Journal of Insect Physiology 57 (2011) 1453–1462

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Journal of Insect Physiology

journal homepage: www.elsevier .com/ locate/ j insphys

AMP-activated protein kinase and metabolic regulation in cold-hardy insects

Mark H. Rider a,⇑, Nusrat Hussain a, Stephen M. Dilworth b, Janet M. Storey c, Kenneth B. Storey c

a Université catholique de Louvain and de Duve Institute, Avenue Hippocrate 75, B-1200 Brussels, Belgiumb Centre for Investigative and Diagnostic Oncology, Middlesex University, The Burroughs, Hendon, London NW4 4BT, UKc Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa K1S 5B6, Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 February 2011Received in revised form 6 July 2011Accepted 6 July 2011Available online 20 July 2011

Keywords:Protein synthesisMetabolic rate depressionAMPKrpS64E-BP1PDHGlycogen phosphorylaseTriglyceride lipase

0022-1910/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.jinsphys.2011.07.006

Abbreviations: 4E-BP1, eukaryotic initiation factoacetyl-CoA carboxylase; AMPK, AMP-activated prote(adipose) triglyceride lipase; eEF2, eukaryotic elongatdehydrogenase; GP, glycogen phosphorylase; LD(m)TORC1, (mammalian) target of rapamycin compleprotein S6 kinase; PDH, pyruvate dehydrogenase; PPrpS6, 40S ribosomal protein S6.⇑ Corresponding author. Address: Université cathol

Institute 75.29, Avenue Hippocrate 75, B-1200 Bruss7485; fax: +32 2 764 7507.

E-mail address: mark.rider@uclouvain.be (M.H. Rid

Winter survival for many insects depends on cold hardiness adaptations as well as entry into a hypomet-abolic diapause state that minimizes energy expenditure. We investigated whether AMP-activated pro-tein kinase (AMPK) could be involved in this adaptation in larvae of two cold-hardy insects, Eurostasolidaginis that is freeze tolerant and Epiblema scudderiana that uses a freeze avoidance strategy. AMPKactivity was almost 2-fold higher in winter larvae (February) compared with animals collected in Sep-tember. Immunoblotting revealed that phosphorylation of AMPK in the activation loop and phosphory-lation of acetyl-CoA carboxylase (ACC), a key target of AMPK, were higher in Epiblema duringmidwinter whereas no seasonal change was seen in Eurosta. Immunoblotting also revealed a significantincrease in ribosomal protein S6 phosphorylation in overwintering Epiblema larvae, and in both Eurostaand Epiblema, phosphorylation of eukaryotic initiation factor 4E-binding protein-1 dramatically increasedin the winter. Pyruvate dehydrogenase (PDH) E1a subunit site 1 phosphorylation was 2-fold higher inextracts of Eurosta larvae collected in February versus September while PDH activity decreased by about50% in Eurosta and 80% in February Eurosta larvae compared with animals collected in September. Glyco-gen phosphorylase phosphorylation was 3-fold higher in Epiblema larvae collected in February comparedwith September and also in these animals, triglyceride lipase activity increased by 70% during winter.Overall, our study suggests a re-sculpting of metabolism during insect diapause, which shifted to a morecatabolic poise in freeze-avoiding overwintering Epiblema larvae, possibly involving AMPK.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Winter survival for many species of insects living in seasonallycold environments includes a radical remodeling of metabolism.This frequently includes the development of cold hardiness, entryinto a hypometabolic state of diapause, and a switch from feedingto the catabolism of stored body fuel reserves to support energyneeds (Lee and Denlinger, 2010). All of these are closely coordi-nated, particularly in univoltine species where a specific life stageoverwinters and entry into that stage is often tied to the initiationof cold hardiness adaptations and an obligate diapause. Winter

ll rights reserved.

r 4E-binding protein-1; ACC,in kinase; (adipose) (A)TGL,ion factor-2; GDH, glutamateH, lactate dehydrogenase;x 1; p70S6K, p70 ribosomal2A, protein phosphatase-2A;

ique de Louvain and de Duveels, Belgium. Tel.: +32 2 764

er).

cold hardiness has been well-studied in the larvae of two speciesof gall-forming insects that live on goldenrod – the goldenrod gallfly, Eurosta solidaginis Fitch (Diptera, Tephritidae) that is freeze tol-erant and the goldenrod gall moth, Epiblema scudderiana Clemens(Lepidoptera, Olethreutidae) that uses the freeze avoidance strat-egy of deep supercooling (reviewed by Storey, 1990; Storey andStorey, 2010). The last instar larvae of both species build up highglycogen reserves during early autumn feeding (September, Octo-ber) and then convert these into massive pools of cryoprotectantsas the autumn progresses, producing glycerol in Epiblema (concen-trations >2 M) or glycerol plus sorbitol in Eurosta (Storey and Sto-rey, 1986; Rickards et al., 1987). Synthesis is modulated by autumnenvironmental factors including plant senescence, photoperiod,and decreasing environmental temperatures. The larvae also entera period of obligate diapause that lasts for 3–4 months until aboutFebruary in the Ottawa area. Subsequently, the larvae pass into aperiod of quiescence where development can continue towardsthe pupal transition, with both the speed of development and therate of degradation of the winter cryoprotectants increasing overtime and with rising environmental temperatures.

