Proc Natl Acad Sci U S A 104(17): 7217-7222

Resveratrol stimulates AMP kinase activity in neurons

Departments of *Pathology and

^{Neurology and}

^{Hope Center for Neurological Disorders, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110}

^{To whom correspondence should be addressed. E-mail:}ude.ltsuw@tdnarblimj

Edited by Richard H. Goodman, Oregon Health & Science University, Portland, OR, and approved March 6, 2007

Author contributions: B.D. designed research; B.D. performed research; B.D. and J.M. analyzed data; and B.D. and J.M. wrote the paper.

Edited by Richard H. Goodman, Oregon Health & Science University, Portland, OR, and approved March 6, 2007

Received 2006 Nov 13

Abstract

Resveratrol is a polyphenol produced by plants that has multiple beneficial activities similar to those associated with caloric restriction (CR), such as increased life span and delay in the onset of diseases associated with aging. CR improves neuronal health, and the global beneficial effects of CR have been postulated to be mediated by the nervous system. One key enzyme thought to be activated during CR is the AMP-activated kinase (AMPK), a sensor of cellular energy levels. AMPK is activated by increases in the cellular AMP:ATP ratio, whereupon it functions to help preserve cellular energy. In this regard, the regulation of dietary food intake by hypothalamic neurons is mediated by AMPK. The suppression of nonessential energy expenditure by activated AMPK along with the CR mimetic and neuroprotective properties of resveratrol led us to hypothesize that neuronal activation of AMPK could be an important component of resveratrol activity. Here, we show that resveratrol activated AMPK in Neuro2a cells and primary neurons in vitro as well as in the brain. Resveratrol and the AMPK-activating compound 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) promoted robust neurite outgrowth in Neuro2a cells, which was blocked by genetic and pharmacologic inhibition of AMPK. Resveratrol also stimulated mitochondrial biogenesis in an AMPK-dependent manner. Resveratrol-stimulated AMPK activity in neurons depended on LKB1 activity but did not require the NAD-dependent protein deacetylase SIRT1 during this time frame. These findings suggest that neuronal activation of AMPK by resveratrol could affect neuronal energy homeostasis and contribute to the neuroprotective effects of resveratrol.

Keywords: caloric restriction, neuronal energy, neuronal protection

Abstract

Resveratrol is a polyphenol that is present at high levels in grapes, nuts, pomegranates, and Polygonum cuspidatum, a component of Chinese herbal medicines. Resveratrol has potent antioxidant and antitumorigenic activities as well as important protective effects on the nervous system (1). For example, resveratrol blocks the accumulation of mutant protein aggregates and improves survival in nematode models of Parkinson's and Huntington's diseases (2). In the short-lived fish Nothobranchius furzeri, resveratrol delays age-dependent declines in locomotor activity and memory and reduces the neurofibrillary degeneration that occurs with normal aging (3). In mammalian neurons, resveratrol delays axonal degeneration after injury (4), blocks accumulation of Aβ peptide in vitro (5), and provides protection from brain ischemia in both adult and neonatal rodents (6). Because of these promising neuroprotective effects, resveratrol is currently being evaluated in clinical trials of patients with Alzheimer's disease. Interestingly, many of the activities of resveratrol are similar to the beneficial effects offered by caloric restriction (CR), including slowed aging and delaying the onset of chronic diseases (7, 8,).

Despite these protective effects on neurons, the mechanism of action of resveratrol is not fully understood. Resveratrol has been reported to alter expression of enzymes such as COX2 and ODC, inhibit cytochrome P450 enzymes, and activate the silent information regulator 2 (Sir2) protein, an NAD-dependent protein deacetylase (1). The activation of Sir2 was an exciting discovery because it provided a molecular link to the effects of resveratrol on longevity. Indeed, increased longevity due to resveratrol in nematodes and Drosophila depends on the presence of functional Sir2 (7). Resveratrol also consistently mimics the protective effects of SIRT1 (a mammalian Sir2 protein) overexpression in cell culture, suggesting that its neuroprotective effects are also mediated through this pathway.

Resveratrol and CR also cause metabolic changes such as decreased insulin/IGF signaling and increased mitochondrial biogenesis (1, 8). Interestingly, alterations in insulin signaling and mitochondrial activity also result from activation of AMP-activated kinase (AMPK), the central energy sensor in the cell (9 –11). AMPK exists as a heterotrimeric complex containing a catalytic α subunit (α1 or α2), a regulatory β subunit (β1 or β2), and a γ subunit (γ1, γ2, or γ3) (12). AMPK is activated by alterations in the AMP:ATP ratio that occur in response to energetic stress and requires phosphorylation of Thr^{in the activation loop of the catalytic α subunit (}13). Two upstream kinases have been identified as activators of AMPK, the tumor suppressor LKB1 (14, 15) and calcium/calmodulin-dependent protein kinase β (CaMKKβ) (16, 17).

