AMPK

AMPK Protein Kinase AMP-Activated Ver. 1.0.

Summary remarks

The AMPK activation mechanism is complex and there are some conflicting data. Part of the controversies in the results of the studies is related to insufficient purification of AMPK, obtained from cells, so these results were not subsequently confirmed.

Different complexes can have different functions and targets. For example, AS160 phosphorylation is associated with activation of α2β2γ1- but not α2β2γ3-AMPK trimeric complex in skeletal muscle during exercise in humans (Treebak 2007) [1]. Also, different targets may be for AMPK complexes containing α1 and α2 isoforms. In mouse muscle, the α2-isoform seems to be the most important α-AMPK isoformin phosphorylating GS because knocking out the α2-subunit completely prevented AICAR-induced deactivation of GS (Jorgensen 2004) [2].

Different complexes can have different activity and kinetics. For example, the α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. The α2-complexes were ∼25- fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172/174). However, α2-complexes were more readily activated by AMP than α1- complexes (Rajamohan 2016) [3].

The number of AMPK outputs (i.e.,identified downstream targets) is expanding rapidly. AMPK has a recognition motif that is among the best defined of any protein kinase, making it potentially useful for predicting novel targets. Since AMPK phosphorylation often triggers 14-3-3 binding, the identification of some of these was aided by screening for candidates that bound 14-3-3 proteins in an AMPK-dependent manner. The availability of phosphorylation site prediction tools should greatly assist in the elucidation of the complete network of downstream targets. However, the current site prediction tools do have some limitations. AMPK α1 and α2 lie in the same branch of the kinome as up to twelve AMPK- related kinases and some of the latter, notably the MARKs [69], have similar recognition motifs. Thus, using the AMPK recognition motif to predict sites may also yield targets for AMPK-related kinases. (Hardie 2016) [4].

Different AMPK trimeric complex can have different localization within the muscle cells. For example, only AMPKα2 was detected in the nucleus of cells. AMPK activation and function depends on its subcellular localization, and that it may be largely dependent on the regulatory γ subunit in the complex.

AMPK subunits are expressed in a fibre type-dependent manner. For example, ~70% lower γ3 AMPK protein content observed in type I vs. II muscle fibres in humans (Kristensen 2015) [5].

In response to training the protein content of different isoforms changes differently. For example, α1, β2 and γ1 increased in the trained leg by 41, 34, and 26%, respectively. In contrast, the protein content of the regulatory γ3-isoform decreased by 62% in the trained leg (P= 0.01), whereas no effect of training was seen for α2, β1, and γ2. (Frøsig 2004) [6].

Activation of AMPK and downstream targets is fibre type-specific. For example, regulation of AMPK downstream targets suggests that interval exercise elicits a fibre type-specific response and activation of α2β2γ3 AMPK complexes is a major contributor to this response (Kristensen 2015) [5].

Structure

The structure with regulation sites is presented on Figure 1.

Figure from (Jeon ‎2016) [7]. Molecular regulation of AMPK and LKB1. (a) Modification of the AMPK α1 (top) and α2 (bottom) subunits by phosphorylation/dephosphorylation, ubiquitination, sumoylation and oxidation/reduction. Pathways marked in red indicate α1- or α2-subunit-specific modifications. Numbers of modified amino acids are based on human proteins, and numbers in parenthesis are those reported in the original research (see text for details). (b) Modification of the AMPK β1 (top) and β2 (bottom) subunits by myristoylation, ubiquitination, sumoylation and glycogen binding. Pathways marked in red indicate β1- or β2-subunit-specific modifications. (c) Modification of the AMPK γ-subunit by AMP, ADP or ATP binding. Binding of AMP to CBS1 induces allosteric activation, and binding of AMP or ADP to CBS3 induces T172 phosphorylation. (d) Modification and regulation of LKB1 by phosphorylation, acetylation, ubiquitination, sumoylation and 4HNE adduction. Arrow indicates activation, and bar-headed line indicates inhibition. α/γ-BD, α/γ-subunit-binding domain; AID, autoinhibitory domain; β-BD, β-subunit-binding domain; CBM, carbohydratebinding module; CBS, cystathionine beta-synthase domain; NLS, nuclear localization signal.

Action

AMPK, a fuel sensor and regulator, promotes ATP-producing and inhibits ATP-consuming pathways in various tissues. AMPK exists as a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits. The kinase is activated in response to stresses that deplete cellular ATP supplies such as low glucose, hypoxia, ischemia and heat shock. Binding of AMP to the γ subunit allosterically activates the complex, making it a more attractive substrate for its major upstream AMPK kinase, LKB1. As a cellular energy sensor responding to low ATP levels, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies. For example, activation of AMPK enhances both the transcription and translocation of GLUT4, resulting in an increase in insulin-stimulated glucose uptake. In addition, it also stimulates catabolic processes such as fatty acid oxidation and glycolysis via inhibition of ACC and activation of PFK2. AMPK negatively regulates several proteins central to ATP consuming processes such as TORC2, glycogen synthase, SREBP-1 and TSC2, resulting in the downregulation or inhibition of gluconeogenesis, glycogen, lipid and protein synthesis. Due to its role as a central regulator of both lipid and glucose metabolism, AMPK is considered to be a key therapeutic target for the treatment of obesity, type II diabetes mellitus, and cancer. The AMP-activated protein kinase (AMPK) has an important role in the regulation of cellular energy homeostasis. The enzyme is activated under conditions of low ATP, often caused by a variety of stresses and regulates signaling pathways that increase the supplies of ATP available. (http://www.sinobiological.com/ampk-signaling-pathwawy.html)

Kinetics

Data from (Xiao 2011) [8]. Equilibrium dissociation constants for the binding of AXPs to phosphorylated AMPK.

Ligand	Kd OjlM)	Kd,i i[iM)	Kd,ii (M'M)
	fsNADH	vs C-AXPs
AMP	1.6 (0.5)	2.5 (0.6)	80 (25)
ADP	1.3 (0.5)	1.5 (0.4)	50(15)
ATP	0.9 (0.3)	1.7 (0.5)	65(15)
Mg-ATP	32(12)	18(7.5)	230 (80)

Dissociation constants were determined at 20°C by competition against NADH or C-AXPs in 25 mM Tris, 1 mM TCEP, 100 mM NaCl (pH 8) with and without 5 mM MgCl2. The Kd values are reported as the mean (± SD) determined from at least three independent measurements. Protective effect of AMP/ADP is mediated by its binding to the weaker of the two exchangeable sites which we have identified as site-3. We have also shown that the α-hook region binds into this site in the presence of AMP and predict that the same situation would occur with ADP. We further suggest that binding of the α-hook acts to restrict the flexibility of the preceding α linker region (residues 300 to 370) and, in so doing, promotes the interaction of the kinase domain with the regulatory fragment seen in our crystal structure. This interaction, which mostly involves contacts between the activation loop and the C-terminal domain of β, would therefore act to protect Thr-172 against dephosphorylation. Since the interaction surface of the α–hook with the regulatory fragment is relatively small it is likely that there is a dynamic equilibrium between the α–hook bound and α–hook unbound species. If, as our structure suggests, AMP/ADP binding favours the α–hook bound species but Mg.ATP binding drives formation of the α–hook unbound species, then the competitive binding of AMP/ADP versus Mg.ATP would control the extent to which the enzyme was protected from dephosphorylation and inactivation.

