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Am J Physiol Heart Circ Physiol 293:457-466, 2007. First published Mar 16, 2007;
doi:10.1152/ajpheart.00002.2007
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Am J Physiol Heart Circ Physiol 293: H457–H466, 2007.
First published March 16, 2007; doi:10.1152/ajpheart.00002.2007.
Zhang L, He H, Balschi JA. Metformin and phenformin ferase 1, the primary transporter and control point for fatty
activate AMP-activated protein kinase in the heart by increasing acyl-CoA entry into the mitochondrion. Thus lower malonyl-
cytosolic AMP concentration. Am J Physiol Heart Circ Physiol CoA results in an increase in the transport of fatty acids into the
293: H457–H466, 2007. First published March 16, 2007; mitochondrion, acceleration of -oxidation of fatty acids, and
doi:10.1152/ajpheart.00002.2007.—AMP-activated protein kinase increased generation of ATP. Fatty acid oxidation by AMPK
(AMPK) acts as a cellular energy sensor: it responds to an increase in
increases during reperfusion after ischemia. Increased fatty
AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP).
Metformin and phenformin, which are biguanides, have been reported acid oxidation in this setting can contribute to ischemic injury
by inhibiting glucose oxidation (9).
Address for reprint requests and other correspondence: J. A. Balschi, 221 The costs of publication of this article were defrayed in part by the payment
Longwood Ave., BLI 247, Boston, MA 02115 (e-mail: jbalschi@rics.bwh. of page charges. The article must therefore be hereby marked “advertisement”
harvard.edu). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
http://www.ajpheart.org 0363-6135/07 $8.00 Copyright © 2007 the American Physiological Society H457
H458 METFORMIN ACTIVATES AMPK BY INCREASING CYTOSOLIC [AMP]
phosphorylation of AMPK-␣. This biguanide-induced increase baseline period for each heart was set to 10.8 mM (2), and the areas
in AMPK activity was preceded by and correlated with an of other resonances, corrected for variable saturation, were calculated
increase in cytosolic [AMP]. The total AMP content of met- on this basis. Intracellular pH (pHi) was calculated from the chemical
formin-treated hearts did not change. shift of intracellular Pi (3). Metabolite measurements are an average of
the 4 min of the 31P-NMR data acquisition, with the midpoint of the
MATERIALS AND METHODS spectrum reported as the measurement time.
Calculation of cytosolic AMP. 31P-NMR measurements of PCr
Preparation of Isolated Perfused Rat Hearts concentration ([PCr]), ATP concentration ([ATP]), and H⫹ concen-
Hearts of male Sprague-Dawley rats (280 –320 g) were isolated and tration ([H⫹]) were entered into the creatine (Cr) kinase reaction
perfused in the isovolumic Langendorff model. Krebs-Henseleit (PCr ⫹ ADP ⫹ H⫹ 7 Cr ⫹ ATP) equilibrium expression to calculate
buffer (KH; in mM: 118 NaCl, 5.9 KCl, 1.2 MgSO4, 25 NaHCO3, cytosolic ADP concentration ([ADP]) using an equilibrium constant
1.75 CaCl2, 0.5 Na-EDTA, 10 D-glucose, and 0.5 pyruvate) was (Keq) of 1.66 ⫻ 109 M⫺1 (23)
equilibrated with 95% O2-5% CO2 to achieve pH 7.4. Phenformin
(Sigma-Aldrich) and metformin (Sigma-Aldrich) were added to KH. 共关ATP兴关Cr兴兲
关ADP兴 ⫽ (1)
A pressure transducer connected to a latex balloon in the left 共关PCr兴关H⫹兴 Keq兲
ventricle (LV) was used to monitor heart function, i.e., LV pressure
and heart rate (HR). LV pressure and HR were recorded using a Cr concentration ([Cr]) is calculated as the difference between PCr
MacLab system (ADInstruments, Colorado Springs, CO). content and total Cr content (3). The 31P-NMR-measured pHi was
