photoperiodic regulation of glycogen metabolism, glycolysis, and glutamine synthesis in tanycytes of...

Photoperiodic Regulation of Glycogen Metabolism,Glycolysis, and Glutamine Synthesis in Tanycytes ofthe Siberian Hamster Suggests Novel Roles ofTanycytes in Hypothalamic Function

KANISHKA NILAWEERA,1,2 ANNIKA HERWIG,1 MATEI BOLBOREA,1,2 GILL CAMPBELL,1

CLAUS D. MAYER,3 PETER J. MORGAN,1 FRANCIS J. P. EBLING,2 AND PERRY BARRETT1*1Rowett Institute for Nutrition and Health, University of Aberdeen, Aberdeen, United Kingdom2School of Biomedical Sciences, University of Nottingham Medical School, Nottingham, United Kingdom3Biomathematics Statistics Scotland, Rowett Institute for Nutrition and Health, University of Aberdeen,Aberdeen, United Kingdom

KEY WORDShypothalamus; seasonality; melatonin; monocarboxylatetransporter

ABSTRACTThe objective of this study is to investigate the impact ofphotoperiod on the temporal and spatial expression of genesinvolved in glucose metabolism in the brain of the seasonalmammal Phodopus sungorus (Siberian hamster). In situhybridization was performed on brain sections obtainedfrom male hamsters held in long photoperiod (high bodyweight and developed testes) or short photoperiod (reducedbody weight with testicular regression). This analysisrevealed upregulation in expression of genes involved inglycogen and glucose metabolism in short photoperiod andlocalized to the tanycyte layer of the third ventricle. On thebasis of these data and a previously identified photoperiod-dependent increase in activity of neighboring hypothalamicneurons, we hypothesized that the observed expressionchanges may reflect alteration in either metabolic fuel orprecursor neurotransmitter supply to surrounding neurons.Gene expression analysis was performed for genes involvedin lactate and glutamate transport. This analysis showedthat the gene for the lactate transporter MCT2 and gluta-mate transporter GLAST was decreased in the tanycytelayer in short photoperiod. Expression of mRNA forglutamine synthetase, the final enzyme in the synthesis ofthe neuronal neurotransmitter precursor, glutamine, wasalso decreased in short photoperiod. These data suggest arole for tanycytes in modulating glutamate concentrationsand neurotransmitter supply in the hypothalamic environ-ment. VVC 2011 Wiley-Liss, Inc.

INTRODUCTION

Most mammals native to temperate and boreal habi-tats display marked annual cycles of body fattening andenergy metabolism as a strategy to promote winter sur-vival and then reproductive success in the followingspring when food availability and environmental condi-tions are favorable. These natural cycles of appetite andenergy expenditure have been widely exploited as modelsystems to understand the hypothalamic mechanisms

underlying control of body weight. The annual cycle inphotoperiod is the major determinant of such seasonalcycles, and profound changes in body weight can be reli-ably generated in Siberian hamsters (Phodopus sungo-rus) housed in the laboratory by simple manipulation ofthe environmental light–dark cycle (Figala et al., 1973;Steinlechner and Heldmaier, 1982). Thus, exposure towinter-like short days initiates a catabolic state, with upto 40% of body weight, mainly representing abdominalfat depots, being lost, with a concomitant decrease infood intake.

Huge advances have been made in the last decade inunderstanding the anatomical pathways and molecularmechanism underlying the recognition of photoperiodand the transduction of daylength information to thebrain. A critical component of the mechanism is theaction of thyroid-stimulating hormone (TSH) derivedfrom the pars tuberalis acting on tanycytes embedded inthe ventral ependymal lining of the third ventricle(Hanon et al., 2008; Nakao et al., 2008; Ono et al.,2008). The origin of tanycytes is not entirely clear, butthey are considered to be derived from radial glia cells,which also give rise to astrocytes during the course ofbrain development (Rodr�ıguez et al., 2005). As a result,they share the properties of astrocytes such as expres-sion of glia fibril acidic protein (GFAP), the glutamatetransporter GLAST, and aquaporin AQP9 (Berger andHediger, 2001; Elkjar et al., 2000). One of the majoractions of TSH is to upregulate type II deiodinase (Dio2)expression and downregulate type III deiodinase (Dio3)

Kanishka Nilaweera and Annika Herwig contributed equally to this work.

Grant sponsor: The Scottish Executive Environment and Rural AffairsDepartment; Grant sponsor: The Biotechnology and Biological Sciences ResearchCouncil (UK); Grant number: BB/E020437/1; Grant sponsor: The European UnionDiabesity Integrated Project (Aberdeen); Grant number: Framework VII: LSHM-CT-2003-503041; Grant sponsor: Marie Curie Individual Fellowship; Grant num-ber: PIEF-GA-2009-235106.

*Correspondence to: Dr. Perry Barrett, Rowett Institute for Nutrition andHealth, University of Aberdeen, Greenburn Road, Buckburn, Aberdeen AB21 9SB,United Kingdom. E-mail: [email protected]

Received 30 March 2011; Accepted 16 June 2011

DOI 10.1002/glia.21216

Published online 18 July 2011 in Wiley Online Library (wileyonlinelibrary.com).

GLIA 59:1695–1705 (2011)

VVC 2011 Wiley-Liss, Inc.

expression in tanycytes. The net effect is to increase thy-roid hormone (T3) availability in the hypothalamus(Barrett et al., 2007; Guadano-Ferraz et al., 1997;Lechan and Fekete, 2005; Tu et al., 1997) in the long-day-induced summer state, and reduce it in the short-day-induced catabolic state. The change in hypothalamicavailability of T3 resulting from these changes in geneexpression underlies the seasonal metabolic cycles; forexample, our studies demonstrated that microimplantsplaced in the hypothalamus, which chronically releasethyroid hormone, can overcome a programmed short-day-induced change in physiology (Barrett et al., 2007).Although the hypothalamic actions of thyroid hormonein regulating energy metabolism remain to be identified,these observations point to a central role for tanycytesin the programmed physiological responses to seasonalvariation in daylength.

