has more accuracy than MALDI-TOF-MS. Some of 33 identified proteins were same as which identified by 2DE and MALDI-TOF-MS. These proteins were classified into antioxidant proteins, molecular chaperon, metabolism enzymes, synapse associated proteins and cytoskeleton proteins. Some of them such as PRD?, GSTM1, PDI and UCHL-1 have close relationship with development of obesity. We also found some new proteins such as Dynamin 1 and SNAP-25 which may be related with obesity.4. Partial differential proteins were validated by immunohistochemistry and western blotting.1) The data of immunohistochemistry assay showed PDI, PRD?and Dynaminl were over expressed as well as SNAP-25 was down regulated in hypothalamus of DIO rats.2) The results of western blotting demonstrated PDI, PRD?, GSTM1 and Dynaminl were significantly over expressed in DIO rats (p<0.05) as well as PDI, PRD?, GSTM1 were down regulated in DR rats. On the contrary, UCHL-1 and SNAP-25 were over expressed in DR rats and down regulated in DIO rats.Conclusions:1. DIO-DR rat model was built up successfully which has many characteristics similar to that of human being.2. Differential proteins were separated and identified successfully by 2D-DIGE integrated with MALDI-TOF-MS/MS. More signaling proteins relating to obesity have been selected by 2D-DIGE, which makes foundation for further research.3. Cellular stress (oxidative stress and ER stress) in hypothalamus of DIO rats induces insulin-resistant and leptin-resistant by interfering signal transduction pathway of insulin and leptin due to chronic high fat diet. |
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