Category: AICAR

The short answer is: YES, you can, but be careful. Some  info below.

There are more and more far east providers of Aicar who offer the substance at fractions of huge worldwide chem manufacturers cost. Most of them has lower purity, check COA and HPLC results.

AICAR is a potent AMPK activator for research. Current price at Sigma-Aldrich for 98% purity Aicar is 300 USD for 25 milligrams!

AICAR ≥98% (HPLC), powder CAS no. 2627-69-2

5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside, Acadesine, N1-(β-D-Ribofuranosyl)-5-aminoimidazole-4-carboxamide

So, how can you buy approximately 1 gram for the same price? – you buy from a smaller, non-worldwide, not so high rated raw chemical supplier THROUGH US.

1000 mg Aicar for $299

There are many peptide and body building sites that sell Aicar in 100 mg doses at around $100. Our price is half of that – but we don’t dilute it into liquid, you get the raw powder – and you can start experimenting with it.

100 mg Aicar for 59 dollars ONLY 

Please note that 100 mg is barely enough for any research if done on mammals. It can be used as a sample or test project on cell cultures. The smallest recommended amount you can test mammals with is 1 gram.


BUY CHEAP GW1516 research material

We are proud to announce that we carry GW1516

This substance drags a lot of attention and currently undergoes a lot of testing. It is a PPAR-β/δ agonist that acts synergistically with exercise to increase running endurance after 4 weeks.

$160 per gram so once again we will bring you the latest research materials at the most affordable prices! Product is shipped in a sealed foil bag.

Researchers at the Salk Institute have shown that agonists of both AMP-activated protein kinase (AMPK ) and a peroxisome proliferator-activated receptor (PPAR) can mimic some of the beneficial effects of exercise in mice. In a treadmill running test, the PPAR-β/δ agonist, GW 1516 (GW 501516), acted synergistically with exercise to increase running endurance after 4 weeks. The AMPK agonist, AICAR, surprisingly enhanced running endurance even in sedentary mice, also after 4 weeks dosing. PPAR-δ and AMPK agonists have the potential to treat diseases such as diabetes, where exercise has been shown to be beneficial and to offer protection against obesity, but also have the more controversial potential to increase endurance in athletes.
Like exercise, AICAR and GW1516 trigger a variety of changes that contribute improved endurance and the ability of muscle cells to burn fat. A phase II clinical trial of GW1516 for the potential treatment of dyslipidemia has been completed.

GW-501516 (also known as GW-501,516, GW1516 or GSK-516) is a PPARδ modulator compound being investigated for drug use by GlaxoSmithKline.[1][2] It activates the same pathways activated through exercise, including PPARδ and AMP-activated protein kinase. It is being investigated as a potential treatment for obesity, diabetes, dyslipidemia and cardiovascular disease.[3][4] GW-501516 has a synergistic effect when combined with AICAR: the combination has been shown to significantly increase exercise endurance in animal studies more than either compound alone. [5][6]

GW-50156 regulates fat burning through a number of widespread mechanisms;[7] it increases glucose uptake in skeletal muscle tissue and increases muscle gene expression, especially genes involved in preferential lipid utilization.[8][9][10] This shift changes the body’s metabolism to favor burning fat for energy instead of carbohydrates or muscle protein, potentially allowing clinical application for obese patients to lose fat effectively without experiencing muscle catabolism or the effects and satiety issues associated with low blood sugar.[11] GW-501516 also increases muscle mass, which improved glucose tolerance and reduced fat mass accumulation even in mice fed a very high fat diet, suggesting that GW-501516 may have a protective effect against obesity [12]

It has been demonstrated at oral doses of 5 mg a day to reverse metabolic abnormalities in obese men with pre-diabetic metabolic syndrome, most likely by stimulating fatty acid oxidation.[13] Treatments with GW-501516 have been shown to increase HDL cholesterol by up to 79% in rhesus monkeys and the compound is now undergoing Phase II trials to improve HDL cholesterol in humans.[14]

Concerns were raised prior to the 2008 Beijing Olympics that GW-501516 could be used by athletes as a performance enhancing drug which was not currently controlled by regulations or detected by standard tests. One of the main researchers from the study on enhanced endurance consequently developed a urine test to detect the drug, and made it available to the International Olympic Committee.[15] The World Anti-Doping Agency has also begun work on a test for GW-501516 and other related PPARδ modulators,[16] and they have been added to the prohibited list from 2009 onwards.[17] The compound has yet to be named a controlled or prohibited substance by any nation’s drug enforcement or regulation agency. To date, no athlete is known to have tested positive for the substance, though the increase in endurance, muscle fiber performance, fat loss and metabolism suggests GW-501516 has the potential for ergogenic use and abuse.




Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR-δ) and the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild-type and PPAR-δ-deficient mice. Administration of telmisartan up-regulated levels of PPAR-δ and phospho-AMPKα in cultured myotubes. However, PPAR-δ gene deficiency completely abolished the telmisartan effect on phospho-AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post-exercise oxygen consumption, and increased slow-twitch skeletal muscle fibres in wild-type mice, but these effects were absent in PPAR-δ-deficient mice. The mechanism is involved in PPAR-δ-mediated stimulation of the AMPK pathway. Compared to the control mice, phospho-AMPKα level in skeletal muscle was up-regulated in mice treated with telmisartan. In contrast, phospho-AMPKα expression in skeletal muscle was unchanged in PPAR-δ-deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR-δ as a potential therapeutic target for the prevention of type 2 diabetes.

© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

[PubMed – indexed for MEDLINE]



The World Antidoping Agency (WADA) has introduced some changes in the 2012 prohibited list. Among the leading innovations to the rules are that both 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (peroxisome proliferator-activated receptor-δ [PPAR-δ]-5′ adenosine monophosphate-activated protein kinase [AMPK] agonist) and GW1516 (PPAR-δ-agonist) are no longer categorized as gene doping substances in the new 2012 prohibited list but as metabolic modulators in the class “Hormone and metabolic modulators.” This may also be valid for the angotensin II receptor blocker telmisartan. It has recently been shown that telmisartan might induce similar biochemical, biological, and metabolic changes (e.g., mitochondrial biogenesis and changes in skeletal muscle fiber type) as those reported for the former call of substances. We suspect that metabolic modulators abuse such as telmisartan might become a tangible threat in sports and should be thereby targeted as an important antidoping issue. The 2012 WADA prohibited list does not provide telmisartan for a potential doping drug, but arguments supporting the consideration to include them among “metabolic modulators” are at hand.

[PubMed – in process]


There are three isoforms of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA, which promotes mitochondrial biogenesis in skeletal muscles. Compared with PGC-1α-a mRNA, PGC-1α-b or PGC-1α-c mRNA is transcribed by a different exon 1 of the PGC-1α gene. In this study, effects of exercise intensity and 5-aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) on isoform-specific expressions of PGC-1α were investigated. All isoforms were increased in proportion to exercise intensity of treadmill running (10–30 m/min for 30 min). Preinjection of β2-adrenergic receptor (AR) antagonist (ICI 118551) inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs, but not the increase in PGC-1α-a mRNA, in response to high-intensity exercise. Although high-intensity exercise activated α2-AMP-activated protein kinase (α2-AMPK) in skeletal muscles, inactivation of α2-AMPK activity did not affect high-intensity exercise-induced mRNA expression of all PGC-1α isoforms, suggesting that activation of α2-AMPK is not mandatory for an increase in PGC-1α mRNA by high-intensity exercise. A single injection in mice of AICAR, an AMPK activator, increased mRNAs of all PGC-1α isoforms. AICAR increased blood catecholamine concentrations, and preinjection of β2-AR antagonist inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs but not the increase in PGC-1α-a mRNA. Direct exposure of epitrochlearis muscle to AICAR increased PGC-1α-a but not the -b isoform. These data indicate that exercise-induced PGC-1α expression was dependent on the intensity of exercise. Exercise or AICAR injection increased PGC-1α-b and PGC-1α-c mRNAs via β2-AR activation, whereas high-intensity exercise increased PGC-1α-a expression by a multiple mechanism in which α2-AMPK is one of the signaling pathways.

peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which was originally identified as a nuclear receptor coactivator, is expressed in brown adipose tissue (BAT), skeletal muscle, heart, kidney, liver, and brain, is markedly upregulated in BAT and skeletal muscle after acute exposure to cold stress, and promotes mitochondrial biogenesis (30). There are three isoforms of PGC-1α mRNA, which promotes mitochondrial biogenesis and angiogenesis in skeletal muscles (6, 25, 46). Compared with PGC-1α-a mRNA (a previously described isoform), PGC-1α-b and PGC-1α-c mRNAs are transcribed by a different exon 1 of the PGC-1α gene and produce slightly smaller-sized proteins. Transgenic mice overexpressing PGC-1α-b or PGC-1α-c in skeletal muscles showed increased gene expression related to mitochondrial biogenesis and fatty acid oxidation (25). In mice, low-intensity exercise (15 m/min for 45 min) or injection of the β2-adrenergic receptor (AR) agonist clenbuterol increased PGC-1α-b and PGC-1α-c mRNA expression more than 40-fold, but not that of PGC-1α-a, in skeletal muscle (25). The increase in PGC-1α mRNA expression was specific to β2-AR; injection of α-, β1-, or β3-AR agonist did not increase PGC-1α mRNA expression (26). Increased expressions in skeletal muscles in response to exercise were inhibited by pretreatment with the β2-AR-specific antagonist ICI 118551 or nonspecific β-AR antagonist propranolol (26). These data indicate that β2-AR activation is a major mechanism in the increase in PGC-1α expression in skeletal muscles and that the increase in PGC-1α mRNAs is isoform specific. However, it is not known which isoform of PGC-1α was increased in response to the high-intensity exercise, by which α2-AMPK was markedly activated (5, 42). Therefore, in the present study, we examined the effects of exercise intensity on increases and contributions of AMPK to isoform-specific increase of PGC-1α mRNA.

5-Aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) is a pharmacological activator of AMPK demonstrating similar effects of exercise for increasing glucose uptake (23) and fatty acid oxidation (27) in isolated skeletal muscles. In vivo, AICAR injection in rodents resulted in an increase in fatty acid oxidation (15), mitochondrial biogenesis (41), PGC-1α mRNA (17), and protein (36). Therefore, we also investigated isoform-specific increases in PGC-1α mRNA in response to AICAR.


Experimental animals.

Eight-week-old male C57BL/6J mice were obtained from Japan SLC (Hamamatsu, Japan). Five-week-old male Wistar rats were obtained from Japan CLEA (Tokyo, Japan). Mice and rats were cared for in accordance with the NIH Guide for the Care and Use of Laboratory Animals and our own institutional guidelines. All animal experiments were conducted with the approval of the National Institute of Health and Nutrition Ethics Committee on Animal Research (No. 1008).

Experimental protocols.

For exercise experiments, mice were subjected to treadmill running at 10 m/min (low-intensity exercise), 20 m/min (medium-intensity exercise), or 30 m/min (high-intensity exercise) for 30 min. For AICAR experiments, mice were injected subcutaneously with 1 g/kg body wt AICAR or the same volume of saline. At 0 h (just after exercise) and at 1, 3, 6, and 24 h after exercise or AICAR injection, skeletal muscles (gastrocnemius) were isolated. For β2-AR antagonist experiments, mice were injected subcutaneously with 10 mg/kg body wt ICI 118551 or the same volume of saline 1 h before exercise or AICAR injection. In all experiments, skeletal muscle was rapidly (within 30–60 s) removed from mice killed by decapitation, frozen immediately in liquid nitrogen, and kept at −80°C.

Measurement of AMPK activity.

Muscles were homogenized in ice-cold lysis buffer containing 50 mM Tris·HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.1% 2-mercaptoethanol, and “Complete Mini EDTA-free” protease inhibitor cocktail (1 tablet per 10 ml; Roche Diagnostics, Mannheim, Germany). Homogenates were centrifuged at 13,000 g for 10 min at 4°C, supernatants were removed, and aliquots were snap-frozen in liquid nitrogen. Muscle lysate protein (0.1 mg) was incubated at 4°C for 30 min with the corresponding antibody conjugated to protein G-Sepharose (GE Healthcare Bio-Science, Uppsala, Sweden). The immunoprecipitates were washed twice with lysis buffer containing 0.5 mM NaCl and twice with buffer A (50 mM Tris·HCl, pH 7.4, 0.1 mM EGTA, and 0.1% 2-mercaptoethanol). Phosphotransferase activity of the immunoprecipitates toward the SAMS peptide was measured as described previously (32). The antibodies against the α1 or α2 catalytic subunits of AMPK (cat. no. 07-350 and 07-363; Upstate Biotechnology, Lake Placid, NY) were purchased.

Transgenic mice.

Transgenic mice were produced expressing a dominant-negative (DN) mutant of α1-AMPK in skeletal muscle (α1-AMPK-DN mice) as previously described (24). Briefly, human skeletal muscle α-actin promoter provided by Drs. E. D. Hardeman and K. Guven (Children’s Medical Research Institute, Westmead, Australia) was used to express α1-AMPK-DN in skeletal muscle. α1-AMPK-DN mice exhibited a 50% reduction in α1-AMPK activity and almost complete loss of α2-AMPK activity in skeletal muscle compared with wild-type littermates (24). Male chimeras (BDF1 background) harboring the α1-AMPK-DN transgene were mated with pure C57BL/6J females to obtain F1 offspring. The heterozygous F1 male offspring from this breeding were then back-crossed with purebred C57BL/6J females to obtain F2 offspring, and this process was continued until the F7 generation of mice was obtained. Heterozygous F7 offspring and wild type, born at the same period were used for the experiments.

Western blot.

Frozen skeletal muscle was powdered under liquid nitrogen and then homogenized in RIPA lysis buffer (Upstate Biotechnology) containing 1 mM sodium orthovanadate, 1 mM sodium fluoride, and “Complete Mini, EDTA-free” protease inhibitor cocktail (1 tablet per 10 ml). After three cycles of freezing and thawing, the supernatant was separated by centrifugation at 20,400 g for 15 min at 4°C. Fifteen micrograms of protein of the supernatant was applied onto SDS-PAGE. Phosphorylated and total acetyl-CoA carboxylase (ACC) protein was measured by Western blotting with anti-phospho-ACC (S79) antibody (cat. no. 3661; Cell Signaling Technology, Beverly, MA) and anti-ACC antibody (cat. no. 3662; Cell Signaling Technology), respectively.

Epitrochlearis preparation.

Male Wistar rats, whose body weights were ∼110 g, were anesthetized with an intraperitoneal injection of pentobarbital sodium, 5 mg/100 g body wt, and the epitrochlearis muscles were dissected out and incubated at 35°C for 5 h in 3 ml of oxygenated Krebs-Henseleit bicarbonate buffer containing 8 mM glucose and 32 mM mannitol (18). The gas phase in the flasks was 95% O2-5% CO2. Epitrochlearis muscles were exposed to 1 μM β2-AR agonist clenbuterol for 5 h or 0.5 mM AICAR for 6 h. After the incubations, muscles were blotted and frozen at −80°C for RNA preparation.

Quantitative real-time RT-PCR.

RNA preparation methods and quantitative real-time RT-PCR were performed as described previously (25). PCR primers to detect all isoforms of PGC-1α (total PGC-1α) were selected to correspond to sequences in the second and third exons of the PGC-1α gene (25). The mouse-specific primer pairs used were as follows: PGC-1α-a forward 5′-GGGACATGTGCAGCCAAGA-3′, reverse, 5′-AAGAGGCTGGTCCTCACCAA-3′; PGC-1α-b forward 5′-GACATGGATGTTGGGATTGTCA-3′, reverse 5′-ACCAACCAGAGCAGCACATTT-3′; PGC-1α-c forward 5′-TGAGTAACCGGAGGCATTCTCT-3′, reverse 5′-TGAGGACCGCTAGCAAGTTTG-3′; 36B4 forward, 5′-GGCCCTGCACTCTCGCTTTC-3′; 36B4 reverse 5′-TGCCAGGACGCGCTTGT-3′. The rat-specific primer pairs used were as follows: PGC-1α-a forward 5′-GGGACATGTGCAGCCAAGA-3′, reverse 5′-AAGAGGCTGGTCCTCACCAA-3′; PGC-1α-b forward 5′-GATATGGATGTCGGGTTTGTCA-3′, reverse 5′-ACCAACCAGAGCAGCACATTT-3′; 36B4 forward 5′-CCTTCCCACTGGCTGAAAAGG-3′, reverse 5′-AGCCGCAGCCGCAAATGCAG-3′. For comparison of the amount of mRNA in each isoform and total PGC-1α, cDNA was amplified by the same primer sets in quantitative real-time RT-PCR. After purification and quantification of the amount, using cDNA from each isoform or total PGC-1α as a standard, we made a standard titration curve of each isoform or total PGC-1α mRNA by quantitative real-time RT-PCR. We then quantified the amount of mRNA in each isoform and total PGC-1α relative to amount of each cDNA. By these means, we were able to compare the amounts of mRNAs derived from each isoform PGC-1α gene (or total PGC-1α). For comparison, relative values of the mean of PGC1α-a mRNA from the control mice are shown in Figs. 15.

Measurements of ZMP contents.

Gastrocnemius was homogenized with 1.0 N perchloric acid and centrifuged at 10,000 g for 15 min. After neutralization of the supernatant with calcium carbonate, ZMP [5-aminoimidazole-4-carboxamide-1β-d-ribofuranosyl 5′-monophosphate (AICAR monophosphate)] concentrations were determined by high-performance liquid chromatogram (Phenomenex Luna 5 μ NH2; mobile phase, sodium phosphate buffer; detector, 260 nm) (33, 39).

Blood glucose and plasma catecholamine.

Blood samples were obtained intravenously under anesthesia, and blood glucose concentration was measured with a glucose analyzer (Glucometer DEx; Bayer Medical, Tokyo, Japan). For catecholamine analysis, plasma samples were separated by centrifugation in the presence of EDTA and snap-frozen at −80°C until analysis. Plasma catecholamine was measured with an automated catecholamine analyzer/combined post-column reactor system HLC-725CA II (Tosoh, Tokyo, Japan).

Statistical analysis.

All values are means ± SE. Data were analyzed by one-way or two-way ANOVA. Where differences were significant, each group was compared with the other by Student’s t-test (JMP 5.1.2; SAS, Cary, NC). Statistical significance was defined as P < 0.05.


Exercise intensity-dependent expression of PGC-1α mRNA isoform.

Total PGC-1α mRNA expression was determined in gastrocnemius obtained at time 0 (just after exercise) and at 1, 3, 6, and 24 h after treadmill running at 10, 20, or 30 m/min for 30 min. PGC-1α expression was not increased just after exercise (0 h), and for each exercise intensity, PGC-1α expression was highest at 1 h after exercise (Fig. 1A). The amounts of these increases were dependent on the intensity of exercise; at 1 h after the exercise, the amount of PGC-1α mRNA was elevated 2.0-, 5.0-, and 7.1-fold after 10, 20, and 30 m/min running, respectively. In the following experiments, PGC-1α mRNA expression was analyzed in gastrocnemius obtained at 1 h after a 30-min exercise, the time showing highest expression. Expression of the three PGC-1α mRNA isoforms was measured using isoform-specific primers by quantitative RT-PCR (Fig. 1B). Although the magnitudes of the response to exercise were different between isoforms, the expressions of each PGC-1α isoform were increased in accordance with the level of exercise intensity. PGC-1α-a was not increased after 10 m/min running but was significantly increased 1.4- and 1.8-fold after 20 and 30 m/min exercise, respectively. In contrast, PGC-1α-b expressions were increased ∼6.0-, 20.0-, and 33.3-fold, and PGC-1α-c expressions were increased 5.5-, 17.7-, and 16.0-fold after 10, 20, and 30 m/min running, respectively.

