these molecules are implicated in several processes , including adult hippocampal neurogenesis , neuronal firing activity , the formation of the synaptic cleft , the enhancement of neuronal plasticity , and the regulation of mitochondrial dynamics [ 1 , 2 ] . in this context , the ligand wnt5a has been described as an important regulator of several neuronal processes , including protection against amyloid aggregation , dendritic spine formation , expression of micrornas , and regulation of synaptic currents . together , these results highlight the importance of wnt5a in neuronal function [ 36 ] . deregulation of wnt signaling by either loss or gain of function is associated with the progression of various diseases , including cancer , diabetes mellitus type ii , and neurodegenerative diseases , such as parkinson 's and alzheimer 's disease ( ad ) [ 7 , 8 ] . in recent years , several reports have suggested a new role for wnt signaling as a regulator of metabolic pathways [ 9 , 10 ] . this idea has been proposed based on the indirect effects of wnt signaling on other regulators / sensors of glucose metabolism , including phosphoinositide 3-kinase ( pi3k ) and amp - activated protein kinase ( ampk ) , which are both involved in sensing the general metabolic state [ 11 , 12 ] . in brain tissue , glucose is oxidized through glycolysis / oxidative phosphorylation to produce atp , most of which is consumed by neurons during the restoration of ion gradients that are disrupted by synaptic transmission [ 13 , 14 ] . decreased glucose utilization by brain cells has been directly correlated with several brain pathologies , including ad [ 15 , 16 ] . by contrast , enhancing glucose utilization in vivo induces significant improvements in cognitive functions , including memory and learning [ 1719 ] . despite the importance of glucose metabolism in brain function , no studies have described the effect of the wnt5a pathway on glucose metabolism in cortical neurons . in the present study , we demonstrate that wnt5a stimulates glucose uptake , increases glycolytic rate , and stimulates the pentose phosphate pathway ( ppp ) in neurons . additionally , wnt5a treatment increases the activity of regulatory enzymes , such as hexokinase ( hk ) and glucose-6-phosphate dehydrogenase ( g6pdh ) . the effect of wnt5a was dependent on the production of nitric oxide ( no ) . these results suggest that the activation of wnt signaling plays a central role in the regulation of neuronal metabolism and that this effect could be important for the correct function of the neuronal network . the dissection was performed on samples immersed in dissection buffer containing 10  mm hepes ( ph 7.4 , 320  mosm / l ) . the tissues were incubated with 0.25% trypsin-0.2% edta ( w / v ) for 15  min at 37c and then triturated to homogeneity with a fire - polished pasteur pipette . the cells were seeded in poly - d - lysine - coated culture dishes at a density of 510  cells / cm and cultured in dulbecco 's modified eagle 's medium ( dmem ) ( invitrogen , usa ) containing 10% ( v / v ) fetal bovine serum ( thermo fisher scientific inc . after 30  min , the culture medium was changed to neurobasal ( neub ) medium supplemented with b27 ( invitrogen , usa ) , 2  mm l - glutamine , 100 u / ml penicillin , 100  mg / ml streptomycin , and 2.5  mg / ml fungizone ( thermo fisher scientific inc . for all the experiments , the cells were used after 7 days in vitro [ 20 , 21 ] . control and wnt5a - conditioned media were prepared from control l cells ( american type culture collection ( atcc ) : crl-2648 ) and l - wnt5a cells ( atcc : crl2814 ) , respectively ; the cells were cultured according to the protocol described by atcc . briefly , the cells were grown to 70% confluence in mem supplemented with 10% fbs . the medium containing the wnt ligand was recovered ( batch 1 ) , and this process was repeated with the same cells after an additional 4 days in culture ( batch 2 ) . then , batches 1 and 2 of the conditioned media were combined [ 20 , 2226 ] . the neurons were cultured in neub supplemented with b27 until the day of the experiments . before the treatment with the wnt ligand , the cells were maintained in neub without b27 for 1  h ( to avoid the possible effects of the growth factors present in b27 ) . then , the cells were incubated with control or wnt5a media ( 1 - 2  h ) . the neurons were also