The effect of an intracerebroventricular injection of metformin or AICAR on the plasma concentrations of melatonin in the ewe: potential involvement of AMPK?
© Menassol et al; licensee BioMed Central Ltd. 2011
Received: 22 March 2011
Accepted: 29 July 2011
Published: 29 July 2011
It is now widely accepted that AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis. Recently, it has been shown to regulate circadian clocks. In seasonal breeding species such as sheep, the circadian clock controls the secretion of an endogenous rhythm of melatonin and, as a consequence, is probably involved in the generation of seasonal rhythms of reproduction. Considering this, we identified the presence of the subunits of AMPK in different hypothalamic nuclei involved in the pre- and post-pineal pathways that control seasonality of reproduction in the ewe and we investigated if the intracerebroventricular (i.c.v.) injection of two activators of AMPK, metformin and AICAR, affected the circadian rhythm of melatonin in ewes that were housed in constant darkness. In parallel the secretion of insulin was monitored as a peripheral metabolic marker. We also investigated the effects of i.c.v. AICAR on the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in brain structures along the photoneuroendocrine pathway to the pineal gland.
All the subunits of AMPK that we studied were identified in all brain areas that were dissected but with some differences in their level of expression among structures. Metformin and AICAR both reduced (p < 0.001 and p < 0.01 respectively) the amplitude of the circadian rhythm of melatonin secretion independently of insulin secretion. The i.c.v. injection of AICAR only tended (p = 0.1) to increase the levels of phosphorylated AMPK in the paraventricular nucleus but significantly increased the levels of phosphorylated ACC in the paraventricular nucleus (p < 0.001) and in the pineal gland (p < 0.05).
Taken together, these results suggest a potential role for AMPK on the secretion of melatonin probably acting trough the paraventricular nucleus and/or directly in the pineal gland. We conclude that AMPK may act as a metabolic cue to modulate the rhythm of melatonin secretion.
The serine-threonine kinase, AMPK integrates energy homeostasis at both cellular  and whole-body levels . It exists as a heterotrimeric complex of a catalytic α subunit and two regulatory β and γ subunits. In mammals there are two or three isoforms of each subunit (α1, α2, β1, β2, γ1, γ2, γ3), encoded by different genes, allowing for a variety of heterotrimeric combinations . The treatment with pharmacological agents such as metformin and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) activates the AMPK complex both in vitro and in vivo [4–6]. This mechanism is central in the action of metformin when used as a insulin-sensitizing agent to treat type II diabetes . Recent evidence points to an emerging role for AMPK as a critical element linking metabolism and reproduction . Indeed, we demonstrated that treatment with either metformin or AICAR significantly inhibited the release of GnRH from immortalised GnRH neurones (GT1-7 cells) and in vivo, it shortened the inter-oestrus interval in mice . In sheep as in all seasonal breeding species, the daily rhythm of melatonin secretion is the critical element mediating the synchronising effect of photoperiod on reproduction. This rhythm is endogenously controlled by the activity of the master circadian pacemaker located in the suprachiasmatic nucleus . However, how this endogenous rhythm of melatonin affects the seasonal activity of the GnRH/LH/gonadal axis remains unclear.
Um et al.  demonstrated that the peripheral activation of AMPK with metformin induced phase-shifts in some of the core clock proteins thus suggesting a role for AMPK in the control of circadian rhythms. In support of these observations we have identified AMPK in the sheep brain and we have tested, the effects of i.c.v. injections of metformin or AICAR on the secretion of melatonin in ewes housed in constant darkness. In parallel, because of the functional interrelationships between melatonin and insulin [11, 12], we also monitored the secretion of insulin as a potential metabolic marker of peripheral AMPK activation . Our results suggest that i.c.v. injections of metformin and AICAR both affect the amplitude of the circadian rhythm of melatonin secretion without altering the patterns of plasma insulin secretion.