Fig. 1.

Effects of exercise intensity on peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) isoform mRNAs. A: changes in total PGC-1α mRNA expression in skeletal muscles (gastrocnemius) at time 0 (just after exercise) and at 1, 3, 6, and 24 h after treadmill running at 10, 20, or 30 m/min for 30 min. Control mice were kept sedentary (Sed). Graph shows percentage of total PGC-1α mRNA level in sedentary mice. Values are means ± SE (n = 3–10). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Sed. B: increases in PGC-1α isoform mRNA expression in skeletal muscles (gastrocnemius) 1 h after treadmill running at 10, 20, or 30 m/min for 30 min. Control mice were kept sedentary. Graph shows percentage in PGC-1α isoform mRNA level relative to the PGC-1α-a mRNA level of sedentary mice. Values are means ± SE (n = 3–8). *P < 0.05, **P < 0.01 vs. Sed.

Exercise-activated β2-AR for the expression of PGC-1α mRNA isoform.

In our previous study, an injection of clenbuterol (β2-AR agonist) in mice markedly increased PGC-1α-b and PGC-1α-c mRNAs in skeletal muscles but not PGC-1α-a mRNA (25). Pretreatment with the β2-AR-specific antagonist ICI 118551 or the nonspecific β-AR antagonist propranolol inhibited increases in PGC-1α-b and PGC-1α-c isoforms in response to low-intensity exercise (15 m/min, 45 min) (25). The effect of β-AR blockade on levels of PGC-1α isoform mRNA in response to high-intensity exercise (30 m/min), by which PGC-1α-a mRNA was increased, was examined using a β2-AR-specific antagonist. Increased expression of PGC-1α-b and PGC-1α-c isoforms in response to high-intensity exercise was markedly inhibited by pretreatment with ICI 118551 at 1 h before the exercise, whereas increased PGC-1α-a expression was not inhibited (Fig. 2). The same results were observed by pretreatment with the nonspecific β-AR antagonist propranolol (data not shown).

Fig. 2.

Effects of β2-adrenergic receptor (AR) antagonist injection on PGC-1α isoform mRNA expressions in response to high-intensity exercise. Mice were injected with 10 mg/kg body wt sc ICI 118551 (Pre-β2-AR antagonist). An identical volume of saline was injected as a control (Pre-Saline). At 1 h after injection, 30 m/min treadmill exercise was performed for 30 min (Run). Control mice were kept sedentary (Sed). Skeletal muscles (gastrocnemius) at 1 h after a 30-min exercise were taken from mice. Graph shows percentage of PGC-1α isoform mRNA level relative to PGC-1α-a mRNA level of sedentary mice. Values are means ± SE (n = 5–6). **P < 0.01, ***P < 0.001 vs. Sed., †††P < 0.001 vs. Pre-Saline.

α2-AMPK is not essential for increased expression of PGC-1α isoforms in response to exercise.

Activation of AMPK in skeletal muscles increases fatty acid oxidation and mitochondrial biogenesis (40). AMPK phosphorylates PGC-1α protein directly and activates the promoter activity of PGC-1α (17). Because activation of AMPK was observed after exercise in skeletal muscle (5, 42), AMPK might be involved in increased expression of PGC-1α mRNAs after exercise. Gastrocnemius was obtained just after treadmill running at 10, 20, or 30 m/min for 30 min, and AMPK activity was measured. Although α1-AMPK activity was not altered, α2-AMPK activity was increased 1.6- and 2.0-fold just after 20 and 30 m/min running for 30 min, respectively (Fig. 3A). To examine whether activation of AMPK in response to a single bout of exercise might lead to increased expression of PGC-1α mRNA isoforms, transgenic mice expressing α1-AMPK-DN in skeletal muscle were subjected to treadmill running. Because α1-AMPK-DN mice cannot maintain a 30 m/min running speed for 30 min (24), the intensity of exercise was set at 20 m/min for 30 min. α2-AMPK activity was severely inhibited in the skeletal muscle of the α1-AMPK-DN mice when sedentary and after running at 10 m/min (24). α2-AMPK activity was also inhibited in the skeletal muscle of the α1-AMPK-DN mice just after running at 20 m/min for 30min (Fig. 3B). In contrast, α2-AMPK activity was increased 2.7-fold in wild-type skeletal muscle after running. To determine AMPK activity in vivo, phosphorylation of ACC, one of the substrates for AMPK in vivo, was measured. In α1-AMPK-DN mice after running, although α2-AMPK activity was almost completely inhibited, phosphorylation of ACC was significantly increased. However, the amount of phosphorylated ACC in α1-AMPK-DN mice was significantly low compared with that in wild-type mice not only when the mice were sedentary but also after running. Although α2-AMPK activity was severely inhibited in the skeletal muscle of the α1-AMPK-DN mice, 30 min of exercise at 20 m/min resulted in increased expression of each isoform of PGC-1α mRNA (Fig. 3C).

Fig. 3.

Effects of AMPK on increases in PGC-1α isoform mRNAs in response to a bout of exercise. A: changes in α1- and α2-AMPK subunit activities in skeletal muscle (gastrocnemius) after exercise. Skeletal muscles were obtained just after treadmill running at 10, 20, or 30 m/min for 30 min. Control mice were kept sedentary. AMPK activity was measured in immunoprecipitates. Each value (pmol/min/mg protein), is the mean ± SE of 4–6 mice. ***P < 0.001 vs. Sed. B: changes in α1- and α2-AMPK subunit activities, and phosphorylation of ACC in skeletal muscle (gastrocnemius) from α1-AMPK-DN (dominant-negative) mice (AMPK-DN) and wild-type littermates (Wt) after running at 20 m/min for 30 min. Skeletal muscles were obtained just after treadmill exercise (Run) or during the resting state (Sed). AMPK activity was measured in immunoprecipitates. Each value is the mean ± SE of 5–6 mice. ***P < 0.001 vs. Sed., †P < 0.05 and †††P < 0.001 vs. Wt. Phospho-ACC/total ACC was measured by Western blotting. Each value, expressed as a ratio of the value for sedentary Wt littermates, is mean ± SE (n = 5–6). Typical blots are also shown. **P < 0.01 vs. Sed., †P < 0.05 vs. Wt. C: AMPK-DN and Wt mice were run for 30 min on a treadmill at 20 m/min side by side (Run). At 1 h after a 30-min exercise, skeletal muscles (gastrocnemius) were taken, and the level of PGC-1α mRNA was analyzed. Control mice were kept sedentary. Graph shows percentage in PGC-1α isoform mRNA level relative to PGC-1α-a mRNA level of sedentary mice. Values are means ± SE (n = 6–7). ***P < 0.001 vs. Sed.

AICAR-induced expression of PGC-1α mRNA isoform.

AICAR, an AMPK activator, increased the expression of PGC-1α mRNA in skeletal muscle in in vivo and ex vivo experiments (17, 38). Total PGC-1α mRNA expression was measured in gastrocnemius obtained at 0, 1, 3, 6, and 24 h after injection of 1 g/kg body wt AICAR under feeding conditions. This dose of AICAR decreased blood glucose concentrations but did not cause overt hypoglycemia; blood glucose concentrations at 0, 60, and 180 min after AICAR injection were 219, 87, and 149 mg/dl, respectively. Total PGC-1α expression was not increased at 1 h but did increase 8.7- and 6.9-fold at 3 and 6 h after the injection, respectively (Fig. 4A). In the following experiments of the effects of AICAR on PGC-1α expression, expression of PGC-1α mRNA was measured in gastrocnemius obtained at 3 h, the time showing highest expression, after AICAR injection. Expression of all three PGC-1α isoforms was increased significantly, similar to that after a bout of exercise (Fig. 4B). Pretreatment with β2-AR antagonist at 1 h before AICAR injection inhibited the increased expression of PGC-1α-b and PGC-1α-c isoforms but not that of the PGC-1α-a isoform (Fig. 4B). The increased expression of PGC-1α-b and PGC-1α-c isoforms in response to AICAR injection was also inhibited by pretreatment with the nonspecific β-AR antagonist propranolol (data not shown).

Fig. 4.

Effect of AICAR injection on increases in PGC-1α isoform mRNAs and blood catecholamine concentrations. A: changes in total PGC-1α mRNA expression in skeletal muscles (gastrocnemius) at 1, 3, 6, and 24 h after 1 g/kg body wt AICAR injected sc (AICAR). An identical volume of saline was injected as a control (Saline). Graph shows percentage of total PGC-1α mRNA level vs. that before injection (Pre). Values are means ± SE (n = 3–4). ***P < 0.001 vs. Saline. B: effects of β2-AR antagonist on increased expression of PGC-1α isoforms in response to AICAR. Mice were preinjected sc with 10 mg/kg body wt of ICI 118551 (Pre-β2-AR antagonist). An identical volume of saline was injected as a control (Pre-Saline). At 1 h after injection, 1 g/kg body wt of AICAR (AICAR) or an identical volume of saline (Saline) was injected sc. Skeletal muscles (gastrocnemius) at 3 h after AICAR or saline injections were taken from mice. Graph shows percentage in PGC-1α isoform mRNA level relative to PGC-1α-a mRNA level of sedentary mice. Values are means ± SE (n = 5). ***P < 0.001 vs. Saline, †P < 0.05 and †††P < 0.001 vs. Pre-Saline. C: skeletal muscle ZMP concentration and phosphorylation of ACC after AICAR injection with or without preinjection of β2-AR antagonist. Mice were preinjected sc with 10 mg/kg body wt of ICI 118551 (Pre-β2-AR antagonist). An identical volume of saline was injected as a control (Pre-Saline). At 1 h after injection, 1 g/kg body wt of AICAR or an identical volume of saline was injected sc. Skeletal muscles (gastrocnemius) at 1 h after AICAR or saline injections were taken from mice. Values are means ± SE (n = 5–6). N.D., not detected. **P < 0.01 vs. Saline, †††P < 0.001 vs. Pre-Saline. D: plasma catecholamine concentrations after AICAR injection. Epinephrine and norepinephrine concentrations were measured in plasma obtained before (Pre) and at 1 and 3 h after injection of 1 g/kg body wt of AICAR (AICAR) or an identical volume of saline (Saline). Values are means ± SE (n = 6). *P < 0.05, ***P < 0.001 vs. Saline.

AICAR is an adenosine analog taken up by skeletal muscle and phosphorylated to form ZMP, which stimulates AMPK activity (8). To examine whether the concentration of ZMP in gastrocnemius was affected by preinjection of β2-AR antagonist, measurement of ZMP in gastrocnemius was performed at 1 h after AICAR injection (2 h before a maximum increase in PGC-1α mRNA was noted) (Fig. 4C). The concentration of ZMP in the mice preinjected with β2-AR antagonist was 44% lower than that in the mice preinjected with saline, suggesting that pretreatment of mice with β2-AR antagonist might decrease delivery of AICAR or phosphorylation of AICAR into ZMP in the skeletal muscle. However, phosphorylation of ACC in gastrocnemius was increased at 1 h after AICAR injection up to 3.0- and 3.4-fold with or without preinjection of β2-AR antagonist (Fig. 4C). These data suggested that the amount of ZMP in the skeletal muscle would be enough to activate AMPK in vivo (shown as phosphorylation of ACC) and upregulate PGC-1α-a mRNA expression irrespectively of preinjection of β2-AR antagonist.

The increased expression of PGC-1α-b and PGC-1α-c isoforms in response to AICAR injection might be due to the increased plasma catecholamine concentrations. Plasma epinephrine concentrations measured at 1 and 3 h after AICAR injection were increased 9.2- and 2.5-fold, respectively (Fig. 4D). However, plasma norepinephrine concentration was only slightly increased at 1 h after AICAR injection.

Basal plasma catecholamine concentrations might be higher due to the stress induced by handling of the mice. Indeed, the pretreatment with β2-AR antagonist reduced the expression of mRNAs of PGC-1α-b and PGC-1α-c isoforms by saline injection (Fig. 4B), in which catecholamines may induce. Even though basal catecholamine concentration might be elevated, injection of AICAR markedly increased epinephrine concentration in the plasma much more than in the control sample collected under the same condition.

Direct effects of β2-AR agonist and AICAR on the expression of PGC-1α mRNA isoform.

Incubation of isolated muscle with the β2-AR agonist clenbuterol increased the expression of total PGC-1α mRNA (26). To determine which isoform of PGC-1α mRNA was increased, epitrochlearis muscle isolated from rat was incubated with 1 μM clenbuterol for 5 h at 35°C and then frozen, and expression levels of PGC-1α mRNA isoforms in epitrochlearis were measured later (Fig. 5A). We used rat epitrochlearis because of its larger size and easier manipulation. PGC-1α-c was not expressed in rat skeletal muscle, because the sequence required for splicing to produce PGC-1α-c does not exist in rat DNA. A significant increase in the expression of PGC-1α-b mRNA was observed by the incubation with clenbuterol, whereas clenbuterol treatment did not significantly increase expression of PGC-1α-a mRNA. In contrast, when epitrochlearis muscle was incubated with 0.5 mM AICAR for 6 h at 35°C, a significant increase in the expression of PGC-1α-a mRNA was observed, whereas expression of PGC-1α-b mRNA was unchanged (Fig. 5B). These data indicated that an increase of PGC-1α-a mRNA in response to AICAR injection might be due, at least in part, to a direct effect of AICAR on skeletal muscle, and an increase of PGC-1α-b mRNA in response to clenbuterol injection might be due to a direct effect of β2-AR stimulation. However, in vivo, AICAR injection in mice activates β2-AR by an increase in plasma epinephrine concentration, which leads to increases in PGC-1α-b and PGC-1α-c mRNAs.

Fig. 5.

Ex vivo effects of β2-AR agonist (A) and AICAR (B) treatment on PGC-1α isoform mRNAs. Isolated rat epitrochlearis was exposed for 5 h to 1 μM clenbuterol or for 6 h to 0.5 mM AICAR and frozen for mRNA analysis. An identical volume of saline was used as a control. Graph shows percentage in PGC-1α isoform mRNA level relative to PGC-1α-a mRNA level of saline-treated epitrochlearis. Values are means ± SE (n = 9). *P < 0.05, **P < 0.01 vs. Saline.


It has been reported that there are several molecular pathways to increase PGC-1α expression after exercise. The activation of AMPK, calcium/calmodulin-dependent protein kinase (CaMK), calcineurin A (CnA), and p38 mitogen-activated protein kinase (MAPK) signaling cascades are well-known upstream regulators of PGC-1α expression in skeletal muscle (2, 17, 43). In addition, we have previously shown that β2-AR activation is required for low-intensity exercise-induced PGC-1α expression (26), especially for that of PGC-1α-b and PGC-1α-c (25). In the present study, we found that low-, medium-, and high-intensity exercise and AICAR injection increased PGC-1α-b and PGC-1α-c transcripts via β2-AR activation, whereas high-intensity exercise or AICAR injection increased PGC-1α-a transcripts independently of β2-AR activation (Fig. 6, A and B). PGC-1α-a is transcripted from exon 1a; PGC-1α-b and PGC-1α-c are transcripted from an alternate, exon 1b (25).

Fig. 6.

Proposed model of PGC-1α gene expression in response to different intensity of exercise (A) or AICAR injection (B). Exercise activates the promoter for the expression of exon 1b via the sympathetic nervous system (SNS), and that magnitude is dependent on the intensity of the exercise. Pretreatment with β2-AR antagonist markedly inhibited activation of this promoter. Exon 1a is activated only by high-intensity exercise. α2-AMPK activation was not essential for either signaling. AICAR injection increases epinephrine secretion. Epinephrine activates β2-AR and increases PGC-1α-b and PGC-1α-c mRNA expressions. Also, AICAR increased exon 1a expression via β2-AR-independent pathways.

We show here that high-intensity exercise was required for an increase in PGC-1α-a expression by exercise and that such expression was not inhibited by β2-AR blockade (Fig. 2). During high-intensity exercise, α2-AMPK activity was increased in the skeletal muscle (Fig. 3A). Furthermore, treatment with AICAR, an activator of AMPK, in vivo and ex vivo induced PGC-1α-a expression (Figs. 4B and 5B). Although from these results it appeared that the high-intensity exercise-induced activation of α2-AMPK was required for PGC-1α-a expression in skeletal muscle, the high-intensity exercise-induced expression of PGC-1α-a was observed in α1-AMPK-DN mice (Fig. 3C), which showed a marked decrease in α2-AMPK activity (Fig. 3B), suggesting that other mechanisms rather than activation of α2-AMPK were involved in the exercise-induced expression of PGC-1α-a. Existence of other mechanisms was also suggested by an observation that medium-intensity exercise could induce phosphorylation of ACC substantially in α1-AMPK-DN mice, up to a level similar to that in wild-type mice in the sedentary state (Fig. 3B). Activation of sucrose nonfermenting AMPK-related kinase (SNARK) by exercise is a candidate for other mechanisms. Muscle contraction- and exercise-induced activation of SNARK, one of the AMPK-related kinases, was observed in mouse skeletal muscle, and muscle contraction-induced phosphorylation of AS160 or TBC1D1, which might be necessary for increase in glucose transport in skeletal muscle, was mediated by SNARK (19). AICAR treatment also could phosphorylate AS160 or TBC1D1 (37). Therefore, phosphorylation of ACC and expression of PGC-1α-a might also be increased by not only AMPK but also AMPK-related kinases such as SNARK. Another mechanism is also possible: CaMK might be involved in the expression of PGC-1α-a, because high-intensity exercise-induced phosphorylation was observed not only in AMPK but also in CaMK II, and these phosphorylations did not occur after low-intensity exercise (11). These results suggest that the high-intensity exercise-induced PGC-1α-a expressions are regulated by a redundant mechanism in which α2-AMPK is one of multiple signaling pathways.

In contrast, exercise-induced expression of PGC-1α-b or PGC-1α-c is mediated by β2-AR irrespective of intensity. β-AR activation increases not only in cAMP but also in Ca2+ concentration (28), which was observed in sympathetic nerve activation. In addition to β-AR activation, muscle contraction also increases in Ca2+ concentration during exercise (4, 31). However, only β2-AR-mediated change might be involved in exercise-induced PGC-1α-b and PGC-1a-c expression, because β2-AR blockade remarkably inhibited such expression in the present study (Fig. 2). AICAR-induced expression of PGC-1α-b or PGC-1α-c in vivo was probably due to an increase in the concentration of plasma epinephrine by direct stimulation of the adrenal glands by AICAR, as shown in PC12 cells (12) and/or by indirect stimulation via a reduction of blood glucose.

