Participation of Hindbrain AMP-Activated Protein Kinase in Glucoprivic Feeding.

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by  Li, Ai-Jun

ORIGINALARTICLE
Participation of Hindbrain AMP-Activated Protein
Kinase in Glucoprivic Feeding
Ai-Jun Li, Qing Wang, and Sue Ritter
OBJECTIVE—To examine the role of AMP-activated protein as well as for glucoprivation-induced corticosterone se-
kinase(AMPK)inthecontrolofglucoprivicfeedingbyhindbrain cretion and suppression of estrous cycles (2–4) and for
catecholamine neurons. glucoprivation-induced increases in arcuate nucleus neu-
ropetideY(Npy)andagouti-relatedprotein(Agrp)mRNAs
RESEARCH DESIGN AND METHODS—Micropunched hind-
(5). A separate population that projects spinally mediates
brainsampleswerecollectedfromcontroland2-deoxy-D-glucose the adrenal medullary response (2,6). Additional work
(2DG)-injected rats for Western blot analysis of phosphorylated
using a number of different approaches has shown that
(activated) AMPK (pAMPK). Samples also were collected from
2DG-injected rats pretreated with anti-dopamine-b-hydroxylase the hindbrain contains cells capable of detecting a glu-
conjugatedtosaporintolesionhindbraincatecholamineneurons. cose deficit and eliciting feeding and other glucoreg-
Inasecondexperiment,ratsweregivenafourth-ventricleinjec- ulatoryresponses(7–11)andsuggeststhepossibilitythat
tion of compound C (CC) or 5-aminoimidazole-4-carboxyamide certain catecholamine neurons may themselves be glu-
ribonucleoside (AICAR), an inhibitor and activator of AMPK, to coreceptive.
identify a role for AMPK in hindbrain neurons required for elic- A number of findings indicate that the specific cate-
itation of2DG-induced feeding.
cholamine neurons required for glucoprivic feeding are
RESULTS—Systemic2DGstimulatedfoodintakeincontrolsbut located in the ventrolateral hindbrain area containing the
notincatecholamine-lesionedrats.Incontrols,butnotcatechol- rostral portion of cell group A1 and the caudal two-thirds
amine-lesioned rats, 2DG also increased phosphorylated Thr172 of group C1 (A1–C1). The A1–C1 neurons are potently
at AMPKa subunits (pAMPKa) in hindbrain micropunches con- activated by glucoprivation (12–14), and they innervate
taining catecholaminergic cell groups A1 through the middle re-
the hypothalamic areas involved in the control of metab-
gion of C1 (A1–C1m). Increased pAMPKa was not observed in
olism and appetite (15). Furthermore, glucoprivic feeding
the adjacent noncatecholaminergic ventromedial medulla or in
the A2–C2 catecholamine cell groups in the dorsal hindbrain. is blocked by simultaneous silencing in A1–C1 neurons
Fourth-ventricleinjectionofCCattenuated2DG-inducedfeeding of the genes encoding the catecholamine biosynthetic en-
during the first 2 h of the test, and AICAR alone increased food zyme, DBH, and the colocalized peptide, NPY, respec-
intakeonlyduringthefirst60min ofthe4-htest. tively (16).
AMP-activated protein kinase (AMPK) is a highly
CONCLUSIONS—Results indicate that AMPK in catecholamin-
ergicA1–C1mneuronsisactivatedbyglucoprivation.Therefore, conserved serine/threonine kinase that is activated by an
increase of the cellular AMP-to-ATP ratio and is consid-
AMPK may contribute to the glucose-sensing mechanism by
which these neurons detect and signal a glucose deficit in the eredtobeasensorofcellularenergystatus(17–20).Upon
serviceofsystemicglucoregulation.Diabetes60:436–442,2011 activation, AMPK inhibits ATP-consuming pathways and
stimulates ATP-generating pathways to restore cellular
energy balance. AMPK is expressed in brain regions in-
A
volved in the regulation of feeding behavior (21,22), and
continuous supply of glucose is essential for a number of studies have shown that brain AMPK con-
the function and survival of neurons in the tributes to the control of food intake and glucose homeo-
brain. Maintaining the brain’s glucose supply stasis. Food deprivation increases AMPK activity in the
depends on a number of glucoregulatory re- hypothalamus, whereas refeeding inhibits it (23). A third-
sponses elicited by a glucose deficit. Systemic injection of ventricle injection of the AMPK activator 5-amino-
2-deoxy-D-glucose (2DG), a competitive inhibitor of intra- imidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) or
cellular glucose utilization (1), induces cellular glucopri- overexpression of AMPK in the hypothalamus increases
vationandtriggersanumberofglucoregulatoryresponses. feeding and body weight (23,24). Activation of AMPK
Increasedfoodintakeisoneofthemostcrucialresponses requires phosphorylation of catalytic a-subunits at Thr172
to a glucose deficit. Studies using a selective retrogradely (pAMPKa). Reports have shown that pAMPKa in the hy-
transported catecholamine immunotoxin, anti-dopamine- pothalamus is upregulated by insulin-induced hypoglyce-
b-hydroxylase (DBH)-saporin (DSAP), have demonstrated mia or 2DG-induced glucoprivation (25–27) and that 2DG
that catecholamine neurons innervating the medial hypo- enhancespAMPKaexpressionthroughdecreasingcellular
thalamusarerequiredforelicitationofglucoprivicfeeding
ATP levels in cultured neuronal cells (28). Thus, we hy-
pothesizedthat AMPKinthehindbrainalsocontributesto
2DG-induced glucoprivic feeding and may be a part of the
FromthePrograms inNeuroscience,WashingtonStateUniversity, Pullman, glucose-sensing transduction mechanism. As a first step
Washington. in testing this hypothesis, we measured pAMPKa in the
Correspondingauthor:Ai-JunLi,[email protected]. hindbrain catecholamine cell group A1–C1 after 2DG in-
Received10March2010andaccepted29November2010.