Hormones and reagents
The metformin and AICAR used for the i.c.v. injections were obtained from Sigma (Saint Quentin Fallavier, France). The dose injected by means of an intracerebroventricular catheter, was 128 mM for metformin (adapted to sheep from ) and 12 mM for AICAR (similar to ) in a total volume of 50 μl. Untreated control ewes were injected with the same volume of an artificial cerebrospinal fluid (CSF) solution, prepared in the laboratory. All solutions had an equivalent osmolarity (≈ 305 mOsm).
Rabbit polyclonal antibodies to phospho-AMPKα Thr172, AMPKα1/2, AMPKβ1/β2, AMPKγ2, phospho-acetyl-CoA carboxylase (ACC) Ser79 and ACC were purchased from New England Biolabs Inc. (Beverly, MA, USA). A mouse monoclonal antibody to vinculin was obtained from Sigma (Saint Quentin Fallavier, France) and used as an internal standard.
Animals and experimental designs
Nineteen multiparous ewes were surgically fitted with cannulae in the anterodorsal region of their third ventricles . Animals were housed in individual pens in a light-sealed room under a lightning program mimicking a long-day photoperiod (LP) (16 hours of light/day). All ewes had free access to food, water and mineral licks.
For the second experiment, two groups of ewes balanced as for experiment 1, were injected with either AICAR (n = 6) or artificial CSF (n = 5). One hour after injection the animals were slaughtered by decapitation, the brain was removed quickly and frozen in isopentane (SDS, Val De Rueil, France). The pituitary (PIT) and pineal (PIN) glands were immediately dissected, frozen in liquid nitrogen and stored at -80°C. Frontal sections (1 mm thick) were sliced from the diencephalon and other structures of interest were dissected using a sterile scalpel, all were frozen in liquid nitrogen and stored at -80°C. The hypothalamic structures of interest (suprachiasmatic nucleus [SCN], arcuate nucleus [ARC], median eminence [ME], paraventricular nucleus [PVN], dorsomedial hypothalamic nucleus [DMH], tanycytes [TAN] and premammilary hypothalamic nucleus [PMH]) were identified using sheep brain atlases [16, 17].
All these procedures were approved by the French Agricultural and Scientific Research Agencies and conducted in accordance with the EU guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching.
Western-blotting procedures were conducted as previously described  using 60 μg of protein per sample. The data for the phosphorylation of AMPK and ACC are expressed as the ratio of phosphorylated AMPK to total AMPK and phosphorylated ACC to total ACC respectively. The data for the AMPK subunits have been normalised against an internal standard (vinculin).
Melatonin was assayed using a radioimmunoassay (RIA) described by Fraser et al. , with an antibody raised by Tillet et al. . The assay sensitivity was 4 pg/mL, the intra-assay coefficient of variation (CV) ranged between 9-14% and the inter-assay CV was 12%. Insulin was also assayed using a RIA that was developed in our laboratory. Plasma concentrations of insulin were determined in duplicate 100 μL samples. The assay buffer was 0.025 M barbital buffer containing 3% bovine serum albumin (Sigma, Saint Quentin Fallavier, France). Plasma samples were incubated (24 hrs at 4°C) with 300 μL of the primary antibody (anti-porcine insulin antibody raised in guinea pig, final dilution 1/80000; Sigma, Saint Quentin Fallavier, France) added with 0.6 μL of Triton X-100 and 0.5 μL of normal rabbit serum. Samples were then incubated (24 hrs) with 20,000 cpm of iodinated porcine insulin. The next day, samples were incubated for 48 hrs at 4°C with 2 mL of the secondary antibody (anti-guinea pig IgG raised in horses, final dilution 1/333), 4 g/L of CaCl2 and 30 g/L of polyethylene glycol. Tubes were then centrifuged (3,000 g for 30 min at 4°C), the supernatant was removed, and the radioactivity in the precipitate was counted with a Wallac Wizard gamma counter (PerkinElmer Inc., Waltham, Massachusetts, USA) for 1 min. The sensitivity of the assay was 0.95 ± 0.06 ng/mL and the intra-assay CV was 12%.