treated with inhibitors of wnt signaling ; the inhibitors were coincubated with the control media or wnt5a media . the inhibitors used were fzcrd-2 ( 5  ng / ml , antagonist of wnt ligands ) , kn-93 ( 10  m , inhibitor of cam kinase ii ) , g6976 ( 200  nm , inhibitor of pkc ) , and 7-nitroindazole ( 7-ni ; 5  m , inhibitor of nitric oxide synthases ) . all the wnt inhibitors were obtained from sigma - aldrich , usa . the neurons were also treated with inhibitors of glucose metabolism , such as cytochalasin b ( cyt b , 20  m , inhibitor of glucose transporters ( gluts ) ) , cytochalasin e ( cyt e , 20  m , control of the action of cyt b ) , 2-deoxy - d - glucose ( 2-dg , 7  mm , competitive inhibitor of hk ) , and dichloroacetate ( dca , 5  mm , inhibitor of glycolysis that blocks the activity of the pyruvate dehydrogenase complex ) . moreover , we treated the neurons with sodium nitroprusside ( snp , 0800  nm ) , a no donor . in all cases , the neurons were treated with the inhibitors for 30  min prior to the experiment with or without the wnt5a media . after the treatment with wnt5a in the presence or absence of wnt inhibitors , the neurons were carefully selected using a microscope to ensure that only plates containing cultures of uniformly growing neurons were used . following incubation with the wnt ligand , the cells were washed with incubation buffer ( 15  mm hepes , 135  mm nacl , 5  mm kcl , 1.8  mm cacl2 , and 0.8  mm mgcl2 ) supplemented with 0.5  mm glucose . uptake was measured at room temperature by the addition of 11.2  ci of 2-deoxy - d-[1,2-(n)h ] glucose ( [ 2-h]-dg ) at a final specific activity of 13 disintegrations / min / pmol ( approximately 1  mci / mmol ) . uptake was stopped by washing the cells with ice - cold pbs supplemented with 1  mm hgcl2 . the cells were incubated with the glucose metabolism inhibitors for 30  min in the presence of a radioactive substrate . for the slice experiments , the slices were incubated with ( [ 2-h]-dg ) for a specific period , and then a standard methodology was used . the kinetic parameters were determined using a single rectangular hyperbola of the form vmax[glc]/(km + [ glc ] ) , which was adjusted to the data by nonlinear regression using sigmaplot 12 . the ( [ 2-h]-dg ) was purchased from perkinelmer , usa . after treatment with the wnt ligand , the neurons were washed with pbs , treated with trypsin / edta , and centrifuged at 500  g for 5  min at 4c . then , the cells were resuspended at a 1  :  3 dilution in isolation medium ( 250  mm sucrose , 20  mm hepes , 10  mm kcl , 1.5  mm mgcl2 , 1  mm edta , 1  mm dtt , 2  mg / ml aprotinin , 1  mg / ml pepstatin a , and 2  mg / ml leupeptin ) , sonicated at 4c , and then centrifuged at 1,500  g for 5  min at 4c . the purified fraction was mixed with the reaction medium ( 25  mm tris - hcl , 1  mm dtt , 0.5  mm nadp / na , 2  mm mgcl2 , 1  mm atp , 2 u / ml g6pdh , and 10  mm glucose ) and incubated for 30  min at 37c . the reaction was stopped by the addition of 10% trichloroacetic acid ( tca ) , and nadph production was measured at 340  nm . after treatment with wnt5a , the cells were detached from the culture plates using trypsin / edta ( sigma - aldrich , usa ) . next , the neurons were placed in tubes containing 5  mm glucose and then washed twice in krebs henseleit solution ( 11  mm na2hpo4 , 122  mm nacl , 3.1  mm kcl , 0.4  mm kh2po4 , 1.2  mm mgso4 , and 1.3  mm cacl2 , ph 7.4 ) , containing the appropriate concentration of glucose . after equilibration in 0.5  ml of hank 's balanced salt solution / glucose at 37c for 10  min , 0.5  ml of hank 's balanced salt solution containing various concentrations of [ 3-h ] glucose was added , with a final specific activity of 13 disintegrations / min / pmol ( approximately 1  mci / mmol ) . aliquots of 100  l were then transferred to another tube , placed inside a capped scintillation vial containing 0.5  ml of water , and incubated at 45c for 48  h. after this vapor - phase equilibration step , the tube was removed from the vial , a scintillation mixture was added , and the h2o content was measured by scintillation counting over a 5 min period . the [ 3-h ] glucose was obtained from perkinelmer , usa . the cell viability after the experiment was approximately 90% . after treatment with the respective compound , the cells were washed with pbs , collected by scraping in 0.25% trypsin-0.2% edta ( w / v ) , and pelleted . subsequently , the pellet was discarded , and the supernatant was further separated by centrifugation at 13,000  g for 30  min at 4c . finally , the supernatant was used to quantify the g6pdh activity in a reaction buffer consisting of 1  mm atp and 10  mm glucose-6-phosphate ( g6p ) after a 30-min incubation at 37c . the reaction was stopped by the addition of 10% tca , and the nadph production was measured at 340  nm . glucose oxidation via the ppp was measured using a previously described method , which is based on the difference in co2 production from [ 1-c ] glucose ( decarboxylated in the 6-phosphogluconate dehydrogenase - catalyzed reaction and in the krebs cycle ) and [ 6-c ] glucose ( only decarboxylated in the krebs cycle ) . after the treatment with wnt5a in the presence or absence of inhibitors , the medium was removed , and the neurons were washed with ice - cold pbs and collected by trypsinization . the cell pellets were resuspended in o2-saturated krebs henseleit buffer ( 11  mm na2hpo4 , 122  mm nacl , 3.1  mm kcl , 0.4  mm kh2po4 , 1.2  mm mgso4 , and 1.3  mm cacl2 , ph 7.4 ) , and 500  l of this suspension ( ~10 cells ) was placed in erlenmeyer flasks with 0.5  ml of the krebs henseleit solution containing 0.5  ci d-[1-c ] glucose or 2  ci d-[6-c ] glucose and 5.5  mm d - glucose ( final concentration ) . the erlenmeyer flasks were equipped with a central well containing an eppendorf tube with 500  l of benzethonium hydroxide . the flasks were flushed with o2 for 20  s , sealed with rubber caps , and incubated for 60  min in a 37c water bath with shaking . the incubations were stopped by the injection of 0.2  ml of 1.75  m hclo4 into the main well , although shaking was continued for an additional 20  min to facilitate co2 trapping by benzethonium hydroxide . both [ 1-c ] glucose and [ 6-c ] glucose were purchased from perkinelmer , usa . after the treatment with wnt5a , we measured the atp levels in whole - cell lysates of primary neurons using an atp determination kit ( invitrogen / molecular probes ) . the animals were housed at the animal house facility of the facultad de ciencias biolgicas , pontificia universidad catlica de chile , in accordance with the guide for the care and use of laboratory animals ( nih - usa publication 86 - 23 ) . transverse slices ( 350  m ) from the dorsal hippocampus were cut in cold artificial cerebrospinal fluid ( acsf , 119  mm nacl , 26.2  mm nahco3 , 2.5  mm kcl , 1  mm nah2po4 , 1.3  mm mgcl2 , and 10  mm glucose ) using a vibroslice microtome ( world precision instruments ) and incubated in acsf for 1  h at room temperature . the experiments were performed in a recording chamber at room temperature ( 2022c ) [ 38 , 39 ] . all the experiments were performed with an n of 5 ; we used triplicates for each condition of each experiment . the data were analyzed by one - way or two - way analysis of variance ( anova ) followed by bonferroni 's post hoc test ; p  0.05 and p  0.01 were considered significant . the activation of the noncanonical wnt pathway was determined by monitoring the p - pkc levels . we observed a 1.5-fold increase in the p - pkc / pkc ratio after 2  h of wnt5a treatment , and this increase was blocked by the wnt scavenger fzcrd-2 ( data not shown ) . the first step of glucose metabolism is the uptake of glucose from the extracellular media into the cell . we used 2-deoxyglucose ( 2-dg ) , a radioactive analog of glucose , to study glucose uptake . in the brain , glucose is taken up by glucose transporters ( gluts ) and phosphorylated by the enzyme hk , but it is not further metabolized . under control conditions , we observed a time - dependent uptake of 2-dg , with a maximum of 19.2  3.5  nmol  10 cells at 90  sec . after the wnt5a treatment , the 2-dg uptake increased , with a maximum of 39.4  5.3  nmol  10 cells at 90  sec . wnt pathway activation triggered a 2-fold increase in 2-dg uptake , with a significant difference at 15  sec . treatment with the glut inhibitor cyt b ( 20  m ) decreased 2-dg uptake to 1.56  0.4  nmol  10 cells at 90  sec ( figure 1(a ) ) . incubation with wnt5a during the initial phase of monitoring induced an increase in uptake from 9.9  1.5  nmol  10 cells to 20.8  2.0  nmol  10 cells ; this increase was blocked by the coincubation with fzcrd-2 . interestingly , we observed that 7-ni , an inhibitor of neuronal no