Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration

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 (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(172) on AMPK-alpha, and phosphorylation of Ser(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 did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not.     

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

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 microM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol.min(-1).mg protein(-1) in normoxic hearts and from 5 to 55 pmol.min(-1).mg protein(-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 (A0.5) was 3 +/- 1 microM for hypoxic hearts and 28 +/- 13 microM for normoxic hearts. The A0.5 for alpha2-isoform AMPK activity was 2 +/- 1 microM for hypoxic hearts and 13 +/- 8 microM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK alpha-subunit. In potassium-arrested hearts perfused with variable O2 content, alpha-subunit Thr172 phosphorylation increased at O2 < or = 21% even though [AMP] was <0.3 microM. Thus hypoxia or O2 < or = 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].     

Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids.

Rat hearts were perfused for 1 h with 5 mm glucose with or without palmitate or oleate at concentrations characteristic of the fasting state. The inclusion of fatty acids resulted in increased activities of the alpha-1 or the alpha-2 isoforms of AMP-activated protein kinase (AMPK), increased phosphorylation of acetyl-CoA carboxylase and a decrease in the tissue content of malonyl-CoA. Activation of AMPK was not accompanied by any changes in the tissue contents of ATP, ADP, AMP, phosphocreatine or creatine. Palmitate increased phosphorylation of Thr172 within AMPK alpha-subunits and the activation by palmitate of both AMPK isoforms was abolished by protein phosphatase 2C leading to the conclusion that exposure to fatty acid caused activation of an AMPK kinase or inhibition of an AMPK phosphatase. In vivo, 24 h of starvation also increased heart AMPK activity and Thr172 phosphorylation of AMPK alpha-subunits. Perfusion with insulin decreased both alpha-1 and alpha-2 AMPK activities and increased malonyl-CoA content. Palmitate prevented both of these effects. Perfusion with epinephrine decreased malonyl-CoA content without an effect on AMPK activity but prevented the activation of AMPK by palmitate. The concept is discussed that activation of AMPK by an unknown fatty acid-driven signalling process provides a mechanism for a 'feed-forward' activation of fatty acid oxidation.     

Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis

Bacterially expressed heterotrimeric (alpha1, beta1, and gamma1) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the alpha-subunit, viz. Thr258 and Ser485 (Ser491 in the alpha2-subunit) by mass spectrometry, in addition to the previously characterized Thr172 site. Also, autophosphorylation sites in the beta1-subunit were identified as Ser96, Ser101, and Ser108. Mutagenesis of Thr172, Thr258, and Ser485 to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr172 was involved in AMPK activation, whereas Thr258 and Ser485 were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the alpha-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser485 phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition.     

噻唑衍生物WSF-SN-10激活AMPK的机制研究

目的:在噻唑衍生物中筛选新型AMPK激活剂并探究其激活AMPK的分子机制,以期找到副作用少的II型糖尿病治疗药物。方法:用14种噻唑衍生物处理293T细胞,用Western Blot筛选能显著提高AMPK磷酸化水平的化合物;用HPLC和FACS分别检测该化合物对细胞内AMP/ATP比值和Ca~(2+)浓度的影响,探究其激活AMPK的分子机制。结果:WSF-SN-10(2-(2-(6,6-二甲基双环[3,1,1]庚-2-亚基)肼基)-4-(4-氰基苯基)噻唑)为14种噻唑衍生物中活性最强的AMPK激活剂;20μM下WSF-SN-10激活AMPK的活性最强;用20μM WSF-SN-10处理293T细胞后,细胞内的AMP/ATP比值和LKB1的磷酸化水平分别上升至空白对照的1.94和3.04倍,同时Ca~(2+)浓度无明显变化,说明WSF-SN-10通过增加细胞内的AMP/ATP比值来激活AMPK。结论:噻唑衍生物WSF-SN-10能抑制细胞内的ATP合成来间接激活AMPK,是治疗II型糖尿病和肥胖症的潜在药物。     

Biochemical regulation of mammalian AMP-activated protein kinase activity by NAD and NADH