Hence, distinct metabolic states can be defined at different sea-sons. In early autumn (mid-September) larvae are still feeding,

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body mass is increasing with the deposition of both carbohydrateand lipid reserves (Storey and Storey, 1986; Rickards et al.,1987), and the metabolic machinery is in place and poised to sup-port high flux unidirectional synthesis of cryoprotectants whentriggered by environmental cues. For example, when larvae aretransferred from moderate to cold temperatures at this time, a ra-pid glycerol synthesis occurs that is not reversed when animals arereturned to the higher temperature (Storey and Storey, 1983;Churchill and Storey, 1989). By late winter (February), however,the metabolic poise of the system has changed – cryoprotectantlevels begin to fall as environmental temperatures warm, onlyabout half of glycerol carbon reappears as glycogen if larvae arewarmed, and subsequent cold exposures produce a progressivelymuted response in glycerol synthesis (Churchill and Storey,1989). The fate of glycerol carbon that is not returned to glycogenis not fully known but possibilities include glycerol use as a sub-strate for aerobic metabolism and/or for biosynthesis supportingthe pupal transition, or conversion to another fuel reserve (lipids,amino acids) that can be used during non-feeding pupal or adultlife (Storey and Storey, 1991). Note, however, that in Eurosta sorbi-tol interconversion with glycogen remains intact throughout as areversible response to cooling/warming (Storey and Storey, 1986).

Various regulatory controls can be applied to alter the meta-bolic poise of a system. A key player in the control of cellular en-ergy homeostasis in eukaryotes is the AMP-activated proteinkinase (AMPK). AMPK is a heterotrimer consisting of a catalytic asubunit and two regulatory subunits, b and c. AMPK can be acti-vated by changes in the intracellular AMP:ATP ratio, due to a dropin energy charge as occurs, for example, under hypoxia/anoxia, oras a result of other metabolic stresses (Hardie et al., 1998). Onceactivated, AMPK decreases ATP-consumption and stimulates ATP-producing processes (Hardie et al., 1998; Hardie, 2007). For exam-ple, its best known substrate is acetyl-CoA carboxylase (ACC).AMPK-mediated phosphorylation inactivates ACC to inhibit lipo-genesis and stimulate fatty acid oxidation under energy stress sit-uations (i.e. increased AMP levels). In addition, AMPK activationstimulates glucose uptake in skeletal muscle and heart. AMPK acti-vation also inhibits glycogen synthase in order to favor glycolysis(Hardie et al., 1998; Hardie, 2007), and decreases protein synthesisin liver (Horman et al., 2002) and heart (Horman et al., 2003), butnot in skeletal muscle (Miranda et al., 2008), via phosphorylation-induced activation of the eukaryotic elongation factor-2 (eEF2) ki-nase, which in turn phosphorylates and inactivates eEF2.

It is now becoming apparent that AMPK plays a role in multipleforms of animal hypometabolism where animals strongly reducemetabolic rate to achieve long-term survival under conditions ofsevere environmental stress (Rider, 2008). AMPK has been impli-cated in metabolic rate depression during freezing (Rider et al.,2006) and anoxia (Bartrons et al., 2004; Rider et al., 2006) in frogsas well as during anoxia in carp (Stensløkken et al., 2008), goldfish(Jibb and Richards, 2008) and turtles (Rider et al., 2009), but prob-ably not during mammalian hibernation (Horman et al., 2005). Indiapausing Caenorhabditis elegans dauers, AMPK was shown to di-rectly phosphorylate and inactivate adipose triglyceride lipase(ATGL), thereby limiting energy expenditure to ensure survival inthe dormant stage (Narbonne and Roy, 2009). The present studywas therefore undertaken to determine whether AMPK might alsohave a role to play in insect diapause and cold hardiness.

2. Materials and methods

2.1. Materials

Routine chemicals were from Sigma, Boehringer or from sourcespreviously cited (Horman et al., 2005; Rider et al., 2006; Miranda

et al., 2008; Rider et al., 2009). Anti-rat phospho Ser14 glycogenphosphorylase (GP) antibody, anti-human phospho Ser293 (site 1),anti-human phospho Ser300 (site 2), anti-total human pyruvatedehydrogenase (PDH) E1a subunit (Pilegaard et al., 2006), andanti-phospho ACC antibody against the AMPK site of the Drosophilamelanogaster protein (all polyclonal anti-peptide antibodies raisedin sheep) were kindly provided by Prof. Grahame Hardie (Universityof Dundee). Anti-rat phospho Ser79 ACC1 antibody (polyclonal anti-peptide antibody raised in rabbit) was from Upstate. Anti-humanphospho Thr172 AMPK a-subunit (rabbit monoclonal anti-peptideantibody), anti-human phospho Ser235/236 rpS6 and anti-mousephospho Thr37/46 (polyclonal anti-peptide antibodies raised in rab-bit) were from Cell Signaling. Anti-human liver GP polyclonal anti-body raised in rabbit against the immunogenic peptide VVAATLQDIIRRFKASKFGSTRGAGTVFDAFPDQVAIQLNDTHPALAIPELMRIFVDIEKLPWSKAWELTQKTFAYTNHTVLPEALERWPVDLVEKLLPRHLEIIYEINQKHLDRIVALFPKDVDRLRRMSLI was from Sigma. Most of the anti-bodies (except the Drosophila anti-phospho ACC antibody) are rou-tinely used in the Rider laboratory to study signaling in rathepatocytes, rat adipocytes and rat cardiomyocytes where relevantsignals are obtained in protein immunoblotting experiments. More-over, the antibodies have been used to study AMPK signaling in thefrog, Rana sylvatica (Rider et al., 2006) and in the turtle, Trachemysscripta elegans (Rider et al., 2009) by Western blot.

