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Altered transcriptional regulation of

phosphoenolpyruvate carboxykinase in rats

following endotoxin treatment.

M Hill, R McCallum

J Clin Invest.

1991;

88(3)

:811-816.

https://doi.org/10.1172/JCI115381

.

The molecular mechanism involved in altered regulation of the rate-limiting enzyme in

hepatic gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), during

endotoxemia is not completely understood. We examined, therefore, the effect of a nonlethal

dose of Escherichia coli endotoxin on PEPCK gene expression in fasted rats. 5 h after

endotoxin treatment, the PEPCK transcription rate and the amount of mRNA(PEPCK) were

significantly decreased at a time when the insulin/glucagon (I/G) molar ratio and plasma

corticosterone levels were significantly increased. Similar results were observed in a time

course study, in which altered cAMP induction of PEPCK gene expression paralleled

changes in the I/G molar ratio. In diabetic rats treated with endotoxin, PEPCK gene

expression was decreased in the absence, however, of an increased I/G molar ratio. This

finding indicates that other factors, such as inflammatory mediators or cytokines, alter

PEPCK gene transcription during endotoxemia. IL-6, a putative mediator of endotoxin action

in the liver, had no effect on PEPCK gene expression in fasted rats, but did decrease cAMP

induction of PEPCK gene expression. These results indicate that, during endotoxemia,

regulation of PEPCK gene expression is influenced by inflammatory mediators in addition

to the classical endocrine hormones. IL-6, however, does not appear to be involved directly

in the altered regulation of the PEPCK gene during endotoxemia.

Research Article

(2)

Altered Transcriptional Regulation

of

Phosphoenolpyruvate

Carboxykinase

in Rats

Following

Endotoxin

Treatment

Molly HillandRoderick McCallum

DepartmentofMicrobiology and Immunology, University ofOklahomaHealth SciencesCenter,OklahomaCity, Oklahoma73190

Abstract

Themolecular mechanism involvedin alteredregulationof the

rate-limiting enzymeinhepatic gluconeogenesis, phosphoenol-pyruvate carboxykinase

(PEPCK), during

endotoxemia isnot

completelyunderstood. We

examined, therefore,

theeffect ofa nonlethal doseofEscherichia coliendotoxin on PEPCK gene expression in fasted rats. 5 h after endotoxin treatment, the

PEPCKtranscriptionrate and the amountof

mRNAPEI'K

were

significantlydecreased atatime when the

insulin/glucagon (I/

G)molarratioand plasma corticosterone levels were

signifi-cantly increased.Similarresults were observed in a time course

study, in which altered cAMPinduction ofPEPCK gene

ex-pressionparalleledchangesin the

I/G

molarratio.In diabetic ratstreated withendotoxin, PEPCKgene expression was de-creased in the absence, however, of an increased I/G molar ratio.Thisfinding indicatesthat otherfactors,such as

inflam-matory mediatorsorcytokines, alter PEPCKgene transcrip-tion during endotoxemia. IL-6, a putative mediator of endo-toxin action in theliver,had noeffectonPEPCKgene

expres-sioninfasted rats,butdid decreasecAMPinduction of PEPCK

geneexpression. Theseresultsindicate that, during endotoxe-mia, regulation ofPEPCK gene expression is influenced by

inflammatory mediatorsin addition to the

classical

endocrine hormones. IL-6, however, does notappear to beinvolved

di-rectlyin the alteredregulation ofthePEPCKgeneduring

endo-toxemia. (J. Clin.Invest.1991.

88:811-816.)

Keywords: insu-lin *glucagon*corticosterone*diabetic*interleukin6

Introduction

Acutebacterialinfectionsand aserious sequela, septic shock, remain

significant

health problems despite appropriate

antimi-crobial and

supportive therapies. Septic

orendotoxic shock isa

complex syndrome characterized by

hypotension,

poor organ

perfusion,

and severesystemic metabolic

derangements,

partic-ularly hypoglycemia. Hypoglycemia results from increased

pe-ripheral

utilization ofglucose, rapid depletion ofliver

glycogen,

anddepressed hepatic gluconeogenesis (1).Itiswellestablished

Aportion of this work was presented at the First International

Endo-toxin Congress in San Diegoon9-12 May 1990.

