Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
Internal Medicine Research Unit, Pfizer Global Research and Development, Cambridge, MA, USA.
Mol Metab. 2021 Sep;51:101259. doi: 10.1016/j.molmet.2021.101259. Epub 2021 May 23.
Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK that bind to the allosteric drug and metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. This study aimed to illuminate the mechanisms by which ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e., ADaM-site binding small molecules and the prodrug AICAR.
The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2 as well as whole-body AMPKγ3-deficient mouse models. In vitro complex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by western blotting in muscle lysate. To investigate the transferability of these studies, we treated diet-induced obese mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle.
Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2β2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2β2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscles. In vivo PF739 treatment lowered blood glucose levels and increased muscle AMPKγ1-complex activity 2-fold, while AMPKα2β2γ3 activity was not affected.
ADaM-site binding AMPK activators increase glucose uptake independently of AMPKγ3. Co-incubation with PF739 or 991 and AICAR potentiates the effects on muscle glucose uptake and AMPK activation. In vivo, PF739 lowers blood glucose and selectively activates muscle AMPKγ1-complexes. Collectively, this suggests that pharmacological activation of AMPKγ1-containing complexes in skeletal muscle can increase glucose uptake and can lead to blood glucose lowering.
骨骼肌是降低血糖的药理学干预的一个有吸引力的靶点。通过口服小分子直接激活 AMPK 的全激活剂，这些激活剂与别构药物和代谢物（ADaM）结合，通过在骨骼肌中的作用降低血糖。负责这种效果的分子机制尚未详细描述。本研究旨在阐明 ADaM 结合 AMPK 激活剂增加骨骼肌葡萄糖摄取的机制。此外，我们研究了用两种类型的 AMPK 激活剂即 ADaM 结合小分子和前药 AICAR 同时刺激肌肉的后果。
在从肌肉特异性 AMPKα1α2 切除的 m. extensor digitorum longus（EDL）中，离体研究 ADaM 结合小分子（PF739 和 991）、AICAR 或 PF739 或 991 与 AICAR 共同刺激对肌肉葡萄糖摄取的影响。在肌肉裂解物中通过免疫沉淀测量特定于复合物的 AMPK 活性，并通过 Western blot 评估分子信号。为了研究这些研究的可转移性，我们用 PF739 体内治疗饮食诱导的肥胖小鼠，并测量骨骼肌中特定于复合物的 AMPK 激活。
PF739 或 991 孵育骨骼肌可剂量依赖性地增加骨骼肌葡萄糖摄取。与最大剂量的 AICAR 共同孵育 PF739 或 991 可增加葡萄糖摄取，其程度大于任何单一处理。PF739 或 991 均未将 AMPKα2β2γ3 活性增加到与 AICAR 相同的程度，而共孵育导致 AMPKα2β2γ3 激活的增强作用。在 AMPKγ3 KO 小鼠的肌肉中，AICAR 刺激的葡萄糖摄取被消除。相比之下，PF739 或 991 对葡萄糖摄取的影响在 WT 和 AMPKγ3 KO 肌肉之间没有差异。体内 PF739 处理可降低血糖水平并使肌肉 AMPKγ1 复合物活性增加 2 倍，而 AMPKα2β2γ3 活性不受影响。
ADaM 结合 AMPK 激活剂可独立于 AMPKγ3 增加葡萄糖摄取。与 PF739 或 991 和 AICAR 共孵育可增强对肌肉葡萄糖摄取和 AMPK 激活的作用。在体内，PF739 降低血糖并选择性激活肌肉 AMPKγ1 复合物。总的来说，这表明骨骼肌中 AMPKγ1 包含复合物的药理学激活可以增加葡萄糖摄取并导致血糖降低。