Data and Statistical analysis
Melatonin profiles were analysed by comparing the timing of resumption and the amplitude, of the circadian rhythm of melatonin secretion. These criteria were determined using the method of Malpaux et al. . Statistical analyses were performed using the statistical software package, R . Data for insulin were analysed using a mixed-effects model and other data, after log transformation for normalization, were analysed using an analysis of variance. All results are presented as means ± sem. Differences were considered statistically significant whenever p < 0.05.
The hypothesis tested in this study, developed from the findings of Um et al. , was that central activation of AMPK could disrupt the circadian machinery located in the SCN and thus, alter the endogenous timing of the resumption of melatonin secretion by the pineal gland. In our study, such an effect was not observed after i.c.v. treatment with metformin or AICAR, both known activators of AMPK. Surprisingly we found that another component of the endogenous rhythm of melatonin, its amplitude, was altered by these compounds.
There are three main possibilities to account for the rejection of our hypothesis. First, the i.c.v. treatment with either metformin or AICAR was ineffective and did not stimulate AMPK in the hypothalamic structures we examined. Indeed, the similarities of the effects between the i.c.v. injections of both drugs on the amplitude of the rhythm of melatonin secretion suggest that they might be mediated by the activation of AMPK, as a common element along their distinct signalling pathways . However, we do not exclude possible AMPK-independent effects . Second, the treatment effectively activated AMPK within the SCN, but in DD when the SCN is released from entrainment by the prevailing LP produced conflicting signals because of the persistence of entrainment by the preceding LP  and the expected SCN-driven phase shift in the resumption of melatonin secretion. Third, the treatment effectively activated AMPK but not within the SCN or without triggering phase-shifts in the master circadian machinery perhaps because of functional differences  between the master circadian clock and the peripheral-slaves clock that was studied by Um et al. .
The biological relevance of the lowering effects of both metformin and AICAR on the amplitude of the endogenous rhythm of melatonin is difficult to interpret. Indeed, in sheep as in most photoperiodic species, the duration of melatonin secretion is the critical feature of the rhythm of melatonin secretion that transmits photoperiodic information to the reproductive axis ; the role played by the amplitude, the other main feature of this rhythm, remains controversial [27–29]. Some authors have suggested that the amplitude of the melatonin rhythm acts as a potent integrator of environmental factors, other than photoperiod for example, temperature and the food supply (for review see ). In relation to this theory, a complementary study (Menassol et al., unpublished data) has established that feed restriction can alter the seasonal reproductive patterns of the Île-de-France ewe as has been reported in a previous study on sheep , and that this alteration is associated with lower concentrations of plasma melatonin. Therefore we speculate that the central activation of AMPK is involved in the integration of metabolic-related cues that alter the amplitude of the rhythm of melatonin secretion. We speculate further that this effect is mediated by neuropeptides or peptidergic hormones that regulate the amplitude of the rhythm of melatonin secretion such as Vasoactive Intestinal Peptide (VIP), Pituitary Adenylate Cyclase Activating Peptide (PACAP) or Vasopressin (VP) (for review see ) and particularly Neuropeptide Y (NPY) which is activated by AMPK .
The effect of metformin and AICAR on the amplitude of the rhythm of melatonin secretion was not associated with immediate changes in insulin secretion and thus, was not attributable to the interrelationship that exists between these two hormones [11, 12, 34]. Interestingly, some studies have reported a decrease in melatonin concentrations associated with the development of diabetes [35, 36]. Thus, assuming that metformin crosses the blood-brain barrier, one can ask if therapeutic treatment with metformin could not have unreported side-effects on the secretion of melatonin.