It has long been recognized that there are also sea-sonal morphological changes in tanycyte end feet suchthat the ensheathment of neuronal terminals in the me-dian eminence changes, which likely relate to alteredneuroendocrine output (Barrett et al., 2006; De Serannoet al., 2010; Kameda et al., 2003). Recently, we andother research groups have identified a number ofsignificant alteration in gene and protein expression intanycytes that may not only underpin these structuralchanges (the intermediate filament proteins nestin andvimentin) but also engender other physiological changes(Barrett et al., 2007; Herwig et al., 2009; Kameda et al.,2003; Ross et al., 2004, 2005; Shearer et al., 2010).

Some of these genes were serendipitously identified instudies that used laser capture microdissection coupledwith a microarray analysis of gene expression to deter-mine seasonal regulation of genes in the hypothalamicarcuate nucleus, a major homeostatic regulatory center forenergy metabolism. In situ hybridization revealed thatspecific changes in gene expression were occurring in theependymal cell layer rather than the neuropil of the arcu-ate nucleus. The genes identified encode important path-ways in glycogen metabolism and glycolysis. These initialdata led to the identification of other genes regulated byphotoperiod in tanycytes involved in glucose metabolism,glutamine synthesis, lactate and glutamate transport sup-porting a view of novel roles for tanycytes in nutrient andmetabolite sensing by the hypothalamus, and in metabolicsupport of adjacent hypothalamic neurons.

MATERIALS AND METHODSAnimals

Male Siberian hamsters (Phodopus sungorus) weredrawn from a breeding colony maintained at the RowettResearch Institute, where the hamsters were gestated,suckled, and reared in long-day photoperiod (LD, 16-hlight and 8-h darkness). All research involving animalswas licensed under the Animals (Scientific Procedure)Act of 1986 and received ethical approval from the Uni-versity of Aberdeen, Rowett Research Institute Ethicscommittee. Food and water were available ad libitum,

unless otherwise stated. Six-months-old hamsters weresubjected to experimental manipulation and were culledin the middle of the light phase. Brains were dissected,frozen on dry ice, and stored at 270�C until required.For analysis of long-day and short-day expression pat-terns, hamsters were individually housed and main-tained in LD or short-day photoperiod (SD, 8-h light and16-h darkness; n 5 6 both groups) for 14 weeks (physio-logical parameters after 14 weeks, LD body weight37.2 6 1.4 g, SD body weight 28 6 1.8 g, LD testesweight 477 6 53 mg, and SD testes weight 35 6 2 mg).For preparation of hypothalamic tissue for electronmicroscopy, hamsters were maintained in LD or SD for8 weeks from weaning by which time they had estab-lished a significant body weight differential (LD 33.6 61.7 g, n 5 5; SD 25.2 6 0.5 g, n 5 6). For analysis ofglycogen phosphorylase (GP) expression in response tofood deprivation, archived slides were used from a previ-ous experiment of 48-h food-deprived long-day andshort-day hamsters (Herwig et al., 2009).

Cloning of cDNA Fragments and Generationof Riboprobes

Hypothalamic blocks were dissected from hamsterbrains. Total RNA was isolated using RNeasy Mini Kit(Qiagen, United Kingdom) and treated with DNase 1(Promega, Southampton, United Kingdom). Complemen-tary DNA was synthesized using 1 lg of total RNA bySuperscript P reverse transcriptase (Invitrogen, Paisley,United Kingdom). The cDNAwas amplified by PCR usingprimers complementary to mRNAs encoding lactate dehy-drogenase subunit B (LDH-B gene name, ldhb), acetyl-CoA carboxylase 1 (ACC1, gene name Acaca) and mono-carboxylase transporter 2 (MCT2, gene Slc16a7), gluta-mine synthetase (GS, gene Glul), (GLT1, gene Slc1a1),and GLAST (gene Slc1a3). The primers used for thesewere: LDH-B Forward 50-ACTTGCCCTGGTGGATGTGTTG, Reverse 50-CTTAGGTAGCCCGCTCAGTTTCC,ACC1 Forward 50-CGGATGGGCGGGATGGTCTC, Reverse50-GTCGATAAATGCGGTCCTCCTC; MCT2 Forward 50-GATGGCTTTTGTTGATATG; Reverse 50-CTCTTTCTCTGTCTGAGGG; Glutamine synthetase Forward 50-GGRGAGAGG-CAGATYTATCAC, Reverse 50-CTCCCTTGTTGGARAACT-CATA; GLT1 Forward 50-CCGCCTAGGCACGAGAGC,Reverse 50-GCCGAAAGCAATAAAGAATCC; GLAST for-ward 50-TGGGGATGCGMGCTGTRGTCTATT, Reverse50-CGCTTGCCCCTGCTCCTTCAT. The products of thePCR reactions were cloned into pGEM-Teasy plasmid(Promega, United Kingdom; LDH-B, ACC1 MCT2, gluta-mine synthetase) or pSC-B-amp/kan (Agilent Technologies,Stockport, United Kingdom; GLT1, GLAST). DNA frag-ments corresponding to mRNAs encoding glycogen phospho-rylase (GP, gene Pygb), phosphofructokinase-C (PFKC, genePfkp) were isolated from a hamster hypothalamic cDNAlibrary (Nilaweera et al., 2009). Plasmid DNAs wereisolated and sequenced to verify the cloned sequence.

Plasmids containing PCR fragments were linearizedusing appropriate restriction enzymes, or DNA fragments

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encompassing the insert together with polymerasebinding sites were amplified with M13 forward andreverse primers. Antisense and sense riboprobes weregenerated by T7 or SP6 RNA polymerase in transcriptionreactions containing 35S-UTP 0.5–1.0 lg of linearizedplasmid or 50–100 ng of amplified DNA fragment.

