L-triiodothyronine (T3) and L-thyroxine (T4) both inhibited Na+-K+-ATPase activity (but not Mg2+-ATPase activity) in similar dose-dependent fashions, while other metabolites of TH were less effective. Although both T3 and the β-adrenergic agonist isoproterenol inhibited Na+-K+-ATPase activity in cerebrocortical synaptosomes in similar ways, the β-adrenergic receptor blocker propranolol did not counteract the effect of T3. Instead, propranolol further inhibited Na+-K+-ATPase activity in a dose-dependent manner, suggesting that the effect of T3 on synaptosomal Na+-K+-ATPase activity was independent of β-adrenergic receptor activation. The effect of T3 on synaptosomal Na+-K+-ATPase activity was inhibited by the α2-adrenergic agonist clonidine and by glutamate. Notably, both clonidine and glutamate activate Gi-proteins of the membrane second messenger system, suggesting a potential mechanism for the inhibition of the effects of TH. In this paper, we provide support for a nongenomic mechanism of action of TH in a neuronal membrane-related energy-linked process for signal transduction in the adult condition.The role of thyroid hormones (TH) in the normal functioning of adult mammalian brain is unclear. Our studies have identified synaptosomal Na Thyroid hormones (TH) exert major influences on the growth and development of the mammalian brain through specific nuclear receptor-mediated gene expression. Although several different isoforms of nuclear receptors for TH have been described in adult mammalian brain, their physiological function is quite unclear –4. Still3) is distributed, concentrated, and metabolized in the synaptosomal fraction of adult rat cerebral cortex [3-binding sites have also been described in cerebrocortical synaptosomes [3 to its synaptosomal receptor binding sites has been correlated with the corresponding inhibition of the Na+-K+-ATPase activities in adult rat brain [in vitro phosphorylation of synaptosomal proteins in a dose-dependent fashion [Recent evidence has demonstrated that L-triiodothyronine  for signal transduction in adult condition. We have used α- and β-adrenoceptor agonists and antagonists for modulation of the activity of Gs- and Gi-proteins of the membrane adenylate cyclase system. Portions of this work have appeared elsewhere in a preliminary form [However, there is a lack of clear understanding of the mechanism(s) of action of TH in the regulation of synaptic functions in adult neurons. The present study investigates the pathways of Tary form , 19.3), L-thyroxine (T4), 3,3′,5′-L-triiodothyronine (reverse T3 or r-T3), 3,5-L-diiodothyronine (T2), Tris-ATP, yohimbine hydrochloride (YOH), dibutyryl 3′,5′-cyclic adenosine monophosphate sodium salt (DB cAMP), and sodium orthovanadate.The following compounds were purchased from Sigma Chemical Company, USA: bovine serum albumin (BSA), clonidine hydrochloride (CLO), disodium-ATP, isoproterenol hydrochloride (ISO), 2-mercaptoethanol, ouabain octahydrate, prazosin hydrochloride (PRA), phenylephrine hydrochloride (PHE), propranolol hydrochloride (PROP), sodium glutamate, 3,5,3′-L-triiodothyronine  were housed at 25 ± 1°C in 12 h dark-12 h light conditions and fed +-K+-ATPase activity.The synaptosomes from thecerebral cortex were prepared as described previously . Briefly+-K+-ATPase activity was assayed as ouabain-sensitive ATP hydrolysis in reaction mixtures of (i) 30 mM imidazole-HCl, 130 mM NaCl, 20 mM KCl, and 4 mM MgCl2 and (ii) 30 mM imidazole-HCl, 4 mM MgCl2, and 1 mM ouabain, at pH 7.4. Both the reaction media (i) and (ii) were first preincubated in vitro with or without simultaneous addition of various concentrations of thyroid hormones  and TH-analogue (T2) (0.001 nM to 1 μM), adrenergic drugs , glutamate (100 μM), DB cAMP (1 μM–5 mM), and sodium orthovanadate (10 nM–2 mM) followed by addition of the synaptosomal lysates, each containing 20–50 μg synaptosomal protein, at 0°C for 60 minutes in dark. To get a steady-state ouabain binding, both the assay media (i) and (ii), with and without ouabain, respectively, as described above, were preincubated for 60 min at 0°C in the dark, followed by a 5-min incubation at 37°C to equilibrate the temperature. The reaction was started by adding 4 mM Tris-ATP and incubated at 37°C for 10 min. An aliquot of 100 μL of 10% sodium dodecylsulfate was added to stop the enzymatic reaction. The inorganic phosphate (Pi  ) formed was determined in the reaction mixture [+-K+-ATPase activity was calculated as difference in the Pi content between media (i) and (ii) and expressed as μmols Pi/h/mg protein [+-K+-Mg2+-ATPase) was determined from the Pi released in the medium (i) minus that in medium (ii). The Pi released from the reaction medium (ii) was used for determination of the synaptosomal Mg2+-ATPase activity. Synaptosomal Mg2+-ATPase activity, therefore, was assayed as the ouabain-insensitive ATP hydrolysis.Synaptosomal Na mixture . Na+-K+- protein . The ouaSynaptosomal protein content was measured using bovine serum albumin as a standard .t-test, considering P < 0.05 as the significance level. The data for multiple groups were also analyzed by one-way ANOVA followed by Student Newman-Keuls post-hoc comparisons using Sigmastat software. Nonlinear regression analysis was performed using GraphPad Prism software.Results are expressed as the mean ± SEM of 3-4 separate experiments or as mentioned. Each experiment was made from six rats. The statistical analysis of the data was performed by Student's In vitro addition of various doses of T3 to the synaptosomal fraction (which is devoid of cell nuclei) confirmed our previous observation [50 = 166.4 ± 55.0 pM; maximal inhibition = 63.2 ± 3.4% at 95% confidence levels) of Na+-K+-ATPase activity. No significant effect of T3 was noticed on the Mg2+-ATPase specific activity , while T2 had minimal effects (−12–10−8 M) of r-T3 did not inhibit either Na+-K+-ATPase or Mg+2-ATPase activities (data not shown).ervation  and showactivity . T4 had  effects . Further3 and the nonselective β-adrenergic agonist ISO were added separately in vitro, inhibited the Na+-K+-ATPase enzyme activity by 41.3% and 42.6%, respectively . The inhibitory action of ISO (1 nM) on the Na+-K+-ATPase activity was counteracted by PROP (1 nM), whereas PROP could not block T3-mediated inhibition of the enzyme activity. Instead PROP potentiated the T3-mediated inhibition of the enzyme activity in a dose-dependent manner. Significant differences in the potentiation of the T3 effect (1 nM) by PROP on Na+-K+-ATPase activity were noticed between 1 nM and 100 nM (P < 0.001) and between 10 nM and 100 nM (P < 0.05) doses (Equimolar doses (1 nM) of Tectively . The non5) doses .in vitro addition of 1 nM doses of PHE (selective α1-adrenergic receptor agonist) and PRA (α1-adrenergic receptor antagonist) on synaptosomal Na+-K+-ATPase activity or Mg2+-ATPase activity were minimal  at different doses did not elicit significant changes in the synaptosomal Na+-K+-ATPase activity  along with 1 nM CLO (100 nM T3 + 1 nM CLO: 15.23 ± 0.4 μmols Pi/h/mg protein); however, 1 nM CLO attenuated the effect of T3 (100 nM) by 32% more towards the control value . The α2-adrenergic receptor antagonist YOH also inhibited synaptosomal Na+-K+-ATPase activity -mediated inhibition of synaptosomal Na+-K+-ATPase activity in adult rat cerebral cortex (3 (10 nM) was chosen, in order to test the effect of glutamate against a greater inhibitory action on the Na+-K+-ATPase activity.l cortex . A highe+-K+-ATPase activity by T3, first a dose response experiment with various concentrations of DB cAMP (0.001 mM to 5 mM) was performed. In vitro addition of DB cAMP showed a typical sigmoidal curve with gradual decrease in the Na+-K+-ATPase activity to a maximal inhibition at 0.2 mM (In vitro addition of DB cAMP (0.2 mM) with and without various doses of T3 (0.001 nM–10 nM) was examined for effects on Na+-K+-ATPase activity  with or without different doses of sodium o-vanadate (3 (10 nM) was chosen from the T3 dose-response curve, considering its greater inhibitory action on the Na+-K+-ATPase activity. T3 caused an inhibition of Na+-K+-ATPase specific activity, and this effect was enhanced by sodium orthovanadate in a dose-dependent way. In general, the effects of sodium orthovanadate and T3 appeared to be additive until the Na+-K+-ATPase specific activity was completely inhibited.The vanadate . A highe+-K+-ATPase activity in adult rat cerebral cortex.The objective of the present investigation was to search for possible mechanisms for the inhibition by TH of synaptosomal NaIn vitro inhibitory effect of T3 on synaptosomal Na+-K+-ATPase activity supported our previous observation and showed nearly the same trend of a dose-dependent inhibition of Na+-K+-ATPase activity [3 on the synaptosomal Mg2+-ATPase specific activity  in the CV-1 line of monkey fibroblasts [αvβ3, the αv monomer is translocated to the nucleus, where it may transcriptionally regulate expression of protein [+-K+-ATPase protein by a MAPK/ERK1/2-dependent pathway [+-K+-ATPase activity could not be due to a mechanism involving transcriptional regulation, since the synaptosomal preparation is devoid of cell nuclei. It is also suggested that some of the effects of T3 stimulation of the integrin αvβ3 could be more direct than the nuclear interaction [Binding of Troblasts . A similroblasts . Followi protein . TH caus protein  and to e pathway . Note, heraction .+-K+-ATPase or a modulatory molecule. It is well known that catecholamine-mediated phosphorylation of Na+-K+-ATPase inhibits enzymatic activity in Chinese hamster ovary (CHO) cells, but not through a process of internalization of the enzyme [α-subunit of Na+-K+-ATPase [A potential mechanism for the inhibitory effects of TH in the present study might be the regulation of phosphorylation of Nae enzyme –32. Intre enzyme , matchin+-ATPase .