It is unlikely that inhibition of the exercise-induced expression of PGC-1α-b or PGC-1α-c by β2-AR antagonist injection before the exercise was due to whole body effects including change in sympathetic tone and the cardiovascular system, because incubation of isolated rat skeletal muscle with β2-AR agonist increased in PGC-1α-b mRNA (Fig. 5A). In addition, exercise performance was not impaired in the presence of β2-AR antagonist in our experimental conditions (data not shown). Contracting muscles are known to be exposed to much higher concentrations of catecholamines from the nerve endings than are noncontracting muscles; stretch (0.5 kg tension) increased muscle interstitial norepinephrine concentration 45% in the stretched leg and 7% in the control leg in rat (20). Therefore, it is expected that during exercise contracting mouse muscle is exposed to a higher concentration of norepinephrine and increased PGC-1α-b or PGC-1α-c expression compared with that in noncontracting muscle. Inhibition of exercise-induced expression of PGC-1α-b or PGC-1α-c by β2-AR antagonist was likely due to the direct effect on contracting muscle.

Extensive promoter analysis of PGC-1α-a has been conducted (2, 9, 10, 13, 45). There are NFAT (nuclear factor of activated T cell), myocyte enhancer factor (MEF)2, forkhead box O (FOXO)1, cAMP-responsive element-binding protein (CREB), and activating transcription factor (ATF)2 binding sites in the PGC-1α-a promoter (13). Among these sites, conserved MEF2 binding site and cAMP-responsive element (CRE) sequences regulate PGC-1α-a transcription in response to muscle contraction (1, 3). It was suggested that transcriptional activation of PGC-1α-a is induced by the interaction on the MEF2 binding site and CRE sequences on the PGC-1α-a promoter with regulatory factors such as MEF2, class IIa histone deacetylases (HDACs), ATF2, and CREB (1). AMPK, CaMK, CnA, and p38 MAPK have been reported to modulate these regulatory factors (2, 7, 9, 22, 29, 34, 35, 38, 44). Modulation of these transcription factors and the regulators by these protein kinases and phosphatase might be involved in high-intensity exercise-induced PGC-1α-a expression. Recently, it was reported that the effect of AICAR on transcription activation is mediated by the overlapping GATA/E-box binding site at −495 in the human PGC-1α-a promoter (16). Furthermore, newly synthesized PGC-1α protein can coactivate MEF2 and thus positively regulates its own transcription (13). AMPK phosphorylates PGC-1α protein at Thr177 and Ser538, which is required for the PGC-1α-dependent induction of the PGC-1α promoter (17). These results suggest that high-intensity exercise or AICAR-induced expression of PGC-1α-a might also be regulated by the GATA/E-box binding site and phosphorylation of PGC-1α protein by AMPK.

For exercise-induced expression of PGC-1α-b or PGC-1α-c, β2-AR-mediated regulation of CREB and its binding to the CRE sequence are important. In analysis of the PGC-1α-b promoter, CRE and E-box sequences are required for activation of human PGC-1α-b transcription (6, 46). The E-box sequence may contribute to the muscle-enriched expression, whereas the CRE sequence likely serves as a key target of exercise-initiated signaling pathways and mediates the induction of PGC-1α-b and PGC-1α-c expression in skeletal muscles. Expression of constitutively activated forms of CaMKIV and CnA or forskolin treatment, which increases the cytosolic concentrations of cAMP, increases PGC-1α-b promoter activity depending on the CRE sequence via activation of CREB (46). There is another putative CRE sequence in the PGC-1α-b promoter that is also essential for β2-AR-induced expression (6). In agreement with our in vivo finding, PGC-1α-b promoter is activated by CREB activation, but not by AICAR treatment, in the luciferase assay of C2C12 myoblasts (46).

AICAR stimulates PGC-1α-a expression via exon 1a, whereas β2-AR activation stimulates PGC-1α-b and PGC-1α-c expression via exon 1b. Exercise uses both pathways, but only high-intensity exercise uses exon 1a. It is unclear why PGC-1α-b and PGC-1α-c expression is much more sensitive to β2-AR activation than is PGC-1α-a expression, although both promoters possess a functional CRE sequence. There are two types of CRE sequences, the palindromic and variant CRE sequences. In the promoter of the human α-subunit gene, the variant CRE sequence binds the same proteins as does the palindromic CRE sequence but with lower affinity (14). A palindromic CRE sequence was found in the promoter for PGC-1α-a, and both the variant and palindromic (antisense) CRE sequences were found in the promoters for PGC-1α-b and PGC-1α-c (Fig. 7). These CRE sequences might cause the difference in sensitivity to the β2-AR stimuli. These findings suggest that the molecular mechanism by which exercise induces expression of PGC-1α might be more complex. Further studies of promoter analysis including the physiological role of the PGC-1α isoform are warranted.

Adenosine Monophosphate-Activated Protein Kinase (AMPK) as a New Target for Antidiabetic Drugs: A Review on Metabolic, Pharmacological and Chemical Considerations

Arie Gruzman, Gali Babai, Shlomo Sasson

Department of Pharmacology, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel
Address correspondence to: Shlomo Sasson, e-mail:

Manuscript submitted April 10, 2009; resubmitted May 7, 2009; accepted May 9, 2009.

Keywords: diabetes, hyperglycemia, AMP, antihyperglycemic, drug target, energy metabolism, glucose transport, skeletal muscle, D-xylose


In view of the epidemic nature of type 2 diabetes and the substantial rate of failure of current oral antidiabetic drugs the quest for new therapeutics is intensive. The adenosine monophosphate-activated protein kinase (AMPK) is an important regulatory protein for cellular energy balance and is considered a master switch of glucose and lipid metabolism in various organs, especially in skeletal muscle and liver. In skeletal muscles, AMPK stimulates glucose transport and fatty acid oxidation. In the liver, it augments fatty acid oxidation and decreases glucose output, cholesterol and triglyceride synthesis. These metabolic effects induced by AMPK are associated with lowering blood glucose levels in hyperglycemic individuals. Two classes of oral antihyperglycemic drugs (biguanidines and thiazolidinediones) have been shown to exert some of their therapeutic effects by directly or indirectly activating AMPK. However, side effects and an acquired resistance to these drugs emphasize the need for the development of novel and efficacious AMPK activators. We have recently discovered a new class of hydrophobic D-xylose derivatives that activates AMPK in skeletal muscles in a non insulin-dependent manner. One of these derivatives (2,4;3,5-dibenzylidene-D-xylose-diethyl-dithioacetal) stimulates the rate of hexose transport in skeletal muscle cells by increasing the abundance of glucose transporter-4 (GLUT-4) in the plasma membrane through activation of AMPK. This compound reduces blood glucose levels in diabetic mice and therefore offers a novel strategy of therapeutic intervention strategy in type 2 diabetes. The present review describes various classes of chemically-related compounds that activate AMPK by direct or indirect interactions and discusses their potential for candidate antihyperglycemic drug development.

The structure of the AMPK complex

The enzyme AMP-activated protein kinase (AMPK) is expressed in all eukaryotic cells as a heterotrimeric complex. The heteromeric structure and functions of this enzyme complex have been widely investigated. In this section, we focus on structural elements in this complex relevant for its interaction with synthetic pharmaceuticals.

The AMPK complex combines two regulatory subunits (β, 30 kDa; γ, 38-63 kDa) and a catalytic subunit (α, 63 kDa). These subunits are encoded by different genes and several isoforms of each have been discovered: α1, α2, β1, β2, γ1, γ2 and γ3 [1]. Theoretically, there are 12 possible heterotrimeric combinations of AMPK. Moreover, variable gene splicing of these gene products also contributes to the diversity of the AMPK complex composition. It appears that different types of cells and tissues express distinct combinations of the complex. For instance, human skeletal muscles display three complexes in which the β2 subunit is conserved: α2β2γ1, α1β2γ1 and α2β2γ3 [2]. Among these three complexes, the latter is predominantly associated with exercise-induced AMPK activation in human skeletal muscles, while the function of the other is less defined [3]. The β1 subunit is more ubiquitously expressed and is found in other tissues such as the liver [4].

The first crystal structure of a heterotrimeric AMPK complex was obtained from Saccharomyces cerevisae [5]. However, a full length crystal structure of mammalian AMPK has not yet been obtained. Therefore, structural analyses of the human AMPK subunits are largely based on partial domain structure identification and analysis of interactions among proteolytic truncated fragments of these subunits [6]. Detailed structural analysis of these complexes will enhance the effort to design, synthesize and test novel molecules that interact with distinct AMPK complexes expressed in different cells and organs.

The α subunit of the AMPK complex

This catalytic subunit contains a classical serine/threonine protein kinase domain close to the N-terminal [7]. Free catalytic α subunits are usually inactive due to the presence of an autoinhibitory domain that is located in the center of this subunit. When the α subunit forms a functional complex with the β and γ subunits, this autoinhibitory function no longer blocks the catalytic function. Interestingly, Pang et al. have recently suggested that small molecules can directly relieve this inherent autoinhibition in the α subunit, rendering the complex constitutively active and possibly resistant to intracellular degradation [8]. Although α1 and α2 subunits share similar substrate specificity [9], α2 is typically localized in the nucleus whereas α1 is predominantly found in the cytoplasm [10]. It has been shown in HeLa cells that certain stressful conditions induce α1 isoform translocation to the nucleus [11]. This compartmentalization and subcellular re-distribution may explain some diverse functions attributed to AMPK in cells.

The β subunit of the AMPK complex

The C-terminus of the β subunit contains recognition sequences that interact with the two other AMPK subunits and serves as the scaffold of the heterotrimeric complex. In addition, the central part of the β subunit contains a specific sequence that binds to glycogen particles. It has been proposed that this interaction is closely associated with a tight regulation of glycogen metabolism [12]. A myristoylation site located in the N-terminus of this subunit functions as a molecular switch for reversible membrane binding. The functional significance of this membrane docking has been demonstrated when a point mutation in the myristoylation site resulted in a 4-fold increase in the activity of the AMPK complex [13]. A direct autoinhibitory domain located in the N-terminal part of the β subunit has a critical role in autoregulating the activity of the complex [14]. In addition, the nuclear localization of the AMPK complex has been associated with targeted serine-phosphorylations in the β subunit [13]. These recent findings indicate that in addition to its established scaffold function, the β subunit also has important autoregulatory functions in the AMPK complex.

The γ subunit of the AMPK complex

Four tandem repeats of cystathionine-β-synthase sequences (CBS) that are located in the N-terminus of this subunit form two Bateman domains, which selectively bind adenosine containing molecules, such as AMP or ATP [15, 16]. Upon binding, the phosphate group of AMP/ATP lies in a groove on the surface of the γ subunit and interacts with basic residues of amino acids there [6]. Mutations in these basic amino acids result in several human hereditary metabolic disorders, such as impaired cardiac glycogen storage [17]. The binding of AMP to these Bateman domains activates AMPK, whereas ATP binding antagonizes this process. The two Bateman domains bind two pairs of AMP molecules in a positive cooperative manner, suggesting that the second domain is inaccessible for binding unless two AMP molecules occupy the CBS binding sites in the first Bateman domain. This allosteric interaction increases the sensitivity of the AMPK complex to minute changes in intracellular levels of AMP. In contrast, the binding of ATP molecules to these domains antagonizes activation of the complex. These disparate allosteric modulations in the complex, induced by the binding AMP or ATP, form the molecular basis for the metabolic switch function of AMPK and a plausible target for synthetic activators [16].

Mutations in the hydrophobic patch in the γ subunit (Arg171-Phe179), which interacts with the glycogen binding loop of the β subunits, substantially impede AMP-dependent activation of the AMPK complex [14]. It seems, therefore, that the activation process of the complex is also influenced by the γ subunit. Of interest is the finding that the γ2 subunit confers the complex with the greatest AMP dependency in comparison with the γ3 subunit, which moderately affects the sensitivity to AMP [18].

Activation of the AMPK complex

Among stressful conditions, which deplete ATP stores and increase AMP content in cells, heat shock, hypoxia, hyperosmolarity, glucose deprivation, ischemia and prolonged contraction of skeletal muscles have been studied most intensively [19, 20]. It is, however, important to note that some conditions known to activate AMPK in cultured cells are physiologically extreme. For instance, exaggerated hyperosmolarity or severe glucose deprivation are not compatible with real life situations. AMPK is activated in an AMP-dependent manner by targeted phosphorylation of Thr172 in the α subunit [21, 22]. The binding of AMP to the CBS domain in the γ subunit induces conformational changes in the hetrotrimeric complex that promote the phosphorylation of this Thr172 moiety by several upstream kinases. The dissociation of the AMP molecules is followed by the binding of ATP, which then increases the susceptibility of pThr172 to dephosphorylation by protein phosphatases [16].

The “energy charge hypothesis” assigns AMP regulatory metabolic functions in eukaryotic cells [23]. AMP is a very sensitive metabolic indicator of cellular energy metabolism due to the function of adenylate kinases in cells. These enzymes interconvert two molecules of ADP to AMP and ATP [21]. Because these kinases function at near equilibrium, the AMP:ATP ratio in cells varies as the square of the ADP:ATP ratio. When ATP is consumed or depleted and not adequately replenished, the ratio of ADP:ATP rises and drives the adenylate kinase reaction forward to generate ATP and AMP. The latter then further activates the AMPK pathway to conserve energy stores in cells. When cellular ATP levels are high enough and the generation of AMP is reduced, the former molecule preferentially binds to AMPK and blocks it [23]. Because the affinity of ATP binding to the Bateman domain in the γ subunit is lower than that of AMP, relatively higher concentrations of the former are required to attenuate the function of the complex [24]. The recent finding that only a small fraction of the total amount of cellular AMPK is amenable to AMP activation at any given time suggests that additional regulatory interactions, such as compartmentalization or covalent modifications (e.g., phosphorylation), are also involved in this binding process [6].

The tumor suppressor LKB1 is the major upstream serine/threonine kinase that phosphorylates Thr172 in the α subunit and activates the AMPK complex. Of importance is the finding that the LKB1 complex is not directly activated by AMP, but that the latter induces conformational changes in AMPK rendering it a preferable substrate to LKB1 [24]. The human LKB1 gene encodes a single 433-amino acid protein [25], which includes three structural domains: an N-terminal nuclear localization domain, a central catalytic domain and a C-terminal prenylation site that targets the enzyme to membranes [26]. This enzyme plays an important role during embryogenesis and is involved in epithelial, glial and neuronal cell polarity determination [27]. LKB1-knockout mouse embryos die in uterus due to multiple abnormalities, including neural tube and vascular defects [25]. LKB1 is most active in a complex with two accessory proteins: the STE20-related adaptor (STRAD) and the mouse embryo scaffolding protein (MO25) [24, 28, 29]. The pseudokinase STRAD, which binds ATP but lacks a phosphorylation function, enhances the catalytic function of LKB1. MO25, which interacts with the C-terminus of STRAD, stabilizes the entire LKB1 complex [24]. Interestingly, LKB1, like AMPK, shuttles between the nucleus and the cytoplasm [25, 30]. Ribosomal S6 kinase (RSK)- and protein kinase A (PKA)-induced phosphorylation of Ser431 enhances the association of LKB1 with intracellular membranes [31]. It is assumed that this membrane targeting is important to maintain enzymatic activity.

Although AMPK activation is lost when LKB1 expression is silenced, the basal activity of AMPK remains intact [32]. The ability of the AMPK complex to maintain an intrinsic Thr172-autophosphorylating capacity is independent of the fluctuation in AMP levels and seems to maintain a basal intrinsic activity of the complex that allows cells to respond immediately to energetic challenges [33].

Calmodulin-dependent protein kinase kinases (CaMKKs), the upstream kinases for calmodulin-dependent protein kinase I and IV, can also phosphorylate Thr172 and activate AMPK. CaMKKβ, rather than CaMKKα, is the main isoform of the enzyme that activates AMPK [3436]. Because in many cases intracellular Ca2+ signaling often precedes energy utilization and demand, it is conceivable that CaMKKβ prepares cells for a significant increase of energy demand by activating AMPK [37].

Transforming growth factor-β-activated kinase-1 (TAK1) directly phosphorylates AMPK in yeast [38]. Recently, the mammalian TAK1 homolog (previously known as AMPK-kinase), which also phosphorylates Thr172 in AMPK has been found in various mammalian tissues, including skeletal muscle [3840]. It has been suggested that tumor necrosis factor-α (TNF-α) and transforming growth factor-β activate TAK1 [41]. In addition, 5-aminoimidazole-4-carboxamide-1β-D-ribofuranoside (AICAR) and the biguanide metformin also activate TAK1. This effect of metformin was lost in mice cardiomyocytes following the disruption of TAK1 expression [42]. Further studies will ascertain the physiological function of TAK1 in cellular energy metabolism. Protein phosphatases 2A and 2C (PP2A and PP2C) inactivate AMPK by dephosphorylating pThr172 [33, 43]. The activity of these enzymes is negatively regulated by AMP and free fatty acids [44].

In summary, changes in energy and calcium metabolism in cells underlie the activation process of AMPK. Among the effectors that act in concert to induce Thr172 phosphorylation, LKB1, CaMKKβ and TAK1 seem to be prominent.

Metabolic Functions of AMPK

AMPK phosphorylates serine moieties in target proteins. It mostly interacts with a serine moiety within a 9-amino acid motif. In human, this motif is Φ-Ψ-X-X-S/T-X-X-X-Φ, where Φ, Ψ and X denote hydrophobic, basic or any other amino acid, respectively [4547]. Many of these target proteins regulate key metabolic functions, such as glucose uptake, glycolysis, fatty acid oxidation, cholesterol synthesis, glycogen synthesis, gluconeogenesis, protein synthesis or lipolysis [48]. These various functions inhibit anabolic processes and conserve ATP, on one hand, and stimulate catabolic pathways to produce ATP, on the other [46].

Skeletal muscles and heart

Skeletal muscle mass is the main target for insulin-stimulated glucose disposal, whereas insulin resistance is a major contributing factor to the development type 2 diabetes. AMPK-dependent stimulation of glucose transport in skeletal muscles is independent of insulin, phorbol ester or passive stretch [19, 46]. In contrast, muscle contractions, hypoxia, hyperosmolar shock, mitochondrial uncouplers and electron transport inhibitors activate AMPK due to relative energy depletion [49]. Both hypoxia and mitochondrial uncoupling increase the rate of glucose transport in mouse skeletal muscles but fail to produce a similar response in skeletal muscles bearing a dominant-negative AMPKα2 subunit [50, 51].