In Situ Hybridization

Fourteen micron sections spanning the hypothalamuswere cut between Bregma coordinates �20.10 to 22.54mm (Franklin and Paxinos, 1997) and collected ontopoly-L-lysine-coated slides (Nilaweera et al., 2002).In situ hybridization was carried out as described(Barrett et al., 2007). Slides were apposed to KodakBiomax MR film for 5–7 days. The hybridization signal(background-corrected optical density integrated over allpixels in the area) in each region of interest was quanti-fied using Image Pro-Plus (Media Cybernetics, MD). Thedata were standardized using 14C autoradiographicmicroscales (Amersham, United Kingdom). Followingquantification, slides were coated with autoradiographicemulsion and developed after 5 weeks’ exposure. Theslides were viewed using Olympus BX50 microscope(Diagnostic Instruments, United Kingdom).

Electron Microscopy

Twelve Siberian hamsters were kept in LD or SD for8 weeks from weaning. The hypothalamus was dissectedand fixed in 2.5% glutaraldehyde in 0.1M phosphatebuffer for 48 h before being processed to Spurr’s resin.The tissue was washed 3 3 15 min in 0.1M phosphatebuffer and subsequently incubated in osmium tetroxide(OsO4). After 3 3 10 min washes in distilled water, spec-imen were transferred to 30% ethanol for 30 min, thenthrough 70%, 95% ethanol and three acetone washes for60 min each. Samples were forwarded to Acetone:Spurr’s Resin solutions with increasing Spurr’s concen-trations (7:1, 3:1, 1:1 for 3 h each, 1:3, 1:6 for 6 h each,1:0 3 h minimum) before finally being embedded inSpurr’s resin in 60�C vacuum oven using embeddingcapsules (TAAB, Aldermaston, United Kingdom). Thinsections of 80–100 nm were cut using a Leica UC6 ultra-microtome and mounted onto mesh nickel grids.

Electron Microscopic Staining for Carbohydrates

Sections were treated with 1% aqueous periodic acid for20–25 min at RT and washed with two rapid changes ofdistilled water then placed in distilled water for 10 min.Afterward specimens were floated on 0.2% thiocarbohydra-zide in 20% acetic acid for 45 min followed by 2 3 15-minwashes on 10% acetic acid and a 5-min wash on 5% as wellas 1%. After 2 3 5-min washes in distilled water, sectionswere treated with 1% silver proteinate solution for 30 minat room temperature in the dark. Finally, samples werewashed on five changes of distilled water.

Statistical Analysis

The data were analyzed using SigmaStat software(Jandel, CA). The normality and the homoscedasticity ofthe distributions were tested as a preliminary step andthen followed by either a student t-test (t-test) or Mann–Whitney (M-W) rank sum test according to the situation.Statistical significance was established at P < 0.05.Data are presented as mean 6 SEM.

RESULTSPhotoperiod Regulates Expression of Enzymes

Involved in Glycogen Metabolism and Glycolysis

In a previous study, we used laser capture microdis-section of the dmpARC coupled with a microarray analy-sis of gene expression as an approach to identify genesaltered by a change in duration of photoperiod in thisregion of the hypothalamus (Nilaweera et al., 2009).Analysis of the array data identified two genes that maybe upregulated in SD that were worthy of further analy-sis. The first sequence encodes for the rate-limitingenzyme of the glycolytic pathway, phosphofructokinaseC (PFKC), which catalyses the phosphorylation of fruc-tose-6-phosphate to fructose1,6-bisphosphate. The sec-ond sequence encodes for brain GP, which catalyses thebreakdown of stored glycogen to glucose (see Fig. 1).These genes were increased in SD by approximately

Fig. 1. An outline of the glycogen–glycolytic pathway and Krebscycle indicating the steps where genes for enzymes analyzed in thisstudy are involved. Encapsulated in a box to represent a tanycyte cell,the lactate (MCT2) and glutamate (GLAST) transporters are indicatedto show the potential relationship to the above pathways. Arrows sum-marize the direction of change in tanycytes of hamsters held in short-day (SD) photoperiod.

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five-fold (P < 0.05) and 1.3-fold, respectively, althoughthe latter difference did not quite reach statistical signif-icance (P 5 0.06). Both sequences were further investi-gated for temporal and spatial expression by in situhybridization. This analysis revealed that both GP andPFKC show a widespread expression in the brain; how-ever, at 14 weeks after transfer to SD, both genes wereupregulated in a region adjacent to the third ventricle(GP 2.4-fold P < 0.008, PFKC 3.8-fold P < 0.001, Fig. 2).

Emulsion coating tissue sections revealed these cells cor-respond to tanycytes (see Fig. 3), where we have previ-ously observed regulated expression of Dio2 and Dio3(Barrett et al., 2007; Herwig et al., 2009). These datapoint to the catabolism of glycogen and an increase inglycolysis in tanycytes following transfer from LD to SD.To assess if the change in GP mRNA expression is regu-lated by photoperiod per se or as a secondary conse-quence of a metabolic challenge due to reduced food

Fig. 2. Analysis of glycogen phosphorylase (GP) and phosphofructo-kinase C (PFKC) gene expression in tanycytes by in situ hybridization.(A) Image and (B) quantification of autoradiographs showing GP geneexpression in tanycytes of LD and SD hamsters held in respectivephotoperiods for 14 weeks. (C) Image and (D) quantification of autora-diographs showing phosphofructokinase C gene expression in tanycytes

of LD and SD hamsters held in respective photoperiods for 14 weeks.(E and F) Section and quantification of autoradiographs demonstratingthe response of GP in tanycytes of hamsters fed ad-libitum orstarved for 48 h in LD and SD hamsters held in respective photoper-iods for 8 weeks. Arrow indicates tanycyte layer *Pt-test < 0.05,***Pt-test < 0.001.


