DTIC ADA422404: Coordinate Expression of the PDK4 Gene: a Means of Regulating Fuel Selection in a Hibernating Mammal

10 Pages·0.45 MB·English

by  Defense Technical Information Center

PhysiolGenomics8:5–13,2002.
FirstpublishedNovember27,2001;10.1152/physiolgenomics.00076.2001.
Coordinate expression of the PDK4 gene: a means of
regulating fuel selection in a hibernating mammal
MICHAEL J. BUCK,1 TERESA L. SQUIRE,1 AND MATTHEW T. ANDREWS2
1Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695-7614;
and 2Department of Biology, University of Minnesota Duluth, Duluth, Minnesota 55812
Received4September2001;acceptedinfinalform12November2001
Buck, Michael J., Teresa L. Squire, and Matthew T. reduction in cardiac pyruvate dehydrogenase activity
Andrews.CoordinateexpressionofthePDK4gene:ameans during hibernation. In the hearts of Djungarian ham-
ofregulatingfuelselectioninahibernatingmammal.Physiol sters,Heldmaieretal.(16)showedthatinactivationof
Genomics8:5–13,2002.FirstpublishedNovember27,2001; pyruvate dehydrogenase closely correlates with meta-
10.1152/physiolgenomics.00076.2001.—Hibernationinmam-
bolic rate reduction seen during daily torpor. Regu-
mals requires a metabolic shift away from the oxidation of
lated long-term inhibition of pyruvate dehydrogenase
carbohydrates and toward the combustion of stored fatty
activityistheresultofphosphorylationofthepyruvate
acids as the primary source of energy during torpor. A key
dehydrogenase complex by the activity of various
element involved in this fuel selection is pyruvate dehydro-
isoenzymesofthepyruvatedehydrogenasekinasefam-
genasekinaseisoenzyme4(PDK4).PDK4inhibitspyruvate
dehydrogenase and thus minimizes carbohydrate oxidation ily (4).
by preventing the flow of glycolytic products into the tricar- In this paper we address the regulation of the gene
boxylic acid cycle. This paper examines expression of the encoding pyruvate dehydrogenase kinase isoenzyme 4
PDK4geneduringhibernationinheart,skeletalmuscle,and (PDK4) in the hibernating 13-lined ground squirrel,
white adipose tissue (WAT) of the 13-lined ground squirrel, Spermophilus tridecemlineatus. We previously identi-
Spermophilus tridecemlineatus. During hibernation PDK4 fiedPDK4asanupregulatedgeneinthegroundsquir-
mRNAlevelsincrease5-foldinskeletalmuscleand15-foldin relheartduringhibernation(1).Inhibitionofpyruvate
WATcomparedwithsummer-activelevels.Similarly,PDK4
dehydrogenasebyPDK4-mediatedphosphorylationre-
protein is increased threefold in heart, fivefold in skeletal
ducesglucoseoxidationbypreventingtheconversionof
muscle,andeightfoldinWAT.Highlevelsofseruminsulin,
pyruvatetoacetyl-CoAandCO ,thusstoppingtheflow
likelytohaveaninhibitoryeffectonPDK4geneexpression, 2
of glycolytic intermediates into the tricarboxylic acid
areseenduringfallwhenPDK4mRNAlevelsarelow.Coor-
dinate upregulation of PDK4 in three distinct tissues sug- (TCA) cycle (24). PDK4 mRNA exhibits its highest
gests a common signal that regulates PDK4 expression and levels of expression in heart and skeletal muscle in
fuelselectionduringhibernation. both humans (24) and rats (4). More moderate levels
arealsoseeninbrain,placenta,lung,liver,kidney,and
hibernation; pyruvate dehydrogenase kinase isoenzyme 4;
insulin; peroxisome proliferator-activated receptor-(cid:1); Sper- pancreasinhumans(24)andinlung,liver,andkidney
in rats (4). Among the PDK isoenzymes, expression of
mophilustridecemlineatus
PDK4 appears to be the most responsive to physiolog-
ical change (reviewed in Ref. 15). Starvation (30–33)
PROFOUND CHANGES in body temperature, metabolism anddiabetes(31,33)aretwoconditionsthatstimulate
and heart rate allow hibernating mammals to survive PDK4 expression and show physiological characteris-
seasonally cold environments for periods of several tics related to the hibernating state.
months with little or no food. To accomplish this feat, Here we report on PDK4 gene expression in 10 dif-
individual organ systems undergo metabolic rate de- ferentgroundsquirreltissuesandquantifymRNAand
protein levels in those tissues showing the highest
pression, minimize the use of carbohydrates, and
levelsofexpression:heart,whiteadiposetissue(WAT),
switch to stored triacylglycerols as their primary
and skeletal muscle from the thigh (quadriceps femo-
source of fuel (reviewed in Ref. 20). In the absence of
ris).Ouranalysisbeginsinlatesummerandcontinues
feeding, conservation of glucose for utilization by the
throughthehibernationseasontospringarousalwhen
brain becomes essential for survival and is controlled
the animal resumes its activities. We also explore the
byinhibitionofthemitochondrialpyruvatedehydroge-
regulationofPDK4byexaminingtheseasonalexpres-
nase complex (23). In hibernating golden-mantled
sionofinsulin.Insulinisakeymetabolichormonethat
groundsquirrels,BrooksandStorey(6)observeda96%
stimulates glucose uptake from the blood, activates
glycogen synthase, inactivates glycogen breakdown,
Article published online before print. See web site for date of and stimulates the storage of excess fuel as fat. In
publication(http://physiolgenomics.physiology.org). diabetic rats an increase in insulin concentration has
Address for reprint requests and other correspondence: M. T.