DOI:10.2337/db10-0352 jectionincontrolratsandinratswithselectiveretrograde
(cid:1)2011bytheAmericanDiabetesAssociation.Readersmayusethisarticleas immunotoxin lesion of these neurons. In addition, we ex-
longastheworkisproperlycited,theuseiseducationalandnotforprofit,
amined the effect of enhancement or blockade of AMPK
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/fordetails. activity in the hindbrain on glucoprivic feeding.
436 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
A.-J.LI,Q.WANG,ANDS.RITTER
RESEARCHDESIGNANDMETHODS Micropunchedsampleswerequicklyplacedintoaradioimmunoprecipitation
MaleSDrats(SimonsenLaboratories,Gilroy,CA)werehousedindividuallyand assay (RIPA) buffer for protein isolation. The accuracy of the punches was
confirmedbyWesternblotusingantibodiesagainstenzymesforcatecholamine
maintainedona12-hlight/darkcycle(lightsonat0600h)withadlibitumaccess
synthesis(DBHandTH).RepresentativeimagesofDBHandTHWesternblots
topelletedrodentfood(F6diet;HarlanTeklad,Madison,WI)andtapwater.All
forA1–C1m,VMM,andA2–C2tissuepuncheswereshowninFig.1.Forallrats,
experimentalprocedureswereapprovedbytheWashingtonStateUniversity
bothDBHandTHexpressionwereexpressedmorestrongly(morethanthree
Institutional Animal Care and Use Committee, which conforms to National
times)inA1–C1mandA2–C2samplesthanintheVMMsamples,indicatingthe
InstitutesofHealthguidelines.
accuracyofthepunches.
Cannula implantation and fourth-ventricle injections. For the fourth-
In AICAR-induced AMPK experiments, two 2-mm-thick coronal sections
ventriclecannulaimplantation,ratswereanesthetizedusing1.0mL/kgbodywt werecut,usingobextolocatethepositionofthecuts(0–2.0and2.0–4.0mm
ofaketamine/xylazine/acepromazinecocktail(5mLketamineHCl,100mg/mL,
rostraltoobex,respectively).Then,thetwoslicesweredividedintothedorsal
FortDodgeAnimalHealth,FortDodge,IA;2.5mLxylazine,20mg/mL,Vedco,
andventralparts.Slice1,thedorsalpart,includedA2,C2,andtheNTS.Slice1,
St.Joseph,MO;1mLacepromazine,10mg/mL,Vedco;and1.5mL0.9%saline theventralpart,includedA1–C1m.Slice2,thedorsalpart,includedA2and
solution)andplacedinastereotaxicdevice.A26Gcannula(15.0mmlong)was
C2.Slice2,theventralpart,includedC1randA5.Tissuesectionswerequickly
implanted1.55mmrostraltotheoccipitalsuture,60mmlateraltomidlineand
placedintoRIPAbufferforproteinisolationandthenusedforWesternblot.
6.3–6.4 mm ventral to the skull surface (29). Experiments were initiated 1 Westernblot.Themethodwasmodifiedfromourpreviousreports(30,31).
weeklater.Fourth-ventricleinjectionsweredeliveredovera3-to5-minperiod HindbraintissueswerefirstincubatedwithRIPAbuffer(50mmol/LTris,pH
usingaPB-600repeatingdispenser(Hamilton,Reno,NV).Thepositionofthe
8.0; 150 mmol/L NaCl; 1.0% (Octylphenoxy)polyethoxyethanol; 0.5% sodium
fourth-ventriclecannulaforeachratwasconfirmedaftertheexperimentsby deoxycholate;and0.1%SDS),with1mmol/Lsodiumortho-vanadate,1mmol/L
staining hindbrain sections with cresylviolet.Only ratswith acannula cor-
okadiac acid, and 1/10 (vol/vol) protease inhibitor cocktail (Roche Diag-
rectlyinsertedintothefourthventriclewereusedfordataanalysis.
nostics, Penzberg, Germany) for 30 min on ice. After centrifugation (14,000
Feeding tests. For glucoprivic feeding tests, rats were injected subcu- rpmfor15minat4°C),anequivalentamountofprotein(20mgperlane)for
taneouslywith2DG(200mg/kgbodywtin0.9%sterilesaline;Sigma-Aldrich,St.
eachsamplewasseparatedbySDS-PAGE.Theresolvedproteinsonthegel
Louis,MO)orwith0.9%sterilesalineasthecontrol.Foodwasremovedat0800
weretransferredtoanitrocellulosemembrane(Amersham,Piscataway,NJ).
h,2hbeforetheinjections.Foodintakewasmeasuredbeginningimmediately
Themembraneswereblockedin5%nonfatdrymilkinTris-bufferedsaline/
aftertheinjectionandatvariousintervalsthereafter,asindicated.TheAMPK
0.05%Tween20andincubatedwithoneofthefollowingprimaryantibodiesin
inhibitor,compoundC(CC;Sigma-Aldrich),wasdissolvedinDMSO(3mg/mL)
andwasinjectedintothefourthventricleatavolumeof1mLperrat10–15
min before 2DG injection. At this concentration and volume, nonspecific
effectsofDMSOonfeedingwerenotobserved.TheAMPKactivatorAICAR
(Sigma-Aldrich)wasdissolvedinsalineandinjectedintothefourthventricle
atadoseof20,100,300(or500)nmoLperratin3(or5)mLsaline.