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein kinase that is activated by a variety of metabolic stresses that lower cellular energy levels. When activated, AMPK modulates a network of metabolic pathways that result in net increased substrate oxidation, generation of reduced nucleotide cofactors, and production of ATP. AMPK is activated by a high AMP:ATP ratio and phosphorylation on threonine 172 by an upstream kinase. Recent studies suggest that mechanisms that do not involve changes in adenine nucleotide levels can activate AMPK. Another sensor of the metabolic state of the cell is the NAD/NADH redox potential. To test whether the redox state might have an effect on AMPK activity, we examined the effect of beta-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by beta-NAD in a dose-dependent manner, whereas NADH inhibited its activity. We explored the effect of NADH on AMPK by systematically varying the concentrations of ATP, NADH, peptide substrate, and AMP. Based on our findings and established activation of AMPK by AMP, we proposed a model for the regulation by NADH. Key features of this model are as follows. (a) NADH has an apparent competitive behavior with respect to ATP and uncompetitive behavior with respect to AMP resulting in improved binding constant in the presence of AMP, and (b) the binding of the peptide is not significantly altered by NADH. In the absence of AMP, the binding constant of NADH becomes higher than physiologically relevant. We conclude that AMPK senses both components of cellular energy status, redox potential, and phosphorylation potential.     

Regulation of AMP-activated protein kinase by cAMP in adipocytes: Roles for phosphodiesterases,protein kinase B,protein kinase A,Epac and lipolysis

AMP-activated protein kinase (AMPK) is an important regulator of cellular energy status. In adipocytes, stimuli that increase intracellular cyclic AMP (cAMP) have also been shown to increase the activity of AMPK. The precise molecular mechanisms responsible for cAMP-induced AMPK activation are not clear. Phosphodiesterase 3B (PDE3B) is a critical regulator of cAMP signaling in adipocytes. Here we investigated the roles of PDE3B, PDE4, protein kinase B (PKB) and the exchange protein activated by cAMP 1 (Epac1), as well as lipolysis, in the regulation of AMPK in primary rat adipocytes. We demonstrate that the increase in phosphorylation of AMPK at T172 induced by the adrenergic agonist isoproterenol can be diminished by co-incubation with insulin. The diminishing effect of insulin on AMPK activation was reversed upon treatment with the PDE3B specific inhibitor OPC3911 but not with the PDE4 inhibitor Rolipram. Adenovirus-mediated overexpression of PDE3B and constitutively active PKB both resulted in greatly reduced isoproterenol-induced phosphorylation of AMPK at T172. Co-incubation of adipocytes with isoproterenol and the PKA inhibitor H89 resulted in a total ablation of lipolysis and a reduction in AMPK phosphorylation/activation. Stimulation of adipocytes with the Epac1 agonist 8-pCPT-2′O-Me-cAMP led to increased phosphorylation of AMPK at T172. The general lipase inhibitor Orlistat decreased isoproterenol-induced phosphorylation of AMPK at T172. This decrease corresponded to a reduction of lipolysis from adipocytes. Taken together, these data suggest that PDE3B and PDE4 regulate cAMP pools that affect the activation/phosphorylation state of AMPK and that the effects of cyclic AMP on AMPK involve Epac1, PKA and lipolysis.     

Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells

We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD⁺-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.     

Low-intensity contraction activates the alpha1-isoform of 5'-AMP-activated protein kinase in rat skeletal muscle

Skeletal muscle expresses two catalytic subunits, alpha1 and alpha2, of the 5'-AMP-activated protein kinase (AMPK), which has been implicated in contraction-stimulated glucose transport and fatty acid oxidation. Muscle contraction activates the alpha2-containing AMPK complex (AMPKalpha2), but this activation may occur with or without activation of the alpha1-containing AMPK complex (AMPKalpha1), suggesting that AMPKalpha2 is the major isoform responsible for contraction-induced metabolic events in skeletal muscle. We report for the first time that AMPKalpha1, but not AMPKalpha2, can be activated in contracting skeletal muscle. Rat epitrochlearis muscles were isolated and incubated in Krebs-Ringer bicarbonate buffer containing pyruvate. In muscles stimulated to contract at a frequency of 1 and 2 Hz during the last 2 min of incubation, AMPKalpha1 activity increased twofold and AMPKalpha2 activity remained unchanged. Muscle stimulation did not change the muscle AMP concentration or the AMP-to-ATP ratio. AMPK activation was associated with increased phosphorylation of Thr(172) of the alpha-subunit, the primary activation site. Muscle stimulation increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, and the rate of 3-O-methyl-d-glucose transport. In contrast, increasing the frequency (>or=5 Hz) or duration (>or=5 min) of contraction activated AMPKalpha1 and AMPKalpha2 and increased AMP concentration and the AMP/ATP ratio. These results suggest that 1) AMPKalpha1 is the predominant isoform activated by AMP-independent phosphorylation in low-intensity contracting muscle, 2) AMPKalpha2 is activated by an AMP-dependent mechanism in high-intensity contracting muscle, and 3) activation of each isoform enhances glucose transport and ACC phosphorylation in skeletal muscle.     

Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPKalpha2 but not AMPKalpha1

Recent studies indicate that the LKB1 is a key regulator of the AMP-activated protein kinase (AMPK), which plays a crucial role in protecting cardiac muscle from damage during ischemia. We have employed mice that lack LKB1 in cardiac and skeletal muscle and studied how this affected the activity of cardiac AMPKalpha1/alpha2 under normoxic, ischemic, and anoxic conditions. In the heart lacking cardiac muscle LKB1, the basal activity of AMPKalpha2 was vastly reduced and not increased by ischemia or anoxia. Phosphorylation of AMPKalpha2 at the site of LKB1 phosphorylation (Thr172) or phosphorylation of acetyl-CoA carboxylase-2, a downstream substrate of AMPK, was ablated in ischemic heart lacking cardiac LKB1. Ischemia was found to increase the ADP-to-ATP (ADP/ATP) and AMP-to-ATP ratios (AMP/ATP) to a greater extent in LKB1-deficient cardiac muscle than in LKB1-expressing muscle. In contrast to AMPKalpha2, significant basal activity of AMPKalpha1 was observed in the lysates from the hearts lacking cardiac muscle LKB1, as well as in cardiomyocytes that had been isolated from these hearts. In the heart lacking cardiac LKB1, ischemia or anoxia induced a marked activation and phosphorylation of AMPKalpha1, to a level that was only moderately lower than observed in LKB1-expressing heart. Echocardiographic and morphological analysis of the cardiac LKB1-deficient hearts indicated that these hearts were not overtly dysfunctional, despite possessing a reduced weight and enlarged atria. These findings indicate that LKB1 plays a crucial role in regulating AMPKalpha2 activation and acetyl-CoA carboxylase-2 phosphorylation and also regulating cellular energy levels in response to ischemia. They also provide genetic evidence that an alternative upstream kinase can activate AMPKalpha1 in cardiac muscle.     

AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit

The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress. It consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, and is regulated both by the allosteric action of AMP and by phosphorylation of the alpha and beta subunits catalyzed by AMPKK(s) and autophosphorylation. The Thr172 site on the alpha subunit has been previously characterized as an activating phosphorylation site. Using bacterially expressed AMPK alpha1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in alpha subunit regulation. Recombinant alpha1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6x His fusion proteins and purified to homogeneity by Ni(2+) chromatography. Both wild-type alpha1(1-312) and alpha1(1-312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKbeta. The corresponding AMPKalpha1(1-392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive. In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK. Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant alpha1(1-312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active.     

Adrenergic regulation of AMP-activated protein kinase in brown adipose tissue in vivo

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK α2 protein and activity were reduced by 45%. In β(1,2,3)-adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr(172) phosphorylation. In contrast, acute α- or β-adrenergic blockade in WT mice resulted in increased AMPK α Thr(172) phosphorylation and AMPK α1 and α2 activity in BAT. But short term blockade of α-adrenergic signaling in β(1,2,3)-adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser(485/491), a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser(485/491) phosphorylation resulting from α-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of α-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr(172) phosphorylation and chronically through changes in the α catalytic subunit protein levels. Furthermore, AMPK α Ser(485/491) phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects.     