+-K+-ATPase activity in vitro by T3 confirmed our previous in vivo observations [3 and ISO (β-adrenergic receptor agonist) showed an analogous but independent  inhibitory effect on the enzyme activity (+-K+-ATPase activity was blocked by PROP (β-adrenergic receptor blocker) indicating that the synaptosomal membrane interaction with ISO was likely a β-adrenoceptor-mediated event, potentially coupled to Gs-protein. However, PROP was completely unable to block T3-mediated inhibition of synaptosomal Na+-K+-ATPase activity. This clearly indicated that T3-mediated inhibition of the enzyme activity was not coupled to β-adrenoceptor, but rather, may have had a similar effect through another kind of receptor. The augmentation of the T3 effect by PROP appeared to be a type of synergistic action, the mechanism of which remains unclear at present. Increased activity of adenylate cyclase caused by THs, independent of propranolol blockade, has been shown in cultured cerebral cells from embryonic mice, suggesting that the effect of T3 was not mediated through a β-adrenergic-dependent system [3-induced increase in sodium current in neonatal rat myocytes also could not be blocked by PROP, whereas it was antagonized by amiodarone, a nonspecific blocker of β-adrenoceptor, suggesting that the effects were not mediated through β-adrenergic signaling pathways [β-adrenoceptor blockade by chronic subcutaneous delivery of PROP for 14 days has been shown to downregulate levels of TH receptor TR α1-mRNA and β1-mRNA in mouse heart, which may influence the genomic effect of the hormone [The significant inhibition of the synaptosomal Narvations . In ordeactivity . ISO-indt system . The T3-pathways . However hormone .α1-adrenergic receptor agonist and antagonist. Agonists for the α1-adrenergic receptor mediate their actions through Gq protein followed by activation of phospholipase C and subsequent production of the second messengers inositol triphosphate and diacylglycerol, an activator of protein kinase C [α1 agonist) nor PRA (α1 antagonist) had an influence on Na+-K+-ATPase activity. Furthermore, neither compound interacted with the effects of T3. Mg2+-ATPase activity remained unaltered when treated with either of these α1-adrenergic drugs (agonist and antagonist) and T3, alone or in combination  followed by the inhibition of cAMP synthesis and the protein phosphorylation cascade mechanism. It is well known that α2-adrenergic agonists act through stimulation of Gi-protein [On the other hand, CLO, an  agonist , and glu agonist , possiblogenates . One pos-protein , 39, 40.+-K+-ATPase in synaptosomes has been implicated by their correlated regulation via protein kinases [i inhibited the effects of mGluRs [1 and mGluR5 subtypes are coupled to phosphatidyl inositol hydrolysis/Ca2+-signal transduction. mGluR1 has also been shown to stimulate release of arachidonic acid and to increase cAMP formation. The mGluR2, mGluR3, mGluR4, and mGluR5 subtypes appear to be coupled to inhibition of cAMP synthesis, but differ in their agonist selectivity. mGluR2 and mGluR3 mRNAs are highly expressed in the cerebral cortex [β-adrenoceptor-mediated inhibition of afterhyperpolarization in hippocampal neurons of the CA1 area. This has been suggested to be by mGluR-mediated activation of protein kinase C, which inhibited adenylate cyclase pathways [3 action in adult rat synaptosomal membrane, ultimately to inhibit the effector molecule Na+-K+-ATPase, might be mediated through Gs stimulation. mGlu receptors may then have some regulatory roles in counteracting T3-induced action.Association of the glutamate transporter with Na kinases . Glutama kinases , 42, 43, kinases  via metaf mGluRs . mGluR1 l cortex , 42, 43.3-like effect on Na+-K+-ATPase activity  had a Tactivity . Furtherpathways , 45. A dl cortex .3, implying that there could be a separate mechanism of action of the two compounds (3-induced inhibition of Na+-K+-ATPase activity is further suppressed by synergistic action by vanadate via keeping the enzyme in its phosphorylated form, causing inhibition of its activity. A point to note here is that the α-subunit is the catalytic subunit and its phosphorylation causes inhibition of this enzyme [3 appears not to have the inhibitory effect on Na+-K+-ATPase activity by an influence on phosphatase activity.The effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate  appearedompounds . Since vs enzyme . T3 appe3 action in relation to the inhibition of synaptosomal Na+-K+-ATPase are consistent with a T3-synaptosomal membrane component binding site interaction sensitive to the activation of Gi-protein. Such a membrane binding component might interact with a Gs-protein, resulting in increased synthesis of cAMP. The membrane Na+-K+-ATPase is involved in several aspects of physiological processes. In the neuron, its inhibition is linked with neurotransmitter release [3 in the mature mammalian synaptosome. Understanding of the mechanism of action of TH in adult mammalian brain has major implications in the higher mental functions and in the regulation of several neuropsychiatric disorders developed in thyroid dysfunctions in adult humans.Our results regarding T release . Hence,


There is only one established drug binding site on sodium channels. However, drug binding of sodium channels shows extreme promiscuity: ∼25% of investigated drugs have been found to potently inhibit sodium channels. The structural diversity of these molecules suggests that they may not share the binding site, and/or the mode of action. Our goal was to attempt classification of sodium channel inhibitors by measuring multiple properties of inhibition in electrophysiology experiments. We also aimed to investigate if different properties of inhibition correlate with specific chemical properties of the compounds.A comparative electrophysiological study of 35 compounds, including classic sodium channel inhibitors , as well as antidepressants, antipsychotics and neuroprotective agents, was carried out using rNav1.2 expressing HEK-293 cells and the QPatch automatic patch-clamp instrument. In the multi-dimensional space defined by the eight properties of inhibition , at least three distinct types of inhibition could be identified; these probably reflect distinct modes of action. The compounds were clustered similarly in the multi-dimensional space defined by relevant chemical properties, including measures of lipophilicity, aromaticity, molecular size, polarity and electric charge. Drugs of the same therapeutic indication typically belonged to the same type. We identified chemical properties, which were important in determining specific properties of inhibition. State-dependence correlated with lipophilicity, the ratio of the neutral form of molecules, and aromaticity: We noticed that the highly state dependent inhibitors had at least two aromatic rings, logP>4.0, and pKa<8.0.50; therefore, recording multiple properties of inhibition may allow improved prediction of therapeutic usefulness.The correlations of inhibition properties both with chemical properties and therapeutic profiles would not have been evident through the sole determination of IC Pharmacological modulation of sodium channels by sodium channel inhibitors (SCIs) is crucial in local anesthesia, in the treatment of certain types of epilepsy and cardiac arrhythmia . Several SCI drugs are also used for the treatment of neuropathic pain, muscle spasms, Alzheimer's disease, amyotrophic lateral sclerosis and as mood stabilizers There are several different toxin binding sites on sodium channels The way the acquisition of multiple biophysical properties was carried out was unlike conventional analyses of the mode of action. These require multiple protocols, where the exact parameters of protocols must be adjusted individually to specific drugs. Furthermore, exploration of the mode of action of a drug is always an iterative process: responses to initial protocols are used to design new protocols, and to adjust parameters of the protocols to individual drugs. This of course compromises comparability of data with different drugs. Our aim is to develop an approach which can be adopted by pharmaceutical companies, therefore we confined ourselves to protocols which require no more cost or time than a single measurement of inhibition, but which can give radically more information regarding the mode of action of the drug. We used two very simple protocols, which can even be applied within the same experiment, the whole process of ‘control measurement’ – ‘measurement of drug effect’ – ‘measurement after washout’ requires no more than 10 minutes (it is limited by the onset rate of slowly acting drugs). However, we took care to extract all possible useful information from this measurement.r), affinity to depolarized states , and state dependence  were extracted from “steady-state” availability curves , and the size of the molecule were proposed to be the most important predictors of properties of inhibition 50 values Lipophilicity can be quantified by either logP, which is the logarithm of the octanol-water partition coefficient of the neutral form of the molecule, or logD (logarithm of the distribution coefficient), which measures the distribution of all forms that are present at a certain pH value. Because the pKa of the studied SCIs are typically between 7.0 and 10.7, most drugs are predominantly positively charged at neutral pH, and lose their charge at higher pH values. For this reason, logD is increasing with higher pH, approaching logP as an upper limit. A linear correlation was found between logP and the logarithm of ICBecause local anesthetics are predominantly charged, basic compounds, a pKa value between 7.5 and 10 was considered a requirement for being an effective SCI. However, SCI anticonvulsants are predominantly neutral, and several very potent neutral SCIs has been discovered recently Molecular weight has been shown to correlate with potency Most of these results were based on studies of a single chemical and therapeutic class of SCIs. In this current study we attempted to detect similar general principles for a larger, more diverse group of SCIs. Our specific questions were the following:Which measure of lipophilicity, logP or logD better predicts potency?r and with Ki? If yes, then we can deduce something regarding the chemical interactions of the drug with resting and inactivated conformations of the channel. High state-dependence is considered to be essential for good therapeutical applicability. Which chemical properties predict high state-dependence?Are there chemical descriptors which specifically predict individual biophysical properties of inhibition? Most importantly, can we find some difference between descriptors which determine KWhich chemical properties determine specific types of inhibition ?r, Ki, IC50, state-dependence, reversibility, use-dependence, onset and offset time constants). We observed that different chemical properties determine low Kr (high pKa and logP), and low Ki . State-dependence, therefore can be predicted