The activation of muscle AMPK by exogenous compounds or by contraction recruits GLUT-4 to the plasma membrane and augments the rate of glucose transport in a non-insulin-dependent manner [52, 53]. AMPK-induced translocation of GLUT-4-containing vesicles to the plasma membrane is preceded by the phosphorylation of the protein AS-160 at Thr642. This phosphorylated form of AS-160 releases the vesicle from intracellular storages and allows their recruitment to the plasma membrane [54, 55]. In addition, AMPK upregulates the expression of genes encoding GLUT-4 and hexokinase II and stimulates glycogen synthesis in muscles by allosteric activation of glucose-6-phosphatase-induced activity [5658]. Various studies link the glucose transport stimulatory effect of AMPK in skeletal muscles to the activation of ERK1/2, p38-MAPK, Pyk2, PLD, αPKC and Grb2 [19]. In addition to the GLUT-4 translocation, AMPK also exerts its anabolic function in skeletal muscles by activating two major citric acid cycle enzymes: citrate synthase and succinate dehydrogenase [5961]. AMPK also stimulates glycolysis in cardiomyocytes (and hepatocyes) by activating 6-phosphofructo-2-kinase (PFK2) [62, 63]. AMPK (predominantly complexes with the α2 isoform) has a cardioprotective role in augmenting glucose transport and glycolysis in ischemic hearts [64, 65]. Increased myocardial ischemia injury due to enhanced post ischemic myocardial apoptosis, extended infarct size and worsened cardiac functional recovery were inflicted in mice bearing a dominant negative AMPKα2 in their cardiomyocytes [66]. In non-insulin-sensitive cells that do not express GLUT-4, AMPK increases glucose uptake possibly by activating the ubiquitous GLUT-1 that resides in the plasma membrane [67].

Although the outcome of insulin action and AMPK activation of the glucose transport system in skeletal muscles is similar, the transduction mechanism employed by insulin to recruit GLUT-4 to the plasma membrane is entirely different and independent of that recruited by AMPK. Indeed, when costimulated, both the AMPK- and the insulin-dependent pathway increase the rate of glucose transport in an additive manner [68]. The interactions between these two main pathways that regulate glucose metabolism in skeletal muscles have been investigated [69]. Disruption of the AMPKα2 function in skeletal muscles in mice resulted in glucose intolerance and insulin resistance, lending more support to the hypothesis that in addition to normal insulin function, the AMPKα2 isoform also plays a role in maintaining normal insulin sensitivity and reactivity of muscles [19]. In cardiac muscle, however, insulin antagonizes the activation of AMPK by an Akt/PKB-dependent phosphorylation of Ser485 or Ser491 in AMPK α1 or α2, respectively, which attenuates the phosphorylation of Thr172 by LKB1 [7072].

The observation that physical activity induces translocation of AMPKα2 from the cytoplasm to the nucleus supports the idea that AMPK also regulates transcriptional functions, such as the increased expression of GLUT-4 in exercising muscles [7375]. AMPK phosphorylates histone deacetylase-5 and releases it from a complex with myocyte enhancer factor-2 (MEF2), rendering the latter accessible to threonine residues phosphorylation by p38-MAPK and allowing it to form an active transcription complex of the GLUT-4 gene [76].

The blood glucose lowering effect of exercise in type 2 diabetic patients results from an insulin-independent increased rate of glucose transport and enhanced lipid oxidation in skeletal muscles [58]. Interestingly, exercise and AMPK activation also render skeletal muscles more sensitive to insulin action. Indeed, preincubation of isolated rat skeletal muscles with AICAR enhanced insulin-stimulated glucose transport [77]. However, such effects were not observed in cultured myotubes (C2C12) or primary human muscle cells, in which pretreatment with AICAR did not alter their sensitivity to insulin [78]. AMPK-dependent inactivation of IRS-1 by Ser789 phosphorylation in these cells may explain this phenomenon [79].

It is important to note that contraction-mediated glucose uptake is only partially increased in isolated muscles from AMPKα2 whole body knockout mice [80]. Moreover, the simultaneous silencing of the α1 and α2 subunits in mouse skeletal muscles led to a modest reduction in the rate of glucose uptake in contracting muscles [19]. Such findings support the claim that AMPK is not required for contraction-mediated augmentation of glucose transport in skeletal muscles. In contrast, others have found that inhibition of the AMPK complex with Compound C decreased contraction-stimulated glucose transport in rat skeletal muscles [81]. Therefore, further investigation of the specific role of AMPK in the regulation of the glucose transport system in resting and contracting skeletal muscles during acute exercise or endurance training is required. This may lead to the discovery of new cellular targets for the development of novel drugs that mimic effects of muscle contraction and augment glucose transport in skeletal muscle in non-insulin and non-AMPK-dependent mechanisms.

Reduced AMPK activity in insulin resistance and type 2 diabetes has recently been associated with mitochondrial dysfunction and impaired metabolism of lipids in skeletal muscles [82]. Glucose storage in skeletal muscles serves as an immediate source for ATP production, especially during exercise and short-term fasting. Free fatty acids provide an abundant and long-term stock of energy for peripheral energy demands. Fatty acid oxidation in working muscles produces energy 3-fold greater than comparable carbohydrate resources [83]. Inhibition of acetyl-CoA carboxylase (ACC) by AMPK in skeletal muscles improves β-oxidation of fatty acid in the same mechanism as described below for hepatocytes.

Peroxisome proliferator-activated receptor γ (PPARγ) coactivators-1α and -1β (PGC-1α/β) are critical stimulators of mitochondrial biogenesis in response to various stimuli and stressful conditions, such as different diets or physical activity [84]. Muscle PGC-1α is activated by AMPK and by NAD+-dependent type III deacetylase (SIRT1). It has recently been shown that AMPK indirectly controls SIRT1 activity by increasing NAD+ content. SIRT1, in turn, deacetylases and activates PGC-1α [85, 86]. This introduces a functional crosstalk between two important energy sensors that synchronize energy production in mitochondria.

Adipose tissue

The activation of AMPK in fat tissues leads to decreased lipogenic flux, massive fatty acid oxidation and decreased triglyceride synthesis. Fasting, physical exercise or treatment with β-adrenergic agonists activates AMPK via a cAMP-dependent mechanism. The α1 catalytic subunit is the predominant isoform expressed in adipocytes and is critical for the major effects of the AMPK complex [87]. Not only that AMPK activation in adipocytes just marginally increases glucose uptake, but active AMPK antagonizes the augmenting effects of insulin on GLUT-4-mediated glucose uptake. The mechanism of this phenomenon is not clear, but these findings agree with the view that unlike skeletal muscles, glucose in adipocytes is predominantly utilized anabolically for lipid storage [88].

AMPK also regulates lipolysis in adipocytes by inactivating hormone-sensitive lipase (HSL) by a targeted serine phosphorylation [87, 89]. Normally, receptor-coupled adenylate cyclase increases lipolysis via cAMP-dependent protein kinase-A1, which activates HSL. Interestingly, exposure of adipocytes to AICAR blocks lipolysis, which is induced by this mechanism [90]. Both basal and isoproterenol-stimulated lipolysis were elevated and the antilipolytic effect of AICAR was lost in adipocytes from AMPKα1 knockout mice [91]. It appears that AMPK prevents recycling and release of fatty acids from triglycerides, a process which consumes ATP. AMPKα2 has a direct or indirect role in adipose tissue function since its total deletion in mice resulted in an excessive weight gain upon a high-fat diet, but did not entail glucose intolerance [92].

Activation of AMPK in human adipose tissue leads to an increased expression of adiponectin, which is a potent insulin sensitizer in skeletal muscles [87]. The cAMP response element binding protein (CREB), which is over-activated in adipocytes of obese mice, triggers the expression of the ATF3. The latter is a transcriptional repressor that binds to and inhibits the transcription of the adiponectin and GLUT-4 genes. These interactions have recently been linked to the development of hyperglycemia in type 2 diabetic patients [93]. The peripheral effects of adiponectin may explain the significant contribution of AMPK activating drugs to the prevention and improvement of insulin resistance in obese diabetic patients.


The main function of AMPK in the liver is to augment fatty acid oxidation and to prevent cholesterol and triglycerides biosynthesis. Liver-specific AMPKα2 deletion in mice enhances hepatic lipogenesis, increases plasma triglyceride levels and hepatic glucose production. Conversely, overexpression of AMPKα2 in hepatocytes decreases plasma triglyceride level [94, 95]. AMPK also reduces mRNA content of the sterol regulatory element binding protein-1 (SREBP-1) [96]. Over-function of this factor has been associated with the increased prevalence of dyslipidemia in type 2 diabetes. Activation of AMPK also reduces the cellular content of the mRNA of the carbohydrate responsive element-binding protein (ChREBP). This factor, otherwise, upregulates lipogenesis and therefore plays a key role in inducing of hyperlipidemia in type 2 diabetes patients [97].

Adipose tissue-derived adiponectin also improves lipid metabolism in the liver of diabetic obese mice by decreasing fatty acid biosynthesis and increasing mitochondrial fatty acid oxidation [98]. The beneficial effects of AMPK in the liver are attenuated when adiponectin synthesis and secretion from adipose tissues is decreased in diabetes and obesity [93].

Type 2 diabetes is characterized by fasting hyperglycemia and impaired peripheral glucose utilization. A key contributing factor to these abnormalities is the failure of insulin to suppress gluconeogenesis and hepatic glucose production. Liver-specific AMPKα2-knockout mice develop hyperglycemia and glucose intolerance and this is associated with an increased hepatic glucose production. Conversely, the stimulation of AMPK in wild type mice dramatically reduces hepatic glucose output [99]. The suppression of gluconeogenesis by AMPK results from the inhibition of the transcription of phosphoenol pyruvate carboxy kinase (PEPCK), the key regulatory gluconeogeneic enzyme [57]. In addition, AMPK attenuates the synthesis of cholesterol and glycogen in hepatocytes by deactivation of HMG-CoA reductase and glycogen synthase, respectively. In addition, AMPK downregulates the expression of enzymes that are centrally involved in fatty acid synthesis and gluconeogenesis by inhibiting the transcription factors SREBP-1c, ChREBP and HNF-4α, and by attenuating the activity of transcriptional coactivators, like p300 and TORC2 [46]. For example, when the latter is phosphorylated it forms a cytoplasmic complex with 14-3-3 protein, which inhibits the transcription of gluconeogenic enzymes [100].

AMPK also phosphorylates and deactivates ACC [101], an enzyme that exists as two isoforms: ACC1 (cytoplasmic) and ACC2 (predominantly mitochondrial) [102]. The inhibition of the former reduces fatty acid synthesis in cells. Malonyl-CoA, the product of ACC2, is a potent blocker of carnitine palmitoyltransferase-1 (CPT1), which transports long chain fatty acids to mitochondria. When ACC2 is inhibited, the flux of these fatty acids to mitochondria and their oxidation is increased. In addition, AMPK directly stimulates free fatty acid uptake to cells by translocating the fatty acid translocase CD38 to the plasma membrane [46].


Low glucose levels activate AMPK in β-cells. Overexpression of wild type AMPK or constitutively active AMPK, or its pharmacological activation attenuate glucose-induced insulin secretion, whereas the expression of a dominant-negative AMPK in cultured β-cells increases it [103]. It has been suggested that the biguanide metformin affects insulin secretion in β-cells by activating AMPK [104, 105].

Unlike peripheral tissues like skeletal muscle, which predominantly express the α2 subunit of AMPK, the α1 subunit is more abundant in rodent β-cells than the α2 subunit. The former is found most frequently in the cytoplasm, while the latter is distributed between the cytoplasm and the nuclear compartment. It has been proposed that the AMPKα1 complex participates in the electrochemical activity of the cells, whereas the nuclear AMPKα2 complex is involved in regulating gene transcription. Interestingly, neutralization of the α2 subunit in β-cells enhances the transcription of the preproinsulin gene [106]. Therefore, it seems that the transcriptional regulation of AMPKα2 in β-cells greatly differs from the transcriptional regulation it mediates in skeletal muscles [76].

Recent studies on the role of AMPK in the regulation of insulin secretion in β-cells have associated it with the mTOR pathway, energy availability, protein synthesis, cell growth and apoptosis [69]. Collectively, these results demonstrate the complexity of AMPK function in regulating insulin secretion in β-cells and the need for thorough investigations of the complexity of such direct and/or indirect interactions.

Non-metabolic functions of AMPK

AMPK is involved in numerous cellular and physiological interactions not directly related to metabolic homeostasis or diabetes. It is clear that non-specific and non-tissue/cell targeted AMPK activators for the treatment of diabetes and metabolic disorders may also affect these processes and produce diverse side effects. The following is a short summary of such critical functions of AMPK.

An important cross-talk between AMPK and endothelial NO synthase (eNOS) regulates vascular endothelial cell dilatory functions. The AMPK activity in vascular endothelial cells is controlled directly by AMP and indirectly by nitric oxide, the product of eNOS. Following the activation of soluble guanylate cyclase by NO, the former activates CaMKK, a positive upstream activator of AMPK [107]. In a positive feedback loop, AMPKα1 complex phosphorylates eNOS at Ser1177 in an Akt/PKB-dependent manner and increases NO production [108]. This reciprocal and positive feedback mechanism integrates stressful stimuli (e.g., hypoxia) and metabolic (e.g., hypoglycemia) signals to maintain an adequate circulation in critical organs.

AMPK inhibits DNA replication in various cell lines by causing a G1/S-phase cell cycle arrest. This phenomenon is accompanied with the accumulation of tumor suppressor factor p53 and the cyclin-dependent kinase inhibitors p21 and p27 [46]. Another mechanism that may explain some antiproliferative effects of AMPK is the targeting of the RNA-binding protein HuR to the nucleus, where it interacts with and decreases the half-life of mRNAs encoding p21, cyclin A, and cyclin B1 [109]. AMPK has also been implicated in downregulating protein synthesis in cells by inhibiting mammalian target of rapamycin (mTOR) [79], and by phosphorylating the elongation factor-2 kinase, which than inhibits the elongation step [110]. A rare familial deactivating mutation in LKB1 has been linked to the development of hamartomas in humans (Cowden syndrome, Peutz-Jeghers syndrome and tuberose sclerosis). Despite the abnormal growth, cells in these hamartomas retain a normal differentiated state. Interestingly, some reports claim that diabetic patients treated with metformin have a decreased risk of cancer-related mortality than patients treated with other antidiabetic drugs or insulin. These beneficial effects of metformin have been attributed to the activation of the AMPK pathway [111]. These and other findings have initiated a great interest in the potential antitumorgenic activity of AMPK-targeted drugs [112].

Critical functions of primary immune cells, such as chemotaxis and cytokine secretion, are also regulated by AMPK [113]. In fact, the stimulation of AMPK in macrophages from diabetic mice was reduced in comparison with normal macrophages. This perhaps underlies the impeded functional capacity (i.e., reduced macropinocytosis) of the former cells in diabetes [114].

It has recently been shown that AMPK also participates in the regulation of appetite and food intake. Constitutive activation of AMPK in the mouse hypothalamus increases significantly food uptake and body weight, whereas its inhibition has the opposite effects [115, 116]. Noteworthy are the observations that some drugs and factors have opposite effects on hypothalamic and peripheral AMPK complexes. For example, leptin and metformin activate AMPK in skeletal muscles while inhibiting it in the hypothalamus. In contrast, ghrelin and cannabionoids stimulate AMPK in the hypothalamus, but inhibit it in the liver and adipose tissues [117]. These disparate functions are not well-investigated, but seem to be related to diverse heteromeric structures of AMPK complexes in various regions of the central nervous system and in peripheral tissues.

An interesting relationship has been found between AMPK and the cystic fibrosis transmembranal conductance regulator (CFTR). Both proteins are colocalized in the apical membrane of lung secretory epithelial cells. AMPK phosphorylates CFTR at two serine moieties that maintain the chloride channel closed. Therefore, systemic activation of AMPK may have detrimental effects whereas site-specific pulmonary inhibition of AMPK might be considered therapeutically relevant for the treatment of cystic fibrosis. The potential of such treatments with AMPK disruptors or inhibitors in the lung remains to be elucidated [118].

Figure 1 depicts the three main kinases that activate AMPK and its multiple downstream targets, classified according to metabolic, cellular and physiological functions.

In summary, the heterotrimeric structure of the AMPK complex does not only differ among tissues and organs, its activation results in an array of cellular and metabolic responses. Therefore, global activators of AMPK aimed at producing favorable therapeutic effects may exert undesirable side effects due to peripheral and central interactions. This leads to the conclusion that more specific drugs need to be developed. Drugs targeted chemically or by way of administration to specific organs expressing distinct AMPK hetrotrimeric complexes present a potential solution to a rational AMPK-targeted therapy.

Figure 1. Metabolic pathways and functions regulated by AMPK. AMPK can be activated directly by three kinases, LKB1, TAK1 and CaMKK. When activated in various tissues and organs different AMPK complexes mediate a variety of cellular and physiological responses by activating cell-specific targets (e.g., enzymes, transcription factors and docking proteins). Major effects of AMPK activations are metabolic (carbohydrate and lipid metabolism), appetite regulation, cell growth and differentiation, vascular function (blood flow) and basic cellular functions (chloride ion transport). Abbreviations: ACC: acetyl-Co-A carboxylase. AS-160: Akt substrate of 160 kDa. CDKIp21&27: cyclin-dependent kinase inhibitors p21 and p27. CFTR: cystic fibrosis transmembrane conductance regulator. ChREBP: carbohydrate responsive element binding protein. CK: creatine kinase. CS: citrate synthase. EF-2-K: elongation factor 2 kinase. eNOS: endothelial nitric oxide synthase. ERK: extracellular signal-regulated kinases. GPAT: glycerol-3-phosphate acyltransferase. GRB2: growth factor receptor-bound protein 2. GS: glycogen synthase. HDAC25: histone deacetylase 25. HSL: hormone sensitive lipase. HMG-CoA reductase: 3-hydroxy-3-methyl-glutaryl-CoA reductase. HNF4-α: hepatocyte nuclear factor 4α. IRS-1: insulin receptor substrate 1. MCD: malonyl-CoA decarboxylase. p38: p38 mitogen-activated protein kinase. PEPCK: phosphoenolpyruvate carboxy kinase. 6-PF-2-Kinase: 6-phosphofructo-2-kinase. PGC-1α: peroxisome proliferator-activated receptor-γ-coactivator-1α. PYK2: proline-rich tyrosine kinase 2. SD: succinate dehydrogenase. SREBP-1: sterol regulatory element binding protein. TORC2: target of rapamycin complex 2.

Chemical activators of AMPK

The search for novel AMPK activators by rational drug design, screening of vast chemical libraries and testing of various plant extracts has produced numerous reports on new compounds. In the following section, we discuss and classify a collection of such drugs and compounds that activate AMPK. We make the classification according to chemical structures and function.

Metformin and thiazolidinediones

Two groups of common antidiabetic drugs, biguanides and thiazolidinediones (TZD), are believed to mediate part of their effects through AMPK activation. Biguanides, and specifically metformin, inhibit hepatic gluconeogenesis and augment the rate of glucose uptake in skeletal muscles [119]. Some of these effects have been attributed to the activation of AMPKα2 in skeletal muscles. However, the pharmacological significance of this mechanism in diabetic patients is questionable because the required effective concentration of metformin in vitro was as high as 0.5 mM, whereas the plasma effective concentration of the drug in man is at least one order of magnitude lower [96]. Since metformin is excreted in the urine unmetabolized, no active metabolites of this drug can be implicated in its action in skeletal muscles and other tissues [120]. Metformin does not induce LKB1 phosphorylation and activity in skeletal muscles, it fails to increase calcium influx and it does not alter protein phosphatase activity [121]. The main antidiabetic effect of metformin is attributed to hepatic activation of AMPK followed by the inhibition of gluconeogenesis.