The experimental protocols were approved by the Harvard Medical used to estimate [H⫹]. The near equilibrium of the adenylate kinase
Area Standing Committee on Animals and followed the recommen- reaction (2ADP 7 AMP ⫹ ATP) converts increased cytosolic [ADP]
Fig. 1. A: systolic pressure (SP, squares) and end-diastolic pressure (EDP, circles) in isolated isovolumic rat hearts. Perfusion began with Krebs-Henseleit
medium (KH, baseline); at 0 min, perfusion medium was maintained as KH (open symbols) or switched to KH ⫹ 0.2 mM phenformin (filled symbols) for 18
min (treatment); after 18 min, perfusion continued with KH only (posttreatment). B: coronary flow (CF) in isolated rat hearts. Perfusion began with KH (baseline);
at 0 min, perfusion medium was maintained as KH (ƒ) or switched to KH ⫹ 0.2 mM phenformin () for 18 min (treatment); after 18 min, perfusion continued
with KH only (posttreatment). C: SP (squares) and EDP (circles) in isolated isovolumic rat hearts. Perfusion began with KH (baseline); at 0 min, perfusion
medium was maintained as KH (open symbols) or switched to KH ⫹ 10 mM metformin (filled symbols, treatment). D: CF in isolated rat hearts. Perfusion began
with KH (baseline); at 0 min, perfusion medium was maintained as KH (ƒ) or switched to KH ⫹ 10 mM metformin (, treatment). Values are means (SD). *P ⬍
0.05 vs. KH at the same time. #P ⬍ 0.05 vs. baseline (⫺2 min).
31
The effect of phenformin on myocardial function is quite P-NMR-measured metabolite content of the isolated rat
rapid and precedes its effect on metabolism. The effect on heart: phenformin and metformin decreased [PCr] and in-
myocardial function also occurs at a lower phenformin con- creased [AMP]. 31P-NMR was used to measure PCr and ATP
centration than the metabolic effect. Finally, the increase in contents of the isolated heart continuously during the experi-
[AMP] and AMPK activity in the phenformin-treated K⫹- mental protocols. In hearts treated for 18 min with phenformin,
arrested heart occurred without changes in CF. Taken together, [PCr] decreased at 22 min relative to baseline and was different
these results suggest that the effects of phenformin on function from [PCr] of KH-perfused hearts at 26 min (Fig. 2A). In
and metabolism are independent. phenformin-treated hearts, [ATP] decreased after 6 min of
The functional response of hearts perfused with KH ⫹ 10 treatment relative to baseline but was never different from that
mM metformin was quite different from that of phenformin- of the KH-perfused group (Fig. 2B). In phenformin-treated
treated hearts. SP, EDP, and CF were unchanged during the hearts, cytosolic [AMP] increased at 26 min (Fig. 2C) relative
first 46 min of the treatment period (Fig. 1, C and D). At 50 to baseline and KH-perfused hearts.
min, SP and CF increased, while EDP was unchanged. These In metformin-perfused hearts, [PCr] decreased relative to base-
changes in LVSP and CF occur concomitantly with the in- line at 10 min and was different from that in KH-perfused hearts
crease in cytosolic [AMP] (Fig. 3C). These data demonstrate at 30 min (Fig. 3A). In metformin-perfused hearts, myocardial
that SP was not compromised in metformin-treated hearts, [ATP] exhibited a sustained decrease at 30 min relative to baseline
even with substantial decreases in [PCr] and increases in but was only different from that in KH-perfused hearts at several
cytosolic [AMP]. The increase in CF may result from the time points (Fig. 3B). Cytosolic [AMP] in metformin-perfused
elevated [AMP], which will result in increased adenosine hearts increased at 42 min relative to baseline and at 50 min
production (19) and vasodilation (22). relative to KH-perfused hearts (Fig. 3C).