The characterization of the expression of AMPK within the hypothalamus, pituitary and pineal glands of the ewes suggests a potentially wide range of action for metformin and AICAR since all subunits were identified in each of the structures that we examined. The differences among structures in the level of expression of the β2 and γ2 subunits may indicate structural specificities in the sensitivity or functionality of the AMPK complex . Indeed, our investigation of the potential central sites of actions of the AMPK complex, point preferentially to the PVN as a structure of particular interest because some of its neurons that express melatonin-regulating peptides project to the pineal gland . In this particular structure, the direct assessment of the phosphorylation rate of AMPK only reveals a trend toward significance (p = 0.1) for a greater activation after i.c.v. injection of AICAR. However the indirect measurement of AMPK activity, trough the assessment of the phosphorylation rate of its downstream target ACC [37, 38], shows that AICAR treatment significantly inhibited ACC activity in both the paraventricular nucleus (p < 0.001) and the pineal gland (p < 0.05). Therefore the PVN is strongly suggested as a key structure that mediates metabolic influences on the rhythm of secretion of melatonin. Moreover, it seems that direct effects of AMPK on the pineal gland activity itself can not be excluded.
In this experiment, the failure to demonstrate significant direct activation of the AMPK complex following treatment with AICAR might be explained by the time elapsed between tissue fixation and injection (1 hr) and the possible action of endogenous phosphatases .
To conclude, the i.c.v. administration of AMPK-activators had an unexpected insulin-independent effect on the amplitude of the endogenous rhythm of secretion of melatonin that retrospectively, might have also been observed in natural photoperiodic conditions, on the daily pattern of melatonin secretion. This effect probably involves the AMPK system trough the paraventricular nucleus and/or directly within the pineal gland. This mechanism remains to be precisely determined but we suggest that it involves the integration of metabolic-related cues with the photoperiodic signal that synchronizes the annual rhythm of reproduction in sheep.
The authors would like to acknowledge Dr Laurence Dufourny for her help with brain dissections as Dr Jean-Claude Thiéry for his help in pre-experimental studies. Dr Sébastien Elis and Christelle Ramé are acknowledged for their technical help. The authors also thank the staff of the experimental unit of INRA Centre de Tours (UEPAO) for the care provided to the animals, as well as the staff of the Experimental Hospital (PRC) for surgical procedures. RJS was the recipient of an EU Marie Curie Chair of Excellence (MEXC-CT-2006-042499) and JBM a PhD grant from the "Ministère de l'Enseignement Supérieur et de la Recherche".
- Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, Small CJ: AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem. 2004, 279 (13): 12005-12008.View ArticlePubMed
- Kahn BB, Alquier T, Carling D, Hardie DG: AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 2005, 1 (1): 15-25. 10.1016/j.cmet.2004.12.003.View ArticlePubMed
- Hardie DG, Scott JW, Pan DA, Hudson ER: Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett. 2003, 546 (1): 113-120. 10.1016/S0014-5793(03)00560-X.View ArticlePubMed
- Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE: Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001, 108 (8): 1167-1174.PubMed CentralView ArticlePubMed
- 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. 1995, 229 (2): 558-565. 10.1111/j.1432-1033.1995.tb20498.x.View ArticlePubMed
- Coyral-Castel S, Tosca L, Ferreira G, Jeanpierre E, Rame C, Lomet D, Caraty A, Monget P, Chabrolle C, Dupont J: The effect of AMP-activated kinase activation on gonadotrophin-releasing hormone secretion in GT1-7 cells and its potential role in hypothalamic regulation of the oestrous cyclicity in rats. J Neuroendocrinol. 2008, 20 (3): 335-346. 10.1111/j.1365-2826.2007.01643.x.View ArticlePubMed
- Long YC, Zierath JR: AMP-activated protein kinase signaling in metabolic regulation. J Clin Invest. 2006, 116: 1776-1783. 10.1172/JCI29044.PubMed CentralView ArticlePubMed
- Tosca L, Chabrolle C, Dupont J: AMPK: a link between metabolism and reproduction?. Med Sci (Paris). 2008, 24 (3): 297-300. 10.1051/medsci/2008243297.View Article
- Malpaux B: Seasonal regulation of reproduction in mammals. Knobil and Neill's Physiology of Reproduction. Edited by: Neill JD. 2006, The University of Alabama, Birmingham, U.S.A.: Elsevier, 1-2: 2231-2281. ThirdView Article
- Um JH, Yang S, Yamazaki S, Kang H, Viollet B, Foretz M, Chung JH: Activation of 5'-AMP-activated kinase with diabetes drug metformin induces casein kinase Iepsilon (CKIepsilon)-dependent degradation of clock protein mPer2. J Biol Chem. 2007, 282 (29): 20794-20798. 10.1074/jbc.C700070200.View ArticlePubMed
- Atkins TW, Bailey CJ, Matty AJ: The effect of melatonin on insulin secretion in rat and mouse. J Endocrinol. 1973, 58: XVII-XVIII.PubMed
- Bailey CJ, Atkins TW, Matty AJ: Melatonin inhibition of insulin secretion in the rat and mouse. Hormone Research. 1974, 5: 21-28. 10.1159/000178615.View ArticlePubMed
- Salt IP, Johnson G, Ashcroft SJ, Hardie DG: AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate insulin release. Biochem J. 1998, 335 (533-539).