been shown to reduce the levels of both PDK4 protein
Andrews, Dept. of Biochemistry and Molecular Biology, Univ. of
and mRNA in heart and skeletal muscle (31, 33). We
Minnesota School of Medicine, 10 Univ. Drive, Duluth, MN 55812
(E-mail:[email protected]). have measured insulin levels during hibernation,
1094-8341/02$5.00Copyright©2002theAmericanPhysiologicalSociety 5
6 PDK4ANDFUELSELECTIONINAHIBERNATINGMAMMAL
cloned the ground squirrel insulin cDNA, and mea- alltissueswasextractedwithphenol:chloroform:isoamylal-
suredinsulingeneexpressioninthepancreasthrough- cohol (25:24:1) followed by addition of sodium acetate to a
outthehibernationseason.Thesedeterminationshave finalconcentrationof0.2M.Thismixturewasextractedonce
morewithacid-phenol:chloroform(1:1).Allextractionswere
allowed us to contrast levels of PDK4 mRNA and
performedusingphase-locktubes(Eppendorf)at4,500gina
protein with insulin mRNA and protein at various
swinging bucket rotor. The aqueous layer was divided into
times of the year such as summer when the heartbeat
aliquots and stored as isopropanol precipitates. Individual
is rapid (200–300 beats/min) and legs are flexing, vs.
aliquotswerecentrifuged,rinsedwith70%ethanol,dissolved
deep hibernation when the heart rate slows (2–10 inwater,andquantifiedbyabsorptionspectrophotometry.
beats/min) and the legs are inactive. Based on the Northern blots. Total RNAs from designated tissues of
results described in this paper and on other recent activeandhibernating13-linedgroundsquirrelsweresepa-
studies on PDK4 regulation, we propose a regulatory rated on agarose gels containing formaldehyde followed by
mechanismthataccountsfortheswitchfromcarbohy- transfer onto MagnaCharge nylon membranes (Osmonics)
dratetofattyacidsastheprimarysourceoffuelduring using 10(cid:3) SSPE according to Sambrook et al. (25). The
hibernation. multi-tissue blot used to examine PDK4 mRNA levels in 10
different hibernating tissues utilized 15 (cid:4)g total RNA per
lane.Individualheart,WAT,andskeletalmuscleblots,used
MATERIALS AND METHODS
to examine PDK4 mRNA levels at various time points
Animals. Animal care and use was in accordance with throughout the hibernation season, utilized 8 (cid:4)g total RNA
Institutional Animal Care and Use Committee guidelines. per lane. These Northern blots were hybridized with a ran-
Thirteen-lined ground squirrels (S. tridecemlineatus) were dom-primed (8, 9) 32P-labeled PDK4 cDNA (accession no.
receivedfromTLSResearch(Bartlett,IL)within3–4daysof AF020845).PancreasNorthernblotsusedfordetectinginsu-
wildcaptureduringthefirstweekofAugust.ActiveAugust linmRNAcontained10(cid:4)goftotalRNAperlane.Theseblots
animalsweremaintainedwithwaterandkilledwithin24h werehybridizedwitha32P-end-labeled(25)insulinoligonu-
of arrival from the supplier. All other animals were main- cleotide (5(cid:5) TTGCAGTAGTTCTCCAGCTGGTAGAGGGAG-
tainedincaptivityonadietofstandardrodentchowsupple- CAGATG3(cid:5)).
mentedwithsunflowerseedsandwateradlibitum.Allsquir- RNAintegritywasexaminedusinga1%agarose,1(cid:3)TAE
relsweremaintainedat23°CinAugust,17°CinSeptember, non-denaturinggelstainedwithethidiumbromide.Loading
11°C in October, and 5°C from November through mid- and integrity of RNA on Northern blots were examined by
March.Animalswerehousedwitha12:12-hlight/darkcycle hybridization with a 32P-end-labeled (25) 18S rRNA oligo-
fromAugustthroughtheendofOctober.FromNovemberto nucleotide(5(cid:5)CGACTTTTACTTCCTCTAGATAGTCAAGTT-
mid-March the animals were housed in total darkness with CGAC 3(cid:5)). Oligonucleotide sequences for insulin and 18S
onlywateradlibitum.Squirrelswereobserveddailyduring rRNA were constructed from conserved domains for each
these months and hibernation patterns were determined gene product as determined by multi-sequence alignment
usingthesawdusttechnique(22).Hibernatinganimalswere with several mammalian homologs. PDK4 and insulin
atleastinday2ofatorporboutwhenkilled,andanimalsin mRNAlevelsweredeterminedbyaSTORMphosphorimager
interbout arousal (IBA) were collected after at least three usingImageQuantsoftware(MolecularDynamics).
previous hibernation bouts of 8 days or more. IBAs are Library construction. A pancreas cDNA library was con-
naturallyoccurringinterruptionsofthedormantstatewhen structedfrompancreaticpoly(A)(cid:6)mRNAisolatedfromboth
hibernatinganimalsraisetheirbodytemperaturesandshow a hibernating and an active 13-lined ground squirrel. A
briefperiodsofactivity(29).ThetimeofdeathwithinanIBA directional cDNA library was created using the Superscript
was not determined. Hibernating animals killed in October Plasmid System for cDNA Synthesis and Plasmid Cloning
were often in their first torpor bout of the season, whereas (GIBCO-BRL, Life Technologies) according to the manufac-
hibernating animals collected from December through early turer’s protocol. The library was screened for the insulin
Marchhadexperiencedatleastthreeprevioustorporboutsof cDNA using the 32P-end-labeled insulin oligonucleotide de-
8 days or more. The hibernation phase of the study was scribedintheNorthernblots,above.
