Chapter 27
Analyzing AMPK Function in the Hypothalamus
Patricia Seoane-Collazo and Miguel Lo ́pez
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.
Key wordsAMPK, Hypothalamus, Protein extraction, Immunoblot1 Introduction
AMP-activated protein kinase (AMPK) acts as a cellular gauge that
under certain conditions, such as low energy status, is activated,
which leads to increased energy production and reduce energy
expenditure [1–5].
AMPK is a highly conserved serine/threonine kinase, formed
by a heterotrimer complex comprising a catalyticα(α1,α2) sub-
unit, with a serine/threonine protein kinase domain, and two
regulatory subunits,β(β1,β2) andγ(γ1,γ2,γ3),encoded by
different genes [3, 5, 6]. AMPK is activated by phosphorylation
on Thr-172 of theαsubunit, a process that can be allosterically
induced by AMP (but not ADP) [7] and catalyzed by several
upstream kinases, such as liver kinase B1 (LKB1) [8–10], the
pseudokinase, STRAD, the scaffold protein, mouse protein-25
(MO25) [11–13], and calmodulin-dependent kinase kinases
(CaMKKs), especially CaMKK2 [14–16]. AMP and ADP enable
phosphorylation at Thr-172 by LKB1 and CaMKK2 [7, 8, 17–19],
but also inhibit dephosphorylation by protein phosphatases, such as
protein phosphatase 2C alpha (PP2Cα)[20–22]. A more exhaus-
tive description of AMPK structure and regulation is beyond the
scope of the present chapter but has been excellently reviewed by
others recently [3, 4, 23–28].Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_27,©Springer Science+Business Media, LLC 2018
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