by calculating these chemical descriptors of molecules.We succeeded to identify chemical descriptors which determine different types of inhibition, and which predict specific biophysical properties of inhibition . Therefore these were excluded from analysis of further properties of inhibition . Of the 44 investigated drugs nine either caused less than 10% inhibition even at the highest concentration used , or their IC50 values as described in on) and offset (τoff). The calculation of IC50, quantification of Rev and UD, and the determination of time constants are illustrated in on should be concentration-dependent if we assume a single binding reaction, we excluded it from the analyses. Reversibility measured by the QPatch instrument did not match reversibility values obtained by classic patch clamp (data not shown). It has been observed that washout of lipophilic compounds is delayed in automated electrophysiology platforms (e.g. on and UD) are in We measured inhibition , and the shift of the availability curves (ΔV1/2) are both concentration-dependent properties, we calculated affinities to hyperpolarized and depolarized states , which, on the other hand, are concentration-independent characteristics of individual drugs. Calculation of Kr and Ki values are described in r/Ki. Values of the eight properties for individual drugs are shown in r and Ki values can be compared to data from the literature in Standard steady-state inactivation protocols were used to measure the shift of steady-state availability curve caused by the drugs. Currents were evoked by a 10 ms test pulse to −10 mV, preceded by 400 ms pre-pulses from −150 to −30 mV. The holding potential was −120 mV. Because the inhibition at hyperpolarized potentials , slow onset and offset kinetics (time constants between 10 and 53 s), partial reversibility (between 0.2 and 0.6) and use-dependence (1.09 to 1.66). Drugs belonging to this type were mostly antidepressants: fluoxetine, sertraline, paroxetine, amitriptyline, imipramine, desipramine and maprotiline, as well as the antipsychotic haloperidol, and the anxiolytic ritanserin.‘Type 1’  drugs hai 17 to 88 µM; IC50 >95 µM), fast kinetics (time constants <27 s) and almost full reversibility (>0.75) Drugs belonging to this type were the three effective anticonvulsants , the Class IB antiarrhythmic lidocaine and mexiletine, as well as diclofenac, venlafaxine, tolperisone, bupropion, ambroxol and memantine. The group can be further divided: lidocaine, mexiletine, ambroxol and tolperisone , 2 compounds between ‘Type 1’ and ‘Type 3’ (the antipsychotics chlorpromazine and chlorprothixene), while the remaining 4 drugs, bupivacaine, flecainide, nefazodone and riluzole seemed to have their own specific type of inhibition: Bupivacaine with high use-dependence and state-dependence; flecainide, with high use-depencence but low state-dependence and low Kr and Ki values , while the color indicates τoff. Non-use-dependent drugs are marked by underlined italic fonts. The types defined above are circled. Note how the relative position of drugs with a high SD changes: Flunarizine (FLR), chlorpromazine (CPM), chlorprothixene (CHX), riluzole (RIL), bupivacaine (BPV) and, most of all, nefazodone (NFZ) all move to the left on the Rev vs. Ki plot as compared to the Rev vs. Kr plot.50 values were typically midway between Kr and Ki, while for many ‘Type 2’ and ‘Type 4’ drugs IC50 was closer to Kr  from the values for individual drugs and dividing by the standard deviation. Results of the cluster analysis are shown in In order to quantify differences and test the validity of the subjective classification, we performed a cluster analysis using the properties of inhibition. We took the logarithm of KThe overall picture represents the subjective description quite well. ‘Type1’ and ‘Type3’ inhibitions were clearly recognized, as well as the separateness of riluzole and nefazodone. Using different amalgamation rules and distance measures resulted in similar, although not identical classifications. The differences between our subjective classification and the result of cluster analysis were the following:50, τoff and Rev values, was consistently clustered into ‘Type 1’ group. However, despite the similar potency and kinetics, this compound has been shown to have a separate mode of action, being an open channel blocker Flecainide, although had somewhat higher IC‘Type 2’ and ‘Type 4’ groups were not clearly separated.Bupivacaine was clustered into a separate subgroup of the ‘Type2’ – ‘Type4’ group together with mexiletine. However, its UD and SD values  were higher than the rest of either ‘Type 2’ or ‘Type 4’ compounds.The biophysical properties of major groups are also shown in r, Ki−Kapp, Ki−ΔV1/2, IC50, SD values and time constants we used logarithmic transformation). All properties significantly correlated with each other, with the exception of UD and SD. Use-dependence only correlated with potency  values, while state-dependence only correlated with Rev and the two Ki values  tended to have slower onset and offset kinetics, tended to be less reversible, and tended to be more use-dependent. High inactivated state affinity (low Ki) predicted high state-dependence, while high resting affinity (low Kr) did not.It is apparent that more potent drugs  different binding sites, ii) different access pathways, iii) different modes of action  values, as well as reversibility of drugs, was logP, as seen in r, Ki, logIC50 and Rev, respectively) and in 7.3 (the distribution coefficient at pH = 7.3) were considerably lower but still significant, except for Kr , which indicates that Kr was not influenced by logD7.3 , but not with Kr (R = −0.21) . These descriptors correlated significantly with SD, K= −0.21) . Measurer was pKa, the correlation (R = −0.68) was higher than with logP (R = −0.53). Molecules with high pKa values, i.e., which are predominantly positively charged at physiological pH, were more potent against resting channels, while inactivated affinity did not correlate with either pKa or N(pKa). ’) was calculated from pKa values as described in r and UD (but not with Ki), was polar surface area (PSA) . Small polar surface area corresponded with higher resting affinity and higher use-dependence.The most important determinant of KTime constants were determined by aromaticity, lipophilicity and the size of the molecule: large, lipophilic molecules with multiple aromatic rings had slower onset/offset kinetics. From the descriptors which quantify different aspects of molecular size, minimum projection area was found to show somewhat higher correlation than molecular weight . Larger r, Ki, IC50, state-dependence, reversibility, use-dependence, onset and offset time constants. While logP is essential for both resting and inactivated state affinity, resting affinity is higher for positively charged drugs, and interestingly, the lipophilicity of this charged form does not seem to be important. One explanation may be that this type of inhibition requires both interaction of the neutral form with the membrane, and interaction of the charged form with an aqueous environment. On the other hand, for inactivated state affinity positive charge is not essential, in fact neutral molecules tend to show higher state-dependence, furthermore lipophilicity of both the neutral and the charged forms are important, as well as aromaticity. This may suggest that the immediate environment of the bound molecule is apolar, and binding probably involves π - π interactions with aromatic residues , polar surface area, and aromatic atom count) was performed . Anticonvulsant SCIs (a subset of ‘Type 2’ drugs), on the other hand, are predominantly neutral , have relatively low logP (1.9 to 2.8), MW between 230 and 260, and PSA between 46 and 91 Å2. Finally ‘Type 3’ drugs have three or four aromatic rings, a very high logP (5.8 to 6.2), high MW (400 to 440), moderate pKa (7.5 to 8.2) and low PSA (<37 Å2). It is yet to be verified experimentally whether other compounds beyond the ones investigated in this study, which fall within these sections of the “chemical space” are necessarily SCIs, and whether they produce the expected type of inhibition.The similarity between clustering based on biophysical or chemical properties is obvious, which allows prediction of biophysical properties with a considerable certainty. Within certain sections of the “chemical space” , however, several experimental data indicate that alternative binding sites and alternative modes of action do exist. From the 35 drugs investigated in this study 10 has been studied on mutant channels. Lidocaine, mexiletine and ranolazine seem to share the binding site; for amitriptyline, lamotrigine, phenytoin, flecainide and bupivacaine the binding site seem to partially overlap with the local anesthetic binding site; and for sertraline and paroxetine an entirely different binding site seems to exist We found that SCIs are diverse in their potency, kinetics, reversibility, use-dependence and state-dependence. We described three major types of inhibition, and besides we identified a number of compounds  which have additional distinct modes of action.We identified specific chemical descriptors which predict particular properties of inhibition.r, Ki and IC50  values; in addition the inhibition was less reversible, with slower onset and offset kinetics. This is in accordance with studies on structure-activity relationships, where a linear relationship between logP and logIC50 values were described based on the inhibition of action potentials 3H]BTX binding Partition coefficient (logP) was a major determinant of potency, kinetics and reversibility. Lipophilic molecules tended to be more potent in terms of Kr values (left panel) for ‘Type 1’ drugs is smaller than the mean Kr of the more lipophilic ‘Type 3’ drugs, while for Ki values (right panel), more lipophilic ‘Type 3’ drugs are more potent than the average of ‘Type 1’ drugs. This may suggest that lipophilic interactions (logP and logD) are more important in inactivated state affinity.Explanation for the correlation between logP and potency can be either of the following two possibilities: (1) SCIs must cross the membrane in order to enter a hydrophilic environment (which may be the intracellular fluid or the binding site itself); therefore, in order to be able to cross the membrane, the drugs must be lipophilic. (2) The binding site itself is hydrophobic. The first explanation