concluded in mid-March by increasing room temperature to Serum insulin assay. Whole blood collected at death was
23°C,re-establishing12:12-hdark/lightcycle,andproviding spunat3,500gfor10min,andpartiallypurifiedserumwas
a diet of standard rodent chow and sunflower seeds ad libi- transferredto1.5-mltubesandspunat3,500gfor15minat
tum. Rectal temperatures were measured at the time of 4°C.Serumwasdividedintoaliquots,frozeninliquidnitro-
death. Ground squirrel tissue was promptly removed and gen, and stored at (cid:2)80°C until time of assay. Serum was
frozenimmediatelyinliquidnitrogen.Pancreatictissuewas thawedonice,andtheinsulinconcentrationwasdetermined
immersed directly in ice-cold guanidinium isothiocyanate, by radioimmunoassay (rat insulin RIA kit, Linco). The min-
andRNAwaspreparedimmediatelybecauseofhighlevelsof imal detection limit of the serum insulin assay is 0.1 ng/ml.
RNase activity in the pancreas. Whole blood was stored on Twodifferentconcentrationsofpurifiedratinsulinprovided
ice,serumwaspreparedpromptly,distributedintoaliquots, by the manufacturer served as quality controls. We mea-
frozen in liquid nitrogen, and stored at (cid:2)80°C until time of suredtheconcentrationandstandarddeviationofthecontrol
assay. samples by replication before beginning the assays. Experi-
RNA isolation. Total RNA was prepared from ground mentalassayscontainedgroundsquirrelserumsamplesand
squirreltissuesusingthemethoddescribedbyAndrewsetal. the quality controls. In this manner we assured ourselves
(1) or by a modification of the protocol provided with the that any assay is repeatable, because both of the quality
Totally RNA kit from Ambion. For the latter method, all controlvalueswerewithintwostandarddeviations.Samples
solutions were from the Totally RNA kit. The tissue was fromthesameanimalswereperformedinduplicate,andthe
homogenized with a rotary-blade homogenizer in guani- differencebetweentheduplicateswaslessthan10%.
diniumisothiocyanate.TheWAThomogenatewassubjected Westernblots.ProteinfromWAT,heart,andskeletalmus-
toaninitialcentrifugationfor10minat3,000g,afterwhich cle (quadriceps femoris) was prepared as acetone powder
theglycerolandlipidlayerwasremoved.Thehomogenatefor (14).Powdersweresolubilizedin0.2MTris(cid:1)HCl(pH9.2)and
PhysiolGenomics•VOL8•www.physiolgenomics.org
PDK4ANDFUELSELECTIONINAHIBERNATINGMAMMAL 7
1Methyleneglycolandreconstitutedbygentleagitationfor
2–3hat4°C.Thetotalsolubilizedproteinconcentrationwas
determined by Bradford assay using dye reagents from Bio-
Radlaboratories.Tenmicrogramsofsolubilizedproteinwas
boiled in loading buffer (1.5 M Tris(cid:1)HCl pH 8.9, 1.2% SDS,
1% DTT) and separated on the basis of size on a 10% poly-
acrylamide gel by discontinuous SDS-PAGE. The separated
proteinsweresubsequentlytransferredelectrophoreticallyto
NitroBindnitrocellulosemembrane(Osmonics).Membranes
werethenblockedovernightatroomtemperaturewithTBST
(75 mM NaCl, 10 mM Tris(cid:1)HCl, 0.5% Tween 20, pH 9.2)
augmented with 3% (wt/vol) bovine serum albumin. For de-
tection, membranes were incubated for 2 h with a 1:10,000
dilutionofpolyclonalantiseraagainstratPDK4(agiftfrom
R. A. Harris). After three 10-min washes with TBST, mem-
branes were incubated with anti-rabbit IgG secondary anti-
body linked with horseradish peroxidase (1:5,000, in TBST)
for 1 h. The blots were given four 10-min washes in TBST,
andboundantibodywasvisualizedusingtheECLdetection
system(Amersham).Theblotswereexposedtofilm,quanti-
fied by scanning densitometry, and analyzed with Image-
Quant software (Molecular Dynamics). Films used for scan-
ning densitometry were not overexposed to assure that all
densitometricquantificationswerewithinthelinearrangeof
thefilm.Exposuresusedfordensitometryinvolved56differ-
ent tissue samples (heart, 19 animals; skeletal muscle, 18
animals;WAT,19animals)quantifiedonfilmsdifferentfrom
thoseshownfordemonstrationpurposesinFig.3.
Statistical analysis. For Northern blots, loading was nor-
malized by 18S rRNA, and relative values were determined
by comparison to the average August value. For Western
blots,anequalamountofsolubleproteinwasloadedforeach
sample, and relative PDK4 protein levels were then deter-
mined by comparison to the average August amount. For
serum insulin levels, absolute concentrations were used for
statistical comparisons. One-way ANOVAs were used to de-
terminesignificantdifferencesinlevelswhencomparedwith
August active values. The ANOVA calculations were pre-
formed using version 7.1 of the SAS system (SAS Institute,
Cary,NC).