Address correspondence and reprint requests to Dr. Molly Reid

Hill,Department ofMicrobiology and Immunology, University ofOk-lahoma Health Sciences Center, P.O. Box 26901, OkofOk-lahoma City, OK 73190.

Receivedfor publication20 March 1991 and inrevisedform29 May

1991.

thatendotoxin

treatment,

inatime-and

dose-dependent

man-ner,elicits

hypoglycemia

anddecreasesthe

activity

of

hepatic

gluconeogenic

enzymes,

particularly

phosphoenolpyruvate

carboxykinase (PEPCK),'

the

rate-limiting

enzyme in

gluco-neogenesis

(2-5).

As

expected,

decreased

hepatic

PEPCK en-zyme

activity

in

endotoxin-poisoned

animalsis

accompanied

by

decreased

gluconeogenesis

(2, 6).

PEPCK

plays

a

pivotal

role in

intermediary

carbohydrate

metabolism and the

complex

multihormonal control of the

PEPCKgenereflects the

importance

of this enzyme in

hepatic

glucose

production (7). Glucagon,

via

cAMP,

and

glucocorti-coids

(to

alesser

extent) positively regulate

PEPCK

primarily

by increasing

the

transcription

rateofthe gene

(8, 9)

and

by

stabilizing mRNAPElCK (mRNA coding

for

PEPCK) (10,

1

1).

Conversely,

insulin

negatively regulates

PEPCK

by

decreasing

the

transcription

rateof the gene

(12),

aneffect that is domi-nant overtheinductiveeffect of cAMP andglucocorticoidsin cellculturewhen all threehormonesarepresent

(13).

Because

all three hormonesare present in

vivo,

the

insulin/glucagon

(I/G)

molar ratioserves as anindexof themetabolicstatusor

gluconeogenic potential

ofthe animal

(14,

15).

Endotoxin

treatmentofratsand mice results inincreased

plasma

levelsof

glucagon,

glucocorticoids,

andinsulin

(16, 17),

resultingina verycomplex hormonal milieu.Thehormonalstatusof endo-toxin-treatedanimals isfurther

complicated by

the appearance in the circulation of

inflammatory

cytokines such as IL-

1,

TNF,

and IL-6

(18).

Human recombinant

IL-l

and

TNF,

as well as

purified

murine IL-6, have been shown to decrease PEPCKenzyme

activity

inReuber hepatoma cells (19, 20), but

fewstudies havebeenconductedtoexamine theeffectof cyto-kinesonthe

regulation

ofPEPCKgeneexpressionin vivo

(21).

The purpose ofthis

study, therefore,

was todetermineifthe

endotoxin-mediated decrease in PEPCKenzyme activity

re-ported previously

is due to decreased transcription of the

PEPCKgene and if theputative decreasein PEPCK gene tran-scriptionis mediated solely by insulin. Our eventual goal isto

determine how

inflammatory cytokines

interact with

endo-crinehormones

(glucagon,

glucocorticoids,andinsulin)to reg-ulate the

hepatic

PEPCK gene and gluconeogenesis

during

acuteinfectionandinflammation.

Methods

Animals.Male, Sprague-Dawleyrats(100-125 g) were purchased from Sasco (Omaha, NE).Animals werehoused and cared for under the National Institutes of Health(NIH) guidelinesfor the care and use of

laboratoryanimals. In some experiments, rats were treated with

strep-tozotocin(Sigma Chemical Co.,St. Louis, MO) to induce diabetes. Afteranovernight fast,ratsweretreated with 90 mg/kg of streptozoto-cin. 5 h later, food was returned to the cages and the animalsweretested

1. Abbreviationsused in this paper:I/G, insulin/glucagon; PEPCK,

cytosolicphosphoenolpyruvate carboxykinase (GTP) (EC 4.1.32).

EndotoxinandPhosphoenolpyruvate

J.Clin. Invest.

© The American Society for Clinical Investigation, Inc.

(3)

7 to 10 d later for plasma glucose levels. Animals were considered diabetic and included in the study if their plasma glucose values were

>500mg/dl. All experimental protocols were approved by the Institu-tional Animal Care and Utilization Committee (IACUC) at the Univer-sity of Oklahoma Health Sciences Center.