Streptavidin-Agarose, pigeon liver acetone powder and glyceryltrioleate were from Sigma, 4-aminoazobenzene-40-sulfonic acidwas from Alfa Aesar, [9,10-3H(N)] triolein was from PerkinElmerand the ‘‘BAY’’ isoxazolone hormone-sensitive lipase inhibitor(Lowe et al., 2004; Claus et al., 2005) was a kind gift from StefanHallén (AstraZeneca, Mölndal, Sweden).

2.2. Preparation of tissue extracts

Galls containing final instar larvae of Eurosta and Epiblema werecollected from fields around Ottawa, Ontario, Canada in Septemberor February. Galls were briefly held in the lab in an incubator set tothe current outdoor temperature and were then quickly openedand the larvae (50–80 mg body mass) were flash frozen in liquidnitrogen. Samples were air-freighted to Belgium on dry ice andstored at �80 �C until use. September animals had not yet been ex-posed to cold temperatures in nature, had minimal levels of cryo-protectants and were not yet cold hardened. February midwinteranimals were fully cold-hardy with maximal cryoprotectant levelshaving experienced subzero temperatures for many weeks.

Samples of larvae (n = 4 samples for each condition, each sam-ple consisting of a pool of four individuals with total mass 200–300 mg) were taken from �80 �C storage but were not allowed tothaw. Samples were quickly homogenized (Ultra-Turrax) in1.3 ml of ice cold extraction buffer containing 50 mM Hepes, pH7.4, 250 mM sucrose, 20 mM NaF, 5 mM sodium pyrophosphate,1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 5 lg/ml pepstatin,1 mM benzamidine–HCl, and 1 mM phenylmethanesulphonyl fluo-ride as described previously (Rider et al., 2006). Extracts were cen-trifuged in an Eppendorf microfuge at full speed for 10 min at 4 �Cand then supernatants were removed and stored at �80 �C prior tomeasurements of AMPK activity and immunoblotting. Protein con-centrations in the extracts estimated with bovine serum albuminas a standard (see below) did not show significant seasonal differ-ences (Eurosta 12.9 ± 0.3 mg/ml in September versus 13.4 ± 1.5 mg/ml in February; Epiblema 18.6 ± 1.9 mg/ml in September versus17.6 ± 1.3 mg/ml in February; n = 4 for each measurement).

2.3. AMPK assay

Aliquots of supernatants (0.25 ml) were mixed with an equalvolume of 20% (w/v) polyethylene glycol 8000 in extraction buffer.

M.H. Rider et al. / Journal of Insect Physiology 57 (2011) 1453–1462 1455

After 30 min on ice, the samples were centrifuged (Eppendorfmicrofuge, full speed for 1 min at 4 �C) and protein precipitateswere resuspended in 25 ll (September larvae), 50 ll (Eurosta, Feb-ruary) or 100 ll (Epiblema, February) of extraction buffer. Aliquotsof polyethylene glycol fractions (2.5 ll) were assayed for AMPKactivity in a final volume of 30 ll for 5 min as described (Rideret al., 2006), except for samples from Epiblema collected in Septem-ber that were first diluted 3-fold prior to AMPK assay.

2.4. PDH, glutamate dehydrogenase (GDH), lactate dehydrogenase(LDH) and TGL assays

Extracts (50 ll) were assayed for PDH (Coore et al., 1971) spec-trophotometrically in a coupled assay using arylamine acetyltrans-ferase, purified from a pigeon liver acetone powder (Tabor et al.,1953), to acetylate 4-aminoazobenzene-40-sulfonic acid from thereaction product, acetyl-CoA. Extracts were assayed for GDH (Mar-tin and Denton, 1970) and LDH (Saggerson, 1974) as indicated.

Extracts (50 ll) were assayed for TGL (Osterlund et al., 1996) bymeasuring the rate of labeled fatty acid release from [3H] triolein inthe presence and absence of 0.3 lM ‘‘BAY’’ hormone-sensitive li-pase inhibitor, a concentration more than 50 times the IC50 re-ported for human recombinant hormone-sensitive lipase (Clauset al., 2005).

