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, 14 (2), e0211568
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Pharmacological AMPK Activation Induces Transcriptional Responses Congruent to Exercise in Skeletal and Cardiac Muscle, Adipose Tissues and Liver

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Pharmacological AMPK Activation Induces Transcriptional Responses Congruent to Exercise in Skeletal and Cardiac Muscle, Adipose Tissues and Liver

Eric S Muise et al. PLoS One.

Abstract

Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.

Conflict of interest statement

All authors are or were employees of Merck & Co., Inc., Kenilworth, NJ, USA, and may own shares of company stock. Merck & Co., Inc., Kenilworth, NJ, USA, provisional patent applications for LA1, LA2, SA1 and SA2 and related AMPK activators were filed on 23 February 2012 (WO2012116145; Novel Cyclic Azabenzimidazole derivatives useful as anti-diabetic agents). All of the authors employed by Merck & Co., Inc., Kenilworth, NJ, USA, have a potential conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Structures and pharmacokinetic parameters of LA1, LA2, SA1 and SA2.
Pharmacokinetic studies were performed in 8 week old db/db mice treated with LA1 (10 mg/kg), LA2 (30 mg/kg), SA1 (30 mg/kg), SA2 (20 mg/kg) for 14 days (QD, PO). Blood samples were collected via tail vein at 0, 1, 2, 4, 7, and 24h post dosing on day 14 (n = 8). Cmax, u is the unbound Cmax (when accounting for plasma protein binding). Blood Glucose at Ctrough (%) is the percent change of blood glucose of mice treated with different compounds compared to vehicle at 24h post dose on day 14. Data are represented as mean ± SEM.
Fig 2
Fig 2. Effects of AMPK activators on glucose clearance and glycogen mobilization in lean C57BL/6 mice.
Effects of LA1 (30 mg/kg, PO) and SA1 (30 mg/kg, PO) on glucose tolerance, target engagement and glycogen at 1h and 24h post-dose (n = 8). (A) Blood glucose/time curve in an ipGTT. (B) AUC of the blood glucose/time curve. (C) pACC/ACC ratio in skeletal muscle. (D–E) Glycogen contents of skeletal muscle and heart. Data are represented as mean ± SEM. *p < 0.05, **p < 0. 01, *** p < 0.001 relative to vehicle.
Fig 3
Fig 3. Comparison of pharmacological AMPK activation and exercise in lean C57BL/6 mice (n = 8).
Effects of pharmacological AMPK activation on fasting blood glucose (FBG) and pACC/ACC ratio 1h (A) and 5h (B) post dose (LA2, 3 and 30 mg/kg, PO and SA2, 3 and 20 mg/kg, PO) in 12-week old lean C57BL/6 mice. The effects of 130 min treadmill exercise on pACC/ACC ratio (treadmill exercise at speed of 10 m/min) are also shown in (B); Data are represented as mean ± SEM. *p < 0.05, **p < 0. 01, ***p < 0.001 relative to vehicle. (C) Acute exercise and pharmacological AMPK activation have robust transcriptional effects in heart, skeletal muscle, and liver (n = 5). Tissues were collected 5 h post dose, or at the end of exercise. Shown are the number of probesets (y-axis) that met the fold change cutoffs (x-axis). Only the probesets with FDR_BH (False Discovery Rate Benjamini & Hochberg) p<0.1 were included. (D) For skeletal muscle gene expression profiling, 789 probesets are shown that met the +/- 1.2 fold change and FDR_BH p<0.1 threshold in both the exercise and LA2 (30 mg/kg, PO) groups (indicated by grey dots). The color gradient represents fold change compared to vehicle treated sedentary mice (-2.0 to 2.0 fold). (E) Pathway analysis of heart, skeletal muscle, liver, BAT, and WAT of C57BL/6 mice (1) exercised on treadmill for 130 min; and (2) treated with LA2 (30 mg/kg, PO). White boxes represent changes that did not reach statistical significance (1.2 fold and p<0.05). (F) Fkbp5 was one of six genes (represented by 7 probesets) that were significantly regulated by both acute exercise and acute pharmacological AMPK activation (LA2, 30 mg/kg, PO) in all 5 tissues profiled. Shown in the box plot are the log2_Intensity values per treatment group. (G) Schematic diagram summarizing the pathways regulated by exercise and direct AMPK activation.