Pharmacokinetic data on metformin also supports the hypothesis that the liver is the main target for this drug. Orally administered metformin is effectively absorbed from the gastrointestinal tract to the portal vein. Thus, due to this first-pass effect, the liver is exposed to high concentration of the drug. Therefore, in contrast to skeletal muscle, the effect of metformin in the liver is mediated by LKB1 as the specific hepatic knockout of LKB1 in diabetic mice completely rendered them insensitive to metformin [116]. Of concern is a recent report that links metformin-induced activation of AMPK to an increased biogenesis of Alzheimer’s amyloid in mice brains [122]. Therefore, novel AMPK-activating drugs devoid of such serious side effects are needed.

The second class of antidiabetic drugs that stimulate AMPK is thiazolidinediones (TZD). Members of this group that bind to and activate PPARγ improve insulin sensitivity in various peripheral tissues. TZD, such as rosiglitazone and pioglitazone, downregulate lipolysis and reduce the content of fatty acids in adipocytes [123]. These drugs also inhibit the release of several adipokines from adipose cells, such as tumor necrosis factor α (TNF-α), interleukin-6 and resistin, which induce muscle insulin resistance. Concomitantly, TZD augment the secretion of the insulin-sensitizing factor adiponectin from adipose tissue in man and rodents [124]. Adiponectin stimulates glucose uptake and fatty acid oxidation in skeletal muscles and inhibits gluconeogenesis in the liver by activating AMPK [98]. Expression of a dominant negative AMPKα1 mutant in adipocytes or treatments with synthetic inhibitors of AMPK abolished these effects of adiponectin [125]. These findings indicate that AMPK also plays a key role in mediating hepatic metabolic effects of adiponectin.

It has also been reported that the skeletal muscle levels of several oxidative phosphorylation enzymes and of PGC-1 were increased in diabetic patients treated with TZD over 6 months [126]. PGC-1 regulates the expression of several genes that are involved in mitochondrial bioenergetics [127]. Moreover, TZD also increase the expression of super oxide dismutase-2 (SOD2) and quinone oxidoreductase-1 (NQO1), providing an efficient antioxidant defense against elevated levels of reactive oxygen species [128]. Importantly, in vitro experiments suggest that TZD-dependent activation of AMPK-dependent pathways in insulin sensitive tissue is not exclusively mediated by adiponectin. Finally, TZD effects were recorded in adipocytes in which PPARγ expression was blocked [129]. It has recently been reported that the TZD derivative BLX-1002, which does not bind to PPARγ, stimulates insulin secretion from isolated normal and diabetic pancreatic islets or dispersed β-cells only upon incubation under high glucose. Part of this effect has been attributed to the activation of AMPK in β-cells [130].

D-xylose and lipophilic D-xylose derivatives

A decade ago Winder et al., introduced AMPK as a target for developing novel drugs for the treatment of type 2 diabetes [131]. Dozens of molecules that activate AMPK, directly or indirectly, were synthesized or extracted from plants. In the course of our work, we found that the pentose D-xylose augmented the rate of glucose transport in L6 and human myotubes under high glucose conditions by activating AMPK [132]. Because this effect of D-xylose was obtained at very high concentrations (10-20 mM), we synthesized more potent lipophilic derivatives of D-xylose. Among these, three highly lipophilic compounds 2,4;3,5-dibenzylidene-D-xylose-diethyl-dithioacetal (Compound 19), 2,4-benzylidene-D-xylose-diethyl-dithioacetal (Compound 21) and 2,4-benzylidene-D-xylose-3-O-methyl-diethyl-dithioacetal (Compound 24) exerted significant glucose transport stimulatory effects at low concentrations (5-100 μM) in rat and human cultured myotubes (Table 1). These effects were decreased in the presence of the AMPK inhibitor Compound C. All three derivatives and the parent compound, D-xylose, induced Thr172 phosphorylation of AMPKα and of Thr642 in the downstream substrate AS-160 (Figure 2 and Figure 3). Our study also shows that the inhibition of the insulin transduction pathway by wortmannin and an AKT inhibitor did not interfere with the glucose transport stimulatory function of these derivatives. The exact mechanism of action of these compounds is still under investigation. Of major importance is the finding that Compound 19 significantly reduced blood glucose levels towards the normoglycemic range in streptozotocin-diabetic mice and in the genetically diabetic KKAy mice. Table 1 depicts the structures of these compounds and their minimal effective concentrations in augmenting the rate of glucose transport in L6 myotubes and in lowering blood glucose levels in streptozotocin-diabetic C57/Black mice. These findings point to the potential of these compounds in the development of novel and long-acting antihyperglycemic compounds.

Table 1. Structure and function of the D-xylose derivative compounds 19, 21 and 24

Zoom (133KB)

Legend: The table shows the structures of three compounds and their minimal effective concentrations (MEC) required to augment the rate of hexose uptake in cultured L6 myotubes and to reduce the blood glucose levels towards normoglycemia in STZ-diabetic C57/black male mice. Mice were injected subcutaneously twice daily, for 5 days, with 50 mg/kg of each compound dispersed in sesame oil. N/D: not determined.

Figure 2. D-Xylose and Compounds 19, 21 and 24 activate AMPK. A: L6 rat myotube cultures were washed and received fresh medium supplemented with 2% (v/v) FCS, 23.0 mM D-glucose supplemented with 20 mM of D-xylose (D-xyl), 5 μM of Compound 19, 150 μM of Compound 21 or 50 μM of Compound 24. These compounds were present in the medium for 40 min, 12 h, 30 min and 2 h, respectively. Control myotubes received the vehicle (V) only. AICAR (4 mM), 100 nM of insulin (Ins) and 0.25 M of D-sorbitol (S) were present for 1h, 20 min and 30 min, respectively. Whole cell lysates were prepared and Western blot analyses were performed with antibodies against AMPKα and pThr172-AMPKα. B: Human myotubes were treated as described above and taken for Western blot analysis of AMPKα and pThr172-AMPKα. Representative blot and a summary of n = 3 (* p < 0.05) in comparison with the respective controls. Reproduced with permission from [135].

Figure 3. D-Xylose and Compounds 19, 21 and 24 activate AS160. Whole cell content of AS160 and pThr642-AS160 was determined by Western blot analysis in samples that were prepared from L6 myotubes, as described in the legend to Figure 1. Representative blot and a summary of n = 3 (*< p < 0.05) in comparison the respective controls. Reproduced with permission from [135].

These three benzylidene derivatives share some structural similarities to other known activators of AMPK. Figure 4 shows compounds that contain three to five aromatic and non-aromatic rings, connected to each other directly or with short linkers. Some of these rings contain heteroatoms, such as oxygen and nitrogen. For example, berberine, an isoquinoline alkaloid derived from Hydrastic Canadensis, and its synthetic derivative dihydroberberine are claimed to be effective antilipogenic and hypoglycemic agents in rodents [133, 134]. Some studies have shown that both are indirect activators of AMPK, probably by inhibiting complex 1 in mitochondria [134, 135]. The furancarboxylic acid derivative compound D942 has also been found to activate AMPK indirectly, due to its ability to bind to NAD(P)H dehydrogenase and attenuate the function of complex 1 [136].

Figure 4. Molecular structures of AMPK activators sharing structural similarities to the D-xylose derivatives, Compounds 19, 21 and 24.


Cilostazol is a selective inhibitor of phosphodiesterase-3 (PDE3) that shares structural similarities with the compounds shown in Table 1 and Figure 4. One of the striking effects of this compound is the enhancement of Thr172 phosphorylation and activity of AMPK in human umbilical vein endothelial cells. This activation is followed by downstream phosphorylations of ACC and eNOS. Cilostazol restores vascular endothelial cell function in diabetic rats by PDE3 inhibition [137]. In a study on the effects of cilostazol on thrombospondin-1 expression in hearts of streptozotocin-diabetic rats, the investigators reported that a 4-week treatment reduced blood glucose levels significantly from 22.1 to 16.7 mM [138]. Others have shown that cilostazol ameliorates metabolic abnormalities in diabetic mice or rats via activation of PPARγ and the suppression of inflammatory markers [139]. This drug also improved arterial compliance in patients with peripheral arterial disease while also improving their lipid profile [140]. Further clinical observations and trials will determine the efficacy of such auxiliary antidiabetic effects this drug may possess.

Recently, Zhou et al. reported the structure of two patented polycyclic activators of AMPK that may belong to the group presented in Figure 4 [141]. Compound 1 (patent WO2006/071095) increased AMPK activity 6-fold via an unknown mechanism [142]. Compound 1 (patent WO2008/083124) activated AMPK indirectly. It has been suggested that this effect is mediated via adiponectin receptors. It remains to be investigated whether these compounds also affect the mitochondrial complex 1 [143].


It has been assumed that phytoestrogens could reduce diabetic complications by improving glucose homeostasis and insulin resistance. For example, antilipogenic effects of the phytoestrogen genistein (Figure 5) and its capacity to decrease adiposity were related to the activation of AMPK [144]. Genistein, quercetin, isoginkgetin and epigallocathechin-3-gallate (Figure 5), contain isoflavone and isoflavone-like moieties in their structures. They activate AMPK in 3T3-L1 preadipocytes and adipocytes, adiposarcoma cells and primary mouse hepatocytes, respectively [145]. The activation of AMPK by epigallocathechin-3-gallate seems to be mediated by CaMKK [146]. Interestingly, the antioxidant properties of the gallic acid polyphenol moiety in epigallocathechin-3-gallate may also contribute to the antidiabetic effects of this compound [147]. The mechanism of AMPK activation of the other compounds is not yet clear.

Figure 5. Molecular structures of phytoestrogens that interact with AMPK.


Momordicosides, such as maslinic acid, cucurbitane and ginsenosides represent another class of natural compounds that activate AMPK and may also possess antidiabetic properties (Figure 6). The main natural source of maslinic acid is olive’s skin; the rest are extracted from Momordica charantia (bitter melon) [148, 149]. In addition to the four ring triterpenoidic structure, the majority of momordicosides contain one or more sugar moieties bound to the ring structure via glycoside bonds. The most abundant sugar moieties are β-D-glucopyranoside, β-D-allopyranoside and β-D-xylopyranoside. Several studies indicate that these compounds increased the rate of glucose transport and induce GLUT-4 translocation to the plasma membrane in L6 or C2C12 myotubes and in 3T3-L1 adipocytes by activating AMPK. When tested in vivo, some momordicosides enhanced fatty acid oxidation and glucose disposal during glucose tolerance tests in insulin-sensitive or insulin-resistant mice or augmented glucose-stimulated insulin secretion in mice [149, 150]. In vitro studies suggest that ginsenosides increase AMPK phosphorylation and activity in 3T3-L1 cells; however, the relevant molecular interactions have not yet been ascertained [151].

Figure 6. Molecular structures of momordicosides representing a class of natural compounds that activate AMPK.


Another large group of AMPK activators have one or two phenol, polyphenol or phenolmethyl ether moieties in their structures (Figure 7). The majority of these compounds have been extracted from plants. For example, capsaicinoids, a group of the natural compounds extracted from hot peppers, have been reported to promote fatty acid oxidation and decrease body fat accumulation in diabetic mice. The synthetic structural ester isomer isodihydrocapsiate activates LKB1 both in vitro and in vivo [152]. This substance also increases glucose uptake in L6 myotubes and when administered orally to diabetic mice it substantially reduces blood glucose. A recent report indicated that salidroside, the active ingredient purified from Rhodiola Rosea, stimulated glucose transport and enhanced insulin sensitivity in L6 myotubes and 3T3-L1 adipocytes. The inhibition of AMPK activity by compound C completely abolished these effects of salidroside in L6 myotubes. This suggests that this compound exerts its effects by activating AMPK [153]. Another member of this group is the antioxidant resveratrol, which increases insulin sensitivity and lowers lipids in blood of diabetic and obese mice, most likely by activating SIRT1 [154]. Several natural analogues of resveratrol also activate AMPK [155]. One derivative, combretastatin A-4, a natural cis-stilbene is isolated from the plant Combretum caffrum. It is the most effective and potent member of this group of compounds in downregulating the expression of gluconeogenic enzymes in the liver and reducing the fasting blood glucose level in diabetic mice. The effect of combretastatin A-4 on AMPK is indirect and seems to result from the inhibition of the mitochondrial respiratory chain and a subsequent reduction in ATP levels [156].

Figure 7. Molecular structures of compounds that have one or two phenol, polyphenol or phenolmethyl ether moieties.

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, has attracted significant attention and is undergoing early clinical trials as a novel anticancer agent [157]. It has been shown that curcumin-induced death of ovarian cancer cells is mediated by activating AMPK [158]. In other studies, curcumin rescued isolated mouse pancreatic islets from cytokine-induced death in vitro and prevented streptozotocin-induced diabetes in vivo [159]. Of particular interest is the finding that curcumin-induced activation of AMPK underlies the reduction in hepatic glucose production [160].

The thienopyridone derivative A769662 possesses antidiabetic effects by direct activation of AMPK [161] (Figure 8). It has been proposed that this compound activates AMPK in an AMP-independent manner, by binding to an alternative allosteric site in the AMPK complex [162]. Others have shown that A769662 binds to the carbohydrate-binding moiety in the γ subunit of the complex and to several amino acid moieties of the β1 subunit, none of which participates in AMP-binding [14]. Noteworthy is the finding that A769662 activates AMPK complexes that exclusively contain the β1 subunit. This property limits the potential therapeutic use of this compound to tissues expressing the ubiquitous β1 subunit, but not to tissue like skeletal muscle that assemble AMPKβ2 complexes. Therefore, the main target of A769662 is most likely the liver in which an activation of β1-containing AMPK complex reduces the expression of gluconeogenic enzymes and hepatic liver production [161]. Another patented thienopyridone derivative recently reported by Zhou et al. is Compound 202 (patent EP1754483A1) [141, 163] (Figure 8). Like A769662, this molecule, which contains a thienopyridone moiety, activates AMPK most likely by interacting with the β1 subunits.

Figure 8. Molecular structure of the thienopyridone derivative A769662 and Compound 202.


While screening chemical libraries for direct activators of the α-subunit of AMPK Pang et al. [8] discovered a furanothiazolidine derivative, PT1 (Figure 9). When tested in hepatoma HepG2 cells it lowered their lipid content and activated AMPK in a dose-dependent manner. It interacted with the autoinhibitory domain in the α1 subunit and converted AMPK to a constitutively active complex [8]. Experiments with L6 myotubes showed that PT1 effectively increased the phosphorylation of AMPK with no apparent changes in the ATP/ADP ratio in the cells. Moreover, this effect was not mediated via LKB1 because it also occurred in the LKB-deficient cells. PT1-induced phosphorylation of AMPK was lost in cells co-treated with STO-609, a specific CaMKKβ inhibitor, suggesting a critical role for Ca2+-dependent CaMKKβ activation in this process. In vivo tests with this compound are required to ascertain its potential as a prototype molecule for the development of novel antidiabetic drugs.

Figure 9. Molecular structure of the furanothiazolidine derivative, PT1, that interacts with the α-subunit of the AMPK complex.


AMP is the natural endogenous activator of the AMPK complex. As explained above, two pairs of AMP molecules interact with two Bateman domains in the γ subunit and allosterically activate the entire AMPK complex. Figure 10 shows AMP and structurally related compounds that induce similar effects. AICAR is metabolized intracellularly to ZMP (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranotide) by adenosine kinase. ZMP is an AMP analog that interacts with Bateman domains in the γ subunit of AMPK and induces allosteric changes in AMPK conformation, allowing kinase activation [52]. Some researchers, however, have claimed that ZMP can also regulate glucose metabolism by direct inhibition of fructose-1,6-biphosphatase in hepatocytes, thereby blocking gluconeogenesis independently of the activation of AMPK [164]. AICAR was tested in animal models of diabetes and exhibited antidiabetic effects as many pathological metabolic parameters in these animals such as, blood glucose level, lipids profile, hepatic glucose output and glucose disposal were improved [165]. AICAR infusion in both healthy and diabetic individuals increased skeletal muscle glucose uptake [166]. It also dramatically reduced the formation of reactive oxygen species in blood and preserved normal endothelial cell function in diabetic patients [167]. In another study, AICAR increased glycogen content in rat skeletal muscles, mostly due to an increased influx of glucose [73].

Figure 10. Molecular structures of AMP and structurally related compounds that induce allosterical activation of the AMPK complex.

AICAR also negatively regulates IL-6 and IL-8 gene expression and their secretion from rat adipocytes and skeletal muscle cells. These two proinflammatory mediators are secreted from adipose tissues and play an important role in the etiology of insulin resistance in obese patients. Thus, these effects of AICAR led to the idea that it may serve as a potent drug for increasing insulin sensitivity in peripheral tissues [168]. Nevertheless, AICAR suffers from unfavorable pharmacokinetic properties (i.e., high effective concentration, poor bioavailability and short half life) and severe metabolic complications (e.g., lactic acidosis and massive uric acid production) [169].

Another serious shortcoming of AICAR is the lack of a strict specificity for AMPK. In fact, AICAR interacts with other enzymes, such as S-adenosylhomocysteine hydrolase and glycogen phosphorylase [170]. To solve these problems many groups have tried to develop more potent derivatives of AICAR. One example is the imidazo[4, 5-b]pyridine derivative S27847, which increased the activity of AMPK 7-fold in hepatocytes at micromolar concentrations (Figure 10) [171]. Other structurally related imidazol analogues of AICAR and S27847 are the patented Compound 4 and Compound 58 [141, 172, 173]. These derivatives are less effective than S27847; at 500 and 200 μM they increased AMPK activity 3- and 2-fold, respectively, in comparison with the control treatments.

Other structurally unrelated compounds

Chromium picolinate

Some other molecules that activate AMPK do not belong to the various classes of chemically and structurally related compounds described above. One such compound is chromium picolinate, which is used as an antidiabetic food supplement. Recent reports suggest that some antidiabetic effects of this compound result from AMPK activation and the inhibition of resistin secretion from adipocytes. Resistin is a 12.5 kDa cysteine-rich adipokine known to induce insulin resistance in rodents [174]. Such effects are not found in man due to the lack of resistin expression and secretion [175]. Therefore, this resistin-dependent mechanism is not involved in the antidiabetic effects of chromium picolinate in man.

α-lipoic acid

α-lipoic acid decreases lipid accumulation in rat skeletal muscle and steatosis in the obese rat liver by augmenting AMPK phosphorylation [176]. It has been recently shown that an incubation of C2C12 myotubes with α-lipoic acid increased the activity and Thr172 phosphorylation of the AMPKα2 subunit, followed by Ser79 phosphorylation in ACC. Furthermore, inhibition of CaMKK with the selective inhibitor STO-609 abolished α-lipoic acid-stimulated AMPK activation, with a concomitant reduction of Ser79 phosphorylation in ACC. When short interfering-RNA against CaMKK was used to silence its expression, it abolished α-lipoic acid-induced AMPK activation. These data indicate that CaMKK is possibly the target for α-lipoic acid-induced AMPK activation in myotubes [177].

Kainic acid

Another interesting molecule that activates AMPK in the brain is kainic acid (extracted from various seaweeds) [178]. Of interest are the reports showing specific receptors for kainic acid in the brain. It remains to be investigated whether kainic acid affects energy metabolism in the brain and possibly some important metabolic functions, like appetite.