2.5. Protein immunoblotting

For protein immunoblot analysis, samples of polyethylene gly-col fractions (prepared as in Section 2.3) were used for assessingAMPK phosphorylation whereas extract supernatants (as in Sec-tion 2.2) were used for phosphorylation analysis of all other pro-teins. Samples of polyethylene glycol fractions and full extractscontained equal amounts of protein and were mixed with SDS–PAGE loading buffer (20 ll), boiled for 5 min, for loading into eachwell of SDS–polyacrylamide gels as indicated in the figure legends.For some analyses, a suspension of Streptavidin Agarose beads(40 ll 1:1 v/v in phosphate-buffered saline) was added to 200 lgof full extract protein, diluted to 500 ll in extraction buffer, andleft for 2 h at 4 �C with agitation to pull-down ACC. The beads werecollected by centrifugation (3000 rpm � 5 min, Eppendorf micro-fuge at 4 �C), washed 3� with extraction buffer, mixed with SDS–PAGE sample buffer (20 ll) and boiled. The percentages of acryl-amide/bisacrylamide (w/v) used in the running gels were 7.5%for the separation of ACC, 10% for AMPK, PDH E1a subunit andGP, and 12.5% for rpS6, 4E-BP1, b-actin and protein phosphatase-2A (PP2A). After protein transfer to polyvinylidene difluoridemembranes, blots were probed with primary antibodies or horse-raddish peroxidase coupled to streptavidin (to detect the biotincomponents of pyruvate carboxylase and ACC) and developed forimaging by enhanced chemiluminescence. Ratios of band intensi-ties relative to signals obtained with a loading control (totalPP2A catalytic subunit, total PDH, total GP, total ACC or total pyru-vate carboxylase) were calculated as described previously (Hor-man et al., 2005).

2.6. Other methods

Protein concentration was estimated in extracts (Bradford,1976) using bovine serum albumin as a standard for protein load-ing and in polyethylene glycol fractions using human c-globulin asstandard for the calculation of AMPK specific activities. Statisticalsignificance of the data was assessed by an unpaired Student’stwo-sided t-test.

3. Results

3.1. AMPK activity and ACC phosphorylation

To understand the potential role of AMPK in winter diapauseand cold hardiness in insects, polyethylene glycol fractions wereprepared from extracts of Eurosta and Epiblema larvae collectedin September and February for AMPK assay. AMPK activity in bothspecies was strongly elevated in February by 88% and 70% in Euro-sta and Epiblema, respectively, as compared with larvae collected inSeptember (Fig. 1). However, AMPK catalytic a-subunit activationloop phosphorylation levels (corresponding to pThr172 in mam-mals) were only significantly elevated in polyethylene glycol frac-tions from Epiblema where an approximate increase of 3-fold wasseen in animals collected in February compared with Septemberlarvae (Fig. 1). The activation loop sequence containing Thr172 ofthe human AMPK a1/a2-subunit (DFGLSNMMSDGEFLRTSCGSP-NYAAPE) is extremely well conserved throughout eukaryotic evo-lution (for example this sequence corresponds toDFGLSNMMLDGEFLRTSCGSPNYAAPE in D. melanogaster, NCBIAccession No. NM_057965, with just one amino acid substitution)explaining the recognition of phosphorylated insect a-AMPK bythe anti-human phospho Thr172 AMPK a-subunit antibody. Like-wise, the mammalian antibody also recognized snail AMPK (Ram-nanan et al., 2010). The phospho a-AMPK band in both Eurosta andEpiblema migrated with a Mr of around 70,000, as recognized bythe anti-human phospho Thr172 AMPK a-subunit antibody, whichis somewhat higher than the expected Mr (about 65,000). However,using Odyssey infrared imaging with differentially labeled fluores-cent secondary antibodies to simultaneously probe phospho andtotal AMPK proteins in extracts from mammalian cells and tissues,the same Mr for phospho versus total AMPK was observed with sig-nal overlay (not shown). Unfortunately, the technique of analyzingphospho-blots by Odyssey imaging cannot be used in insects be-cause the anti-full length AMPK a-subunit antibodies do not recog-nize the insect protein, which is why we had to resort tochemiluminescence with a suitable loading control.

To study downstream ACC phosphorylation, full extracts werepulled-down onto Streptavidin Agarose beads, taking advantageof the fact that ACC is a biotin-containing enzyme. When expressedrelative to total ACC detected with streptavidin-coupled peroxi-dase, ACC phosphorylation in Epiblema increased 2-fold in Febru-ary compared with September larvae, as detected with anti-ratphospho Ser79 ACC1 antibody (Fig. 2). No band corresponding tophosphorylated ACC was detected in pull-downs of extracts fromEurosta larvae (Fig. 2), presumably due to lack of recognition bythe anti-rat phospho Ser79 ACC1 antibody.

