14 research outputs found
Elucidating the role of Agl in bladder carcinogenesis by generation and characterization of genetically engineered mice
Amylo-\u3b1-1,6-glucosidase,4-\u3b1-glucanotransferase (AGL) is an enzyme primarily responsible for glycogen debranching. Germline mutations lead to glycogen storage disease type III (GSDIII). We recently found AGL to be a tumor suppressor in xenograft models of human bladder cancer (BC) and low levels of AGL expression in BC are associated with poor patient prognosis. However, the impact of low AGL expression on the susceptibility of normal bladder to carcinogenesis is unknown. We address this gap by developing a germline Agl knockout (Agl-/-) mouse that recapitulates biochemical and histological features of GSDIII. Agl-/- mice exposed to N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) had a higher BC incidence compared with wild-type mice (Agl+/+). To determine if the increased BC incidence observed was due to decreased Agl expression in the urothelium specifically, we developed a urothelium-specific conditional Agl knockout (Aglcko) mouse using a Uroplakin II-Cre allele. BBN-induced carcinogenesis experiments repeated in Aglcko mice revealed that Aglcko mice had a higher BC incidence than control (Aglfl/fl) mice. RNA sequencing revealed that tumors from Agl-/- mice had 19 differentially expressed genes compared with control mice. An 'Agl Loss' gene signature was developed and found to successfully stratify normal and tumor samples in two BC patient datasets. These results support the role of AGL loss in promoting carcinogenesis and provide a rationale for evaluating Agl expression levels, or Agl Loss gene signature scores, in normal urothelium of populations at risk of BC development such as older male smokers
Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK
葡萄糖是生物中最基本、最主要的营养物质,它不仅是机体能量的主要来源,也是生物质合成的主要原料。因此,葡萄糖的水平对于生物体是极其重要的。然而,在生活中,体内葡萄糖水平的波动是十分常见的,这是因为我们不可能每时每刻都在摄入葡萄糖:睡一大觉、剧烈运动几个小时或者太忙了没时间吃饭,都会引起葡萄糖水平的显著下降。这时,机体能够触发一套有效的过程应对这类“不利情况”,其中最为关键的就是激活“代谢的核心调节”——AMPK。在葡萄糖水平下降时,被激活的AMPK能够迅速启动脂肪、蛋白质的分解代谢,关闭它们的合成代谢,从而起到维持机体的能量和物质代谢的平衡,弥补机体因葡萄糖不足引起的胁迫压力。那么,机体如何感受葡萄糖水平下降,并“传递”给AMPK使其激活呢?林圣彩教授课题组的这项研究正是发现了生理状态下机体感受葡萄糖水平的机制。通过研究他们发现,无论在不含葡萄糖的细胞培养条件下,还是在饥饿的低血糖的动物体内,都不能观测到AMP水平的上升,这充分说明了机体有一套尚不为人知的、独立于AMP的感应葡萄糖水平的机制。在进一步的研究中他们揭示了这一完整过程:葡萄糖水平下降将引起的葡萄糖代谢中间物——果糖1,6-二磷酸(fructose-1,6-bisphosphate)水平的下降,该过程进一步地被糖酵解通路上的代谢酶——醛缩酶(aldolase)感应,因为醛缩酶正是将含有6个碳原子的果糖1,6-二磷酸裂解成三碳糖的酶,一旦醛缩酶“吃不到”由葡萄糖衍生的果糖1,6-二磷酸,它便“翻脸”,传递给也正是林圣彩教授课题组先前发现的溶酶体途径进而激活AMPK。该过程完全不涉及AMP水平,即能量水平的变化,是一条全新的、完全建立在实际的生理情况上的通路。林圣彩教授进一步地把葡萄糖水平总结为一种“状态信号”,以区别于传统的“能量信号”。据悉,该葡萄糖感知通路的发现对开发用于治疗肥胖症,乃至延长寿命的药物具有深远的意义。【Abstract】The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK)1, but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK2, 3, 4, 5. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation6, 7. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.D.G.H. was supported by an Investigator Award from the Wellcome Trust (097726) and a Programme Grant from Cancer Research UK (C37030/A15101). S.-C.L. was supported by grants from the National Key Research and Development Project of China (2016YFA0502001) and the National Natural Science Foundation of China (#31430094, #31690101, #31571214, #31601152 and #J1310027)