DSAPmicroinjectionandDBHimmunohistochemistry.Theimmunotoxin
DSAP(82ng/200nL;AdvancedTargetingSystems,SanDiego,CA)orcontrol
unconjugatedsaporin(SAP)(17.2ng/200nL),dissolvedin0.1mol/LPBS(pH
7.4),wasinfusedbilaterallythroughapulled-glasscapillarypipette(30-mmtip
diameter) positioned stereotaxically just dorsal to the targeted site in the
paraventricularhypothalamicnucleus(PVH).Solutionsweredeliveredusinga
picospritzer (Parker, Cleveland, OH). Stereotaxic coordinates for the PVH in-
jectionsitewere1.8mmcaudaland60.45mmlateraltobregmaand7.3–7.4mm
ventraltoduramater(29).TheamountofunconjugatedSAPinthecontrol
solution approximated the amount of SAP present in the DSAP conjugate
(21%),asindicatedinthemanufacturer’sproductinformation.Previouswork
comparingSAPanduninjectedcontrolsdemonstratedthatSAPdidnotpro-
duce behavioral or significant histological signs of toxicity (2). Glucoprivic
feedinginducedby2DG(200mg/kg)wastested3weeksafterDSAPorSAP
injections.Oneweeklater,expressionofpAMPKaandtotalAMPKainhind-
brainafterthe2DGinjectionwasmeasured.TheDSAPlesionwasevaluated
byWesternblotanalysisoftyrosinehydroxylase(TH)expressioninhindbrain
sitesofinterestandinaseparategroupoflesionedratsusingimmunohisto-
chemistry (IHC) staining to detect DBH-positive cell bodies. Two methods
were used to confirm the DSAP lesion because we knew that Western blot
analysiswoulddetecttheTHpresentinthepunchedsamplefromterminals
and processes of unlesioned catecholamine neurons not targeted by PVH
DSAPinjection.Thus,WesternblotsofTHwouldnotreflectthedestructionof
catecholaminecellbodiesinthelesionsiteasaccuratelyasIHC.
For IHC, brains were sectioned coronally into four serial sets (40-mm
thickness)andstainedusingstandardavidin-biotin-peroxidaseIHCtechniques
(13).CellbodiesexpressingDBHweredetectedbymurineanti-DBHantibody
(1:10,000;Millipore,Billerica,MA)andcountedinthreeconsecutivesections
(29)ateachoffourhindbrainregions.
Micropunch and dissection of hindbrain tissues. Tissue sites containing
catecholaminecellgroupsweremicropunchedordissectedfromhindbrainfor
Westernblot.CatecholaminecellgroupsaredefinedaccordingtothePaxinos
andWatson(1998)ratbrainatlas(29).However,werefertothemiddleportion
ofC1asC1m,whichextendsfrom12.72to12.30mmcaudaltobregma.
At designated times after treatment, rats were anesthetized deeply with
halothane (Halocarbon Products, North Augusta, SC) and were quickly de-
capitated.Thebrainwasremovedandfrozenondryice.In2DG-inducedAMPK FIG. 1. Tissue micropunches from the hindbrain slices. A: Coronal
experiments,a2-mm-thickcoronalsectionwascut,usingobex(definedhereas sectionofrathindbrain(PaxinosandWatson[29])showingtheposi-
thecaudalborderoftheareapostrema)tolocatethecaudalborderoftheslice tionsofeach1.0-mm-diametercircularpunchesusedforWesternblots.
(0–2.0mm rostral toobex).Then, thedesiredregionsfromthis 2-mm-thick Punches1,2,or3includedA1–C1m,VMM,orA2–C2, respectively. B
sliceweremicropunchedwithastainlesssteelsamplecorerthathada1.0-mm and C: Immunoblots of DBH, TH, and b-actin from different tissue
micropunchesareindicatedinA.HigherexpressionlevelsofDBHand
inner diameter (Fine Science Tools, Foster City, CA). Tissue punches were TH were found in punch 1 (A1–C1m) tissue (B) or punch 3 (A2–C2)
collectedfromthreeareas(fromA1–C1m,froman[noncatecholamine]area tissue(C)thanintheadjacentpunch2(VMM)tissueinallindividual
medial to A1–C1m [ventromedial medulla (VMM)], and from A2–C2 in the rats. AP, area postrema; CC, central canal; py, pyramidal tract; sp5,
nucleusofthesolitarytract[NTS]). spinaltrigeminaltract.
