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Analyzing AMPK Function in the Hypothalamus

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AMPK

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1732))

Abstract

Hypothalamic AMPK plays a key role in the control of energy homeostasis by regulating energy intake and energy expenditure, particularly modulating brown adipose tissue (BAT) thermogenesis. The function of AMPK can be assayed by analyzing its phosphorylated protein levels in tissues, since AMPK is activated when it is phosphorylated at Thr-172. Here, we describe a method to obtain hypothalamic (nuclei-specific) protein extracts and the suitable conditions to assay AMPK phosphorylation by Western blotting.

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References

  1. Kahn BB, Alquier T, Carling D, Hardie DG (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1:15–25

    Article  CAS  PubMed  Google Scholar 

  2. Lage R, Diéguez C, Vidal-Puig A, López M (2008) AMPK: a metabolic gauge regulating whole-body energy homeostasis. Trends Mol Med 14:539–549

    Article  CAS  PubMed  Google Scholar 

  3. Carling D, Mayer FV, Sanders MJ, Gamblin SJ (2011) AMP-activated protein kinase: nature's energy sensor. Nat Chem Biol 7:512–518

    Article  CAS  PubMed  Google Scholar 

  4. Hardie DG, Schaffer BE, Brunet A (2016) AMPK: an energy-sensing pathway with multiple inputs and outputs. Trends Cell Biol 26:190–201

    Article  CAS  PubMed  Google Scholar 

  5. López M, Nogueiras R, Tena-Sempere M, Dieguez C (2016) Hypothalamic AMPK: a canonical regulator of whole-body energy balance. Nat Rev Endocrinol 12:421–432

    Article  PubMed  Google Scholar 

  6. Hardie DG, Ross FA, Hawley SA (2012) AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol 13:251–262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gowans GJ, Hawley SA, Ross FA, Hardie DG (2013) AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation. Cell Metab 18:556–566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Woods A, Johnstone SR, Dickerson K et al (2003) LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13:2004–2008

    Article  CAS  PubMed  Google Scholar 

  9. Hardie DG (2013) The LKB1-AMPK pathway-friend or foe in cancer? Cancer Cell 23:131–132

    Article  CAS  PubMed  Google Scholar 

  10. Hardie DG, Alessi DR (2013) LKB1 and AMPK and the cancer-metabolism link - ten years after. BMC Biol 11:36

    Article  PubMed  PubMed Central  Google Scholar 

  11. Hawley SA, Boudeau J, Reid JL et al (2003) Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2:28

    Article  PubMed  PubMed Central  Google Scholar 

  12. Shaw RJ, Kosmatka M, Bardeesy N et al (2004) The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 101:3329–3335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Alessi DR, Sakamoto K, Bayascas JR (2006) LKB1-dependent signaling pathways. Annu Rev Biochem 75:137–163

    Article  CAS  PubMed  Google Scholar 

  14. Hawley SA, Pan DA, Mustard KJ et al (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2:9–19

    Article  CAS  PubMed  Google Scholar 

  15. Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, Witters LA (2005) The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280:29060–29066

    Article  CAS  PubMed  Google Scholar 

  16. Woods A, Dickerson K, Heath R et al (2005) Ca(2+)/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2:21–33

    Article  CAS  PubMed  Google Scholar 

  17. Woods A, Vertommen D, Neumann D et al (2003) Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis. J Biol Chem 278:28434–28442

    Article  CAS  PubMed  Google Scholar 

  18. Oakhill JS, Steel R, Chen ZP et al (2011) AMPK is a direct adenylate charge-regulated protein kinase. Science 332:1433–1435

    Article  CAS  PubMed  Google Scholar 

  19. Gowans GJ, Hardie DG (2014) AMPK: a cellular energy sensor primarily regulated by AMP. Biochem Soc Trans 42:71–75

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Davies SP, Helps NR, Cohen PT, Hardie DG (1995) 5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC. FEBS Lett 377:421–425

