Neuropeptides xxx (2016) xxx–xxx

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Insulin and IGF-1 regularize energy metabolites in neural cells expressing full-lengthmutant huntingtin

Luana Naia a,b,1, Márcio Ribeiro a,1, Joana Rodrigues a,1, Ana I. Duarte a,c, Carla Lopes a,c, Tatiana R. Rosenstock a,c,Michael R. Hayden d, A. Cristina Rego a,b,⁎a CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3004-504, Portugalb Faculty of Medicine, University of Coimbra, Coimbra 3004-504, Portugalc Institute for Interdisciplinary Research, University of Coimbra (IIIUC), Polo II, Coimbra, Portugald Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, Canada

Abbreviations: ADP, adenosine diphosphate; Akt, promonophosphate; ATL, alanine aminotransferase; ATPcreatine; DG, 2-deoxy-D-glucose; FBS, fetal bovine serumlike growth factor 1; IR/IGF-1R, insulin/IGF-1 receptor; LDHuntington's disease; mHTT, mutant huntingtin; PC, pyrucreatine; PDH, pyruvate dehydrogenase; PI-3K, phosphoin⁎ Corresponding author at: CNC-Center for Neuroscien

of Medicine, University of Coimbra, Rua Larga, 3004-504 CE-mail addresses: a.cristina.rego@gmail.com, arego@fm

(A.C. Rego).1 The authors contributed equally for this work.

http://dx.doi.org/10.1016/j.npep.2016.01.0090143-4179/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article as: Naia, L., et al., InsulNeuropeptides (2016), http://dx.doi.org/10.1

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 September 2015Received in revised form 7 January 2016Accepted 31 January 2016Available online xxxx

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to the expression ofmu-tant huntingtin. Bioenergetic dysfunction has been described to contribute to HD pathogenesis. Thus, treatmentparadigms aimed to ameliorate energy deficits appear to be suitable candidates inHD. In previous studies, we ob-served protective effects of insulin growth factor-1 (IGF-1) in YAC128 and R6/2 mice, two HD mouse models,whereas IGF-1 and/or insulin haltedmitochondrial-driven oxidative stress inmutant striatal cells andmitochon-drial dysfunction in HD human lymphoblasts. Here, we analyzed the effect of IGF-1 versus insulin on energymet-abolic parameters using striatal cells derived from HD knock-in mice and primary cortical cultures from YAC128mice. STHdhQ111/Q111 cells exhibited decreased ATP/ADP ratio and increased phosphocreatine levels. Moreover,pyruvate levelswere increased inmutant cells,most probably in consequence of a decrease in pyruvate dehydro-genase (PDH) protein expression and increased PDH phosphorylation, reflecting its inactivation. Insulin andIGF-1 treatment significantly decreased phosphocreatine levels, whereas IGF-1 only decreased pyruvate levelsin mutant cells. In a different scenario, primary cortical cultures derived from YAC128 mice also displayed ener-getic abnormalities.Weobserved a decrease in both ATP/ADP and phosphocreatine levels, whichwere preventedfollowing exposure to insulin or IGF-1. Furthermore, decreased lactate levels in YAC128 cultures occurredconcomitantly with a decline in lactate dehydrogenase activity, which was ameliorated with both insulin andIGF-1. These data demonstrate differential HD-associatedmetabolic dysfunction in striatal cell lines and primarycortical cultures, both of which being alleviated by insulin and IGF-1.

© 2016 Elsevier Ltd. All rights reserved.

Keywords:Huntington's diseaseEnergy metabolismPyruvate dehydrogenaseLactate dehydrogenaseLactateInsulinIGF-1

1. Introduction

Huntington's disease (HD) in an autosomal dominant neurodegener-ative disorder caused by an abnormal expansion of CAG triplets in theHTT gene, encoding for mutant huntingtin (mHTT in humans, or mHttinmice). HD clinical symptoms include involuntarymovements, demen-tia, dramatic weight loss, and eventually death. Neuropathologically, HDis characterized by selective dysfunction and death of GABAergic

tein kinase B; AMP, adenosine, adenosine triphosphate; Cr,; GLUC, glucose; IGF-1, insulin-H, lactate dehydrogenase; HD,

vate carboxylase; PCr, phospho-ositide 3-kinase.ce and Cell Biology, and Facultyoimbra, Portugal.ed.uc.pt, acrego@cnc.uc.pt

in and IGF-1 regularize energy016/j.npep.2016.01.009

projection medium spiny neurons in the striatum. Moreover, the degreeof striatal atrophy correlates with the degeneration of cerebral cortexduring the latest stages (Gil and Rego, 2008). Although striatal deathunderlies many symptoms in advanced stages of the disease (Vonsatteland DiFiglia, 1998), early deficits, which seem to occur years before theevident movement disorder, are more likely associated with neuronaland synaptic dysfunction in the cortex (e.g. Rosas et al., 2005). Anexpansion ofmore than 39 CAG repeats in theHTT gene underlies severalmechanisms of neurodegeneration, such as oxidative stress, mitochon-drial dysfunction, and deficits in energy metabolism (Naia et al., 2011).In particular, mHTT impairs mitochondrial respiration and ATP produc-tion (Milakovic and Johnson, 2005; Seong et al., 2005; Silva et al.,2013). Elevated levels of lactate were also detected in the striatum andcerebral cortex of HD patients (Koroshetz et al., 1997; Jenkins et al.,1998), aswell as decreased ATP and phosphocreatine (PCr) levels follow-ing glycolysis inhibition in HD cybrids, exhibiting bioenergeticallydysfunctional mitochondria (Ferreira et al., 2011). Additionally, a diversi-ty of metabolic enzymes are altered in HD, namely, pyruvate dehydroge-nase (PDH) (Perluigi et al., 2005; Ferreira et al., 2011), pyruvate

metabolites in neural cells expressing full-length mutant huntingtin,

2 L. Naia et al. / Neuropeptides xxx (2016) xxx–xxx

carboxylase (PC) (Butterworth, 1986; Lee et al., 2013), glucose-6-phosphate dehydrogenase (Ferreira et al., 2011), aconitase (Tabriziet al., 2000), and aspartate aminotransferase (Perluigi et al., 2005).