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 437
HINDBRAINAMPKANDGLUCOPRIVICFEEDING
blockingsolutionfor16–18hat4°C.Themembraneswerethenwashedwith (Fig. 3B)inthe hindbrain.SAP rats atefivefold more food
Tris-buffered saline/0.05% Tween 20, incubated in goat anti-rabbit IgG, goat during the 4-h period after 2DG injection than after the
anti-murineIgG,ordonkeyanti-goatIgGantibodyconjugatedwithperoxidase salineinjection(P,0.001),whereasDSAPratsatealmost
(1:4,000; DakoCytomation, Glostrup, Denmark) and developed with the en- the same amount of food in the two tests (P . 0.1). In
hancedchemiluminescencemethod(Amersham)andexposedtoX-OMATfilm
(Kodak,Rochester,NY).Thefollowingantibodieswereused:murineanti–b- aseparateexperiment,foodintakewasmeasuredatboth2
actin (1:10,000; Sigma-Aldrich), rabbit anti-AMPKa(1:1,000; Santa Cruz Bio- and 4 h after 2DG injection. Between 0 and 2 h, SAP rats
tech,SantaCruz,CA),rabbitanti-pAMPKa(Thr172)(1:1,000;Millipore),goat ate 4.8 6 1.1 g vs. 0.2 6 0.1 g for DSAP rats, whereas
anti-DBH(1:600;SantaCruzBiotech),andmurineanti-TH(1:6,000;Millipore). between2and4h,DSAPratsate0.460.1gvs.0.560.1g
Densitometric analysis of blots was performed using Scion Image software for SAP rats (n = 5 for each group). 2DG increased the
(Scion, Frederick, MD). The intensity of each band for each protein was pAMPKa expression in A1–C1m in the SAP rats at 45 min
quantifiedandnormalizedwithhousekeepinggeneb-actinexpressioninthe
samemembrane.ThelevelsoftotalAMPKaorpAMPKawereexpressedas after2DGinjection(P,0.01),whereas2DGhadnoeffect
apercentageoverthatintheratsinjectedwithsaline. on pAMPKa expression in the DSAP rats (Fig. 3C). DSAP
Statisticalanalysis.Allresultsarepresentedasmeans6SEM.Forstatistical rats, treated with saline or 2DG, had slightly higher
analysisofdata,weusedunpairedttestorone-wayANOVAusingSigmaStat pAMPKa expression levels than SAP saline rats, but there
(SystatSoftware,SanJose,CA).AftersignificancewasdeterminedbyANOVA, wasnosignificantdifferencebetweenthetwoDSAPgroups.
multiplecomparisonsbetweenindividualgroupsweretestedusingaposthoc No significant changes were found in the total AMPKa ex-
Fisherprotectedleastsignificantdifference(PLSD)test.P=0.05orP,0.05
wasconsideredtobestatisticallysignificant. pression levels between SAP and DSAP rats, regardless of
whether they were injected with saline or 2DG (Fig. 3D).
In DSAP rats, TH expression in A1–C1m, by Western
RESULTS blot, was decreased to 44.2% of the level in SAP controls
2DG increased pAMPKa expression in A1–C1m. As (P,0.001)(Fig.3B).THexpressiondidnotdifferforA1–
shown in Fig. 2A, pAMPKa was significantly increased C1minSAPsalineandnon–SAPsalinerats(100.0610.2%
by 2DG in ventrolateral medulla punches, which included vs.94.364.6%;n=6–7rats;P.0.6),indicatingthatSAP
A1–C1m, 45 min after the injection (193.6 6 13.8% in the injections did not retrogradely lesion hindbrain catechol-
2DGgroupvs.100.066.1%inthesalinegroup;P,0.001) amine neurons. Parallel IHC analysis of the lesion (Fig. 4)
and returned to near-control values 2 h after the injection showedthat PVH DSAP injection eliminatedDBH-positive
(113.1 6 8.1% of control). This enhancement was site fibers in the PVH and significantly reduced the number
specific. In the VMM, a region adjacent to the A1–C1m, of DBH-positive cells in the A1–C1m, which heavily in-
pAMPKa was unchanged by 2DG (Fig. 2A). In the A2–C2 nervatethePVH,asshownpreviously(15,32).Thenumber
region, pAMPKa was somewhat increased at both 45 min ofDBH-positivecellswasreducedintheA1–C1mto21.5%
and 2 h after 2DG injection, but the increased levels did of the number present in SAP rats.
not differ significantly from control values (P . 0.4 be- InhibitionofAMPKactivityinthehindbrainimpaired
tween groups). Total AMPKa expression was not altered glucoprivic feeding. Preinjection of CC significantly
by 2DG in any of the sampled regions at the time points suppressed glucoprivic feeding during the first 2 h after
tested (Fig. 2B). 2DG injection to 70.6% of the amount consumed by rats
LesionofhindbrainneuronsbyDSAPabolished2DG- injected with 2DG alone (P , 0.05) but did not suppress
induced pAMPKa expression. After PVH DSAP injec- 2DG-induced feeding at 4 h (81%, P . 0.1) or 7 h (97.1%,
tions,catecholamineneuronsinthehindbrainweregreatly P . 0.8) after injection (Fig. 5).
reduced, as indicated by the attenuation of 2DG-induced Effect of hindbrain activation of AMPK on food
glucoprivicfeeding(Fig.3A)anddecreasedTHexpression intake. To reveal whether activation of hindbrain AMPK
FIG.2.TotalandphosphorylatedformsofAMPKainthreehindbrainregionsafter2DGinjection.Tissuehomogenateswereobtained45or120min
aftersaline(control)or2DG(200mg/kgs.c.)injection.EquivalentamountsofproteinweresubjectedtoSDS-PAGEandimmunoblottedwithanti-
pAMPKa,anti-totalAMPKa,oranti–b-actinantibody.A:Upperpanel:immunoblotsofpAMPKaandb-actininA1–C1m,VMM,andA2–C2regions
withsalineor2DGinjection.Lowerpanel:quantitativedata.B:Upperpanel:immunoblotsoftotalAMPKaandb-actininA1–C1mandA2–C2
regions.Lowerpanel:quantitativedata.Tocompareexpressionlevelsoftheseproteins,theintensityofeachbandwasquantifiedandnormalized
withb-actinandexpressedasthepercentageofsalinecontrolrats.*P<0.01vs.thecontrolsaline-injectedrats.FisherPLSDtestafterone-way
ANOVA(n=eightratsforeachgroup).
438 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
A.-J.LI,Q.WANG,ANDS.RITTER
FIG.3.GlucoprivicfeedingandAMPKexpressioninDSAP-orSAP-injectedrats.A:Foodintakeduringthe4-hperiodafter2DG(200mg/kgs.c.)or
saline(Sal)injectioninSAPandDSAPrats.*P<0.001vs.SAPratswithsalineinjection(n=16ratspergroup).B:THexpressionlevelsinA1–
C1mregionsinSAPandDSAPrats,measuredbyWesternblot.ThelevelofTHinSAPrats,normalizedtob-actin,wasusedas100%.*P<0.001vs.