AJP-Heart Circ Physiol • VOL 293 • JULY 2007 • www.ajpheart.org
METFORMIN ACTIVATES AMPK BY INCREASING CYTOSOLIC [AMP] H461
AMPK activity and AMPK and ACC phosphorylation in [AMP] measured at 53 min (Fig. 5A) includes the five hearts
isolated rat heart: effects of phenformin and metformin. clamped at 53 min.
AMPK activity and phosphorylation were measured in hearts The relationship between AMPK activity and cytosolic
that were freeze-clamped just after the end of phenformin [AMP] is shown in Fig. 6 for hearts treated with phenformin
treatment at 21 min (Phen) and at the end of the posttreatment and metformin. Data from all KH-perfused hearts and hearts
period at 37 min (arrows in Fig. 2C). After the treatment treated with phenformin, phenformin-treated and KH-perfused
period, [AMP], AMPK activity, phosphorylated AMPK, and hearts, and hearts treated with metformin for 53, 61, and 70
phosphorylated ACC of the KH- and phenformin-perfused min (Figs. 2–5 and 7) are included. Measurements from hearts
hearts were equal (Fig. 4). In hearts perfused for another 20 treated with phenformin and metformin under conditions that
min with KH after 18 min of phenformin treatment, [AMP], surpass the threshold increase in AMPK activity are included
AMPK activity, phosphorylated AMPK, and phosphorylated as well.
ACC were increased (Fig. 4).
In metformin-treated hearts, AMPK activity and phosphor- DISCUSSION
ylation were measured at 53 and 61 min (arrows in Fig. 3C). In
hearts perfused for 53 min with metformin, [AMP] (Fig. 5A, 50 The cause of increased AMPK activity resulting from phen-
min), AMPK activity (Fig. 5B), phosphorylated AMPK (Fig. formin and metformin exposure has not been defined. There
5C), and phosphorylated ACC (Fig. 5D) were equal to those in are two known pathways for AMPK regulation in mammalian
KH-perfused hearts. In hearts perfused for another 8 min with cells. The first and best defined pathway involves increased
metformin (61 min of perfusion) [AMP], AMPK activity, AMP binding to the cystathione -synthase domains of
phosphorylated AMPK, and phosphorylated ACC were in- AMPK-␥. This increases Thr172 phosphorylation of AMPK-␣
creased. In metformin-treated hearts, cytosolic [AMP] was by AMPK kinase, which is required for increased AMPK
significantly different than that in KH-perfused hearts at 50 activity. In the heart, LKB1 is the AMPK kinase that phos-
min. Measurement of [AMP] at 50 min includes all 10 hearts. phorylates AMPK-␣2, but not AMPK-␣1, heterotrimers during
AJP-Heart Circ Physiol • VOL 293 • JULY 2007 • www.ajpheart.org
H462 METFORMIN ACTIVATES AMPK BY INCREASING CYTOSOLIC [AMP]
ischemia and hypoxia, both of which increase AMP (32). We nase-1 (TAK1) has been reported to function as an AMPK
have demonstrated in the isolated rat heart that AMPK activity kinase in yeast and HeLa cells (27). In cultured fibroblasts,
responds to cytosolic [AMP] (11), but in a cytosolic [AMP]- TAK1 was activated by a number of stimuli, including met-
independent manner to hypoxia (12). The pathway for regula- formin, that are known to increase AMPK activity (36). Loss of
tion by LKB1 after an increase in [AMP] corresponds to the TAK1 prevented the increase in Thr172 phosphorylation of
cellular energy sensor function of AMPK. The second pathway AMPK-␣ induced by metformin and interfered with activation
for AMPK regulation is directed by Ca2⫹/calmodulin-depen- of LKB1. These results suggest that TAK1 increases LKB1
dent protein kinase kinase (CaMKK) activity (18, 21, 35). activity and does not directly phosphorylate AMPK. It appears
CaMKK also phosphorylates AMPK-␣ at Thr172. AMP bind- that TAK1 modulates the AMPK energy sensor pathway.
