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AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts (n = 5-7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and [sup.31]P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of [Thr.sup.172] on AMPK-[alpha], and phosphorylation of [Ser.sup.79] on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP d id not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not. [sup.31]P-NMR spectroscopy; HPLC; total AMP content; AMP/ATP; biguanides doi:10.1152/ajpheart.00002.2007

AMP-activated protein kinase (AMPK) responds to impaired cellular energy status by stimulating substrate metabolism for ATP generation. Mutation of the γ2 regulatory subunit of AMPK in humans renders the kinase [...]

AMP-activated protein kinase (AMPK) is a cellular energy sensor whose activity responds to AMP concentration ([AMP]). An agent that activates AMPK in cells is 5-aminoimidazole-4-carboxamide-1-riboside (AICA-riboside). Phosphorylated AICA-riboside or AICA-ribotide (ZMP) is an AMP analog. It is generally assumed that ZMP accumulation does not alter [AMP]. Additionally, the effect of AICA-riboside on AMPK activity of the heart is uncertain. Two hypotheses were tested in the isolated mouse heart: 1) sufficient ZMP concentration ([ZMP]) forms to increase AMPK activity, and 2) [ZMP] accumulation increases [AMP]. Perfusion of isolated mouse hearts with KrebsHenseleit buffer containing 0.15-2 mM AICA-riboside concentration resulted in [ZMP] of 2-8 mM. ZMP accumulation reduced phosphocreatine concentration, which increased cytosolic [AMP]. In hearts with [ZMP] less than ~3 mM, in vivo AMPK allosteric activity effects of ZMP were observed; AMPK phosphorylation and [AMP] were not increased. With [ZMP] between 3 and 5 mM, in vitro AMPK activity and phosphorylation increased with unchanged [AMP]. This occurred in hearts perfused with 0.25 mM AICA-riboside for 48 min and 0.5 mM AICA-riboside for 24 min. The [ZMP] resulting in 50% AMPK activity (covalent phosphorylation of AMPK) was 4.1 [+ or -] 0.6 mM. Hearts with [ZMP] >5 mM displayed increased [AMP] and AMPK activity that was not different from hearts with similar [AMP] with no [ZMP]; the half-maximal activity of AMP was 5.6 [+ or -] 1.6 [micro]M. Thus, in mouse hearts, AICA-riboside was metabolized to [ZMP] adequately to increase AMPK activity. Higher [ZMP] also increased cytosolic [AMP], which affects AMPK activity. signal transduction; acetyl-CoA carboxylase; 5'-aminoimidazole-4-carboxamide- 1-[beta]-D-riboside

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 [micro]M using metabolic inhibitors during both normal oxygenation (95% [O.sub.2]-5% C[O.sub.2], normoxia) and limited oxygenation (95% [N.sub.2]-5% C[O.sub.2], hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol x [min.sup.-1] x mg [protein.sup.-1] in normoxic hearts and from 5 to 55 pmol x [min.sup.-1] x mg [protein.sup.-1] in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ([A.sub.0.5]) was 3 [+ or -] 1 [micro]M for hypoxic hearts and 28 [+ or -] 13 [micro]M for normoxic hearts. The [A.sub.0.5] for [[alpha].sub.2]-isoform AMPK activity was 2 [+ or -] 1 [micro]M for hypoxic hearts and 13 [+ or -] 8 [micro]M for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the [Thr.sup.172] residue of the AMPK [alpha]-subunit. In potassium-arrested hearts perfused with variable [O.sub.2] content, [alpha]-subunit [Thr.sup.172] phosphorylation increased at [O.sub.2] [less than or equal to] 21% even though [AMP] was signal transduction; [sup.31]P-nuclear magnetic resonance spectroscopy; acetyl-CoA carboxylase

This study tests the hypothesis that a decrease of the free energy of ATP hydrolysis ([DELTA][G.sub.ATP]) below a threshold value will inhibit [Na.sup.+]-[K.sup.+]-ATPase ([Na.sup.+] pump) activity and result in an increase of intracellular [Na.sup.+] concentration ([[Na.sup.+].sub.i]) in the heart. Conditions were designed in which hearts were solely dependent on ATP derived from oxidative phosphorylation. The only substrate supplied was the fatty acid butyrate (Bu) at either low, 0.1 mM (LowBu), or high, 4 mM (HighBu), concentrations. Escalating work demand reduced the [DELTA][G.sub.ATP] of the LowBu hearts. [sup.31]P, [sup.23]Na, and [sup.87]Rb NMR spectroscopy measured high-energy phosphate metabolites, [[Na.sup.+].sub.i], and [Rb.sup.+] uptake. [Rb.sup.+] uptake was used to estimate [Na.sup.+] pump activity. To measure [[Na.sup.+].sub.i] using a shift reagent for cations, extracellular [Ca.sup.2+] was reduced to 0.85 mM, which eliminated work demand [DELTA][G.sub.ATP] reductions. Increasing extracellular [Na.sup.+] ([Na.sup.+.sub.e]) to 200 mM restored work demand [DELTA][G.sub.ATP] reductions. In response to higher [[NA.sup.+].sub.e], [[Na.sup.+].sub.i] increased equally in LowBu and HighBu hearts to ~8.6 mM, but [DELTA][G.sub.ATP] decreased only in LowBu hearts. At lowest work demand the LowBu heart [DELTA][G.sub.ATP] was -53 kJ/mol, [Rb.sup.+] uptake was similar to that of HighBu hearts, and [[Na.sup.+].sub.i] was constant. At highest work demand the LowBu heart [DELTA][G.sub.ATP] decreased to -48 kJ/mol, the [[Na.sup.+].sub.i] increased to 25 mM, and [Rb.sup.+] uptake was 56% of that in HighBu hearts. At the highest work demand the HighBu heart [DELTA][G.sub.ATP], was -54 kJ/mol and [[Na.sup.+].sub.i] increased only ~10%. We conclude that a [DELTA][G.sub.ATP] below -50 kJ/mol limits the [Na.sup.+] pump and prevents maintenance of [[Na.sup.+].sub.i] homeostasis. energy metabolism; intracellular sodium