to use or not to use metformin in cerebral ischemia: a ...phosphorylation has been described in...

Review ArticleTo Use or Not to Use Metformin in Cerebral IschemiaA Review of the Application of Metformin in Stroke Rodents

Isaac Arbelaacuteez-Quintero andMauricio Palacios

Centro de Estudios Cerebrales Facultad de Salud Universidad del Valle Cali Colombia

Correspondence should be addressed to Isaac Arbelaez-Quintero isaacarbelaezcorreounivalleeducoand Mauricio Palacios mauriciopalacioscorreounivalleeduco

Received 7 August 2016 Revised 22 October 2016 Accepted 26 October 2016 Published 28 May 2017

Academic Editor Xiang-Ping Chu

Copyright copy 2017 Isaac Arbelaez-Quintero and Mauricio Palacios This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Ischemic strokes are major causes of death and disability Searching for potential therapeutic strategies to prevent and treat strokeis necessary given the increase in overall life expectancy Epidemiological reports indicate that metformin is an oral antidiabeticmedication that can reduce the incidence of ischemic events in patients with diabetes mellitus Its mechanism of action has notbeen elucidated but metformin pleiotropic effects involve actions in addition to glycemic control AMPK activation has beendescribed as one of the pharmacological mechanisms that explain the action of metformin and that lead to neuroprotective effectsMost experiments done in the cerebral ischemia model via middle cerebral artery occlusion in rodents (MCAO) had positiveresults favoring metforminrsquos neuroprotective role and involve several cellular pathways like oxidative stress endothelial nitric oxidesynthase activation activation of angiogenesis and neurogenesis autophagia and apoptosis We will review the pharmacologicalproperties of metformin and its possible mechanisms that lead to neuroprotection in cerebral ischemia

1 Introduction

Metformin is an antidiabetic drug from the dimethyl-biguanide family It was synthesized in 1920 It originates fromthe EuropeanGalega officinalis plant which has been used fordifferent pathologies for approximately 400 years This plantis rich in guanidine compounds that produce hypoglycemiceffects in animals In the early twentieth century researchshowed that guanidines produce hypoglycemic effects inanimals but human use was avoided due its toxic effects [1]

Thediscovery of insulin and its subsequent extraction andpurification from bovine pancreas decreased the interest inoral antidiabetic drugs until the mid-twentieth century JeanSterne a French physician was the first one that described thehypoglycemic effects of metformin after using it in diabetespatients [2ndash4] Metformin was associated with lactic acidosisbecause it belongs to the same family that is phenforminwhich prevented FDA approval of metformin as an oralhypoglycemic agent until 1995 [5] Currently metformin isone of themost produced oral antidiabetic drugs in the worldand is considered the first-line treatment option managing

type 2 diabetes mellitus Recently additional therapeuticactions have been described in other pathologies such aspolycystic ovary syndrome obesity and hepatic steatosis andpotential uses in breast cancer [4 6 7]

A prospective follow-up of diabetic patients through theUKPDS (United Kingdom Prospective Study) indicated adecreased risk of death from all causes including heartattack and diabetes-related deaths in patients treated withmetformin deaths comparedwith patients treated using otherhypoglycemic agents An associated decrease in stroke riskwas reported but the decrease was not statistically significant[8] These observations are consistent with other reports thatdescribe similar results In 2003 Scarpello described positiveeffects wherein independent of glycemic control mortalitydue to cardiovascular events and diabetes was reduced inpatients takingmetformin [9] Roussel andTravert followed up19691 diabetic patients for one year These authors observedlower mortality in a group of patients taking metformin [10]

Cheng et al conducted a prospective study inThailand for4 years based on its electronicmedical recordsThey followedup 14856 patients and estimated a nearly 50 reduction in

HindawiStroke Research and TreatmentVolume 2017 Article ID 9756429 13 pageshttpsdoiorg10115520179756429

2 Stroke Research and Treatment

stroke episodes (adjusted hazard ratio 046 95 CI 0424ndash0518 119901 lt 0001) in patients with diabetes treated with met-formin versus patients who did not use metformin [11] Theclinical benefit of metformin in diabetic patients for strokeprevention was described in a retrospective analysis byMimaet al who reported a reduction in the severity of neurologicalsymptoms [12]

Opposing results have also been reported for otherpathologies of nervous system In a case-control studypatients in the group treated with metformin alone versusother oral antidiabetic agents exhibited an increased riskof Alzheimerrsquos disease (adjusted OR 171 95 CI 112ndash260versus other oral agents such as sulphonylurea adjusted OR101 95 CI 072ndash142) [13]

These epidemiological observations have increased inter-est in elucidating the mechanisms of action of metformin toexplain these positive effects In neuroscience the search for aneuroprotective agent has been intense with several promis-ing cellular models although unsuccessful in its applicabilityin humans partly due to the great distance separating thesebiological experiments with the human brain Metformin isone of these molecules with neuroprotective potential thathas been studied in various experimental models such asneuronal cultures andmodels of cerebral ischemia in rodentsThese have allowed observation of their cellular effects atthe neuronal glial and endothelial levels as well as cellularpathways such as apoptosis neurogenesis angiogenesis andinflammationThis review focuses on the effect of metforminin brain ischemia in rodents and themolecular targets identi-fied as part of the processes that explain the epidemiologicalphenomena discussed

2 Pharmacological Aspects of Metformin

Metformin is a small amphoteric hydrophilic molecule withlow lipid solubility and 124 pKa which favors the acid (pro-tonated) state in the intestine Metformin requires an organiccationic transporter (OCT12) to enter the hepatocytes [14]In humans intestinal absorption yields a final bioavailabil-ity of approximately 50 An inverse relationship betweenabsorption and dose has been observed which suggests asaturable process [15] Metformin is not metabolized in thekidney from which it is excreted without change and witha half-life of 4-5 hours Compared with other biguanidessuch as phenformin metformin is less potent and does notproduce its pharmacological effects in humans until it reaches2700mgday This low potency may explain its superiorsafety profile compared with other biguanides especially itsrelationship with lactic acidosis [16]The cationic transporterOCT2 is expressed in the kidney endothelium which isinvolved in metformin elimination This transporter hasalso been identified in neurons [17] and involves passage ofmetformin from systemic circulation to the neuronal interiorToxicity was observed in rats at doses of metformin greaterthan 900mgkgday delivered by gavage [18]

Metformin must cross the blood-brain barrier (BBB) toproduce its neuroprotective actions For this issue observa-tions in obese patients with and without diabetes treated with

metformin show a decreased in food intake [14 19] Lv et alassumed that these effects could be mediated by orexigenicandor anorectic peptide expression or inhibition Thereforethe authors evaluated metformin concentrations in cere-brospinal fluid (CSF) and in the hypothalamic region of obesediabetic male rats and observed concentrations at approx-imately four percent (4) in the CSF compared with theplasma concentration [20] Łabuzek et al also demonstratedthe presence of metformin in brains of Wistar rats treatedwith acute and chronic doses of 150mgkg metformin in amodel of systemic inflammation induced by adding lowdosesof lipopolysaccharide (LPS) at 2120583gkgday Groups treatedwith metformin (acute and chronic) without LPS showed thehighest concentration of metformin in the pineal gland andcerebellum and low concentrations in the striatum In theLPS-treated groups a higher metformin concentration wasobserved in the hypothalamus and striatum suggesting thatmild inflammatory conditions which have been describedfor diabetes and atherosclerotic disease facilitate metformintransport into the brain [15]

3 Metformin Action Mechanism

Althoughmetformin has been approved in Europe since 1957for treating type 2 diabetesmellitus and several cellular path-ways studies showed changes in the presence of metforminthe exactmetformin actionmechanism is unknown (Table 2)Its hypoglycemic action is due to reduced gluconeogenesisincreased insulin receptor sensitivity especially in musclecells and decreased glucose uptake in the intestine [4 20]

31 The Effects on AMPK Zhou et al (2001) determined thatAMP-activated protein kinase (AMPK) is a cellular target ofmetformin by observing that AMPK activation was requiredfor the metformin inhibitory effect on hepatocyte endoge-nous glucose production and for the decrease expression ofthe lipogenic gene SREBP-1 Zhou et al found that AMPKactivation could explain metforminrsquos pleiotropic effects [21]AMPK is a highly conserved serinethreonine kinase withthree domains 120572 120573 and 120574 The 120572 subunit contains thecatalytic site two isoforms have been described 1205721 and 1205722The 120574 subunit comprises the regulatory sector that preventspermanent phosphorylation of a threonine (Thr172) in the 120572subunit [22] AMPKmonitors energy to protect cellular func-tions through sensing the AMPATP and ADPATP rates ofchange AMPK is activated by increased cytoplasmic concen-trations of AMP which have been shown in nutrient depri-vation vigorous exercise and ischemia The physiologicalchanges observed duringAMPKactivation induce a catabolicstate and inhibit cellular processes that require high ATPconsumption such as protein glucose and lipid biosynthesis[23] AMPK activation by both changes in the AMPATPintracellular rate and the AMPK upstream activator LKB1leads to decreased gluconeogenic gene transcription

32 The effects on Complex I of the Mitochondria RespiratoryChain Foretz et al showed that the hypoglycemic effects of

Stroke Research and Treatment 3

metformin were maintained in AMPK and LKB1 knockoutmice which indicated that metformin produces such effectthrough an indirect mechanism [24] Previously El-Mir etal observed less mitochondria oxygen consumption in thepresence of biguanide which suggests that this organelleespecially complex I of themitochondrial respiratory chain isthe site of pharmacological action for metformin [25] Inter-ference with electron flow and a decrease in endogenous glu-cose production suggest that metformin-dependent cellularenergy depletion leads to insufficient levels of ATP for hepaticgluconeogenesis [19] Viollet et al propose that inhibitingATP production in mitochondria increases AMP levels witha consequent activation of AMPK This AMPK activationdecreases gluconeogenesis because an energy substrate forthis metabolic process is not available [14] At the neuronallevel the absence of enzymes in the glycolytic pathwaydecreases the neuronal ability to store nutrients Intense andgeneralized AMPK activation was observed during cerebralischemia in both ipsilateral and contralateral regions [23]

An increase in lactate and glycerol plasma levels hasbeen observed in animals treated with metformin but theincrease cannot be attributed to reduced activity for theenzymes involved in pyruvate metabolism such as pyruvatecarboxylase citrate synthase and alanine aminotransferaseThe increase in plasma lactate may indicate the cytosolicredox state by affecting the lactatepyruvate rate Madiraju etal showed that acute and chronic metformin actions reduceendogenous glucose production by raising the cytosolic redoxstate and decreasing the mitochondrial redox state throughnoncompetitive inhibition of mitochondrial glycerophos-phate dehydrogenase [26] As a result the balance betweenglycerophosphatedihydroxyacetone phosphate NADHNAD+ and lactatepyruvate in cytoplasm is affected whichdecreases gluconeogenesis and releases excess lactate andglycerol to the plasma (Figure 1) [27]

