Abstract
The small molecule kinase inhibitor SBI-0206965 was originally described as a specific inhibitor of ULK1/2. More recently, it was reported to effectively inhibit AMPK and several studies now report its use as an AMPK inhibitor. Currently, we investigated the specificity of SBI-0206965 in incubated mouse skeletal muscle, measuring the effect on analog 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-stimulated AMPK-dependent glucose transport and insulin-stimulated AMPK-independent glucose uptake. Pre-treatment with 10 µM SBI-0206965 for 50 min potently suppressed AICAR-stimulated glucose transport in both the extensor digitorum longus (EDL) and soleus muscle. This was despite only a modest lowering of AICAR-stimulated AMPK activation measured as ACC2 Ser212, while ULK1/2 Ser555 phosphorylation was prevented. Insulin-stimulated glucose transport was also potently inhibited by SBI-0206965 in soleus. No major changes were observed on insulin-stimulated cell signaling. No general effect of SBI-0206965 on intracellular membrane morphology was observed by transmission electron microscopy. As insulin is known to neither activate AMPK nor require AMPK to stimulate glucose transport, and insulin inhibits ULK1/2 activity, these data strongly suggest that SBI-0206965 has a non-specific off-target inhibitory effect on muscle glucose transport. Thus, SBI-0206965 is not a specific inhibitor of the AMPK/ULK-signaling axis in skeletal muscle, and data generated with this inhibitor must be interpreted with caution.
Highlights
The Unc-51 like autophagy activating kinases (ULK)1/2 are best known as being essential regulators of the initiation of autophagy [1]
We observed a similar effect of SBI-0206965 on insulinDuring our initial testing, we found SBI-0206965 to be an extremely potent inhibitor of stimulated glucose transport
[9].,this this dose chosen forinitial our initial in incubated adult kinase dose andand timetime waswas chosen for our testingtesting in incubated adult mouse mouse skeletal muscle
Summary
The Unc-51 like autophagy activating kinases (ULK)1/2 are best known as being essential regulators of the initiation of autophagy [1]. ULK1/2 are themselves regulated by phosphorylation by upstream kinases AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1. The catabolism-promoting AMPK-complex is canonically activated in response to energy deficiency by an increased AMP/ATP ratio and perhaps AMP-independently by glucose starvation [3], and phosphorylates ULK1/2 on Ser555 to increase its activity [4]. AMPK-dependent phosphorylation of ULK1/2 has been suggested to regulate autophagy in the contexts of exercise and starvation [5,6]. AMPK is a mediator of glucose transport induced by the AMP analog 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or alternative.
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