Cannabinoids interact with AMPK complexes in various tissues in opposing manners. They activate AMPK in the hypothalamus and the heart, and inhibit it in the liver and adipose tissues. It has been assumed that cardioprotective effects of cannabinoids result from activating AMPK-dependent metabolic pathways [179]. Nevertheless, no significant benefits of these compounds in terms of blood glucose normalization in diabetes have yet been reported.

Long chain-fatty acids

It has also been proposed that long chain-fatty acids activate AMPK [180]. However, these effects are short-lived and transient due to efficient β-oxidation. Notwithstanding, such effects of long chain fatty acid have been documented in rats and mice and isolated insulin-sensitive tissues. Substituted α,ω-dicarboxylic acids of 14-18C fatty acids (MEDICA analogs), which are not metabolized beyond their acyl-CoA thioesters, also activate recombinant AMPK in a free cell system, in cell lines such as HepG2 and 3T3-L1 and in diabetic db/db mice. Activation of AMPK in the latter animal model normalized blood glucose and suppressed hepatic glucose production [180]. On interest is the recent finding that oral administration of the short chain fatty acid butyric acid also activated AMPK in skeletal muscles and brown adipocytes of dietary-obese C57 black mice. This treatment also prevented the development of insulin resistance and obesity, decreased blood glucose level and reduced body fat content in the treated animals [181]. Thus, both long and short chain fatty acid may activate AMPK.

Reactive oxygen species

Reactive oxygen species (ROS) such as peroxynitrite (ONOO) have been associated with the activation of AMPK in cells, especially in tissues with a high oxygen demand [182]. Pathologically relevant concentrations of peroxynitrite are capable of activating AMPK independently of variations in the AMP/ATP ratio. It has been suggested that this process is significantly enhanced following hypoxia and reoxygenation in the heart [183]. Similar AMPK stimulatory activity has also been assigned to the free radical hydrogen peroxide [182]. The idea that activated AMPK prevents oxidative stress associated with diabetes by upregulating mitochondrial uncoupling protein-2 (UCP-2) presents an attractive pathway for antidiabetic effects of such AMPK activators [167]. Similarly, the finding that nitric oxide (NO) per se activates AMPK via the Ca2+-dependent CaMKK pathway in vascular endothelial cells points to another mechanism for AMPK activation, especially in cells and tissues where this radical generation is increased due to an of induction of NOS isotypes.


Leptin is a 16 kDa peptide that is secreted from adipocytes and regulates energy metabolism and satiety. Several reports show that it activates AMPK in skeletal muscles but inhibits its activation in the hypothalamus. These disparate tissue-specific effects are remarkably related to the overall metabolic regulation in the organism: activation of AMPK in skeletal muscles potentiates the rates of glucose transport and fatty acid oxidation, whereas in the hypothalamus, the inhibition of AMPK reduces the appetite and food intake. Leptin-overexpressing mice exhibit reduced tissue triacylglycerol content and an increased energy expenditure, due to the peripheral phosphorylation of AMPKα2 isoform [184, 185]. This finding was confirmed when leptin infusion increased AMPKα2 activity in skeletal muscles of control and STZ-diabetic mice [184]. The molecular mechanism of leptin-induced activation of AMPK is not yet clear.


Ghrelin has the opposite effects of leptin on AMPK. It is secreted from the gastric mucosa and acts as a growth hormone-releasing- and an appetite stimulating factor. Several studies have found that ghrelin activates AMPK in the hypothalamus and in the heart and inhibits it in the liver and adipose tissues [186]. It seems to lack any effect on AMPK phosphorylation in skeletal muscles [187]. The upstream kinase by which ghrelin exerts its effects on AMPK has not yet been identified. However, ghrelin has been shown to induce CaMKKβ-dependent signaling in hypothalamic neurons [188].


Interleukin-6 (IL-6) rapidly and markedly increases AMPK activity in skeletal muscles. It also increases fatty acid oxidation and basal and insulin-stimulated glucose uptake by translocation of glucose transporter-4 to the plasma membrane of L6 myotubes. These effects are lost in L6 myotubes overexpressing the dominant negative form of AMPKα2 [189]. The relevance of these finding to diabetes treatment is still under investigation.

AMPK inhibitors

Iodotubercidin and arabinose-adenosine (AraA)

The inhibitor iodotubercidin is a synthetic adenosine derivative that has been used to inhibit AMPK in vitro. It should be noted that this compound also interacts with and inhibits other enzymes, such as glycogen synthase, phosphokinase A, phosphokinase C or casein kinase [190193].

Arabinose-adenosine (AraA) was originally isolated from the Caribbean sponge Tehya crypta. This compound, which has some antiviral activity, also inhibits various kinases including AMPK [194]. Another shortcoming of these two inhibitors is their inability to inhibit contraction-induced AMPK activation in skeletal muscles [195]. Thus, results on AMPK inhibition that were obtained from experiments with these inhibitors should be reevaluated.

Compound C

The synthetic Compound C is a reversible and an AMP-competitive inhibitor of AMPK [196]. When tested in cells, it fails to completely block all AMPK-dependent stimuli. For instance, while AICAR-dependent activation of AMPK in skeletal muscles was blocked by Compound C, the dinitrophenol-induced activation of AMPK was not affected by it. Moreover, some reports claim that Compound C inhibits adenosine transport into the cells. Thus, when presented together with AICAR it can limit the influx of the latter and therefore prevent its potential to activate AMPK intracellularly [196]. An additional limitation of Compound C is its inconsistent level of inhibition of AMPK activity in different tissues: for example, it is very effective in hepatocytes but much less so in skeletal muscles [81]. This may indicate an AMPK-subunit specificity of this inhibitor. Recent studies show that Compound C also inhibits other protein kinases, such as, ERK1/2 and PHK. Therefore, cellular effects mediated by Compound C should be carefully studied and evaluated.


New compounds that activate AMPK in a complex- and tissue-specific manner may eventually become novel antidiabetic and antiobesity drugs. The ideal AMPK activator should have several properties. It should specifically activate AMPK at a much lower concentration than AICAR. The activation should be targeted to AMPK subunits specifically expressed in the liver, adipocytes or skeletal muscles, but not in the central nervous centers where AMPK activation increases appetite and food consumption. Obviously, oral formulation of drugs that produce minimal side effects is essential.

This review describes some classes of direct and indirect activators of AMPK. Further studies are required to understand the molecular interactions of these compounds and structural requirements for their specificity to the various subunits of AMPK or its upstream effectors. Such ongoing efforts may provide a novel class of drugs to treat diabetes and related metabolic abnormalities in the future.

Conflict of interest statement: The authors declare that they have no competing conflict of interests with respect to financial or other issues.

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Arctigenin Efficiently Enhanced Sedentary Mice Treadmill Endurance

Physical inactivity is considered as one of the potential risk factors for the development of type 2 diabetes and other metabolic diseases, while endurance exercise training could enhance fat oxidation that is associated with insulin sensitivity improvement in obesity. AMP-activated protein kinase (AMPK) as an energy sensor plays pivotal roles in the regulation of energy homeostasis, and its activation could improve glucose uptake, promote mitochondrial biogenesis and increase glycolysis. Recent research has even suggested that AMPK activation contributed to endurance enhancement without exercise. Here we report that the natural product arctigenin from the traditional herb Arctium lappa L. (Compositae) strongly increased AMPK phosphorylation and subsequently up-regulated its downstream pathway in both H9C2 and C2C12 cells. It was discovered that arctigenin phosphorylated AMPK via calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase 11(LKB1)-dependent pathways. Mice treadmill based in vivo assay further indicated that administration of arctigenin improved efficiently mice endurance as reflected by the increased fatigue time and distance, and potently enhanced mitochondrial biogenesis and fatty acid oxidation (FAO) related genes expression in muscle tissues. Our results thus suggested that arctigenin might be used as a potential lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases.
Physical inactivity is considered as one of the risk factors for the development of type 2 diabetes and other metabolic diseases. It is known that endurance exercise training could lead to fiber type transformation, mitochondrial biogenesis, angiogenesis and other adaptive changes in skeletal muscle [1], [2], thus further enhancing fat oxidation that is associated with improvement of insulin sensitivity in obesity [3]. Currently, at least 60% of the global population fails to achieve the daily minimum recommendation of 30 min moderate intensity of physical activity, and within these people the risk of getting the related chronic diseases including type 2 diabetes increases by 1.5 times [4]. Therefore, it has become valuable to discover active agents that would mimic the effects of exercise training to prevent or treat metabolic diseases.
AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase with three subunits (α, β, γ) [5]. As the major molecular sensor for AMP/ATP ratio in cells, AMPK plays a pivotal role in the regulation of energy metabolism [6]. AMPK activation switches on ATP-producing processes (such as glucose uptake, mitochondrial biogenesis and glycolysis) and inhibits ATP-consuming anabolic processes (such as protein synthesis and sterol synthesis) [7]. AMPK phosphorylation is regulated by a series of upstream AMPK kinases, including serine/threonine kinase (LKB1) [8], Tak1 kinase and two calmodulin-dependent protein kinase kinases (CaMKKα and CaMKKβ) [9], [10].
Recently, it was reported that AMPK activation could improve mice endurance in the absence of exercise training [11]. Under endurance training condition, skeletal muscle suffers a number of changes, such as glucose consumption decreasing, main energy source transition from glucose to fatty acid utilization, mitochondrial biogenesis increasing and fiber-type switch [11], [12]. AMPK activation could increase peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) gene expression or the direct phosphorylation of PGC-1α in skeletal muscle [6], [13]. PGC-1α is known to be involved in the regulation of mitochondrial biogenesis, respiration, hepatic gluconeogenesis and other biological processes by interaction with several transcription factors, such as ERRα, NRF1, NRF2 and PPARs [14], [15]. PGC-1α null mice showed muscle dysfunction, abnormal weight control and hepatic steatosis [16], [17]. Although skeletal muscle was not an active lipogenic organ as liver, exercise training or AMPK activation has been also reported to promote fatty acid synthesis and oxidation as evidenced by up-regulation of varied main enzymes such as pyruvate dehydrogenase kinase 4 (PDK4), stearoyl-CoA desaturase-1 (SCD-1), fatty acid synthetase (FAS) and muscle carnitine palmitoyltransferase I (mCPT1b) within related pathways in skeletal muscles [18][20]. Oxidation of fatty acid exports more energy than metabolism of glucose, and alteration of utilizing this energy substrate could contribute to exercise tolerance [11], [21]. Additionally, AMPK activation changes skeletal muscle myofiber type composition, which mimics the fiber type switch induced by endurance training [11], [22]. Therefore, all the above findings suggested that AMPK might act as a key mediator of endurance training-induced changes, which was also confirmed further by the result that treatment of AMPK agonist AICAR at a dose of 500 mg/kg/day could induce metabolic genes expression and enhance running endurance [11].
Arctigenin (ATG, Fig. 1A) is a phenylpropanoid dibenzylbutyrolactone lignan extracted from the traditional herb Arctium lappa L. (Compositae) with anti-cancer and anti-inflammatory effects [23][26]. As a new type of antitumor agent, arctigenin could block the unfolded protein response (UPR) by reducing the expression of UPR-related genes, such as C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4) and glucose-regulated protein of 78 kDa (GRP78) under glucose deprivation [23][25]. Its anti-inflammatory effect mainly acts by inhibiting type I-IV allergic inflammation and pro-inflammatory enzymes in vitro and in vivo [26].
Figure 1
Figure 1

Arctigenin (ATG) increased AMPK phosphorylation in H9C2 and C2C12 cells.
In the present study, we discovered that arctigenin could increase AMPK phosphorylation and up-regulate its downstream-pathway related genes mRNA levels to promote mitochondrial biogenesis and fatty acid synthesis and oxidation in vitro and in vivo, subsequently leading to the mice treadmill endurance enhancement. Cell based assay revealed that arctigenin increased AMPK phosphorylation targeting the CaMKK and LKB1-dependent pathways. Our results thus demonstrated that arctigenin might be used as a lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases.
Arctigenin increased AMPK phosphorylation in H9C2 and C2C12 cells
As reported, AMPK activation switched on ATP-producing processes to enhance endurance [7], [11]. With this information, we thus constructed the phospho-AMPK activator screening platform (Text S1), based on which our lab in-house natural product library was screened out and the natural product arctigenin (Fig. 1A) was finally identified to efficiently activate AMPK phosphorylation (Fig. S1). As shown in Fig. 1B and C, arctigenin dose-dependently increased AMPK phosphorylation in both H9C2 and C2C12 muscle cells, while had no effects on total AMPK.
Arctigenin activated PGC-1α transcription via up-regulating AMPK phosphorylation
PGC-1α was known to act as the master regulator in mitochondrial biogenesis and skeletal muscle adaptation [27][29]. Since AMPK activation by actual exercise or pharmacological treatment could lead to up-regulation of PGC-1α gene expression [30], [31], we investigated the potential effects of arctigenin on PGC-1α mRNA level in both H9C2 and C2C12 cells. Compared with DMSO-treated group, arctigenin incubation could dose-dependently up-regulate PGC-1α mRNA levels in both cell lines (Fig. 2A). By considering that AMPK phosphorylated PGC-1α directly at theronine-177 and serine-538, which are required for induction of PGC-1α promoter [6], we thus examined whether arctigenin could increase PGC-1α transcription through regulating its promoter activity by luciferase assay. As shown in Fig. S2A, the relative PGC-1α promoter activity was highly increased in arctigenin treated group compared with the DMSO group in HEK293T cells.
Figure 2
Figure 2

Arctigenin (ATG) increased PGC-1α transcription via enhancing AMPK phosphrylation.
PGC-1α as a key regulator in energy metabolism could respond to varied physical stimuli, such as muscle contraction, cold stress and overfeeding [32][35]. It could be regulated by AMPK, p38 MAPK or NF-κB pathway [11], [36][38]. Since arctigenin has been determined to increase AMPK phosphorylation and PGC-1α transcription, we thus wondered whether the effect of arctigenin on PGC-1α was dependent on its effect on AMPK. Therefore, we examined the effects of arctigenin on PGC-1α mRNA level and PGC-1α promoter activity together with AMPK inhibitor compound C incubation in the related cells. As shown in Fig. 2B–E and Fig. S2B, treatment of compound C almost inhibited the arctigenin-induced AMPK phosphorylation and completely blocked the arctigenin-induced up-regulation of PGC-1α mRNA level and promoter activity, implying that the effect of arctigenin on PGC-1α transcription regulation was dependent on its role in AMPK phosphorylation.
These results thereby indicated that arctigenin could induce PGC-1α transcription in skeletal muscle and cardiac muscle cell lines via up-regulating AMPK phosphorylation.
Arctigenin increased mitochondrial biogenesis and fatty acid oxidation genes expression
Since arctigenin has been determined to induce PGC-1α transcription via up-regulating AMPK phosphorylation, this result thereby implied that arctigenin might play a potential role in promoting mitochondrial biogenesis and function. To further evaluate this hypothesis, the mRNA levels of the related genes were detected in both H9C2 and C2C12 cell lines with results listed in Fig. 3A and B. Estrogen-related receptor α (ERRα), a nuclear receptor activated by interacting with PGC-1α, was reported to control the expression of nuclear genes encoding mitochondrial proteins (NUGEMPs) [39], [40]. We found ERRα mRNA level was obviously elevated by arctigenin treatment. At the same time, as a typical NUGEMP and significant component of electron transport chain (ETC) in mitochondrial, cytochrome c mRNA was also induced by arctigenin administration. Furthermore, as also indicated in Fig. 3A, arctigenin significantly activated the mRNA levels of PDK4, SCD1, FAS and mCPT1b which are key enzymes in fatty acid synthesis and oxidation for promotion of energy source transforming from glucose to fatty acid in C2C12 cells at 40 µM, while the regulation of arctigenin in these genes was not effectively in H9C2 cells for lack of significant difference in SCD1 and FAS mRNA levels between high dose of arctigenin administration group (40 µM) and DMSO control (Fig. 3B).
Figure 3
Figure 3

Arctigenin (ATG) promoted mitochondrial biogenesis and FAO related gene expression.
To clarify whether arctigenin regulated mitochondrial biogenesis and FAO related genes mRNA levels through its effect on AMPK phosphorylation, we examined the influence of arctigenin on these genes together with AMPK inhibitor (compound C) incubation in C2C12 and H9C2 cells. As indicated in Fig. S3 and S4, treatment of compound C almost inhibited the arctigenin-induced genes expression except mCPT1b in H9C2 and SCD1 in C2C12 cells. These results thus demonstrated that arctigenin increased mitochondrial biogenesis and fatty acid oxidation genes mRNA levels mainly through its effect on AMPK phosphorylation.
Therefore, all our results suggested that arctigenin could activate PGC-1α transcription and increase mitochondrial biogenesis and fatty acid oxidation genes expression in both skeletal muscle and cardiac muscle cells.
Arctigenin regulated the related genes expression not via affecting transcriptional activity of PPARδ
As reported, AMPK catalytic subunit over-expression evidently promoted basal and ligand-dependent transcription of PPARδ [11], indicative of that PPARδ might be also involved in AMPK related gene expression. The fact that AICAR (AMPK activator) synergistically increased mice endurance and gene expression with GW501516 (PPARδ agonist) further implied the potential of AMPK-PPARδ signaling axis. Here, we also examined whether arctigenin could induce the related PPARδ involved gene expression with mammalian one-hybrid and transcriptional activation assay systems. As shown in Fig. S5, arctigenin failed to regulate the co-activator recruitment or transcriptional activity of exogenous or endogenous PPARδ.
Therefore, all above-mentioned results suggested that arctigenin regulating the relative gene expression is related to the enhancement of AMPK phosphorylation, while might not to the promotion of PPARδ transcriptional activity.
Arctigenin enhanced AMPK phosphorylation through CaMKK and LKB1-dependent pathways
Considering that arctigenin could activate AMPK phosphorylation, we subsequently investigated the potential regulation of this natural product against the relevant AMPK-involved pathways. As determined, directly activating AMPK in an allosteric manner and/or indirectly promoting Thr172 phosphorylation of AMPK both contributed to the AMPK activation. Bear that in mind, we examined the recombinant AMPK enzyme activity with or without arctigenin incubation in vitro to investigate whether arctigenin could influence the conformation of AMPK thus inducing AMPK activity. As indicated in Fig. 4A, arctigenin had no effect on the recombinant AMPK enzyme activity, while AMPK agonist A-769662 as a positive control significantly increased the phosphorylation of its substrate SAMS [41], [42], implying that arctigenin was not an AMPK ligand and indirectly activated AMPK.
Figure 4
Figure 4

