Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Oct 1;311(4):E706-E719.
doi: 10.1152/ajpendo.00237.2016. Epub 2016 Aug 30.

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

Laurent Bultot  1 , Thomas E Jensen  2 , Yu-Chiang Lai  3 , Agnete L B Madsen  2 , Caterina Collodet  4 , Samanta Kviklyte  3 , Maria Deak  1 , Arash Yavari  5 , Marc Foretz  6 , Sahar Ghaffari  5 , Mohamed Bellahcene  5 , Houman Ashrafian  5 , Mark H Rider  3 , Erik A Richter  2 , Kei Sakamoto  7
Affiliations

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

Laurent Bultot et al. Am J Physiol Endocrinol Metab. .

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.

Keywords: 5-aminoimidazole-4-carboxamide riboside; 991; A769662; AMP-activated protein kinase; LKB1; compound 13; ex229.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Validation of AMP-activated protein kinase (AMPK) isoform-specific antibodies for immunoblot (IB) analysis. A and B: IB analysis was performed with 20 μg liver tissue protein extracts from wild-type (WT) and AMPKα1/2 knockout (KO) mice (A) as well as AMPKβ1/β2 KO mice (B) using the indicated antibodies. IP, immunoprecipation. C: FLAG-tagged human AMPKγ1, AMPKγ2, and AMPKγ3 were ectopically expressed in COS1 cells, and cell extracts were generated. All three AMPKγ isoforms were immunoprecipitated with anti-FLAG antibody and immunoblotted with the indicated antibodies. D–F: IB analysis was performed with 20 μg of the indicated tissue extract proteins from WT and isoform-specific AMPKγ KO mice using the indicated antibodies. Gas, gastrocnemius; EDL, extensor digitorum longus; Sol, soleus. G: a panel of mouse tissue extracts (male C57BL/6NTac mice) was immunoblotted with the indicated antibodies. Sk, skeletal PKB, protein kinase B. Representative blots are shown from n = 1–3 mice/tissues.
Fig. 2.
Fig. 2.
Validation of AMPK isoform-specific antibodies for IP. A–E: Endogenous AMPK complexes were immunoprecipitated using the indicated antibodies from 200 μg extract protein from the gastrocnemius muscle (A and C) or liver (B, D, and E) of the indicated AMPK WT or KO mice. IB analysis was performed on the input (10% = 20 μg), the supernatant (SN; 10% = 20 μg), and the immunoprecipitates (IP) with the indicated antibodies. F: summary table of the isoform-specific antibodies used in the present study.
Fig. 3.
Fig. 3.
Effect of the small-molecule AMPK activator 991 on activity of a panel of 139 protein kinases. A: a kinase screen was performed by the International Centre for Kinase Profiling (University of Dundee). Assays were performed in the presence of 1 μM 991. Results are shown in rank order as mean percent activity compared with controls in the absence of compound ± SD. B–E: mouse embryonic fibroblasts were treated with vehicle (0.1% DMSO) or 991 (10 μM) for 1 h (3 independent plates for each condition). Cell lysates were immunoblotted with the indicated antibodies (B). The indicated kinases were immunoprecipitated from cell lysates, and in vitro kinase activity toward substrate peptide was measured (C–E) as described in materials and methods. ACC, acetyl-CoA carboxylase; RAPTOR, regulatory associated protein of mechanistic target of rapamycin; SIK3, salt-inducible kinase. Results are expressed as means ± SD. *P < 0.05.
Fig. 4.
Fig. 4.
991 treatment enhances AMPK activity induced by 5-aminoimidazole-4-carboxamide riboside (AICAR) or C13 in hepatocytes. Hepatocytes were isolated from male C57BL/6NTac mice and cultured overnight as described in materials and methods. Hepatocytes were untreated (control) or treated with AICAR (0.1 mM) or C13 (10 μM) for 1 h in the presence of the indicated concentrations of 991. A: IB analysis was performed with 20 μg lysates using the indicated antibodies. Representative blots are shown; n = 3. Shorter (1 min) and longer (5 min) exposure indicate the exposure time with X-ray film. B--G: AMPK complexes were immunoprecipitated from 50 μg lysates with the indicated antibodies. AMPK activity (in duplicate) was measured in vitro as described in materials and methods. Results are expressed as means ± SE; n = 3. *Significance of AICAR or C13 versus the respective control condition (0, 0.01, or 0.1 μM 991); #significance of 991 versus the respective compound (AICAR or C13) or vehicle. P < 0.05.
Fig. 5.
Fig. 5.