Basal AMPK activity depends on AMP (AMPfree) content in resting muscles. Typical values are present in Table below:

VO2max	Tissue	Fiber Type	ExDuration (min)	ExIntencity (%VO2max)	CoFactor	Target	FoldChange	[Target0] Rest	[Target1] After Ex	Measure Unit	Commentary	Ref
50	vastus lateralis		60	44		ADP	1,07	2,70	2,90	mmol/kg dry wt	low intensity cycling exercise was performed until exhaustion	Wojtaszewski 2002
50	vastus lateralis		120	44		ADP	1,07	2,70	2,90	mmol/kg dry wt	low intensity cycling exercise was performed until exhaustion	Wojtaszewski 2002
50	vastus lateralis		208	44		ADP	1,07	2,70	2,90	mmol/kg dry wt	low intensity cycling exercise was performed until exhaustion	Wojtaszewski 2002
65	vastus lateralis		10	70	Low glycogen	ADP	1,03	3,50	3,60	mmol/kg dry wt		Wojtaszewski 2003
65	vastus lateralis		60	70	Low glycogen	ADP	0,94	3,50	3,30	mmol/kg dry wt		Wojtaszewski 2003
65	vastus lateralis		10	70	High glycogen	ADP	1,03	3,10	3,20	mmol/kg dry wt		Wojtaszewski 2003
65	vastus lateralis		60	70	High glycogen	ADP	0,97	3,10	3,00	mmol/kg dry wt		Wojtaszewski 2003
66	vastus lateralis		20	80		ADP	1,10	3,07	3,37	mmol/kg dry wt	20 min of cycle exercise at 80% of peak O2 uptake	Nielsen 2003
44	vastus lateralis		20	80		ADP	1,14	3,45	3,94	mmol/kg dry wt	20 min of cycle exercise at 80% of peak O2 uptake	Nielsen 2003
				Average		ADP		3,09		mmol/kg dry wt
48	vastus lateralis		20	40		ADPfree	1,32	108,90	143,60	μmol/kg dry wt	cycling 20 min at 40% of VO2 peak	Chen 2003
48	vastus lateralis		20	59		ADPfree	2,74	108,90	298,30	μmol/kg dry wt	cycling 20 min at 60% of VO2 peak	Chen 2003
48	vastus lateralis		20	79		ADPfree	5,83	108,90	634,50	μmol/kg dry wt	cycling 20 min at 80% of VO2 peak	Chen 2003
40	vastus lateralis		0,5	300		ADPfree	1,43	86,00	123,00	μmol/kg dry wt		Chen 2000
				Average		ADPfree		97,45		μmol/kg dry wt
50	vastus lateralis		60	44		AMP	1,10	0,10	0,11	mmol/kg dry wt	low intensity cycling exercise was performed until exhaustion	Wojtaszewski 2002
50	vastus lateralis		120	44		AMP	1,20	0,10	0,12	mmol/kg dry wt	low intensity cycling exercise was performed until exhaustion	Wojtaszewski 2002
50	vastus lateralis		208	44		AMP	1,10	0,10	0,11	mmol/kg dry wt	low intensity cycling exercise was performed until exhaustion	Wojtaszewski 2002
65	vastus lateralis		10	70	Low glycogen	AMP	1,04	0,26	0,27	mmol/kg dry wt		Wojtaszewski 2003
65	vastus lateralis		60	70	Low glycogen	AMP	1,08	0,26	0,28	mmol/kg dry wt		Wojtaszewski 2003
65	vastus lateralis		10	70	High glycogen	AMP	1,09	0,23	0,25	mmol/kg dry wt		Wojtaszewski 2003
65	vastus lateralis		60	70	High glycogen	AMP	1,00	0,23	0,23	mmol/kg dry wt		Wojtaszewski 2003
53	vastus lateralis		40	80		AMP	1,00	0,25	0,25	mmol/kg dry wt	no changes	Kristensen 2007
66	vastus lateralis		20	80		AMP	0,88	0,42	0,37	mmol/kg dry wt	20 min of cycle exercise at 80% of peak O2 uptake	Nielsen 2003
44	vastus lateralis		20	80		AMP	0,86	0,37	0,32	mmol/kg dry wt	20 min of cycle exercise at 80% of peak O2 uptake	Nielsen 2003
				Average		AMP		0,23		mmol/kg dry wt
48,5	vastus lateralis		120	65		AMPfree	17,67	0,90	15,90	μmol/kg dry wt		Lee-Young 2006
48,5	vastus lateralis		120	65	CHO supplementation	AMPfree	11,75	0,80	9,40	μmol/kg dry wt	a bolus of 44.3 mmol/kg of the glucose tracer was administered intravenously immediately before commencement of a 120-min preexercise constant infusion (0.58 mmol/kg-1/min-1), which was continued throughout the exercise bout	Lee-Young 2006
49	vastus lateralis		60	70		AMPfree	17,88	0,16	2,86	μmol/kg dry wt		Steinberg 2006
49	vastus lateralis		60	70	Low glycogen	AMPfree	44,20	0,10	4,42	μmol/kg dry wt		Steinberg 2006
52	vastus lateralis		20	77		AMPfree	2,14	0,70	1,50	μmol/kg dry wt	20 min of bicycling at 80% VO2peak (77±3%)	Birk 2006
66	vastus lateralis		20	80		AMPfree	2,81	0,96	2,70	μmol/kg dry wt	20 min of cycle exercise at 80% of peak O2 uptake	Nielsen 2003
44	vastus lateralis		20	80		AMPfree	15,23	0,60	9,14	μmol/kg dry wt	20 min of cycle exercise at 80% of peak O2 uptake	Nielsen 2003
52	vastus lateralis		2	110		AMPfree	2,36	1,10	2,60	μmol/kg dry wt	120s at 110% of peak work rate	Birk 2006
40	vastus lateralis		0,5	300		AMPfree	3,00	0,37	1,11	μmol/kg dry wt		Chen 2000
52	vastus lateralis		0,5	300		AMPfree	1,75	0,80	1,40	μmol/kg dry wt	30s sprint	Birk 2006
48	vastus lateralis		20	40		AMPfree	1,67	0,60	1,00	μmol/kg dry wt	cycling 20 min at 40% of VO2 peak	Chen 2003
48	vastus lateralis		20	59		AMPfree	9,67	0,60	5,80	μmol/kg dry wt	cycling 20 min at 60% of VO2 peak	Chen 2003
48	vastus lateralis		20	79		AMPfree	46,17	0,60	27,70	μmol/kg dry wt	cycling 20 min at 80% of VO2 peak	Chen 2003
				Average		AMPfree		0,64		μmol/kg dry wt

Different complexes can have different activity and kinetics. For example, the α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. The α2-complexes were ∼25- fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172/174). However, α2-complexes were more readily activated by AMP than α1- complexes (Rajamohan 2016) [3]. See Table below:

Specific activities of α1- and α2-containing AMPK complexes are shown below:

Pathways

In general, upstream pathways presented at Figure from (Jeon ‎2016) [7].

In general, downstream pathways presented at Figure from (Jeon ‎2016) [7].

AMPK Regulate Multiple Metabolic Processes in Cells.

Figure 3. Substrates of AMPK Regulate Multiple Metabolic Processes in Cells. AMPK is phosphorylated and activated by LKB1 and CAMKK2 in response to stimuli that increase AMP/ADP levels (energy stress) or Ca2+ flux, respectively. Once active, AMPK induces metabolic changes through the phosphorylation of substrates. Some of the best-established metabolic processes regulated by AMPK are shown, together with the relevant substrates (Garcia 2017) [9].