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intake, in situ hybridization was performed on brain sec-tions of hamsters held in LD or SD for 8 weeks beforebeing food-deprived for 48 h. However, food deprivationdid not induce expression of GP mRNA in tanycytes inLD or affect the increase in expression in SD (Fig. 2C).

Photoperiod Regulates Expression of KeyEnzymes Involved in the Fate of Pyruvate

Pyruvate is the final product of the glycolytic pathway,and several options are available for metabolism of thisproduct (see Fig. 1). We hypothesized that a function of

this pathway may be the provision of lactate to sur-rounding neurons (Dringen et al., 1993; Swanson, 1992;Swanson and Choi, 1993), in particular those of theregion of the dmpARC that shows increased neuronalactivity in SD. Pyruvate and lactate are reversibly inter-converted by isoforms of the enzyme lactate dehydrogen-ase (LDH). The enzyme composed of subunits of isoformA drives the conversion from pyruvate to lactate. Theenzyme composed of subunits of isoform B drives theconversion from lactate to pyruvate. In situ hybridiza-tion with riboprobes complementary to transcripts ofLDH subunit genes A and B encoding functional homo-or heterodimers of LDH enzymes revealed expression ofboth transcripts in tanycytes. Following 14 weeks in SDexpression of LDH-B subunit was found to be increasedby 50% (P < 0.05) (Fig. 4A).

In addition to the effect of photoperiod on LDH subu-nit expression, analysis was performed on the expres-sion of monocarboxylate transporters MCT2 and MCT4that participate in lactate and pyruvate transport to andfrom cells in the brain (Bergersen, 2007). Of thesetransporters, only MCT2 expression was detected in theependymal layer, which included both tanycytes andependymocytes. Expression in the tanycytes was signifi-cantly reduced during SD by 40% (P < 0.001, Fig. 4B).

An alternative hypothesis for the fate of pyruvate isentry into the Krebs cycle to form the Krebs cycle inter-mediate, a-ketoglutarate, an intermediate in astrocytesfor the synthesis of glutamine, a key neurotransmitterexported by astrocytes to neuronal cells (Bak et al.,2006; Hertz et al., 1999). The final step in the pathwayis the synthesis performed by glutamine synthetase(GS). In situ hybridization analysis reveals widespreadGS expression in the brain, including the ependymallayer in the location of tanycytes, where expression isreduced in SD hamsters (Fig. 4C). This suggests gluta-mine synthesis is decreased in SD hamster tanycytes.

Preliminary analysis was also performed for theexpression of glutamate dehydrogenase (preceding stepin glutamine synthesis), malate dehydrogenase, andpyruvate carboxylase. While, as expected, these showedwidespread expression in the brain, no variation inexpression was observed in the ependymal layer, andthese genes were not investigated further.

A further fate of pyruvate is decarboxylation to acetylCoA (see Fig. 1). Acetyl CoA is a key substrate involved inde novo fatty acid synthesis (Wolfgang and Lane, 2006).The first step in this synthetic pathway is carboxylation ofacetyl CoA by acetyl-CoA carboxylase 1 (ACC1), leading tothe production of malonyl-CoA. In situ hybridization of aprobe for ACC1 detected expression in tanycytes, where itincreased by 50% in SD (P < 0.05) (Fig. 4D).

Regulation of the Glutamate Transporter GeneExpression

As tanycytes and astrocytes share a common origin, wehypothesized that the regulation of Glul gene expressionmay be related to glutamate recycling, a function performed

Fig. 3. Dark-field images of emulsion-coated brain sections fromhamsters held in either LD or SD for 14 weeks showing ependymal cellsconstituting the tanycyte layer following hybridization of riboprobes forthe genes indicated. �V� in the SD panel for each gene indicates theposition of the third ventricle. Arrow in each LD panel indicates thetanycyte cell layer.


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by astrocytes for the provision of glutamine to neurons forneurotransmitter synthesis (Bak et al., 2006). GlutamatetransportersGLT1 andGLAST have previously been shownto be highly expressed in rat tanycytes (Berger and Hediger,2001). In situ hybridization of riboprobes for these genesrevealed widespread expression of both genes throughoutthe brain. However, GLAST was found to be highlyexpressed in the hamster tanycyte and ependymal layer.GLAST expression quantified in the tanycyte layerin hamsters held in LD and SD revealed a decrease of 48%(P< 0.05) in SD hamsters (Fig. 5A). In addition to photoper-iod regulation in tanycytes, GLAST expression was alsoobserved to change in the arcuate nucleus, decreasing by31% (P< 0.05) in SD hamsters (Fig. 5B).

Glycogen Presence in Tanycytes

Electron microscopy confirmed the presence of glycogenparticles in the tanycyte cells. Electron micrographs wereexamined for three juvenile hamsters in LD and three

juvenile hamsters in SD. Glycogen particles wereobserved in tanycytes of hamsters in LD but not SD. Lipiddroplets were observed in both LD and SD tanycytes.Interestingly, glycogen particles could be observedsurrounding lipid droplets in tanycytes of LD hamsters,in addition to free-standing groups of variable number(see Fig. 6).

DISCUSSION

There is growing evidence that tanycytes of the hypo-thalamic ependymal layer are a major relay station in apathway transmitting the circannual variation in photo-period to the hypothalamus in birds and mammalsthrough a mechanism involving seasonal availability ofthyroid hormone (Barrett et al., 2007; Hanon et al.,2008; Yoshimura et al., 2003).

Tanycytes of the Siberian hamster demonstrate one ofthe highest number of changes in gene expression with

Fig. 4. Section of an autoradiograph and quantification of autoradiographic signal for (A) lactate de-hydrogenase-B (LDH-B), (B) lactate transporter MCT2, (C) glutamine synthetase (GS), and (D) acetylCoA carboxylase 1 (ACC1) by in situ hybridization in tanycytes of LD and SD Siberian hamsters.Arrows indicate the tanycyte layer. *Pt-test < 0.05.