    Article  CAS  PubMed  Google Scholar 

  21. Steinberg GR, Michell BJ, van Denderen BJ et al (2006) Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling. Cell Metab 4:465–474

    Article  CAS  PubMed  Google Scholar 

  22. Xiao B, Sanders MJ, Underwood E et al (2011) Structure of mammalian AMPK and its regulation by ADP. Nature 472:230–233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Carling D, Thornton C, Woods A, Sanders MJ (2012) AMP-activated protein kinase: new regulation, new roles? Biochem J 445:11–27

    Article  CAS  PubMed  Google Scholar 

  24. Hardie DG (2014) AMPK–sensing energy while talking to other signaling pathways. Cell Metab 20:939–952

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hardie DG (2014) AMP-activated protein kinase: maintaining energy homeostasis at the cellular and whole-body levels. Annu Rev Nutr 34:31–55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hardie DG, Ashford ML (2014) AMPK: regulating energy balance at the cellular and whole body levels. Physiology (Bethesda) 29:99–107

    CAS  Google Scholar 

  27. Hardie DG (2014) A new protein kinase cascade. Nat Rev Mol Cell Biol 15:223

    Article  CAS  PubMed  Google Scholar 

  28. Ross FA, Jensen TE, Hardie DG (2016) Differential regulation by AMP and ADP of AMPK complexes containing different gamma subunit isoforms. Biochem J 473:189–199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Minokoshi Y, Alquier T, Furukawa N et al (2004) AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428:569–574

    Article  CAS  PubMed  Google Scholar 

  30. López M, Lage R, Saha AK et al (2008) Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin. Cell Metab 7:389–399

    Article  PubMed  Google Scholar 

  31. Andrews ZB, Liu ZW, Walllingford N et al (2008) UCP2 mediates ghrelin's action on NPY/AgRP neurons by lowering free radicals. Nature 454:846–851

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lage R, Vázquez MJ, Varela L et al (2010) Ghrelin effects on neuropeptides in the rat hypothalamus depend on fatty acid metabolism actions on BSX but not on gender. FASEB J 24:2670–2679

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Al MO, López M, Tschop M, Dieguez C, Nogueiras R (2017) Current understanding of the hypothalamic ghrelin pathways inducing appetite and adiposity. Trends Neurosci 40:167–180

    Article  Google Scholar 

  34. Cui H, López M, Rahmouni K (2017) The cellular and molecular bases of leptin and ghrelin resistance in obesity. Nat Rev Endocrinol 13:338–351

    Article  CAS  PubMed  Google Scholar 

  35. Ramírez S, Martíns L, Jacas J et al (2013) Hypothalamic ceramide levels regulated by CPT1C mediate the orexigenic effect of ghrelin. Diabetes 62:2329–2337

    Article  PubMed  PubMed Central  Google Scholar 

  36. López M, Varela L, Vázquez MJ et al (2010) Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance. Nat Med 16:1001–1008

    Article  PubMed  PubMed Central  Google Scholar 

  37. Alvarez-Crespo M, Csikasz RI, Martinez-Sanchez N et al (2016) Essential role of UCP1 modulating the central effects of thyroid hormones on energy balance. Mol Metab 5:271–282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Martinez-Sanchez N, Moreno-Navarrete JM, Contreras C et al (2017) Thyroid hormones induce browning of white fat. J Endocrinol 232:351–362

    Article  CAS  PubMed  Google Scholar 

  39. Martínez-Sánchez N, Seoane-Collazo P, Contreras C et al (2017) Hypothalamic AMPK-ER stress-JNK1 axis mediates the central actions of thyroid hormones on energy balance. Cell Metab 26:212–219

    Article  PubMed  PubMed Central  Google Scholar 

  40. Martínez de Morentin PB, Gónzalez-García I, Martins L et al (2014) Estradiol regulates brown adipose tissue thermogenesis via hypothalamic AMPK. Cell Metab 20:41–53

