P-glycoprotein在第一型及第二型糖尿病小鼠大腦微血管上表現量及功能之探討

Abstract

糖尿病是一種全身性的代謝疾病，可能造成視網膜、腎臟、神經和血管方面的病變。近來有研究指出糖尿病可能改變腦部的血流和血腦障壁間物質的運送，P-glycoprotein (P-gp, MDR1) 表現在腦部微血管上的luminal side，並且分布在caveolae上，主要功能是排除其受質並保護腦部組織。本研究主要是探討在糖尿病的情形下，caveolin-1與caveolin-2的蛋白質表現量和P-gp功能的關係；在第二部分的研究中，我們探討糖尿病是否影響P-gp的寡聚化。 本研究所使用的自發性第一型和第二型糖尿病動物模式分別為NOD (non-obese diabetic) 和NZO (New Zealand obese) 小鼠，對照組則是NON (non-obese, non-diabetic) 小鼠。西方墨點法的結果顯示NZO公鼠腦中P-gp的表現量大約是NON公鼠的1.5倍，而NOD母鼠的P-gp表現量則是有些微的降低。組織免疫染色的結果顯示NZO公鼠腦部微血管上P-gp表現量上升，而NOD母鼠腦部微血管上P-gp表現量沒有顯著的改變。雖然西方墨點法的結果顯示NOD母鼠和NZO公鼠腦中caveolin-1和caveolin-2的蛋白質表現量都上升，但是在組織免疫染色中，NOD母鼠和NZO公鼠大腦紋狀體微血管上caveolin-1和caveolin-2的蛋白質表現量反而是下降的。我們選用P-gp典型的受質rhodamine 123從靜脈給藥並利用體內大腦微透析系統 (in vivo brain microdialysis technique) 來分析P-gp在血腦障壁上排除受質的能力，結果發現在NOD母鼠和NZO公鼠腦細胞外液中藥物濃度大約只有NON小鼠的一半，顯示糖尿病小鼠腦中P-gp的功能是增加的。 在第二部分的研究中，我們利用blue native polyacrylamide gel電泳 (BN-PAGE) 合併西方墨點法來分析自然型態的P-gp，結果顯示在NON小鼠腦中的P-gp是以二聚體的型態存在，但是在NOD公鼠、NOD母鼠和NZO公鼠腦中，P-gp會形成單體及二聚體。 糖尿病小鼠腦部微血管上P-gp的功能除了蛋白質本身表現量的影響之外，caveolin-1和caveolin-2蛋白質的表現量也扮演重要調控的角色；另外，糖尿病也可能改變P-gp在腦中的寡聚化，不過P-gp聚合的程度在生理上的意義為何仍需進一步的研究。綜合以上的實驗結果，我們發現不管是在自發性第一型糖尿病或是第二型糖尿病小鼠大腦微血管上，P-gp的功能都有顯著的增加。

Diabetes mellitus is a systematic metabolic disease and is related to a number of complications, including retinopathy, nephropathy, neuropathy, and vascular diseases. Recent studies show that diabetic pathology may alter cerebral blood flow and blood brain transport. P-glycoprotein (P-gp, MDR1) is highly expressed in the luminal side of brain capillary and is located in the caveolae. The current study is to investigate brain expression of caveolin-1 and caveolin-2, the major proteins of caveolae, and P-gp function in diabetes. On the other hand, this study is also to investigate P-gp oligomer formation in diabetes. Inbred non-obese diabetic (NOD) and New Zealand obese (NZO) mice were used as the models of type 1 and type 2 diabetes, respectively, in comparison with non-obese non-diabetic (NON) mice. Western blots showed that P-gp protein expression was significantly higher (about 1.5-fold) in the brain of male NZO mice and slightly lower in the brain of female NOD mice than in male and female NON mice. Immunohistochemical staining showed higher P-gp expression in the brain capillaries of male NZO mice, whereas expression in female NOD mice was comparable to that in NON mice. Although levels of caveolin-1 and caveolin-2 were significantly higher in the brain of female NOD and male NZO mice on Western blots, immunochemical analysis showed lower caveolin expression in the capillaries in brain striatum of female NOD and male NZO mice compared to NON mice of the same gender. P-gp efflux pump activity in the blood-brain barrier (BBB) was measured by an in vivo brain microdialysis technique. Brain extracellular concentrations of intravenously injected rhodamine 123, a known P-gp substrate, were decreased by half in female NOD and male NZO mice, showing higher P-gp efflux pump activity. To investigate P-gp oligomerization, blue native polyacrylamide gel electrophoresis (BN-PAGE) was used to separate native forms of P-gp. P-gp exists as a dimer in the brain of male and female NON mice. However, mono- and dimeric forms of P-gp were both detected in the brain of male and female NOD and male NZO mice. In conclusion, P-gp activity in brain capillaries in diabetic mice is not only due to P-gp protein expression, but may also be affected by caveolin expression. On the other hand, P-gp oligomeric formation may be affected by diabetes, and further studies are still required to understand the physiological role of P-gp multimerization. Inbred mice with type 1 or type 2 diabetes show increased P-gp activity in the blood-brain barrier.