33 Other Possible Targets of Metformin Metformin interac-tion with metals has been described since the last centuryjust after its synthesis and even before the description of itshypoglycemic effects [28] Recently a possible mechanism ofaction associatedwith copper ion binding has been describedCopper is essential for aerobic life given its presence asa catalytic cofactor for mitochondrial enzymes responsiblefor ATP synthesis [29 30] The available crystallographicand spectroscopic evidence that demonstrates the binding ofmetal ions to metformin contrasts with the lack of informa-tion about its affinity or its regulatory actions over certainproteins [19] However it is uncertain whether the effects ofmetformin on the mitochondrial complex I are related to itsaffinity for copper atoms that make part of metalloenzymesor cofactors in the respiratory chain In relation to this Logieet al (2012) published a study on the properties of metforminrelative to metal binding particularly mitochondrial copperand the effects of metformin relative to AMPK activationdependence in the presence of metals The authors suggestthat the cellular effects of metformin depend on its metal-binding properties [28]

4 Studies on Metforminrsquos NeuroprotectiveEffects in Rodent Animal Models

Themodel of cerebral ischemia of the middle cerebral arteryin rodents (MCAO) was initially proposed by Koizumi 30years ago (with some later variations suggested by Longa etal) and consists in the introduction of amonofilament (4-0 or5-0 with a poly-L-lysine or silicone-rubber cover at the tip) bycarotid internal artery to ascend it to the origin of the middlecerebral artery and producing a localized cerebral ischemia[31ndash33] This model has been used to study different cellularpathways using metformin as described below

41 AMPK Actions Neurons are sensitive to energy deficitsthus the high levels of AMPKexpression observed in the cen-tral nervous system during ischemic events are unsurprisingHowever the role of AMPK in neurons and its protective orharmful effects on brain ischemia remain controversial [34]Harada et al observed neuroprotective effects of intraperi-toneal administration of metformin (250mgkg) through theperipheral activation of AMPK after suppressing the glucoseintolerance 24 hours after reporting a less alteration in themnemonic tests after three days of focal cerebral ischemia inmice [35] In this study Harada et al also injected intraven-tricular metformin and observed an increase in the neuronaldamage with neurological alterations which suggests thatcentral versus peripheral activation of AMPK may explaindifferences between neuroprotective and harmful metformineffects after ischemia

Kuramoto et al also described neuroprotective effectsupon AMPK activation in a cerebral ischemia rodent modelThese researchers observed enhanced activation of themetabotropic receptor GABAB and its inhibitory actionsthrough coupling to a postsynaptic G protein receptor(GiGo) with neuronal hyperpolarization which reducedsecondary excitotoxicity during cerebral ischemia [36] (Fig-ure 1) McCullough et al described opposing results in 2005upon observing neuroprotective effects using an inhibitor ofAMPK referred to as compound C and knockout mice forAMPK1205721 and AMPK1205722 [37] In this study McCullough et alwere the first to report an association between the effects ofactivated AMPK and neuronal nitric oxide synthase (nNOS)in a cerebral ischemia mouse model (see below) Duringischemic events AMPK activation in neurons producesdeleterious effects that are likely secondary to its reduced gly-colytic capacity [19]Therefore prolonged production of ATPmolecules through anaerobic glycolysis by astrocytes can leadto acidosis and inhibit lactate use by neurons as an energysubstrate Li et al weremore specific in describing the activityof each isoform AMPK1205721 and AMPK1205722 to demonstrate thatinhibition or acute inactivation of the 1205722 isoform is responsi-ble for the neuroprotective effects in cerebral ischemiamousemodels [38]

Later Li et al (2010) tested the neuroprotective actionsof metformin using a mouse model of cerebral ischemia for90minutes (ischemiareperfusion) and reported AMPK acti-vation with acute metformin treatment and downregulationwith chronicmetformin treatmentThe authors also reportedan increase in infarct size in the groups that received acute


R1

Metformin

R2

G3PDHAP

mGDP

NADH ATP

Gluconeogenesis

FBPasaACC

Lipogenesis

aPKC-CBP

Neurogenesis

NOS

ON

mTOR

Growth andproliferation

Glut4

Glucose capture

Mitochondria

Nucleus

C

p-AMPKactivated

C

N N

Complex I

+

＋+

TNF-

LKB1CaMKK

darr

darr Gene expression

NF-

uarr AMP

GABAb４12

Figure 1 Different cellular pathways described for metformin function Gluconeogenesis is inhibited due to changes in gene expression(such as for lipogenesis) FBPase inhibition and changes in the ATPAMPG3PDHAP and lactatepyruvate ratios AMPKmay exert differentactions in neurons because they donot have glycolytic enzymes and thus a reduced ability to store nutrients and respond to energy deficit witha higher risk of lactic acidosis The GABAB receptor is involved in the effects of metformin through inducing hyperpolarization secondary toK+ release and inhibition of Ca2+ entry secondary to AMPK activation OCT organic cation (proton) transporter G3P glycerol-3-phosphatemGDP mitochondrial glycerophosphate dehydrogenase DHAP dihydroxyacetone phosphate eNOS endothelial nitric oxide synthase ONnitric oxide aPKC atypical protein kinase C CBP CREB binding protein NF-KB nuclear factor kappa beta TNF-120572 tumor necrosis factor-alpha FBPasa fructose 16 bisphosphatase ACC acetyl CoA carboxylase CaMKK120573 protein calmodulin-dependent kinase beta mTORtarget of rapamycin in mammalian cells

doses ofmetformin (24 and 72 hours before the stroke) versusa decrease in infarct area with treatment for 3 weeks prior toischemia [39] No differences were observed in the cerebralinfarction volume with the acute metformin treatment inAMPK1205722 knockout mice versus the control group Theincrease in infarct size from the acute ischemic events wasnot observed in neuronal nitric oxide synthase- (nNOS-)knockout mice This effect led the authors to suppose thatthe neuroprotective effects were mediated by nitric oxide(see below) However a metformin treatment for three weeksbefore ischemia decreased AMPK activation and the infarctsize [39] Based on these results and the assumption that lessAMPK activation may yield neuroprotective effects Mccul-lough and Benashski investigated ischemic preconditioning

with 3minutes of ischemia 72 hours before definitiveMCAOwhich showed neuroprotective effects in mice secondary tothe AMPK inhibition and increased HSP70 expression [40]

42 Nitric Oxide Production and Oxidative Stress After focalischemia events that cause damage or cell death involve aquick fall of adenosine triphosphate concentration (ATP)oxidative stress changes in membrane potential with axonaldepolarization and massive entrance of sodium chlorideand calcium ions and exchange of potassium ions [41] Theintracellular increase in calcium concentration promotes theproduction of nitric oxide through the activation of constitu-tive synthases (endothelial and neuronal) which contributesto the initial damage The generation of reactive oxygen


species (ROS) became worse during reperfusion throughthe production of synthases involved in these processes[42] Cerebral ischemia induces changes in the nitric oxideconcentration in the brain 20 times the basal value aftermiddle cerebral artery occlusion for 30 minutes [43] Duringischemia NO concentration decreases by the reduction ofoxygen availability so the constitutive endothelial isoformseNOS and nNOS synthetize low levels of NO [44] HowevermRNA levels that encode nNOS increase in the neurons ofthe core and the penumbra zone after the cerebral ischemia[45 46] In the first hours after ischemia high levels of NOare observed mainly during reperfusion by overactivation ofnNOS [47] The inducible isoform of the nitric oxide syn-thase (iNOS) nondependent on calcium concentration is notexpressed in nervous cells but its activity increases mainlyin glial cells 12 hours after ischemia and maintains highconcentrations ofON in the brain at least for 8 days especiallyin ischemic region [42 48]The transformation of nitric oxideinto superoxides during reperfusion could be the cause of theadditional damage to the nervous tissue so the inhibition ofthe inducible nitric oxide (iNOS) may have neuroprotectiveeffects by decreasing the nitrative stress [44 49]

Therefore the role of NO in the ischemic event may bedual due to its neuroprotective or neurotoxic effects [42 4345 48 50] NO synthetized at low concentrations by eNOShas local vasodilatory effects with good results in experimentsthat induce cellular damage after controlled ischemia [51]These vasodilatory properties also have been described fornNOS when placed in the aortic endothelium [52] Inendothelial cells a relation between cellular exposure to met-formin and the activation of AMPK with secondary eNOSphosphorylation has been described in cardiac ischemicmodels and in muscle cells [53] Although this results cannotbe extrapolated to brain tissue the supply of L-arginine (NOprecursor) and the specific inhibitors of nNOS and iNOS inthe murine cerebral ischemia model produce a reduction inthe size of the infarct In the case of eNOS knockout micefor eNOS showedwide infarct areas [50] During reperfusionNO concentrations increased at the beginning due to theactivation of nNOS and iNOS damaging neurons at thepenumbra zone [42 47 54] The effects of NO produced bythe early activation of eNOS could be neuroprotective duringthe first 2 hours by promoting the collateral circulationinhibiting platelet aggregation and decreasing the leukocyteadhesion and by antiapoptotic effects [45 48] Hence earlyeNOS activation could be a therapeutic target given biologicalchance of its neuroprotective effects An initial approachwith cellular studies in the endothelium showed at this levelmetformin granted the activation of eNOS and the bioactivityof NO secondary to the activation of AMPK in cell cultures ofaorticmice endothelial cells [55] At the neuron level in post-stroke mice it has been noticed that the activation of AMPKdepends on the production of NO by nNOS [39] As wemen-tioned before in rats subjected to ischemia with reperfusionneuroprotective effects of metformin were observed duringthe chronic treatment before ischemia These effects werenot observed in knockout rats for nNOS [39] These findingssuggest that the selective inhibition of iNOS and activation ofnNOS and eNOS may have neuroprotective effects

43 Oxidative Stress The excess NO production after cere-bral ischemia and its deleterious effects on the activationof NOS and iNOS as well as the continued neuronal deathseveral hours and days after the stroke suggest thatmoleculeswith antioxidant propertiesmay have a neuroprotective effectThus the nitrative stress described in the ischemiareper-fusion murine models suggests nitration increases of the p58regulatory subunit of AKT (protein kinase B) favoring theactivation of the apoptotic p38MAPK kinase route By theapplication of metformin Correia and Carvalho observeda functional recovery of cerebral vessels in Goto-Kakizakidiabetic rats after focal cerebral ischemia by a reduction ofnitrotyrosine in the endothelial cells of the microvasculatureand an increase of AKT phosphorylation suggesting addi-tional antioxidant effect [56] Correia and Carvalho also eval-uated the antioxidant action of metformin in Goto-Kakizakirats by measuring levels of protein oxidation using perox-ynitrite glutathione vitamin E glutathione peroxidase andsuperoxide dismutase enzymes After comparing treatmentgroups they reported a protective effect against oxidativestress through lower levels of lipid peroxidation markers aswell as glutathione peroxidase and glutathione reductase activ-ity [56]

Moustaf and Mohamed observed similar results asreported by Correia using an ischemiareperfusion Wistarrat model by occluding both common carotid arteries for30 minutes and then reperfusing for 60 minutes The ratstreated with metformin administered via a gavage tube(500mgkgday) exhibited less oxidative stress with lowersuperoxide dismutase glutathione peroxidase and catalaseactivities than the sham operated group [57] These resultsshow that metformin may impact oxidative stress inducedby cerebral ischemia and may impact antioxidant propertiesuseful in controlling the side effects of ROS