During the last few years, our group analyzed the effects of insulinand insulin-like growth factor 1 (IGF-1) inHD, fromperipheral to neuralcells and also in in vivo models. The insulin/IGF-1 signaling has beenstudied for many years in several neurodegenerative disorders linkedto toxic protein aggregation, but it has generated conflicting results.While some studies demonstrated that IGF-1 modulates the clearanceof brain aggregation-prone proteins, such as amyloid-β peptide (Carroet al., 2002) or mHTT (Humbert et al., 2002), other authors claimedthat reduced insulin/IGF-1 signaling regulate disaggregation ofamyloid-β peptide, ataxin-3, and other polyglutamine proteins to pro-mote cell survival (Morley et al., 2002; Cohen et al., 2006; Kapperleret al., 2008; Teixeira-Castro et al., 2011). Indeed, IGF-1 signalingwas de-scribed to be deleterious in the regulation of lifespan in nematodes(Kenyon et al., 1993) and mouse models (Holzenberger et al., 2003);however, the ablation of IGF-1 or its receptor promoted brain growthretardation (D'Ercole et al., 2002), indicating that IGF-1 may stimulateneuronal development. Amore recent study found nomeasurable effectbetween the inhibition of insulin/IGF-1 pathway and the decreasedmHTT aggregation (Jakubik et al., 2014). Even so, IGF-1 plasma levelswere shown to be increased in YAC128 mice (expressing human full-length mHTT), correlating with increased body weight (Pouladi et al.,2010), and high IGF-1 levels were associated with cognitive decline inHD patients (Saleh et al., 2010).

Counterweighting the previous data, recent findings in our groupshowed that peripheral administration of IGF-1 prevented metabolicabnormalities in a hemizygous R6/2 mouse model of HD, such asimpaired glucose tolerance and age-related decrease in body weightby enriching blood insulin and IGF-1 levels (Duarte et al., 2011). Indeed,both insulin and IGF-1 may activate insulin/IGF-1 receptor (IR/IGF-1R),stimulating PI-3K/Akt signaling pathways, through direct phosphoryla-tion of HTT by Akt at serine 421 (Humbert et al., 2002),which appears topromote mitochondrial function and modulate the expression of pro-teins involved in glucose metabolism and anti-apoptotic mechanisms(Duarte et al., 2008; Naia et al., 2015). The activation of the same path-way also counteracted the increase in lactate/pyruvate ratio in bothYAC128 mice (Lopes et al., 2014) and HD human lymphoblasts (Naiaet al., 2015). In addition, we showed that the activation of IGF-1/insulin signaling pathways in striatal cells expressing 111 glutaminesprecludes mitochondrial generation of reactive oxygen species andmitochondrial dysfunction, largely reducing apoptotic and senescentcells induced by mHtt expression (Ribeiro et al., 2014). Nevertheless,it remained unknownwhether the improvement inmitochondrial func-tion would also be linked to an improvement in cell bioenergetics.Therefore, we hypothesized that insulin or IGF-1 treatment could ame-lioratemetabolic function in the context of HD. Here, we show that bothinsulin and IGF-1 can rescue energy deficits in YAC128 primary corticalcells by ameliorating ATP, PCr, and lactate levels, the later involvingmodified lactate dehydrogenase (LDH) activity. Furthermore, IGF-1alleviates anomalous pyruvate levels in homozygous STHdhQ111/Q111

striatal cells derived from HD knock-in mice.

2. Materials and methods

2.1. Materials

Fetal bovine serum (FBS), B27 supplement and penicillin/strepto-mycin were from Gibco (Paisley, Scotland, UK). Insulin from porcinepancreas, DMEM medium, trypsin-type IX-S from porcine pancreas,IGF-1, glucose-6-phosphate, deoxy-D-glucose (DG), L-glutamine,poly-L-lysine, fatty acid free bovine serum albumin (BSA), trypanblue (0.4%), resazurin, adenine dinucleotide phosphate hydrate(NADP), nicotinamide adenine dinucleotide hydrate (NAD), ADP,glucose 6 phosphate dehydrogenase (G6P-DH), hexokinase, creatine

Please cite this article as: Naia, L., et al., Insulin and IGF-1 regularize energyNeuropeptides (2016), http://dx.doi.org/10.1016/j.npep.2016.01.009

kinase, cytosine β-D-arabinofuranoside, CoASH, and thiaminepyrophosphate (TPP) from Sigma Aldrich (St. Louis, MO, USA). Bio-Rad protein assay was from Bio-Rad (Hemel Hempstead, UK). Lactate,pyruvate, and phospho-PDH in-cell ELISA kit from Abcam (Cambridge,UK). All other reagents were of analytical grade.