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altered photoperiod (Barrett et al., 2006, 2007; Herwiget al., 2009; Kameda et al., 2003; Ross et al., 2004, 2005;Watanabe et al., 2004). These gene expression changeswould suggest tanycytes may have multiple roles inrespect to hypothalamic functions that are required tobe modified as part of the response to changing daylength, with probable consequences for neuroendocrineinteractions (Kameda et al., 2003; Prevot et al., 2007,2010). In this report, we provide evidence for anotherfacet of tanycyte function namely the storage and mobi-lization of glucose in the form of glycogen.

Microarray analysis of gene expression on RNA cap-tured from brain tissue in the region of the dmpARC(Nilaweera et al., 2009) and in situ hybridization unex-pectedly revealed increased expression of GP and phos-phofructose kinase C in tanycytes of the ependymallayer. This is most likely to have occurred as a result of

capturing a small amount of the adjacent tanycyte layerduring capture of the dmpARC (Nilaweera et al., 2009).Both these enzymes are important rate-limiting enzymesin the metabolism of glycogen to glucose and on to pyru-vate in the glycolytic pathway (see Fig. 1).

In the brain, glycogen is mainly found in astrocytesand may serve a role in the provision of a metabolic fuelin response to glucose deprivation or during increasedneuronal transmission (Swanson, 1992; Swanson andChoi, 1993). Glycogen for neuronal functions is mobi-lized from astrocytes via the glycolytic pathway to lac-tate (Brown and Ransom, 2007; Dringen et al., 1993).We have previously shown that neurons of the dmpARCshow a four- to five-fold increase in activity in SD(Barrett et al., 2009), and other investigators havenoted a potential decrease in nonfasting glucose levelsin SD-housed Siberian hamsters (Bartness et al., 1995).Therefore, given the proximity of tanycytes to thedmpARC neurons, our initial hypothesis was that tany-cytes mobilize glycogen to lactate in SD to provide ametabolic fuel for surrounding hypothalamic neurons.

The interconversion of pyruvate to lactate is depend-ent upon the isoform of LDH present, wherein LDH-Aconverts pyruvate to lactate and the LDH-B subunitconverts lactate to pyruvate. In situ hybridization forLDH-A showed widespread distribution in the brain butno indication of a LD–SD difference in tanycytes. How-ever, LDH-B was increased in SD implicating drive topyruvate formation in SD. Parallel screening of potentialtransporters of lactate, MCT2, and MCT4 revealed a40% decrease in MCT2 expression in tanycytes in SD.MCT2 expression was also detected in the ependymo-cytes located dorsally to tanycytes in the ependymallayer but did not change in response to photoperiod. Thepresence of MCT2 in tanycytes is somewhat unexpectedas this lactate transporter is considered to be the neuro-nal transporter for the uptake of lactate by neurons(Bergersen, 2007; Br€oer et al., 1997). These data wouldimplicate a greater lactate uptake by tanycytes in LD.Further work is required to establish if there is a rolefor MCT2 in tanycytes beyond photoperiod-dependentphysiology, for example in the broader context of lactateregulation of food intake and glucose homeostasis (Lamet al., 2005, 2008).

An increase in tanycyte glycolysis could be a responseto the photoperiod-induced reduction in food intake andtherefore could be a general response of tanycytes toreduced food availability at anytime of the year. How-ever, GP expression was not increased in food-restrictedhamsters in either photoperiod, a treatment in whichmeasures to counter energy deficits occur to stimulatefood intake (Coppola et al., 2007; Herwig et al., 2009).Therefore, it would seem unlikely that glycogen storedin tanycytes provides an energy source for surroundinghypothalamic neurons during periods of reducedfood intake.

Neurons are unable to synthesize de novo the keyneurotransmitters glutamate and GABA (Bak et al.,2006) but rely on astrocytes for a source of glutaminefor conversion to glutamate and GABA to replenish

Fig. 5. Analysis and quantification of the glutamate transporterGLAST gene expression in (A) the arcuate nucleus (arrow) *PM-W < 0.05and (B) tanycyte layer (arrow) of the third ventricle by in situhybridization. *Pt-test < 0.05.


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these neurotransmitter pools. Astrocytic glutamine mayarise from two sources: either de novo synthesis fromglucose via the Krebs cycle intermediate a-ketoglutarate(Hertz et al., 1999) or synthesis from glutamate takenup from the extracellular environment (the glutamate–glutamine shuttle, Bak et al., 2006). In respect of thelatter route, tanycytes of rats have been demonstratedto express both glutamate transporters GLT-1 andGLAST, indicating a role in glutamate uptake (Bergerand Hediger, 2001). DmpARC neurons of the Siberianhamster are four- to five-fold more active in SD (Barrettet al., 2009) and therefore may require replenishment ofglutamate for which tanycytes may provide the source ofglutamine. Consistent with this idea is the noted pres-ence of glycogen and lipid droplets in tanycytes of otherspecies (Akmayev and Popov, 1977; Lima et al., 2010;Wittkowski and Mueller, 1976) and a seasonal change inthe accumulation of glycogen in the hedgehog where gly-cogen accumulates during hibernation (Wittkowski and

Mueller, 1976), a period when neuronal signaling maybe greatly diminished (Beckman and Stanton, 1982).

The common step in both pathways of glutamine pro-duction is amidiation of glutamate catalyzed by theenzyme GS. In situ hybridization of a RNA probe for GSdemonstrated a decrease in expression in SD, implicat-ing a reduction in the production of glutamine. Further-more, the glutamate transporter GLAST was found to beexpressed in tanycytes, which also decreased expressionin SD. Therefore, our data would indicate that gluta-mate uptake and production of glutamine as a neuro-transmitter precursor may be an active process in LDbut does not support such a role in relation to the activ-ity of dmpARC neurons in SD.