44 The Blood-Brain Barrier and Vascular Actions of Met-formin The blood-brain barrier (BBB) constituted byendothelial cells perivascular cells (pericytes) and astrocytesfeed endings performed a complex interphase between bloodcirculation and the brain parenchyma which keep thehomeostasis of the central nervous system through theregulation of the ionic exchange neurotransmitters macro-molecules and nutrients [58 59] The breakdown of BBBduring focal cerebral ischemia is associated with cerebraledema which favors the hemorrhagic transformation ofictus [60] Given the diverse functions of the BBB and thefew drugs with therapeutic actions at this level Takata etal observed metformin action dependent of the activationof AMPK in BBB functions by increasing transendothelialelectrical resistance and decreasing sodium permeability invitro using a culture of endothelial cells obtained from ratbrains suggesting a strength of the endothelial cells tightjunctions [61] Farbood et al evaluated the actions of met-formin in the BBB in a global transitory ischemic model for30 minutes through the extravasation of Evans blue and thereactive hyperemia secondary to the ischemiaThey observedthat metformin decreases the BBB rupture and the neuronalexcitability increases in the hippocampus [62] Metforminactions and their relationship with AMPK activation


at the blood-brain barrier have been studied Liu et aldescribed the protective effects of metformin (200mgkgintraperitoneally) through altering the blood-brain barriersubsequent to MCAO for 90 minutes in mice by decreasingits permeability

Additionally the authors of this study evaluated theanti-inflammatory effects of cerebral ischemia followed bymetformin which is important considering the secondaryinflammation that hypoxia triggers and neuronal death bynecrosis in the affected brain parenchyma (or ischemiccore) Previous studies show that metformin induces cellularchange from an inflammatory to an anti-inflammatory phe-notype Consistentwith these data researchers have observeda decrease in expression of proinflammatory cytokines (IL-1120573 IL-6 and TNF-120572) and neutrophil infiltration subsequentto cerebral ischemia (the third day) through inhibitingexpression of intercellular adhesion molecule (ICAM-1) sec-ondary to AMPK activation [63] Interestingly unalteredoccludin structure in the tight junctions area has beenobserved after metformin application as in the zonula occlu-dens in the duodenal endothelium secondary to hepaticsteatosis due to an elevated fructose diet [64]

These results show a contradiction in the neuroprotectiveeffects ofmetformin since at the level of BBB theAMPKacti-vation favors the tight junctions and reduces the efflux of fluidto the interstitial space but at the neuronal level the acuteAMPK activation during stroke has shown negative effects inthese cells It is possible that not only temporary factors likechronic versus acute treatment but also the activation of dif-ferent cellular pathways for instance the tyrosine-dependentphosphorylation the age of the biomodels or even the pres-ence of diabetes can be the cause of these paradoxical results

In diabetic rats neovascularization of the brain vesselswith a major number of collaterals and an increase in thevascular tortuosity especially at the cerebral cortex havebeen observed However this increase in angiogenesis is notrelated to the proper maturation of the blood vessels wallbecause a decrease in the number of pericytes is observedaround the endothelial cells and also an increased amountof nonperfused vessels [65] Prakash et al assessed the vas-cularization pattern in both hemispheres using diabetic ratsundergoing cerebral ischemia for 90minutesThey comparedthe effects of metformin administered orally 24 hours afterischemia at 300mgkgday They reported a decrease in thereparative neovascularization pattern in diabetic rats exposedtometformin after ischemia with a better functional outcomein the group with glycemic control [66] The reduction of theosmotic changes secondary to the ischemic events whethercaused by damage to the BBB or by neuronal death cancause distant effects due to the secondary edema in somecases Control of edema and BBB damage may explain thepositive results after metformin use in reducing the size ofthe infarct and on the improved motor performance duringthe postischemia period in murine stroke models

45 Vascular Actions The protective actions by metforminat the vascular level were assessed by Elgebaly et al in acerebral ischemia diabetic ratmodelThismodel was exposedto minocycline and metformin for 4 weeks to determine

their effects on vascular remodeling under such conditionsthe role of matrix metalloproteinases (MMP) 2 and 9 inthese vascular changes and the risk of hemorrhagic trans-formation after MCAO ischemia The authors observed anincrease in the vascular remodelingmarkers indicating vesseltortuosity number of medial (MCA) collaterals numberof anastomoses diameter of the collateral arteries to theMCA and activity of MMP-9 in diabetic rats The rate ofvascular remodeling was significantly reduced in the groupsexposed to metformin which also showed an associationwith reduced MMP-9 enzymatic activity a smaller infarctionarea compared with controls and a vasoprotective effectthrough preventing subsequent hemorrhagic transformation[67] This ldquovascular antiremodelingrdquo effect could explainthe significant reduction of ECV events in diabetic patientstreatedwithmetformin as seen in the diabetic patient follow-up study by Cheng et al [11]

46 Neuronal Apoptosis Secondary to Cerebral Ischemia Oneof the most interesting aspects of metformin is its antipro-liferative action by inhibiting the mTOR complex in certaincancers But the mechanism by which this effect may explainits neuroprotective action is contradictory [6 14] sincein terms of neuroprotection avoiding neuronal death byapoptosis inhibition may be beneficial El-Mir et al reportedneuroprotective effects from metformin through preventingcell death in cortical primary neurons by inhibiting poreopening and mitochondrial permeability in the presence ofneuronal death inducer (etoposide) [25]Theneuroprotectiveactions of metformin alone (or in combination with thymo-quinone) were also described for a primary culture of corticalneurons exposed to ethanol (100mM) The actions includeless neuronal death inhibition of impaired cytoplasmiccalcium homeostasis inhibition of an altered mitochondrialgradient increased Bcl-2 (antiapoptotic) expression anddecreased Bax (proapoptotic) expression which reduced thecytochrome c release and repressed caspase-9 [68] Ashabiet al used a global cerebral ischemia rat model (with 4cerebral vessels occluded) and observed that precondition-ing with metformin at 200mgkgday by gavage for twoweeks attenuated apoptosis of neurons located at CA1 in thehippocampus through measuring the effects on BaxBcl-2caspase-3 and PARP levels Further metformin use favoredmitochondrial biogenesis of certain proteins (PGC-1120572 pro-liferator activator receptor gamma coactivator-1120572) Theseeffects were eliminated with compound C (selective andreversible AMPK inhibitor) The metformin doses were 100200 and 400mgkgday orally better neuroprotective resultswere observed for the 200mg dose which based on theabove-described pharmacokinetic properties is the concen-tration that should exhibit better bioavailability (Table 1) [69]

47 Neurogenesis and Angiogenesis after Cerebral IschemiaNeurogenesis is a characteristic of certain neuronal typesthat remain with mitotic activity throughout their life spanThe generation of new neurons after an ischemic event mayhave neuroprotective effects as long as order and stabilityare maintained to provide integration of new neuronal tissueand its functionality Jin et al found that through the


Table 1 Different cellular pathways studied on cultured neurons using metformin in vitroin vivo model

Biomodel Culture Effects Postulated mechanism Doses Ref

Rat Culture of primaryneurons of day 17 Antiapoptotic Pore opening inhibits mitochondrial permeability

transition 100 120583g for 24 hours [25]

Mice Immunostaininghippocampal sections Neurogenesis

Metformin activates the aPKC-CBP pathway inneurons and induces neurogenesis of humanneurons in culture In adult mice it induces

neurogenesis in the hippocampus and olfactorybulb Spatial learning tests improved in mice

Daily injectedmetformin (200mgkg)and BrdU for 3 days andthen only metformin for

9 days

[70]

Rat

Culture of corticalneurons from

embryos obtained ongestation day 175

Antiapoptotic

Decreases neuronal death inhibits apoptosisactivation maintains the mitochondrial gradient

inhibits cytochrome c release and regulatesinternal calcium homeostasis

Culture at a highconcentration of ethanoland groups receivedmetformin (100mM)andor thymoquinone

[68]

RatPrimary cultures ofrat brain endothelial

cells

Maintainedintegrity of

BBB

Decreases sodium pass and maintainstransmembrane electric gradient

Metformin dissolved1mM [61]

aPKC-CBP pathway metformin promotes neurogenesis inthe hippocampus of adult mice with improved performancein spatial memory testing [77]

The production of new blood vessels is a phenomenonthat has been observed in studies with metformin as wellA rapid angiogenesis could be a valuable therapeutic toolbut angiogenic therapies must have a time frame or windowthat allows the development of new blood vessels prior tothe stroke In accordance with this Venna et al observedneurological improvement and revascularization of ischemictissue in mice after the administration of intraperitonealmetformin during 3 weeks prior to the stroke [72] Theseangiogenic effects were dependent on AMPK1205722 During theacute administration of metformin angiogenesis can haveneuroprotective effects after the stroke Liu et al observedthat after 14 days of metformin administration to ischemicmice angiogenesis increased in the subventricular areaminimizing cerebral injury [74] These effects were mediatedby the activation of AMPK

48 Inflammatory Pathway Actions Metformin actions bysuppressing the activation of inflammatory routes has beenstudied in different cell types like hepatocytes adipocytesand neurons [78 80]

As mentioned previously inflammation control sec-ondary to ischemic events is a therapeutic target that hasbeen studied for its potential neuroprotective effect In 2003Jung et al used a AMP analogue AMPK-activator referredto as AICAR and observed proapoptotic effects in neurob-lastoma cells associated with activation of this pathway [81]Thereafter several cellular targets have been described forAICAR which may explain these contradictory results withthe neuroprotective actions of metformin [38] Moiseeva etal previously described metformin actions in inhibiting asenescent phenotype through inhibitingNF-B120581 translocationto the nucleus and repressing transcription of inflammatorycytokines genes [82]

Zhu et al demonstrated that chronic preconditioningwith metformin provides neuroprotection in permanent

cerebral ischemia in rats by reducing the cerebral infarc-tion area The data showed improved neurological deficitthrough suppressing the inflammatory pathway mediated bynuclear factor kappa beta (NF-120581B) with reduced expressionof proinflammatory cytokines as well as astrocytosis andsecondary microgliosis during the first 24 hours of ischemia[71] Ashabi et al showed similar results when they analyzedanti-inflammatory and antioxidant actions after applicationof metformin in a global ischemic transitory model in rats (4vessels) They determined reduced levels of TNF-120572 NF-120581BCOX-2 Nrf2 catalase and hemooxigenase-1 when comparedwith control group and sham rats When an inhibitor ofAMPK was used the effect of metformin was counteracted[78]

5 Concluding Remarks

No currently available drug has a neuroprotective-approvedindication for use in humans To date primary preventionis the best option to avoid complications associated withstroke However due to its high morbidity and mortalitythe impaired quality of life of the surviving patients andtheir families and the increase in life expectancy of humansinvestigating drugs for neuroprotective effects is necessary forpatients with a high stroke risk