2.2. Primary culture of cortical cells and treatments

Heterozygous YAC128 (containing the full-length human HTT genewith 128 CAG repeats) mice embryos with 16–17 days and age-matched WT mice (as controls) (FVB/N strain) embryos were used.YAC128 mice were previously described by Slow et al. (2003). These an-imals were obtained from our local colony (CNC/Faculty of Medicine An-imal Facility, University of Coimbra) with breeding couples provided byDr. Michael Hayden (University of British Columbia, Vancouver,Canada). Animals were kept under controlled light and humidity condi-tions, being sacrificed by cervical displacement and decapitation (EUguideline 86/609/EEC); the studieswere performed according to the Hel-sinki Declaration and Guide for the Care and Use of Laboratory Animals(NIH, USA). Cortical cells were isolated using a previously described pro-cedure (Duarte et al., 2008), with some minor modifications. Briefly,mouse brains were dissected out and the cortex was digested with0.6mg/mL trypsin for 5min, at 37 °C in Ca2+- andMg2+-free Hank's bal-anced salt solution containing (in mM): 137 NaCl, 5.36 KCl, 0.44 KH2PO4,0.34 Na2HPO4.2H2O, 4.16 NaHCO3, 5 glucose, 1 sodium pyruvate and 10HEPES, at pH 7.2. Cells were then plated at a density of 9 × 104 cells/cm2 in poly-L-lysine (0.1 mg/mL) coated 96- or 12-well plates, accordingto the experimental procedure. Cells were cultured for 13 days in vitro(DIV) in 95% air and 5% CO2, in serum-free neurobasal medium supple-mented with 2% B27, 0.5 mM glutamine, and 0.12 mg/mL gentamicin.The glial cellmarkerGFAPwasused to confirm thepresence of astrocytes.According to this characterization, the percentage of neurons in ourprimary culture was about 60%, indicating the presence of a co-cultureof neuronal and non-neuronal cells.

Treatment consisted in 1 nM insulin or IGF-1, added to corticalcultures 48 h before collection. For hyperglycemia induction,neurobasal medium was supplemented with 6 mM D-glucose (BASAL)and 56 mM D-glucose (+GLUC), 24 h before collection. For hypoglyce-mia conditions, neurobasal medium was replaced by Krebs mediumcontaining the following (in mM): 132 NaCl, 4 KCl, 1.2 NA2HPO4, 1.4MgCl2, 1 CaCl2 supplemented with 6 mM D-glucose (BASAL), or in theabsence of glucose plus 2 mM deoxy-D-glucose (−GLUC + DG)20 min before the experiment.

2.3. Cell lines culture and treatments

Striatal cells derived from mice expressing normal HTT (STHdhQ7/Q7

or wild-type cells; clone 2aA5) or homozygous knock-in mice express-ing mHTT with 111 glutamines (STHdhQ111/Q111 or mutant cells; clone109-1 A) were used. The cell lines were kindly donated by Dr. MarcyE.MacDonald (Department of Neurology,Massachusetts GeneralHospi-tal, Boston, USA). The cells were maintained as described previously(Trettel et al., 2000). Striatal cells were plated on poly-L-lysine coatedglass coverslips, multiwell chambers, or flasks at a density of0.06 × 106 cells/cm2 48 h before the experiments in order to allow thedesired confluence. Twenty-four hours before the experiment, cellswere incubated with insulin (0.1 nM) or IGF-1 (1 nM).

2.4. Measurement of intracellular adenine nucleotides, phosphocreatine,and pyruvate levels

Cells were washed with ice-cold PBS and centrifuged at 145×g, for5 min (4 °C). Extracts were performed with 0.6 M perchloric acid sup-plemented with 25 mM EDTA-Na+ and then centrifuged at 20,800×gfor 2 min at 4 °C to remove cell debris. The resulting pellet was solubi-lized with 1 M NaOH and further analyzed for total protein content by

metabolites in neural cells expressing full-length mutant huntingtin,

3L. Naia et al. / Neuropeptides xxx (2016) xxx–xxx

the Bio-Rad Protein assay. After neutralizationwith 3MKOH/1.5M Tris,samples were centrifuged at 20,800×g for 5 min, at 4 °C. The resultingsupernatants were assayed for the following:

(1) ATP, ADP, and AMP determination by separation in a reverse-phase high-performance liquid chromatography column(Merck, Darmstadt, Germany), with detection at 254 nm, as de-scribed previously (Stocchi et al., 1985). The chromatographicapparatus used was a Beckman-System Gold controlled by acomputer. The detection wavelength was 254 nm, and the col-umn used was a Lichrospher 100 RP-18 (5 μm). An isocratic elu-tion with 100 mM phosphate buffer (KH2PO4), pH 6.5, and 1%methanol was performed with a flow rate of 1 ml/min. Peakidentity was determined by following the retention time of stan-dards.

(2) Intracellular phosphocreatine levels, by following NADP+ reduc-tion at 339 nm, mediated by ATP production by creatine kinase,in the presence of hexokinase and glucose-6-phosphate dehy-drogenase (G6PD), by using a Microplate SpectrophotometerSpectraMax Plus 384 (Molecular Devices, USA), according to apreviously described method (Lamprecht et al., 1974).

(3) Intracellular pyruvate levels by using the pyruvate assay kit(Abcam). In this assay, pyruvate is oxidized by pyruvate oxidase,further generating a fluorescent compound. The fluorescencewas measured by fluorimetry (Ex/Em = 535/587 nm), and theintensity was proportional to pyruvate content.

2.5. Assessment of intracellular lactate

Cells were washed with ice-cold PBS and extracted with 0.6 Mperchloric acid supplemented with 25 mM EDTA-Na+. Cell extractswere centrifuged at 20,800×g for 5min at 4 °C to remove cell debris. In-tracellular lactate levels were determined by using the lactate assay kit(Abcam) according tomanufacturer's instructions. Lactate was oxidizedby lactate dehydrogenase to generate pyruvate and NADH, which inturn interacts with the WST probe to produce a colored product(formazan; OD= 450 nm).

2.6. Assessment of intracellular lactate dehydrogenase (LDH) activity

Cells were washed with ice-cold PBS and scraped in 10 mM HEPES(pH 7.4) plus 0.01% Triton X-100. Cell extracts were obtained by freez-ing and thawing at−80° and 37 °C, respectively, and then centrifugedat 20,800×g for 5 min, at 4 °C (Eppendorf Centrifuge 5417R) to removecell debris. One hundred threemicroliters of each sample was distribut-ed in a 96-well plate, and 16 μL pyruvate 19.5mMwas added. The reac-tion started with the addition of 81 μL NADH 0.48 mM in each well.Intracellular LDH was determined spectrophotometrically by followingthe rate of conversion for reduced nicotinamide adenine dinucleotide(NADH) to oxidized NAD+, at 340 nm.