Fig. 1. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) gene
RESULTS expressionin13-linedgroundsquirrels.A:Northernblotcontaining
totalRNA(15(cid:4)g/lane)isolatedfrommultiplegroundsquirreltissues
During hibernation in the 13-lined ground squirrel duringhibernation.Tissuesweretakenfrommultipleanimalskilled
(S. tridecemlineatus) PDK4 mRNA was seen at rela- inDecemberandJanuarywithbodytemperaturesrangingfrom6to
10°C.Tissuesareindicatedaboveeachlane,andthe(cid:8)-actinmRNA
tivelylowlevelsintheadrenals,brainstem,cerebrum,
(“actin”)and18SrRNA(“18S”)profilesareshowndirectlybelow.B:
kidney,liver,pancreas,andtestes;andatmuchhigher Northern blot of total RNA (8 (cid:4)g/lane) from heart (Hrt), skeletal
levelsinheart(1),skeletalmuscle,andWAT(Fig.1A). muscle(Skl),andwhiteadiposetissue(WAT)ofthesameanimalfor
PDK4 mRNA was also detected during hibernation in eachtimepoint.Ambienttemperature(Ta),bodytemperature(Tb),
andmonthofdeathareindicatedaboveeachlane.Thestateofeach
the heart of the golden-mantled ground squirrel (S.
animal is indicated as active (A), hibernating (H), or in interbout
lateralis) and in arctic ground squirrel (S. parryii)
arousal(I).RNAintegrityandgelloadingwascontrolledbyhybrid-
heart and WAT (data not shown). Our investigation of izationwith18SrRNA(notshown).
PDK4expressionin13-linedgroundsquirrelswascon-
ducted throughout the hibernation season to deter-
mine whether there was evidence for common regula- nificant increase in expression in all three tissues rel-
tion among the three tissues showing the highest ative to August values as shown graphically for
mRNA levels. Total RNA from heart, skeletal muscle skeletal muscle (Fig. 2A) and WAT (Fig. 2B). Later in
from the thigh (quadriceps femoris), and abdominal the hibernation season (beginning in December) there
WAT were prepared from individual animals at vari- wasafurtherincreaseinmRNAlevels,withthegreat-
ous states of activity from August through March. est increase relative to August values seen during
Expression of the PDK4 gene was similar in heart, IBAs. After hibernation ceased in the spring, PDK4
skeletal muscle and WAT throughout the year (Fig. message dropped back to near August levels. In skele-
1B). PDK4 mRNA levels were relatively unchanged talmuscle,PDK4messagewasincreased5-foldduring
until the animal entered hibernation in October. Hi- hibernation and 7-fold during IBAs, compared with
bernating animals in October showed a slight nonsig- Augustactivevalues(P(cid:7)0.01;Fig.2A).InWAT,there
PhysiolGenomics•VOL8•www.physiolgenomics.org
8 PDK4ANDFUELSELECTIONINAHIBERNATINGMAMMAL
Fig.2. SummariesofPDK4mRNAlevelsinskeletalmuscle(A)and
WAT(B).SimilarmeasurementsofPDK4mRNAlevelsinheartare
published(1).Ambienttemperature(T ),bodytemperature(T ),and
a b
month(s) of death are indicated below each bar. The state of each
animalisindicatedaboveasactive(A),hibernating(H),orininter-
bout arousal (I). The PDK4 level was normalized to the average
August level. The number of animals (n) for each time point is
indicated. Statistically significant comparisons to August mRNA
levelsweredeterminedbyone-wayANOVA:*0.01(cid:7)P(cid:7)0.05and
**P(cid:7)0.01.
was a 15-fold increase in PDK4 mRNA during hiber-
nation and a 20-fold increase during IBAs (P (cid:7) 0.05;
Fig.2B).RelativelevelsofPDK4mRNAinheartwere
quantified previously (1) and closely resemble those
Fig.3. Westernblotsoftotalprotein(10(cid:4)g/lane)isolatedfromheart
seen in WAT.
(A),skeletalmuscle(B),andWAT(C).Theblotswereprobedwith
Western blot analysis using antiserum against rat
anti-ratPDK4antiserum.Ambienttemperature(T ),bodytemper-
a
PDK4 (a gift from R. A. Harris) was employed to ature (T ), and month of death are indicated above each lane. The
b
measure 13-lined ground squirrel PDK4 protein levels stateofeachanimalisindicatedasactive(A),hibernating(H),orin
in heart, skeletal muscle, and WAT (Fig. 3). PDK4 interbout arousal (I). The size of PDK4 in kilodaltons (kDa) is
indicatedontheleftofeachblot.
protein levels in these three tissues were consistent
with their mRNA profiles. In active August animals,
PhysiolGenomics•VOL8•www.physiolgenomics.org
PDK4ANDFUELSELECTIONINAHIBERNATINGMAMMAL 9
PDK4proteinwasfoundatlowlevelswithinheartand levelofPDK4proteininallthreetissues(Fig.3).This
skeletal muscle but was barely detectable in WAT. difference was most evident in WAT in which the
Compared with August squirrels, PDK4 protein de- PDK4 protein, barely detectable during summer and
clined significantly in hearts from active September- fall, was seen at high levels after animals entered
October animals (P (cid:7) 0.01) and then increased three- hibernationinOctoberandthenremainedhighduring
fold in hibernating hearts (P (cid:7) 0.01; Fig. 4A). In hibernation (Fig. 3C). After hibernation concluded in
skeletalmuscle,PDK4proteinremainedrelativelyun- spring, the relative concentration of PDK4 protein
changed from August until entrance into hibernation, droppedtosummerlevelsinallthreetissuesexamined
whenitslevelincreasedfivefold(P(cid:7)0.05)andreached (Figs. 3 and 4).