supposes that drug molecules have to partition into the membrane, and then out of the membrane toward the intracellular fluid or the binding site. Too high lipophilicity would prevent effective partitioning out of the membrane environment; therefore logP should have a definite optimum. Although it cannot be statistically proven from our data, note that in r/Ki ratio) is of particular importance. It is thought to be essential for the safety of SCIs: state-dependent drugs are able to selectively inhibit excessive/pathological firing patterns, such as in epilepsy, neuropathic pain or cardiac arrhythmia, while their effect on normal firing activity is minimal.Among the eight properties of inhibition state-dependence  was the best predictor of state-dependence, and one of the best predictors of Ki. Drugs with high logD7.3 were more state-dependent, and had high inactivated affinity, while this property was irrelevant for resting affinity (see 7.3 of a drug can be high: Some of the compounds had a logP so high (>4.0), that even in their charged form they were still strongly lipophilic . Some other compounds had a low pKa value (<8.2), which means that a considerable fraction (>10%) of the molecules is neutral at pH = 7.3, therefore there is not much difference between logP and logD7.3 (less than 1 unit). Some examples are carbamazepine, lamotrigine, phenytoin, mirtazapine, trazodone, bupivacaine and ranolazine. Finally, there were five drugs, where both the logP was high and the pKa was low: nefazodone, riluzole, ritanserin, flunarizine and lifarizine. These drugs (with the exception of ritanserin) had the highest state dependence values among the 35 drugs investigated.Distribution coefficient at pH = 7.3 , the ratio of neutral molecules probably affects state-dependence directly.From this reasoning it follows, that the ratio of neutral form, N(pKa) also had to be a major determinant of state-dependence. Indeed, less charged molecules were found to be more state-dependent. This is contrary to the widely held view that SCIs should be positively charged. Because there was no significant correlation between N(pKa) and logDr did not depend on logD7.3), suggests that in resting inhibition an interaction of the charged form and the channel occurs within a polar environment. The major determinant of resting affinity was found to be pKa, indicating the importance of positive charge in resting inhibition. It was also the most important determinant of use-dependence, which is in accordance with previous studies, where positively charged molecules had slower kinetics and showed more use-dependence On the other hand, if we consider resting affinity, we can notice that positively charged SCIs tended to be more potent. The finding, that positively charged molecules are better inhibitors of resting channels irrespectively of the lipophilicity of the charged form  are important in binding to inactivated state. Aromaticity also determined time constants and reversibility. This may be one explanation of the finding that dimers of lidocaine (containing two aromatic rings) show both increased potency and decreased reversibility The role of aromaticity of the most important residue of the local anesthetic binding site (Phe1710 in rNav1.3) in use-dependent- but not resting inhibition has been shown by different amino acid substitutions. For resting inhibition hydrophobicity of the residue was sufficient, while effective use-dependent inhibition required that the residue was aromatic 7.3 >3.0, and pKa <8.0 . We have identified a couple of highly state-dependent compounds, which could be used as a basis for further drug development. The SCI property of bupivacaine, riluzole, flunarizine and lifarizine are well known, but we would like to call attention to the attractive properties of nefazodone, chlorpromazine and chlorprothixene, as highly state-dependent SCIs.In summary, we propose that drugs with high state-dependence are more likely to be found among compounds which contain more than one aromatic rings, and which have logD7.3, and aromaticity in determining Ki, but the correlations were less convincing. Furthermore, the role of positive charge in resting affinity, and neutrality in inactivated affinity could not be detected. The advantages of using identical experimental conditions for all drugs – as in our current study – are: improved reliability of data, the possibility of correlating chemical properties with multiple biophysical properties, and the possibility of detecting distinct types of inhibition within the “multi-dimensional space” defined by both biophysical properties of inhibition and chemical properties of the molecules.Our data confirm our previous results based on a meta-analysis of the literature Therapeutic applicability is not determined solely by the potency. In fact some of the most widely used drugs  are among the least potent SCIs. However, we expect that SCIs acting by similar mechanisms will have similar therapeutic action . In this respect it is important to find out about different types of inhibition, to locate novel drugs in the “biophysical space”, and to learn how this location is determined by chemical properties. This study, of course, have not accomplished mapping of the entire “biophysical space” for SCIs, but we hope the concept has been introduced, and at least we have identified three basic types of inhibition and a couple of additional drugs with interesting properties. Antiarrhythmics and local anesthetics  were a diverse group, while the three anticonvulsants  were found to be similar. A group of antidepressants: selective serotonin reuptake inhibitors, tricyclic antidepressants and maprotiline formed a fairly homogenous group, while the remaining antidepressants were diverse in both chemical and biophysical properties. It is worth noting that flunarizine and lifarizine . The challenge is to maximize the information content that can be obtained from more complex protocols, while not increasing the cost of measurements, and keeping the analysis manageable.50 value will help drug discovery, especially if we can determine the specific chemical properties which predict individual types of inhibition. This concept may be particularly profitable in the study of certain ion channels, which are notorious of their promiscuity in drug binding.We believe that this new approach of mapping drugs in the “biophysical space”, rather than determining a single ICHEK-293 cells stably expressing rNav1.2 sodium channels were obtained from NeuroSearch . The cells were grown in Dulbecco's modified Eagle medium  supplemented with 10% FBS. Prior to use, the cells were trypsinized  and subsequently kept in suspension in the QPatch cell storage facility in CHO-S- SFM-II medium .2, 1 MgCl2, 0.1 CdCl2, 20 TEA-Cl, 5 HEPES, adjusted to pH 7.3, Osmolality: ∼320 mOsm. The intracellular solution consisted of the following (in mM): 135 CsF, 10 NaCl, 1 EGTA, 10 HEPES, adjusted to pH 7.3 with CsOH (∼5 mM), Osmolality: ∼320 mOsm.Cells were automatically prepared for application to the chips  as described previously Drugs were obtained from Sigma, Tocris, or synthesized in Gedeon Richter Plc. . The list of drugs together with the three-letter codes, as well as their therapeutic indication, main mechanism of action, and human plasma concentration are shown in All electrophysiological experiments were conducted on Qpatch-16 or QPatch HT instruments using QPlate™ chips. For a detailed description of the instrument and the patch-clamp chips see 1/2 values and slope factors are shown in Chip properties, distribution of gigaseal properties and stability of whole-cell parameters were essentially the same as described before for HEK-293 cells ctr – amplitudedrug)/amplitudectrInh  =   * cc/InhIC. Reliability of the calculation was verified by measuring the effect of different concentrations of the same drug (in the case of 5 out of the 35 drugs). The process is illustrated using the example of lidocaine in From this protocol we extracted five parameters, as illustrated in r) values were calculated from the inhibition at −150 mV pre-pulse potential (Inh−150), using the rearranged Hill equation as described above (i) values were calculated assuming a simple four-state model of drug binding and channel gating 1/2), the slope of the Boltzmann fit of the availability curve (k) and Kr, using the formula described by Bean, Cohen and Tsien i−ΔV1/2; see app) at a certain pre-pulse potential, which was calculated from the inhibition (e.g. Inh−60) as described above, and the availability (h) at the same pre-pulse potential, using the formula from Kuo and Bean i were minimal, Ki−ΔV1/2/Ki−Kapp ratios were 0.98±0.098. For further analysis Ki values obtained with the latter method were used, because these seemed more reliable . Geometric means of measured Kr, Ki−ΔV1/2 and Ki−Kapp values are shown in Resting affinity (Ked above . InactivpH/(10pH+10pKa).Chemical descriptors were generated using JChem for Excel 1.1.1 and Marvin 5.2 software from ChemAxon . Neutral fraction of drugs at pH = 7.3 was calculated using the rearranged Henderson-Hasselbalch equation: N(pKa) = 10Exponential fitting was made using Origin 8 . Statistical analysis was performed using Statistica 8.0 .Materials and Methods S1Source of drugs and stock solutions.(PDF)Click here for additional data file.Materials and Methods S2Main electrical properties of cells.(PDF)Click here for additional data file.Materials and Methods S350 calculation from single-concentration inhibition values.Reliability of IC(PDF)Click here for additional data file.Results S1Comments on biophysical properties of inhibition.(PDF)Click here for additional data file.Results S2Detailed discussion of state-dependence.(PDF)Click here for additional data file.Results S3Cross-correlations of 58 chemical descriptors, based on the chemical properties of the 44 drugs we used.(PDF)Click here for additional data file.Results S4Classification of 58 chemical descriptors based on their correlations with individual properties of inhibition.(PDF)Click here for additional data file.Results S5Correlation coefficients between chemical descriptors and biophysical properties.(PDF)Click here for additional data file.Results S6Values of selected chemical descriptors for the drugs studied.(PDF)Click here for additional data file.Results S7r, Ki−ΔV1/2 and Ki−Kapp.Geometric and arithmetic mean values of K(PDF)Click here for additional data file.