- Foretz M, Hebrard S, Leclerc J, Zarrinpashneh E, Soty M, Mithieux G, Sakamoto K, Andreelli F, Viollet B: Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state. J Clin Invest. 2010, 120 (7): 2355-2369. 10.1172/JCI40671.PubMed CentralView ArticlePubMed
- Thiery JC, Lomet D, Bougoin S, Malpaux B: Turnover rate of cerebrospinal fluid in female sheep: changes related to different light-dark cycles. Cerebrospinal Fluid Res. 2009, 6: 9-10.1186/1743-8454-6-9.PubMed CentralView ArticlePubMed
- Richard P: Atlas stéréotaxique du cerveau de brebis pré-alpes du sud. 1967, Paris: INRA
- Yoshikawa T: The brain of the sheep. Atlas of the Brains of Domestic Animals. 1968, Tokyo: University of Tokyo Press
- Fraser S, Cowen P, Franklin M, Franey C, Arendt J: Direct radioimmunoassay for melatonin in plasma. Clin Chem. 1983, 29 (2): 396-397.PubMed
- Tillet Y, Ravault JP, Selve C, Evin G, Castro B, Dubois MP: [Conditions for the use of specific antibodies for immunohistochemical visualization of serotonin and melatonin in the pineal gland of sheep]. C R Acad Sci III. 1986, 303 (3): 77-82.PubMed
- Malpaux B, Wayne NL, Karsch FJ: Termination of the breeding season in the Suffolk ewe: involvement of an endogenous rhythm of reproduction. Biol Reprod. 1988, 39 (2): 254-263. 10.1095/biolreprod39.2.254.View ArticlePubMed
- R-Development-Core-Team: R: A language and environment for statistical computing. 2010, Vienna, Austria: R Foundation for Statistical Computing
- Fryer LG, Parbu-Patel A, Carling D: The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem. 2002, 277 (28): 25226-25232. 10.1074/jbc.M202489200.View ArticlePubMed
- Guigas B, Bertrand L, Taleux N, Foretz M, Wiernsperger N, Vertommen D, Andreelli F, Viollet B, Hue L: 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and metformin inhibit hepatic glucose phosphorylation by an AMP-activated protein kinase-independent effect on glucokinase translocation. Diabetes. 2006, 55 (4): 865-874. 10.2337/diabetes.55.04.06.db05-1178.View ArticlePubMed
- Matthews CD, Seamark RF, Guerin MV: Plasma melatonin profiles of Romney Marsh sheep in natural photoperiod and in acutely extended darkness. J Reprod Fertil. 1992, 95 (3): 869-875. 10.1530/jrf.0.0950869.View ArticlePubMed
- Challet E, Caldelas I, Graff C, Pevet P: Synchronization of the molecular clockwork by light- and food-related cues in mammals. Biol Chem. 2003, 384 (5): 711-719. 10.1515/BC.2003.079.View ArticlePubMed
- Wayne NL, Malpaux B, Karsch FJ: How does melatonin code for day length in the ewe: duration of nocturnal melatonin release or coincidence of melatonin with a light-entrained sensitive period?. Biol Reprod. 1988, 39 (1): 66-75. 10.1095/biolreprod39.1.66.View ArticlePubMed
- Vivien-Roels B, Pitrosky B, Zitouni M, Malan A, Canguilhem B, Bonn D, Pevet P: Environmental control of the seasonal variations in the daily pattern of melatonin synthesis in the European hamster, Cricetus cricetus. Gen Comp Endocrinol. 1997, 106 (1): 85-94. 10.1006/gcen.1996.6853.View ArticlePubMed