itsmaximumduringIBAs(P(cid:7)0.01;Fig.4B).InWAT, The coordinate expression of PDK4 in different tis-
PDK4 protein increased twofold (P (cid:7) 0.05) in Septem- sues during hibernation prompted us to measure cir-
ber-October animals and eightfold (nonsignificant) in culating insulin levels because of the long-standing
hibernatinganimals(Fig.4C).Whencomparinghiber- observation that insulin has a controlling influence on
natingandnonhibernatinganimalskilledonthesame pyruvate dehydrogenase kinase activity (17). More re-
dayinOctober,thehibernatinganimalhasanelevated cent experiments with rat hearts (33) and skeletal
Fig.4. SummariesofPDK4proteinlevelsinheart(A),skeletalmuscle(B),andWAT(C).Ambienttemperature
(T ),bodytemperature(T ),andmonth(s)ofdeathareindicatedbeloweachbar.Theactivitystateisindicated
a b
aboveasactive(A),hibernating(H),orininterboutarousal(I).Thenumberofanimals(n)foreachtimepointis
indicated.ThePDK4levelwasnormalizedtotheaverageAugustvalue.Statisticallysignificantcomparisonsto
Augustproteinlevelsweredeterminedbyone-wayANOVA:*0.01(cid:7)P(cid:7)0.05and**P(cid:7)0.01.
PhysiolGenomics•VOL8•www.physiolgenomics.org
10 PDK4ANDFUELSELECTIONINAHIBERNATINGMAMMAL
muscle (31) have shown high levels of PDK4 mRNA to control levels. In ground squirrels, we found the
were induced in animals rendered diabetic by treat- serum insulin concentration below 1 ng/ml for most of
ment with an antibiotic that destroys the insulin-pro- the year except during September and October, when
ducing capacity of the pancreas. Administration of in- the insulin concentration was increased fourfold (P (cid:7)
sulintothesediabeticratsloweredPDK4mRNAback 0.01) compared with August active animals (Fig. 5A).
Fig.5. Seasonalexpressionofinsulin.A:seruminsulinlevelsatvarioustimesoftheyear.Insulinconcentration
wasdeterminedbyradioimmunoassay.Ambienttemperature(T ),bodytemperature(T ),andmonth(s)ofdeath
a b
areindicatedbeloweachbar.Theactivitystateisindicatedaboveasactive(A),hibernating(H),orininterbout
arousal (I). The number of animals (n) for each time point is indicated. Statistically significant comparisons to
August serum insulin levels were determined by one-way ANOVA: **P (cid:7) 0.01. B: insulin gene expression in
pancreas.NorthernblotoftotalRNA(10(cid:4)gperlane)isolatedfrompancreasandprobedwithaninsulin-specific
oligonucleotide.Ambienttemperature(T ),bodytemperature(T ),andmonthofdeathareindicatedaboveeach
a b
lane.Thestateofeachanimalisindicatedasactive(A),hibernating(H),orininterboutarousal(I).The18SrRNA
(18S)profileinthepancreasisshowndirectlybelow.Thesizeoftheinsulinmessageisindicatedinkilobases(kb).
C:summaryofrelativeinsulinmRNAlevelinthepancreas.TheinsulinmRNAlevelwasnormalizedtotheaverage
Augustlevel.Thenumberofanimals(n)foreachtimepointisindicated.Statisticallysignificantcomparisonsto
AugustmRNAlevelsweredeterminedbyone-wayANOVA:*0.01(cid:7)P(cid:7)0.05.
PhysiolGenomics•VOL8•www.physiolgenomics.org
PDK4ANDFUELSELECTIONINAHIBERNATINGMAMMAL 11
When serum insulin is high in September-October, inhibits carbohydrate oxidation by phosphorylating
active animals show significantly lower PDK4 protein the pyruvate dehydrogenase complex, thus impairing
levelsinheartcomparedwithAugustanimals,andall theflowofglycolyticintermediatesintotheTCAcycle.
threetissuesshowlowerPDK4levelsthanhibernating WefoundthatthegeneencodingPDK4isupregulated
animals (Fig. 4). duringhibernationinheart,WAT,andskeletalmuscle
Afull-lengthinsulincDNA(accessionno.AY038604) of the 13-lined ground squirrel, S. tridecemlineatus.
wasisolatedfroma13-linedgroundsquirrelpancreatic Hibernation-associated expression of the PDK4 gene
cDNA library derived from hibernating and nonhiber- results in increased mRNA and protein levels in all
nating animals. Ground squirrel preproinsulin is 110 three tissues.
aminoacidslongand89%identicaltotheunprocessed Identification of specific genes associated with the
human protein (accession no. P01308). To investigate hibernating state allows researchers to identify and
insulin gene expression, total RNA from pancreas was test endogenous and exogenous factors that control
prepared from individual animals at various states of differential gene expression during hibernation. Fac-
activity from August through June, immobilized on tors important in hibernation-related gene activity
Northern blots, and probed with an end-labeled oligo- maybeenvironmentalsuchascoolerambienttemper-
nucleotide complementary to mammalian insulin atures and fewer daylight hours; and/or internal fac-
mRNA (Fig. 5B). Examination of insulin expression in tors such as circannual rhythms and changes in me-
the pancreas across the hibernation season revealed tabolites and hormone levels. Coordinate activation of
thatmRNAlevelswithinthepancreasdidnotcorrelate the gene encoding PDK4 in different tissues through-
with serum insulin concentrations. When serum insu- out the body points to circulating effector molecules
lin levels were near their lowest point in December- that regulate its expression during hibernation. We
Januaryhibernatinganimals(Fig.5A),insulinmRNA proposeamodel(Fig.6)showinghowchangesinserum
was upregulated fourfold (P (cid:7) 0.05) compared with insulinandfattyacidlevelsregulatePDK4expression
summer active animals (Fig. 5C). Elevated levels of and ultimately the switch from carbohydrate- to fat-
insulinmRNAinthepancreasduringhibernationmay basedcatabolismduringhibernation.Figure6extends
be held in reserve so that insulin protein can be re- our previous model (27) on the regulation of genes
leasedwhenneededsuchasresumptionoffeedingand controlling metabolism during hibernation and is
subsequent glucose utilization at spring arousal. based on results reported in this paper and recent
experimental findings described in the following para-
DISCUSSION
graphs.