SAPrats(n=16ratspergroup).C:Upperpanel:immunoblotsofpAMPKaandb-actininA1–C1mregions45minaftersalineor2DGinjection.
Lowerpanel:quantitativepAMPKadata,normalizedtob-actin.*P<0.05vs.saline-injectedSAPrats;#P<0.05vs.2DG-injectedSAPrats.Fisher
PLSDtestafterone-wayANOVA(n=eightratspergroup).D:Upperpanel:immunoblotsoftotalAMPKaandb-actininA1–C1mregions45min
aftersalineor2DGinjection.Lowerpanel:quantitativedata,normalizedtob-actin(n=eightratspergroup).
can induce feeding, an AMPK activator, AICAR, was AMPK is a universal cellular energy sensor, present in
injected into the fourth ventricle. Figure 6 shows that both peripheral tissues and throughout the brain. There-
AICAR at 20 and 100 nmol per rat increased feeding only fore,onequestionthatimmediatelyarisesfromourresults
during the first 60 min after the injection (all P = 0.05 or
P , 0.05). The higher dose, 500 nmol, did not appear to
alter foodintake atanytimepoint.The timecourseofthe
AICAR response was much more transient than that pro-
duced by 2DG or insulin-induced hypoglycemia. Western
blot was performed to check whether such doses of
AICARefficientlyincreaseAMPKactivityinthehindbrain.
As shown in Table 1, AICAR doses $100 nmol per rat
significantly increased pAMPKa expression in the hind-
brain 35–45 min later. AICAR was more effective in in-
creasing pAMPKa in the ventral than in the dorsal
hindbrain (Table 1), in both slice 1 (0–2.0 mm rostral to
obex) and slice 2 (2.0–4.0 mm rostral to obex).
DISCUSSION
In the current study, we found that systemic 2DG treat-
ment increased hindbrain AMPK phosphorylation at the
Thr172ofthea-subunitdetectedbyourantibody.pAMPKa
expression was significantly elevated only in the A1–C1m
region of the ventrolateral medulla but not in neighbor-
ing noncatecholaminergic VMM. Expression of pAMPKa
also tended to increase in A2–C2, but there the increase
was not statistically significant. The fact that pAMPKa
expression levels were not increased by 2DG in the A1–
C1mregioninratsinwhichcatecholamineneuronsinthis
region were lesioned retrogradely by PVH DSAP injection
suggests that the AMPK phosphorylation induced by 2DG
inSAPcontrolsoccursinA1–C1mcatecholamineneurons. FIG.4.LesioneffectofDSAPonhindbraincatecholamineneurons.A:
The fact that this same DSAP lesion also abolished the RepresentativeIHCimagesofDBHstaininginaSAP-injected(aandc)
orDSAP-injected(bandd)ratinPVH(aandb)orthehindbrainA1/C1
glucoprivic feeding response is consistent with a func- region(candd).Scalebar=0.5mm.B:DBH-positivecellsineachofthe
tional role for AMPK in mediation of glucoprivic feeding. hindbrainregions.*P<0.01vs.SAPrats(n=fiveratsineachgroup).
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 439
HINDBRAINAMPKANDGLUCOPRIVICFEEDING
glucoprivation than other brain neurons, including those
that are downstream in the glucoregulatory circuitry.
Therefore, a potential answer to this question is that cat-
echolamine neurons in the A1–C1m region are more sen-
sitive to a glucoprivic challenge than most neurons. This
possibilityisconsistentwithpreviousreportsshowingthat
A1–C1m neurons are more sensitive than most other cat-
echolamine neurons to glucoprivation-induced increases
in expression of c-Fos, Npy, and Dbh mRNA (2,12,13).
These results suggest that AMPK may be phosphorylated
at a higher glucose level in these catecholamine neurons
thaninmanyotherneuronsinpartbecausetheiractivityis
increased, as opposed to reduced or not altered, by a glu-
cosedeficit,causingthemtogenerateanATPdeficitmore
rapidly.
Theabovequestionisimportantinconsideringwhether
the pAMPK is a consequence of increased neuronal ac-
tivity during glucoprivation or whether this signal is a
componentoftheglucoreceptivetransductionmechanism.