In recent years metformin has garnered growing interestdue to its likely neuroprotective actions and epidemiolog-ical observations on the mortality associated with its usein diabetes patients Rodent models studies indicate thatmetformin produces favorable effects for preventing strokeand aftermath recovery actions that are independent of itshypoglycemic effects Metformin effects at the vascular levelhave been described also in experiments withmyocardial andendothelial cells In all of them it is suggested that the effectof metformin is via enhancing focal eNOS expression [74]AMPK activation in hepatocytes and neurons involves anindirect mechanism relative to both its actions in glycemiccontrol and its neuroprotective effects Directly or indirectlyAMPK activation in the presence of metformin is associated


Table2

Differentinvivo

studiesusingm

etform

ininam

odeloffocalorg

lobalischemiawith

orwith

outreperfusio

nMostofthe

studiesshow

neurop

rotectivee

ffectsPO

oralI

Pperiton

eal

injection

ICV

intraventricularand

IPC

ischemicprecon

ditio

ning

Biom

odel

Strain

Ischem

icfocal

orglob

alTimeo

fisc

hemicreperfusio

nNeuroprotectio

nPo

stulated

mechanism

Doses

androuteo

fadministratio

nMetform

intre

atmenttim

eRe

f

Rat

Wistar

Global

30min1h

Yes

Redu

ctionsuperoxide

dism

utase

activ

ityandglutathion

eperoxidase

500m

gkg

POOne

weekbefore

stroke

[57]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Supp

ressionof

theN

F-120581B

inflammatorypathway

50mgkg

IP3weeks

before

stroke

[71]

Mice

C57B

L6N

Focal

60min72h

Yes

Ang

iogenesis

andim

provec

erebral

dopaminergicton

e

02m

Lkg

IPo

50mgkgday

IP

50mgkgday

beginn

ing

24haft

erstr

okefor

3weeks

[72]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Indu

cesa

utop

hagy

10mgkgIP

Sing

ledo

se24

hbefore

stroke

[73]

Mice

CD-1

Focal

90min14

days

Yes

Prom

otes

neurogenesisand

angiogenesisin

subventricular

zone

200m

gkg

IPFo

r14days

after

stroke

[74]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Redu

celevelsof

nitro

tyrosin

e300m

gkgday

POFo

r14days

after

stroke

[75]

Mice

C57B

L6N

Focal

3min

(IPC

)at72

hours9

0min24h

No

AMPK

activ

ationantagonizes

neurop

rotectivee

ffectof

ischemic

precon

ditio

ning

100m

gkg

IPSing

ledo

sesa

fterstro

ke[76]

Mice

CD-1

Focal

60min14

days

Yes

Favorschange

inph

enotypeM

2microgliaastr

ocyte

50mgkgday

IPBe

ginn

ing24

haft

erstroke

[77]

Mice

C57B

L6

Focal

90min24y

72ho

urs

Noacute

treatment

Yeschronic

treatment

Inchronictreatmentlessactiv

ation

ofAMPK

Acute50

a100m

gkgdıaIP

Chronic50

a100m

gkday

Acute24

hbefore

Chronic3weeks

before

[39]

Rat

Wistar

Global

30min72h

Yes

AMPK

activ

ationinhibitsapop

tosis

inhipp

ocam

paln

eurons

200m

gkgday

PO2weeks

before

ischemia

[69]

Rat

Goto-Ka

kizaki

Focal

90min21h

Yes

Redu

cedvascular

remod

elingand

severityof

hemorrhagic

transfo

rmationin

diabetes

300m

gkgday

POStartin

gwith

theo

nsetof

diabetes

[67]

Rat

Wistar

Global

30min72h

Yes

Attenu

ates

cellu

larlevels

ofNF-kB

andincreasedlevelsof

Nrf2

inhipp

ocam

pus

200m

gkgday

PO2weeks

before

ischemia

[78]

Rat

Wistar

Global

30min72h

Yes

Decreases

reactiv

ehyperem

iaand

perm

eabilityo

fBBB

inisc

hemicrats

200m

gkgday

PO2weeks

before

ischemia

[62]

Mice

ddY

Focal

60min72h

Noacuteinjectio

nintraventricular

YeschronicIP

Centralversus

perip

heralactivation

ofAMPK

250m

gkgIP

three

times

or2510

0120583gICVthree

times

ford

ay

Afte

rstro

keun

tildeath

[35]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Improved

vascularizationin

diabeticgrou

pachievee

uglycemia

300m

gkgday

POAfte

rstro

keun

tildeath

[66]

Mice

C57B

L6

Focal

60min24h

Yes

Inseven-daypretreatmentless

activ

ationof

AMPK

10mgkgday

IPAfte

rstro

keun

tildeath

[79]


with neuroprotective effects that involve the cellular pathwaysrelated to autophagia microglial activation a decrease in themovement of sodium through the BBB angiogenesis andneurogenesis

In humans this activation may produce endothelialprotective effects that explain the lower cardiovascular riskobserved in diabetic patients that use metformin [83] andsome of these effects depend on the nitric oxide productionby its endothelial isoform (eNOS)

Acute AMPK activation at the neuronal level producesharmful effects with an increased cerebral infarct area com-pared with chronic treatments for at least 2 to 3 weeksbefore ischemia in experimental biomodels [39] Chronictreatment with metformin reduces AMPK activation andmay produce similar effects as in studies on preconditioningbefore ischemia However activation of AMPK after cerebralischemia for several days had demonstrated neuroprotectiveeffects The literature remains vague on the timing of begin-ning metformin for the acute condition in the early hours ofan ischemic eventmetformin canproduce deleterious effectsbut long-term postinfarction administration may improvefunctional recovery in rodents [72 77]

The different results for the cellular pathways studied incerebral ischemia indicate that thismolecule produces intrin-sic neuroprotective factors because it affects the expression ofneuroprotective genes such as vascular endothelial growthfactor (VEGF) [72] affects oxidative stress by activatingantioxidant enzymes such as glutathione peroxidase [5784] induces endogenous transcription factors through theaPKC-CBPpathway [70] favors theM2microglial phenotypeassociated with tissue repair [77] inhibits cytochrome crelease from the mitochondria and induces neurogenesis inthe hippocampus and subventricular zone [25 72]

Biomodels tests in rodents are recommended to investi-gate potential drug candidates with potential neuroprotectiveeffects because a standardized protocol is available whichfacilitates correlationswith brain cuts in specific areas Unfor-tunately many molecules have not proven equally effectivewhen attempting to extrapolate the results in humans [85]One reason could be the unidentified bias of using ischemiaanimal models without a clinical correspondence to humanssuch as age race weight sex use of anesthetics for MCAOsuch as ketamine and the time of ischemia Comparing theresults of neuronal cell culture versus in vivo results therelationship between different types of cells in the nervoussystem such as glia and neurons cannot be simulated andthe drug concentrations used for irrigating a cell culture aremuch higher compared with bioavailability in a biomodelMany studies focus on neuronal cell bodies in the cerebralcortex the striatum andor hippocampus without consider-ing axonal injury (white matter) which may also producea major clinical impact This consideration is relevant inhumans since the involvement of the internal capsule is veryfrequent and affects mainly the axons that go through

The neuroprotective effects of metformin can beexplained by its vascular actions (including its effects onthe blood-brain barrier) neuronal actions and glial actionsbased on the reported results For example its actions at the

cerebral endothelium during ischemia indicate that it atten-uates damage to blood-brain barrier through maintainingtight junctions reducing sodium permeability and reducingcerebral edema Could this infarct area reduction effect withmetformin in these biomodels be related to more than itsneuronal actions In addition other pleiotropic metforminactions may also function in cerebral ischemia such assuppressing mTOR by which AMPK controls autophagycell growth and secondary inflammation [63]

Thus it should be considered that AMPK activation mayproduce temporary (acute and chronic) effects on cellularcomponents of tissue (neurons glia and endothelium) Thisnotion implies that neurons and glia activated pathways geneexpression and cellular actions may differ especially for thelowglycolytic capacity of neurons andpermanent postmitoticstate [23] Further expression of the catalytic subunit ofAMPK (1205721 y 1205722) was described with a specific locationamong different cell types which is important because the 1205722subunit is involved in the neuroprotective effects Howevereven though there is evidence that suggests that the effectsof metformin could be mediated by its actions at a vascularlevel the 1205721 subunit is mainly expressed in the endotheliumand the 1205722 subunit is mainly in the nucleus of neurons andthe cytoplasmic region of astrocytes [86]

It seems paradoxical that the observed effects of met-formin in certain cancer types (lung prostate ovarian andmelanoma) where it induces apoptosis of malignant cellsoppose the results described at the neuronal level [68 87ndash89] which exhibit repression of this programmed cell deathprocess and activation of neuronal signaling in stem cellsMetformin activation of the FOXO3-AMPK pathway thateliminates cells which initiate gliomas by inducing differen-tiation into nontumor cells has been described in the brain[90]One explanation for these opposing effects ofmetforminin different tissues could be the postmitotic phase or cellcycle arrest in neurons whichmaintainmetabolicenzymaticmachinery that differs from and responds differently to thepresence of biochemical signals such as cyclins [91]

Metformin has pharmacological properties that shouldbe more precisely defined in biomodels like rodents todetermine the effectiveness of each clinical trial To produceits hypoglycemic effects in humans high doses of metforminranging up to approximately 2700mgday are typicallyrequired This low pharmacological potency (compared withother oral antidiabetics) may impact research results inrodents especially given the absence of a determined dose ora threshold where neuroprotective effects are observed Thedoses used in rats and mice range between 10mgkgday and500mgkgday with different administration routes (gavageand intraperitoneal) which can lead to significant changesin bioavailability of the molecule because given its lowabsorption in the intestine and the high dose required toproduce cellular effects studies wheremetforminwas admin-istered by gavage can produce different results comparedwithintraperitoneal injection Studies of these possible differencesin bioavailability based on the administration route formetformin are still necessary It is interesting however thatthe dosage used in humans of approximately 30mgKgday


when given intraperitoneally to rats 7 days before the strokedoes not have the neuroprotective effect [79]

Finally other available oral antidiabetic agents such assulfonylureas and inhibitors of dipeptidyl peptidase-4 (DPP-4) show similar actions to metformin which are outlined inthis review as candidates due to antiapoptotic neuroprotec-tive anti-inflammatory and antioxidant actions and attenu-ated microglial reactivity [92 93]

Competing Interests

The authors declare no conflict of interests with the publica-tion of this article

References

[1] C K Watanabe ldquoStudies in the metabolism changes inducedby administration of guanidine bases I Influence of injectedguanidine hydrochloride upon blood sugar contentrdquo The Jour-nal of Biological Chemistry vol 33 pp 253ndash265 1918

[2] K S Polonsky ldquoThe past 200 years in diabetesrdquo The NewEngland Journal ofMedicine vol 367 no 14 pp 1332ndash1340 2012

[3] C Bailey and C Day ldquoMetformin its botanical backgroundrdquoPractical Diabetes International vol 21 no 3 pp 115ndash117 2004

[4] A Maric ldquoMetforminmdashmore than lsquogold standardrsquo in the treat-ment of type 2 diabetes mellitusrdquoDiabetologia Croatica vol 39no 3 pp 95ndash104 2010