SLC22A1, SLC22A2, or SLC22A3), which are tissue specifically expressed at significant levels in various organs such as liver, muscle, and kidney. Because metformin does not undergo hepatic metabolism, drug-drug interaction by inhibition of OCT transporters may be important. So far, comprehensive data on the interaction of proton pump inhibitors (PPIs) with OCTs are missing although PPIs are frequently used in metformin-treated patients. Using in silico modeling and computational analyses, we derived pharmacophore models indicating that PPIs  are potent OCT inhibitors. We then established stably transfected cell lines expressing the human uptake transporters OCT1, OCT2, or OCT3 and tested whether these PPIs inhibit OCT-mediated metformin uptake in vitro. All tested PPIs significantly inhibited metformin uptake by OCT1, OCT2, and OCT3 in a concentration-dependent manner. Half-maximal inhibitory concentration values (IC50) were in the low micromolar range (3–36 µM) and thereby in the range of IC50 values of other potent OCT drug inhibitors. Finally, we tested whether the PPIs are also transported by OCTs, but did not identify PPIs as OCT substrates. In conclusion, PPIs are potent inhibitors of the OCT-mediated metformin transport in vitro. Further studies are needed to elucidate the clinical relevance of this drug-drug interaction with potential consequences on metformin disposition and/or efficacy.Metformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT) 1, 2, and 3 (encoded by  Metformin  is an oral insulin-sensitizing agent commonly used either alone or in combination with other antihyperglycemic drugs in patients with type 2 diabetes The glucose-lowering effect of metformin is largely attributable to inhibition of hepatic gluconeogenesis, and additionally, insulin-stimulated glucose uptake into skeletal muscle cells and adipocytes is increased by metformin in vitro has been reported Since metformin does not undergo hepatic metabolism Hundreds of xenobiotics including drugs potentially inhibiting OCTs were tested in the past and several new inhibitors have been identified in vitro the drug-drug interaction potential of PPIs with OCTs. We first used in silico pharmacophore modeling to assess the inhibitory potential of PPIs. We then generated cell lines stably expressing recombinant human OCT1 (encoded by the SLC22A1 gene), OCT2 (SLC22A2), or OCT3 (SLC22A3) to experimentally confirm the inhibitory potential of PPIs on OCT-mediated metformin transport. Finally, we assessed whether PPIs are transported by OCTs. Altogether, we identified PPIs as potent inhibitors of OCT-mediated metformin transport.The aim of the present study was to investigate systematically Pharmacophore models were constructed for all 3 OCTs. For this, the 15 most potent inhibitors of OCT1, OCT2, or OCT3 were included as training compounds to test whether the PPIs omeprazole, pantoprazole, lansoprazole, rabeprazole, and tenatoprazole are OCT inhibitors . This liFor all 5 PPIs high pharmacophore fit values were calculated  and eachin vitro, we stably transfected HEK cells with cDNAs encoding human OCT1, OCT2, or OCT3. All 3 transfected cell lines showed time-dependent uptake of the prototypic substrate TEA  analysis. OCT1 inhibition was best explained by a two-component model. The cumulative Q2 value of 0.74 indicated a good predictive power of the model and the R2 value of 0.99 indicated that variation in the IC50 values was largely modeled by the two components. For comparison, a one-component model resulted in a Q2 value of 0.19 explaining 89% of variation in the IC50 values. The variable influence on projection (VIP) function was then used to identify the physicochemical parameters most relevant for explaining the IC50 values in the two-component model. The number of H-bond acceptor sites (VIP = 1.61) and the ClogP value (VIP = 1.15) contributed most to the two-component model for OCT1 inhibition. For OCT2 and OCT3 inhibition, the PLS analysis resulted in one-component models. Although the R2 values were 0.53 for OCT2 and 0.56 for OCT3, the Q2 values were <0 for both suggesting that the models cannot predict IC50 values for OCT2 and OCT3.We next determined the relationship between the ICIn order to assess whether the 5 PPIs are transported by OCTs, PPI uptake by OCT-expressing HEK cells was compared with that by control cells after 1 and 5 min of incubation . None ofWith the recent advances in the understanding of the role of drug transporters in pharmacokinetics it has become critical to elucidate drug-drug interactions that are rooted in transporters. Drug transporters can be generally classified as either uptake or efflux transporters characterizing whether they facilitate drug entry into a cell or efflux out of a cell.2 receptor antagonists .In the present paper we focused on the uptake transporter proteins OCT1, OCT2, and OCT3 since the antidiabetic drug metformin is a substrate for each and there is already evidence that e.g. the antidiabetics repaglinide or rosiglitazone The pharmacophore models described for OCT1 silico pharmacophore modeling, we generated cell systems stably expressing recombinant human OCT1, OCT2, or OCT3. All 3 transfected HEK cell lines expressed functionally active organic cation transporters as demonstrated by time-dependent TEA and metformin uptake  with ICus blood , Table 1us blood . The facus blood .Moreover, OCT1- and OCT3-mediated metformin uptake appears to be activated by low concentrations of selected PPIs  reported by Ahlin et al. 50 values in the low nanomolar range; 50 values from 6–25 µM) and the NRTIs as training compounds. Because for OCT3 no comprehensive inhibition analysis is available, compounds with the lowest IC50 values (0.8–20 µM) were included from several studies  and [14C]metformin hydrochloride (4074 MBq⋅mmol−1) were from PerkinElmer  and Hartmann Analytic , respectively. [14C]Omeprazole (470 MBq⋅mmol−1) and [14C]pantoprazole (550 MBq⋅mmol−1) were generously supplied by Byk Gulden  as described metformin, 100 µM [14C]TEA, 10 µM [14C]omeprazole, or 10 µM [14C]pantoprazole. Uptake was stopped at indicated time points by removing the uptake buffer and immediately washing the cells 3 times with ice-cold uptake buffer. Cells were lysed with 0.2% SDS and intracellular radioactivity was determined by liquid scintillation counting . For measuring inhibition of OCT-mediated metformin uptake by PPIs, 5 µM [14C]metformin uptake was measured in the presence of different PPI concentrations and stopped after 5 min  or 5 sec (OCT2).HEK cells (400000 cells/well) were seeded into 24-well cell culture plates  and grown for 48 h. Cell adherence was improved by coating plates with poly-L-lysine  prior to cell plating. All uptake studies were carried out at 37°C as described For determination of intracellular accumulation of unlabeled lansoprazole, rabeprazole, and tenatoprazole, cells were incubated for 1 or 5 min with 5 µM PPI at 37°C and then immediately washed three times with ice-cold uptake buffer and twice with ice-cold phosphate-buffered saline. Cells were harvested by scraping them off in 500 µl 0.1 M sodium carbonate buffer (pH 9.3) : methanol 4∶1 (v/v) and then transferred into Eppendorf tubes. Cells were disrupted by three cycles of shock freezing/thawing  and further by ultra-sonification, three times 3 sec, at 4°C. Disrupted cell solutions were centrifuged 5 min (15000 g) at 4°C and supernatants were transferred into Eppendorf tubes and stored at −20°C for analytic determination of the PPIs.Protein content of lysed cells was determined in 25-µl aliquots using the bicinchoninic acid assay as described −1. Gradient runs with the mobile phases (A) 10 mM ammonium acetate in water and (B) acetonitrile were programmed as described previously −1 nitrogen, drying gas temperature 350°C, and nebulizer pressure 30 psig (207 kPa gauge). The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 370 for lansoprazole, m/z 360 for rabeprazole, m/z 347 for tenatoprazole, and m/z 346 for the internal standard omeprazole. The fragmentor was set at 80 V for lansoprazole, rabeprazole and omeprazole, and at 60 V for tenatoprazole.Lansoprazole, rabeprazole, and tenatoprazole were determined by LC-MS using omeprazole as internal standard similar to a method described for omeprazole 2) linear regression for the peak area ratio of the analyte to the internal standard against the amount of the analyte. The concentration in unknown samples was obtained from the regression line.Calibration samples were prepared in 0.1 M sodium carbonate buffer pH 9.3 : methanol 4∶1 (v/v) containing 1 µM omeprazole as internal standard, in the concentration range 2.5 to 250 nM for lansoprazole and rabeprazole, and from 5 to 250 nM for tenatoprazole. Calibration samples were worked up as the samples, and analyzed together with the unknown samples. Calibration curves based on internal standard calibration were obtained by weighted . Values in OCT-specific metformin uptake was obtained by subtracting metformin uptake in the presence of 2 mM unlabeled 1-methyl-4-phenylpyridinium (MPP) from that in its absence. MPP was used because it is transported by all 3 OCTs with high-affinity  and “charge at pH 7.4” were calculated using MarvinView . Seven other computed physicochemical properties  were from the publicly available PubChem Compound database  and P values (two-tailed) were calculated with GraphPad Prism 4. Normality of data was confirmed by the Kolmogorov-Smirnov test. Statistical significance was defined as P<0.05. Multivariate analyses were performed with the Excel-based add-in tool XLSTAT  using the partial least square analysis module. The 7 physicochemical parameters molecular weight, ClogP, TPSA, H-bond acceptor count, rotatable bond count, tautomer count, and heavy atom count were included as quantitative descriptive x values and the IC50 values as quantitative dependent y values. The R2 value indicates how much of variation of the IC50 values is explained by the components. The quality of the fit is estimated by the Q2 value. A Q2>0 indicates that the model has a better predictive power than no model.For the analysis of the relationship between physicochemical properties of PPIs and ICFigure S1Molecular structures of the 5 tested PPIs.(TIF)Click here for additional data file.Figure S2Structural formula of the 5 PPIs with those chemical features marked that were found by LigandScout to be relevant for the OCT1, OCT2, or OCT3 pharmacophore model.(TIF)Click here for additional data file.Table S1Chemical feature coordinates and tolerance radii (in Ångstroms) found by LigandScout for the pharmacophore models of OCT1, OCT2, and OCT3.(DOC)Click here for additional data file.Table S2Pharmacophore fit values of training sets and PPIs as test set.(DOC)Click here for additional data file.Table S3Physicochemical properties of the tested PPIs.(DOC)Click here for additional data file.Table S450 values of OCT1, OCT2, and OCT3 as determined by univariate analysis.Relationship of physicochemical properties of the tested PPIs and IC(DOC)Click here for additional data file.