Fig 4
Fig 4. Effects of AMPK activators and exercise on glycogen accumulation and mobilization in liver, skeletal muscle, and heart.
Lean mice, sedentary or exercised on treadmill, were treated with vehicle or LA1 (10 mg/kg, PO). (A) Exercise paradigm (n = 8). (B) Effects on plasma glucose. (C) Effects on plasma FFA. (D–F) Effects on glycogen contents of liver, skeletal muscle, and heart. *p < 0.05, **p < 0. 01, *** p < 0.001 relative to vehicle.
Fig 5
Fig 5
Short-acting AMPK activators reduce hyperglycemia, at trough, without inducing cardiac hypertrophy in rodent models of diabetes; db/db (A-E), eDIO (F), and ob/ob (G) mice and fZDF (H) rats. (A) Glucose measured at trough (approximately 24 hours post last dose) on day 12 of 8 week old male db/db mice treated with vehicle, LA1, and SA1 (n = 10). (B–C) pACC/ACC ratio of skeletal muscle at 2h (n = 7) and 7h (n = 3) post dose on day 14. (D) Heart weight of db/db mice on day 14. (E) Heart glycogen content of db/db mice on day 14. (F). Plasma levels of fasting glucose and insulin in 26 week old eDIO mice (average body weight of 48 grams) treated with vehicle or SA2 (3 mg/kg, QD, PO) for 4 weeks (n = 7). (G) Glucose levels of 7 week old ob/ob mice at 24h post compound administration on day 1 and day 7. (H) Glucose levels at 24h post dose (trough) after 21-day treatment of SA2 (1 and 3 mpk, QD, PO) in 8 week old fZDF (n = 10) and lean control rats (n = 6). Data are represented as mean ± SEM. *p < 0.05, **p < 0. 01, *** p < 0.001 relative to vehicle.
Fig 6
Fig 6
Effect of AMPK activators on FAO in db/db mice (A-C) and energy expenditure, respiratory exchange ratio and FAO in lean C57BL/6 mice (D-F, n = 8). (A–C) Plasma level of β-hydroxybutyrate and FAO index determined by D2O-labeled oleate oxidation in 8 week old db/db mice treated with vehicle, LA1 (30 mg/kg), SA2 (30 mg/kg (A), 10–120 mg/kg (B-C)), CPT-1 inhibitor (CPT-1i) (50 mg/kg), and SA2 (120 mg/kg) + CPT-1i (50 mg/kg), all QD, PO. (D) Time curve of whole body energy expenditure in 12 week old lean C57BL/6 mice (average body weight of 25 grams) housed in metabolic cages at baseline and after treatment with vehicle (0.25% MC, 5% Tween-80, 0.02% SDS, 5mM HCl, and 60mg/kg HPMCP polymer) or SA1 at 30 mg/kg (QD, PO indicated by arrows) or mice pair-fed to the SA1 group. (E—F) Respiratory quotient (RQ, or respiratory exchange ratio, RER) in mice after treatment with vehicle or SA1 at 30 mg/kg (QD, PO indicated by arrows) or mice pair-fed to the SA1 group. Data are represented as mean ± SEM. *p < 0.05, **p < 0. 01, *** p < 0.001 relative to vehicle.

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Grant support

This work was fully funded by Merck & Co., Inc., Merck Research Laboratories, Kenilworth, NJ, USA. The strategic goal of Merck & Co., Inc., was to explore the drug development potential of an AMPK activator for the treatment of diabetes mellitus and potentially other metabolic disorders. The studies presented in this manuscript accordingly represent investigations undertaken toward this purpose, to gain preclinical proof of concept that long- and short-acting AMPK activators could influence diabetes mellitus in rodent and murine models of this disorder and further, to gain deeper insight into the mechanisms by which salutary effects were obtained through this mechanism. The investigators listed as authors designed the experiments, carried these out, including analysis and made the decision to prepare and publish the manuscript. Senior leaders within Merck & Co., Inc., specifically within Merck Research Laboratories, did periodically review the progress of the AMPK activator program, and as is customary, assign prioritization to the AMPK program. Additionally, through a standard internal review process, senior leaders within Merck authorized the submission of the manuscript for publication.
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