synthase ( nnos ) [ 41 , 42 ] , blocked the effect of wnt5a , suggesting that the effect of this ligand was mediated by the intracellular release of ca followed by the downstream generation of no . we treated the cells with inhibitors of several downstream targets of noncanonical wnt signaling to further investigate this effect . neither kn-93 ( inhibitor of calcium / calmodulin - dependent protein kinase ii ( camkii ) ) nor g6976 ( inhibitor of pkc ) blocked the effect of wnt5a . by contrast , treatment with the no donor snp alone increased the 2-dg uptake to 17.8  4.3  nmol  10 cells ( figure 1(b ) ) . the kinetic parameters of glucose uptake were estimated for a more comprehensive analysis of the effect of wnt signaling . under control conditions , we observed a km value of 7.1  0.8  mm and a vmax of 9.4  0.4  pmol10  cells / min ( figure 1(c ) ) . in the wnt5a - treated cells , the km and vmax values were 2.5  0.1  mm and 8.7  0.2  pmol10 cells / min , respectively ( figure 1(d ) ) . figure 1(e ) illustrates how exposure to the wnt5a ligand increased the apparent affinity of glucose in the neuronal transport pathway . after being transported into the cell , glucose is converted to g6p by hk , the first regulatory enzyme of glycolysis . we observed that the wnt5a treatment induced a robust increase in activity from 2.2  0.17  units / mg to 4.4  0.8  units / mg after 2  h of treatment ( figure 2(a ) , ( i ) ) . the effect of wnt5a was prevented by the coincubation with fzcrd-2 and 7-ni ( figure 2(a ) , ( ii ) ) . our results thus far show that treating neurons with wnt5a stimulates the uptake of 2-dg and hk activity . the next step was to determine whether this increased uptake was correlated with an increase in the glucose utilization through glycolysis . first , we measured the glycolytic rate at several time points after treatment with wnt5a . under control conditions , we observed rates of 1.1  0.15  pmol / mg protein . wnt5a increased the glycolytic rate in a time - dependent manner , with a maximum rate of 2.9  0.3  pmol / mg protein at 2  h versus a control rate of 1.3  0.19  pmol / mg protein . this effect was partially blocked by the coincubation with fzcrd-2 ( figure 2(b ) , ( i ) ) . using a pharmacological approach , we studied the effects of several inhibitors at 2  h and observed that the effect of wnt5a was blocked by the coincubation with fzcrd-2 and 7-ni ( figure 2(b ) , ( ii ) ) . our previous results suggest that the downstream generation of no following the activation of noncanonical wnt signaling may be important for the effect of wnt5a on the glycolytic rate . therefore , we analyzed the effect of snp , a no donor , by treating cells with several concentrations of snp for 2  h ; we observed a strong increase in the glycolytic rate , with a maximum of 150  12  pmol / mg protein in the cells that were treated with 700  nm snp . the effect of snp was partially abolished by the coincubation with 7-ni . as a control , the cyt b treatment markedly decreased the glycolytic rate in neurons ( figure 2(b ) , ( iii ) ) . our results indicate that the activation of wnt signaling stimulates glucose uptake and the utilization of this molecule by glycolysis , suggesting an increase in the levels of atp . we measured the neuronal atp levels following a 2 h treatment with wnt5a to confirm this prediction . under control conditions , we measured a value of 36.02  10.03  nmol atp / mg of protein . after the treatment with wnt5a , the atp levels increased to 61.78  6.11  nmol atp / mg of protein ; this increase in the atp levels was blocked when cells were coincubated with wnt5a and the antagonist fzdcrd-2 ( figure 2(c ) ) . as described above , several metabolic pathways could use the g6p generated by hk , including the ppp . we measured the effect of wnt5a on g6pdh activity and observed an increase in the enzymatic activity after 1  h of wnt5a treatment ( figure 3(a ) , ( i ) ) . the effect of wnt5a on g6pdh activity was blocked by the coincubation with fzcrd-2 and 7-ni ( figure 3(a ) , ( ii ) ) . then , we measured the ppp activity using radioactive glucose and observed that the wnt5a treatment increased the ppp activity from 0.33  0.02  nmol / min  mg protein in the control condition to 0.77  0.07  nmol / min  mg protein . the effect of wnt5a on the ppp activity was blocked by the coincubation with fzdcrd-2 ( figure 3(b ) ) . we studied