AMPK is activated by a number of pathological stresses, including hypoxia, oxidative stress, glucose deprivation, as well as exercise and dietary hormones, such as leptin and adiponectin (12). AMPK activation plays a protective role against stress, in particular ischemia, where it decreases infarct size (11, 18 –20). AMPK is also activated in the hypothalamic neurons under diet-restricted conditions (21). Because some of the metabolic changes caused by resveratrol mimic those observed in response to AMPK activation, we hypothesized that AMPK activation might be an important mediator of resveratrol actions in neurons. Our results show that resveratrol is a potent activator of AMPK in neuronal cell lines, primary neurons, and the brain. Furthermore, many of the actions of resveratrol, including mitochondrial biogenesis and neurite outgrowth, depended on the presence of a functional AMPK complex and its upstream regulator LKB1. However, resveratrol-mediated AMPK activation during this time period was independent of SIRT1. These results indicate that AMPK influences neuronal differentiation and that at least some of the actions of resveratrol in neurons are mediated by AMPK activation.

Click here to view.

Acknowledgments

We thank Eugene Johnson, Craig Press, Sanjay Jain, Robert Baloh, and Yo Sasaki for fruitful discussions and reading of the manuscript and Yo Sasaki for ATP and AMP measurements by HPLC. This work was supported by National Institutes of Health (NIH) Neuroscience Blueprint Core Grant NS057105 (to Washington University), the HOPE Center for Neurological Disorders, and NIH Grants AG13730 and NS39358 (to J.M.).

Acknowledgments

Abbreviations

ACC	acetyl-CoA carboxylase
AICAR	5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside
AMPK	AMP-activated kinase
ca	constitutively active
CaMKKβ	calcium/calmodulin-dependent protein kinase β
CC	Compound C
CR	caloric restriction
dn	dominant-negative
DRG	dorsal root ganglia
En	embryonic day n
PGC-1α	peroxisome proliferator-activated receptor γ coactivator 1α
Sir	silent information regulator
Tfam	mitochondrial transcription factor A.

Abbreviations

Footnotes

Conflict of interest: J.M. and Washington University have a financial interest in Sirtris Pharmaceuticals. Sirtris Pharmaceuticals did not support this work.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0610068104/DC1.