Upstream

AMPK is phosphorylated and activated by LKB1 (LKB1/STRAD/MO25 complex), CaMKKβ

AMPK is phosphorylated and ingibited by AKT, ROS, RNS, PKD1

AMPK is dephosphorylated by protein PP1A, PP2A, PP2C

(Morales-Alamo 2016) [10]. Reactive oxygen and nitrogen species (RONS) may activate AMPK by direct an indirect mechanisms. Directly, RONS may activate or deactivate AMPK by modifying RONS-sensitive residues of the AMPK-α subunit. Both too high (hypoxia) and too low (ingestion of antioxidants) RONS levels may lead to Ser485-AMPKα1/ Ser491-AMPKα2 phosphorylation causing inhibition of Thr172-AMPKα phosphorylation.

(Kjøbsted 2018) [11]. After exercise and energy repletion, AMPK is converted back to an inactive form by dephosphorylation catalyzed by protein phosphatases (PP1A, PP2A, and PP2C) and undergoes inhibition by glycogen via binding to the GBD of the β subunit.

In vitro or in non human muscle cells in some additional factors was identified:

(Heathcote 2016) [12]. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491 respectively as an inhibitory regulation mechanism. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells.

(Valentine 2014) [13]. Insulin and IGF-1 diminish AMPK activity in hepatocytes and muscle, most likely through Akt activation and the inhibitory phosphorylation of Ser485/491 on its α-subunit.

Coughlan 2016 [14]. PKD1 inhibits AMPK directly by phosphorylating it at Ser491 of the α2 subunit, thus diminishing AMPK activity in muscle cells.

(Viollet ‎2010) [15]. Similarly, it has been shown that IL-6 activates AMPK in skeletal muscle by increasing the concentration of cAMP and, secondarily, the AMP:ATP ratio (Kelly et al., 2009). Recent data revealed a new mechanism that regulates AMPK activity independently of AMP and of phosphorylation or dephosphorylation processes. Modulation of AMPK complex stability via ubiquitination-mediating degradation has emerged through a complex containing cell death-inducing DNA fragmentation factor α-like effector A (Cidea) and AMPK (Qi et al. , 2008 ). Cidea and AMPK have been shown to co-localize in the endoplasmic reticulum and form a complex in vivo through specific interaction with the AMPKβ subunit to promote ubiquitin-mediated AMPK degradation and down-regulation of its activity.

AMPK in general can be ingibited by several initial factors (Viollet ‎2010) [15] :

• Inhibition by lipid overload
• Inhibition by high glucose concentration
• Inhibition by glycogen accumulation
• Inhibition by amino acids
• Inhibition by insulin in the heart (Rem: what about skeletal miscles ???)
• Inhibition by inflammatory signals

Downstream

Direct phosphorylation of AMPK occurs on: ACC1, ACC2, TBC1D1, TBC1D4, RPTOR, ULK1, PRKAA2, PGC-1α, FOXO family, CREB, HDAC5, nNOSµ, Nrf2, GS, AKAPI, STIM1, VAPA, AGPAT9, SLC12A2, CTNND1, AKAP13, GPHN, G0LGA4, IGF2R, TNIK, MINK1, PPP1R12B (See links below)

In general in human tissue direct phosphorylation of AMPK can occurs on many others proteins. After discovering (screening) extended pool of exercise induced genes, potential AMPK targets can be associated with such genes pathways. For example, in (Schaffer 2015) [16] next AMPK substrates was listed: ABLIM1, ACACA, ACACB, AGPAT9, ARHGEF2, ATP2B1, ATP2B2, BAIAP2, BAIAP2L1, BRAF, C19orf21, CDC27, CDC42EP1, CDKN1B, CLASP1, CLIP-170, CRTC2, CRY1, CTNND1, eEF2K, ELFN1, EML3, EP300, ERBB2IP, FAM65B, FAM71D, FOXO3a, FRYL, GABABR2, GBF1, GFPT1, GOLGA4, GPHN, GYS1, GYS2, H2B, HDAC5, HEATR5B, HMGCR, IRS1, ITGB1BP1, JPH3, KCNB1, KLC2, KLC3, KLC4, MAPT, MDM4, MLTK, MTFR1L, MYO18A, NDRG1, NET1, NUMA1, p53, PAK2, PEA15, PER3, PFKFB2, PGC1A, PIKFYVE, PKP2, PLD1, PPP1R12A, PPP1R12C, PPP1R13L, PRKCQ, RAB11FIP2, RAG1, RALGAPA1, RBM14, RICTOR, RPTOR, RRN3, SGK223, SH3BP4, SH3PXD2A, SLC35C2, SLC4A7, SMCR8, SNAP29, SNX17, SPANXE, SREBP-1c, SSH3, STIM1, STK31, TBC1D1, TBC1D4, TJP2, TMEM201, TNNI3, TP53BP2, TP53I11, TP73, TSC2, TTN, TXNIP, ULK1, VASP, WDFY3

See details on “List_of_109_AMPK_Substrates” in file https://www.cell.com/cms/attachment/2039832072/2053421466/mmc7.xlsx

Direct phosphorylation of AMPK occurs, for example, on the coactivator PGC-1α (Thr177 and Ser538, (Jäger 2007) [17]), the FOXO family of transcription factors (for example, FOXO3 is phosphorylated by AMPK in up to 6 residues [106]), CREB (Ser133 (Thomson 2008) [18]), which is also phosphorylated and activated by AMPK. Phosphorylation of HDAC5 (Ser259 and Ser498 [124]) by AMPK relieves the inhibition on the MEF2/GEF complex and allows transcriptional activation (Cantó 2010) [19].

(Chen 2003) [20]. We have shown that nNOSµ is a target for phosphorylation by AMPK in human skeletal muscle (17). We found a significant increase in phosphorylation of nNOSµ at Ser-1451 immediately after maximal sprint exercise (17) but only modest effects during lower-intensity exercise at 60% VO2 peak (16). In the current study, nNOSµ phosphorylation was also quite variable between subjects and only achieved statistically significant increases at the highest levels of exercise intensity.

(Joo 2016) [21]. AMPK phosphorylates Nrf2 at Ser550 residue, which in conjunction with AMPK-mediated GSK3β inhibition promotes nuclear accumulation of Nrf2 for antioxidant response element (ARE)-driven gene transactivation.

(McBride 2009) [22]. AMPK phosphorylates muscle glycogen synthase (mGS) at site 2 (Ser-7) (Carling and Hardie, 1989). Phosphorylation at this site causes a decrease in activity at low concentrations of the allosteric activator glucose-6-phosphate (Skurat et al., 1994).

(Hoffman 2015) [23]. Global phosphoproteomic analysis of human skeletal muscle detect next AMPK substrates:

Target	(Target)	Site	Species	Source	Link
ACACA	(ACC1)	S80	Human Exercise	Hoffman 2015	[23]
ACACB	(ACC2)	S222	Human Exercise	Hoffman 2015	[23]
TBC1D1		S237	Human Exercise	Hoffman 2015	[23]
TBC1D4		S704	Human Exercise	Hoffman 2015	[23]
RPTOR		S722	Human Exercise	Hoffman 2015	[23]
ULK1		S694	Human Exercise	Hoffman 2015	[23]
PRKAA2		S491	Human Exercise	Hoffman 2015	[23]
AKAPI		S107	Human Exercise	Hoffman 2015	[23]
STIM1		S257, S521	Human Exercise	Hoffman 2015	[23]
VAPA		S164	Human Exercise	Hoffman 2015	[23]
AGPAT9		S68	Human Exercise	Hoffman 2015	[23]
SLC12A2		S77	Human Exercise	Hoffman 2015	[23]
CTNND1		S268	Human Exercise	Hoffman 2015	[23]
AKAP13		S2563	Human Exercise	Hoffman 2015	[23]
GPHN		S305	Human Exercise	Hoffman 2015	[23]
G0LGA4		S41	Human Exercise	Hoffman 2015	[23]
IGF2R		S2347	Human Exercise	Hoffman 2015	[23]
TNIK		S1021	Human Exercise	Hoffman 2015	[23]
MINK1		S900	Human Exercise	Hoffman 2015	[23]
PPP1R12B		S29	Human Exercise	Hoffman 2015	[23]

‘‘‘Other downstream targets, discovered not on human muscles. ‘‘‘

(Cantó 2010) [19]. AMPK has also been shown to directly phosphorylate CBP/ p300 at Ser89 [93]. This phosphorylation presumably alters the structure of the N-terminal region of the protein, impeding its interaction with nuclear receptors, such as PPARs.