[5] F J Gainza I Gimeno and RMuniz ldquoAcidosis lactica asociadacon la utilizacion de metformina Papel de la hemodialisis en eltratamientordquo Nefrologia vol 18 no 5 pp 427ndash430 1998

[6] A M Gonzalez-Angulo and F Meric-Bernstam ldquoMetformina therapeutic opportunity in breast cancerrdquo Clinical CancerResearch vol 16 no 6 pp 1695ndash1700 2010

[7] M Aghahosseini A Aleyaseen L Safdarian S Moddaress-Hashemi B Mofid and L Kashani ldquoMetformin 2500mgdayin the treatment of obese women with polycystic ovary syn-drome and its effect on weight hormones and lipid profilerdquoArchives of Gynecology and Obstetrics vol 282 no 6 pp 691ndash694 2010

[8] S Genuth R Eastman R Kahn et al ldquoImplications of theUnited Kingdom Prospective Diabetes Studyrdquo Diabetes Carevol 25 no 1 pp S28ndashS32 2002

[9] J H B Scarpello ldquoImproving survival with metformin theevidence base todayrdquoDiabetes ampMetabolism vol 29 no 4 part2 pp 6S36ndash6S43 2003

[10] R Roussel and F Travert ldquoMetformin use and mortalityamong patients with diabetes and atherothrombosisrdquo Archivesof Internal Medicine vol 170 no 21 pp 1892ndash1899 2010

[11] Y-Y Cheng H-B Leu T-J Chen et al ldquoMetformin-inclusivetherapy reduces the risk of stroke in patients with diabetes a4-year follow-up studyrdquo Journal of Stroke and CerebrovascularDiseases vol 23 no 2 pp e99ndashe105 2014

[12] Y Mima T Kuwashiro M Yasaka et al ldquoImpact of metforminon the severity and outcomes of acute ischemic stroke inpatients with type 2 diabetes mellitusrdquo Journal of Stroke andCerebrovascular Diseases vol 25 no 2 pp 436ndash446 2016

[13] Y Chen K Zhou R Wang et al ldquoAntidiabetic drug metformin(GlucophageR) increases biogenesis of Alzheimerrsquos amyloidpeptides via up-regulating BACE1 transcriptionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 10 pp 3907ndash3912 2009

[14] B Viollet B Guigas N S Garcia J Leclerc M Foretz and FAndreelli ldquoCellular and molecular mechanisms of metforminan overviewrdquo Clinical Science vol 122 no 6 pp 253ndash270 2012

[15] K Łabuzek D Suchy B Gabryel A Bielecka S Liber and BOkopien ldquoQuantification of metformin by the HPLC methodin brain regions cerebrospinal fluid and plasma of rats treatedwith lipopolysacchariderdquo Pharmacological Reports vol 62 no5 pp 956ndash965 2010

[16] P Imfeld M Bodmer S S Jick and C R Meier ldquoMetforminother antidiabetic drugs and risk of Alzheimerrsquos disease apopulation-based case-control studyrdquo Journal of the AmericanGeriatrics Society vol 60 no 5 pp 916ndash921 2012

[17] J W Jonker and A H Schinkel ldquoPharmacological and physio-logical functions of the polyspecific organic cation transportersOCT1 2 and 3 (SLC22A1-3)rdquo Journal of Pharmacology andExperimental Therapeutics vol 308 no 1 pp 2ndash9 2004

[18] M P Quaile D H Melich H L Jordan et al ldquoToxicity andtoxicokinetics of metformin in ratsrdquo Toxicology and AppliedPharmacology vol 243 no 3 pp 340ndash347 2010

[19] G Rena E R Pearson and K Sakamoto ldquoMolecular mecha-nismof action ofmetformin old or new insightsrdquoDiabetologiavol 56 no 9 pp 1898ndash1906 2013

[20] W-S Lv J-P Wen L Li et al ldquoThe effect of metformin on foodintake and its potential role in hypothalamic regulation in obesediabetic ratsrdquo Brain Research vol 1444 pp 11ndash19 2012

[21] G Zhou R Myers Y Li et al ldquoRole of AMP-activated proteinkinase in mechanism of metformin actionrdquo The Journal ofClinical Investigation vol 108 no 8 pp 1167ndash1174 2001

[22] V G Ronnett S Ramamurthy A M Kleman L E Landreeand S Aja ldquoAMPK in the brain its roles in energy balance andneuroprotectionrdquo Journal of Neurochemistry vol 109 no s1 pp17ndash23 2009

[23] J Li and L D McCullough ldquoEffects of AMP-activated proteinkinase in cerebral ischemiardquo Journal of Cerebral Blood Flow ampMetabolism vol 30 no 3 pp 480ndash492 2010

[24] M Foretz S Hebrard J Leclerc et al ldquoMetformin inhibits hep-atic gluconeogenesis inmice independently of the LKB1AMPKpathway via a decrease in hepatic energy staterdquo The Journal ofClinical Investigation vol 120 no 7 pp 2355ndash2369 2010

[25] M Y El-Mir D Detaille G R-Villanueva et al ldquoNeuropro-tective role of antidiabetic drug metformin against apoptoticcell death in primary cortical neuronsrdquo Journal of MolecularNeuroscience vol 34 no 1 pp 77ndash87 2008

[26] A K Madiraju D M Erion Y Rahimi et al ldquoMetforminsuppresses gluconeogenesis by inhibiting mitochondrial glyc-erophosphate dehydrogenaserdquo Nature vol 510 no 7506 pp542ndash546 2014

[27] E G Phimister and E Ferrannini ldquoThe target of metformin intype 2 diabetesrdquoThe New England Journal of Medicine vol 371no 16 pp 1547ndash1548 2014

[28] L Logie J Harthill K Patel et al ldquoCellular responses to themetal-binding properties of metforminrdquoDiabetes vol 61 no 6pp 1423ndash1433 2012

[29] D Horn and A Barrientos ldquoMitochondrial copper metabolismand delivery to cytochrome C oxidaserdquo IUBMB Life vol 60 no7 pp 421ndash429 2008

[30] X Quan R Uddin A Heiskanen et al ldquoThe copper bind-ing properties of metforminmdashQCM-D XPS and nanobeadagglomerationrdquo Chemical Communications vol 51 no 97 pp17313ndash17316 2015


[31] E Z Longa P R Weinstein S Carlson and R CumminsldquoReversible middle cerebral artery occlusion without craniec-tomy in ratsrdquo Stroke vol 20 no 1 pp 84ndash91 1989

[32] R J Laing J Jakubowski and R W Laing ldquoMiddle cerebralartery occlusion without craniectomy in rats which methodworks bestrdquo Stroke vol 24 no 2 pp 294ndash298 1993

[33] A Durukan and T Tatlisumak ldquoCardiovascular genomicsrdquo inMethods inMolecular Biology K DiPetrillo Ed Humana PressTotowa NJ USA 2009

[34] D G Hardie and B G Frenguelli ldquoA neural protection racketAMPK and theGABA119861 receptorrdquoNeuron vol 53 no 2 pp 159ndash162 2007

[35] S Harada W Fujita-Hamabe and S Tokuyama ldquoThe impor-tance of regulation of blood glucose levels through activationof peripheral 51015840-AMP-activated protein kinase on ischemicneuronal damagerdquo Brain Research vol 1351 pp 254ndash263 2010

[36] N Kuramoto M E Wilkins B P Fairfax et al ldquoPhospho-dependent functional modulation of GABA119861 receptors by themetabolic sensor AMP-dependent protein kinaserdquoNeuron vol53 no 2 pp 233ndash247 2007

[37] L D McCullough Z Zeng H Li L E Landree J McFaddenand G V Ronnett ldquoPharmacological inhibition of AMP-activated protein kinase provides neuroprotection in strokerdquoJournal of Biological Chemistry vol 280 no 21 pp 20493ndash20502 2005

[38] J Li Z Zeng B Viollet G V Ronnett and L D McCul-lough ldquoNeuroprotective effects of adenosine monophosphate-activated protein kinase inhibition and gene deletion in strokerdquoStroke vol 38 no 11 pp 2992ndash2999 2007

[39] J Li S E Benashski V R Venna and L D McCulloughldquoEffects of metformin in experimental strokerdquo Stroke vol 41no 11 pp 2645ndash2652 2010

[40] L DMccullough and S E Benashski ldquoPreconditioning inducessustained neuroprotection by down regulation of AMPKrdquoNeuroscience vol 10 no 201 pp 280ndash287 2012

[41] A Kunz U Dirnagl and P Mergenthaler ldquoAcute pathophysi-ological processes after ischaemic and traumatic brain injuryrdquoBest Practice amp Research Clinical Anaesthesiology vol 24 no 4pp 495ndash509 2010

[42] M Godınez-Rubı A E Rojas-Mayorquın and D Ortuno-Sahagun ldquoNitric oxide donors as neuroprotective agents afteran ischemic stroke-related inflammatory reactionrdquo OxidativeMedicine and Cellular Longevity vol 2013 Article ID 29735716 pages 2013

[43] NA TerpolilliMAMoskowitz andN Plesnila ldquoNitric oxideconsiderations for the treatment of ischemic strokerdquo Journal ofCerebral Blood Flow ampMetabolism vol 32 no 7 pp 1332ndash13462012

[44] M J L Eliasson Z Huang R J Ferrante et al ldquoNeuronalnitric oxide synthase activation and peroxynitrite formation inischemic stroke linked to neural damagerdquo Journal of Neuro-science vol 19 no 14 pp 5910ndash5918 1999

[45] C Iadecola ldquoBright and dark sides of nitric oxide in ischemicbrain injuryrdquo Trends in Neurosciences vol 20 no 3 pp 132ndash1391997

[46] W Wu F J Liuzzi F P Schinco et al ldquoNeuronal nitric oxidesynthase is induced in spinal neurons by traumatic injuryrdquoNeuroscience vol 61 no 4 pp 719ndash726 1994

[47] Y Ito T Ohkubo Y Asano et al ldquoNitric oxide productionduring cerebral ischemia and reperfusion in eNOS- and nNOS-knockout micerdquo Current Neurovascular Research vol 7 no 1pp 23ndash31 2010

[48] M Khan B Sekhon S Giri et al ldquoS-nitrosoglutathione reducesinflammation and protects brain against focal cerebral ischemiain a rat model of experimental strokerdquo Journal of Cerebral BloodFlow amp Metabolism vol 25 no 2 pp 177ndash192 2005

[49] Y Gursoy-Ozdemir H Bolay O Saribas and T DalkaraldquoRole of endothelial nitric oxide generation and peroxynitriteformation in reperfusion injury after focal cerebral ischemiardquoStroke vol 31 no 8 pp 1974ndash1981 2000

[50] Z Huang P L Huang J Ma et al ldquoEnlarged infarcts inendothelial nitric oxide synthase knockout mice are attenu-ated by nitro-L-argininerdquo Journal of Cerebral Blood Flow andMetabolism vol 16 no 5 pp 981ndash987 1996