The effects of CC on glucoprivic feeding suggest that the
elevated pAMPK may be related more to activation of the
neurons than to the transduction mechanism for sensing
FIG.5.EffectofCC,anAMPKinhibitor,on2DG-inducedfeeding.CCor
thecontrolsolvent,DMSO,wasinjectedintothefourthventricle10–15 glucoprivation. CC-induced attenuation of 2DG-induced
minbefore2DG(200mg/kgs.c.)orsaline(Sal)injection.Foodintake feeding had a transient effect, and rats completely com-
wasmeasuredforthenext7h.*P<0.05vs.DMSO-Salgroup;#P<0.05
pensated for the brief reduction of food intake during the
vs.DMSO-2DGgroup.FisherPLSDtestafterone-wayANOVA(n=eight
ratsineachgroup). ensuing hours of the feeding test. If AMPK phosphory-
lation represented an adequate stimulus for detecting
glucoprivation, one might have expected a reversal or at-
is why increased pAMPK induced by systemic glucopri- tenuation of feeding that was uncompensated. Thus, it
vationwasapparentonlyintheA1–C1mtissuepunch.One appears as if the CC did not block a transduction process
would expect that ubiquitous reduction in glucose utiliza- or reduce glucoprivic signal strength but rather reduced
tionaftersystemic2DGtreatmentwouldactivateAMPKin therateoftranslationtoabehavioralresponse,possiblyby
many neurons. However, based on Fos expression as an reducing the cellular metabolic rate of the essential cate-
indicatorofactivation,relativelyfewneuronsareactivated cholamine neurons. Likewise, a fourth-ventricle injection
by glucoprivation. Furthermore, increased Fos in many of AICAR stimulated feeding only during the first 60 min
other brain areas is reduced or abolished when hindbrain after injection. The brevity of these effects may be attrib-
catecholamineneuronsaredestroyedbyDSAP(2).Hence, utable to differing pharmacokinetics of the 2DG, CC, and
it is clear that A1–C1 neurons are more responsive to AICAR. However, this explanation does not account for
thefactthat2DGinjectionproduceslong-lastingeffectson
feeding but produced a relatively transient activation of
pAMPKa expression in the A1–C1m region. Indeed, it is
known that the feeding response and increased activation
of hindbrain catecholamine neurons (as measured by hy-
pothalamic norepinephrine turnover) persists even after
the return of normoglycemia, .6 h after 2DG- or insulin-
inducedhypoglycemia,ifnutrientsarewithheldduringthe
glucoprivic episode (33–36). Additional work will be re-
quired to determine how AMPK phosphorylation and its
downstream effects in A1–C1m and other neurons are re-
lated to the stimulation and time course of glucoprivic
feeding.
Although the results of pharmacological AMPK manip-
ulations do not provide strong support for a role in
transduction of the glucoprivic signal, several experimen-
tal observations have linked AMPK activation with tran-
scriptional changes appropriate to increasing food intake.
First, overexpression of the dominant-negative AMPKa
subunits in the ventromedial hypothalamus suppresses
food intake, reduces body weight, and decreases expres-
sion oftheorexigenicneuropetidegenes Npyand Agrpin
the arcuate nucleus, whereas expression of constitutively
active AMPKa subunits in that region increases feeding,
FIG. 6. Effect of hindbrain AMPK activation on food intake. Saline bodyweight,andenhancesthefasting-inducedincreaseof
(Sal)ordifferentdosesoftheAMPKactivator,AICAR,wereinjected Npy and Agrp in the arcuate nucleus (23). Injection of
intothefourthventricle.Foodintakewasmeasuredaftertheinjection. AICAR into the lateral ventricle enhances Npy expression
*P=0.05orP<0.05vs.thesalinegroup.FisherPLSDtestafterone-
wayANOVA(n=5–8ratspergroup). in thehypothalamus, asshownby Northern blot (37). Our
440 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
A.-J.LI,Q.WANG,ANDS.RITTER
TABLE1
ExpressionlevelsofpAMPKaandtotal AMPKainthehindbrainafter AICARinjection
DoseofAICAR(nmolperrat)
Region 0 20 100 300–500
Slice 1,dorsalpart
pAMPKa 100.062.6 100.3615.8 116.0622.5 100.3612.8
TotalAMPKa 100.061.5 111.363.1 124.366.3 111.168.6
Slice 1,ventral part
pAMPKa 100.065.1 115.468.6 159.5619.1* 191.8624.8†
TotalAMPKa 100.061.4 101.6610.3 108.366.5 106.265.8
Slice 2,dorsalpart
pAMPKa 100.064.2 87.3618.5 111.368.7 120.0617.0
TotalAMPKa 100.067.3 104.6613.2 118.967.5 107.862.3
Slice 2,ventral part
pAMPKa 100.065.9 111.468.1 143.9611.7* 159.5617.4†
TotalAMPKa 100.060.6 103.062.3 110.062.6 105.665.2
Dataaremeans6SEM.ExpressionlevelsofpAMPKaandtotalAMPKa(bothnormalizedtob-actinexpression)quantifiedbyWesternblot
fromdissectedhindbraintissuescollected35–45minafteraninjectionofAICAR(0,20,100,or300–500nmolperratinsaline)intothefourth
ventricle.*P,0.05;†P,0.01vs.thecontrolsaline-injectedrats(as100%)byFisherPLSDtestafterone-wayANOVA(n=4ratspergroup).
previous work is consistent with the possibility that Npy pAMPKa expression was increased by ~30% in the A2–C2
geneexpressionalsoisatargetofAMPKinthehindbrain. micropunches at both 45 min and 2 h after the 2DG
Studies have shown that Dbh and Npy are colocalized in treatment.
A1–C1 catecholamine neurons (15,32) and are both re- Basal levels of pAMPKa in DSAP rats were slightly en-
quired for glucoprivic feeding (2,3,12–14,38). Glucopriva- hanced in the A1–C1m compared with SAP rats. The
tionenhancesexpressionofthesegenesinA1–C1neurons mechanisms underlying this apparent slight enhancement
(12,13). Simultaneous silencing Npy and Dbh by injection and the cell types contributing to this increase were un-
of small-interfering mRNA into the A1–C1 region blocks clear inthe current study.One possibility is that activated
feeding in response to systemic glucoprivation but fails to glia cells, which persist for prolonged periods after neu-
block feeding when either of these genes is blocked sep- ronal destruction, were present in the A1–C1 area as a
arately (16). Npy in A1–C1 catecholamine neurons would correlate of catecholamine neuron degeneration and may
be a feasible target of AMPK because it appears to be in have contributed to enhancement of basal AMPK phos-
the hypothalamus. phorylationlevels.Regardlessofthecelltyperesponsible,
In the current study, we found a significant enhance- it is clear from the current study that this enhanced basal
ment ofpAMPKaby 2DG inthe ventral (includingA1, A1/ pAMPKa did not enhance, and therefore presumably was
C1 overlap, and C1m), but not in dorsal, hindbrain not related to, glucoprivic feeding.