The presence of tight junction proteins in ventraltanycytes and the exclusion of peripheral administeredEvans blue dye suggest ventral tanycytes have a role inblood–brain barrier regulation (Mullier et al., 2010).Tanycyte end feet makes contact with brain capillaries

Fig. 6. Electron microscopy analysis of tanycytes lining the third ventri-cle. Electron micrographs showing: (A) At 33,400 magnification, tanycyteswith their elongated nuclei. N indicates a tanycyte nucleus. Scale bar 5 lm.(B) At higher resolution (36,400), glycogen clusters are visible in the tany-

cyte layer of LD hamsters (arrows), scale bar 0.2 lm. (C) Glycogen clusters(arrows) in LD tanycytes at higher resolution (magnification 394,000),scale bar 200 nm. (D) Lipid droplet (L) surrounded by glycogen particles(arrows) in a LD tanycyte. Magnification394,000 scale bar 200 nm.


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and is likely to be involved in the regulation of transportacross the endothelial layer. In this respect, transport oflarge neutral amino acids by brain microvessels acrossthe blood–brain barrier involves the exchange of theneutral amino acid for glutamine (Cangiano et al.,1983). One possible function of tanycytes may be theprovision of a localized source of glutamine for such atransport system. In such a role, it would follow thatthis transport would be reduced in SD.

Pyruvate is also an intermediate in hypothalamic denovo lipogenesis with an early intermediate being malo-nyl CoA. Evidence would now support a role for de novosynthesis of malonyl CoA within hypothalamic neuronsin a mechanism to reduce appetite and food intake(He et al., 2006). A key step in this process is theexpression and activity of the enzyme acetyl CoA carbox-ylase 1 to catalyze the carboxylation of acetyl CoA toform malonyl CoA. In turn, malonyl CoA inhibits carni-tine palmitoyltransferase I transfer of long-chain fattyacid across the mitochondrial membrane for oxidationand energy production. In situ hybridization analysis ofACC1 mRNA expression showed upregulation in SD.This observation indicated increased fatty acid biosyn-thesis, which is consistent with the presence of lipiddroplets in tanycytes, and intriguingly, electron micros-

copy analysis showed glycogen particles in close associa-tion with some lipid droplets in LD; alternatively, malo-nyl CoA could play a role to reduce energy expenditurein tanycytes by inhibiting fatty acid oxidation forming apart of a mechanism to alter function and morphology oftanycytes in SD.

An important role is emerging for tanycytes as meta-bolic sensors. The expression of glucose and other trans-porters, glucokinase, active lactate transport, anincrease in intracellular calcium and ATP release follow-ing direct application of glucose (Cortes-Campos et al.,2011; Frayling et al., 2011; Rodr�ıguez et al., 2005), andimpairment of counter-regulatory responses to glucosefollowing tanycyte ablation indicate the importance oftanycytes in glucose sensing (Sanders et al., 2004). Wenow provide evidence for an additional role for glucosein tanycyte function. Furthermore, with lactate playingan important role as a neuronal energy source (Schurret al., 1999; Wyss et al., 2011), our data indicate tany-cytes may make an important contribution to surround-ing lactate and glutamate pools with potential conse-quences for adjacent hypothalamic neurons.

In Fig. 7, we have provided a model as working hy-pothesis for the involvement of the proteins identified bygene expression in this study.

Fig. 7. A diagrammatic outline of a model for the role of tanycytes inthe regulation of glutamate and lactate in the hypothalamic mileu duringexposure to long days and short days. During long days, glutamate and lac-tate uptake into tanycytes may contribute to the production of glutaminefor export to surrounding hypothalamic neurons. Active tanycytes withextended tanycyte end feet use long-chain fatty acids for energy productionin mitochondria. In short photoperiod, glutamate and lactate uptake

together with glutamine synthesis is reduced. Glycogen is mobilized andconverted to pyruvate, which provides a substrate for the production ofmalonyl CoA, an inhibitor of CPT1, a long-chain fatty acid transporter.This reduces supply of long-chain fatty acids to mitochondria reducingenergy production or provides the basis for fatty acid synthesis that consti-tutes the lipid droplets that can be observed in tanycytes. In the figure,bold arrows and text indicate greater activity of the pathways outlined.


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ACKNOWLEDGMENTS

This work was supported by the Scottish ExecutiveEnvironment and Rural Affairs Department, the Bio-technology and Biological Sciences Research Council(UK) project grant BB/E020437/1, the European Unionas part of Framework VII: LSHM-CT-2003-503041, ‘‘Dia-besity Integrated Project (Aberdeen),’’ and PIEF-GA-2009-235106 Marie Curie individual fellowship toAnnika Herwig. The authors thank P. Young for assis-tance with DNA sequencing, Gillian Smith for electronmicroscope sample preparation and guidance on micro-scope use, and Dana Wilson for technical assistance.

REFERENCES

Akmayev IG, Popov AP. 1977. Morphological aspects of the hypothala-mic hypophyseal system. VII. the tanycytes: Their relation to thehypophyseal adrenocorticotrophic function: An ultrastructural study.Cell Tissue Res 180:263–282.

Bak LK, Schousboe A, Waagepetersen HS. 2006. The glutamate/GABA-glutamine cycle: Aspects of transport, neurotransmitter homeostasisand ammonia transfer. J Neurochem 98:641–653.

Barrett P, Ebling FJP, Schuhler S, Wilson D, Ross AW, Warner A, Jet-hwa P, Boelen A, Visser TJ, Ozanne DM, Archer ZA, Mercer JG, Mor-gan PJ. 2007. Hypothalamic thyroid hormone catabolism acts as agatekeeper for the seasonal control of body weight and reproduction.Endocrinology 148:3608–3617.

Barrett P, Ivanova E, Graham ES, Ross AW, Wilson D, Pl�e H, MercerJG, Ebling FJ, Schuhler S, Dupr�e SM, Loudon A, Morgan PJ. 2006.Photoperiodic regulation of cellular retinoic acid-binding protein 1,GPR50 and nestin in tanycytes of the third ventricle ependymal layerof the siberian hamster. J Endocrinol 191:687–698.