Endotoxin andIL-6. Endotoxin was prepared from Escherichiacoli

serotype 011l:B4by the Westphal method (22) and was essentially protein and RNA free. The

LD.o

ofthis preparation, in Sprague-Daw-ley rats, was 35.5 mg/kg as determined by the Reed-Muench method (23). No deaths were observed during the course of the experiments described below, using a dose of 10 mg/kg. Recombinant human IL-6 was purchased from Boehringer-Mannheim Biochemicals (Indianapo-lis, IN). The specific activity of IL-6 was greater than 2X10 U/mg, as determined by stimulation of 7TDl mouse-mouse hybridoma cells (24).

Nuclear run-on assay. The transcription elongation assay was carried out according to the procedure of Sasaki et al. (13), which was modified from the method of McKnight and Palmiter (25). After ho-mogenization of liver tissue, nuclei were isolated through a series of centrifugations in sucrose buffers. Isolated nuclei (200 ug) were incu-batedat260Cfor 10 min in 75 mM HEPES at pH 8.0, 100 mM KC1, 2.5mMMgC12, 25% glycerol, 0.1 mM PMSF, 0.05 mM EDTA, 4 mM DTT, 25 U RNasin, 0.5 mM each ofATP, CTP, and GTP, and 200MCi ofa-[32P]UTP(400-600 Ci/mmol). The reaction was terminated by

digestionwith DNAseI and proteinase K and after phenol extraction and alcoholprecipitation,the RNA samples were hybridized with ni-trocellulose-bound pPCI 12.R3(providedbyDr.Daryl Granner, Van-derbilt University, Nashville, TN) or pBR322 as control. pPCl 12.R3 is

aplasmid witha 5.8-kb EcoRI fragment of genomic PEPCK DNA inserted into the EcoRI site of pPR322 (26). All of the insert represents PEPCK DNA that is transcribed into precursor mRNA. The results are expressedasparts of PEPCKmRNAtranscribed per one million parts of totalRNAtranscribed.

Quantitation ofmRNA. After experimental treatment, total liver RNA wasisolated by the Chirgwin method (27). RNA samples were denatured in 20X standard saline citrate plus 37% formaldehyde. After heating at 60°C for 15 min, 2.5Mlof each sample were applied under vacuum toanylon membrane with an ABN slot blot apparatus

(Ameri-canBionetics, Hayward,CA). The blotswereairdried and bakedat

80°C for 1 h. The blotswereprehybridizedfor 6-18 h andhybridized overnightwithacDNAprobefor PEPCK(pPCl16, suppliedbyDr.

Daryl Granner) (26) or actin(Drosophila melanogaster,obtained from Dr.Sally Tobin, University of Oklahoma Health Sciences Center) la-beled with32[P]dCTPbyrandompriming usingtheAmersham Prime-A-Gene kit(Amersham Corp, Arlington Heights, IL). The blotswere

washed under stringent conditions and autoradiographed. Intensity of bandswasdetermined by laserscan densitometry and expressed

as relative integrator units adjusted accordingto the OD260 of the

RNAsamples. Northern analysis confirmed that the results obtained with the slot blot methodology were due to specific hybridization of

mRNAPEP with PC1 16 (datanotshown).

Biochemicalassays. PEPCK enzymeactivitywasmeasured by the

'4C-radiometricmethod of Chang andLane(28)asdescribed

previ-ously(2). Units of enzyme activity correspondtonanomolesof

oxalo-acetateformed fromphosphoenolpyruvateper 15 min per mg of cyto-solprotein. Proteinwasquantitated usingthe BCAproteinassay kit (29) from Pierce Chemical Co. (Rockford,IL). Plasma glucagon and insulin levels were measured usingradioimmunoassaykits from