micropunches (including A2, C2, and the NTS). The neu- In summary, we found that 2DG-induced glucoprivation
rons in the ventral hindbrain regions also were more sen- activated pAMPKa in the hindbrain region containing
sitive to AMPK activation because AICAR injection into A1–C1m catecholamine neurons. This activation was site
the fourth ventricle increased pAMPKa expression in the specific(itdidnotoccurinadjacentnoncatecholaminergic
ventralbutnotthedorsalpartofthehindbrain.Incontrast, tissue). Both 2DG-induced glucoprivic feeding and the
Hayes et al. (39) have reported an enhancement of enhancementofpAMPKaintheA1–C1mwerecompletely
pAMPKainratNTSafter24–48hoffooddeprivation.They abolished by retrograde DSAP lesion of A1–C1m neurons,
did not report results from ventral hindbrain samples. suggesting a major contribution of these neurons to the
However,thefact that their dorsal hindbrain results differ pAMPK enhancement. Activation of AMPK by fourth ven-
fromoursdeservescomment.First,thedifferencesmaybe tricular AICAR induced a brief feeding response, and
attributed to the differing treatments used in these two preinhibition of hindbrain AMPK activity by CC briefly
studies.Fooddeprivationfor24–48hdoesnotnecessarily impaired the feeding response to 2DG. These results sug-
produceglucoprivation. Fooddeprivation overthis period gest that AMPK may contribute to the functions of A1–C1
altersnutrientandgastrointestinalsignalsknowntoacton catecholamine neurons during glucoprivation and may in-
the vagus nerve and/or the NTS (40) and are a likely stim- dicate a role for AMPK as a glucosensory transduction
ulus for AMPK activation, whereas the major responses mechanism for the initiation of systemic glucoregulatory
to glucoprivation occur by direct effects of glucopriva- responses.
tion within the brain. In addition, differences between
these studies may be attributed to tissue sampling. Our
dorsalhindbraintissuepunchesincludedtheentireextent ACKNOWLEDGMENTS
of the NTS in order to sample both the A2 and C2 cell This work was supported by Public Health Service Grants
groups. Hayes et al. (39) found, however, that the caudal DK040498 and DK00345072 (to S.R.).
NTS at the level of the area postrema was sensitive to CC No potential conflicts of interest relevant to this article
exposure, whereas the rostral NTS was not. If this differ- were reported.
entialresponsealsooccursduringglucoprivation,thenour A.-J.L. and S.R. researched data, contributed to discus-
sampling method may have diluted the pAMPK signal to sion, and wrote and reviewed the manuscript. Q.W. re-
a nonsignificant level. In support of this, we found that searched data and contributed to discussion.
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 441
HINDBRAINAMPKANDGLUCOPRIVICFEEDING
REFERENCES 21. Turnley AM, Stapleton D, Mann RJ, Witters LA, Kemp BE, Bartlett PF.
1. BrownJ.Effectsof2-deoxyglucoseoncarbohydratemetablism:reviewof Cellular distribution and developmental expression of AMP-activated
theliteratureandstudiesintherat.Metabolism1962;11:1098–1112 protein kinase isoforms inmouse centralnervous system. J Neurochem
2. RitterS,BugarithK,DinhTT.Immunotoxicdestructionofdistinctcate- 1999;72:1707–1716
cholamine subgroups produces selective impairment of glucoregulatory 22. CulmseeC,MonnigJ,KempBE,MattsonMP.AMP-activatedproteinki-
responsesandneuronalactivation.JCompNeurol2001;432:197–216 naseishighlyexpressedinneuronsinthedevelopingratbrainandpro-
3. RitterS,Watts AG,DinhTT,Sanchez-WattsG,PedrowC.Immunotoxin motes neuronal survival following glucose deprivation. J Mol Neurosci
lesion of hypothalamically projecting norepinephrine and epinephrine 2001;17:45–58
neurons differentially affects circadian and stressor-stimulated cortico- 23. Minokoshi Y, Alquier T, Furukawa N, et al. AMP-kinase regulates food
steronesecretion.Endocrinology2003;144:1357–1367 intakebyrespondingtohormonalandnutrientsignalsinthehypothala-
4. I’AnsonH,SundlingLA,RolandSM,RitterS.Immunotoxicdestructionof mus.Nature2004;428:569–574
distinct catecholaminergic neuron populations disrupts the reproductive 24. AnderssonU,FilipssonK,AbbottCR,etal.AMP-activatedproteinkinase
responsetoglucoprivationinfemalerats.Endocrinology2003;144:4325– plays a role in the control of food intake. J Biol Chem 2004;279:12005–
4331 12008
5. FraleyGS,DinhTT,RitterS.Immunotoxiccatecholaminelesionsatten- 25. KimMS,ParkJY,NamkoongC,etal.Anti-obesityeffectsofalpha-lipoic
uate2DG-inducedincreaseofAGRPmRNA.Peptides2002;23:1093–1099 acidmediatedbysuppressionofhypothalamicAMP-activatedproteinki-
6. MaddenCJ,StockerSD,SvedAF.Attenuationofhomeostaticresponsesto nase.NatMed2004;10:727–733
hypotension and glucoprivation after destruction of catecholaminergic 26. HanSM,NamkoongC,JangPG,etal.HypothalamicAMP-activatedprotein
rostral ventrolateral medulla neurons. Am J Physiol Regul Integr Comp kinase mediates counter-regulatory responses to hypoglycaemia in rats.