991 treatment enhances AMPK activity induced by AICAR in C2C12 muscle cells. A: IB analysis was performed with 20 μg lysates from C2C12 myoblasts or myotubes with the indicated antibodies. Representative blots are shown; n = 2 for each condition. Creatine kinase was used as a differentiation marker. B: IB analysis was performed with 20 μg lysates from C2C12 myotubes untreated (control) or treated with AICAR (2 mM) in the presence or absence of the indicated dose of 991 for 30 min. Representative blots are shown; n = 3 for each condition. C: schematic representation of the sequential immunoprecipitation protocol for the isolation of AMPKγ3 and AMPKγ1 from tissue lysates (top). AMPKγ3 and AMPKγ1 immune complexes were sequentially immunoprecipitated from 200 μg mouse gastrocnemius muscle lysate protein. IB analysis was then performed on the input (10% = 20 μg), the SN (10% = 20 μg), and the immunoprecipitates (IP) from the first and second immunoprecipitation using the indicated antibodies (bottom). D: AMPKγ3 and AMPKγ1 activity (in duplicate) from 200 μg C2C12 myotube lysate protein were measured after sequential immunoprecipitations as described in materials and methods. Results are expressed as means ± SE; n = 3. *Significance of AICAR versus the respective control condition (0 or 10 μM 991); #significance of 991 versus the respective control (0 or 2 mM AICAR). P < 0.05.
Fig. 6.
Fig. 6.
Dual compound treatment further increases AMPK activity and glucose transport in mouse skeletal muscle compared with single compound treatment. EDL muscles from C57BL/6 mice were isolated and incubated in the presence or absence of the indicated compounds (and concentrations) for 1 h followed by the 2-deoxyglucose (2-DG) transport assay as described in materials and methods. A: IB analysis was performed with 20 μg muscle lysate protein with the indicated antibodies. Representative blots are shown; n = 4. B: 2-DG transport (n = 4). C and D: AMPK complexes were immunoprecipitated from 200 μg muscle lysate protein using either AMPKγ1 (C) or AMPKγ3 (D) antibody. AMPK activity was measured as described in materials and methods. Results are expressed as means ± SE; n = 4. *Significance of AICAR versus the respective control condition (0, 5, or 30 μM 991); #significance between 991 and the respective vehicle condition (0 or 0.3 mM AICAR). P < 0.05.
Fig. 7.
Fig. 7.
991 and contraction additively increase AMPK activity and glucose transport in mouse skeletal muscle. EDL muscles from C57BL/6 mice were isolated and incubated in the presence or absence of 991 (5 μM) for 45 min. Muscles were then either electrically stimulated to induce contraction or further incubated (no electrical stimulation) for 15 min. 2-DG transport was measured during the last 10 min of contraction or incubation as described in materials and methods. A: IB analysis was performed on 20 μg muscle lysate protein with the indicated antibodies. Representative blots are shown; n = 8. Shorter (20 s) and longer (2 min) exposure indicate the exposure time with X-ray film. B: 2-DG transport (n = 8). C and D: AMPK complexes were immunoprecipitated from 200 μg muscle lysate protein using either AMPKγ1 (C) or AMPKγ3 (D) antibody. AMPK activity was measured as described in materials and methods. Results are expressed as means ± SE. *Significance of contraction versus the respective control condition (0 or 5 μM 991); #significance of 991 versus the respective vehicle condition with or without 1-Hz contraction. P < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alessi DR, Sakamoto K, Bayascas JR. LKB1-dependent signaling pathways. Annu Rev Biochem 75: 137–163, 2006. - PubMed
    1. Barnes BR, Marklund S, Steiler TL, Walter M, Hjalm G, Amarger V, Mahlapuu M, Leng Y, Johansson C, Galuska D, Lindgren K, Abrink M, Stapleton D, Zierath JR, Andersson L. The 5′-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle. J Biol Chem 279: 38441–38447, 2004. - PubMed
    1. Birk JB, Wojtaszewski JF. Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle. J Physiol 577: 1021–1032, 2006. - PMC - PubMed
    1. Calabrese MF, Rajamohan F, Harris MS, Caspers NL, Magyar R, Withka JM, Wang H, Borzilleri KA, Sahasrabudhe PV, Hoth LR, Geoghegan KF, Han S, Brown J, Subashi TA, Reyes AR, Frisbie RK, Ward J, Miller RA, Landro JA, Londregan AT, Carpino PA, Cabral S, Smith AC, Conn EL, Cameron KO, Qiu X, Kurumbail RG. Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms. Structure 22: 1161–1172, 2014. - PubMed
    1. Cheung PC, Salt IP, Davies SP, Hardie DG, Carling D. Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem J 346: 659–669, 2000. - PMC - PubMed

Publication types

MeSH terms