2.7. Assessment of pyruvate dehydrogenase (PDH) activity

PDH activity was measured as described previously (Zhou et al.,2008). Cells were lysed in 25.0 mM KH2PO4 and 0.5 mM EDTA,pH 7.25, containing 0.01% Triton X-100. Enzyme activity wasdetermined by following the rate of NADH formation at 340 nm. Fivemicrograms of cell sample was mixed with 2.5 μM rotenone, 0.5 mMof NAD+, 0.2 mM thiamine pyrophosphate (TPP+), 0.04 mM CoASH,and 4 mM pyruvate. The activity was calculated as the absorbanceunits per min per mg protein.

2.8. Determination of PDH E1α subunit protein levels and phosphorylation

PDH expression and phosphorylation were assessed using an in-cellELISA kit from Abcam (Cambridge, UK) following the manufacturer's

Please cite this article as: Naia, L., et al., Insulin and IGF-1 regularize energyNeuropeptides (2016), http://dx.doi.org/10.1016/j.npep.2016.01.009

instructions and using the solutions provided in the kit. Briefly, cellswere plated at confluent density, allowed to adhere, and incubatedwith insulin of IGF-1. On the day of collection, cells were fixed with3.7% formaldehyde/4% sucrose. Cells were permeabilized, blocked, andincubated with total PDH E1α antibody combined with anotherantibody against the phosphorylated forms of PDH E1α at pSer232,pSer293, pSer300, and developed by dual colorimetric detection. Celldensity was normalized after Janus Green staining. Absorbance wasmonitored using a microplate reader Spectra Max Plus 384 (MolecularDevices, USA).

2.9. AlamarBlue assay

Resazurin (1 mg/ml), diluted in neurobasal medium supplementedwith B27 (1:100), was added to cultured cells 90 min before samplepreparation. Then, the absorbancewas spectrophotometrically detectedat 570 nm and 600 nm. Resazurin reduction was calculated uponsubtraction of the absorbance at 570 nm by the absorbance at 600 nm,and adjusted to protein levels.

2.10. Data analysis and statistics

Results are themean±SEMof the indicatednumber of independentexperiments in figure and table legends. The F testwas performed to an-alyze the interaction term, as described in the figure and table legends.Comparisons among multiple groups were performed by two-wayANOVA, followed by Bonferroni post hoc test. Student's t-test was alsoperformed for comparison between two Gaussian populations, asdescribed in figure legends. p b 0.05 was considered significant.

3. Results

3.1. Insulin and IGF-1 restore energy metabolic parameters in primarycortical cultures derived from YAC128 mice

We started by characterizing the energy levels in YAC128 versuswild-type mice primary cortical cells exhibiting similar levels of HTTprotein (Supplementary Fig. S1). In these cells, we found a significantdecrease in ATP/ADP ratio (Fig. 1A) that was accompanied by anincrease in AMP levels (data not shown). Decreased ATP/ADP ratiowas restored after insulin or IGF-1 (1 nM) exposure (Fig. 1A). Impor-tantly, due to structural homology, insulin and IGF-1 can bind andactivate both IR and IGF-1R, with insulin binding to the IR with highaffinity (~1 nM), while this concentration increases 100–500-fold forIGF-1; similarly, IGF-1R preferentially binds IGF-1 (~1 nM), in whichconcentration increases 100–500-fold in the case of insulin (Rechleret al., 1980). Thus, low nanomolar concentrations of insulin and IGF-1may selectively activate each receptor subtype. Additionally, 1 nM insu-lin or IGF-1 showed no toxicity after assessing cell viability by theAlamarBlue assay (not shown).

Furthermore, we evaluated the role of glycolysis in ATP generation,providing cultured cortical cells to medium without glucose plus2-deoxyglucose (−GLUC + DG). We observed a significant decreasein ATP/ADP ratio in WT cortical cells, in relation to WT cells exposedto normal glucose (6 mM), but not in cultures derived from YAC128mice (Fig. 1B), suggesting that the glycolytic pathwaywas already com-promised inmutant cells under basal conditions.We also analyzed cells'reducing capacity by following resazurin reduction (AlamarBlue assay),an indirect measure of activity of cellular dehydrogenases. In YAC128mice-derived cortical cells, we observed a significant increase in cell re-ducing capacity (Table 1); this result was further confirmed by theMTTassay (data not shown). In order to evaluate whether the effect wasmaximal, cortical cultures were exposed to an excess of glucose(56 mM). Remarkably, WT cells responded by significantly increasingcellular reduction status by almost 2-fold, whereas YAC128 cells didnot show significant changes in resazurin reduction. These data suggest

metabolites in neural cells expressing full-length mutant huntingtin,

Fig. 1. IGF-1 and insulin restore energymetabolic parameters inYAC128primary cortical cultures. (A, C, D, E)WTand YAC128 cortical cellswere preincubated inneurobasalmedium in theabsence (BASAL) or presence of 1 nM insulin (INS) or IGF-1, for 48 h at 37 °C. (B) Primary cortical cells were incubated in Krebs medium without glucose plus deoxyglucose (2 mM)(−GLUC + DG), for 20 min at 37 °C. Energy metabolism was evaluated by determining ATP/ADP ratio, pyruvate, lactate, and phosphocreatine levels. Data are the mean ± SEM of fourindependent experiments performed in duplicates, expressed as the percentage of wild-type control cells, considering (A) 2.32 ± 0.61, (B) 1.69 ± 0.32, (C) 3.60 ± 1.44 pmol/mg prot,(D) 1290.40 ± 625.51 pmol/mg prot, and (E) 7.0 ± 2.60 pmol/mg prot as 100%. Statistical significance: (A) two-way ANOVA analyses revealed no interaction between genotype andtreatment [F(2,44) = 3.03, p = 0.0586]; however, unpaired t-test revealed a ttp = 0.0054 significance between untreated WT and YAC128 cortical cells; interaction found betweengenotype and treatment in (B) [F(1,31) = 6.37, p = 0.017], (C) [F(2,29) = 3.33, p = 0.0498], and (E) [F(2,26) = 10.02, p = 0.0006]; (D) no interaction between genotype andtreatment was found [F(2,29) = 0.39, p = 0.681]. ⁎p b 0.05, &p b 0.05, #p b 0.05, ##p b 0.01 and ###p b 0.001 by two-way ANOVA for multiple groups using Bonferroni as post hoc test.ttpb0.01 when compared to the WT control, by the Student's t-test. ns – non-significant.