    Article  PubMed  PubMed Central  Google Scholar 

  41. Tanida M, Yamamoto N, Shibamoto T, Rahmouni K (2013) Involvement of hypothalamic AMP-activated protein kinase in leptin-induced sympathetic nerve activation. PLoS One 8:e56660

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Beiroa D, Imbernon M, Gallego R et al (2014) GLP-1 agonism stimulates brown adipose tissue thermogenesis and browning through hypothalamic AMPK. Diabetes 63:3346–3358

    Article  CAS  PubMed  Google Scholar 

  43. Whittle AJ, Carobbio S, Martíns L et al (2012) Bmp8b increases brown adipose tissue thermogenesis through both central and peripheral actions. Cell 149:871–885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Martins L, Seoane-Collazo P, Contreras C et al (2016) A functional link between AMPK and orexin mediates the effect of BMP8B on energy balance. Cell Rep 16:2231–2242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Martínez de Morentin PB, Whittle AJ, Ferno J et al (2012) Nicotine induces negative energy balance through hypothalamic AMP-activated protein kinase. Diabetes 61:807–817

    Article  PubMed  PubMed Central  Google Scholar 

  46. Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B (2014) Metformin: from mechanisms of action to therapies. Cell Metab 20:953–966

    Article  CAS  PubMed  Google Scholar 

  47. Foretz M, Viollet B (2015) Therapy: metformin takes a new route to clinical efficacy. Nat Rev Endocrinol 11:390–392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ikegami M, Ikeda H, Ishikawa Y et al (2013) Olanzapine induces glucose intolerance through the activation of AMPK in the mouse hypothalamus. Eur J Pharmacol 718:376–382

    Article  CAS  PubMed  Google Scholar 

  49. Ikegami M, Ikeda H, Ohashi T et al (2013) Olanzapine increases hepatic glucose production through the activation of hypothalamic adenosine 5′-monophosphate-activated protein kinase. Diabetes Obes Metab 15:1128–1135

    Article  CAS  PubMed  Google Scholar 

  50. Skrede S, Martins L, Berge RK, Steen VM, Lopez M, Ferno J (2014) Olanzapine depot formulation in rat: a step forward in modelling antipsychotic-induced metabolic adverse effects. Int J Neuropsychopharmacol 17:91–104

    Article  CAS  PubMed  Google Scholar 

  51. He M, Zhang Q, Deng C, Wang H, Lian J, Huang XF (2014) Hypothalamic histamine H1 receptor-AMPK signaling time-dependently mediates olanzapine-induced hyperphagia and weight gain in female rats. Psychoneuroendocrinology 42:153–164

    Article  PubMed  Google Scholar 

  52. Breen DM, Sanli T, Giacca A, Tsiani E (2008) Stimulation of muscle cell glucose uptake by resveratrol through sirtuins and AMPK. Biochem Biophys Res Commun 374:117–122

    Article  CAS  PubMed  Google Scholar 

  53. Vingtdeux V, Giliberto L, Zhao H et al (2010) AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism. J Biol Chem 285:9100–9113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Ahn J, Lee H, Kim S, Park J, Ha T (2008) The anti-obesity effect of quercetin is mediated by the AMPK and MAPK signaling pathways. Biochem Biophys Res Commun 373:545–549

    Article  CAS  PubMed  Google Scholar 

  55. Lu J, Wu DM, Zheng YL et al (2010) Quercetin activates AMP-activated protein kinase by reducing PP2C expression protecting old mouse brain against high cholesterol-induced neurotoxicity. J Pathol 222:199–212

    Article  CAS  PubMed  Google Scholar 

  56. Martínez de Morentin PB, González CR, López M (2010) AMP-activated protein kinase: ‘a cup of tea’ against cholesterol-induced neurotoxicity. J Pathol 222:329–334