Isoforms

AMPK is present in only three heterotrimers in human skeletal muscle: α1/β2/γ1, α2/β2/γ1 and α2/β2/γ3. The approximate distribution of the three AMPK heterotrimers can be estimated to be ∼15% α1/β2/γ1, ∼65% α2/β2/γ1 and∼20% α2/β2/γ3 (Birk 2006) [24].

AMPK α2/β2/γ3 heterotrimer is predominantly activated during exercise in human skeletal muscle (Birk 2006) [24].

Figure from (Jensen 2009) [25]. Skeletal muscle AMPK expression and regulation. (a) Molecular regulation of AMPK activity. (b) AMPK expression in human quadriceps and rodent oxidative and glycolytic muscles and their tentative activation profiles during contraction/exercise.

(Cantó 2010) [19]. Trimers containing the γ3 subunit are responsible for the majority of the effect of AMPK on PGC-1α deacetylation and activation upon exercise or fasting [65]. This is an interesting finding with longreaching consequences, as the γ3 subunit is enriched in fast glycolytic muscle, while it is almost absent in oxidative muscle [42]. This helps explaining why PGC-1α is not deacetylated in the oxidative soleus muscle or in the heart upon AMPK activation, but only in glycolytic skeletal muscle [62, 72]. Similarly, trimers containing the γ3 subunit are the ones more sensitive to exercise-induced energy stress in mouse muscle [28], making them the more apt to fine-tune exercise intensity/duration to transcriptional outputs.

(Cheung 2000) [26]. Expression of the γ3 subunit appears highly specific to glycolytic skeletal muscle whereas γ1 and γ2 show broad tissue distribution.

(Viollet ‎2010) [15].

AMPKα1 is hence likely to phosphorylate cytosolic and plasma membrane substrates, whereas AMPKα2 may be primarily involved in the conversion of metabolic signals into transcriptional regulation (Salt et al. , 1998a ).

Location

From (Pinter ‎2013) [27]. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca2+ influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be coordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

(Salt 1998) [28]. Within the individual muscle fibres, α2 AMPK has been reported in both the cytosol and nucleus, while α1 seems expressed exclusively in the cytosol.

(Calabrese 2014) [29]. a-N-myristoylation of the β subunit may facilitate reversible binding to membranes and activation by upstream kinases (Oakhill et al., 2010; Steinberg and Kemp, 2009; Warden et al., 2001)/

(Sanz 2013) [30]. Myristoylation consists of the addition of myristic acid (a 14-carbon saturated fatty acid) to a Gly residue in position 2, following processing of N-terminal Met residue. The myristoyl group acts as a lipid anchor, recruiting modified protein to cellular membranes [41]. Human AMPKβ subunits contain the consensus motif for myristoylation (MGNXXS/T) [42], because they have at their N-terminus the protein sequence MGNTSS (AMPKβ1) or MGNTTS (AMPKβ2). More importantly, using MS analysis it has been demonstrated that both AMPKβ subunits are myristoylated in vivo at Gly2 [38,43].

(Viollet ‎2010) [15]. AMPKα2 bound to AMPK β1 is anchored in the cytoplasm at the outer mitochondrial membrane through the myristoylation site of β1 subunit. In contrast, AMPKα2 bound to AMPKβ2 translocates to the nucleus in a manner driven by a nuclear localization signal present in AMPKα2 but not in AMPKα1 subunit (Suzuki et al., 2007).

Activity

AMPK phosphorylation (Thr172) and AMPK activity in mammalian muscles are usually strongly positively related. For example, for rat muscle, see figure (Park 2002) [31].

For human muscle see AMPK y3 associated activity after exercise at figure from (Birk 2006) [24]:

There is substantial constitutive AMPK kinase activity at rest. AMPK phosphorylation in resting muscle is possible because significant amount of AMPfree is present in resting muscle, for example see data from (Birk 2006) [24]:

In resting muscle most or all of the phosphorylated AMPK heterotrimers must be α1/β2/γ1 and α2/β2/γ1 (Birk 2006) [24].

Activity for isoforms AMPK-α1 and –α2

The measured values of AMPK activity, possibly depends on substrates and methods. For example in two works with the same exercise (30-s bicycle sprint) different activation patterns was shown:

1. (Chen 2000) [32].30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK- α1 and - α2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser79 (AMPK phosphorylation site) 8.5-fold.

2. (Birk 2006) [24]. In response to high-intensity exercise protocol, there was, surprisingly, no detectable increase in p-AMPK. In line with this, α1-associated activity also decreased significantly, whereas the α2 activity was unchanged. Between the two α2 complexes, only the α2/β2/γ3 activity increased in response to exercise.

Typical values for AMPK α1 and α2 activity are shown in Table:

Mode	AMPK-α1 activity	SD	measure unit	AMPK-α2 activity	SD	Substrate	Link	Ref
at rest	0,3	0,05	pmol/mg/min	0,27	0,05	phosphate incorporated into the ACC(73-87)A77	[32]	Chen 2000
at rest	0,5	0,08	pmol/mg/min	0,4	0,02	activity against the AMARA peptide	[24]	Birk 2006
at rest	0,75	0,05	pmol/mg/min	0,55	0,03	activity against the AMARA peptide	[24]	Birk 2006
at rest	0,85	0,05	pmol/mg/min	0,4	0,03	activity against the AMARA peptide	[24]	Birk 2006
at rest	0,9	0,02	pmol/mg/min	0,5	0,02	phosphate transferred to the SAMS peptide	[20]	Chen 2003
at rest	2,9	0,3	pmol/mg/min	1,1	0,1	SAMS-peptide (200 mol/l) as substrate	[33]	Wojtaszewski 2003
30-s sprint	0,8	0,15	pmol/mg/min	0,7	0,1	phosphate incorporated into the ACC(73-87)A77	[32]	Chen 2000
30-s sprint	0,6	0,05	pmol/mg/min	0,4	0,05	activity against the AMARA peptide	[24]	Birk 2006
120-s sprint	0,4	0,05	pmol/mg/min	0,8	0,1	activity against the AMARA peptide	[24]	Birk 2006
20 min at 80% VO2peak	0,7	0,05	pmol/mg/min	1,4	0,2	activity against the AMARA peptide	[24]	Birk 2006
20 min at 80% VO2peak	1,8	0,02	pmol/mg/min	6	2	pmol of phosphate transferred to the SAMS peptide	[20]	Chen 2003
10 min at 70% VO2peak	3	0,3	pmol/mg/min	1,35	0,3	SAMS-peptide (200 mol/l) as substrate	[33]	Wojtaszewski 2003
60 min at 70% VO2peak	3,5	0,3	pmol/mg/min	1,5	0,3	SAMS-peptide (200 mol/l) as substrate	[33]	Wojtaszewski 2003

Even at rest in one article different values (0.5-0.85 pmol/mg/min) of AMPK activity were measured for 3 ocassions (Birk 2006).

So data and conclusions about AMPK activity from different articles must be accepted with cautions.

AMPK α1- and α2- associated activity shows different time course during exercise (1 h at 70% peak VO2) and depends on muscle glycogen content (Wojtaszewski 2003) [33].

