Benzimidazole derivative small-molecule 991 enhances AMPK activity and glucose uptake induced by AICAR or contraction in skeletal muscle

Abstract

AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory β (β1 and β2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 μM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle.

AMPK signalling in health and disease

“…Less is known about protein expression of g2 in either human or mouse tissues. In a recent study, however, it was reported that g2 protein was barely detectable in mouse heart, with much higher levels detected in liver, white adipose tissue and brain [18], reflecting reasonably well the previously reported mRNA expression profile in mouse. A possible explanation for the phenotypic differences could simply be due to differences in tissue expression of g2 between human and mouse.…”

AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR

“…3 and Supplemental Table S1). This observation confirms the nearly exclusive specificity of 991 for targeting AMPK in MEFs and hepatocytes and is consistent with the in vitro (cell‐free) observation in our previous study (4). In contrast, AICAR induced a much greater transcriptional response with a majority (~50%) of the transcripts differentially regulated in the absence of AMPK (1026 out of 2053 in MEFs and 754 out of 1718 in primary hepatocytes, respectively) (Fig.…”

AMPK promotes survival of c‐Myc‐positive melanoma cells by suppressing oxidative stress

“…In mammalian cells, there are two genes encoding the AMPKα catalytic subunit (α1, α2), two β genes (β1, β2), and three γ subunits (γ1, γ2, γ3) (Hardie et al , ). As expression of some of these isoforms can be tissue specific (Bultot et al , ), we characterized the expression of the different AMPK isoforms in mM1 and mM2 cells with and without genetic inactivation or pharmacological inhibition of c‐Myc . Protein extracts from mouse liver and skeletal muscle tissues were used as positive controls to account for different cell type‐specific AMPK isoform expression.…”

“…In mammalian cells, there are two genes encoding the AMPKa catalytic subunit (a1, a2), two b genes (b1, b2), and three c subunits (c1, c2, c3) (Hardie et al, 2012). As expression of some of these isoforms can be tissue specific (Bultot et al, 2016), we characterized the expression of the different AMPK isoforms in mM1 and mM2 cells 8 of 20…”