whether the effects of the wnt5a ligand could be recapitulated in a more complex model to further analyze the effect of wnt pathway activation on glucose metabolism . for this experiment , we used cortical - hippocampal slices from mouse brains ( figure 4(a ) ) . after a 30 min treatment , we observed significant time - dependent differences in the accumulation of 2-dg , with a maximum of 6.9  0.9  nmol  10 after 2  h of wnt5a treatment compared with 3.4  0.4  nmol  10 for the control condition ( figure 4(b ) , ( i ) ) . the effect of a 1 h wnt5a treatment was blocked by treatments with fzdcrd-2 and 7-ni in a similar manner to that observed in neuronal cultures ( figure 4(b ) , ( ii ) ) . after the 1 h incubation with wnt5a , we observed an increase in the glycolytic rate in the slices from 1.07  0.11  pmol / mg protein to 2.12  0.08  pmol / mg protein ; this effect was blocked by the coincubation with 2-dg and fzdcrd-2 ( figure 4(c ) ) . finally , we measured the ppp activity in slices and observed an activity of 0.32  0.01  nmol / min  mg protein in the controls . the wnt5a treatment induced an increase in the ppp activity to 0.78  0.02 01  nmol / min  mg protein ; this effect was blocked by the coincubation with fzdcrd-2 and 7-ni ( figure 4(d ) ) . in the present work , we studied the effect of wnt5a on glucose metabolism in cortical neurons . we report that wnt5a treatment stimulates glucose uptake in a time - dependent manner ; this increase was correlated with an increase in both the hk activity and the glycolytic rate . moreover , the treatment with wnt5a increased the activity of the g6pdh enzyme and the ppp . together , these results suggest that the activation of wnt signaling by wnt5a regulates cellular glucose metabolism in neurons . wnt signaling can basically be divided into two pathways : the canonical pathway ( wnt/-catenin ) and the noncanonical pathway . wnt5a has been described as a noncanonical wnt ligand , and the noncanonical pathway includes the planar cell polarity ( wnt / pcp ) and wnt / ca pathways . in the wnt / pcp pathway , the wnt ligand binds to the fzd receptor and activates small gtpases that subsequently induce the expression of genes related to the reorganization of the cytoskeleton . in the wnt / ca pathway , ligand binding to the fzd receptor activates the enzyme phospholipase c ( plc ) , which increases the production of inositol triphosphate ( ip3 ) , generating an increase in the intracellular ca concentration that leads to the activation of calcium - dependent proteins [ 4547 ] . subsequently , wnt5a signaling increases the intracellular levels of several second messengers , including ca and no , which are both considered regulators of glucose metabolism , a critical process required for brain function [ 4851 ] . the importance of the wnt pathway in regulating glucose metabolism has been increasingly recognized in recent years due to studies in humans , where several components of the wnt pathway have been identified as risk factors for metabolic diseases , including age - related dementia ; however , the final effect depends on whether the canonical or noncanonical wnt pathway is affected [ 52 , 53 ] . activation of the wnt/-catenin pathway promotes a decrease in the plasma glucose levels in vivo , and this decrease modulates the localization and expression of glut4 in adipocytes and increases glucose uptake in these cells . similarly , patients with diabetes mellitus type ii have been shown to exhibit increased expression of sfrp5 , a wnt inhibitor , which has been correlated with a decrease in wnt5a levels . furthermore , activation of the wnt / ca pathway has been reported to modulate mitochondrial dynamics , which may affect atp generation [ 56 , 57 ] . approximately 20% of the oxygen and 25% of the glucose consumed by the human body are dedicated to cerebral functions , even though the brain only accounts for 2% of the total body mass . the atp generated by glucose oxidation is used to maintain and restore the ion gradients dissipated by signaling processes , such as postsynaptic stimulation and action potentials , and for neurotransmitter uptake and recycling . glucose is the main energy substrate of the adult brain , and its metabolism is mainly divided into two stages : glucose uptake and intracellular oxidative metabolism . our data also showed that wnt5a increases the apparent affinity of the