PLC-β1及丙酮酸媒介部分葡萄糖誘發之訊息傳導

Abstract

丙酮酸(Pyruvate)為糖解(Glycolysis)作用的終產物，並可以再繼續進行發酵反應產生乳酸或乙醇，或進入粒線體進行檸檬酸循環產生更大的能量。如同其他的α酮酸(α-keto acids)，丙酮酸具有抗氧化的性質，例如以非酵素作用方式與過氧化氫作用產生乙酸、二氧化碳及水。過去認為丙酮酸是醣類代謝的重要調控分子，藉由異位調節(Allosteric regulation)來調控糖解作用及檸檬酸循環的速率。隨著近年的代謝體學發展，許多代謝小分子被發現除了在本身代謝途徑中的調控，同時也具有其他生理上的意義。我們藉由串連疏水性層析及質譜儀分析，發現丙酮酸和PLC-β1可能具有極佳之結合力，當HEK(Human embryonic kidney)細胞經過glucose starvation並以丙酮酸處理後，會造成PLC-β1活化，並由細胞質轉移至細胞核中，在核內活化下游的PKC pathway。此一現象與HEK細胞經不同濃度之葡萄糖處理後之表現相符，也佐證了葡萄糖代謝會引發特定訊息傳導的想法，而且丙酮酸可能是媒介為醣類代謝訊息傳導的關鍵物。同時外加入的丙酮酸，也會活化ERK-MAPK pathway，並可能在核內活化PLC-β1，利用抑制劑，我們確認丙酮酸是同時活化PLC-β1及ERK-MAPK pathway，而非單一的藉由ERK 1/2來活化核內的PLC-β1。當外加丙酮酸時，不論是在正常細胞或過度表現PLC-β1的細胞，都擁有較佳的存活率，而過度表現PLC-β1的細胞又稍高於正常細胞，因此由丙酮酸所誘發的PLC-β1活化路徑，可能對於細胞生存上有著正面意義。

Pyruvate is the end product of glycolysis. It may undergo the fermentation process leading to lactate or ethanol production, or enter the TCA cycle in the mitochondria to produce more energy. Like the other α-keto acids, pyruvate has the antioxidative property in which pyruvate reacts with hydrogen peroxide in a non-enzymatic way yielding acetate, carbon dioxide and water. Pyruvate has been considered as an important regulatory molecule in the carbohydrate metabolism affecting the velocity of glycolysis and TCA cycle by allosteric regulation. Recently many metabolites are considered to exert other physiological functions besides serving as metabolic intermediates. Using Affinity Elution from Tandem Hydrophobic Interaction Chromatography(AETHIC) and MS analysis, we discovered that PLC-β1 may be a pyruvate-binding protein. In addition, pyruvate induces PLC-β1 activation and translocation from cytosol to nucleus in glucose-starved HEK cells, leading to PKC activation. Similar result have been found in starved cells treated with glucose. These data confirmed that glucose-induced signal transduction may be mediated by pyruvate and PLC-β1. Treatment of pyruvate also activates the ERK-MAPK pathway. However we have found that activation of PLC-β1 and ERK are two independent pathways. In the presence of exogenous pyruvate, both normal cells and cells over-expressing PLC-β1 survive better than cells without pyruvate. Therefore, pyruvate serves as a signal molecules in cells with a high flux of glycolysis.