Dendranthema × grandiflorum cv. “Mottenohoka” that promote neurite outgrowth of PC12 cells were identified and the mechanism of neurite outgrowth stimulated by isolated components was studied. Components that promoted the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) of PC12 cells were isolated. From various structural analyses, the active components were identified as acacetin and luteolin. The effects of acacetin or luteolin on PC12 cells were evaluated by electro-blotting and immunostaining. Slight neurite outgrowth in PC12 cells was observed within 2 days of culture after stimulation by luteolin or acacetin. However, NGF-stimulation induced remarkable neurite outgrowth in comparison. Neurite outgrowth by luteolin or acacetin was significantly enhanced by pretreatment with SB203580 (a p38MAPK inhibitor). The results of this study into the phosphorylation of ERK 1/2 and p38MAPK by flavonoids suggest that the inhibition of p38MAPK phosphorylation may effectively enhance neurite outgrowth.Components from  Dendranthema × grandiflorum cv. “Mottenohoka,” a distinctive edible chrysanthemum with a beautiful color, pleasant aroma, and crunchy texture, is a special product of north-eastern Japan and has traditionally been eaten as a delicacy around the country. The pharmacological effects of Chrysanthemum morifolium, including Mottenohoka, are various and include sleep induction [Chrysanthemum morifolium that may be associated with these pharmacological effects are luteolin and apigenin [O-dicaffeoylquinic acid, 3′,4′,5-trihydroxyflavanone 7-O-glucuronide [O-β-d-(4′-caffeoyl)glucuronide and apigenin 7-O-β-d-glucuronide [O-β-d-galactopyranoside [ Spring cherry blossoms and the chrysanthemums of autumn are typical ornamental and edible flowers of Japan. nduction , protectnduction , toxicitnduction , suppresnduction , protectnduction , cardiacnduction , anti-HInduction , 8, and nduction . Componeapigenin , 11, aciapigenin , 12, dicapigenin , chlorogcuronide , apigenicuronide , and acaWe tested the effects of extracts from various specialty vegetable products of northeastern Japan on the intracellular signaling of PC12 cells and found that Mottenohoka extracts increased the level of phosphorylated extracellular signal-regulated kinases 1/2 (ERK 1/2). It was important to determine whether the Ras/ERK 1/2 pathway is involved in ERK 1/2 phosphorylation induced by Mottenohoka extracts because the activation of the pathway leads to neuritogenesis followed by neuronal differentiation of PC12 cells. Thus, the substances that can activate Ras/ERK 1/2 might be promising for protection against or therapy for some neurological disorders with suspected neurotrophic factor involvement, such as depression or Alzheimer's disease. These considerations prompted us here to identify the components from Mottenohoka that activate mitogen-activated protein kinase (MAPK) of PC12 cells and to clarify the intracellular signals so that their possible therapeutic use in neurological disorders may be explored.3--2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich . Hexane, chloroform, and methanol were obtained from Wako Pure Chemical Industries . A specific inhibitor for tyrosine kinase of epidermal growth factor receptor (EGFR) (AG1478), an inhibitor of Trk-type tyrosine kinases (K252a), MEK inhibitor (U0126), and p38 mitogen activated protein kinase (p38MAPK) inhibitor (SB203580) were purchased from Calbiochem  or Nacalai Tesque Inc. .Raw Mottenohoka was purchased commercially from Yamagata prefecture and the components were fractionated according to a flow sheet . In brieμm particle size; Nomura Chemical Co., Ltd., Aichi, Japan), a PU-800 pump , and an evaporative light scattering detector . The column was run using a water : methanol : trifluoroacetic acid = 30 : 70 : 0.1 (v/v/v) mobile phase at a flow rate of 10 mL/min. The eluted substances were collected separately by referring to the signal detected by the evaporative light scattering detector.Methanol extract (10 g) was applied for Sephadex LH-20 column chromatography (CC) and eluted with methanol into 100 fractions. The activity of each fraction was evaluated in cultured PC12 cells. An aliquot of the active fractions was injected into preparative high performance liquid chromatography equipped with a Develosil ODS 60–10 column  NMR spectra were recorded with a JEOL ECX 400 spectrometer, using tetramethylsilane as an internal standard.In the present study, we used rat pheochromocytoma cell line PC12 because previous reports suggested the suitability of this cell line as a model for studying mechanisms of neuronal differentiation including neuritogenesis . The cel6 cells/well) in serum-containing medium for 2 days at 37°C in an atmosphere of 95% air/5% CO2. Culture medium was replaced with 50 μL of serum-free medium containing each test agent after washing with PBS, and the cells were cultured with each test agent over a range of days. Cell viability was determined by the 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay [μL of 50% (v/v) dimethylformamide (DMFA) containing 20% (w/v) sodium dodecyl sulfate (SDS). The amount of MTT formazan product was determined photometrically using a microplate reader  by measuring the absorbance at 562 nm with a reference wavelength of 630 nm. The number of cells in each well was calculated on the basis of a known relationship between absorbance and cell number.PC12 cells were cultured on collagen-coated 96-well plates  supplemented with the serum-containing medium and cultured for 2 days at 37°C in an atmosphere of 95% air/5% CO2. The cells were then washed with phosphate-buffered saline (PBS) and incubated with the above mentioned serum-free culture medium containing the various agents for 10 min at 37°C. The culture plates were then placed on ice and each well was washed with 3 mL of 2 mM Tris-HCl buffer (pH 8.0) containing 0.33 M NaF and 6.25 M Na3VO4, and subsequently lysed with 150 μL of 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (w/v), 1% sodium deoxycholate (w/v), 0.1% SDS (w/v), 50 mM NaF, 0.1% aprotinin (w/v), 0.1% leupeptin (w/v), 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride (PMSF). Cell lysates were collected by using a cell scraper and were centrifuged at 15000 ×g for 30 min at 4°C. The supernatant was collected and the overall protein concentration was determined by a BCA Protein Assay Reagent Kit  with BSA as the standard.Each test compound was suspended in serum-free medium and sonicated until fully emulsified. PC12 cells were seeded at 2 × 10μg) were mixed with lithium dodecyl sulfate (LDS) sample buffer  and were incubated for 5 min at 80°C. Proteins in the samples were separated on SDS-polyacrylamide gel electrophoresis, and the proteins in gels were electroblotted onto polyvinylidene fluoride (PVDF) filters . Immunoblotting analysis was performed by using monoclonal antibodies against p44/42 ERK, phospho-p44/42 ERK, p38MAPK, phospho-p38MAPK, stress-activated protein kinase/Jun amino-terminal kinase (JNK/SAPK), phospho-JNK/SAPK, Akt, phospho-Akt, neurofilament-M, or β-actin  as the primary antibodies, followed by reaction with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies from Promega Corporation  as the secondary antibody. The blots were developed by the enhanced chemiluminescence method .Supernatant fluids containing proteins  or 10 μM (U0126) in serum-containing medium from a preliminary evaluation test for dose responsiveness between the concentration of inhibitor and the level of phosphorylated protein. PC12 cells were seeded at 2 × 106 cells/well supplemented with serum-containing medium and precultured for 2 days at 37°C in an atmosphere of 95% air/5% CO2. Cells were preincubated for 3 h in the serum-free medium containing each inhibitor, washed with PBS, and incubated with the medium containing the corresponding active substance for 10 min at 37°C. Cells were then collected and the cell lysates were used for immunoblot analyses of phosphorylation or expression of proteins.Each inhibitor was used at a final concentration of 1 μg/mL (data not shown).The method of solvent fractionation is shown in The levels of phosphorylated ERK 1/2 of PC12 cells after treatment with solvent extracts of Mottenohoka are shown in 1–3) from the mixture of fractions no. 40 to 100.Methanol extract was applied to a Sephadex LH-20 column equilibrated with methanol and eluted with methanol as the mobile phase. The fractions were collected in the order of elution (no. 1 to 100). Enhanced ERK 1/2 activation was found in fractions no. 40 to 100. The active fractions were pooled, lyophilized, and applied to an ODS column for reverse phase preparative chromatography. We obtained three kinds of active components  showed the existence of a 1,3,5-trisubstituted aromatic ring. These findings led us to determine that the structure of compound 1 was 3′,5,5′,7-tetrahydroxyflavanone.The 1H NMR of component 2 showed the characteristic signals of 5,7-dihydroxyflavone; the singlet at δH 6.59 was assigned to the H-3 position of the flavone skeleton. In addition, three signals  showed the existence of a 1,3,4-trisubstituted aromatic ring. Thus, the structure of component 2 was established as luteolin. The 1H NMR of component 3 exhibited signals similar to those of component 2. The spectrum clearly showed the signals of a para-substituted aromatic ring (δH 8.04 and 7.14) and a methoxy group (δH 3.92). The structure of component 3 was established as acacetin : δ 7.04 , 6.88 , 5.97 , 5.95 , 5.40 , 3.14 , 2.72 .3′,5,5′,7Luteolin. 1H NMR : δ 7.52 , 7.48 , 7.01 , 6.59 , 6.54 , 6.26 .Acacetin. 1H NMR : δ 8.04 , 7.14 , 6.69 , 6.57 , 6.28 , 3.92 .The MAPK family is a group of serine/threonine protein kinases, including ERK 1/2, p38MAPK, JNK/SAPK, and ERK 5, known to be involved in various cellular events such as survival/death, differentiation, and migration . InitialIt has been found that the activation of Akt as well as the MAPK family is required for neuronal differentiation . TherefoμM, while there was no such effect on ERK 1/2 (data not shown).We evaluated the effects of K252a (an inhibitor of tyrosine kinase of high-affinity NGF receptor Trk), AG1478 (an inhibitor of tyrosine kinase of EGF receptor), and U0126 (an inhibitor of MEK (ERK kinase)) on luteolin- or acacetin-induced phosphorylation of ERK 1/2 in PC12 cells . PhosphoPC12 cells were exposed to luteolin, acacetin, NGF, or EGF for 2 days in serum-free medium. Prominent neurite outgrowth was elicited by NGF and slight outgrowth by luteolin or acacetin . InteresChrysanthemum morifolium Dendranthema × grandiflorum [μM but had no such effect on Akt. NGF, which stimulates neuronal differentiation of PC12 cells, facilitated the phosphorylation of ERK 1/2, SAPK/JNK, and Akt but not that of p38MAPK. EGF, which stimulates cell proliferation of PC12 cells, promoted significant phosphorylation of ERK 1/2 and slight phosphorylation of Akt. However, it did not promote the phosphorylation of SAPK/JNK and p38MAPK.The well-known neurotrophic factor, NGF, acts on cultured PC12 cells and induces many biological events including neurite outgrowth for neuronal differentiation that gives rise to sympathetic neuron-like cells. These NGF actions require phosphorylation of the classical Ras/MAPK pathway. Based on this fact, extracts of Mottenohoka were tested and found to effectively enhance the level of phosphorylated ERK 1/2 of PC12 cells. The active components were isolated from the extracts and identified as 3′,5,5′,7-tetrahydroxyflavanone, acacetin, and luteolin. Acacetin and luteolin are known components of diflorum –11; howeLuteolin and acacetin slightly but significantly stimulated neurite outgrowth, although this effect was much weaker than that of NGF . The intIt has been suggested that sustained activation of ERK 1/2 is required for neuronal differentiation and neurite outgrowth in PC12 cells . HoweverWhile the phosphorylation of p38MAPK is necessary for neural differentiation , 28, theK252a, an inhibitor of the Trk family of tyrosine kinases of high affinity neurotrophin receptors, is known to induce neurite outgrowth . Since tLuteolin- or acacetin-induced phosphorylation of ERK 1/2 was inhibited by U0126 (MEK phosphorylation inhibitor) treatment, leading to the assumption that the classical MAPK pathway was activated by luteolin or acacetin-stimulation. On the other hand, since the levels of luteolin- or acacetin-induced phosphorylated ERK 1/2 were unchanged by treatment with K252a or EGR receptor inhibitor, it can be deduced that luteolin or acacetin activates the classical MAPK cascade via receptors other than Trks or EGFR. Neurotrophic factors such as neurotrophins, including NGF, were predicted to be suitable therapeutic tools for some neurological disorders or as supplements for brain health. However, they are macromolecules that cannot cross the blood brain barrier and are unstable in the blood stream and tissues, thus studies have produced unsuccessful results. In the present study, we identified components in Mottenohoka that effectively activated Ras/MAPK(ERK 1/2) pathway of PC12 cells and led to neuritogenesis. The active entities were flavonoids, acacetin, and luteolin. A number of studies have dealt with flavonoids and it has been demonstrated that a component of ingested flavonoids moves into the plasma bound to albumin [In order to escape starvation during famines that have occurred regularly, plants that are not eaten in other regions are still cultivated and consumed in Yamagata Prefecture, Japan. Interestingly, it is possible that the flavonoids in such plants are useful in maintaining mental health because the number of suicides due to depression in Yamagata Prefecture is low compared with a neighboring prefecture.Our results suggested that the inhibition of p38MAPK phosphorylation enhances neurite outgrowth stimulated by luteolin or acacetin. Thus, perhaps the combined use of previously examined natural components that promote neurite outgrowth and components that regulate p38MAPK phosphorylation could bring us a step closer to the commercialization of natural products for maintaining the health of the central nervous system.