4 L. Naia et al. / Neuropeptides xxx (2016) xxx–xxx

that HD cortical cells are under a maximal metabolic rate (through theactivity of cellular dehydrogenases) in the presence of a normal glucoseconcentration to compensate the energy deficit. The response ofmutantneural cells may occur independently of mitochondrial function since,

Table 1Effect of glucose overload or insulin/IGF-1 treatment on cellular reducing capacity of wild-type and YAC128 primary cortical cells.

STIMULUS CELL GENOTYPE

GLUC INS IGF-1 WT cortical cells YAC128 cortical cells

- - - 100.0 ± 4.3 150.6 ± 10.1 **

+ - - 194.2 ± 12.5 &&& 118.7 ± 16.0 **

- + - 89.8 ± 7.5 94.7 ± 16.7 ##

- - + 120.0 ± 12.0 140.8 ± 21.4

Cellular reducing capacity was measured by following resazurin reduction (AlamarBlueassay) and normalized for protein content, as described in the Materials and methodssection. Primary cortical cultureswere incubated in Krebsmediumwith 6mM(non-treated;in gray) or 56mMglucose (GLUC) for 24 h or with 1 nM insulin (INS) or IGF-1, for 48 h, at37 °C. Data are the mean ± SEM of three to five independent experiments performed induplicates, expressed as the percentage of wild-type control cells, considering 0.11 ±0.02 O.D./mg prot as 100%. Statistical significance: two-way ANOVA analyses revealed asignificant interaction between genotype and GLUC treatment [F(1,26) = 24.09, p b

0.0001]; moreover, there is a significant effect of INS and/or IGF-1 treatment [F(2,32) =

4.36, p= 0.0293]. ⁎⁎p b 0.01 vs. respective WT cortical cells; &&&p b 0.001 vs. non-treatedWT cortical cells; ##p b 0.01 vs. non-treated YAC128 cortical cells by two-way ANOVA,followed by Bonferroni post hoc test, as indicated in the Table. +, presence; −, absence.

Please cite this article as: Naia, L., et al., Insulin and IGF-1 regularize energyNeuropeptides (2016), http://dx.doi.org/10.1016/j.npep.2016.01.009

in addition to mitochondrial dehydrogenases, AlamarBlue can be re-duced by dehydrogenases located in the cytosol (e.g., Rampersad,2012), such as those involved in glycolysis and the pentose phosphatepathway or other oxidoreductase reactions, including NADPH dehydro-genases. Insulin, at 1 nM, was able to reduce the increased metabolicrate in YAC128 cortical cells, while no significant effects could be attrib-uted to IGF-1 (Table 1).

The PCr levels were also significantly reduced in YAC128 corticalcells; insulin promoted a slight (but non-significant) increase in PCr,whereas treatment with IGF-1 completed restored PCr levels (Fig. 1C).Moreover, we observed a decrease in lactate levels (Fig. 1E) and in lac-tate/pyruvate ratio (not shown) in YAC128 cells that were completelyrecovered by insulin or IGF-1, while no significant alterations weredetected in pyruvate levels (Fig. 1D).

3.2. Insulin and IGF-1 ameliorate phosphocreatine and pyruvate levels instriatal cells from HD knock-in mice

Previously,we have shown that lownanomolar concentrations of in-sulin and IGF-1 were able to activate PI-3K/Akt signaling pathway inSTHdhQ111/Q111 HD knock-in striatal cells, counteracting mitochondrialdysfunction and reactive oxygen species formation, and decreasingapoptosis (Ribeiro et al., 2014). Similarly to YAC128 cortical cells,STHdhQ111/Q111 striatal cells showed a significant decrease in ATP/ADPratio (Fig. 2A). However, neither insulin (0.1 nM) nor IGF-1 (1 nM)were able to restore the ATP levels in the HD striatal cells (Fig. 2A).Mutant striatal cells exhibited a significant decrease in HTT protein

metabolites in neural cells expressing full-length mutant huntingtin,

Fig. 2. STHdhQ111/Q111 striatal cells evidence a deficit in ATP levels and increased phosphocreatine and pyruvate levels. STHdhQ7/Q7 and STHdhQ111/Q111 striatal cells were preincubated inDMEM medium in the absence (BASAL) or presence of 0.1 nM insulin (INS) or 1 nM IGF-1, for 24 h at 33 °C. Energy metabolism was evaluated by determining (A) ATP/ADP ratio,(B) phosphocreatine, (C) pyruvate, and (D) lactate levels. Data are the mean ± SEM of three independent experiments performed in duplicates, expressed as the percentage of Q7control cells, considering (A) 21.41 ± 1.82, (B) 0.084 ± 0.003 pmol/mg prot, (C) 8640.0 ± 480.20 pmol/mg prot, and (D) 14.65 ± 0.73 pmol/mg prot as 100%. Statistical significance:(A) two-way ANOVA analyses revealed a significant effect of genotype [F(1,12) = 69.58, p b 0.0001], but no interaction between genotype and treatment was found [F(2,12) = 0.02,p = 0.985]; (B) two-way ANOVA analyses revealed a significant effect of IGF-1 treatment [p b 0.01], but no interaction between genotype and treatment was found [F(2,28) = 2.47,p = 0.103]; (C) interaction between genotype and treatment was found [F(2,30) = 10.21, p = 0.0004]; (D) no interaction between genotype and treatment was found [F(2,12) =0.27, p= 0.771]. ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001, ##p b 0.01 and ###p b 0.001 by two-way ANOVA for multiple groups using Bonferroni as post hoc test.