Suppression of 5'-nucleotidase enzymes promotes AMP-activated protein kinase (AMPK) phosphorylation and metabolism in human and mouse skeletal muscle

The 5'-nucleotidase (NT5) family of enzyme dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. We hypothesized that gene silencing of NT5 enzymes to increase the intracellular availability of AMP would increase AMP-activated protein kinase (AMPK) activity and metabolism. We determined the role of cytosolic NT5 in metabolic responses linked to the development of insulin resistance in obesity and type 2 diabetes. Using siRNA to silence NT5C2 expression in cultured human myotubes, we observed a 2-fold increase in the AMP/ATP ratio, a 2.4-fold increase in AMPK phosphorylation (Thr(172)), and a 2.8-fold increase in acetyl-CoA carboxylase phosphorylation (Ser(79)) (p < 0.05). siRNA silencing of NT5C2 expression increased palmitate oxidation by 2-fold in the absence and by 8-fold in the presence of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside. This was paralleled by an increase in glucose transport and a decrease in glucose oxidation, incorporation into glycogen, and lactate release from NT5C2-depleted myotubes. Gene silencing of NT5C1A by shRNA injection and electroporation in mouse tibialis anterior muscle reduced protein content (60%; p < 0.05) and increased phosphorylation of AMPK (60%; p < 0.05) and acetyl-CoA carboxylase (50%; p < 0.05) and glucose uptake (20%; p < 0.05). Endogenous expression of NT5C enzymes inhibited basal lipid oxidation and glucose transport in skeletal muscle. Reduction of 5'-nucleotidase expression or activity may promote metabolic flexibility in type 2 diabetes.     

Myocardial ischemia differentially regulates LKB1 and an alternate 5'-AMP-activated protein kinase kinase

During myocardial ischemia, activation of 5'-AMP-activated protein kinase (AMPK) leads to the stimulation of glycolysis and fatty acid oxidation. Together these metabolic changes contribute to cardiac dysfunction. Although AMPK signaling in the ischemic heart is well characterized, the relative contribution of phosphorylation by AMPK kinase (AMPKK), and positive allosterism by the ratios of AMP:ATP and creatine (Cr):phosphocreatine (PCr), in stimulating AMPK during ischemia are unknown. In hearts subjected to severe ischemia, the ratios of AMP:ATP and Cr:PCr were significantly elevated as compared with aerobic hearts. Severe ischemia stimulated AMPK signaling, as demonstrated by an increase in both AMPK activity and acetyl-CoA carboxylase phosphorylation. Although AMPK phosphorylation was increased by severe ischemia, the protein abundance and activity of the recently identified AMPKK, LKB1, were similar between aerobic and severely ischemic hearts. However, in contrast to LKB1, the activity of AMPKK was stimulated in severely ischemic hearts. To further delineate the relative roles of positive allosterism and AMPKK in the regulation of AMPK during ischemia, hearts were subjected to mild ischemia. Although mild ischemia did not alter the ratios of AMP:ATP and Cr:PCr, mild ischemia increased AMPK activity and increased AMPK phosphorylation. Mild ischemia also stimulated the activity of AMPKK. In summary, we demonstrate that myocardial ischemia stimulates AMPK via an AMPKK other than LKB1. Additionally, we show that changes in high energy phosphates are not essential for the activation of AMPK by ischemia. Our data emphasize the critical role AMPKK plays in mediating AMPK signaling during myocardial ischemia.     

Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by alpha-lipoic acid in C2C12 myotubes

alpha-Lipoic acid (ALA) widely exists in foods and is an antidiabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by the activation of AMP-activated protein kinase (AMPK) in skeletal muscle, but the underlying mechanism for AMPK activation is unknown. Here, we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. Incubation of C2C12 myotubes with 200 and 500 microM ALA increased the activity and phosphorylation of the AMPK alpha-subunit at Thr(172). Phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC), at Ser(79) was also increased. No difference in ATP, AMP, and the calculated AMP-to-ATP ratio was observed among the different treatment groups. Since the upstream AMPK kinase, LKB1, requires an alteration of the AMP-to-ATP ratio to activate AMPK, this data showed that LKB1 might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased the intracellular Ca(2+) concentration measured by fura-2 fluorescent microscopy (P < 0.05), showing that ALA may activate AMPK through enhancing Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) signaling. Indeed, chelation of intracellular free Ca(2+) by loading cells with 25 microM BAPTA-AM for 30 min abolished the ALA-induced activation of AMPK and, in turn, phosphorylation of ACC at Ser(79). Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA-stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser(79). In addition, ALA treatment increased the association of AMPK with CaMKK. To further show the role of CaMKK in AMPK activation, short interfering RNA was used to silence CaMKK, which abolished the ALA-induced AMPK activation. These data show that CaMKK is the kinase responsible for ALA-induced AMPK activation in C2C12 myotubes.     