Oncorhynchus mykiss) possesses paralogs of the three known AMPK subunits that co-diversified, that the AMPK protein is present in the liver and in isolated hepatocytes, and it does change in response to physiological (fasting-re-feeding cycle) and pharmacological (AICAR and metformin administration and incubations) manipulations. Moreover, the phosphorylation of AMPK results in the phosphorylation of acetyl-CoA carboxylase, a main downstream target of AMPK in mammals. Other findings include changes in hepatic glycogen levels and several molecular actors involved in hepatic glucose and lipid metabolism, including mRNA transcript levels for glucokinase, glucose-6-phosphatase and fatty acid synthase both in vivo and in vitro. The fact that most results presented in this study are consistent with the recognized role of AMPK as a master regulator of energy homeostasis in living organisms supports the idea that these functions are conserved in this piscine model.AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is proposed to function as a “fuel gauge” to monitor cellular energy status in response to nutritional environmental variations. However, in fish, few studies have addressed the metabolic consequences related to the activation of this kinase. This study demonstrates that the rainbow trout ( Intracellular ATP concentrations must be maintained within a narrow range to sustain appropriate metabolic function. Recent evidence indicates that the coordination of this task is achieved through the AMP-activated protein kinase (AMPK) 172) activation loop whose phosphorylation is required for full activation. Homologues of all three subunits exist in mammals, invertebrates, yeast, plants and the primitive protozoa, with a high degree of conservation suggesting that this signaling pathway evolved over one billion years ago to regulate metabolic homeostasis, although the exact evolutionary dynamics of AMPK are unclear The AMPK of mammals exists as a heterotrimeric complex consisting of a catalytic α subunit and two regulatory subunits β and γ Carassius auratus) Danio rerio) Carassius carassius) Salmo salar) . Additionally, numerous submissions to gene databases complete the list of species in which this kinase is reported including puffer fish , stickleback (Gasterosteus aculeatus), and the Japanese ricefish (Oryzias latipes). Despite this apparent wide distribution in the piscine model, only a few studies have dealt with AMPK functions in fish or have established a phylogenetic relationship of AMPK subunits as found in fish and other vertebrates. Three different studies published nearly simultaneously addressed the role of AMPK in hypoxia-related functions all in hypoxic-insensitive cyprinids The existence of AMPK is reported in several fish species including goldfish . We also conducted complementary experiments to establish whether physiological conditions modify trout liver AMPK, including feeding trout with metformin-supplemented diets or subjecting trout to a fasting-re-feeding cycle. Furthermore, we assessed hepatic glycogen levels and, at the molecular level, the activity of several representative genes of glucose and lipid metabolism in liver and isolated hepatocytes to explore the potential metabolic effects of AMPK activation.The present study places the rainbow trout AMPK subunits into a phylogenetic context and shows that all three subunits diversified at approximately the same geological time. We employ two known activators of AMPK, AICAR and metformin  to explore the metabolic effects of the activation of AMPK in rainbow trout liver. Using mammalian antibodies, we identified in both trout liver and isolated hepatocytes the phosphorylation status of AMPK and indirectly assessed its activity by examining the phosphorylation of the AMPK downstream target acetyl-CoA carboxylase (ACC) at the serine residue  were obtained from the INRA experimental fish farm facilities of Donzacq . Fish were maintained in tanks kept in open circuits supplied with 17°C well aerated water under a controlled photoperiod (LD 12:12) and fed with a commercial diet . Fish weighed 33±2 g for the AICAR experiments and 84±4 g for the metformin experiments. The experiments were conducted following the Guidelines of the National Legislation on Animal Care of the French Ministry of Research  and were approved by the Ethics Committee of INRA .Rainbow trout (www.ncbi.nlm.nih.gov). The human sequence of each paralog of each subunit was used as a BLASTn query to find all other sequences used in this study. A number of sequences from the chordates were retrieved, including a relatively large number of mammal sequences (i) to be able to use some of the fossil divergences as calibration priors (see below) and (ii) in order to check that accurate species trees were obtained for each paralog. Only sequences with an E-value <10−500 were used. The rainbow trout sequences were accessed by routine 5’RACE PCR and PCR experiments using human AMPK isoform primers. The resulting trout sequences were uploaded to GenBank (accession numbers: HQ403672-HQ403678).Sequences were downloaded from GenBank at the National Center for Biotechnology Information (4 (four discrete rate categories) and NNI + SPR in PhyML ver. 3.0 The protein-coding parts of these sequences were extracted, and aligned with Muscle Amniota/Amphibia , Mammalia/Sauropsida , Hominoidea/Cercopithecoidea (Homo/Macaca: 0.020 BYA), Theria/Prototheria (Homo/Ornithorhynchus: 0.200 BYA) and Actinopterygii/Sarcopterygii . These fossil dates were used as minimum ages in the model, and mean 0.05 exponential prior distributions were used (note that times are in units of billion years). A vague lognormal prior (mean 0.5 and standard deviation 0.5) was placed on the root of each tree, with a minimum age of 0.450 BYA. The prior on rates was lognormal, and that on times was a pure-birth (Yule) process. The Markov chain Monte Carlo (MCMC) samplers were run in duplicates (to check convergence) for 100 million generations with a thinning of 2000  or saline containing either AICAR  or metformin . Sampling was initiated 2 h after the injection when six fish per group were randomly sampled and their liver immediately frozen in liquid nitrogen and kept at −80°C pending analyses.For drug administration, 48 h-fasted fish were lightly anesthetized (0.05% (v/v) 2-phenoxyethanol), weighed, and intraperitoneally (ip) injected with 5 ml·kgThe fasting-re-feeding trial was done with rainbow trout (fish mass 95±3 g) fasted for 10 days and then re-fed with a semi-purified diet . Liver samples were taken before the re-feeding at 1 and 24 h after the meal. The nutritional study followed the experimental design described in Panserat et al. in situ, excised and minced with a razor blade. After filtration and centrifugation , the resulting cell pellets were resuspended three successive times in a modified Hanks’ medium  and centrifuged . Cells were finally suspended in the modified Hanks’ medium supplemented with 1.5 mM CaCl2, 1% defatted BSA, 4·10−9 M bovine insulin (Sigma), 20 mM glucose, MEM essential amino acids , MEM non-essential amino acids  and antibiotic antimycotic solution . Hepatocytes were plated in six well culture dishes at a density of 3·106 cells per well and incubated at 18°C. The incubation medium was replaced every 24 h over the 72 h primary cell culture period. For protein analysis, 48 h-cultured hepatocytes were stimulated using either metformin or AICAR at 0.5 mM for 16 h, doses used previously for mammalian cell culture studies Isolated liver cells were prepared from four day-fasted rainbow trout as previously described by Mommsen and colleagues 172 and anti-AMPK antibodies  that recognize both α-1 and α-2 isoforms of the enzyme. For ACC, Akt and β-tubulin blots, anti-phosphor-ACC Ser79 and anti-β-tubulin antibodies  were used as noted previously. Frozen livers or hepatocytes were homogenized on ice with an Ultraturrax homogenizer in a buffer containing 150 mM NaCl, 10 mM Tris, 1 mM EGTA, 1 mM EDTA (pH = 7.4), 100 mM sodium fluoride, 4 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% Triton X-100, 0.5% NP-40-IGEPAL and a protease inhibitor cocktail . Homogenates were centrifuged for 15 min at 12,000 g and the resulting supernatants stored at −80°C. Protein concentrations were determined using the Bio-Rad protein assay kit . Protein lysates (20 µg of protein) were subjected to SDS-PAGE and Western blotting using the appropriate antibody. After washing, membranes were incubated with an IRDye infrared secondary antibody . Bands were visualized by Infrared Fluorescence using the Odyssey® Imaging System  and quantified by Odyssey infrared imaging system software .Protein extraction (20 µg) and Western blotting were undertaken as in ef1α gene expression as reference, which was found to be stably expressed in this study. Relative quantification of the target gene transcript with the ef1α reference gene transcript was made following the Pfaffl method Liver mRNA levels for GK (glucokinase), G6Pase (glucose 6-phosphatase), FAS (fatty acid synthase), PEPCK (phosphoenolpyruvate carboxykinase) and FBPase  were estimated by real-time quantitative RT-PCR (q-PCR) 2O, and 1 µl of 5x diluted cDNA template. Cycling conditions were as follows: 10 min initial denaturation at 95°C, 40 cycles at 95°C for 30 s, and 60°C for 1 min. To account for differences in amplification efficiency between different cDNAs, standard curves were constructed for each target gene using serial dilutions of quantified liver and muscle cDNA. To account for differences in cDNA production and loading differences, all samples were normalized to the expression level of the housekeeping gene β-actin, which did not change over the course of the experimental treatments. Gene expression data were calculated using the 2ΔΔ- method of Pfaffl 2O and non-reverse transcribed RNA were assayed on each plate to ensure the absence of contamination in the reagents and primers used. Primers were designed using Integrated DNA Technologies online assay design program and can be found in Due to the similarities of the AMPKα1 and AMPKα2 isoforms, probe-based qPCR was employed to detect differences in expression from liver samples. Briefly, first strand cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen). mRNA expression was quantified