3.2. rpS6 and 4E-BP1 phosphorylation

Phosphorylation of ribosomal S6 protein (rpS6) was investi-gated by immunoblotting larval extracts with anti-human phosphoSer235/236 rpS6 antibody using anti-PP2A catalytic subunit anti-body as a loading control as in previous studies (Rider et al.,2009). The sequence surrounding Ser235/236 of human rpS6(KRRRLSSLRAST) is similar in insect species such as D. melanogasterwhere the sequence is KRRRSASIRESK (NCBI Accession No.NP_727213) with conservation of only the Ser236 site of the hu-man protein. Phosphorylation of rpS6 Ser235/236 increased by 4-fold in Epiblema larvae collected in February compared with ani-mals in September (Fig. 3). However, no seasonal differences inrpS6 phosphorylation in Eurosta were seen. The use of anti-b-actinas a loading control in these immunoblotting experiments was notpossible because its expression levels decreased by about 20% and70%, respectively, in larval extracts of Eurosta and Epiblema col-

Eurosta

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Fig. 1. AMPK activity and phosphorylation state in polyethylene glycol fractions of Eurosta and Epiblema larvae collected in September and February. Polyethylene glycolfractions of extracts from whole larvae collected in September or February were prepared and assayed for AMPK as described in Section 2. Polyethylene glycol fractions werealso subjected to SDS–PAGE (50 lg of protein in each lane) for immunoblotting with anti-rat phospho Thr172 AMPK a-subunit antibody followed by streptavidin coupled tohorse-raddish peroxidase to detect the biotin component of pyruvate carboxylase as a loading control by chemiluminescence. The data are means ± SEM, n = 4 samples (eachsample made from a pool of four larvae) for September (white bars) or February (black bars) larvae. ⁄⁄,⁄⁄⁄Significant increase compared with the September values (P < 0.01and P < 0.001, respectively).

pS79 ACC

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Fig. 2. Phosphorylation state of ACC in Eurosta and Epiblema larvae collected in September and February. Extracts (200 lg of protein) prepared from September or Februarylarvae were pulled-down onto Streptavidin Agarose beads for SDS–PAGE and immunoblotting. Blots were probed with anti-rat phospho Ser79 ACC1 antibody followed bystreptavidin coupled to horse-raddish peroxidase to detect the biotin component of total ACC as a loading control by chemiluminescence. The histograms show mean bandintensities ± SEM (n = 4) obtained with the anti-phospho antibodies relative to the loading control for September (white bars) or February (black bars) larvae. Theimmunoblots are shown above the histograms. ⁄⁄Significantly different compared with the September value (P < 0.01).

1456 M.H. Rider et al. / Journal of Insect Physiology 57 (2011) 1453–1462

lected in February versus September (data not shown). Surpris-ingly, in larval extracts from insects collected in September, 4E-BP1 phosphorylation was undetectable by immunoblotting withanti-mouse phospho Thr37/46 4E-BP1 antibody, whereas a strongsignal appeared both in Eurosta and Epiblema larvae gathered in

February (Fig. 3). Cross-reactivity should not have been a problem,since the sequence around Thr37/46 of mouse 4E-BP1 (PPG-DYSTTPGGTLFSTTPGGTR) is conserved in insects such as D. melano-gaster (sequence MPEVYSSTPGGTLYSTTPGGTK; NCBI Accession No.NP_477295.1).

Fig. 3. Phosphorylation state of rpS6 and 4E-BP1 in extracts of Eurosta and Epiblema larvae collected in September and February. Extracts of whole larvae collected inSeptember or February were subjected to SDS–PAGE (40 lg of protein in each lane) and immunoblotting. Blots were probed with anti-human phospho Ser235/236 rpS6 andanti-mouse phospho Thr37/46 4E-BP1 antibodies along with anti-total PP2A catalytic subunit antibody as a loading control for detection by chemiluminescence. Thehistograms show mean band intensities ± SEM (n = 4) obtained with the anti-phospho antibodies relative to the loading control for September (white bars) or February (blackbars) larvae. The immunoblots are shown above the histograms. ⁄,⁄⁄⁄Significant increase compared with the September values (P < 0.05 and P < 0.001, respectively).

M.H. Rider et al. / Journal of Insect Physiology 57 (2011) 1453–1462 1457

3.3. PDH activity, E1a subunit phosphorylation and expression

To further explore metabolic changes in the cold-hardy insectlarvae, we examined the phosphorylation state of PDH. Extractswere blotted with anti-human PDH antibodies to study phosphor-ylation of the equivalent two main regulatory sites on the PDH E1asubunit, Ser293 (site 1) and Ser300 (site 2) relative to the total PDHE1a protein. The sequences of the peptides used to raise antibodiesagainst total human PDH and phospho sites 1 and 2 wereDPGVSYRTREEIQE, YRYHGH(pS)MSDPGV and MSDPGV(pS)YRTREE,respectively (Pilegaard et al., 2006). These sequences are highlyconserved in D. melanogaster (DPGTSYRTREEIQE, YR-YSGHSMSDPGT and MSDPGTSYRTREE; NCBI Accession No.NP_726946.1, respectively). Phosphorylation at site 1 was 2-foldhigher in larvae of Eurosta collected in February compared to ani-mals taken in September, but no seasonal changes in phosphoryla-tion at site 2 of PDH E1a in Eurosta or at PDH E1a sites 1 and 2 inEpiblema were observed (Fig. 4). When expressed relative to PP2Acatalytic subunits as a loading control, there was a large decreasein total PDH E1a expression in Eurosta and a tendency towards adecrease in PDH expression levels in Epiblema larvae collected inFebruary compared with animals collected in September (Fig. 4).This was supported by enzyme assay indicating decreases of about50% and 80% in the specific activities of PDH in Eurosta and Epibl-ema larvae, respectively, collected in winter versus September(Fig. 5). Interestingly, there was also a decrease of about 50% inactivity of the mitochondrial marker enzyme GDH in extracts fromboth overwintering animals compared with those collected in Sep-tember (Fig. 5).