- Garidou ML, Vivien-Roels B, Pevet P, Miguez J, Simonneaux V: Mechanisms regulating the marked seasonal variation in melatonin synthesis in the European hamster pineal gland. Am J Physiol Regul Integr Comp Physiol. 2003, 284 (4): R1043-1052.View ArticlePubMed
- Zarazaga LA, Malpaux B, Chemineau P: Amplitude of the plasma melatonin nycthemeral rhythms is not associated with the dates of onset and offset of the seasonal ovulatory activity in the Ile-de-France ewe. Reprod Nutr Dev. 2003, 43 (2): 167-177. 10.1051/rnd:2003015.View ArticlePubMed
- Vivien-Roels B: Seasonal variations in the amplitude of the daily pattern of melatonin secretion in mammalian and non-mammalian vertebrates: Possible physiological consequences. Comparative Endocrinology and Reproduction. Edited by: Joy KP, Krishna A, Haldar C. 1999, New-Dehli: Narosa Publishing House, 529-542.
- Forcada F, Abecia JA, Sierra I: Seasonal changes in oestrus activity and ovulation rate in Rasa Aragonesa ewes maintained at two different body condition levels. Small Ruminant Res. 1992, 8 (4): 313-324. 10.1016/0921-4488(92)90212-M.View Article
- Simonneaux V, Ribelayga C: Generation of the melatonin endocrine message in mammals: a review of the complex regulation of melatonin synthesis by norepinephrine, peptides, and other pineal transmitters. Pharmacol Rev. 2003, 55 (2): 325-395. 10.1124/pr.55.2.2.View ArticlePubMed
- Claret M, Smith MA, Batterham RL, Selman C, Choudhury AI, Fryer LG, Clements M, Al-Qassab H, Heffron H, Xu AW, Speakman JR, Barsh GS, Viollet B, Vaulont S, Ashford ML, Carling D, Withers DJ: AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. J Clin Invest. 2007, 117 (8): 2325-2336. 10.1172/JCI31516.PubMed CentralView ArticlePubMed
- Conti A, Maestroni GJ: Role of the pineal gland and melatonin in the development of autoimmune diabetes in non-obese diabetic mice. J Pineal Res. 1996, 20: 164-172. 10.1111/j.1600-079X.1996.tb00253.x.View ArticlePubMed
- Peschke E, Stumpf I, Bazwinsky I, Litvak L, Dralle H, Muhlbauer E: Melatonin and type 2 diabetes - a possible link?. J Pineal Res. 2007, 42 (4): 350-358. 10.1111/j.1600-079X.2007.00426.x.View ArticlePubMed
- Peschke E, Frese T, Chankiewitz E, Peschke D, Preiss U, Shreyer U, Spessert R, Mülbauer E: Diabetic Goto Kakizaki rats as well as type 2 diabetic patients show a decreased diurnal serum melatonin level and an increased pancreatic melatonin-receptor status. J Pineal Res. 2006, 40 (135-143).
- Carling D, Zammit VA, Hardie DG: A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett. 1987, 223 (2): 217-222. 10.1016/0014-5793(87)80292-2.View ArticlePubMed
- Park SH, Gammon SR, Knippers JD, Paulsen SR, Rubink DS, Winder WW: Phosphorylation-activity relationships of AMPK and acetyl-CoA carboxylase in muscle. J Appl Physiol. 2002, 92 (6): 2475-2482.View ArticlePubMed
- Voss M, Paterson J, Kelsall IR, Martin-Granados C, Hastie CJ, Peggie MW, Cohen PTW: Ppm1E is an in cellulo AMP-activated protein kinase phosphatase. Cell Signal. 2011, 23: 114-124. 10.1016/j.cellsig.2010.08.010.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.