During hibernation, carbohydrates are spared and Injectionofinsulinintodiabeticratshasbeenshown
fatty acids become the primary source of fuel as ob- toreducethelevelsofbothPDK4proteinandmRNAin
served by a respiratory quotient (RQ) of 0.7 (reviewed heart and skeletal muscle (31, 33). In 13-lined ground
in Ref. 20). RQ is a unitless value representing the squirrels we found high levels of serum insulin in fall
moles of CO respired per moles of O consumed. A activeanimalscoincidewithlowlevelsofPDK4expres-
2 2
value of 1.0 indicates combustion of carbohydrates; sioninheart,skeletalmuscle,andWAT.Anexception
however, an RQ of 0.7 indicates that fat is the major is seen in hibernating October animals when the level
substrate for energy production. The enzyme PDK4 of PDK4 mRNA increases while the concentration of
Fig. 6. Model showing regulation of
PDK4geneexpressionandtheswitch
fromcarbohydratetofattyacidsasthe
primarysourceoffuelduringhiberna-
tion: effects of serum levels of insulin
and free fatty acids (ffa) on PDK4 ex-
pression, carbohydrate oxidation, and
fattyacidoxidationinheartandskel-
etal muscle of active (PRE-HIB; Sep-
tember-October) and hibernating (HIB;
December-January)animals.Longlines
with arrowheads indicate upregulation
oractivation,andlineswithbluntends
indicate downregulation or inhibition.
Longsolidlinesshowthepredominant
mode of regulation, and dashed lines
show potential secondary regulation.
Short vertical arrows pointing up indi-
cateanincreaseinconcentrationorac-
tivity. Short vertical arrows pointing
down indicate a decrease in concentra-
tionoractivity.HSL,hormone-sensitive
lipase; PPAR(cid:1), peroxisome proliferator
activated receptor-(cid:1); TG, triacylglycer-
ols.
PhysiolGenomics•VOL8•www.physiolgenomics.org
12 PDK4ANDFUELSELECTIONINAHIBERNATINGMAMMAL
serum insulin remains high. Despite the lack of feed- erols stored in WAT (2). Unlike hormone-sensitive
ing,highseruminsulinlevelsinOctoberpersistforthe lipase, which is the primary lipolytic enzyme in WAT,
firstfewdaysafterhibernationbeginsandmaybethe pancreatic triacylglycerol lipase is not inhibited by
result of reduced insulin turnover due to low body insulin and can therefore provide a steady supply of
temperatures(12–13°C).TheincreaseinPDK4mRNA free fatty acids before and during hibernation. The
is probably due to a competing PDK4-activation path- suggestion that free fatty acids can activate PDK4
way (Fig. 6; described below) and/or by the onset of expressioninthepresenceofinsulinisstrengthenedby
insulin resistance like that observed in another hiber- a recent line of evidence showing that insulin is not
nator, the yellow-bellied marmot (Marmota flaviven- effectiveinopposingorreversingWY-14,643activation
tris; Ref. 10). Insulin resistance is a reduction in the of the PDK4 gene in Morris 7800C1 hepatoma cells
abilityofinsulintoregulateglucosehomeostasis,caus- (15). This result, combined with potential insulin re-
ing an increase in serum insulin or hyperinsulinemia. sistance(10),suggeststhatfreefattyacidactivationof
Florantandcolleagues(10)suggestthathyperinsulin- PPAR(cid:1) can initiate PDK4 gene expression near the
emiaandperipheralinsulinresistancearemaximalas onset of hibernation despite relatively high serum in-
the animal ceases to feed and enters hibernation. sulin levels.
Ourobservationthatlevelsofseruminsulinincrease Another activity of PPAR(cid:1) can be seen in the acti-
in active fall animals (September-October) and then vation of genes involved in lipid metabolism (26). Of
decline to less than 1 ng/ml in hibernating December- key importance to hibernation is the potential role of
January animals is also seen in the yellow-bellied PPAR(cid:1)incoordinatingPDK4geneexpressionwiththe
marmot (28). Seasonal insulin concentrations in the expression of genes responsible for extracellular and
marmot are similar to those shown in Fig. 5A for intracellular lipid transport and mitochondrial (cid:8)-oxi-
13-lined ground squirrels. Despite the depressed se- dationoffattyacids(reviewedinRef.27).Asshownin
rum insulin levels in December-January, we found Fig. 6, activation of PPAR(cid:1) provides a mechanism
that insulin mRNA levels in the pancreas are the linking the inhibition of carbohydrate oxidation with
highest in hibernating animals collected December increased fatty acid oxidation, thus accounting for the
throughMarch(Fig.5C).Thisobservationisanalogous switch in fuel selection during hibernation. PPAR(cid:1)
to high insulin protein levels retained in the pancreas activity has also been associated with expression of
of the hibernating little brown bat (Myotis lucifugus; uncoupling protein-3 (UCP3; Ref. 7). This finding con-
3). During hibernation the level of bat insulin protein necting PPAR(cid:1) to UCP gene expression is especially
steadily increases in the pancreas, reaching its maxi- relevant with respect to hibernation and nonshivering
malleveljustpriortospringarousal.Bauman(3)notes thermogenesis (5). In consideration of its role in the
that the increase in pancreatic insulin concentration, activation of genes associated with carbohydrate utili-
from early to late hibernation, may provide a rapidly zation, lipid catabolism, and nonshivering thermogen-
releasable storage pool required for immediate secre- esis, PPAR(cid:1) is likely to act as a common regulator of
tion during spring arousal. This suggestion could ex- the metabolic response during hibernation.