Ven-trexLaboratories(Cambridge, MA) and plasma corticosterone levels

weremeasuredusing a radioimmunoassay kit from ICN Bio Medicals,

Inc.(Costa Mesa, CA). Plasma glucose levels were measured usinga

colorimetric glucose oxidaseassay kit fromSigmaChemical Co. InductionofPEPCK gene transcription in vivo. There are primarily

twowaystoincrease thetranscriptionrateof the PEPCK gene in ani-mals. The simplest method is by fasting overnight, which lowers the

insulin/glucagon (I/G) molar ratio and results in anapproximately twofold inductionin therateoftranscription.Another method isby dietarymanipulationwhich involvessubmittingthe animalto a

fast-ing-refeedingcycle followed byinjectionwith cAMP(30).This

experi-mentalprotocol, described originallybylynedjianetal.(30),wasused tomanipulatethe hormonal response inrats sothatchangesin the PEPCKtranscriptionrate could be examinedafter endotoxin

treat-ment. Briefly, animals were fastedovernight,refed with glucose(0.5 g)

by gavage, and 2 h laterdibutyrylcAMP(30 mg/kg)andtheophylline

(30mg/kg)wereadministeredintraperitoneally.Animals received di-butyryl-cAMP andtheophylline (referredtohenceascAMP)every 90 minthereafter.Glucose feeding stimulates insulinrelease,which dein-duces PEPCK geneexpression (31, 32).Any increase in the PEPCK

transcriptionratecan,therefore,be observedafteradministration of inducer(cAMP) (30,32-33). The advantageofdietarymanipulation

overfastingis that the induction of PEPCK genetranscriptionisrapid

and large. We have used both methods in the resultsdescribedbelow.

Statistics. Statistical analysisofthe data wasperformedbyusing

Student'sttest(34).

Results

Effect

of

endotoxin on PEPCK gene expression. Previous work

has shown thatendotoxin treatment decreasesPEPCK enzyme

activity in mice4 to6hafterchallenge (35, 36). The effect of

endotoxin ontranscriptional regulation ofthe PEPCKgene,

however,

has not beenexamined directly.Changes in PEPCK gene

expression

and circulating levels of insulin, glucagon,and

corticosteronewereassessed, therefore, in fastedrats 5 h after

endotoxin

treatment (10 mg/kg) (Table I). Endotoxin treat-mentsignificantly decreasedthe PEPCKtranscriptionrate and amountof mRNAPEPc toapproximately one-third of control.

No changein

mRNA1

was observed (data notshown). As

expected, the hormonal status of endotoxin-treated animals

wassignificantly altered,asindicatedby aninefold increasein

the

I/G

molar ratio andafourfold increase in plasma

corticoste-rone levels,compared with saline-treated controls.Inrats given

endotoxin, insulinvalues rose 18-fold (control=2.4±0.7

,uU/

ml vs.endotoxin=44.0±3.4

,U/ml)

and glucagon values

dou-bled (control = 1,352±97 pg/mlvs.endotoxin = 2,734±251 pg/ml). SimilarchangesinthePEPCK transcriptionrate were

observed inratsgiven freeaccess tofood and treated with saline

or endotoxin (control = 2,437 ppm vs. endotoxin = 1,355

ppm, n =2).

Altered cAMP

induction of

PEPCKgene expression

after

endotoxin

treatment. To examine furtherthe mechanism of altered PEPCKgeneexpression in endotoxic animals, the ef-fect ofendotoxintreatment on cAMPinduction

of

PEPCKwas

tested.

Endotoxin

was

injected

2 h before cAMP treatment becauseithas beendemonstratedthat serum levelsofmany

inflammatory cytokines appear and/orpeak 2 h

after

endo-TableI.Endotoxin Action in Fasted Rats

PEPCK mRNAPFaCX

Treatment* Transcription (relative I/G molar Plasma

(5h) rate integratorunits) ratio corticosterone

ppm jug/dl

Controls

3572±100*

201±26 0.042±0.014 15.6±2.0 Endotoxin 1145±270§ 66±14§ 0.381±0.0245 65.9±8.2§

* Ratswerefasted overnight, followed by an intraperitoneal injection of either 0.5 mlof sterile salineor E.coli endotoxin (10 mg/kg). * Meanvalue±SEM,n = 4.1Statistically different from control (P

(4)

160- mRNAPCK Figure 1. Timecourse >120- / f of PEPCK gene expres-o . / _Induced sion in

endotoxin-a

80

-2

/ /control Itreatedratsafterglucose

/ PEPCK

feeding

and induction

40-

T

Transciptionraze withdibutyryl-cAMP

o T treatment.Rats were

'