3.4. GP phosphorylation and expression

Glycogen is the fuel used to synthesize cryoprotectants in bothgoldenrod gall formers (as well as many other cold-hardy species)and it has long been known that low temperature in the autumntriggers a strong increase in the amount of active phosphorylatedGP due at least in part to rapid-acting differential temperature ef-fects on the activities of phosphorylase kinase versus phosphory-lase phosphatase (reviewed in Storey and Storey, 1991). Toassess the phosphorylation state of GP in the larvae, extracts wereimmunoblotted with anti-rat phospho Ser14 GP antibody versusan anti-total GP antibody recognizing the human liver protein.The sequence surrounding Ser14 of GP in rat (EKRRQISIRGIV) iswell conserved in D. melanogaster (DRRKQISVRGIA) as is the immu-nogenic peptide used for raising the commercial anti-total GP anti-body (residues 300–432 of human liver GP) in the D. melanogastersequence (NCBI Accession No. NP_722762). While seasonal totalGP expression levels remained relatively constant, the phosphory-lation state of GP was more than 3-fold higher in Epiblema larvaecollected in February compared with larvae collected in Septem-ber, but in Eurosta there was no significant difference in GP phos-phorylation (Fig. 6).

3.5. TGL activity

Since AMPK in C. elegans has been shown to block ATGL-medi-ated triglyceride mobilization to ensure long-term dauer survival,we measured TGL activity in larval extracts of Eurosta and Epibl-ema. The assays were performed in the presence and absence of

Fig. 4. Phosphorylation state and expression level of the PDH E1a subunit in extracts of Eurosta and Epiblema larvae collected in September and February. Extracts of wholelarvae collected in September or February were subjected to SDS–PAGE (40 lg of protein in each lane) and immunoblotting. Blots were probed with anti-human phosphoSer293 (site 1) and anti-human phospho Ser300 (site 2) PDH E1a subunit antibodies along with anti-total PDH E1a or anti-total PP2A catalytic subunit antibodies as a loadingcontrol for detection by chemiluminescence. The histograms show mean band intensities ± SEM (n = 4) obtained with the anti-phospho antibodies relative to total PDH E1aand total PDH E1a relative to PP2A catalytic subunits for September (white bars) or February (black bars) larvae. The immunoblots are shown above the histograms.⁄Significantly different compared with the September values (P < 0.05).

Eurosta Epiblema0.0

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Fig. 5. PDH and GDH activities in extracts of Eurosta and Epiblema larvae collected in September and February. Extracts of whole larvae collected in September (white bars) orFebruary (black bars) were assayed for PDH (50 ll of extract) or GDH (10–20 ll of extract) as described in Section 2. The histograms show specific activities relative to totalextract proteins measured with bovine serum albumin as a standard. ⁄,⁄⁄Significant decrease compared with the September values (P < 0.05 and P < 0.01, respectively).

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the ‘‘BAY’’ hormone-sensitive lipase inhibitor, which in our handsreduces total TGL activity measured in adipocyte extracts by 60–70% at the concentrations used here (not shown). Inclusion of the‘‘BAY’’ inhibitor in TGL assays of the insect larval extracts barely af-fected lipase activity, suggesting that HSL and other neutral lipases

using a Ser-His-Asp catalytic triad, such as the fat body lipase char-acterized and cloned from Manduca sexta (Arrese et al., 2010),make little contribution to overall TGL activity and that the insectATGL homolog would be the major lipase in Eurosta and Epiblemalarvae. When expressed relative to the activity of the cytosolic en-

Total GP

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Fig. 6. Phosphorylation state of GP in extracts of Eurosta and Epiblema larvae collected in September and February. Extracts of whole larvae collected in September orFebruary were subjected to SDS–PAGE (40 lg of protein in each lane) and immunoblotting. Blots were probed with anti-rat phospho Ser14 GP antibody along with anti-totalGP antibody as a loading control for detection by chemiluminescence. The histograms show mean band intensities ± SEM obtained with the anti-phospho antibodies relativeto the loading control for September (white bars) or February (black bars) larvae. The immunoblots are shown above the histograms. ⁄Significant increase compared with theSeptember value (P < 0.05).

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zyme marker LDH to correct for differences in amount and recov-ery, there was no significant difference in TGL activity in extractsof Eurosta larvae collected in September compared with larvae col-lected in February (Fig. 7). However in winter, a significant 70% in-crease in TGL activity in extracts from Epiblema was observed(Fig. 7). There was an increase in specific activity of LDH in latewinter (Eurosta: 72.2 ± 4.4 mU/mg of protein September versus88.8 ± 5.8 mU/mg of protein February; Epiblema: 19.9 ± 2.2 mU/mg of protein September versus 28.2 ± 1.4 mU/mg of protein Feb-ruary, n = 4 for each measurement). The increase was statisticallysignificant only for Epiblema LDH (P < 0.02), such that the increasein TGL activity was amplified in extracts of animals harvested inFebruary compared with those taken in September when ex-pressed on a protein basis: 143 ± 35.4 pmol/min/mg of protein ver-sus 60.7 ± 18.2 pmol/min/mg of protein when assayed in theabsence of HSL inhibitor, respectively, but the increase was notquite significant (P = 0.08).