glut3 transporter , the main glut isoform expressed in neurons . because we did not observe changes in the vmax value , we can disregard the expression of other glut isoforms in our experiments . the effect of wnt5a on glucose uptake will increase the bioavailability of intracellular glucose , with a subsequent increase in the g6p levels generated by hk , another target of the wnt5a pathway . the generated g6p is mainly used by three different metabolic pathways : glycolysis , the ppp , and the glycogen synthesis pathway . glycolysis is coupled to the krebs cycle and oxidative phosphorylation to generate substrates for atp production in mitochondria through oxidative phosphorylation . by contrast , the ppp is required to protect neurons from oxidative stress through the generation of nadph [ 61 , 62 ] . however , the neuronal utilization of glucose for glycogen synthesis has been accepted for years ; neurons have recently been shown to use glucose for glycogen synthesis , a function that is typically attributed to astrocytes . however , this glycogen synthesis could induce neuronal cell death through the formation of glycogen aggregates called lafora bodies [ 64 , 65 ] . neuronal glycogen may be important for promoting neuronal survival under pathological conditions , such as oxidative stress and hypoxia [ 6668 ] . in the present work , we did not evaluate the effect of wnt5a on glycogen synthesis , but we can not discard this possibility because the importance of glycogen in neurons has recently been reported [ 67 , 69 ] . we observed that wnt5a increases the glycolytic rate and ppp activity , thus promoting atp generation and increasing the levels of the nadph cofactor . the increase in the nadph levels resulting from ppp activity could be a protective mechanism against oxidative stress ( generated by the increased ros levels produced in the mitochondria ) , because this molecule is critical for the recycling of antioxidant agents , such as ascorbic acid and glutathione [ 69 , 70 ] . additionally , we observed that the effects of wnt5a on 2-dg uptake and the glycolytic rate were blocked by an inhibitor of nnos . this finding suggests a role for intracellular no , a molecule generated downstream of the wnt / ca pathway , because ca release activates calcineurin , a phosphatase that leads to nnos activation and no production ( figure 5 ) [ 41 , 71 ] . no has been previously shown to stimulate glucose metabolism in neurons ; however , the specific mechanism underlying this effect remains unknown [ 72 , 73 ] . because the brain includes other cells , such as astrocytes , which play critical roles in the regulation of neuronal metabolism [ 7476 ] , we tested the effects of wnt5a on the more complex system of hippocampal slices . here , we observed results similar to those of the in vitro studies , suggesting that the effects of wnt5a are mainly localized to neurons ; however , further studies are required to confirm this hypothesis . our results suggest a novel function for wnt signaling as an activator of glucose metabolism in neurons . this novel role of wnt signaling in neuronal physiology might be an interesting topic in the search for new treatments for neurological disorders .
in the last few years , several reports have proposed that wnt signaling is a general metabolic regulator , suggesting a role for this pathway in the control of metabolic flux . 
 wnt signaling is critical for several neuronal functions , but little is known about the correlation between this pathway and energy metabolism . 
 the brain has a high demand for glucose , which is mainly used for energy production . 
 neurons use energy for highly specific processes that require a high energy level , such as maintaining the electrical potential and synthesizing neurotransmitters . 
 moreover , an important metabolic impairment has been described in all neurodegenerative disorders . despite the key role of glucose metabolism in the brain , little 
 is known about the cellular pathways involved in regulating this process . 
 we report here that wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway ; the effects of wnt5a require the intracellular generation of nitric oxide . 
 our data suggest that wnt signaling stimulates neuronal glucose metabolism , an effect that could be important for the reported neuroprotective role of wnt signaling in neurodegenerative disorders .