[51] J W Calvert S Gundewar S Jha et al ldquoAcute metformintherapy confers cardioprotection against myocardial infarctionvia AMPK-eNOS- mediated signalingrdquo Diabetes vol 57 no 3pp 696ndash705 2008

[52] L S Capettini S F Cortes and V S Lemos ldquoRelative contri-bution of eNOS and nNOS to endothelium-dependent vasodi-lation in the mouse aortardquo European Journal of Pharmacologyvol 643 no 2-3 pp 260ndash266 2010

[53] N Takahashi R Shibata N Ouchi M Sugimoto T Muro-hara and K Komori ldquoMetformin stimulates ischemia-inducedrevascularization through an eNOS dependent pathway in theischemic hindlimb mice modelrdquo Journal of Vascular Surgeryvol 61 no 2 pp 489ndash496 2015

[54] B P Carreira C M Carvalho and I M Araujo ldquoRegulationof injury-induced neurogenesis by nitric oxiderdquo Stem CellsInternational vol 2012 Article ID 895659 15 pages 2012

[55] B J Davis Z Xie B Viollet and M-H Zou ldquoActivationof the AMP-activated kinase by antidiabetes drug metforminstimulates nitric oxide synthesis in vivo by promoting theassociation of heat shock protein 90 and endothelial nitric oxidesynthaserdquo Diabetes vol 55 no 2 pp 496ndash505 2006

[56] S Correia and C Carvalho ldquoMetformin protects the brainagainst the oxidative imbalance promoted by type 2 diabetesrdquoMedicinal Chemistry vol 4 no 4 pp 358ndash364 2008

[57] A A Moustaf and A M Mohamed ldquoModulation of theoxidative stress bymetformin in the cerebrumof rats exposed toglobal cerebral ischemia and ischemiareperfusionrdquo EuropeanReview for Medical and Pharmacological Sciences vol 18 pp2387ndash2392 2014

[58] V Nischwitz A Berthele and B Michalke ldquoSpeciation analysisof selected metals and determination of their total contents inpaired serum and cerebrospinal fluid samples an approach toinvestigate the permeability of the human blood-cerebrospinalfluid-barrierrdquo Analytica Chimica Acta vol 627 no 2 pp 258ndash269 2008

[59] N J Abbott A A Patabendige D E Dolman S R Yusof andD J Begley ldquoStructure and function of the blood-brain barrierrdquoNeurobiology of Disease vol 37 no 1 pp 13ndash25 2010

[60] M Kuntz C Mysiorek O Petrault et al ldquoStroke-induced brainparenchymal injury drives blood-brain barrier early leakagekinetics a combined in vivoin vitro studyrdquo Journal of CerebralBlood Flow ampMetabolism vol 34 no 1 pp 95ndash107 2014

[61] F Takata SDohgu JMatsumoto et al ldquoMetformin induces up-regulation of blood-brain barrier functions by activating AMP-activated protein kinase in rat brain microvascular endothelialcellsrdquo Biochemical and Biophysical Research Communicationsvol 433 no 4 pp 586ndash590 2013

[62] Y Farbood A Sarkaki L Khalaj F Khodagholi M Badaviand G Ashabi ldquoTargeting adenosinemonophosphate-activated


protein kinase by metformin adjusts post-ischemic hyperemiaand extracellular neuronal discharge in transient global cerebralischemiardquoMicrocirculation vol 22 no 7 pp 534ndash541 2015

[63] Y Liu G Tang Y Li et al ldquoMetformin attenuates blood-brainbarrier disruption in mice following middle cerebral arteryocclusionrdquo Journal of Neuroinflammation vol 11 article 1772014

[64] A Spruss G Kanuri C Stahl S C Bischoff and I BergheimldquoMetformin protects against the development of fructose-induced steatosis inmice role of the intestinal barrier functionrdquoLaboratory Investigation vol 92 no 7 pp 1020ndash1032 2012

[65] A Ergul M Abdelsaid A Y Fouda and S C Fagan ldquoCerebralneovascularization in diabetes implications for stroke recoveryand beyondrdquo Journal of Cerebral Blood Flow ampMetabolism vol34 no 4 pp 553ndash563 2014

[66] R Prakash W Li Z Qu M A Johnson S C Fagan and AErgul ldquoVascularization pattern after ischemic stroke is differentin control versus diabetic rats relevance to stroke recoveryrdquoStroke vol 44 no 10 pp 2875ndash2882 2013

[67] M M Elgebaly R Prakash W Li et al ldquoVascular pro-tection in diabetic stroke role of matrix metalloprotease-dependent vascular remodelingrdquo Journal of Cerebral Blood FlowampMetabolism vol 30 no 12 pp 1928ndash1938 2010

[68] I Ullah N Ullah M I Naseer H Y Lee and M O KimldquoNeuroprotection with metformin and thymoquinone againstethanol-induced apoptotic neurodegeneration in prenatal ratcortical neuronsrdquo BMC Neuroscience vol 13 article 11 2012

[69] G Ashabi F Khodagholi L Khalaj M Goudarzvand andM Nasiri ldquoActivation of AMP-activated protein kinase bymetformin protects against global cerebral ischemia in malerats interference of AMPKPGC-1120572 pathwayrdquo Metabolic BrainDisease vol 29 no 1 pp 47ndash58 2014

[70] J Wang D Gallagher L M DeVito et al ldquoMetformin activatesan atypical PKC-CBP pathway to promote neurogenesis andenhance spatial memory formationrdquo Cell Stem Cell vol 11 no1 pp 23ndash35 2012

[71] X-C Zhu T Jiang Q-Q Zhang et al ldquoChronic metforminpreconditioning provides neuroprotection via suppression ofNF-120581B-mediated inflammatory pathway in rats with permanentcerebral ischemiardquo Molecular Neurobiology vol 52 no 1 pp375ndash385 2015

[72] V R Venna J Li M D Hammond N S Mancini and LD McCullough ldquoChronic metformin treatment improves post-stroke angiogenesis and recovery after experimental strokerdquoEuropean Journal of Neuroscience vol 39 no 12 pp 2129ndash21382014

[73] T Jiang J Yu X Zhu et al ldquoAcute metformin preconditioningconfers neuroprotection against focal cerebral ischaemia by pre-activation of AMPK-dependent autophagyrdquo British Journal ofPharmacology vol 171 no 13 pp 3146ndash3157 2014

[74] Y Liu G Tang Z Zhang YWang and G-Y Yang ldquoMetforminpromotes focal angiogenesis and neurogenesis in mice follow-ing middle cerebral artery occlusionrdquo Neuroscience Letters vol579 pp 46ndash51 2014

[75] M Abdelsaid R Prakash W Li et al ldquoMetformin treatmentin the period after stroke prevents nitrative stress and restoresangiogenic signaling in the brain in diabetesrdquo Diabetes vol 64no 5 pp 1804ndash1817 2015

[76] L McCullough V Venna and J Li ldquoPreconditioning inducessustained neuroprotection by down regulation of AMPKrdquoNeuroscience vol 201 pp 280ndash287 2012

[77] Q Jin J Cheng Y Liu et al ldquoImprovement of functionalrecovery by chronic metformin treatment is associated withenhanced alternative activation of microgliamacrophages andincreased angiogenesis and neurogenesis following experimen-tal strokerdquo Brain Behavior and Immunity vol 40 pp 131ndash1422014

[78] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[79] T Deng Y-R Zheng W-W Hou et al ldquoPre-stroke metformintreatment is neuroprotective involving AMPK reductionrdquoNeu-rochemical Research vol 41 no 10 pp 2719ndash2727 2016

[80] O Q Russe C V Moser K L Kynast et al ldquoActivationof the AMP-activated protein kinase reduces inflammatorynociceptionrdquo Journal of Pain vol 14 no 11 pp 1330ndash1340 2013

[81] J E Jung J Lee J Ha et al ldquo5-Aminoimidazole-4-carbox-amide-ribonucleoside enhances oxidative stress-induced apop-tosis through activation of nuclear factor-120581B in mouse Neuro2a neuroblastoma cellsrdquoNeuroscience Letters vol 354 no 3 pp197ndash200 2004

[82] O Moiseeva X Deschenes-Simard E St-Germain et al ldquoMet-formin inhibits the senescence-associated secretory phenotypeby interfering with IKKNF-120581B activationrdquo Aging Cell vol 12no 3 pp 489ndash498 2013

[83] M-H Zou andYWu ldquoAMP-activated protein kinase activationas a strategy for protecting vascular endothelial functionrdquoClinical and Experimental Pharmacology and Physiology vol 35no 5-6 pp 535ndash545 2008

[84] T YamashitaNNonoguchiN Ikeda S Kawabata SMiyatakeandTKuroiwa ldquoBrain poster session neuroprotectionrdquo Journalof Cerebral Blood Flow ampMetabolism vol 29 supplement 1 ppS420ndashS454 2009

[85] P Perel I Roberts E Sena et al ldquoComparison of treatmenteffects between animal experiments and clinical trials system-atic reviewrdquo BMJ vol 334 no 7586 p 197 2007

[86] A M Turnley D Stapleton R J Mann L A Witters B EKemp and P F Bartlett ldquoCellular distribution and develop-mental expression of AMP-activated protein kinase isoforms inmouse central nervous systemrdquo Journal of Neurochemistry vol72 no 4 pp 1707ndash1716 1999

[87] K Janjetovic L Harhaji-Trajkovic M Misirkic-Marjanovic etal ldquoIn vitro and in vivo anti-melanoma action of metforminrdquoEuropean Journal of Pharmacology vol 668 no 3 pp 373ndash3822011

[88] S W Cho K H Yi S K Han et al ldquoTherapeutic potentialof metformin in papillary thyroid cancer in vitro and in vivordquoMolecular and Cellular Endocrinology vol 393 no 1-2 pp 24ndash29 2015

[89] A Yasmeen M-C Beauchamp E Piura E Segal M Pollakand W H Gotlieb ldquoInduction of apoptosis by metforminin epithelial ovarian cancer involvement of the Bcl-2 familyproteinsrdquo Gynecologic Oncology vol 121 no 3 pp 492ndash4982011

[90] A Sato J Sunayama M Okada et al ldquoGlioma-initiating cellelimination by metformin activation of FOXO3 via AMPKrdquoStemCells TranslationalMedicine vol 1 no 11 pp 811ndash824 2012

[91] P H Becerra Lina ldquoApoptosis neuronal la diversidad de senalesy de tipos celularesrdquo Colomb Meeica vol 40 pp 124ndash133 2009


[92] D Magkou and K Tziomalos ldquoAntidiabetic treatment strokeseverity and outcomerdquo World Journal of Diabetes vol 5 no 2pp 84ndash88 2014

[93] V Darsalia M Larsson D Nathanson T Klein T Nystromand C Patrone ldquoGlucagon-like receptor 1 agonists and DPP-4 inhibitors potential therapies for the treatment of strokerdquoJournal of Cerebral Blood Flow amp Metabolism vol 35 no 5 pp718ndash723 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


stroke episodes (adjusted hazard ratio 046 95 CI 0424ndash0518 119901 lt 0001) in patients with diabetes treated with met-formin versus patients who did not use metformin [11] Theclinical benefit of metformin in diabetic patients for strokeprevention was described in a retrospective analysis byMimaet al who reported a reduction in the severity of neurologicalsymptoms [12]