Barrett P, Van Den Top M, Wilson D, Mercer JG, Song CK, BartnessTJ, Morgan PJ, Spanswick D. 2009. Short photoperiod-induceddecrease of histamine H3 receptors facilitates activation of hypothala-mic neurons in the siberian hamster. Endocrinology 150:3655–3663.

Bartness TJ, Morley JE, Levine AS. 1995. Effects of food deprivationand metabolic fuel utilization on the photoperiodic control of foodintake in Siberian hamsters. Physiol Behav 57:61–68.

Beckman AL, Stanton TL. 1982. Properties of the CNS during hiberna-tion. In: Beckman AL, editor. The neural basis of behavior. NewYork: Spectrum. pp 19–45.

Berger UV, Hediger MA. 2001. Differential distribution of the gluta-mate transporters GLT-1 and GLAST in tanycytes of the third ventri-cle. J Comp Neurol 433:101–114.

Bergersen LH. 2007. Is lactate food for neurons? Comparison of mono-carboxylate transporter subtypes in brain and muscle. Neuroscience145:11–19.

Br€oer S, Rahman B, Pellegri G, Pellerin L, Martin JL, Verleysdonk S,Hamprech B, Magistretti PJ. 1997. Comparison of lactate transportin astroglial cells and monocarboxylate transporter 1 (MCT 1)expressing xenopus laevis oocytes. Expression of two different mono-carboxylate transporters in astroglial cells and neurons. J Biol Chem272:30096–30102.

Brown AM, Ransom BR. 2007. Astrocyte glycogen and brain energy me-tabolism. Glia 55:1263–1271.

Cangiano C, Cardelli-Cangiano P, James JH. 1983. Brain microvesselstake up large neutral amino acids in exchange for glutamine. Cooper-ative role of Na1-dependent and Na1-independent systems. J BiolChem 258:8949–8954.

Coppola A, Liu Z, Andrews ZB, Paradis E, Roy MC, Friedman JM, Ric-quier D, Richard D, Horvath TL, Gao X, Diano S. 2007. A centralthermogenic-like mechanism in feeding regulation: An interplaybetween arcuate nucleus T3 and UCP2. Cell Metab 5:21–33.

Cortes-Campos C, Elizondo R, Llanos P, Uranga RM, Nualart F, GarciaM. 2011. A MCT expression and lactate influx/efflux in tanycytesinvolved in glia-neuron metabolic interaction. PLoS One 6:e16411

De Seranno S, D’Anglemont de Tassigny X, Estrella C, Loyens A, KasparovS, Leroy D, Ojeda SR, Beauvillain J-C, Prevot V. 2010. Role of estradiol inthe dynamic control of tanycyte plasticity mediated by vascular endothe-lial cells in the median eminence. Endocrinology 151:1760–1772.

Dringen R, Gebhardt R, Hamprecht B. 1993. Glycogen in astrocytes:Possible function as lactate supply for neighboring cells. Brain Res623:208–214.

Elkjar M, Vajda Z, Nejsum LN, Kwon TH, Jensen UB, Amiry-Moghad-dam M, Frokiar J, Nielsen S. 2000. Immunolocalization of AQP9 inliver, epididymis, testis, spleen, and brain. Biochem Biophys ResCommun 276:1118–1128.

Figala J, Hoffmann K, Goldau G. 1973. The annual cycle in the djun-garian hamster phodopus sungorus pallas. Oecologia 12:89–118.

Franklin K, Paxinos G. 1997. The mouse brain sterotactic coordinates.San Diego: Academic Press.

Frayling C, Britton R, Dale N. 2011. ATP-meidated glucosening byhypothalamic tanycytes. J Physiol 589:2275–2286.

Guadano-Ferraz A, Obregon MJ, St Germain DL, Bernal J. 1997. Thetype 2 iodothyronine deiodinase is expressed primarily in glial cellsin the neonatal rat brain. Proc Natl Acad Sci 94:10391–10396.

Hanon EA, Lincoln GA, Fustin JM, Dardente H, Masson-P�evet M,Morgan PJ, Hazlerigg DG. 2008. Ancestral TSH mechanism signalssummer in a photoperiodic mammal. Curr Biol 18:1147–1152.

He W, Lam TKT, Obici S, Rossetti L. 2006. Molecular disruption ofhypothalamic nutrient sensing induces obesity. Nat Neurosci 9:227–233.

Hertz L, Dringen R, Schousboe A, Robinson SR. 1999. Astrocytes: Glu-tamate producers for neurons. J Neurosci Res 57:417–428.

Herwig A, Wilson D, Logie TJ, Boelen A, Morgan PJ, Mercer JG, Bar-rett P. 2009. Photoperiod and acute energy deficits interact on compo-nents of the thyroid hormone system in hypothalamic tanycytes ofthe Siberian hamster. Am J Physiol Regul Integr Comp Physiol296:R1307–R1315.

Kameda Y, Arai Y, Nishimaki T. 2003. Ultrastructural localization ofvimentin immunoreactivity and gene expression in tanycytes andtheir alterations in hamsters kept under different photoperiods. CellTissue Res 314:251–262.

Lam CKL, Chari M, Wang PYT, Lam TKT. 2008. Central lactate metab-olism regulates food intake. Am J Physiol Endocrinol Metab295:E491–E496.

Lam TKT, Gutierrez-Juarez R, Pocai A, Rossetti L. 2005. Medicine:Regulation of blood glucose by hypothalamic pyruvate metabolism.Science 309:943–947.

Lechan RM, Fekete C. 2005. Role of thyroid hormone deiodination inthe hypothalamus. Thyroid 15:883–897.