Footnotes

References

1. Baur JA, Sinclair DA. Nat Rev Drug Discov. 2006;5:493–506.[PubMed]
2. Parker JA, Arango M, Abderrahmane S, Lambert E, Tourette C, Catoire H, Néri C. Nat Genet. 2005;37:349–350.[PubMed]
3. Valenzano DR, Terzibasi E, Genade T, Cattaneo A, Domenici L, Cellerino A. Curr Biol. 2006;16:296–300.[PubMed]
4. Araki T, Sasaki Y, Milbrandt J. Science. 2004;305:1010–1013.[PubMed]
5. Han YS, Zheng WH, Bastianetto S, Chabot JG, Quirion R. Br J Pharmacol. 2004;141:997–1005.
6. Wang Q, Xu J, Rottinghaus GE, Simonyi A, Lubahn D, Sun GY, Sun AY. Brain Res. 2002;958:439–447.[PubMed]
7. Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D. Nature. 2004;430:686–689.[PubMed]
8. Zhang J. Biochem J. 2006;397:519–527.
9. Wang CZ, Wang Y, Di A, Maqnuson MA, Ye H, Roe MW, Nelson DJ, Bell GI, Philipson LH. Biochem Biophys Res Commun. 2005;330:1073–1079.[PubMed]
10. Bergeron R, Ren JM, Cadman KS, Moore IK, Perret P, Pypaert M, Young LH, Semenkovich CF, Shulman GI. Am J Physiol. 2001;281:E1340–E1346.[PubMed]
11. Kukidome D, Nishikawa T, Sonoda K, Imoto K, Fujisawa K, Yano M, Motoshima H, Taguchi T, Matsumura T, Araki E. Diabetes. 2006;55:120–127.[PubMed]
12. Hardie DG, Scott JW, Pan DA, Hudson ER. FEBS Lett. 2003;546:113–120.[PubMed]
13. Hardie DG, Salt IP, Hawley SA, Davies SP. Biochem J. 1999;338:717–722.
14. Hong SP, Leiper FC, Woods A, Carling D, Carlson M. Proc Natl Acad Sci USA. 2003;100:8839–8843.
15. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, Hardie DG. J Biol. 2003;2:28.1–28.16.
16. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenquelli BG, Hardie DG. Cell Metab. 2005;2:9–19.[PubMed]
17. Woods A, Dickerson K, Heath R, Hong S, Momcilovic M, Johnstone SR, Carlson M, Carling D. Cell Metab. 2005;2:21–33.[PubMed]
18. Culmsee C, Monnig J, Kemp BE, Mattson MP. J Mol Neurosci. 2001;17:45–48.[PubMed]
19. Terai K, Hiramoto Y, Masaki M, Sugiyama S, Kuroda T, Hori M, Kawase I, Hirota H. Mol Cell Biol. 2005;25:9554–9575.
20. Shibata R, Sato K, Pimentel DR, Takemura Y, Kihara S, Ohashi K, Funahashi T, Noriyuki O, Walsh K. Nat Med. 2005;11:1096–1103.
21. Minokoshi Y, Alquier T, Furukawa N, Kim Y, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, et al Nature. 2004;428:569–574.[PubMed][Google Scholar]
22. Motoshima H, Goldstein BJ, Igata M, Araki E. J Physiol (London) 2006;574:63–71.
23. Lopez-Lluch G, Hunt N, Jones B, Zhu M, Jamieson H, Hilmer S, Cascajo MV, Allard J, Ingram DK, Navas P, et al Proc Natl Acad Sci USA. 2006;103:1768–1773.[Google Scholar]
24. Nisoli E, Tonello C, Cardile A, Cozzi V, Bracale R, Tedesco L, Falcone S, Valerio A, Cantoni O, Clementi E, et al Science. 2005;310:314–317.[PubMed][Google Scholar]
25. Zong H, Ren JM, Young LH, Pypaert M, Mu J, Birnbaum MJ, Shulman GI. Proc Natl Acad Sci USA. 2002;99:15983–15987.
26. Kelly DP, Scarpulla RC. Genes Dev. 2004;18:357–368.[PubMed]
27. Hartman RE, Shah A, Fagan AM, Schwetye KE, Parsadanian M, Schulman RN, Finn MB, Holtzman DM. Neurobiol Dis. 2006;24:506–515.[PubMed]
28. Virgili M, Contestabile A. Neurosci Lett. 2000;281:123–126.[PubMed]
29. Prolla TA, Mattson MP. Trends Neurosci. 2001;24(Suppl 11):S21–S31.[PubMed]
30. Pallottini V, Montanari L, Cavallini G, Bergamini E, Gori Z, Trentalance A. Mech Age Dev. 2004;125:633–639.[PubMed]
31. Maswood N, Young J, Tilmont E, Zhang Z, Gash DM, Gerhardt GA, Grondin R, Roth GS, Mattison J, Lane MA, et al Proc Natl Acad Sci. 2004;101:18171–18176.[Google Scholar]
32. Anson RM, Guo Z, de Cabo R, Iyun T, Rios M, Hagepanos A, Ingram DK, Lane MA, Mattson MP. Proc Natl Acad Sci. 2003;100:6216–6220.
33. Duan W, Guo Z, Jiang H, Ware M, Li XJ, Mattson MP. Proc Natl Acad Sci. 2003;100:2911–2916.
34. Bartke A. Endocrinology. 2005;146:3718–3723.[PubMed]
35. Patel NV, Gordon MN, Connor KE, Good RA, Engelman RW, Mason J, Morgan DG, Morgan TE, Finch CE. Neurobiol Aging. 2005;26:995–1000.[PubMed]
36. Kaeberlein M, McVey M, Guarente L. Genes Dev. 1999;13:2570–2580.
37. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL, et al Nature. 2003;425:191–196.[PubMed][Google Scholar]
38. Kaeberlein M, Steffen KK, Hu D, Dang N, Kerr EO, Tsuchiya M, Fields S, Kennedy BK. Science. 2006;312:1312.[PubMed]
39. Kaeberlein M, Kirkland KT, Fields S, Kennedy BK. PLoS Biol. 2004;2:1381–1387.[PubMed]
40. Kim J, Yoon M, Choi S, Kang I, Kim S, Kim Y, Choi Y, Ha J. J Biol Chem. 2001;276:19102–19110.[PubMed]
41. Apfeld J, O'Connor G, McDonagh T, DiStefano PS, Curtis R. Genes Dev. 2004;18:3004–3009.
42. Gidday JM. Nat Rev Neurosci. 2006;7:437–448.[PubMed]
43. Long Y, Zierath JR. J Clin Invest. 2006;116:1776–1783.
44. Viollet B, Andrelli F, Jorgensen SB, Perrin C, Geloen A, Flamez D, Mu J, Lenzner C, Baud O, Mennoun M, et al J Clin Invest. 2003;111:91–98.[Google Scholar]
45. Tschäpe JA, Hammerschmied C, Mühlig-Versen M, Athenstaedt K, Daum G, Kretzschmar D. EMBO J. 2002;21:6367–6376.
46. Stahmann N, Woods A, Carling D, Heller R. Mol Cell Biol. 2006;26:5933–5945.
47. Tamas P, Hawley SA, Clarke RG, Mustard KJ, Green K, Hardie DG, Cantrell DA. J Exp Med. 2006;203:1665–1670.
48. Zang M, Xu S, Maitland-Toolan KA, Zuccollo A, Hou X, Jiang B, Wierzbicki M, Verbeuren TJ, Cohen RA. Diabetes. 2006;55:2180–2191.[PubMed]