Data from (Birk 2006) [24] shows that AMPK α2/β2/γ3 heterotrimer is predominantly activated during exercise in human skeletal muscle, only the α2/β2/γ3 subunit is phosphorylated and activated during high-intensity exercise in vivo.

AMPK activity associated with the α2/β2/γ3 heterotrimer was strongly correlated to γ3-associated α-Thr-172 AMPK phosphorylation (r2 =0.84, P <0.001) (Birk 2006) [24].

The phosphorylated γ3 heterotrimers in the exercised state accounted only for 32±5% (n =11) of all the γ3 heterotrimers, still leaving the majority of these unphosphorylated and available for activation (Birk 2006) [24].

Data predict a unique role of the α2/β2/γ3 heterotrimer in human skeletal muscle, as only this heterotrimer is phosphorylated and activated during the three high-intensity exercise regimes investigated. The α2/β2/γ3 heterotrimer only constitutes one-fifth of all AMPK heterotrimers, and only one-third of these heterotrimers contain phosphorylated α2 protein, indicating a large pool of spare AMPK signalling within the cell even during high-intensity exercise. Accordingly, only a small fraction (10%) of the total α2 protein pool is phosphorylated during exercise (Birk 2006) [24].

Interestingly, in human skeletal muscle exercise training has been shown to reduce mRNA and γ3 protein expression (Nielsen et al. 2003; Frosig et al. 2004;Wojtaszewski et al. 2005). As hypothesized, the present finding, that only γ3 heterotrimers are activated in response to exercise, is in accordance with the observation of attenuated AMPK activation during acute exercise after a period of exercise training, even when exercise is performed at the same relative intensity (Nielsen et al. 2003; Yu et al. 2003; Frosig et al. 2004; McConell et al. 2005) from (Birk 2006) [24].

Short term stimulation of rat L6 myotubes with the AMPK activator (AICAR), activates AMPK and promotes translocation of the Na+,K+ -ATPase a1-subunit to the plasma membrane and increases Na+,K+-ATPase activity (Benziane 2012) [34].

Activity regulation

AMPK activity regulation is very complex process.

From (Garcia 2017) [9].

AMPK becomes fully activated through a three-pronged mechanism. First, binding of AMP or ADP to the γ subunit promotes Thr172 phosphorylation in the activation loop in the KD by upstream kinases. The main upstream kinase responsible for Thr172 phosphorylation in response to energy stress is the serine/threonine kinase LKB1 (liver-kinase-B1) (Hawley et al., 2003; Shaw et al., 2004; Woods et al., 2003). Phosphorylation of Thr172 in the a subunit is the principal event required for full activation of AMPK. This phosphorylation event can increase AMPK activity up to 100-fold in vitro, although fold activation in intact cells is usually more modest (Gowans et al., 2013; Oakhill et al., 2011; Suter et al., 2006). Second, binding of AMP or ADP to the γ subunit induces a conformational change that protects against Thr172 dephosphorylation by protein phosphatases (Gowans et al., 2013; Xiao et al., 2011). Notably, the phosphatases that normally dephosphorylate AMPK under physiological conditions remain largely unknown, though recent reports implicate roles for different phosphatases (Garcia-Haro et al., 2010; Joseph et al., 2015). Lastly, binding of AMP, but not ADP, results in up to 10-fold allosteric activation of AMPK (Gowans et al., 2013). Of note, ATP inhibits all three mechanisms. In addition to changes in adenine nucleotide levels, it has become increasingly clear that there are other important, non-canonical modes of AMPK regulation (Figure 2). The bestcharacterized nucleotide-independent regulation of AMPK is phosphorylation of Thr172 by CAMKK2 (calcium/calmodulindependent kinase kinase 2, also known as CAMKKb), the other major upstream kinase of AMPK (Hawley et al., 2005; Hurley et al., 2005; Woods et al., 2005). CAMKK2 is activated by increases in intracellular Ca2+ levels. Indeed, Ca2+-mediated CAMKK2 activation of AMPK is a frequent mechanism by which metabolically relevant hormones induce transient activation of AMPK. Thus, although CAMKK2 does not itself sense cellular energy status, it is nonetheless critical for the regulation of many aspects of whole-body metabolism by AMPK.

Besides phosphorylation of Thr172, phosphorylation of the ST-loop has emerged as an important site for the regulation and inhibition of AMPK by other kinases (Hardie, 2014). Examples of this include phosphorylation of Ser485 in AMPKa1 and the equivalent, Ser491, in AMPKa2 by PKA (cyclic-AMP-dependent protein kinase), which was proposed to be important for counteracting AMPK activity during gluconeogenic periods (Hurley et al., 2006), and by AKT (insulin-activated protein kinase), which was proposed to be a mechanism by which insulin inhibits AMPK (Horman et al., 2008). Similarly, S6K (p70S6 kinase) has been reported to inhibit AMPK activity by phosphorylating Ser491 in AMPKa2, and this phosphorylation was proposed as the mechanism by which leptin inhibits AMPK in the hypothalamus (Dagon et al., 2012).

Other kinases reported to inhibit AMPK by phosphorylating various residues in the ST-loop are GSK3 (glycogen synthesis kinase 3) (Suzuki et al., 2013), PKD1 (protein kinase D) (Coughlan et al., 2016), and PKC (protein kinase C) (Heathcote et al., 2016), though it remains to be determined which of these kinases are relevant in different tissues in vivo. Although the mechanism of AMPK inhibition is not entirely clear, it seems that phosphorylation of the STloop reduces net phosphorylation of Thr172, either by physically interfering with its phosphorylation or by promoting its dephosphorylation (Hawley et al., 2014). Collectively, these phosphorylation events on the ST-loop may represent an important mechanism to keep AMPK activity low during periods when anabolic metabolism is required.

Ubiquitin ligase, called WWP1, was reported to degrade AMPKa2 under high-glucose conditions in muscle cells (Lee et al., 2013), suggesting that ubiquitin-dependent degradation of AMPK may be a more broad mechanism for the regulation of AMPK than is currently recognized.

Another context that is increasingly appreciated as important for the regulation of AMPK activity is its localization to cellular membranes. Indeed, myristoylation of the b subunit can promote localization of AMPK to membranes (Liang et al., 2015; Oakhill et al., 2010). Interestingly, LKB1 is farnesylated and can also localize to membranes, which raised the question of whether co-localization of AMPK and LKB1 into two-dimensional surfaces of cellular membranes could promote activation of AMPK by LKB1.

N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. These may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high (Ali 2016) [35].

(Suzuki 2013) [36].

Glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK β regulatory subunit and phosphorylates the AMPK α catalytic subunit (Thr479). This phosphorylation enhances the accessibility of the activation loop of the a subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels.

The role of AMP to promote phosphorylation has proved controversial.

(Carling 2012) [37].

Our own studies indicated that the stimulatory effect of AMP on phosphorylation of Thr172 was due to contamination of the upstream kinase preparation with PP2C [43]. More recently, Kemp and colleagues reported that AMP can promote phosphorylation of Thr172, but only when the β subunit is myristoylated at the N-terminus [44]. However, another study using native AMPK purified from rat liver in which the β subunit is N-terminally myristoylated [45] failed to detect a stimulatory effect of AMP on Thr172 phosphorylation [26]. Further studies seem warranted in order to resolve these conflicting reports. A third independent mechanism by which AMP was proposed to increase Thr172 phosphorylation is by protecting the kinase from dephosphorylation [32,43,46]. In this case, there appears to be no dispute and recent data from structural studies provides strong evidence underlying a mechanistic basis for the protective effect [47].

