Direct small molecule ADaM-site AMPK activators reveal an AMPKγ3-independent mechanism for blood glucose lowering

Abstract

Objective 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. Methods 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. Results 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. Conclusions 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.

Regulation of yeast Snf1 (AMPK) by a polyhistidine containing pH sensing module

“…This is probably because of the distal part of the PKR and AMPKβ (Snf4 in S . cerevisiae ) forming the (allosteric drug and metabolite) ADaM pocket ( Calabrese et al., 2014 ; Jørgensen et al., 2021 ). This pocket centered around G53 may also explain why the hyperactive Sc Snf1 G53R mutant suppresses snf4 ( Estruch et al., 1992 ).…”

AMPKγ3 Controls Muscle Glucose Uptake in Recovery From Exercise to Recapture Energy Stores

“…AMPKγ3 ( PRKAG3 ) knockout (KO) mice were generated at The Jackson Laboratory (Bar Harbor, ME) using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology as previously described ( 11 ). In short, Cas9 protein with a single guide RNA was used to induce a frameshift deletion in exon 6, resulting in a premature truncation of the PRKAG3 gene and KO of the AMPKγ3 protein.…”

“…Pharmacological activation of AMPK in skeletal muscle increases glucose uptake ( 7 , 8 ), and observations from rodent models and nonhuman primates have confirmed that targeting AMPK in skeletal muscle by selective activators is a viable therapeutic approach to reverse hyperglycemia ( 9 , 10 ). We and others have shown that ADaM-site–binding small-molecule activators of AMPK, including PF739, 991, and MK-8722, increase muscle glucose uptake via an AMPKα-dependent but AMPKγ3-independent mechanism ( 11 , 12 ). In contrast, the nonspecific AMPK activator AICAR (AMP mimetic) has repeatedly been shown to increase muscle glucose uptake via an AMPKγ3-dependent mechanism ( 11 – 13 ).…”

“…We and others have shown that ADaM-site–binding small-molecule activators of AMPK, including PF739, 991, and MK-8722, increase muscle glucose uptake via an AMPKα-dependent but AMPKγ3-independent mechanism ( 11 , 12 ). In contrast, the nonspecific AMPK activator AICAR (AMP mimetic) has repeatedly been shown to increase muscle glucose uptake via an AMPKγ3-dependent mechanism ( 11 – 13 ). Physiologically, AMPK is activated in skeletal muscle during exercise in a time- and intensity-dependent manner ( 14 – 17 ).…”

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

“…Recent in vivo studies have shown that MK-8722, PF-739 and 991 induced skeletal muscle glucose uptake independently of α2β2γ3 [29,30]. This important finding, combined with our previous report showing β2-dependent increases in ex vivo glucose uptake in mouse soleus and extensor digitorum longus (EDL) muscles by the imidazopyridine SC4, implicate α2β2γ1 complexes as the physiological target for ADaM site drugs in skeletal muscle [21,29,30]. Furthermore, SC4 activates all six α2 complexes in vitro but demonstrates poor activation potential against α1β2γ1.…”