5L. Naia et al. / Neuropeptides xxx (2016) xxx–xxx

andmRNA levels (Supplementary Fig. S1), but neither IGF-1 nor insulinlargely affected HTT expression in the mutant cells.

Opposing to YAC128 cell cultures, striatal mutant cells showed highlevels of PCr (Fig. 2B). Moreover, the mutant cells exhibited a largeincrease in pyruvate levels (Fig. 2B); of note, IGF-1 treatment partially,but significantly, reduced abnormal PCr and pyruvate levels inSTHdhQ111/Q111 cells (Fig. 2B, C). Of relevance, increased pyruvatelevels in mutant striatal cells were accompanied by unchangedlactate levels (Fig. 2D), and thus decreased lactate/pyruvate ratio (notshown).

3.3. YAC128 cortical cells and STHdhQ111/Q111 striatal cells show downregu-lated LDH and PDH activities

Data described above suggest that LDH activity, the enzyme that cat-alyzes the interconversion of pyruvate and lactate, and/or PDH activity,the enzyme complex responsible for irreversibly converting pyruvateinto acetyl-CoA, might be altered in HD cortical and striatal cells.Concordantly, we found a significant decrease in LDH activity in bothuntreated mutant cells (Figs. 3A and 4A). Both insulin and IGF-1restored LDH activity in YAC128 cortical cells to the levels of WT cells(Fig. 3A). Indeed, the recovery in lactate levels described in YAC128cortical cells could be promoted by the rescue in LDH activity.

We further analyzed PDH E1alpha (PDHE1α) subunit levels andphosphorylation, which reflect its inactivation. In YAC128 cells we ob-served a slight, but significant, decrease in PDHE1α levels; although

Please cite this article as: Naia, L., et al., Insulin and IGF-1 regularize energyNeuropeptides (2016), http://dx.doi.org/10.1016/j.npep.2016.01.009

insulin has further exacerbated this reduction, IGF-1 completely recov-ered YAC128 PDHE1α expression to the levels of WT cortical cells(Fig. 3B). Moreover, an increase in PDHE1α phosphorylation onSer232 and Ser293 was observed in untreated YAC128 cortical cells,with no alterations in Ser300 phosphorylation, evidencing a decline inPDH activity (Fig. 3C), as described before in our laboratory in anotherHD cell model (Ferreira et al., 2011). Unexpectedly, IGF-1 further in-creased PDH phosphorylation at Ser293 (Fig. 3Cii), without changingthe phosphorylation status of the other serines.

Interestingly, a great decrease in both PDHE1α subunit and PDHenzyme activity, accompanied by an increase in phosphorylation atthe three serines studied (Fig. 4B, C, D) appears to underlie the largeincrease in pyruvate levels observed in the mutant striatal cell line. De-spite these changes, and similarly to the cortical cultures, neither insulinnor IGF-1were able to significantly restore PDH activity or PDHE1α sub-unit levels, or significantly modify PDHE1α phosphorylation at Ser232,Ser293, or Ser300 in STHdhQ111/Q111 cells (Fig. 4B–D), suggesting thatthe decrease in pyruvate levels following IGF-1 treatment was not dueto an increase in its conversion into acetyl-CoA.

4. Discussion

Under a great controversy (Carro et al., 2002; Morley et al., 2002;Cohen et al., 2006; Kapperler et al., 2008), insulin and IGF-1 havedemonstrated important effects in the central nervous system, includ-ing neuronal survival, learning and memory, and animal life span

metabolites in neural cells expressing full-length mutant huntingtin,

Fig. 3. Insulin and IGF-1 regularize LDH activity, but not PDH expression/phosphorylation in YAC128 cortical cultures. Cortical cells were preincubated in neurobasal medium in theabsence (BASAL) or presence of 1 nM insulin (INS) or IGF-1, for 48 h at 37 °C. (A) LDH activity, (B) PDHE1α protein levels, and (Ci, Cii, Ciii) phosphorylated PDHE1α were measured asdefined in the Materials and methods section. Phospho(Ser232/Ser293/Ser300)-PDHE1α were normalized for total PDHE1α, whereas total PDHE1α was normalized for proteindensity (by janus green staining, as described in the Materials and methods section). Data are the mean ± SEM of three to four independent experiments performed in duplicates,expressed as the percentage of wild-type control cells, considering (A) 2.35 ± 0.60 mol/s/mg prot, (B) 2.52 ± 0.33 O.D. (optical density), (Ci) 1.79 ± 0.24 O.D., (Cii) 0.63 ± 0.08 O.D.,and (Ciii) 0.48 ± 0.04 O.D. as 100%. Statistical significance: (A) two-way ANOVA analyses revealed no interaction between genotype and treatment [F(2,39) = 1.44, p = 0.25];however, unpaired t-test revealed a ttp = 0.0015 significance between untreated WT and YAC128 cortical cells; (B) interaction between genotype and treatment was found[F(2137) = 9.55, p = 0.0001]; in Ci, there is a significant effect of genotype [F(1,36) = 17.69, p = 0.0002], and the unpaired Student's t-test revealed a tp = 0.037 significancebetween untreated WT and YAC128 cortical cells; in Cii, two-way ANOVA analyses revealed a significant effect of IGF-1 treatment [p b 0.001], but no interaction between genotypeand treatment was found [F(1,38) = 0.83, p = 0.4441]; in Ciii, there is no interaction between genotype and treatment [F(2,38) = 0.07, p = 0.9342]. &p b 0.05, ⁎p b 0.05, ⁎⁎p b 0.01,⁎⁎⁎p b 0.001 and ###p b 0.001 by two-way ANOVA for multiple groups using Bonferroni as post hoc test.