(Suter 2006) [38]. AMP does not appear to augment AMPK phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK α-subunits at Thr-172 by protein phosphatase 2Cα is attenuated by AMP.

(Sanders 2007) [39]. We were unable to detect any effect of AMP on Thr172 phosphorylation by either LKB1 or CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver, there was an apparent AMP-stimulation of Thr172 phosphorylation by LKB1, but this was blocked by the addition of NaF, a PP inhibitor. Western blotting of partially purified rat liver AMPK and LKB1 revealed the presence of PP2Cα in the preparations. We suggest that previous studies reporting that AMP promotes phosphorylation of Thr172 were misinterpreted. A plausible explanation for this effect of AMP is inhibition of dephosphorylation by PP2Cα, present in the preparations of the kinases used in the earlier studies.

AMPK works like a delicate instrument. α-Thr172 is just like a start button, once it is phosphorylated, it achieves the initial activity. The γ subunit works like a sensor which senses the allosteric signal of AMP and induces the long range allosteric activation. The allosteric signal will be transduced to the engine, the α subunit, and fully activate the holoenzyme. When ATP and other molecules replace AMP, the allosteric activation is disturbed (Li 2017) [40].

At the same time opposite causal relationship described in review (Gowans 2014) [41] : “Only binding of AMP promotes phosphorylation of Thr172 by LKB1.”

(Gowans 2013) [42].

We report that: AMP is 10-fold more potent than ADP in inhibiting Thr172 dephosphorylation; only AMP enhances LKB1-induced Thr172 phosphorylation; and AMP can cause >10-fold allosteric activation even at concentrations 1–2 orders of magnitude lower than ATP. We also provide evidence that allosteric activation by AMP can cause increased phosphorylation of acetyl-CoA carboxylase in intact cells under conditions in which there is no change in Thr172 phosphorylation. Thus, AMP is a true physiological regulator of AMPK, and allosteric regulation is an important component of the overall activation mechanism.

Another important conclusion from our results is that simply estimating Thr172 phosphorylation as a marker for AMPK activation, which is common in the literature, can yield misleading results in that it completely ignores the effects of allosteric activation. We would suggest that it is advisable to also monitor the phosphorylation of at least one validated downstream target for AMPK, such as ACC.

(Zhang 2013) [43]. These findings demonstrate an initiating role of AMP and demonstrate that AXIN directly transmits AMP binding of AMPK to its activation by LKB1, uncovering the mechanistic route for AMP to elicit AMPK activation by LKB1.

From (Hardie 2016) [4]. It is now clear that AMP binding has three effects on AMPK that activate the system in a synergistic manner, making the final response very sensitive to even smallchanges in AMP:

(i) promotion of phosphorylation by LKB1, but not CaMKKβ although this selectivity for LKB1 has been disputed;

(ii) protection against dephosphorylation of Thr172 by protein phosphatases;

(iii) allosteric activation of the phosphorylated kinase.

Of these three effects, it has been reported that mechanisms (i) and (ii) are also mimicked by binding of ADP. Given that ADP is present in unstressed cells at concentrations ten times higher than AMP,and that allosteric activation (which is only caused by AMP binding) is often reported as being small in magnitude (<2-fold), this led top roposals that ADP rather than AMP might be the crucial activator of AMPK. However,our group reported that while mechanism (ii) can indeed be caused by binding of ADP, AMP is about 10-fold more potent. Thus, while ADP may contribute to activation, we would argue that AMP remains the primary regulator of AMPK.

Allosteric activation mechanisms of AMPK by AMP

A proposed allosteric activation model

The energy status holds a dynamic equilibrium in eukaryotic cells. In the resting state, AMPK stays in the ATP-bound form and ATP occupies γ-site 1 and γ-site 4. The conformational changes of the γ subunit and the collective upwards movement of the inter-subunit β-sheet make the holoenzyme less compact and also restrict the interaction between α-RIMs and the γ-sites. The α-linker is flexible and the AID stably binds to the kinase domain and maintains it in a relative open, inactive conformation. When the energy level decreases, the concentration of AMP will increase and AMP competitively binds to the AMPK at γ-site 1, γ-site 3 and γ-site 4. This induces substantial conformational changes that enable α-RIM 1 and α-RIM 2 to be recruited to the γ-site 2 and γ-site 3, respectively. Then, the constrained α-linker forces the disassociation of the adjacent AID from the KD, then the AID moves towards and directly binds to the γ-subunit. Thus, the KD is in a more closed, active conformation and the AMPK is allosterically activated (Fig 2D).

Figure 2D from (Li 2017) [40]. In the resting state, the concentration of ATP is high and AMPK is saturated with ATP at γ-site1 and 4, the holoenzyme adopts an relative loose conformation. The AID stably binds to the backside of KD and keeps it in an inactive state, and the α-linker is flexible and disordered in the structure (dotted line). Once the AMP/ATP ratio increases, the AMP will competitively bind to the γ-site1, 3 and 4 and induces lots of conformational changes, then recruits α-RIM1/2 and the induced β-loop. The constrained α-linker forces the disassociation of AID from KD, therefore releases its inhibition effect to KD, and the AID binds to the N-terminus of γ-subunit. The holoenzyme becomes more compact and is allosterically activated.

From review (Oakhill ‎2012) [44]. The calculated free AMP and ADP values (expressed in mM) can be used to ask how they compare with Kd values for the binding of adenine nucleotides to site 3, which controls AMPK a-Thr172 phosphorylation [20]. With metabolic stress, free AMP levels in the perfused heart increase 136-fold, from 0.2 mM to 27.9 mM, whereas ADP levels increase 10.2-fold, from 41.9 mM to 425 mM. However, both ADP and AMP bind to site 3 with similar Kd values of 50–80 mM, respectively (Table 1). Thus, despite the large fold-increase in AMP at its maximum, concentrations only approach approximately 50% of the Kd value for site 3. By contrast, ADP at its maximum concentration is approximately eightfold the Kd value for site 3. It is clear from Figure 3 that rises in ADP correlate well with AMPK activation, whereas AMP levels do not exceed the Kd for site 3. Similar results are seen in three human exercise studies [58–60].

Figure 3(b) from (Oakhill ‎2012) [44]. Exercising human skeletal muscle (exercise regimes – low int.: 40 +-2% VO2 peak, 20 min; med. int.: 59+-1% VO2 peak, 20 min; high int.: 79+-1% VO2 peak, 20 min) [59]. Over low-, moderate-, and high-intensity exercise regimes, free AMP levels increased 46-fold, from 0.2 mM to 9.3 mM, whereas free ADP levels increased 5.8-fold, from 36.5 mM to 212.9 mM. Again, the rise in AMP concentration in response to exercise is dramatic relative to resting levels, but the maximum concentration is well short of the Kd for site 3. By contrast, the peak in free ADP concentration is 15.5-fold the Kd for site 3. Hence, the activation of AMPK in response to exercise clearly correlates with the rise in ADP. The Km for AMP binding at the deaminase active site is ~50 mM [62], similar to the Kd for AMP at AMPK g site 3. Therefore, deamination of AMP ensures that its concentration is unlikely to exceed the Kd for site 3 binding, thus preventing it from playing a significant role in regulating a-Thr172 phosphorylation in vivo. Is AMP direct allosteric activation likely to play a role in vivo? The Kd for AMP binding to site 1, which is responsible for direct allosteric activation of a-Thr172-phosphorylated AMPK, is 2.5 mM [20] and well within the range of free AMP levels that occur in metabolically stressed tissue (~30 mM). However, Mg2+-free ATP and ADP also bind to site 1 with similar Kd values and would out-compete AMP, thus minimizing any direct allosteric activation.

In exercising skeletal muscle AMP is readily deaminated to IMP, which does not participate in AMPK regulation [19].