Physiol2006;291:R751–R759 Diabetologia2005;48:2170–2178
7. Flynn FW, GrillHJ. Insulin elicits ingestion indecerebrate rats. Science 27. AlquierT,KawashimaJ,TsujiY,KahnBB.Roleofhypothalamicadenosine
1983;221:188–190 59-monophosphate-activated protein kinase in the impaired counter-
8. Darling RA, Ritter S. 2-Deoxy-D-glucose, but not mercaptoacetate, in- regulatory response induced by repetitive neuroglucopenia. Endocrinol-
creasesfoodintakeindecerebraterats.AmJPhysiolRegulIntegrComp ogy2007;148:1367–1375
Physiol2009;297:R382–R386 28. LeeK,LiB,XiX,SuhY,MartinRJ.Roleofneuronalenergystatusinthe
9. Ritter RC, Slusser PG, Stone S. Glucoreceptors controlling feeding and regulation of adenosine 59-monophosphate-activated protein kinase,
bloodglucose:locationinthehindbrain.Science1981;213:451–452 orexigenic neuropeptides expression, and feeding behavior. Endocrinol-
10. RitterS,DinhTT,ZhangY.Localizationofhindbrainglucoreceptivesites ogy2005;146:3–10
controllingfoodintakeandbloodglucose.BrainRes2000;856:37–47 29. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. San
11. AndrewSF,DinhTT,RitterS.Localizedglucoprivationofhindbrainsites Diego,CA,AcademicPress,1998
elicitscorticosteroneandglucagonsecretion.AmJPhysiolRegulIntegr 30. LiAJ,SuzukiM,SuzukiS,IkemotoM,ImamuraT.Differentialphosphor-
CompPhysiol2007;292:R1792–R1798 ylationatserinesitesinglutamatereceptor-1withinneonatalrathippo-
12. Li AJ, Ritter S. Glucoprivation increases expression of neuropeptide Y campus.NeurosciLett2003;341:41–44
mRNA in hindbrain neurons that innervate the hypothalamus. Eur J 31. LiAJ,RitterS.FunctionalexpressionofneuropeptideYreceptorsinhu-
Neurosci2004;19:2147–2154 manneuroblastomacells.RegulPept2005;129:119–124
13. LiAJ,WangQ,RitterS.Differentialresponsivenessofdopamine-beta-hy- 32. EverittBJ,HökfeltT,TereniusL,TatemotoK,MuttV,GoldsteinM.Dif-
droxylase gene expression to glucoprivation in different catecholamine ferentialco-existenceofneuropeptideY(NPY)-likeimmunoreactivitywith
cellgroups.Endocrinology2006;147:3428–3434 catecholamines in the central nervous system of the rat. Neuroscience
14. Ritter S, Llewellyn-Smith I, Dinh TT. Subgroups of hindbrain catechol- 1984;11:443–462
amine neurons are selectively activated by 2-deoxy-D-glucose induced 33. BellinSI,RitterS.Disparateeffectsofinfusednutrientsondelayedglu-
metabolicchallenge.BrainRes1998;805:41–54 coprivic feeding and hypothalamic norepinephrine turnover. J Neurosci
15. SawchenkoPE,SwansonLW,GrzannaR,HowePR,BloomSR,PolakJM. 1981;1:1347–1353
Colocalization of neuropeptide Y immunoreactivity in brainstem cate- 34. BellinSI,RitterS.Insulin-inducedelevationofhypothalamicnorepineph-
cholaminergicneuronsthatprojecttotheparaventricularnucleusofthe rineturnoverpersistsafterglucorestorationunlessfeedingoccurs.Brain
hypothalamus.JCompNeurol1985;241:138–153 Res1981;217:327–337
16. LiAJ,WangQ,DinhTT,RitterS.SimultaneoussilencingofNpyandDbh 35. GrannemanJ,FriedmanMI.Feedingafterrecoveryfrom2-deoxyglucose
expressioninhindbrainA1/C1catecholaminecellssuppressesglucoprivic injection: cerebral and peripheral factors. Am J Physiol 1983;244:R383–
feeding.JNeurosci2009;29:280–287 R388
17. HardieDG,CarlingD,CarlsonM.TheAMP-activated/SNF1proteinkinase 36. RitterRC,RoelkeM,NevilleM.Glucoprivicfeedingbehaviorinabsenceof
subfamily:metabolicsensorsoftheeukaryoticcell?AnnuRevBiochem othersignsofglucoprivation.AmJPhysiol1978;234:E617–E621
1998;67:821–855 37. KimEK,MillerI,AjaS,etal.C75,afattyacidsynthaseinhibitor,reduces
18. KimMS,LeeKU.Roleofhypothalamic59-AMP-activatedproteinkinasein foodintakeviahypothalamicAMP-activatedproteinkinase.JBiolChem
theregulationoffoodintakeandenergyhomeostasis.JMolMed2005;83: 2004;279:19970–19976
514–520 38. Hudson B, Ritter S. Hindbrain catecholamine neurons mediate consum-
19. RamamurthyS,RonnettGV.Developingaheadforenergysensing:AMP- matoryresponsestoglucoprivation.PhysiolBehav2004;82:241–250
activatedproteinkinaseasamultifunctionalmetabolicsensorinthebrain. 39. HayesMR,SkibickaKP,BenceKK,GrillHJ.Dorsalhindbrain59-adenosine
JPhysiol2006;574:85–93 monophosphate-activated protein kinase as an intracellular mediator of
20. XueB,KahnBB.AMPKintegratesnutrientandhormonalsignalstoreg- energybalance.Endocrinology2009;150:2175–2182
ulatefoodintakeandenergybalancethrougheffectsinthehypothalamus 40. Ritter RC. Gastrointestinal mechanisms of satiation for food. Physiol
andperipheraltissues.JPhysiol2006;574:73–83 Behav2004;81:249–273
442 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org