6 L. Naia et al. / Neuropeptides xxx (2016) xxx–xxx

(D'Ercole et al., 2002; Zemva and Schubert, 2014). Moreover, these pep-tides have been previously described to stimulate glucose uptake andintracellular metabolism in peripheral tissues (Andreassen et al., 2002;Duarte et al., 2011; Naia et al., 2015); however, its role in brain metab-olism is not well defined. Previously, we have shown that YAC128mousemodel presented centralmetabolic deficits. In that respect, intra-nasal IGF-1 administration enhanced IGF-1 cortical levels, partially re-covering energy levels (energy charge and lactate/pyruvate ratio) inthe brain of theseHDmice,whichwas linked to enhancedAkt activation(Lopes et al., 2014). Indeed, IGF-1/Akt signaling was previously shownto prevent HTT-induced toxicity (Humbert et al., 2002) and mitochon-drial dysfunction (Ribeiro et al., 2014; Naia et al., 2015) through HTTphosphorylation at Ser421. Nevertheless, the direct link to energy me-tabolism is unknown. Here, we present evidence showing thatYAC128 primary cortical cultures and STHdhQ111/Q111 HD knock-instriatal cells exhibit a differentmetabolic profile; however, bothmodelssupport some bioenergetic dysfunctions that have been described inHD. Remarkably, the activation of insulin/IGF-1 pathway totally orpartially counteract the changes in energy metabolites in HD corticalcultures, while the effect in striatal cells is not so evident. The lesseffectiveness to the treatment (at low nM concentrations)may be relat-ed to striatal cell growth in a culture media richer in embryonic growthpromoting factors, such as insulin, IGF-1, and also IGF-2.

One of the most described metabolic changes in HD is the deficit inATP/ADP ratio (Gines et al., 2003; Seong et al., 2005). A decrease inATP/ADP ratio implicates that either ATP is being less produced or

Please cite this article as: Naia, L., et al., Insulin and IGF-1 regularize energyNeuropeptides (2016), http://dx.doi.org/10.1016/j.npep.2016.01.009

more consumed. In both cortical and striatal HD models, weobserved a significant decrease in ATP/ADP levels, due to a decreasein ATP; this was accompanied by an increase in ADP and AMP levelsin cortical cells and by an increase in ADP levels in striatal cells (notshown). Indeed, lower ATP production was previously reported inSTHdhQ111/Q111 HD knock-in striatal cells and in R6/2 mice striatum(Dedeoglu et al., 2002; Milakovic and Johnson, 2005; Ryu et al.,2005). We also found that PCr levels are highly increased in striatalcells, in accordance with Mochel et al. (2012). Indeed, exacerbatedlevels of PCr observed inHD striatal cells may be a plausible explanationfor decreased ATP pool. Nevertheless, a slight reduction in PCr levels in-duced by IGF-1 in mutant striatal cells was not sufficient to counterbal-ance the change in ATP/ADP ratio. Conversely, HD cortical primarycultures presented a large decrease in PCr levels and in ATP/ADP ratio,as described before in other HD models (Jenkins et al., 1998; Lodiet al., 2000). Data suggest that under these conditions, decreased ATPlevels may abrogate the maintenance of a pool of PCr. Interestingly,the levels of PCr in primary cortical cells (3.60± 1.44 pmol/mg protein)were higher than in the striatal cell line (0.084 ± 0.003 pmol/mg pro-tein). Importantly, ATP/ADP and PCr levels in HD primary cultureswere almost completely recovered following treatment with insulinand IGF-1, supporting our previous results performed in lymphoblastsderived from symptomatic HD patients where IR/IGF-1R activationcompletely recovered mitochondrial and metabolic function in aPI-3K/Akt-dependent manner (Naia et al., 2015). In contrast, otherstudies showed that stimulation of IR or IGF-1R increased oxidative

metabolites in neural cells expressing full-length mutant huntingtin,

Fig. 4. STHdhQ111/Q111 cells show decreased LDH activity and PDH E1alpha subunit expression and increased PDH phosphorylation. STHdhQ7/Q7 and STHdhQ111/Q111 striatal cells werepreincubated in DMEM medium in the absence (BASAL) or presence of 0.1 nM insulin (INS) or 1 nM IGF-1, for 24 h at 33 °C. (A, B) LDH and PHD activity, (C) PDHE1alpha proteinlevels and (Di, Dii, Diii) phosphorylated PDHE1alpha were measured as defined in the Materials and methods section. Phospho(Ser232/Ser293/Ser300)-PDHE1α were normalized fortotal PDHE1α, whereas total PDHE1α was normalized for protein density (by janus green staining, as described in the Materials and methods section). Data are the mean ± SEM ofthree to five independent experiments performed in duplicates, expressed as the percentage of STHdhQ7/Q7 control cells, considering (A) 1.17 ± 0.14 mol/s/mg prot, (B) 1177.54 ±142.80 slope/mg prot, (C) 3.60 ± 0.19 O.D., (Di) 4.32 ± 0.47 O.D., (Dii) 3.94 ± 0.39 O.D., and (Diii) 3.28 ± 0.21 O.D. as 100%. Statistical significance: (A) two-way ANOVA analysesrevealed no interaction between genotype and treatment [F(2,29) = 0.85, p = 0.436]; however, unpaired t-test revealed a tp = 0.0168 significance between untreated WT and HDstriatal cells; a significant effect of genotype was found in B [F(2,12) = 34.94, p b 0.0001], Di [F(1,36) = 46.95, p b 0.0001], Dii [F(1,35) = 51.43, p b 0.0001], and Diii [F(1,35) = 32.35,p b 0.0001]; (C) significant effect of both genotype [F(1111) = 129.06, p b 0.0001] and treatment [F(1111) = 5.21, p = 0.0069]; however, there is no interaction between them[F(1111) = 2.37, p = 0.098]. &&p b 0.01, ⁎p b 0.05, ⁎⁎p b 0.01, and ⁎⁎⁎p b 0.001 by two-way ANOVA for multiple groups using Bonferroni as post hoc test.