Opposing results have also been reported for otherpathologies of nervous system In a case-control studypatients in the group treated with metformin alone versusother oral antidiabetic agents exhibited an increased riskof Alzheimerrsquos disease (adjusted OR 171 95 CI 112ndash260versus other oral agents such as sulphonylurea adjusted OR101 95 CI 072ndash142) [13]

These epidemiological observations have increased inter-est in elucidating the mechanisms of action of metformin toexplain these positive effects In neuroscience the search for aneuroprotective agent has been intense with several promis-ing cellular models although unsuccessful in its applicabilityin humans partly due to the great distance separating thesebiological experiments with the human brain Metformin isone of these molecules with neuroprotective potential thathas been studied in various experimental models such asneuronal cultures andmodels of cerebral ischemia in rodentsThese have allowed observation of their cellular effects atthe neuronal glial and endothelial levels as well as cellularpathways such as apoptosis neurogenesis angiogenesis andinflammationThis review focuses on the effect of metforminin brain ischemia in rodents and themolecular targets identi-fied as part of the processes that explain the epidemiologicalphenomena discussed

2 Pharmacological Aspects of Metformin

Metformin is a small amphoteric hydrophilic molecule withlow lipid solubility and 124 pKa which favors the acid (pro-tonated) state in the intestine Metformin requires an organiccationic transporter (OCT12) to enter the hepatocytes [14]In humans intestinal absorption yields a final bioavailabil-ity of approximately 50 An inverse relationship betweenabsorption and dose has been observed which suggests asaturable process [15] Metformin is not metabolized in thekidney from which it is excreted without change and witha half-life of 4-5 hours Compared with other biguanidessuch as phenformin metformin is less potent and does notproduce its pharmacological effects in humans until it reaches2700mgday This low potency may explain its superiorsafety profile compared with other biguanides especially itsrelationship with lactic acidosis [16]The cationic transporterOCT2 is expressed in the kidney endothelium which isinvolved in metformin elimination This transporter hasalso been identified in neurons [17] and involves passage ofmetformin from systemic circulation to the neuronal interiorToxicity was observed in rats at doses of metformin greaterthan 900mgkgday delivered by gavage [18]

Metformin must cross the blood-brain barrier (BBB) toproduce its neuroprotective actions For this issue observa-tions in obese patients with and without diabetes treated with

metformin show a decreased in food intake [14 19] Lv et alassumed that these effects could be mediated by orexigenicandor anorectic peptide expression or inhibition Thereforethe authors evaluated metformin concentrations in cere-brospinal fluid (CSF) and in the hypothalamic region of obesediabetic male rats and observed concentrations at approx-imately four percent (4) in the CSF compared with theplasma concentration [20] Łabuzek et al also demonstratedthe presence of metformin in brains of Wistar rats treatedwith acute and chronic doses of 150mgkg metformin in amodel of systemic inflammation induced by adding lowdosesof lipopolysaccharide (LPS) at 2120583gkgday Groups treatedwith metformin (acute and chronic) without LPS showed thehighest concentration of metformin in the pineal gland andcerebellum and low concentrations in the striatum In theLPS-treated groups a higher metformin concentration wasobserved in the hypothalamus and striatum suggesting thatmild inflammatory conditions which have been describedfor diabetes and atherosclerotic disease facilitate metformintransport into the brain [15]

3 Metformin Action Mechanism

Althoughmetformin has been approved in Europe since 1957for treating type 2 diabetesmellitus and several cellular path-ways studies showed changes in the presence of metforminthe exactmetformin actionmechanism is unknown (Table 2)Its hypoglycemic action is due to reduced gluconeogenesisincreased insulin receptor sensitivity especially in musclecells and decreased glucose uptake in the intestine [4 20]

31 The Effects on AMPK Zhou et al (2001) determined thatAMP-activated protein kinase (AMPK) is a cellular target ofmetformin by observing that AMPK activation was requiredfor the metformin inhibitory effect on hepatocyte endoge-nous glucose production and for the decrease expression ofthe lipogenic gene SREBP-1 Zhou et al found that AMPKactivation could explain metforminrsquos pleiotropic effects [21]AMPK is a highly conserved serinethreonine kinase withthree domains 120572 120573 and 120574 The 120572 subunit contains thecatalytic site two isoforms have been described 1205721 and 1205722The 120574 subunit comprises the regulatory sector that preventspermanent phosphorylation of a threonine (Thr172) in the 120572subunit [22] AMPKmonitors energy to protect cellular func-tions through sensing the AMPATP and ADPATP rates ofchange AMPK is activated by increased cytoplasmic concen-trations of AMP which have been shown in nutrient depri-vation vigorous exercise and ischemia The physiologicalchanges observed duringAMPKactivation induce a catabolicstate and inhibit cellular processes that require high ATPconsumption such as protein glucose and lipid biosynthesis[23] AMPK activation by both changes in the AMPATPintracellular rate and the AMPK upstream activator LKB1leads to decreased gluconeogenic gene transcription

32 The effects on Complex I of the Mitochondria RespiratoryChain Foretz et al showed that the hypoglycemic effects of











R1

Metformin

R2

G3PDHAP

mGDP

NADH ATP

Gluconeogenesis

FBPasaACC

Lipogenesis

aPKC-CBP

Neurogenesis

NOS

ON

mTOR


Glut4

Glucose capture

Mitochondria

Nucleus

C

p-AMPKactivated

C

N N

Complex I

+

＋+

TNF-

LKB1CaMKK

darr


NF-

uarr AMP

GABAb４12





























9 days

[70]

Rat



Antiapoptotic




[68]


cells


BBB













Table2

Differentinvivo

studiesusingm

etform

ininam

odeloffocalorg

lobalischemiawith

orwith

outreperfusio

nMostofthe

studiesshow

neurop

rotectivee

ffectsPO

oralI

Pperiton

eal

injection

ICV

intraventricularand

IPC

ischemicprecon

ditio

ning

Biom

odel

Strain

Ischem

icfocal

orglob

alTimeo

fisc

hemicreperfusio

nNeuroprotectio

nPo

stulated

mechanism

Doses

androuteo

fadministratio

nMetform

intre

atmenttim

eRe

f

Rat

Wistar

Global

30min1h

Yes

Redu

ctionsuperoxide

dism

utase

activ

ityandglutathion

eperoxidase

500m

gkg

POOne

weekbefore

stroke

[57]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Supp

ressionof

theN

F-120581B

inflammatorypathway

50mgkg

IP3weeks

before

stroke

[71]

Mice

C57B

L6N

Focal

60min72h

Yes

Ang

iogenesis

andim

provec

erebral

dopaminergicton

e

02m

Lkg

IPo

50mgkgday

IP

50mgkgday

beginn

ing

24haft

erstr

okefor

3weeks

[72]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Indu

cesa

utop

hagy

10mgkgIP

Sing

ledo

se24

hbefore

stroke

[73]

Mice

CD-1

Focal

90min14

days

Yes

Prom

otes

neurogenesisand

angiogenesisin

subventricular

zone

200m

gkg

IPFo

r14days

after

stroke

[74]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Redu

celevelsof

nitro

tyrosin

e300m

gkgday

POFo

r14days

after

stroke

[75]

Mice

C57B

L6N

Focal

3min

(IPC

)at72

hours9

0min24h

No

AMPK

activ

ationantagonizes

neurop

rotectivee

ffectof

ischemic

precon

ditio

ning

100m

gkg

IPSing

ledo

sesa

fterstro

ke[76]

Mice

CD-1

Focal

60min14

days

Yes

Favorschange

inph

enotypeM

2microgliaastr

ocyte

50mgkgday

IPBe

ginn

ing24

haft

erstroke

[77]

Mice

C57B

L6

Focal

90min24y

72ho

urs

Noacute

treatment

Yeschronic

treatment


ation

ofAMPK

Acute50

a100m

gkgdıaIP

Chronic50

a100m

gkday

Acute24

hbefore

Chronic3weeks

before

[39]

Rat

Wistar

Global

30min72h

Yes

AMPK

activ

ationinhibitsapop

tosis

inhipp

ocam

paln

eurons

200m

gkgday

PO2weeks

before

ischemia

[69]

Rat

Goto-Ka

kizaki

Focal

90min21h

Yes

Redu

cedvascular

remod

elingand

severityof

hemorrhagic

transfo

rmationin

diabetes

300m

gkgday

POStartin

gwith

theo

nsetof

diabetes

[67]

Rat

Wistar

Global

30min72h

Yes

Attenu

ates

cellu

larlevels

ofNF-kB


Nrf2

inhipp

ocam

pus

200m

gkgday

PO2weeks

before

ischemia

[78]

Rat

Wistar

Global

30min72h

Yes

Decreases

reactiv

ehyperem

iaand

perm

eabilityo

fBBB

inisc

hemicrats

200m

gkgday

PO2weeks

before

ischemia

[62]

Mice

ddY

Focal

60min72h

Noacuteinjectio

nintraventricular

YeschronicIP

Centralversus

perip

heralactivation

ofAMPK

250m

gkgIP

three

times

or2510

0120583gICVthree

times

ford

ay

Afte

rstro

keun

tildeath

[35]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Improved

vascularizationin

diabeticgrou

pachievee

uglycemia

300m

gkgday

POAfte

rstro

keun

tildeath

[66]

Mice

C57B

L6

Focal

60min24h

Yes


activ

ationof

AMPK

10mgkgday

IPAfte

rstro

keun

tildeath

[79]















Competing Interests


References







































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























R1

Metformin

R2

G3PDHAP

mGDP

NADH ATP

Gluconeogenesis

FBPasaACC

Lipogenesis

aPKC-CBP

Neurogenesis

NOS

ON

mTOR


Glut4

Glucose capture

Mitochondria

Nucleus

C

p-AMPKactivated

C

N N

Complex I

+

＋+

TNF-

LKB1CaMKK

darr


NF-

uarr AMP

GABAb４12





























9 days

[70]

Rat



Antiapoptotic




[68]


cells


BBB













Table2

Differentinvivo

studiesusingm

etform

ininam

odeloffocalorg

lobalischemiawith

orwith

outreperfusio

nMostofthe

studiesshow

neurop

rotectivee

ffectsPO

oralI

Pperiton

eal

injection

ICV

intraventricularand

IPC

ischemicprecon

ditio

ning

Biom

odel

Strain

Ischem

icfocal

orglob

alTimeo

fisc

hemicreperfusio

nNeuroprotectio

nPo

stulated

mechanism

Doses

androuteo

fadministratio

nMetform

intre

atmenttim

eRe

f

Rat

Wistar

Global

30min1h

Yes

Redu

ctionsuperoxide

dism

utase

activ

ityandglutathion

eperoxidase

500m

gkg

POOne

weekbefore

stroke

[57]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Supp

ressionof

theN

F-120581B

inflammatorypathway

50mgkg

IP3weeks

before

stroke

[71]