ing to AMPK is not required. CaMKK activity increases in All studies that have attempted to define the regulation of
response to increased Ca2⫹. This pathway for AMPK regula- AMPK activity by phenformin and metformin have measured
tion by CaMKK, which is dependent on intracellular Ca2⫹, total AMP content or the ratio of total AMP content to total
corresponds to a metabolic regulation function for AMPK at ATP content (total AMP/ATP). In muscle cells (13) and
the organism level (34). Chinese hamster ovary fibroblasts and rat hepatoma cells (17),
A third AMPK kinase has been postulated and may indicate metformin activated AMPK in the absence of any increase in
a third pathway for AMPK regulation. TGF--activated ki- AMP/ATP. There are a few exceptions to these reports. In
AJP-Heart Circ Physiol • VOL 293 • JULY 2007 • www.ajpheart.org
METFORMIN ACTIVATES AMPK BY INCREASING CYTOSOLIC [AMP] H463
human skeletal muscle, ATP and PCr content decreased with abolically active fractions of the total content of these two
chronic metformin administration (28). Decreases in PCr typ- nucleotides. Hence, AMPK detects cytosolic [AMP], not total
ically result in increased AMP. High concentrations of phen- AMP or total AMP/ATP.
formin (1–5 mM) increased AMP/ATP in rat brain slices (18). To test this prediction, cytosolic [AMP] and total AMP were
In the heart, total AMP content is much greater than cyto- measured (Fig. 7) in metformin-treated and KH-perfused
solic AMP content. For example, conversion of HPLC-mea- hearts. Cytosolic [AMP] and AMPK activity were increased in
sured total AMP content of the well-perfused rat heart to the metformin-treated hearts. Total AMP and total AMP/ATP
[AMP] would result in ⬃350 M AMP; yet 31P-NMR-esti- were not different between the metformin-treated and KH-
mated cytosolic [AMP] is ⬃0.4 M. Cytosolic [AMP] is perfused hearts. Thus total [AMP] and total AMP/ATP did not
estimated from measured [PCr], [ATP], and pHi: the creatine correlate with AMPK activity, whereas cytosolic [AMP] cor-
kinase equilibrium expression (Eq. 1) is used to calculate related with increased AMPK activity.
cytosolic [ADP], and cytosolic [ADP] and measured [ATP] In the heart, acute decreases in [PCr] typically indicate a
are used to calculate cytosolic [AMP] using the adenylate reduction in ATP synthesis (imbalance of synthesis and use).
kinase equilibrium expression (Eq. 2). These calculations are The decreased [PCr] and increased cytosolic [AMP] are con-
valid, because the creatine kinase and adenylate kinase reac- sistent with biguanides acting as metabolic inhibitors. Owen
tions are in states of near equilibrium in vivo and both enzymes et al. (29) reported that metformin inhibited complex 1 of the
are abundant. Cytosolic [ADP] and [AMP] represent the met- respiratory chain in liver mitochondria. El-Mir et al. (10) also
AJP-Heart Circ Physiol • VOL 293 • JULY 2007 • www.ajpheart.org
H464 METFORMIN ACTIVATES AMPK BY INCREASING CYTOSOLIC [AMP]
Fig. 7. A: 31P-NMR-measured cytosolic [AMP] in rat hearts. KH hearts (n ⫽ 8) were perfused with KH. Met hearts (n ⫽ 12) were perfused with KH during
baseline and then with KH ⫹ 10 mM metformin until they were freeze-clamped at 70 min. B: total AMP content measured by HPLC in KH and Met hearts.
C: ratio of total AMP content to ATP content measured by HPLC in KH and Met hearts. D: AMPK activity in KH and Met hearts. Measurements from individual
hearts are shown along with mean (dashed line) and SD (solid lines). *P ⬍ 0.05 vs. KH.
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