in duplicate on a Stratagene MX3000P real-time PCR machine using probe based (FAM labeled) PrimeTime Mini qPCR Assays (Integrated DNA Technologies). Each reaction contained 12.5 µl SYBR green mix, 1 µl primer/probe mix, 0.375 µl ROX reference dye (1∶500 dilution), 10.125 µl RNase/DNase-free Hn = 6) and were analyzed by one-way ANOVA to compare groups receiving drug administration or the vehicle using Student-Newman-Keuls a posteriori multiple comparison test. The level of significance was set at α<0.05.Results are expressed as means ± s.e.m. , β subunit  and γ subunit . The dot notation for the fish-specific paralogs is ours. No γ3 sequences were obtained. The actual identification of these sequences was performed through a phylogenetic analysis, conducted as described in the Branchiostoma sp. and Saccoglossus sp. sequences as “α2” (as they are) or even “α1”, as they are neither. The dearth of fish sequences for the α subunit makes it difficult to interpret the phylogenetic dynamics of this subunit here. On the other hand, the β subunit clearly shows evidence for the fish-specific WGD event, here dated between ∼0.560 and 0.180 BYA, as all the gene duplicates seem to have been preserved  was lower than that of AICAR (up to 10-fold increase). No changes were found in the total level of AMPK.The phosphorylation status of AMPK (threonine-172) and the total AMPK levels in liver of fasted rainbow trout after ip injection of AICAR or metformin is reported on 79, although we did observe significant variability between fish for this analysis. In contrast, metformin injection resulted in a significant two-fold up-regulation of the ACC phosphorylation status compared to the saline control group.The phosphorylation status of ACC (serine-79) in liver of fasted rainbow trout after ip-injected AICAR or metformin is reported on Hepatic glycogen levels in trout ip-injected with either AICAR or metformin are shown in Analysis of the phosphorylation status of AMPK and ACC as well as total AMPK and β-tubulin levels in cultured hepatocytes of rainbow trout is shown in 172 and total AMPK levels in trout fasted for 10 days and re-fed is shown in The phosphorylation status of AMPK at Thr172 and the total AMPK protein levels, mRNA levels of AMPK α1 and α2 as well as glycogen levels in the liver of rainbow trout fed with a high carbohydrate diet with or without metformin are shown in The phosphorylation status of AMPK at Thrin vivo and in vitro) are shown in in vivo and in vitro in the presence of AICAR, which probably was also responsible for AMPK phosphorylation. However, when metformin was administrated, GK mRNA expression level changed only in the in vivo experiments, possibly as a result of the differences between the hepatocytes that were cultured in media containing insulin and glucose, while the fish used in the in vivo trial were fasted for 48 h. Similar to the changes found for GK, G6Pase mRNA transcript abundance was also reduced when compared with the control in both AICAR trials. In contrast, no changes were observed with the metformin treatment, either in vivo or in vitro. Two enzymes involved in gluconeogenesis, FBPase and PEPCK, demonstrated no changes for any treatment or for either in vivo or in vitro experiments. A contrary effect of the AICAR treatment was found comparing the data obtained in vivo and in vitro regarding the FAS mRNA levels. Thus, FAS mRNA expression levels were significantly up-regulated in vivo, but down-regulated in vitro. No significant differences in FAS mRNA abundance were found with the metformin treatment, although a general trend to down-regulate was observed.mRNA transcript levels for proteins involved in glucose and lipid metabolism after AICAR or metformin treatments . Although we present here the first attempt at a phylogenetic analysis of this important nutrient sensor, deeper sequencing efforts are required to unravel the complex history of AMPK subunits in fish models such as the rainbow trout, and its relationship with the emergence of AMPK functions.Our phylogenetic analysis clearly shows the near-simultaneous emergence of the AMPK subunit paralogs at the occasion of the second vertebrate-specific WGD event. The fish-specific WGD event led to the emergence of additional paralogs, retained for the β1, γ1 and γ2 subunits. We could not find evidence for any other fish-specific paralogs, be it in GenBank or through our sequencing effort in rainbow trout, which does not mean however that these sequences have been lost. Note that no fish-specific paralogs were found in the α subunit, which might be linked to the presence of the activation site at Thr172 of the α subunit. The binding of AMP to the γ subunit activates AMPK via a complex mechanism involving direct allosteric activation and phosphorylation of the α subunit on the Thr172 by upstream kinases such as the protein kinase LKB1 and the CaMKKIIβ in vivo experiments, injecting fasted trout with two mammalian activators of AMPK, AICAR 172) was found (see GenBank accession HQ403672 and 403673). The fact that these same drugs are able to activate AMPK in other animals including birds −1·day−1) the phosphorylation of this kinase was not detected 428 was evaluated in the present study, but no bands for LKB1 were found in trout liver or hepatocytes homogenates (data not shown).Our study demonstrates for the first time in rainbow trout the presence of AMPK in its total form and, more importantly, the phosphorylation at the Thrin vivo study, AICAR after a 16 h incubation induced AMPK phosphorylation. However, no AMPK phosphorylation was found in the metformin-stimulated hepatocytes in clear contrast with the ip in vivo results. Since the antibody used in the present study is not specific for trout, it is possible that AMPK phosphorylation was below the detection level of the particular antibody used. However, we can also hypothesize that the dose used for the stimulation was too low since studies with mammalian hepatocytes show maximum AMPK phosphorylation was achieved at 2 mM metformin Further support for the induction of AMPK phosphorylation in trout liver was found using hepatocytes primary cultures stimulated with either AICAR or metformin. Consistent with the Finally, we assessed the phosphorylation status of liver AMPK in trout subjected to a fasting-re-feeding trial. After 10 days of food-deprivation trout were re-fed and 1 h later, the AMPK phosphorylation status was significantly decreased, while 24 h after this first meal the levels trend to recover to those of the fasted fish. Although we are unaware whether a 10-day fast is adequate to cause energy stress in this species which is able to fast for weeks in vivo trials induced a significant increase in phospho-ACC protein levels while a slight increase was noted in phospho-ACC in trout hepatocytes, suggesting some degree of AMPK activation, possibly not detected in our present conditions as discussed above. The in vivo effect of metformin is not only consistent with the increased phosphorylation status of the AMPK, but also with the ability of this drug to repress lipogenesis in the liver of mammalian species in vivo induction of ACC phosphorylation was found in the present study, although as noted the AICAR trial showed considerable variability. However, the results obtained in AICAR-stimulated hepatocytes strongly support an increased ACC phosphorylation that resulted from an AICAR-dependent AMPK activation. As stated above, the ACC phosphorylation is consistent with a decreased ACC activity and also with the down-regulated mRNA levels of FAS observed in our previous in vivo studies in vivo after AICAR injection is surprising and could be also related with the pharmacological AICAR dose used in the present study. Other metabolic markers addressed here have not previously been explored in fish before with AMPK activation, and constitute important data concerning potential metabolic AMPK functions in fish.One of the main downstream targets of mammalian AMPK is the lipogenic enzyme ACC. ACC-serine79 is directly phosphorylated by this kinase to inhibit its activity in vivo and in vitro are internally consistent, re-enforcing the idea that the AICAR-induced AMPK activation is the main mediator of such a regulation. Moreover, our results are in agreement with the main function of AMPK in mammals where it acts as a master regulator of energy production, repressing anabolic and stimulating catabolic processes AICAR effects on metabolic-related genes in liver and hepatocytes of trout other than FAS discussed above can be summarized as the down-regulation of GK and G6Pase mRNA expression levels, while the gluconeogenic genes assessed (PEPCK and FBPase) were unaffected. The results obtained both Although these data are consistent with the results obtained for glycogen levels in AICAR-injected trout, they are in clear disagreement with those reported in mammals where AMPK α activation leads to the inhibition of glycogen synthase 172, and changes in phosphorylation pattern under physiological and pharmacological conditions. Moreover, the phosphorylation of this kinase by two recognized AMPK activators (AICAR and metformin) does induce changes in the main downstream target, the phosphorylation of ACC, as well as changes in glycogen levels and in several molecular actors involved in hepatic glucose and lipid metabolism. Further confirmation of our results can be found in the effects exerted mainly by AICAR in trout hepatocytes and the decreased AMPK phosphorylation in re-fed trout. The fact that many of our in vivo and in vitro results are consistent with the recognized role of AMPK as a master regulator of energy homeostasis in organisms We clearly demonstrate in this paper the presence of AMPK in rainbow trout. In particular we present evidence regarding the retention of paralogs that emerged during the fish-specific whole-genome duplication event for the β and γ subunits, while the α subunit does not seem to show any evidence of such a duplication event, presumably because of the loss of the corresponding paralog. This loss might be in relation to the functional role of the α subunit, a possibility that should be investigated by extended sequencing studies. We also show for the first time evidence for AMPK activation through the phosphorylation of the AMPK α at Thr