.. fasted for18h and then

0 2 3.5 5 6.5 8 10 given 500mg glucose

it

f

f

f

bygavageat0 h.At2h

cAMP cAMP cAMP cAMP andevery90minthere-Endotoxin TIME(h) after,ratswereinjected

intraperitoneallywith

dibutyryl-cAMP(30 mg/kg) and theophylline (30 mg/kg). Rats were treated with endotoxin (10mg/kgi.p.) at 0 h (simultaneously with glucose) and were killed at the indicated times. Nuclei and total RNA were isolated from the livers and were assayed for PEPCK transcrip-tion rates andmRNAPE't levels, as described under Methods. Re-sults are expressed as the mean value±SEM and plotted as a percent-ageof induced control. Each time point represents data from four to six animals. Closed circles represent the PEPCK transcription rate and opencircles represent

mRNAPIP.

*Valuessignificantly

differ-entfrominducedcontrols (P<0.05), as determined by Student'st

test.

toxin administration (18). The resultsare presentedas a

per-centage ofinducedcontrolduetovariation from experimentto

experiment.

As canbe seenin Fig. 1, adynamic responseto

endotoxinwasobservedoverthe courseof 8 h. By 3.5h after

endotoxin treatment,no

significant

change in the PEPCK

tran-scriptionrate or

mRNAPE'

levelwasobserved in endotoxin plus cAMP-treated

animals

comparedwith induced controls.

Significant inhibition

of the PEPCK

transcription

rate and

mRNA"E'P

level by endotoxin treatment, however, was

ob-served at 5 h (control =

2,833±794

ppm vs. endotoxin

=644±24 ppm,P<0.05).At8 h, PEPCKgeneexpressionwas not

different

from the

induced

controls.PEPCK enzyme activ-itywas also

significantly

decreased in the endotoxin-treated group, compared with the induced controls at the 5-h time point. PEPCKenzymeactivitywas

increased

overdeinduced controls

(animals receiving

glucose only) by 262 U/mg

com-pared with 134 U/mg in endotoxic animals (51% of induced controls,P <0.05). 6haftercAMPtreatment (8hafter

endo-toxin),

PEPCK enzyme

activity

was not

significantly

different from

induced

controls.

The plasma profiles of

insulin,

glucagon,andthe

I/G

molar ratio, in controlandendotoxic animals subjectedto

fasting-re-feeding

and cAMP

injection,

are

illustrated

in Fig. 2. Endo-toxin treatment

significantly increased

insulin levels after 3.5

and 5 h,

however,

by 8 h, the insulin values in control and endotoxicrats weresimilar.In contrast totheinsulin response, glucagon levelswere

increased

by2 hafter endotoxin treatment andremained elevated by 8 h. At3.5 and 5 h, the

I/G

molar ratiowas

increased

in endotoxic

animals

(althoughnot statisti-cally

different from induced

controlsat 5h),yetby 8hthe

I/G

molarratiowassimilartothe

induced

controls. The

increased

insulin

levels andthe

I/G

molarratio correlatedwellwith de-creased PEPCKgeneexpression in

endotoxin-treated

animals

and suggested a rolefor insulin in downregulation of PEPCK

gene

expression during

endotoxemia.

PEPCKgene expression in diabetic rats afterendotoxin treatment.Inordertoexaminefurther theroleofinsulinin the

downregulation

of PEPCK gene expression during

endotoxe-100

-75

-

E-02

25 *_

0 3000-1

=

0 08

2000-=_ z C.

E

-Q

1000-2.0 -

1.5-L._

a 1.0

E

>

0.5-Gluc

I-0

SCr

'I / ±1

Figure

2. Timecourse

/o / of insulin, glucagon,

andI/Gmolar ratio in g * * * * * * * § * endotoxin-treatedrats 2 3.5 5 6.5 8 after glucosefeeding

and induction of

I PEPCK with

dibutyryl-*Endotoxin cAMPtreatment.Rats

oControl / weretreatedas

de-scribed in the

legend

to Fig. 1.Plasmawas col-lectedfor determination

of

glucagon

and insulin levels

by

radioimmuno-.'