Relationship between 5-aminoimidazole-4-carboxamide-ribotide and AMP-activated protein kinase activity in the perfused mouse heart

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 Krebs-Henseleit 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 approximately 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 +/- 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 +/- 1.6 microM. 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.     

Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogated ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activated PKCzeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.     

Berberine suppresses neuroinflammatory responses through AMP‐activated protein kinase activation in BV‐2 microglia

The AMPK cascade is a sensor of cellular energy change, which monitors the AMP/ATP ratio to regulate cellular metabolism by restoring ATP levels, but its regulation of neuroinflammation mechanism remains unclear. Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been shown to improve several metabolic disorders, such as obesity and type II diabetes. However, the effect of berberine on neuroinflammatory responses in microglia are poorly understood. This study shows that berberine represses proinflammatory responses through AMP‐activated protein kinase (AMPK) activation in BV‐2 microglia. Our findings also demonstrate that berberine significantly down‐regulates LPS‐ or interferon (IFN)‐γ‐induced nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) expression in BV‐2 microglia cells. Berberine also inhibited LPS‐ or IFN‐γ‐induced nitric oxide production. In addition, berberine effectively inhibited proinflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 expression. On the other hand, upon various inflammatory stimulus including LPS and IFN‐γ, berberine suppressed the phosphorylated of ERK but not p38 and JNK in BV‐2 microglia. AMPK activation is catalyzed by upstream kinases such as LKB1 and Ca2+/calmodulin‐dependent protein kinase kinase‐II (CaMKK II). Moreover, berberine induced LKB1 (Ser428), CaMKII (Thr286), and AMPK (Thr172) phosphorylation, but not AMPK (Ser485). Furthermore, the inhibitory effect of berberine on iNOS and COX‐2 expression was abolished by AMPK inhibition via Compound C, an AMPK inhibitor. Berberine‐suppressed ERK phosphorylation was also reversed by Compound C treatment. Our data demonstrate that berberine significantly induces AMPK signaling pathways activation, which is involved in anti‐neuroinflammation. J. Cell. Biochem. 110: 697–705, 2010. © 2010 Wiley‐Liss, Inc.     

Possible involvement of the alpha1 isoform of 5'AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle

Recent studies have suggested that 5'AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H(2)O(2), AMPKalpha1 activity increased in a time- and dose-dependent manner, whereas AMPKalpha2 activity remained unchanged. The activation of AMPKalpha1 was associated with phosphorylation of AMPK Thr(172), suggesting that an upstream kinase is involved in the activation process. H(2)O(2)-induced AMPKalpha1 activation was blocked in the presence of the antioxidant N-acetyl-l-cysteine (NAC), and H(2)O(2) significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H(2)O(2) did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H(2)O(2) increased 3-O-methyl-d-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKalpha1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKalpha1-mediated mechanism.     

Berberine improves glucose metabolism through induction of glycolysis

Berberine, a botanical alkaloid used to control blood glucose in type 2 diabetes in China, has recently been reported to activate AMPK. However, it is not clear how AMPK is activated by berberine. In this study, activity and action mechanism of berberine were investigated in vivo and in vitro. In dietary obese rats, berberine increased insulin sensitivity after 5-wk administration. Fasting insulin and HOMA-IR were decreased by 46 and 48%, respectively, in the rats. In cell lines including 3T3-L1 adipocytes, L6 myotubes, C2C12 myotubes, and H4IIE hepatocytes, berberine was found to increase glucose consumption, 2-deoxyglucose uptake, and to a less degree 3-O-methylglucose (3-OMG) uptake independently of insulin. The insulin-induced glucose uptake was enhanced by berberine in the absence of change in IRS-1 (Ser307/312), Akt, p70 S6, and ERK phosphorylation. AMPK phosphorylation was increased by berberine at 0.5 h, and the increase remained for > or =16 h. Aerobic and anaerobic respiration were determined to understand the mechanism of berberine action. The long-lasting phosphorylation of AMPK was associated with persistent elevation in AMP/ATP ratio and reduction in oxygen consumption. An increase in glycolysis was observed with a rise in lactic acid production. Berberine exhibited no cytotoxicity, and it protected plasma membrane in L6 myotubes in the cell culture. These results suggest that berberine enhances glucose metabolism by stimulation of glycolysis, which is related to inhibition of glucose oxidation in mitochondria. Berberine-induced AMPK activation is likely a consequence of mitochondria inhibition that increases the AMP/ATP ratio.