4. Discussion

Studies with nematodes, C. elegans, have indicated that AMPK isrequired for survival in the dormant ‘‘dauer’’ stage (Narbonne andRoy, 2009) and AMPK is also activated and linked with inhibition ofselected anabolic events during estivation in land snails, Otala lac-tea (Ramnanan et al., 2010). However, the role of AMPK in a com-parable hypometabolic state, diapause in insects, has not yet beeninvestigated. The AMPK a, b and c subunits are coded by singlecopy genes in insects and deletion of the a-subunit in D. melano-gaster was lethal resulting in severe defects in embryonic cellpolarity and cell structure (Lee et al., 2007). Moreover, RNAi inhibi-

tion of Drosophila AMPK in muscle reduced longevity and stressresistance (Tohyama and Yamaguchi, 2010). Here we show AMPKactivation in polyethylene glycol fractions of diapausing larvae ofEurosta and Epiblema by direct activity measurement (Fig. 1). How-ever, only in Epiblema larvae could we detect a significant increasein AMPK activation loop phosphorylation in winter animals (Fig. 1),raising the possibility that the increase in activity seen in polyeth-ylene glycol fractions from Eurosta harvested in February com-pared to September might reflect activation of AMPK-relatedkinases which are known to be able to phosphorylate the ‘‘SAMS’’substrate peptide. After pulling-down ACC on Streptavidin Agarosebeads, a winter increase in ACC phosphorylation was seen in Epibl-ema (Fig. 2). Recognition of downstream phosphorylation of ACC inEpiblema using anti-rat phospho Ser79 ACC1 antibody was fortu-itous since no signal was seen in blots of pull-downs from Eurostaeven though total ACC was detected (Fig. 2). With the exception ofthe key basic and hydrophobic residues flanking the phosphory-lated Ser residue in the AMPK recognition motif, the other sur-rounding amino acids in the single ACC enzyme of insects whosegenomes are available (e.g. silkworm, jewel wasp, pea aphid, flourbeetle, honey bee) are quite variable (not shown). Indeed no anti-phospho ACC signal was obtained for blots of Eurosta or Epiblemapolyethylene glycol fractions probed with an anti-phospho ACCantibody generated to recognize the AMPK site in D. melanogaster(Pan and Hardie, 2002). However, as in Epiblema, ACC phosphory-lation in snails O. lactea was readily detected using a mammalianantibody (Ramnanan et al., 2010). AMPK activation (Fig. 1) anddownstream ACC phosphorylation (Fig. 2) in overwintering Epibl-ema larvae could play a role in metabolic rate suppression duringdiapause. A similar conclusion about a role in hypometabolismwas drawn from results for AMPK and ACC responses when estivat-

Eurosta Epiblema

September February0

1

2

3

4

TGL

activ

ity (n

mol

/min

/Uni

t LD

H)

September February0

2

4

6

8 - BAY+ BAY** *

TGLa

ctiv

ity(n

mol

/min

/Uni

tLD

H)

Fig. 7. TGL activities in extracts of Eurosta and Epiblema larvae collected in September and February. Extracts (50 ll) of whole larvae collected in September (white bars) orFebruary (black bars) were assayed for TGL in the presence or absence of 0.3 lM ‘‘BAY’’ inhibitor and for LDH (5 ll of extract) as described in Section 2. The histograms showTGL activities expressed relative to LDH activity. ⁄,⁄⁄Significant increase compared with the September values (P < 0.05 and P < 0.01, respectively).

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ing versus active land snails were compared (Ramnanan et al.,2010). Phosphorylation of ACC by AMPK would lead to enzymeinactivation, thereby inhibiting de novo fatty acid synthesis in thewinter months. This correlates with the observed suppression ofactivities of other enzymes associated with lipid biosynthesis(e.g. ATP-citrate lyase, malic enzyme) seen over the winter in bothspecies (Joanisse and Storey, 1996). Inactivation of ACC would alsolead to lowered malonyl-CoA levels, which would be expected tostimulate mitochondrial long chain fatty acid oxidation as a fuelfor winter survival. Fatty acids would be a major fuel for freeze-avoiding Epiblema throughout the winter and for Eurosta wheneverit is not frozen. Freezing in Eurosta would lead to a lack of oxygenand thus inability to catabolize lipids as fuels.

During winter diapause, anabolic energy consuming processessuch as protein synthesis should be suppressed. AMPK activationis inhibitory for protein synthesis at both initiation and elongationsteps (Proud, 2007). Unfortunately, we could not evaluate the reg-ulation of translation elongation since meaningful blots could notbe obtained from extracts using anti-rat phosphoThr56 eEF2 anti-body. AMPK activation seen in February Epiblema larvae comparedwith September animals was associated with increases in 4E-BP1and rpS6 phosphorylation (Fig. 3), which seems contradictory, gi-ven that activated AMPK has been proposed to reduce mTORC1 sig-naling by phosphorylating tuberous sclerosis complex-2 (TSC2),mTOR kinase (reviewed by Proud, 2007) and raptor (Gwinn et al.,2008). However, although global protein synthesis should be sup-pressed during the winter, cap-dependent translation of specificmRNA species required for adaptation may be ongoing, requiringphosphorylation of 4E-BP1 and rpS6 to be maintained. Similarobservations were made in studying tissues from frozen frogs (Ri-der et al., 2006) or anoxic turtles (Rider et al., 2009) where AMPKactivation was seen concomitant with increases in 4E-BP1phosphorylation.