plain the increase in insulin mRNA that we report in
hibernating13-linedgroundsquirrels(Fig.5,BandC). WearegratefultoJayitaGuhaniyogiforconstructionandscreen-
ing of the ground squirrel pancreas cDNA library. We thank R. A.
While insulin acts as an inhibitor of PDK4 expres-
HarrisforthegiftofPDK4antiserum,B.B.BoyerandS.L.Martin
sion, a member of the peroxisome proliferator-acti- for the gifts of tissues from other Spermophilus species, M. M.
vated receptor (PPAR) family is known to activate the TredreaforassemblyofthegroundsquirrelinsulincDNAsequence,
PDK4 gene. PPARs are ligand-dependent nuclear re- andL.B.Mamofortechnicalassistance.
Thenewsequencereportedinthispaperhasbeendepositedinthe
ceptors that heterodimerize with the retinoid X recep-
GenBankdatabase(accessionno.AY038604).
tor (19, 21) and thereby act as transcription factors.
ThisworkwassupportedbyUSArmyResearchGrantDAAD19-
PDK4 expression is induced in skeletal muscle of rats 01-1-0014 and Augmentation Awards for Science and Engineering
and the hearts of mice by a hypolipidemic drug and TrainingDAAG55-97-1-0175.
known peroxisome proliferator, WY-14,643 (31, 32).
This finding indicates a role for PPAR(cid:1) in controlling REFERENCES
PDK4 gene expression due to the fact that WY-14,643 1. Andrews MT, Squire TL, Bowen CM, and Rollins MB.
isaselectivePPAR(cid:1)-activatingligand(12).Thisroleof Low-temperature carbon utilization is regulated by novel gene
PPAR(cid:1) in activating the PDK4 gene has also recently activityintheheartofahibernatingmammal.ProcNatlAcad
SciUSA95:8392–8397,1998.
been confirmed using PPAR(cid:1)-null mice (32).
2. BauerVW,SquireTL,LoweME,andAndrewsMT.Expres-
NaturalPPAR-activatingligandsincludelong-chain sion of a chimeric retroviral-lipase mRNA confers enhanced
freefattyacids,suchaslinoleicacid,linolenicacid,and lipolysis in a hibernating mammal. Am J Physiol Regulatory
arachidonicacid(18).Elevatedlevelsoffattyacidsare IntegrativeCompPhysiol281:R1186–R1192,2001.
3. BaumanWA.Seasonalchangesinpancreaticinsulinandglu-
observed in serum both before and during hibernation
cagon in the little brown bat (Myotis lucifugus). Pancreas 5:
(11, 13). This elevation is likely a consequence of in- 342–346,1990.
creased food intake in the months leading up to hiber- 4. Bowker-Kinley MM, Davis WI, Wu P, Harris RA, and
nationwhentheanimalisfatteningandpreparingfor PopovKM.Evidenceforexistenceoftissue-specificregulation
ofthemammalianpyruvatedehydrogenasecomplex.BiochemJ
dormancy. In 13-lined ground squirrels, elevated se-
329:191–196,1998.
rum fatty acid levels can also result from pancreatic
5. BoyerBB,BarnesBM,LowellBB,andGrujicD.Differen-
triacylglycerol lipase-mediated lipolysis of triacylglyc- tialregulationofuncouplingproteingenehomologuesinmulti-
PhysiolGenomics•VOL8•www.physiolgenomics.org
PDK4ANDFUELSELECTIONINAHIBERNATINGMAMMAL 13
pletissuesofhibernatinggroundsquirrels.AmJPhysiolRegu- proliferatorsignallingpathwaysthroughheterodimerformation
latoryIntegrativeCompPhysiol275:R1232–R1238,1998. oftheirreceptors.Nature358:771–774,1992.
6. BrooksSPJandStoreyKB.Mechanismsofglycolyticcontrol 20. LymanCP,WillisJS,MalanA,andWangLCH.Hibernation
duringhibernationinthegroundsquirrelSpermophiluslatera- and Torpor in Mammals and Birds. New York, NY: Academic
lis.JCompPhysiol[B]162:23–28,1992. Press,1982.
7. BrunS,CarmonaMC,MampelT,VinasO,GiraltM,Igle- 21. MangelsdorfDJ,ThummelC,BeatoM,HerrlichP,Schutz
siasR,andVillarroyaF.Activatorsofperoxisomeproliferator- G,UmesonoK,BlumbergB,KastnerP,MarkM,Chambon
activatedreceptor-alphainducetheexpressionoftheuncoupling P,andEvansRM.Thenuclearreceptorsuperfamily:thesec-
protein-3geneinskeletalmuscle:apotentialmechanismforthe onddecade.Cell83:835–839,1995.
lipid intake-dependent activation of uncoupling protein-3 gene 22. PengelleyETandFisherKC.Rhythmicalarousalfromhiber-
expressionatbirth.Diabetes48:1217–1222,1999. nation in the gold-mantled ground squirrel, Citellus lateralis
8. FeinbergAPandVogelsteinB.Atechniqueforradiolabeling tescorum.CanJZool39:105–120,1961.
DNArestrictionendonucleasefragmentstohighspecificactivity. 23. Randle PJ. Fuel selection in animals. Biochem Soc Trans 14:
AnalBiochem132:6–13,1983. 799–806,1986.