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stress and mitochondrial dysfunction in different models, including inR6/2 HDmice brain (Holzenberger et al., 2003; Sadagursky et al., 2011).

Altered levels of pyruvate and lactate were also reported in severalHD models. We previously described increased lactate/pyruvate levels,both in HD cybrids (Ferreira et al., 2011) and in YAC128 mice brain(Lopes et al., 2014). Similar results were observed in the cortex of HDpatients (Koroshetz et al., 1997; Martin et al., 2007) and in R6/2 micebrain (Tsang et al., 2006). In opposition to previous studies, the presentwork shows decreased lactate/pyruvate ratio in both HD cell models.Reduced intracellular lactate/pyruvate ratio in HD cells may beaccounted for by changes in the glycolytic pathway (which was provenbe severely altered since neither glycolysis inhibition nor stimulationhad an effect onmutant cells), and/or altered activity of PDH or LDH. In-deed, we previously observed dysfunctional PDH E1alpha subunit levelsand activity in HD cybrids (Ferreira et al., 2011). In agreement, STHdh-Q111/Q111 cells exhibited a significant decrease in LDH and PDH activitiesand increased PDH E1alpha phosphorylation/inactivation, justifying theincrease in pyruvate levels. Although IGF-1 significantly reduced pyru-vate levels, it was unable to rescue PDH activity or E1alpha subunitlevels and phosphorylation in striatal cells, suggesting that the decreasein pyruvate levels observed in the presence of IGF-1 was not due to itsirreversible conversion into acetyl-CoA or an increased generation oflactate through LDH. Therefore, we hypothesize that in these cells IGF-1may favor pyruvate conversion into alanine through reversible alanineaminotransferase (ALT) or into oxaloacetate through PC.

In YAC128 cortical cells there is a different scenario; in contrast tomost published studies (Koroshetz et al., 1997; Jenkins et al., 1998;

Please cite this article as: Naia, L., et al., Insulin and IGF-1 regularize energyNeuropeptides (2016), http://dx.doi.org/10.1016/j.npep.2016.01.009

Martin et al., 2007), decreased lactate/pyruvate ratio was originatedfrom an ~50% decrease in lactate levels. Reduced LDH activity anddysfunctional astrocytic metabolism (Jenkins et al., 1993; Gårsethet al., 2000) may account for this decrease. Indeed, lactate producedfrom anaerobic glycolysis in astrocytes (as glial cells constitute about40% of our cortical culture) provides the primary metabolic fuel forneurons. Thus, decreased lactate levels in YAC128 cortical culturesmay account for by enhanced levels of PC (an astrocyte specific mito-chondrial enzyme), as described in an HD transgenic mice (Lee et al.,2013), decreasing the astrocytic pool of pyruvate. Importantly, both in-sulin and IGF-1 recover lactate and LDH levels. Enhanced lactate levelsfollowing insulin and IGF-1 treatment may result from increased mito-chondrial hexokinase II, a key mediator of glycolysis (Wolf et al., 2011;Ribeiro et al., 2014). Remarkably, despite decreased PDH and LDH activ-ities in YAC128 cortical cells, pyruvate levels were maintained. Indeed,other important metabolic enzymes that are markedly affected in HDcaudate nucleus, namely, ALT and PC (Carter, 1984; Butterworth,1986; Lee et al., 2013), catalyze processes that represent alternativefates for pyruvate. In this perspective, alanine could re-establish thepyruvate pool in neurons through ALT.

Over the years several studies have deepened the metabolic path-ways in different HDmodels and, although they are far from resemblingthe human brain, they help to define the intricate mechanisms regulat-ing cellular fate in HD. Previously, we have shown that IGF-1 protectedagainst diabetic features in the R6/2 model of HD (Duarte et al., 2011);we also showed that intranasal administration of IGF-1 ameliorates glu-cose brainmetabolism andmotor function in 6month-old YAC128mice

metabolites in neural cells expressing full-length mutant huntingtin,

8 L. Naia et al. / Neuropeptides xxx (2016) xxx–xxx

(Lopes et al., 2014). Here, we provide evidence that, in addition toimproving mitochondrial function in HD cells (Ribeiro et al., 2014;Naia et al., 2015), insulin/IGF-1 differentially counteract the metabolicimpairment observed in the twoHDcellmodels studied, by regularizingenergy metabolites, namely ATP, PCr and lactate/pyruvate, and LDHactivity, supporting the hypothesis that these peptides alleviatebioenergetics deficits in HD.

Fig. S1 showsHTT protein levels in YAC128 cortical cultures and HTTprotein and mRNA levels in HD striatal cells treated with insulin or IGF-1. The materials and methods related to Fig. S1 are also described inSupplementary section. Supplementary data associatedwith this articlecan be found in the online version, at http://dx.doi.org/10.1016/j.npep.2016.01.009.

Conflict of interest

The authors declare that there are no conflict of interests regardingthis study.

Acknowledgements

This work was supported by “Fundação para a Ciência e Tecnologia”(FCT), Portugal, grant referencePTDC/SAU-FCF/66421/2006 and PTDC/SAU-FCF/108056/2008, and co-financed by COMPETE-“ProgramaOperacional Factores de Competitividade”, QREN, and the EuropeanUnion (FEDER-“Fundo Europeu de Desenvolvimento Regional”). CNCwas supported by project PEst-C/SAU/LA0001/2013-2014. L. Naia, M.Ribeiro, and C. Lopes are/were supported by Ph.D. fellowships fromFCT (SFRH/BD/86655/2012, SFRH/BD/41285/899/2007, and SFRH/BD/51192/2010, respectively). A.I. Duarte and T.R. Rosenstock were sup-ported by postdoctoral fellowships from FCT (SFRH/BPD/26872/2006and SFRH/BPD/44246/2008, respectively).

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