Arctigenin (ATG) enhanced AMPK phosphorylation through CaMKK and LKB1-dependent pathway.
As reported, AMPK has an obligate requirement for phosphorylation by an upstream kinase on Thr-172 in the α-subunit catalytic domain [5]. Since the above assay has indicated that arctigenin activated AMPK in an indirect manner, we further explored the potential signaling responsible for arctigenin-induced AMPK activation. Regarding the fact that LKB1 and CaMKK are within the main identified kinases in AMPK upstream pathway and play pivotal roles in regulation of AMPK phosphorylation [43], we thus addressed these two kinases related assays. Firstly, we determined whether CaMKK activation was necessary for arctigenin-induced AMPK phosphorylation. As shown in Fig. 4B, pretreatment of HEK293T, a model cell usually used in mechanism studies, with the selective CaMKK inhibitor STO-609 [9] obviously attenuated the arctigenin-induced AMPK phosphorylation. To investigate whether LKB1 pathway might participate in the arctigenin-induced AMPK activation, we carried out LKB1 knock-down assay in HEK293T cells as reported [44]. The results in Fig. 4C revealed that LKB1 knock-down in HEK293T cells efficiently down-regulated the arctigenin-induced AMPK phosphorylation.
Taken together, our results thereby suggested that arctigenin stimulated AMPK phosphorylation via CaMKK and LKB1-dependent pathways.
Arctigenin efficiently enhanced sedentary mice treadmill endurance
AMPK was reported as an “exercise mimetic”, whose pharmacological activator-AICAR was ever tested to provide several benefits of exercise in sedentary mice [11]. Since arctigenin has been determined able to effectively activate AMPK phosphorylation, we thereby evaluated whether this natural product could enhance mice endurance. To address this issue, we performed the treadmill exhaustion test among the selected mice (All mice were examined regarding the treadmill performance before arctigenin administration, and those mice whose running time was too long or too short compared with the average were eliminated to reduce the potential effects by the inherent variation) after 6-week arctigenin administration (8 mg/kg). Running time and distance till fatigue were estimated as maximal endurance capacity. As shown in Fig. 5A and B, arctigenin administration brought on approximately an increase of 40% in mean fatigue time and 65% in mean fatigue distance, further indicating that arctigenin efficiently enhanced sedentary mice treadmill endurance.
Figure 5
Figure 5

Arctigenin (ATG) elevated mice treadmill performance without inducing myofiber type conversion in skeletal muscle.
During the treatment, mice showed similar basal behavior, food consumption and body weight (Fig. S6A and B). In addition, to investigate the preliminary toxicity of arctigenin, TNFα and IL-6 levels in mice serum were tested. The results revealed that there was no difference in those two inflammatory factors comparing arctigenin with vehicle groups (Fig. S6C and D). At the same time, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also detected. As shown in Fig. S6E and F, no change was found for AST while ALT elevated with arctigenin administration, suggestive of the potential hepatotoxicity of arctigenin in ip administration. Therefore, these results demonstrated that arctigenin elevated sedentary mice endurance efficiently while no significant toxicity was observed.
Arctigenin could not induce skeletal muscle fiber-type changes
As reported, endurance exercise, AMPK mutation with persistent activation and PGC-1α over-expression in transgenic mice driven by muscle creatine kinase (MCK) promoter could induce myofiber type switch [12], [22], [45]. We thereby considered that arctigenin might change myofiber type construction in skeletal muscle tissues. However, we failed to find any obvious conversions in the mRNA levels of four different myosin heavy chain (MHC) isoforms (MHCI as a marker to represent type I myofibers, MHCIIa, MHCIIx and MHCIIb as markers to represent type II myofibers) in gastrocnemius (Fig. 5C) and quadriceps (Fig. 5D) comparing arctigenin administration group with vehicle group. Additionally, no obvious changes were found in myofibers compositions between arctigenin treatment group and vehicle group in ATPase staining (Fig. S7). These results thus suggested that arctigenin could not affect myofiber type proportion.
Arctigenin induced AMPK phosphorylation, mitochondrial biogenesis and FAO pathway in vivo
To further investigate the potential regulative mechanism of arctigenin regarding its improvement of mice treadmill endurance, we addressed the relevant tissue-based assays. Compared with vehicle group, arctigenin (8 mg/kg) administration enhanced AMPK phosphorylation in gastrocnemius (Fig. 6A), quadriceps (Fig. 6B) and cardiac muscles (Fig. 6C). The results shown in Fig. 6D–F suggested that arctigenin up-regulated the mRNA levels of PGC-1α and ERRα and the protein levels of cytochrome c in gastrocnemius, quadriceps and cardiac muscles. Additionally, mRNA levels of SCD1, PDK4 and mCPT1b were also determined to be obviously elevated in gastrocnemius and quadriceps muscles with arctigenin treatment, consistent with the cell based results. Notably, there was also a tendency for higher average FAS mRNA levels in arctigenin treated group but without significance. To further identify the regulation of arctigenin on FAO pathway, the protein level of uncoupling protein 3 (UCP3) that is also involved in FAO induction [46] was examined. As shown in Fig. 6A–C, UCP3 expression was obviously elevated in gastrocnemius and quadriceps muscles.
Figure 6
Figure 6

Arctigenin (ATG) enhanced AMPK phosphorylation, mitochondrial biogenesis and FAO pathway in vivo.
As fatty acid synthesis and storage related genes were up-regulated, we thereby hypothesized that arctigenin treated mice might contain more fatty acid in skeletal muscle for instant oxidation to supply energy source during exercise. Bearing that in mind, we thus detected fatty acid levels in skeletal muscles (gastrocnemius and quadriceps) and found that arctigenin treatment could enhance fatty acid storage in gastrocnemius evidently while in quadriceps without significance (Fig. S8A and B).
Therefore, all those in vitro and in vivo results thereby suggested that arctigenin could enhance AMPK phosphorylation, mitochondrial biogenesis and FAO pathway related genes expression, finally leading to the enhancement of exercise-free endurance.
Over the past decades, aerobic endurance exercise has been potently highlighted concerning its significance in the clinical amelioration of many disease symptoms, such as glucose metabolism in type 2 diabetes [47], dyslipidemia in atherosclerosis [48] and hypertension in stroke, acute myocardial infarction or cardiac insufficiency [49], [50]. Nevertheless, the inability to afford definite intensity of physical exercise has been always the obstacle to make profits of exercises [51]. Discovery of active agents that would mimic the reprogramming metabolism induced by exercise training is one of the effective strategies to overcome these obstacles.
Actual exercises could result in activation of kinases/phoshatases signaling pathway, nuclear translocation of transcription/translation factors, up-regulation of NUGEMPs and mtDNA-encoded proteins, and augmentation of muscle aerobic capacity [52]. AMPK is activated during physical activities to promote down-stream metabolic reprogramming (e.g. mitochondria biogenesis, fatty acid oxidation promotion and myofiber type switch), and considered as a significant mediator in skeletal muscle adaptations [6], [7], [22]. Therefore, pharmacological activation of AMPK may provide benefits of endurance exercise without actual physical activities, which was ever confirmed by endurance enhancing activity of its activators AICAR [11]. It was found that AMPK activator-AICAR, which is metabolized to an AMP mimetic in cell, could increase sedentary mice treadmill endurance at a high dosage of 500 mg/kg/day via intraperitoneal injection and up-regulate genes linked to oxidation metabolism via AMPK-PPARδ signaling axis [11]. Resveratrol, a natural polyphenolic product derived from grapes, could also increase mice aerobic capacity without exercise at a dose of 400 mg/kg/day orally [53] targeting SIRT1 and subsequently regulating its downstream pathway, which was reported to tightly couple with AMPK [54].
Compared with synthetic compounds, small molecules from natural sources are featured by their large-scale of structure diversity [55]. Therefore, we performed the screening of the efficient phosphor-AMPK activator targeting our in-house natural product library and finally discovered that arctigenin dose-dependently increased AMPK phosphorylation in vitro (Fig. 1B and C) and in vivo (Fig. 6A–C).
Arctigenin was extracted from Arctium lappa L., which has been widely used in traditional Chinese medicine [56]. Previous studies have illustrated that arctigenin was active in anti-viral infection, anti-tumor, anti-inflammation, and neuroprotection [23][26], [57][59]. Here we reported that arctigenin promoted AMPK phosphorylation on Thr172 site through CaMKK and LKB1-dependent pathways (Fig. 4). Recently, AMPK Ser485/491 phosphorylation was also reported involving in regulation of AMPK activity [60], [61]. To clarify whether arctigenin could affect AMPK phosphorylation on Ser485/491, we examined Ser485/491 phosphorylation levels of AMPK in arctigenin treated cells. As shown in Fig. S9, arctigenin did not change AMPK Ser485/491 phosphorylation in HEK293T, H9C2 or C2C12 cell lines, implying that arctigenin activated AMPK phosphorylation on Thr172 without impacting AMPK Ser485/491 phosphorylation.
We also found that arctigenin significantly enhanced sedentary mice running endurance at a dose of 8 mg/kg/day (Fig. 5A and B). Furthermore, cytochrome c protein and mRNA levels were typically increased in mitochondrial biogenesis, and FAO related genes (SCD1, PDK4, FAS and mCPT1b) mRNA levels were also obviously elevated in muscle tissues (Fig. 6). Fatigue induced by treadmill running was primarily developed from periphery tissues and featured by rapid clearance of intracellular ATP and relative insufficiency of oxidation metabolism in cardiovascular and skeletal muscle system [62]. We thus concluded that promotion of mitochondrial biogenesis and FAO linked gene expression induced by arctigenin attributed to mice prolonged running time and distance.
Myofiber type switch induced by endurance training was ever considered as one of the reasons for exercise tolerance [12], [22], [45], but we could not find any differences of fiber type composition in skeletal muscle tissues (gastrocnemius and quadriceps) between arctigenin-treatment and vehicle groups (Fig. 5C–D and Fig. S5). Compared with AMPK persistent activating mutation that led to myofiber type switch in vivo [45], the indirect phosphorylation of AMPK by arctigenin might only share part of the down-stream genes response with AMPK persistent activating mutation, which resulted in unchangeable myofiber type composition in arctigenin administrated group. Meanwhile, PGC-1α was also reported playing a pivotal role in myofiber type transformation via activating calcineurin signaling pathway [22]. Although arctigenin obviously up-regulated PGC-1α transcription in vitro (Fig. 2A and B) and in vivo (Fig. 6D–F), it might not mobilize calcium/calcineurin pathway or totally activate PGC-1α interaction with related proteins for promotion of the myofiber type switch. It is noted that arctigenin up-regulated mitochondrial biogenesis related genes (such as ERRα and cytochrome c) both in cardiac and skeletal muscle tissues, but elevated FAO related genes only in skeletal muscle tissues, which might be tentatively attributed to the tissue specificity responding to arctigenin in fatty acid metabolism.
In summary, we demonstrated that arctigenin could efficiently increase rodent sedentary treadmill endurance via enhancing AMPK phosphorylation. This natural product induced the accommodation of mitochondrial biogenesis and FAO pathway to promote mitochondrial oxidative capacity without actual physical activities as summarized in Fig. 7. Our results have provided additional understanding of pharmacological functions for arctigenin and traditional Chinese medicine Arctium lappa L., and suggested the potential of arctigenin as a lead compound for anti-chronic metabolic disease (e.g. obesity or type 2 diabetes) drug discovery.
Figure 7
Figure 7

A proposed model demonstrating arctigenin (ATG)-induced endurance enhancement mechanism.
Materials and Methods
Ethics Statement
All animal experiments were carried out in accordance with the Regulations of Experiments Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. Permit numbers: SCXK (HU) 2008-0017; SYXK (HU) 2008-0049. This study was approved by Science and Technology Commission of Shanghai Municipality.
Restriction enzymes were purchased from New England Biolabs. Cell culture plastic ware was purchased from Corning Inc. DMEM, fetal bovine and horse serums were purchased from Invitrogen. Compound C and STO609 were obtained from Sigma. Calcium Phosphate Cell Transfection Kit was obtained from Beyotime. RNAiso, RT reagent Kit and SYRB Premix Ex Taq were purchased from TaKaRa. Dual Luciferase Assay System was obtained from Promega. Anti-cytochrome c, anti-phospho-AMPK (Thr172), anti-AMPKα1/α2, and anti-LKB1 antibodies were purchased from Cell Signaling Technology. Anti-CaMKK antibody was purchased from Senta Cruz Biotechnology. HEK293T, H9C2 and C2C12 cells were obtained from ATCC.
Cell culture and differentiation
As a typical cardiac muscle cell line, H9C2 was derived from embryonic BD1X rat heart tissue and not differentiated in our study. C2C12 was a subclone of the mouse myoblast cell line and differentiated in DMEM with 2% horse serum, forming contractile myotubes and expressing characteristic muscle proteins. Differentiated myotubes were used in our experiments.
H9C2 and C2C12 cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and the cells were grown at 37°C in an environment of 5% CO2. To induce myoblast fusion and myotubes differentiation, C2C12 myoblasts were switched to differentiation medium when 100% confluent in 6-well plate. Differentiation medium was exchanged every 2 days for 6 days before experimental manipulation.
Western Blot analysis
Tissues were lysed with lysis buffer containing 25 mmol/L Tris-HCl (PH 7.5), 150 mmol/L NaCl, 1 mmol/L Na3VO4, 1% Triton X-100 and a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of lysates or supernatants of the whole cell extracts were fractionated by SDS-PAGE and transferred to Hybond-c nitrocellulose membrane (Amersham Bioscience). The membranes were blocked for one hour at room temperature and then incubated overnight at 4°C in TBST buffer (5% milk) containing related antibody. The membranes were then incubated for an hour at room temperature in TBST buffer (5% milk) containing anti-rabbit IgG or anti-mouse IgG (Jackson-ImmunoResearch, West Grove, PA). Blots were visualized by incubation with SuperSignal West Dura chemiluminescence kit (Pierce Biotechnology) and exposing to light-sensitive film.
The bands were quantified as “intensity×area” using Image-Pro Plis software (MediaCybernetics) and statistically analyzed. SE was calculated from three repeats of the experiments or five individuals of arctigenin treated and vehicle groups.
RT-PCR and quantitative real-time PCR
Total RNA was extracted from administrated cells or mice tissues using RNAiso (TaKaRa) reagent in accordance with the Kit instruction. cDNA was synthesized by RT reagent Kit (TaKaRa) and real-time PCR was performed using SYRB Premix Ex Taq (TaKaRa) on DNA Engene Opticon TM2 system (MJ Research, Waltham, MA, USA). The primers were listed as follows:
PGC-1α: sense 5′- gcccggtacagtgagtgttc-3′, anti 5′- ctgggccgtttagtcttcct-3′.
ERRα: sense 5′- ctcagctctctacccaaacgc-3′, anti 5′- ccgcttggtgatctcacactc-3′.
Cytochrome c: sense 5′- cagcttccattgcggacac-3′, anti 5′- ggcactcacggcagaatgaa-3′.
PDK4: sense 5′- agggaggtcgagctgttctc-3′, anti 5′- ggagtgttcactaagcggtca-3′.
FAS: sense 5′- ggaggtggtgatagccggtat-3′, anti 5′- tgggtaatccatagagcccag-3′.
mCPT1b: sense 5′- tgggactggtcgattgcatc-3′, anti 5′- tcagggtttgtcggaagagaga-3′.
SCD1: sense 5′- ttcttgcgatacactctggtgc-3′, anti 5′- cgggattgaatgttcttgtcgt-3′.
GAPDH: sense 5′- acagcaacagggtggtggac-3′, anti 5′- tttgagggtgcagcgaactt-3.
MHCI: sense 5′- ccttggcaccaatgtcccggctc-3′, anti 5′- gaagcgcaatgcagatgcggtg-3.
MHCIIa: sense 5′- atgagctccgacgccgag-3′, anti 5′- tctgttagcatgaactggtaggcg-3.
MHCIIx: sense 5′- aaggagcaggacaccagcgccca-3′, anti 5′- atctctttggtcactttcctgct-3.
MHCIIb: sense 5′- gtgatttctcctgtcacctctc-3′, anti 5′- ggaggaccgcaagaacgtgctga-3.
Luciferase assay
For evaluation of the effects of arctigenin on PGC-1α promoter, HEK293T cells (24-well plate) were transfected with pGL3-PGC-1α-luc together with PRL-SV40 and refreshed with normal medium 5 hours later. After transfection, HEK293T cells were incubated with indicated concentration of arctigenin for 24 h. Luciferase activity was measured using Dual Luciferase Assay kit.
AMPK enzymatic activity assay
The recombinant AMPK activity was assayed using the modified conventional approach in non-radioactive way (such as Jun N-terminal kinase 2 (Jnk2α2), casein kinase 1, Protein kinase A (PKA), etc.) [63], as outlined in Fig. S10.
In the assay, the substrate SAMS was used according to the literature methods [41], [42]. The recombinant AMPK isoform α2β1γ1 was purchased from Invitrogen. AMPK was diluted to 400 ng/ml in assay buffer (pH 7.4, containing 15 mM HEPES, 20 mM NaCl, 1 mM EGTA, 0.02% Tween) and pre-incubated with arctigenin (0.2, 2, 20 µM) or A-769662 (1 µM) for 30 min on ice. The kinase reaction was initiated by adding ATP (50 µM) and SAMS (50 µM) at room temperature for 30 min. The produced ADP reflecting the AMPK enzyme activity was thus measured by the ADP Hunter Plus Assay kit (DiscoverX), and the fluorescent signal was detected with an M5 Multi-Detection Reader using excitation and emission wavelengths of 530 and 590 nm.
Animal experiment
C57BL/6J male mice at 6 weeks of age were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences, and acclimated to SPF microisollators for 2 days before any experimental intervention. Mice were accommodated under standard conditions (strict 12:12-h light-dark cycle, 22°C, 60% humidity) in plastic cages and provided with water and food ad libitum.
All 30 mice were adapted to treadmill running for 10 min at 10 m/min at week -1 (5 days/week) avoiding unexpected accidents and the first fatigue test was conducted at week 0. For fatigue test, mice ran at 10 m/min for 5 min and 15 m/min for 10 min. After the initial warm-up period, exercise intensity was increased by 5 m/min every 30 min from 20 m/min until mice could not be prompted to continue running by moderate electric stimulation (less than 0.1 milliampere) and stayed at electrode for at least 10 sec. After the first fatigue test, 20 mice with moderate endurance capacity were selected from total 30 mice and divided into arctigenin administration and vehicle treatment groups (n = 10/group). Before final fatigue assay, sedentary mice were treated with arctigenin (8 mg/kg, body weight/day) or vehicle (sterilized 0.9% Sodium Chloride containing 5% Tween-80) daily via intraperitoneal injection for 6 weeks. Mice endurance capacity was estimated by treadmill (Litai Science and Technology Inc. Exer6, Hangzhou, China) running time and distance until fatigue [11], [62]. After 6-week arctigenin administration, the last fatigue test was performed according to the same protocol as before (n = 8/group). For investigation of the relevant gene changes in tissues, two mice in each group as control were chosen escaping the fatigue test.
Tissue collection
Animals were euthanized 72 h after the last bout of exercise. Gastrocnemius, quadriceps and heart muscles were thus isolated, frozen, and stored at −80°C until further analysis.
Statistical analysis
All data were reported as mean ± standard deviation of the mean (SD). Data were analyzed in either one-way ANOVA with an appropriate post hoc test for comparison of multiple groups or unpaired student’s t test for comparison of two groups as described in figure legends (Graphpad Prism software).
Supporting Information
Figure S1