9. FeinbergAPandVogelsteinB.Atechniqueforradiolabeling 24. Rowles J, Scherer SW, Xi T, Majer M, Nickle DC, Rom-
DNArestrictionendonucleasefragmentstohighspecificactivity mens JM, Popov KM, Harris RA, Riebow NL, Xia J, Tsui
(Addendum).AnalBiochem137:266–267,1984. LC,BogardusC,andProchazkaM.Cloningandcharacter-
10. FlorantGL,LawrenceAK,WilliamsK,andBaumanWA. izationofPDK4on7q21.3encodingafourthpyruvatedehydro-
Seasonal changes in pancreatic B-cell function in euthermic
genase kinase isoenzyme in human. J Biol Chem 271: 22376–
yellow-bellied marmots. Am J Physiol Regulatory Integrative
22382,1996.
CompPhysiol249:R159–R165,1985.
25. SambrookJ,FritschEF,andManiatisT.MolecularCloning.
11. Florant GL, Nuttle LC, Mullinex DE, and Rintoul DA.
NewYork:ColdSpringHarborLaboratoryPress,1989.
Plasmaandwhiteadiposetissuelipidcompositioninmarmots.
26. Schoonjans K, Staels B, and Auwerx J. The peroxisome
AmJPhysiolRegulatoryIntegrativeCompPhysiol258:R1123–
proliferator activated receptors (PPARS) and their effects on
R1131,1990.
lipidmetabolismandadipocytedifferentiation.BiochimBiophys
12. Forman BM, Chen J, and Evans RM. Hypolipidemic drugs,
Acta1302:93–109,1996.
polyunsaturated fatty acids, and eicosanoids are ligands for
27. SquireTLandAndrewsMT.Geneticcontrolofcarbonutili-
peroxisome proliferator-activated receptors alpha and delta.
zation during hibernation: mechanistic considerations. In: Life
ProcNatlAcadSciUSA94:4312–4317,1997.
in the Cold: Eleventh International Hibernation Symposium,
13. GalsterWAandMorrisonP.Seasonalchangesinserumlipids
edited by Heldmaier G and Klingenspor M. Berlin: Springer-
and proteins in the 13-lined ground squirrel. Comp Biochem
Verlag,2000,p.325–337.
Physiol18:489–501,1966.
28. Tokuyama K, Galantino HL, Green R, and Florant GL.
14. GarfinkelASandSchotzMC.Separationofmolecularspecies
Seasonalglucoseuptakeinmarmots(Marmotaflaviventris):the
oflipoproteinlipasefromadiposetissue.JLipidRes13:63–68,
role of pancreatic hormones. Comp Biochem Physiol A Physiol
1972.
100:925–930,1991.
15. HarrisRA,HuangB,andWuP.Controlofpyruvatedehydro-
29. WangLCH.Timepatternsandmetabolicratesofnaturaltorpor
genasekinasegeneexpression.AdvEnzymeRegul41:269–288,
2001. in the Richardson’s ground squirrel. Can J Zool 57: 149–155,
16. Heldmaier G, Klingenspor M, Werneyer M, Lampi BJ, 1979.
Brooks SP, and Storey KB. Metabolic adjustments during 30. Wu P, Blair PV, Sato J, Jaskiewicz J, Popov KM, and
dailytorporintheDjungarianhamster.AmJPhysiolEndocri- HarrisRA.Starvationincreasestheamountofpyruvatedehy-
nolMetab276:E896–E906,1999. drogenasekinaseinseveralmammaliantissues.ArchBiochem
17. HutsonNJ,KerbeyAL,RandlePJ,andSugdenPH.Con- Biophys381:1–7,2000.
version of inactive (phosphorylated) pyruvate dehydrogenase 31. Wu P, Inskeep K, Bowker-Kinley MM, Popov KM, and
complex into active complex by the phosphatase reaction in HarrisRA.Mechanismresponsibleforinactivationofskeletal
heartmitochondriaisinhibitedbyalloxan-diabetesorstarvation musclepyruvatedehydrogenasecomplexinstarvationanddia-
intherat.BiochemJ173:669–680,1978. betes.Diabetes48:1593–1599,1999.
18. KliewerSA,SundsethSS,JonesSA,BrownPJ,WiselyGB, 32. WuP,PetersJM,andHarrisRA.Adaptiveincreaseinpyru-
KobleCS,DevchandP,WahliW,WillsonTM,LenhardJM, vate dehydrogenase kinase 4 during starvation is mediated by
andLehmannJM.Fattyacidsandeicosanoidsregulategene peroxisome proliferator-activated receptor alpha. Biochem Bio-
expression through direct interactions with peroxisome prolif- physResCommun287:391–396,2001.
erator-activatedreceptorsalphaandgamma.ProcNatlAcadSci 33. WuP,SatoJ,ZhaoY,JaskiewiczJ,PopovKM,andHarris
USA94:4318–4323,1997. RA. Starvation and diabetes increase the amount of pyruvate
19. Kliewer SA, Umesono K, Noonan DJ, Heyman RA, and dehydrogenasekinaseisoenzyme4inratheart.BiochemJ329:
Evans RM. Convergence of 9-cis retinoic acid and peroxisome 197–201,1998.
PhysiolGenomics•VOL8•www.physiolgenomics.org