Mice

C57B

L6N

Focal

60min72h

Yes

Ang

iogenesis

andim

provec

erebral

dopaminergicton

e

02m

Lkg

IPo

50mgkgday

IP

50mgkgday

beginn

ing

24haft

erstr

okefor

3weeks

[72]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Indu

cesa

utop

hagy

10mgkgIP

Sing

ledo

se24

hbefore

stroke

[73]

Mice

CD-1

Focal

90min14

days

Yes

Prom

otes

neurogenesisand

angiogenesisin

subventricular

zone

200m

gkg

IPFo

r14days

after

stroke

[74]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Redu

celevelsof

nitro

tyrosin

e300m

gkgday

POFo

r14days

after

stroke

[75]

Mice

C57B

L6N

Focal

3min

(IPC

)at72

hours9

0min24h

No

AMPK

activ

ationantagonizes

neurop

rotectivee

ffectof

ischemic

precon

ditio

ning

100m

gkg

IPSing

ledo

sesa

fterstro

ke[76]

Mice

CD-1

Focal

60min14

days

Yes

Favorschange

inph

enotypeM

2microgliaastr

ocyte

50mgkgday

IPBe

ginn

ing24

haft

erstroke

[77]

Mice

C57B

L6

Focal

90min24y

72ho

urs

Noacute

treatment

Yeschronic

treatment


ation

ofAMPK

Acute50

a100m

gkgdıaIP

Chronic50

a100m

gkday

Acute24

hbefore

Chronic3weeks

before

[39]

Rat

Wistar

Global

30min72h

Yes

AMPK

activ

ationinhibitsapop

tosis

inhipp

ocam

paln

eurons

200m

gkgday

PO2weeks

before

ischemia

[69]

Rat

Goto-Ka

kizaki

Focal

90min21h

Yes

Redu

cedvascular

remod

elingand

severityof

hemorrhagic

transfo

rmationin

diabetes

300m

gkgday

POStartin

gwith

theo

nsetof

diabetes

[67]

Rat

Wistar

Global

30min72h

Yes

Attenu

ates

cellu

larlevels

ofNF-kB


Nrf2

inhipp

ocam

pus

200m

gkgday

PO2weeks

before

ischemia

[78]

Rat

Wistar

Global

30min72h

Yes

Decreases

reactiv

ehyperem

iaand

perm

eabilityo

fBBB

inisc

hemicrats

200m

gkgday

PO2weeks

before

ischemia

[62]

Mice

ddY

Focal

60min72h

Noacuteinjectio

nintraventricular

YeschronicIP

Centralversus

perip

heralactivation

ofAMPK

250m

gkgIP

three

times

or2510

0120583gICVthree

times

ford

ay

Afte

rstro

keun

tildeath

[35]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Improved

vascularizationin

diabeticgrou

pachievee

uglycemia

300m

gkgday

POAfte

rstro

keun

tildeath

[66]

Mice

C57B

L6

Focal

60min24h

Yes


activ

ationof

AMPK

10mgkgday

IPAfte

rstro

keun

tildeath

[79]















Competing Interests


References







































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































9 days

[70]

Rat



Antiapoptotic




[68]


cells


BBB













Table2

Differentinvivo

studiesusingm

etform

ininam

odeloffocalorg

lobalischemiawith

orwith

outreperfusio

nMostofthe

studiesshow

neurop

rotectivee

ffectsPO

oralI

Pperiton

eal

injection

ICV

intraventricularand

IPC

ischemicprecon

ditio

ning

Biom

odel

Strain

Ischem

icfocal

orglob

alTimeo

fisc

hemicreperfusio

nNeuroprotectio

nPo

stulated

mechanism

Doses

androuteo

fadministratio

nMetform

intre

atmenttim

eRe

f

Rat

Wistar

Global

30min1h

Yes

Redu

ctionsuperoxide

dism

utase

activ

ityandglutathion

eperoxidase

500m

gkg

POOne

weekbefore

stroke

[57]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Supp

ressionof

theN

F-120581B

inflammatorypathway

50mgkg

IP3weeks

before

stroke

[71]

Mice

C57B

L6N

Focal

60min72h

Yes

Ang

iogenesis

andim

provec

erebral

dopaminergicton

e

02m

Lkg

IPo

50mgkgday

IP

50mgkgday

beginn

ing

24haft

erstr

okefor

3weeks

[72]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Indu

cesa

utop

hagy

10mgkgIP

Sing

ledo

se24

hbefore

stroke

[73]

Mice

CD-1

Focal

90min14

days

Yes

Prom

otes

neurogenesisand

angiogenesisin

subventricular

zone

200m

gkg

IPFo

r14days

after

stroke

[74]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Redu

celevelsof

nitro

tyrosin

e300m

gkgday

POFo

r14days

after

stroke

[75]

Mice

C57B

L6N

Focal

3min

(IPC

)at72

hours9

0min24h

No

AMPK

activ

ationantagonizes

neurop

rotectivee

ffectof

ischemic

precon

ditio

ning

100m

gkg

IPSing

ledo

sesa

fterstro

ke[76]

Mice

CD-1

Focal

60min14

days

Yes

Favorschange

inph

enotypeM

2microgliaastr

ocyte

50mgkgday

IPBe

ginn

ing24

haft

erstroke

[77]

Mice

C57B

L6

Focal

90min24y

72ho

urs

Noacute

treatment

Yeschronic

treatment


ation

ofAMPK

Acute50

a100m

gkgdıaIP

Chronic50

a100m

gkday

Acute24

hbefore

Chronic3weeks

before

[39]

Rat

Wistar

Global

30min72h

Yes

AMPK

activ

ationinhibitsapop

tosis

inhipp

ocam

paln

eurons

200m

gkgday

PO2weeks

before

ischemia

[69]

Rat

Goto-Ka

kizaki

Focal

90min21h

Yes

Redu

cedvascular

remod

elingand

severityof

hemorrhagic

transfo

rmationin

diabetes

300m

gkgday

POStartin

gwith

theo

nsetof

diabetes

[67]

Rat

Wistar

Global

30min72h

Yes

Attenu

ates

cellu

larlevels

ofNF-kB


Nrf2

inhipp

ocam

pus

200m

gkgday

PO2weeks

before

ischemia

[78]

Rat

Wistar

Global

30min72h

Yes

Decreases

reactiv

ehyperem

iaand

perm

eabilityo

fBBB

inisc

hemicrats

200m

gkgday

PO2weeks

before

ischemia

[62]

Mice

ddY

Focal

60min72h

Noacuteinjectio

nintraventricular

YeschronicIP

Centralversus

perip

heralactivation

ofAMPK

250m

gkgIP

three

times

or2510

0120583gICVthree

times

ford

ay

Afte

rstro

keun

tildeath

[35]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Improved

vascularizationin

diabeticgrou

pachievee

uglycemia

300m

gkgday

POAfte

rstro

keun

tildeath

[66]

Mice

C57B

L6

Focal

60min24h

Yes


activ

ationof

AMPK

10mgkgday

IPAfte

rstro

keun

tildeath

[79]















Competing Interests


References







































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2

Differentinvivo

studiesusingm

etform

ininam

odeloffocalorg

lobalischemiawith

orwith

outreperfusio

nMostofthe

studiesshow

neurop

rotectivee

ffectsPO

oralI

Pperiton

eal

injection

ICV

intraventricularand

IPC

ischemicprecon

ditio

ning

Biom

odel

Strain

Ischem

icfocal

orglob

alTimeo

fisc

hemicreperfusio

nNeuroprotectio

nPo

stulated

mechanism

Doses

androuteo

fadministratio

nMetform

intre

atmenttim

eRe

f

Rat

Wistar

Global

30min1h

Yes

Redu

ctionsuperoxide

dism

utase

activ

ityandglutathion

eperoxidase

500m

gkg

POOne

weekbefore

stroke

[57]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Supp

ressionof

theN

F-120581B

inflammatorypathway

50mgkg

IP3weeks

before

stroke

[71]

Mice

C57B

L6N

Focal

60min72h

Yes

Ang

iogenesis

andim

provec

erebral

dopaminergicton

e

02m

Lkg

IPo

50mgkgday

IP

50mgkgday

beginn

ing

24haft

erstr

okefor

3weeks

[72]

Rat

Sprague-

Daw

ley

Focal

Not

reperfusion

Yes

Indu

cesa

utop

hagy

10mgkgIP

Sing

ledo

se24

hbefore

stroke

[73]

Mice

CD-1

Focal

90min14

days

Yes

Prom

otes

neurogenesisand

angiogenesisin

subventricular

zone

200m

gkg

IPFo

r14days

after

stroke

[74]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Redu

celevelsof

nitro

tyrosin

e300m

gkgday

POFo

r14days

after

stroke

[75]

Mice

C57B

L6N

Focal

3min

(IPC

)at72

hours9

0min24h

No

AMPK

activ

ationantagonizes

neurop

rotectivee

ffectof

ischemic

precon

ditio

ning

100m

gkg

IPSing

ledo

sesa

fterstro

ke[76]

Mice

CD-1

Focal

60min14

days

Yes

Favorschange

inph

enotypeM

2microgliaastr

ocyte

50mgkgday

IPBe

ginn

ing24

haft

erstroke

[77]

Mice

C57B

L6

Focal

90min24y

72ho

urs

Noacute

treatment

Yeschronic

treatment


ation

ofAMPK

Acute50

a100m

gkgdıaIP

Chronic50

a100m

gkday

Acute24

hbefore

Chronic3weeks

before

[39]

Rat

Wistar

Global

30min72h

Yes

AMPK

activ

ationinhibitsapop

tosis

inhipp

ocam

paln

eurons

200m

gkgday

PO2weeks

before

ischemia

[69]

Rat

Goto-Ka

kizaki

Focal

90min21h

Yes

Redu

cedvascular

remod

elingand

severityof

hemorrhagic

transfo

rmationin

diabetes

300m

gkgday

POStartin

gwith

theo

nsetof

diabetes

[67]

Rat

Wistar

Global

30min72h

Yes

Attenu

ates

cellu

larlevels

ofNF-kB


Nrf2

inhipp

ocam

pus

200m

gkgday

PO2weeks

before

ischemia

[78]

Rat

Wistar

Global

30min72h

Yes

Decreases

reactiv

ehyperem

iaand

perm

eabilityo

fBBB

inisc

hemicrats

200m

gkgday

PO2weeks

before

ischemia

[62]

Mice

ddY

Focal

60min72h

Noacuteinjectio

nintraventricular

YeschronicIP

Centralversus

perip

heralactivation

ofAMPK

250m

gkgIP

three

times

or2510

0120583gICVthree

times

ford

ay

Afte

rstro

keun

tildeath

[35]

Rat

Goto-Ka

kizaki

Focal

90min14

days

Yes

Improved

vascularizationin

diabeticgrou

pachievee

uglycemia

300m

gkgday

POAfte

rstro

keun

tildeath

[66]

Mice

C57B

L6

Focal

60min24h

Yes


activ

ationof

AMPK

10mgkgday

IPAfte

rstro

keun

tildeath

[79]















Competing Interests


References







































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Competing Interests


References







































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















to use or not to use metformin in cerebral ischemia: a ...phosphorylation has been described in...

Documents