Arctigenin (ATG) enhanced AMPK phosphorylation in HEK293T cells. HEK293T cells were incubated with indicated concentrations of arctigenin (0-40 µM) for 30 min, phospho- and total AMPK were then detected by western blotting. The results shown are representative of three independent experiments. The bands were quantified using Image-Pro Plus software. Values are means ± SE. **, p<0.05; ***, p<0.005; one-way ANOVA.
Figure S2

Arctigenin (ATG) activated PGC-1α transcription via up-regulating AMPK phosphorylation. A. When the confluence reached 30∼40% (24-well plate), HEK293T cells were transiently transfected with pGL3-PGC-1α promoter-Luc and SV40. 5 hours later, cells were refreshed with medium supplemented with arctigenin (1, 10, 40 µM) or DMSO and incubated for 24 hours before Luciferase assays as described in “Materials and methods”. B. After transfection, HEK293T cells were administrated with or without 20 µM compound C for 1 hour before and during the incubation with actigenin (40 µM) for 24 hours before Luciferase assays as described in “Materials and methods”. **, p<0.01. ##, p<0.01: for compound C and arctigenin co-incubation group versus arctigenin treated group; student’s t test.
Figure S3

Effects of arctigenin (ATG) on ERRα, cytochrome c, PDK4, SCD1, FAS and mCPT1b were subjective to AMPK phosphorylation in H9C2. H9C2 cells were treated with or without 20 µM compound C for 1 hour before and during the incubation with actigenin (20 µM) for 24 hours. After harvested, mRNA levels of ERRα (A), cytochrome c (B), SCD1 (C), PDK4 (D), FAS (E) and mCPT1b (F) were analyzed. The results shown are representative of three independent experiments. Values are means ± SD. *, p<0.05. #, p<0.05: for compound C and arctigenin co-incubation group versus arctigenin treated group; student’s t test.
Figure S4

Effects of arctigenin (ATG) on ERRα, cytochrome c, PDK4, SCD1, FAS and mCPT1b were subjective to AMPK phosphorylation in C2C12. Differentiated C2C12 cells were administrated with or without 20 µM compound C for 1 hour before and during the incubation with actigenin (20 µM) for 24 hours. After harvested, mRNA levels of ERRα (A), cytochrome c (B), SCD1 (C), PDK4 (D), FAS (E) and mCPT1b (F) were analyzed. The results shown are representative of three independent experiments. Values are means ± SD. *, p<0.05; **, p<0.01; ***, p<0.005. #, p<0.05; ##, p<0.01; ###, p<0.005: for compound C and arctigenin co-incubation group versus arctigenin treated group; student’s t test.
Figure S5

Arctigenin (ATG) failed to regulate the co-activator recruitment and transcriptional activity of PPARδ. A. HEK293T cells were transfected with UAS-TK-Luc, pCMX-Gal4DBD-PPARδ-LBD and pRL-SV40 followed by treatment of DMSO, GW501516 (PPARδ agonist), and varied concentrations of arctigenin for 24 hours. B. HEK293T cells were transfected with pAdTrack-PPARδ, pcDNA3.1-RXRα, pSV-PPRE-Luc and pRL-SV40 and then incubated with DMSO, GW501516 (PPARδ agonist), and varied concentrations of arctigenin for 24 hours. C. HEK293T cells were transfected with pSV-PPRE-Luc and pRL-SV40, and incubated with DMSO, GW501516 (PPARd agonist), and varied concentrations of arctigenin for 24 hours. Relative luciferase activities were measured as described in Text S1. The results shown are representative of three independent experiments. Values are means ± SD. *, p<0.05; **, p<0.01; ***, p<0.005; one-way ANOVA.
Figure S6

Effects of arctigenin (ATG) on diet, weight, inflammation and liver toxicity of mice. A. Daily food intake of each group was analyzed (n = 10/group). B. Weight change in each group was measured (n = 10/group). C. D. Serum from mice in each group was collected and levels of TNFα (C) and IL-6 (D) were analyzed (n = 10/group). E. F. Activities of ALT (E) and AST (F) were measured (n = 10/group). Values are means ± SE. *, p<0.5; **, p<0.01; student’s t test.
Figure S7

Arctigenin (ATG) failed to induce skeletal muscle fiber-type change. Metachromatical staining of frozen cross-sections from gastrocnemius and quadriceps in vehicle and arctigenin treated groups. The results shown are representative of three independent experiments. Dark-brown stained type I fibers were indicated by arrows.
Figure S8

Arctigenin (ATG) enhanced fatty acid storage in gastrocnemius. Free fatty acid in gastrocnemius (A) or quadriceps (B) of each group was analyzed (n = 7/group). Values are means ± SD. *, p<0.5; student’s t test.
Figure S9

Arctigenin (ATG) failed to impact the phosphorylation of AMPK on Ser485/491 sites. HEK293T, H9C2 and differentiated C2C12 cells were incubated with indicated concentrations of arctigenin (0–40 µM) for 30 min, AMPK (Thr172), AMPK (Ser485/491) and total AMPK were then detected by western blotting. The results shown are representative of three independent experiments.

Figure S10

A scheme demonstrating recombinant AMPK activity assay approach.


The increased energy required for acute moderate exercise by skeletal muscle (SkM) is derived equally from enhanced fatty acid (FA) oxidation and glucose oxidation. Availability of FA also influences contracting SkM metabolic responses. Whole body glucose turnover and SkM glucose metabolic responses were determined in paired dog studies during 1) a 30-min moderate exercise (maximal oxygen consumption of ∼60%) test vs. a 60-min low-dose 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) infusion, 2) a 150-min AICAR infusion vs. modest elevation of FA induced by a 150-min combined intralipid-heparin (IL/hep) infusion, and 3) an acute exercise test performed with vs. without IL/hep. The exercise responses differed from those observed with AICAR: plasma FA and glycerol rose sharply with exercise, whereas FA fell and glycerol was unchanged with AICAR; glucose turnover and glycolytic flux doubled with exercise but rose only by 50% with AICAR; SkM glucose-6-phosphate rose and glycogen content decreased with exercise, whereas no changes occurred with AICAR. The metabolic responses to AICAR vs. IL/hep differed: glycolytic flux was stimulated by AICAR but suppressed by IL/hep, and no changes in glucose turnover occurred with IL/hep. Glucose turnover responses to exercise were similar in the IL/hep and non-IL/hep, but SkM lactate and glycogen concentrations rose with IL/hep vs. that shown with exercise alone. In conclusion, the metabolic responses to acute exercise are not mimicked by a single dose of AICAR or altered by short-term enhancement of fatty acid supply.

Exercise vs. AICAR Infusion

With 30 min of moderate exercise, plasma glucose, insulin [4.1 ± 1.0 (before) vs. 3.0 ± 0.5 mU/l (after)], and glucagon [75 ± 17 (before) vs. 93 ± 21 ng/l (after); P = not significant (NS)] were unchanged; however, plasma FFA, lactate, and glycerol all rose significantly (P > 0.01) (Fig. 1). In contrast, with the 60-min AICAR infusion, plasma glucose fell (P = 0.05), insulin and glucagon were unchanged [insulin = 4.7 ± 0.5 (before) vs. 6.1 ± 0.8 mU/l (after), P = NS; glucagon = 53 ± 11 (before) vs. 68 ± 11 ng/l (after), P = NS], plasma FFA decreased significantly (P < 0.01), with no change in glycerol, and lactate rose sharply (P < 0.01) (Fig. 1). Thus the incremental responses of FFA and glycerol to exercise and AICAR were significantly different and in the opposite directions (Fig. 1). There was a greater than twofold increase (P < 0.02–0.01) in HGP, Rd, MCRg, and GFexog after the 30-min moderate exercise test (Fig. 2). In contrast, although these parameters also increased after the 60-min AICAR infusion (P < 0.02), the responses were considerably less (∼30–40%) and significantly (P < 0.01) smaller for the AICAR than for the exercise responses (Fig. 2). The metabolic responses to AICAR at 150 min were similar to those obtained at 60 min, with only a minor attenuation of MCRg by 150 min. SkM substrate concentrations of intracellular glucose and lactate did not change with exercise alone, whereas G6P significantly increased (change of 1.0 ± 0.3 mmol/l; P = 0.05) and glycogen decreased (change of –35 ± 36 mmol/kg dry wt; P = 0.06). In contrast, during AICAR infusion, a decrease in intracellular SkM glucose (change of –0.20 ± 0.0.9 mmol/l; P = 0.06) was noted, but no significant changes occurred in G6P, lactate, or glycogen. However, the absolute decrease of the glycogen concentration was significantly different for exercise compared with that for AICAR (change of –35 ± 36 vs. 9 ± 36 mmol/kg dry wt; P = 0.06). There were no changes in AMPK-α1 and AMPK-α2 activities with exercise or AICAR infusion (data not shown), but ACCβ-pSer221 was elevated by ∼35% [from basal level of 1.05 ± 0.02 to 1.38 ± 0.15 (P ≤ 0.05) with exercise and to 1.32 ± 0.15 (arbitrary units) (P = 0.06) with AICAR].

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Fig. 1. Paired comparisons of absolute plasma substrate responses and incremental changes (Δ) after either 30-min moderate exercise [exer; ∼60% maximal oxygen consumption (O2max); open bars] or 60-min low-dose 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) infusion (hatched bars) in 6 dogs. A: plasma (P) glucose. B: plasma free fatty acid (FFA). C: lactate. D: glycerol. *P = 0.05; **P < 0.01 vs. the within-group basal state; ††P < 0.01 between groups.

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Fig. 2. Paired comparisons of absolute responses and incremental changes (Δ) of hepatic glucose production (HGP; A), metabolic clearance rate of glucose (MCRg; B), Rd (C), and glycolytic flux (GFexog; D) to 30-min moderate exercise (∼60% O2max) (open bars) or 60-min AICAR infusion (hatched bars) in 6 dogs. *P < 0.02; **P < 0.01 vs. the within-group basal state; ††P < 0.01 between groups.

IL/Hep Infusion vs. Saline and AICAR Infusions

The 150-min IL/hep infusion alone raised plasma FFA by ∼60% and glycerol by ∼80% (P < 0.05–0.01) (Table 1). This resulted in a small but significant fall in fasting glucose (P ≤ 0.05), with no changes in insulin or glucagon (Table 1). There were also no changes induced by the IL/hep infusion in HGP, Rd, and MCRg, but GFexog fell significantly (P < 0.05) by ∼20% (Table 1). The effects of increasing FA supply compared with the AICAR infusion at 150 min were different (Table 1). Plasma glucose did not change; however, in the IL/hep group, the change in plasma lactate was markedly reduced (P < 0.001) and, as expected, FFA and glycerol were raised, compared with the responses observed in the AICAR studies (P ≤ 0.05–0.01) (Table 1). The modest significant increments observed for HGP, Rd, MCRg, and GFexog with AICAR were not observed in the IL/hep studies (Table 1). There was no relationship between the changes in plasma glucagon and HGP with the combined AICAR and IL/hep groups (r = 0.40, P = 0.22). After the IL/hep infusion, there were no changes (Δ) in intramuscular substrates (Δglucose = –0.05 ± 0.27 mmol/l, ΔG6P = 0.7 ± 0.7 mmol/kg dry wt, Δlactate = 0.51 ± 0.50 mmol/l, Δglycogen = 15 ± 33 mmol/kg dry wt, and ΔACCβ-pSer221 = 0.03 ± 0.05 arbitrary units).

Table 1. Comparison of the absolute and incremental effects of 60-min and 150-min AICAR infusion and 150-min saline or IL/heparin infusion on plasma substrates and hormones, glucose turnover, and skeletal muscle substrates in 6 resting normal dogs

Exercise With or Without IL/Hep Infusion

The exercise-induced responses of plasma substrates in the presence or absence of excess FA supply were similar for plasma glucose, glycerol, lactate, insulin, and glucagon. No exercise-induced increments for plasma FA were observed in the IL/hep group, since the FFA levels were already raised preexercise above those seen postexercise for the non-IL/hep group (Fig. 3). On the background of the lipid-induced lower basal GFexog (Table 1), the glucose turnover responses to exercise were similar for the IL/hep and non-IL/hep groups for HGP, Rd, MCRg, and GFexog (Fig. 4). Only small responses in the SkM substrates with exercise performed with and without IL/hep infusion were noted [Δglucose = 0.3 ± 0.1 vs. 0.0 ± 0.2 mmol/l, ΔG6P = 0.8 ± 0.6 vs. 1.0 ± 0.3 mmol/l, Δlactate = 2.6 ± 1.1 vs. 0.0 ± 0.3 mmol/l (P = 0.07), Δglycogen = 30 ± 37 vs. –35 ± 36 mmol/kg dry wt (P = 0.09), and ΔACCβ-pSer221 = 0.29 ± 0.06 vs. 0.23 ± 0.15 arbitrary units, respectively].

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Fig. 3. Paired comparisons of absolute responses and incremental changes of plasma substrates to 30-min moderate exercise (∼60% O2max), either in the absence [open bars, with saline (Sal)] or presence (hatched bars) of intralipid-heparin (IL/hep) infusion in 6 dogs. A: plasma glucose. B: plasma FFA. C: plasma lactate. D: plasma glycerol. *P < 0.05; **P < 0.01 vs. the within-group basal state; †P ≤ 0.06 between groups.

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Fig. 4. Paired comparisons of absolute responses and incremental changes of HGP (A), MCRg (B), Rd (C), and GFexog (D) to moderate exercise (∼60% O2max), either in the absence (open bars) or presence (hatched bars) of IL/hep infusion in 6 dogs. **P < 0.01 vs. the within-group basal state; †P ≤ 0.05 vs. the corresponding basal study without IL/hep.


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AICAR, aminoimidazole carboxamide ribonucleotide, acts as an agonist to AMP-activated protein kinase; AMP-activated protein kinase, also known as AMPK, is an enzyme with an important role in cellular homeostasis and energy regulation.[1]  AMPK acts through a variety of means to ultimately stimulate liver fatty oxidation, ketogenesis, beta-cell modulation of insulin secretion, and other functions within the body.  AICAR has been shown to stimulate glucose uptake and reduce apoptosis by reducing reactive oxygen compounds within cells.[2][3]

In a breakthrough study in 2008, Narkar et al of the Salk Institute discovered that AICAR significantly improves the performance of mice in endurance-type exercise by converting fast-twitch muscle fibers to the more energy-efficient, fat-burning, slow-twitch type. They also found that AICAR and GW1516, when given to “sedentary” mice, activated 40% of the genes that were turned on when mice were given GW1516 and made to exercise. As a result a publicity storm about “exercise pills” and “exercise in a pill” ensued.  The World Anti-Doping Agency now lists both compounds on their prohibited list (since 2009), and the lead researcher of the breakthrough study cooperated in providing data to make possible a urinalysis test to detect AICAR.[4][5]

Figure 5 Likely effects of AICAR (and other AMPK activators) on glucose homoeostasis in vivo

AICAR (1) stimulates glucose uptake into muscle through the membrane recruitment of Glut4, (2) inhibits hepatic glucose output and triacylglycerol synthesis, (3) inhibits both glucose uptake and lipolysis by adipose tissue, (4) acutely suppresses insulin release from pancreatic islets, and (5) activates glucose-responsive neurons in the paraventricular and arcuate nuclei of the hypothalamus, potentially stimulating appetite. Effects (1)–(4) probably explain the glucose-lowering effects of AICAR (and contribute to the effects of metformin and glitazones) and are likely to be beneficial in Type II diabetes. Effects (4) and (5) may be contra-indicated. FA, fatty acids.

AICAR has a number of other experimental/clinical and research chemical uses as it is expressed in a variety of tissue types.  Bai et al found that “data demonstrate that AICAR-initiated AMPK activation may represent a promising alternative to our current approaches to suppressing intestinal inflammation in IBD.”[6]

Guo et al found “results suggest[ing] a mechanism by which AICAR inhibits the proliferation of EGFRvIII expressing glioblastomas and point toward a potential therapeutic strategy for targeting EGFR-activated cancers.”[7]

An original study by Pold et al offers additional hope that AICAR could offer important treatment potential for humans:

Five-week-old, pre-diabetic ZDF rats underwent daily treadmill running or AICAR treatment over an 8-week period and were compared with an untreated group. In contrast to the untreated, both the exercised and AICAR-treated rats did not develop hyperglycemia during the intervention period. Whole-body insulin sensitivity, as assessed by a hyperinsulinemic-euglycemic clamp at the end of the intervention period, was markedly increased in the exercised and AICAR-treated animals compared with the untreated ZDF rats (P < 0.01). In addition, pancreatic beta-cell morphology was almost normal in the exercised and AICAR-treated animals, indicating that chronic AMPK activation in vivo might preserve beta-cell function. Our results suggest that activation of AMPK may represent a therapeutic approach to improve insulin action and prevent a decrease in beta-cell function associated with type 2 diabetes.[8]


[1]Corton JM, Gillespie JG, Hawley SA, Hardie DG. “5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?”. Eur. J. Biochem. 229 (2): 558–65.  1995.

[2]Lemieux K, Konrad D, Klip A, Marette A. “The AMP-activated protein kinase activator AICAR does not induce GLUT4 translocation to transverse tubules but stimulates glucose uptake and p38 mitogen-activated protein kinases alpha and beta in skeletal muscle”. Faseb J. 17 (12): 1658–65. 2003.

[3]Kim JE, Kim YW, Lee IK, Kim JY, Kang YJ, Park SY.  “AMP-activated protein kinase activation by

5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) inhibits palmitate-induced endothelial cell apoptosis through reactive oxygen species suppression”. J. Pharmacol. Sci. 106 (3): 394–403. 2008.

[4]Narkar VA, Downes M, Yu RT, Embler E, Wang YX, Banayo E, Mihaylova MM, Nelson MC, Zou Y, Juguilon H, Kang H, Shaw RJ, Evans RM. “AMPK and PPARdelta agonists are exercise mimetics”. Cell 134 (3): 405–15. 2008. [5]WADA 2009 Prohibited List: WADA PROHIBITED LIST PDF (PDF Document).

[6]Bai A, Yong M, Ma Y, Ma A, Weiss C, Guan Q, Bernstein C, Peng Z. Novel Anti-Inflammatory Action of 5-Aminoimidazole-4-carboxamide ribonucleoside with protective effect in DSS-induced acute and chronic colitis. J Pharmacol Exp Ther. 2010 Mar 17.

[7]Guo D, Hildebrandt IJ, Prins RM, Soto H, Mazzotta MM, Dang J, Czernin J, Shyy JY, Watson AD, Phelps M, Radu CG, Cloughesy TF, Mischel PS.  The AMPK agonist AICAR inhibits the growth of EGFRvIII-expressing glioblastomas by inhibiting lipogenesis.Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):12932-7.

[8]Pold R, Jensen LS, Jessen N, Buhl ES, Schmitz O, Flyvbjerg A, Fujii N, Goodyear LJ, Gotfredsen CF, Brand CL, Lund S. Long-term AICAR administration and exercise prevents diabetes in ZDF rats.  Diabetes. 2005 Apr;54(4):928-34.

*The latter article is intended for educational / informational purposes only. THIS PRODUCT IS INTENDED AS A RESEARCH CHEMICAL ONLY. This designation allows the use of research chemicals strictly for in vitro testing and laboratory experimentation only. Bodily introduction of any kind into humans or animals is strictly forbidden by law.