2 3.5 5 | assayasdescribedunder

6.5 8

Methods.Resultsare

expressedasthemean value±SEM.Each time

*

Endooxin

point represents data

oControl * fromfourtosix

ani-4 N - ¢ mals.Opencircles

rep-/ X ,Xe 1 resent saline treated an-It ;/>

N+

fimalsand closed circles

representendotoxin treated animals. *Values |2-3.5 65| significantlydifferent

s 6.5 8 from induced

controls

t

f

t

t

(P<0.05),as

deter-cAMP cAMP cAMP cAMP mined by Student'st

TIME(h) test.

mia,

diabetic

(plasma

glucose

>500

mg/dl)

rats were fasted

overnight,

treated with sterilesalineorendotoxin

(10

mg/kg),

and killed 5 hlater. PEPCKgene expression and

circulating

hormone levelswereexamined. Theresultsareshownin Ta-ble II. In thediabetic rats, endotoxin treatmentresulted ina

significant

decrease in the PEPCK transcription rate and

mRNAPEi'K

toatleastone-third of thevalues obtained from diabeticrats

given

saline. Plasma corticosterone levelsin

dia-beticrats

receiving

endotoxinwere

significantly

elevated

com-pared

withdiabeticcontrols andweresimilartothelevels ob-servedin thenondiabetic animals

given

endotoxin. A

signifi-cantdecreaseinPEPCKgene

expression

occurred indiabetic

animals, however,

in theabsenceofincreased plasma levelsof

insulinor

glucagon.

Indiabeticratsgiven endotoxin, therewas

no

significant

change

ininsulin values

(control

=3.3±1.5

ALU/

mlvs.endotoxin=3.7±3.7

,U/ml)

orglucagonvalues

(control

=

2,539±729 pg/ml

vs.endotoxin =2,786±653 pg/ml).

Thus,

PEPCKwas

downregulated despite

nochangein theI/Gmolar ratio.

Altered cAMP induction ofPEPCK gene expression

after

IL-6treatment.Basedontheobservationindiabeticrats,that insulinalone didnotdecrease PEPCKgeneexpression

during

endotoxemia,

we

investigated

the role ofhuman recombinant IL-6in theregulationofPEPCKgeneexpression. IL-6 modu-latestheexpressionof manyhepaticacutephasegenes

during

acuteinfectionandinflammationand may serve as a second

signal

of IL-I andTNF during endotoxemia (18).Whenfasted

(5)

Table II. EndotoxinAction inDiabeticRats

PEPCK mRNAPEPCK Plasma

Treatment* Transcription (relative I/Gmolar

cortico-(5 h) rate integratorunits) ratio sterone

ppm Ag/dl

Diabetic controls 2580±39t 468±70 0.029±0.007 32.3±2.5 Diabetic

+endotoxin 956±134§

249±5l0

0.025±0.01 65.3±3.2§

*Rats were fasted overnight, followed by an intraperitoneal injection

of either 0.5 ml of sterile saline or E. coliendotoxin (10mg/kg).

*Mean value±SEM, n = 4. § Statistically different from control (P

<0.05), determined by Student's t test.

rats were injected with human recombinant IL-6 (10,000U) and killed 5 h later, there was no change, compared with saline injected controls, in any of the parameters ofPEPCK gene expression (PEPCK transcription rate, mRNAPE" , or PEPCK enzyme activity), the

I/G

molar ratio, or plasma glu-cose levels (data not shown). Plasma corticosterone values, however, were elevated threefold (control= 15.6±2.0 jug/dlvs. IL-6 = 51.1±9.4

/Ag/dl,

P < 0.05). Because glucocorticoids alone can increase PEPCK gene expression (8), we tested whether IL-6, via increased corticosterone levels, could en-hance the cAMP induction of PEPCK gene expression, using the experimental protocol described in the legend to Fig. 1. Animals were treated with IL-6 (10,000 U) simultaneously with glucose and killed 5 h later (after 3 h of cAMP treatment). Interestingly, IL-6 treatment resulted in asignificantdecrease in the PEPCK transcription rate andmRNAPE1 levels, com-pared with induced controls (Table III). Nosignificantchange in PEPCK enzyme activity was observed,however,in the IL-6-treated animals, compared with induced controls (induced controls = 262±13 U/mg vs. IL-6 = 214±21 U/mg). In addi-tion, no significant change occurred in the

I/G

molar ratio,

comparedwiththe induced controls.