    Article  PubMed  Google Scholar 

  57. Eid HM, Nachar A, Thong F, Sweeney G, Haddad PS (2015) The molecular basis of the antidiabetic action of quercetin in cultured skeletal muscle cells and hepatocytes. Pharmacogn Mag 11:74–81

    Article  PubMed  PubMed Central  Google Scholar 

  58. López M, Tena-Sempere M (2017) Estradiol effects on hypothalamic AMPK and BAT thermogenesis: a gateway for obesity treatment? Pharmacol Ther 178:109. https://doi.org/10.1016/j.pharmthera.2017.03.014

    Article  PubMed  Google Scholar 

  59. López M (2017) EJE PRIZE 2017: hypothalamic AMPK: a golden target against obesity? Eur J Endocrinol 176:R235–R246

    Article  PubMed  PubMed Central  Google Scholar 

  60. Minokoshi Y, Kim YB, Peroni OD et al (2002) Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339–343

    Article  CAS  PubMed  Google Scholar 

  61. Vázquez MJ, González CR, Varela L et al (2008) Central resistin regulates hypothalamic and peripheral lipid metabolism in a nutritional-dependent fashion. Endocrinology 149:4534–4543

    Article  PubMed  Google Scholar 

  62. Ferno J, Varela L, Skrede S et al (2011) Olanzapine-induced hyperphagia and weight gain associate with orexigenic hypothalamic neuropeptide signaling without concomitant AMPK phosphorylation. PLoS One 6:e20571

    Article  PubMed  PubMed Central  Google Scholar 

  63. Sangiao-Alvarellos S, Varela L, Vázquez MJ et al (2010) Influence of ghrelin and GH deficiency on AMPK and hypothalamic lipid metabolism. J Neuroendocrinol 22:543–556

    Article  CAS  PubMed  Google Scholar 

  64. Velasquez DA, Martinez G, Romero A et al (2011) The central Sirtuin 1/p53 pathway is essential for the orexigenic action of ghrelin. Diabetes 60:1177–1185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Varela L, Martínez-Sánchez N, Gallego R et al (2012) Hypothalamic mTOR pathway mediates thyroid hormone-induced hyperphagia in hyperthyroidism. J Pathol 227:209–222

    Article  CAS  PubMed  Google Scholar 

  66. Martinez de Morentin PB, Lage R, Gonzalez-Garcia I et al (2015) Pregnancy induces resistance to the anorectic effect of hypothalamic malonyl-CoA and the thermogenic effect of hypothalamic AMPK inhibition in female rats. Endocrinology 156:947–960

    Article  CAS  PubMed  Google Scholar 

  67. Bain J, Plater L, Elliott M et al (2007) The selectivity of protein kinase inhibitors: a further update. Biochem J 408:297–315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Paxinos G, Watson C (eds) (1986) The rat brain in stereotaxic coordinates. Academic Press, Sydney

    Google Scholar 

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Author information

Authors and Affiliations

  1. NeurObesity Group, Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain

    Patricia Seoane-Collazo & Miguel López

  2. CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Santiago de Compostela, Spain

    Patricia Seoane-Collazo & Miguel López

Authors
  1. Patricia Seoane-Collazo
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  2. Miguel López
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Corresponding authors

Correspondence to Patricia Seoane-Collazo or Miguel López .

Editor information

Editors and Affiliations

  1. Department of Pathology CARIM School for Cardiovascular Diseases, Maastricht University, Maastricht, The Netherlands

    Dietbert Neumann

  2. Department EMD, Inserm, U1016, Institut Cochin, CNRS, UMR8104, Université Paris Descartes, Paris, France

    Benoit Viollet

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Seoane-Collazo, P., López, M. (2018). Analyzing AMPK Function in the Hypothalamus. In: Neumann, D., Viollet, B. (eds) AMPK. Methods in Molecular Biology, vol 1732. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7598-3_27

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  • DOI: https://doi.org/10.1007/978-1-4939-7598-3_27

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7597-6

  • Online ISBN: 978-1-4939-7598-3

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