Department of Experimental Medicine, Section of Biochemistry, School of Medical and Pharmaceutical Sciences, University of Genova, Viale Benedetto XV 1, 16132, Genova, Italy.
Institute for Macromolecular Studies, National Research Council, Via De Marini 6, 16149, Genova, Italy.
Mol Metab. 2021 Nov;53:101263. doi: 10.1016/j.molmet.2021.101263. Epub 2021 Jun 5.
Abscisic acid (ABA) is a plant hormone also present and active in animals. In mammals, ABA regulates blood glucose levels by stimulating insulin-independent glucose uptake and metabolism in adipocytes and myocytes through its receptor LANCL2. The objective of this study was to investigate whether another member of the LANCL protein family, LANCL1, also behaves as an ABA receptor and, if so, which functional effects are mediated by LANCL1.
ABA binding to human recombinant LANCL1 was explored by equilibrium-binding experiments with [H]ABA, circular dichroism, and surface plasmon resonance. Rat L6 myoblasts overexpressing either LANCL1 or LANCL2, or silenced for the expression of both proteins, were used to investigate the basal and ABA-stimulated transport of a fluorescent glucose analog (NBDG) and the signaling pathway downstream of the LANCL proteins using Western blot and qPCR analysis. Finally, glucose tolerance and sensitivity to ABA were compared in LANCL2 and wild-type (WT) siblings.
Human recombinant LANCL1 binds ABA with a K between 1 and 10 μM, depending on the assay (i.e., in a concentration range that lies between the low and high-affinity ABA binding sites of LANCL2). In L6 myoblasts, LANCL1 and LANCL2 similarly, i) stimulate both basal and ABA-triggered NBDG uptake (4-fold), ii) activate the transcription and protein expression of the glucose transporters GLUT4 and GLUT1 (4-6-fold) and the signaling proteins AMPK/PGC-1α/Sirt1 (2-fold), iii) stimulate mitochondrial respiration (5-fold) and the expression of the skeletal muscle (SM) uncoupling proteins sarcolipin (3-fold) and UCP3 (12-fold). LANCL2 mice have a reduced glucose tolerance compared to WT. They spontaneously overexpress LANCL1 in the SM and respond to chronic ABA treatment (1 μg/kg body weight/day) with an improved glycemia response to glucose load and an increased SM transcription of GLUT4 and GLUT1 (20-fold) of the AMPK/PGC-1α/Sirt1 pathway and sarcolipin, UCP3, and NAMPT (4- to 6-fold).
LANCL1 behaves as an ABA receptor with a somewhat lower affinity for ABA than LANCL2 but with overlapping effector functions: stimulating glucose uptake and the expression of muscle glucose transporters and mitochondrial uncoupling and respiration via the AMPK/PGC-1α/Sirt1 pathway. Receptor redundancy may have been advantageous in animal evolution, given the role of the ABA/LANCL system in the insulin-independent stimulation of cell glucose uptake and energy metabolism.
脱落酸（ABA）是一种植物激素，也存在于动物体内并发挥作用。在哺乳动物中，ABA 通过其受体 LANCL2 刺激脂肪细胞和肌细胞中胰岛素非依赖性的葡萄糖摄取和代谢，从而调节血糖水平。本研究的目的是探讨 LANCL 蛋白家族的另一个成员 LANCL1 是否也作为 ABA 受体发挥作用，如果是，哪些功能效应是由 LANCL1 介导的。
通过 [H]ABA 的平衡结合实验、圆二色性和表面等离子体共振研究了 ABA 与人类重组 LANCL1 的结合。使用过表达 LANCL1 或 LANCL2 的大鼠 L6 成肌细胞，或沉默两种蛋白表达的 L6 成肌细胞，用 Western blot 和 qPCR 分析研究荧光葡萄糖类似物（NBDG）的基础和 ABA 刺激转运以及 LANCL 蛋白下游的信号通路。最后，在 LANCL2 和野生型（WT）兄弟姐妹中比较了葡萄糖耐量和对 ABA 的敏感性。
人重组 LANCL1 与 ABA 结合的 Kd 值在 1 到 10 μM 之间，具体取决于测定方法（即在 LANCL2 的低亲和性和高亲和性 ABA 结合位点之间的浓度范围内）。在 L6 成肌细胞中，LANCL1 和 LANCL2 同样地，i）刺激基础和 ABA 触发的 NBDG 摄取（4 倍），ii）激活葡萄糖转运蛋白 GLUT4 和 GLUT1 的转录和蛋白表达（4-6 倍）和信号蛋白 AMPK/PGC-1α/Sirt1（2 倍），iii）刺激线粒体呼吸（5 倍）和骨骼肌（SM）解偶联蛋白肌浆素（3 倍）和 UCP3（12 倍）的表达。与 WT 相比，LANCL2 小鼠的葡萄糖耐量降低。它们在 SM 中自发过表达 LANCL1，并对慢性 ABA 治疗（1μg/kg 体重/天）做出反应，表现为对葡萄糖负荷的血糖反应改善和 SM 中 GLUT4 和 GLUT1（20 倍）的转录增加，以及 AMPK/PGC-1α/Sirt1 通路和肌浆素、UCP3 和 NAMPT（4-6 倍）的转录增加。
LANCL1 作为 ABA 受体发挥作用，与 LANCL2 相比，其对 ABA 的亲和力略低，但具有重叠的效应功能：通过 AMPK/PGC-1α/Sirt1 通路刺激葡萄糖摄取和肌肉葡萄糖转运体以及线粒体解偶联和呼吸。鉴于 ABA/LANCL 系统在胰岛素非依赖性刺激细胞葡萄糖摄取和能量代谢中的作用，受体冗余在动物进化中可能具有优势。
