CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.
CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.
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DOI：
10.1016/j.molmet.2023.101761
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发表时间：
2023-09
影响因子：
8.1
通讯作者：
Sakamoto, Kei
中科院分区：
文献类型：
--
作者：
Negoita, Florentina;Addinsall, Alex B.;Hellberg, Kristina;Bringas, Conchita Fraguas;Hafen, Paul S.;Sermersheim, Tyler J.;Agerholm, Marianne;Lewis, Christopher T. A.;Ahwazi, Danial;Ling, Naomi X. Y.;Larsen, Jeppe K.;Deshmukh, Atul S.;Hossain, Mohammad A.;Oakhill, Jonathan S.;Ochala, Julien;Brault, Jeffrey J.;Sankar, Uma;Drewry, David H.;Scott, John W.;Witczak, Carol A.;Sakamoto, Kei
关键词：
AMP激活的蛋白激酶（AMPK）响应于能量应激（例如收缩）而被激活，并且在调节各种代谢过程（例如骨骼肌中的胰岛素非依赖性葡萄糖摄取）中起重要作用。在骨骼肌中通过α-AMPK Thr 172磷酸化激活AMPK的主要上游激酶是LKB 1，然而一些研究表明Ca 2 +/钙调素依赖性蛋白激酶激酶2（CaMKK 2）作为激活AMPK的替代激酶。我们的目的是确定钙调蛋白激酶2是否参与激活AMPK和促进骨骼肌收缩后的葡萄糖摄取。使用最近开发的CaMKK 2抑制剂（SGC-CAMKK 2 -1）以及结构相关但无活性的化合物（SGC-CAMKK 2 - 1 N）以及CaMKK 2敲除（KO）小鼠。进行体外激酶抑制选择性和功效测定，以及CaMKK抑制剂（STO-609和SGC-CAMKK 2 -1）的细胞抑制功效分析。在用/不用CaMKK抑制剂处理或从野生型（WT）/CaMKK 2 KO小鼠分离的小鼠骨骼肌中，评估收缩（离体）后AMPK的磷酸化和活性。通过qPCR测量小鼠组织中的Camkk 2 mRNA。CaMKK 2蛋白的表达进行了评估，通过免疫印迹与或不事先富集钙调蛋白结合蛋白的骨骼肌提取物，以及通过基于质谱的蛋白质组学的小鼠骨骼肌和C2 C12肌管。STO-609和SGC-CAMKK 2 -1在无细胞和基于细胞的测定中在抑制CaMKK 2方面同样强效和有效，但SGC-CAMKK 2 -1的选择性要高得多。收缩刺激的磷酸化和激活AMPK不受影响与CaMKK抑制剂或在CaMKK 2空肌肉。WT和CaMKK 2 KO肌肉之间的收缩刺激葡萄糖摄取相当。CaMKK抑制剂（STO-609和SGC-CAMKK 2 -1）和无活性化合物（SGC-CAMKK 2 - 1 N）均显著抑制收缩刺激的葡萄糖摄取。SGC-CAMKK 2 -1还抑制由药理学AMPK激活剂或胰岛素诱导的葡萄糖摄取。在小鼠骨骼肌中检测到相对低水平的Camkk 2 mRNA，但在小鼠骨骼肌组织中检测不到CaMKK 2蛋白及其衍生肽。我们证明，药理学抑制或遗传丢失的CaMKK 2不影响收缩刺激的AMPK磷酸化和激活，以及骨骼肌中的葡萄糖摄取。先前观察到的STO-609对AMPK活性和葡萄糖摄取的抑制作用可能是由于脱靶效应。CaMKK 2蛋白在成年鼠骨骼肌中不存在或低于目前可用方法的检测限。在骨骼肌中，CaMKK 2不是AMPK的上游调节剂。与STO-609相比，SGC-CAMKK 2 -1是一种更具选择性的CaMKK抑制剂。CaMKK 2抑制通过脱靶效应减少收缩诱导的葡萄糖摄取。CaMKK 2蛋白在骨骼肌中检测不到。
The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods. CaMKK2 is not an upstream regulator of AMPK in skeletal muscle. SGC-CAMKK2-1 is a more selective CaMKK inhibitor compared to STO-609. CaMKK2 inhibition reduces contraction-induced glucose uptake via off-target effects. CaMKK2 protein is undetectable in skeletal muscle.
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影响因子：
29
作者：
Gowans GJ;Hawley SA;Ross FA;Hardie DG
通讯作者：
Hardie DG
高强度间隔训练重塑了人骨骼肌的蛋白质组和乙酰基组。
影响因子：
7.7
作者：
Hostrup, Morten;Lemminger, Anders Krogh;Stocks, Ben;Gonzalez-Franquesa, Alba;Larsen, Jeppe Kjaergaard;Quesada, Julia Prats;Thomassen, Martin;Weinert, Brian Tate;Bangsbo, Jens;Deshmukh, Atul Shahaji;James, David E.
通讯作者：
James, David E.
影响因子：
7.3
作者：
Eduful BJ;O'Byrne SN;Temme L;Asquith CRM;Liang Y;Picado A;Pilotte JR;Hossain MA;Wells CI;Zuercher WJ;Catta-Preta CMC;Zonzini Ramos P;Santiago AS;Couñago RM;Langendorf CG;Nay K;Oakhill JS;Pulliam TL;Lin C;Awad D;Willson TM;Frigo DE;Scott JW;Drewry DH
通讯作者：
Drewry DH
影响因子：
29
作者：
Blazev, Ronnie;Carl, Christian S.;Parker, Benjamin L.
通讯作者：
Parker, Benjamin L.
DOI：
10.1074/mcp.m114.044222
发表时间：
2015-04
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Deshmukh AS;Murgia M;Nagaraj N;Treebak JT;Cox J;Mann M
通讯作者：
Mann M