The 2-fold increase in PDH E1a subunit site 1 phosphorylationobserved in February larvae of Eurosta compared with Septemberanimals (Fig. 4) would be expected to decrease PDH activity anddecrease carbohydrate oxidation. Also the immunoblotting exper-iments indicated a significant decrease in PDH E1a subunit expres-sion levels in overwintering larvae of Eurosta compared withanimals collected in September. Direct assays showed significantdecreases in PDH and GDH specific activities in extracts from bothspecies of larvae harvested in February compared with those col-lected in September (Fig. 5), suggestive of a decrease in mitochon-drial content during overwintering. These data agree well withprevious studies that examined mitochondrial changes duringoverwintering. Both species showed reduced activities of many

mitochondrial enzymes over the winter months, including GDH,with the notable exception of the increased activities of enzymesof fatty acid oxidation in Epiblema (Joanisse and Storey, 1994,1996; McMullen and Storey, 2008). Studies of other mitochondrialparameters (DNA, RNA, cytochrome c oxidase) indicated that mito-chondrial numbers were maintained in Epiblema over the winter(hence, reduced enzyme activities probably result largely from reg-ulated suppression) whereas the analysis for freeze tolerant Euro-sta indicated an approximately 50% reduction in mitochondrialnumbers over the winter (Levin et al., 2003; McMullen and Storey,2008; Storey and Storey, 2010). An increase in pyruvate after rapidcold hardening of flesh flies reported in a metabolomics study (Mi-chaud and Denlinger, 2007) would be consistent with the decreasein PDH activity observed in diapausing insects here.

GP is typically held in a low activity state in autumn larvae be-fore cold stimulated cryoprotectant synthesis begins (Churchill andStorey, 1989; Storey and Storey, 1981, 1991) and this would ac-count for the low level of GP phosphorylation detected in Septem-ber. By contrast, a generally higher phosphorylation state occurredin the winter months when larvae are at subzero temperatures, aswas also reported by Churchill and Storey (1989). Indeed ourimmunoblotting experiments indicated an increase in phosphory-lation state of GP in Epiblema larvae collected in February com-pared to larvae collected in September (Fig. 6). This wouldmaintain a metabolic poise towards glycogen breakdown and to-gether with AMPK phosphorylation-induced inhibition of glycogensynthase (Hardie et al., 1998), inhibition of phosphofructokinase(Storey, 1982) and reduced PDH activity (see above), could helpto keep carbohydrates in the cryoprotectant pool (glycerol, sorbi-tol) over the winter months.

Surprisingly in overwintering Epiblema larvae, AMPK activationwas associated with elevated TGL activity (Fig. 7), presumably viathe insect homolog of ATGL, which could have been the result ofincreased enzyme expression or control by covalent modification.In C. elegans, AMPK has been shown to inactivate ATGL via phos-phorylation of Ser303 (Narbonne and Roy, 2009), but this residueis not conserved in the insect ATGL homologs. The fact that TGLactivity is not suppressed in these winter diapausing larvae wouldfacilitate mobilization of lipid reserves and coupled with loweredmalonyl-CoA levels due to AMPK-induced ACC inactivation, allowATP levels to be maintained via fatty acid oxidation. Epiblema lar-vae have huge reserves of lipid and the amount that is used overthe winter at cold temperatures would not necessarily be signifi-cant compared with the whole reserve. This is because the fuel re-serves in the insect’s body at the start of the winter not only haveto fuel survival over the winter (at very low temperatures) but also

M.H. Rider et al. / Journal of Insect Physiology 57 (2011) 1453–1462 1461

need to fuel the whole rest of the insect’s life including pupation,adult development, flight, reproduction, egg development andegg laying, all done at much higher environmental temperatures(the adults do not eat). So the percentage of stored fuels that gointo winter viability is likely very low compared to consumptionat other life stages.

Overall, our data suggest a re-sculpting of metabolism in diap-ausing cold-hardy insect larvae to a more catabolic poise that wasmore extensive in freeze-avoiding Epiblema than in freeze tolerantEurosta. In Epiblema, AMPK activation could promote catabolism inwinter (as compared with early autumn when fuel reserves are stillbeing accumulated) to maintain ATP levels and at the same timeminimize anabolic processes such as fatty acid biosynthesis there-by contributing to survival over the long-term in the hypometabol-ic state of diapause.

Acknowledgments

The work was supported by the Interuniversity Attraction PolesProgram – Belgian Science Policy (P6/28), the Directorate GeneralHigher Education and Scientific Research, French Community ofBelgium, the Fund for Medical Scientific Research (Belgium), theEXGENESIS Integrated Project (LSHM-CT-2004-005272) from theEuropean Commission and a Natural Sciences and Engineering Re-search Council of Canada grant (#6793).

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