Discussion

The molecular mechanisms involved indecreased gluconeo-genesis after endotoxin treatment have not been fully eluci-dated. The data presented here demonstrate that the previously observed decrease in PEPCK enzyme activityafterendotoxin

Table III. Effect of IL-6 Treatment on cAMPInduction

of PEPCK Gene Expression

PEPCK mRNA1"

Transcription (relative integrator I/Gmolar

Treatment* rate units) ratio

ppm

Control 2833±797$ 149.1±12.1 0.59±0.17 IL-6 344±127§ 87.2±9.61 0.82±0.15

*Rats were treated as described in the legend to Fig. 1, except that

IL-6 (10,000 U) was injected instead of endotoxin and the animals were killed at the 5-h time point.*Mean value±SEM, n=4.1

Statis-tically different from control (P <0.05), determined by Student'st test.

treatmentresultsfrom decreasedgene

expression.

With the re-cent

availability

of

appropriate

cDNA

probes (7),

wewereable to

demonstrate,

in endotoxic

animals,

a decrease in the

PEPCK

transcription

rate,whichwasreflected

subsequently

in a decreased amount of

mRNAPEcK.

After endotoxin

treat-ment,the

magnitude

of change inmRNA

'"C

corresponded

well with the

magnitude

of

change

in the

transcription

rate,

suggesting

that the decreasein

mRNAPEPc§

wasdueto a

corre-sponding

decrease inthePEPCK

transcription

rate. Itis

inter-esting

to notethat decreased PEPCKgene

expression

was ob-servedinthis

study

in endotoxicratsthatwere

(a)

fasted

over-night

and

during

the

experiment,

(b)

fed (allowed access to

food),

or

(c)

fasted

overnight,

refed with

glucose,

andtreated

with cAMP.

Considering

the dominant role of insulin in

downregulating

the PEPCKgene

(7),

the observed elevation in the

I/G

molarratio couldaccount for the decrease in PEPCK

gene

expression during

endotoxemia. In ordertodetermine if insulin was the

only

hormone

downregulating

PEPCK gene expression

during

endotoxemia,

weuseddiabeticratsthatwere hyperglycemic and had low, but detectable, levels of

plasma

insulin.

Using

a nonlethaldose of endotoxin in diabetic rats,

we demonstrated that decreased PEPCKgene expression

oc-curred in the absence ofan increase in

plasma

insulin. It is

interesting

tonotethat plasma glucagon levelswereelevatedto

asimilar

degree

in controlandendotoxin-treated diabeticrats

(which

wastwice thevalueobserved innormalfasted rats),yet

the observed

hyperglucagonemia

failedtopreventthe decrease in PEPCK gene

expression

due to endotoxin. These results

support previous reports

that,

in addition to

glucocorticoids,

PEPCKis

nonresponsive

to

regulation by glucagon

in

endo-toxic animals (

16).

These resultsfurthersuggestthatother fac-tors, in addition to

insulin,

may be involved in the

negative

regulation

ofPEPCK

during

acuteinfection orinflammation.

Ithas beenwelldocumentedthattheeffect of endotoxinto inhibit hepatic gluconeogenesis and PEPCKisnot adirect

ef-fect of endotoxin onthe liver

(1,

2, 6, 37).

IL-1, TNF,andIL-6 have been

implicated

as mediators of the endotoxin effect

on PEPCK because they decrease enzyme activity and

mRNAPE'

in vitro in Reuber H-4-II-E rat hepatoma cells

(19,

20, 38). Multiple cytokines

appearin the circulationafter

endotoxin treatment and may elicit the

production

of addi-tional

cytokines, resulting

inacascadeof

inflammatory

media-tors

(18).

WechosetoexamineIL-6as a

putative

mediatorof

endotoxin actionon PEPCKgene

expression

because IL-6 is the dominant

cytokine

involved in induction of the

hepatic

acute

phase

proteins (39-41)

and because endotoxin,

TNF,

andIL- 1 elicitIL-6

production (42, 43).

IL-6actsviaa

specific

membrane receptor (44) and after

binding

there is increased

transcription

of

hepatic

acute

phase

genes, suchas

a2-macro-globulin, haptoglobin, cysteine proteinase inhibitor,

and a1-acid

glycoprotein.