(Oakhill 2011) [45].

The contribution of AMP deaminase in muscle, which rapidly and irreversibly removes AMP from the system to form inosine monophosphate (IMP), a clearance mechanism that may drive the adenylate kinase reaction to reduce ADP that would otherwise inhibit protein kinase signaling (27). Concentrations of IMP increase more than 350-fold in skeletal muscle after stimulated contraction or exercise, with a corresponding drop in total adenine nucleotide content (27–29). We found no AMPK regulatory function for IMP, and IMP did not antagonize AMPenhanced phosphorylation of Thr172. Presumably, IMP cannot form critical N6 hydrogen bonds to backbone residues in sites 1 and 3.

(Kim 2016) [46]. In contrast to the LKB1 complex, another upstream AMPK kinase, CaMKKβ, can activate AMPK in response to increases in cellular Ca2+ without any significant change in ATP/ADP/AMP levels.

Possible regulation of AMPK by creatine phosphate

(Ponticos 1998) [47].

A key finding which supports a physiological role for the control of MM-CK by AMPK was the elucidation of a novel and complex mechanism of AMPK regulation by PCr and Cr. We have shown in vitro that, at concentrations which occur within muscle, PCr inhibits AMPK and Cr antagonizes this inhibition. This implies that AMPK is sensitive to the PCr:Cr ratio, rather than to the concentration of either metabolite per se.

(Taylor 2006) [48].

Inhibition of skeletal muscle AMPK by creatine phosphate was greatly reduced or eliminated with increased AMPK purity. In conclusion, these results suggest that creatine phosphate is not a direct regulator of LKB1 or AMPK activity. Creatine phosphate may indirectly modulate AMPK activity by replenishing ATP at the onset of muscle contraction.

Neverless, PCr / (Cr + PCr) ratio is closely related to increasing of AMPK phosphorylation, see unpublished data from meta analysis (Vertyshev 2017).

AMPK regulation by Thr172 phosphorylation

From review (Gowans 2014) [41]. The major kinase responsible for phosphorylation of Thr172 is a complex containing the tumour suppressor LKB1 [18,19]. Although this complex appears to be constitutively active [20], binding of AMP to AMPK not only causes allosteric activation, but also makes AMPK a better substrate for LKB1 [21], and a worse substrate for protein phosphatases [22], thus promoting net phosphorylation and activation. Thr172 can also be phosphorylated by CaMKKs (Ca2+ /calmodulin-activated protein kinase kinases), especially CaMKKβ [23–25], providing an alternative Ca2+ - dependent pathway for AMPK activation. This can occur in the absence of any changes in adenine nucleotides, although changes in cellular AMP can amplify the effect of the Ca2+ - activated pathway due to the protective effect of AMP on Thr172 dephosphorylation [26].

Binding of ADP, as well as AMP, protects AMPK against dephosphorylation by protein phosphatases, but AMP was approximately 10-fold more potent than ADP [38].

Only binding of AMP promotes phosphorylation of Thr172 by LKB1.

Our laboratory originally reported that AMP binding to AMPK promoted phosphorylation of Thr172 by LKB1, but not CaMKKβ [23]. However, it was reported more recently that binding of AMP promoted phosphorylation by both LKB1 and CaMKKβ, whereas ADP also promoted phosphorylation by CaMKKβ, as long as the β subunit in the AMPK complex was N-myristoylated [33,39]. We have reinvestigated this and confirmed our original findings that AMP promoted phosphorylation only by LKB1, and not CaMKKβ. We were unable to detect effects of ADP on the rate of phosphorylation by either kinase. The effect of AMP to promote phosphorylation by LKB1 appears to require higher concentrations than its effects on dephosphorylation, suggesting that it may be caused by binding to a different site.

Despite the fact that phosphorylation of Thr172 by upstream kinases can cause >100-fold activation when fully dephosphorylated AMPK complexes are incubated with upstream kinases in cell-free assays, we showed that the effects in intact cells are much more modest [38].

(Kim 2016) [46]. The effect of AMP on Thr172 phosphorylation of the AMPK α-subunit appears to be dependent on N‐terminal myristoylation of the β-subunit, although the underlying mechanism remains to be demonstrated [45].

AMPK regulation by phosphorylation, hormones

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser487 in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase- α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. (Hawley 2014) [49].

From review (Hardie 2016) [50].

Hormones that increase intracellular Ca2+ activate AMPK via phosphorylation of Thr172 by the calmodulin-dependent proteinkinase CaMKKβ [33–35]. This represents an alternative, Ca2+-dependent pathway for AMPK activation that is independent of changes in adenine nucleotides and is therefore considered to be non canonical. However, because AMP binding inhibits Thr172 dephosphorylation, the two pathways can act synergistically [36].

AMPK regulation by ROS and NOS

From review (Morales-Alamo 2016) [10]. Reactive oxygen and nitrogen species (RONS) may activate AMPK by direct an indirect mechanisms. Directly, RONS may activate or deactivate AMPK by modifying RONS-sensitive residues of the AMPK-α subunit. Indirectly, RONS may activate AMPK by reducing mitochondrial ATP synthesis, leading to an increased AMP:ATP ratio and subsequent Thr172-AMPK phosphorylation by the two main AMPK kinases: LKB1 and CaMKKβ. In presence of RONS the rate of Thr172-AMPK dephosphorylation is reduced. RONS may activate LKB1 through Sestrin2 and SIRT1 (NAD+/NADH.H+-dependent deacetylase). RONS may also activate CaMKKβ by direct modification of RONS sensitive motifs and, indirectly, by activating the ryanodine receptor (Ryr) to release Ca2+. Both too high (hypoxia) and too low (ingestion of antioxidants) RONS levels may lead to Ser485-AMPKα1/ Ser491-AMPKα2 phosphorylation causing inhibition of Thr172-AMPKα phosphorylation.

From (Morales-Alamo 2013) [51]. Antioxidant ingestion 2 h before sprint exercise abrogates the Thr172-AMPKα phosphorylation response observed after the ingestion of placebo by reducing CaMKII and increasing Ser485-AMPKα1/Ser491-AMPKα2 phosphorylation. Sprint performance, muscle metabolism, and AMP-to-ATP and NAD+-to- NADH.H+ ratios are not affected by the acute ingestion of antioxidants.

AMPK regulation by glycogen

Hypotetically, glycogen depletion causes the release of AMPK to the cytosol, which occurs in conjunction with AMPK activation (Oligschlaeger 2015) [52].

AMPK β subunits contain a central conserved region that we refer to as the glycogen-binding domain (GBD), which causes AMPK complexes to associate with glycogen in cultured cells and cell-free systems (Hudson et al., 2003; Polekhina et al., 2003). Glycogen availability is known to affect AMPK regulation in vivo. We show that AMPK is inhibited by glycogen, particularly preparations with high branching content. AMPK, as well as monitoring immediate energy availability by sensing AMP/ATP, may also be able to sense the status of cellular energy reserves in the form of glycogen. Even a modest reduction in glycogen content might cause release of significant quantities of AMPK from the polysaccharide so that more of the kinase becomes available to phosphorylate targets (McBride 2009) [22].

The isolated β1- and β2-CBM (carbohydrate-binding module) domains of AMPK show different affinities for carbohydrate, with β2-CBM always binding with a higher affinity than β1-CBM. The most marked difference observed was the 15-fold increase in affinity by β2-CBM for glucosyl-maltoheptaose (18). The differences observed for the isolated domain are generally maintained in the AMPK heterotrimer, suggesting that AMPKs containing the β2-subunit will be localized to glycogen more readily than those containing the β1-subunit. Compared with the isolated CBMs, the affinity differences of the AMPK heterotrimers for carbohydrate are more pronounced, with AMPK β2-subunit binding ~10-fold more tightly to maltoheptaose and ~30-fold more tightly to glucosyl-maltoheptaose. The AMPK b2-subunit is highly expressed in skeletal muscle, contributing to α1β2γ1, α2β2γ1, α2β2γ3 heterotrimers (Bieri 2012) [53].