Lima SS, Lima Dos Santos MC, Sinder MP, Moura AS, Barradas PC,Ten�orio F. 2010. Glycogen stores are impaired in hypothalamicnuclei of rats malnourished during early life. Nutr Neurosci 13:21–28.

Mullier A, Bouret SG, Prevot V, Dehouck B. 2010. Differential distribu-tion of tight junctions proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the mouse brain. J Comp Neurol518:943–962.

Nakao N, Ono H, Yamamura T, Anraku T, Takagi T, Higashi K, YasuoS, Katou Y, Kageyama S, Uno Y, Kasukawa T, Iigo M, Sharp PJ, Iwa-sawa A, Suzuki Y, Sugano S, Niimi T, Mizutani M, Namikawa T, Ebi-hara S, Ueda HR, Yoshimura T. 2008. Thyrotrophin in the parstuberalis triggers photoperiodic response. Nature 452:317–322.

Nilaweera KN, Ellis C, Barrett P, Mercer JG, Morgan PJ. 2002. Hypo-thalamic bHLH transcription factors are novel candidates in the reg-ulation of energy balance. Eur J Neurosci 15:644–650.

Nilaweera KN, Archer ZA, Campbell G, Mayer CD, Balik A, Ross AW,Mercer JG, Ebling FJP, Morgan PJ, Barrett P. 2009. Photoperiod reg-ulates genes encoding melanocortin 3 and serotonin receptors andsecretogranins in the dorsomedial posterior arcuate of the Siberianhamster. J Neuroendocrinol 21:123–131.

Ono H, Hoshino Y, Yasuo S, Watanabe M, Nakane Y, Murai A, EbiharaS, Korf HW, Yoshimura T. 2008. Involvement of thyrotropin in photo-periodic signal transduction in mice. Proc Natl Acad Sci U S A105:18238–18242.

Prevot V, Bellefontaine N, Baroncini M, Sharif A, Hanchate NK, Par-kash J, Campagne C, de Seranno S. 2010. Gonadotrophin-releasinghormone nerve terminals, tanycytes and neurohaemal junctionremodelling in the adult median eminence: Functional consequencesfor reproduction and dynamic role of vascular endothelial cells.J Neuroendocrinol 22:639–649.

Prevot V, Dehouck B, Poulain P, Beauvillain JC, Bu�ee-Scherrer V,Bouret S. 2007. Neuronal-glial-endothelial interactions and cell plas-ticity in the postnatal hypothalamus: Implications for the neuroendo-crine control of reproduction. Psychoneuroendocrinology 32:S46–S51.

Rodr�ıguez EM, Bl�azquez JL, Pastor FE, Pel�aez B, Pe~na P, Peruzzo B,Amat P. 2005. Hypothalamic tanycytes: A key component of brain-en-docrine interaction. Int Rev Cytol 247:89–164.

Ross AW, Bell LM, Littlewood PA, Mercer JG, Barrett P, Morgan PJ.2005. Temporal changes in gene expression in the arcuate nucleusprecede seasonal responses in adiposity and reproduction. Endocri-nology 146:1940–1947.

Ross AW, Webster CA, Mercer JG, Moar KM, Ebling FJ, Schuhler S,Barrett P, Morgan PJ. 2004. Photoperiodic regulation of hypothalamic


GLIA

retinoid signaling: Association of retinoid X receptor g with bodyweight. Endocrinology 145:13–20.

Sanders NM, Dunn-Meynell AA, Levin BE. 2004. Third ventricularAlloxan reversibly impairs glucose counterregulatory responses. Dia-betes 53:1230–1236.

Schurr A, Miller JJ, Payne RS, Rigor BM. 1999. An increase in lactateoutput by brain tissue serves to meet the energy needs of glutamate-activated neurons. J Neurosci 19:34–39.

Shearer KD, Goodman TH, Ross AW, Reilly L, Morgan PJ, McCafferyPJ. 2010. Photoperiodic regulation of retinoic acid signaling in thehypothalamus. J Neurochem 112:246–257.

Steinlechner S, Heldmaier G. 1982. Role of photoperiod and melatoninin seasonal acclimatization of the Djungarian hamster, Phodopussungorus. Int J Biometeorol 26:329–337.

Swanson RA. 1992. Physiologic coupling of glial glycogen metabolismto neuronal activity in brain. Can J Physiol Pharmacol 70:S138–S144.

Swanson RA, Choi DW. 1993. Glial glycogen stores affect neuronalsurvival during glucose deprivation in vitro. J Cereb Blood FlowMetab 13:162–169.

Tu HM, Kim SW, Salvatore D, Bartha T, Legradi G, Larsen PR, LechanRM. 1997. Regional distribution of type 2 thyroxine deiodinasemessenger ribonucleic acid in rat hypothalamus and pituitary and itsregulation by thyroid hormone. Endocrinology 138:3359–3368.

Watanabe M, Yasuo S, Watanabe T, Yamamura T, Nakao N, Ebihara S,Yoshimura T. 2004. Photoperiodic regulation of type 2 deiodinasegene in djungarian hamster: Possible homologies between avian andmammalian photoperiodic regulation of reproduction. Endocrinology145:1546–1549.

Wittkowski W, Mueller K. 1976. Researches on the infundibulum of thehedgehog. Verh Anat Ges 70:49–54.

Wolfgang MJ, Lane MD. 2006. Control of energy homeostasis: Role ofenzymes and intermediates of fatty acid metabolism in the centralnervous system. Annu Rev Nutr 26:23–44.

Wyss MT, Jolivet R, Buck A, Magistretti PJ, Webster B. 2011. In Vivo evi-dence for lactate as a neuronal energy source. J Neurosci 31:7477–7485.

Yoshimura T, Yasuo S, Watanabe M, Iigo M, Yamamura T, Hirunagi K,Ebihara S. 2003. Light-induced hormone conversion of T4 to T3 regu-lates photoperiodic response of gonads in birds. Nature 426:178–181.


GLIA

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