Concomitantly, synthesis

of several acute

phase proteins, such asalbumin, is decreasedaftertreatment

with IL-6 (39-41). The doseofIL-6(10,000 U/100grat) used in thesestudieswaschosenbased onapreviousstudy by Mar-inkovicet al.(45)who reported that 40,000 U IL-6 per 400 grat

werenecessary toelicitthe same acute phase response

quantita-tively

as an acute

inflammatory

agent,

turpentine.

The resultsweobtained with IL-6treatmentare

interesting,

but somewhat

perplexing.

IL-6 treatment, after 5 h, had no effectonPEPCKgene

expression

infastedrats,indicating that

the

cytokine

alone, attheindicated dose, isnotcapable of

(6)

treatment, however, decreased the PEPCK transcription rate

and the amount of

mRNAPEP'

after cAMP treatment in fasted, refed rats. The magnitude of decrease inthe PEPCK transcriptionrate wasmuch greater than theobserved decrease in

mRNAPE"c-,

which suggeststhat increased stabilization of

mRNA'E1

may have occurred. Although glucocorticoids have been shown tostabilize

mRNAPEPC

in Reuber hepatoma cells (11), it is not known if the observed increase in corticoste-rone levels after IL-6 treatment contributed to increased mRNAPEICR stabilization. The observation that no significant change in PEPCK enzyme activity occurred in the IL-6-treated animals lends support to this idea. One explanation for the IL-6 inhibition of cAMP-mediated induction of PEPCK gene

ex-pression might be that IL-6 acts as an insulin-like factor (46) or that thecytokine prolongs the action of insulin when it is ad-ministered at the same time as glucose. Other investigators have reported that IL-6 enhances glucose-stimulated produc-tion of insulin from isolatedpancreatic ,8-cells (47), however, we observed nochange ininsulin levels after IL-6 treatment. Although an IL-6 response element hasnotbeen mappedon

the promoterregion of the PEPCK gene,a corehexanucleotide

CTGGGAsequence found in the promoterregionof IL-6

re-sponsivegenes, is found in the5-flanking regionofthePEPCK

gene described as theglucocorticoid responseunit(GRU) (48). AportionoftheGRU also overlaps with the recentlydescribed

insulinresponseelement (IRE) (49).This raises the possibility, although purely speculative,thatputativenuclear elements or

factors mediating theIL-6 effect on the PEPCKgene, under certain conditions, interact at theGRU, IRE, orthe cAMP

response element.

AlthoughinsulindecreasesPEPCKsynthesis after the

ap-propriate dietary stimulusin normalanimals, itis clear from ourresults that otherfactors in addition toinsulin contributeto the negative regulationofthePEPCK gene during

endotoxe-mia.Weobservedinthisstudy thatendotoxin eliciteda

tran-sientchangein PEPCKgeneexpression,however, the change occurred at acritical timein the developmentof progressive hypoglycemia (2). When larger dosesofendotoxin

(LDsO)

are

administered, PEPCK enzyme activity is significantly de-creased atatimecoincidentwithdeveloping hypoglycemia (4-6h)and remainsdepressed throughoutthe course ofthe

exper-iment(2). Although decreased PEPCK enzymeactivityis not solelyresponsible forthe observed hypoglycemiainseptic ani-mals,itisanimportantfactor inthefailure ofthe

gluconeo-genic pathway to provide sufficientglucose to satisfythe in-creased energydemandsoftheinjured host.Furtherstudiesare

requiredtoidentify putative inflammatory mediators involved

and to elucidate how they interact withinsulin in negatively regulating PEPCKgeneexpression duringacuteinfectionand

inflammation.

Wegratefullyacknowledge theassistance of Dr. DarylGranner for

allowingone of the authors(Dr. Hill)tospendvaluabletime in his

laboratory studyingthetranscriptional regulationof PEPCK and for

providingthe necessary cDNAprobestocarryoutthesestudies. The

enlighteningdiscussions of Dr. Richard Printzaregreatlyappreciated.

The expert technical assistance of BradKingis alsogratefully

acknowl-edged. The authors thank Dr. RobertSteinberg(Departmentof

Bio-chemistry, Universityof Oklahoma Health SciencesCenter)for theuse

of the laserdensitometer,whichwaspurchasedwith fundsprovided by

the OklahomaCenterforAdvancement of Science and Technology (OCAST)andPresbyterianHealthFoundation.

We would also like to thank OCAST and Presbyterian Health

Foundation forsupportingthis work.

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