(Steinberg 2006) [54].

Low muscle glycogen was associated with elevated AMPK α2 activity and acetyl-CoA carboxylase β phosphorylation, increased translocation of AMPK α2 to the nucleus, and increased GLUT4 mRNA.

(Sanz 2013) [30]. AMPKβ subunits are also critical players in AMPK function, because they can regulate the phosphorylation status and activity of the AMPK complex. It has been reported that phosphorylation of AMPK β1 subunit at Ser24/25 and Ser182 residues, although it does not affect AMPK activity, is necessary for the nuclear exclusion of the β1 subunit, whereas phosphorylation of Ser108, although it does not affect subcellular distribution of AMPKβ, increases AMPK activity [28].

(McBride 2009) [22]. A major problem with the study of glycogen as a regulatory molecule is that it does not have a defined structure, so different preparations may behave quite differently. This is illustrated by the dramatic difference in inhibitory effect between the preparations of bovine and rat liver glycogen. This may also account for the findings of Polekhina et al. (2003), who reported that glycogen did not inhibit purified rat liver AMPK, and for the failure of Parker et al. (2007) to find any AMPK associated with glycogen purified from rat liver.

AMPK regulation by exercise

(Frøsig 2004) [6].

In response to training the protein content of α1, β2 and γ1 increased in the trained leg by 41, 34, and 26%, respectively (α1 and β2 P<0.005, γ1 P<0.05). In contrast, the protein content of the regulatory γ3-isoform decreased by 62% in the trained leg (P= 0.01), whereas no effect of training was seen for α2, β1, and γ2. AMPK activity associated with the α1- and the α2-isoforms increased in the trained leg by 94 and 49%, respectively (both P< 0.005). In agreement with these observations, phosphorylation of α-AMPK- (Thr172) and of the AMPK target acetyl-CoA carboxylase-β(Ser221) increased by 74 and 180%, respectively (both P< 0.001). Essentially similar results were obtained in four additional subjects studied 55 h after training. This study demonstrates that protein content and basal AMPK activity in human skeletal muscle are highly susceptible to endurance exercise training. Except for the increase in γ1 protein, all observed adaptations to training could be ascribed to local contraction-induced mechanisms, since they did not occur in the contralateral untrained muscle.

(Kristensen 2015) [5].

The ~70% lower γ3 AMPK protein content observed in type I vs. II muscle fibres is more pronounced than previously reported (Lee-Young et al. 2009). Immunohistochemical detection of γ3 AMPK in muscle cryosections showed that the expression level of γ3 AMPK was highest in type IIx > IIa > I muscle fibres (approximately 14% lower in type I vs. IIa muscle fibres and 33% lower in type I vs. IIx muscle fibres) (Lee-Young et al. 2009). In human skeletal muscle γ3 selectively associates with α2 and β2 (Wojtaszewski et al. 2005) and thus our findings indicate a lower expression level of the α2β2γ3 AMPK complex in human type I vs. II muscle fibres. γ3 AMPK protein in muscle is decreased by exercise training and increased by detraining (muscle denervation) and associations with MHC expression have been reported in both conditions (Nielsen et al. 2003; Frøsig et al. 2004; Wojtaszewski et al. 2005; Mortensen et al. 2009; Kostovski et al. 2013). Confirming previous observations (Birk & Wojtaszewski, 2006; Treebak et al. 2007), α2β2γ3 AMPK was the only complex activated during exercise in whole muscle biopsies, in the present study. As the glycogen degradation pattern suggests a lower activation/recruitment of type I vs. type II muscle fibres during exercise in INT, we expected a lower α2β2γ3 AMPK activity in type I compared to type II fibres during exercise in INT. Fibre type-specific AMPKThr172 phosphorylation during exercise supports this assumption. However, since the α2β2γ3 AMPK complex accounts for only ~20% of all AMPK complexes in human skeletal muscle (Birk & Wojtaszewski, 2006), phosphorylation of AMPKThr172 is not necessarily a precise measure of AMPK complex activation. Thus, we cannot exclude the possibility of a differentiated AMPK activation in type I and II muscle fibres during exercise in CON based on this measurement alone. Since AMPK activity is also regulated allosterically, and to further delineate AMPK activation in type I and II muscle fibres during exercise, we measured site-specific phosphorylation of several confirmed AMPK targets: ACCSer221, TBC1D1Ser231, TBC1D4Ser704 and GS2+2a. In skeletal muscle, these targets have been verified using pharmacological and exercise-induced AMPK activation in various transgenic animal models in which AMPK expression has been ablated (Carling & Hardie, 1989; Ha et al. 1994; Jørgensen et al. 2004; Pehmøller et al. 2009; Treebak et al. 2010; Frøsig et al. 2010). In line with this, phosphorylation of ACCSer221, TBC1D1Ser231 and TBC1D4Ser704 were closely associated with α2β2γ3 AMPK activity measured in whole muscle lysate in the present study (Fig. 7A–C). Furthermore, these three estimates of endogenous AMPK activity showed no fibre type-specific regulation during exercise in CON whereas all depicted this during exercise in INT in agreement with the phosphoregulation of AMPKThr172. Altogether, regulation of these AMPK downstream targets suggests that interval exercise elicits a fibre type-specific response and we propose that activation of α2β2γ3 AMPK complexes is a major contributor to this response.

Outstanding Questions

(Hardie 2016b) [55].

Outstanding Questions Given that site 3 on the AMPKγ subunit seems to be the crucial site where binding of AMP causes activation, what is the function of AMP binding at the other two sites, 1 and 4?

How does binding of AMP or ADP to the AMPKγ subunit promote phosphorylation of the AMPKα subunit at Thr172 by upstream kinases such as LKB1?

Are there any naturally occurring metabolites in mammalian cells that bind to the ADaM-binding pocket between the a and p subunits and thus activate or inhibit AMPK?

How are responses to different inputs affected by the numerous post-trans-lational modifications that have been reported to occur on each AMPK subunit?

Does AMPK phosphorylate a different subset of substrates depending on the inputs via which it was activated?

Are there subsets of primary or secondary structures, beyond the defined window from amino acid -5 to +4 in the AMPK recognition motif, that can help better predict substrates?

How much flexibility is there in the recognition motif surrounding any given AMPK phosphorylation site? For example, can more distal features, such as the N-terminal amphipathic a helices that are observed in acetyl-CoA carboxylase and HMG-CoA reductase, determine hew rigidly the motif from P-5 to P+4 must adhere to the canonical recognition motif?

Expression

PRKAA1 Protein Kinase AMP-Activated Catalytic Subunit Alpha 1 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAA1)

PRKAA2 Protein Kinase AMP-Activated Catalytic Subunit Alpha 2 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAA2)

PRKAB1 Protein Kinase AMP-Activated Non-Catalytic Subunit Beta 1 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAB1)

PRKAB2 Protein Kinase AMP-Activated Non-Catalytic Subunit Beta 2 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAB2)

PRKAG1 Protein Kinase AMP-Activated Non-Catalytic Subunit Gamma 1 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAG1)

PRKAG2 Protein Kinase AMP-Activated Non-Catalytic Subunit Gamma 2 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAG2)

PRKAG3 Protein Kinase AMP-Activated Non-Catalytic Subunit Gamma 3 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAG3)

http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD006182

References

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