AMPK

AMPK Protein Kinase AMP-Activated Ver. 0.2.

Structure

The structure with regulation sites is presented on Figure 1 from [1].

Figure from [1]. 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 is 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)

Pathways

In general, upstream pathways presented at Figure from [1].

In general, downstream pathways presented at Figure from [1].

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 [2].

Downstream

(Chen 2003) [3]. 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.

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 [4].

AMPK α2/β2/γ3 heterotrimer is predominantly activated during exercise in human skeletal muscle [4].

Figure from [5]. 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.

Location

Within the individual muscle fibres, α2 AMPK has been reported in both the cytosol and nucleus, while α1 seems expressed exclusively in the cytosol [6].

Activity

AMPK phosphorylation (Thr172) and AMPK activity in mammalian muscles are usually strongly positively related. For example, for rat muscle, see figure [7].

For human muscle see figure from [4]:

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 [4]:

In resting muscle most or all of the phosphorylated AMPK heterotrimers must be α1/β2/γ 1 and α2/β2/γ 1 [4].

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) [8].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) [4]. 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 phosphorylation and activity increased in response to exercise.

Typical values for AMPK activity are shown in Table:

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

Even at rest and one article different values 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 show different time course during exercise (1 h at 70% peak VO2) and depends on muscle glycogen content [9].

Data from (Birk 2006) [4] 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) [4].

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 [4].

Data predict a unique role of the α2/β2/γ 3 heterotrimer in human skeletal muscle, as only this heterotrimer is phosphorylated and activated during the three high-intensity exercise regimes investigated. The α2/β2/γ 3 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 [4].

Interestingly, in human skeletal muscle exercise training has been shown to reduce mRNA and γ 3 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) [4].

Activity regulation

(b) 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].

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)

References

1. Jeon SM. Regulation and function of AMPK in physiology and diseases. Exp Mol Med. 2016 Jul 15;48(7):e245. DOI:10.1038/emm.2016.81 |
2. Garcia D and Shaw RJ. AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance. Mol Cell. 2017 Jun 15;66(6):789-800. DOI:10.1016/j.molcel.2017.05.032 |
3. Chen ZP, Stephens TJ, Murthy S, Canny BJ, Hargreaves M, Witters LA, Kemp BE, and McConell GK. Effect of exercise intensity on skeletal muscle AMPK signaling in humans. Diabetes. 2003 Sep;52(9):2205-12. DOI:10.2337/diabetes.52.9.2205 |
4. Birk JB and Wojtaszewski JF. Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle. J Physiol. 2006 Dec 15;577(Pt 3):1021-32. DOI:10.1113/jphysiol.2006.120972 |
5. Jensen TE, Wojtaszewski JF, and Richter EA. AMP-activated protein kinase in contraction regulation of skeletal muscle metabolism: necessary and/or sufficient?. Acta Physiol (Oxf). 2009 May;196(1):155-74. DOI:10.1111/j.1748-1716.2009.01979.x |
6. Salt I, Celler JW, Hawley SA, Prescott A, Woods A, Carling D, and Hardie DG. AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform. Biochem J. 1998 Aug 15;334 ( Pt 1)(Pt 1):177-87. DOI:10.1042/bj3340177 |
7. Park SH, Gammon SR, Knippers JD, Paulsen SR, Rubink DS, and Winder WW. Phosphorylation-activity relationships of AMPK and acetyl-CoA carboxylase in muscle. J Appl Physiol (1985). 2002 Jun;92(6):2475-82. DOI:10.1152/japplphysiol.00071.2002 |
8. Chen ZP, McConell GK, Michell BJ, Snow RJ, Canny BJ, and Kemp BE. AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation. Am J Physiol Endocrinol Metab. 2000 Nov;279(5):E1202-6. DOI:10.1152/ajpendo.2000.279.5.E1202 |
9. Wojtaszewski JF, MacDonald C, Nielsen JN, Hellsten Y, Hardie DG, Kemp BE, Kiens B, and Richter EA. Regulation of 5'AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle. Am J Physiol Endocrinol Metab. 2003 Apr;284(4):E813-22. DOI:10.1152/ajpendo.00436.2002 |