overcoming diabetes-induced hyperglycemia through inhibition of hepatic phosphoenolpyruvate...

ARTICLEdoi101016jymthe200508026

Overcoming Diabetes-Induced Hyperglycemia throughInhibition of Hepatic Phosphoenolpyruvate

Carboxykinase (GTP) with RNAi

Alicia G Gomez-Valades1 Anna Vidal-Alabro1 Maria Molas1 Jordi Boada1 Jordi Bermudez1

Ramon Bartrons2 and Jose C Perales1

1Biophysics Unit and 2Biochemistry Unit Department of Physiological Sciences II IDIBELLndashUniversity of Barcelona

Feixa Llarga sn 08907 LTHospitalet del Llobregat Spain

To whom correspondence and reprint requests should be addressed at the Departament de Ciencies Fisiologiques II

Universitat de Barcelona Feixa Llarga sn 08907 LTHospitalet Spain Fax +34 93 4024268 E-mail jperalesubedu

Available online 3 November 2005

Abbrevia

NIDDM

dependengen PKR

ycytosine)

fatty acid

protein 1c

MOLECULA

Copyright C

1525-0016$

Phosphoenolpyruvate carboxykinase (PEPCK EC 41132) is the rate-controlling enzyme ingluconeogenesis In diabetic individuals altered rates of gluconeogenesis are responsible forincreased hepatic glucose output and sustained hyperglycemia Liver-specific inhibition of PEPCKhas not been assessed to date as a treatment for diabetes We have designed a therapeuticvector-based RNAi approach to induce posttranscriptional gene silencing of hepatic PEPCK usingnonviral gene delivery A transient reduction of PEPCK enzymatic activity (76 F 06 vs 97 F 11mUmg P bbb 005) that correlated with decreased protein content of up to 50 was achievedusing this strategy in diabetic mice PEPCK partial silencing was sufficient to demonstrate loweredblood glucose (218 F 26 vs 364 F 33 mgdl P bbb 0001) and improved glucose tolerance togetherwith decreased circulating FFA (089 F 010 vs 144 F 011 mEqdl P bbb 0001) and TAG (65 F 11 vs102 F 16 mgdl P bbb 001) in the absence of liver steatosis or lactic acidosis SREBP1c was down-regulated in PEPCK-silenced animals suggesting a role for this pathway in the alterations of lipidmetabolism These data reinforce the significance of PEPCK in sustaining diabetes-inducedhyperglycemia and validate liver-specific intervention at the level of PEPCK for diabetes genetherapy

Key Words phosphoenolpyruvate carboxykinase diabetes mellitus hydrodynamic gene transferRNA interference gluconeogenesis

tion

non

t dipro

A

syn

R TH

The A

3000

INTRODUCTION

Gluconeogenesis is responsible for sustained productionof glucose in fasting animals [12] Phosphoenolpyruvatecarboxykinase (PEPCK) catalyzes the formation of phos-phoenolpyruvate from oxaloacetate the rate-controllingstep in the gluconeogenic pathway In addition asignificant role of the enzyme in the regulation of energyhomeostasis and flux through the TCA cycle has beenrecently demonstrated in a liver-specific gene-deletion

s used PEPCK phosphoenolpyruvate carboxykinase

-insulin-dependent diabetes mellitus IDDM insulin

abetes mellitus TZD thiazolidinediones Gly glycotein kinase R poly(dIdC) poly(deoxyinosinedeox

CC-P phosphorylated acetyl-CoA carboxylase FAS

thase SREBP1c sterol regulatory element binding

ERAPY Vol 13 No 2 February 2006

merican Society of Gene Therapy

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mouse model [3] PEPCK is also involved in the control ofacidndashbase in the kidney [45] glyceroneogenesis inadipose tissue [56] and glutamine metabolism in smallintestine [57] In diabetic individuals altered rates ofgluconeogenesis are responsible for increased hepaticglucose output (HGO) and therefore sustained hyper-glycemia observed in both IDDM and NIDDM [89]Expression from the gene for cytosolic phosphoenolpyr-uvate carboxykinase (PEPCK-C) is induced during diabe-tes both in animals and in human patients and thisinduction correlates with the increased rate of gluconeo-genesis in liver and kidney [10]

Several pathways involved in energy metabolism arealtered in the liver adipose tissue and muscle duringdiabetes These alterations are secondary to a resistance toinsulin signaling (NIDDM) or a reduced concentration ofinsulin (IDDM) The mechanism of action of current

401

ARTICLE doi101016jymthe200508026

drugs for the treatment of NIDDM biguanides (egmetformin) and thiazolidinediones (eg rosiglitazone)[1112] induces an increase in insulin sensitivity inperipheral tissues and the liver The antihyperglycemicaction of thiazolidinediones is a direct result of activationof the nuclear receptor peroxisome proliferator-activatedreceptor-g (PPAR-g) [13] whereas metformin has beenlinked directly to inhibition of liver gluconeogenesis[1415] However these drugs activate upstream regula-tors (eg PPAR-g or AMP-K) that are involved in a broadrange of physiological functions [1316ndash19]

Because of the theoretical potential of blocking HGOby means of a specific inhibition of a key enzymatic stepin the pathway we have aimed at down-regulatinghepatic PEPCK-C To accomplish this goal we havedeveloped specific short-hairpin RNA (shRNA) reagentsto knock down PEPCK-C and achieved organ specificityusing liver-directed transfection Silencing of endogenousgenes mediated by RNA interference is a conservedbiological process that can be used to induce experimen-tal sequence-specific gene silencing with either smallinterference duplexes (siRNA) or shRNA expression vec-tors This kind of vector drives shRNA expression from ahuman U6 promoter and their efficacy has been demon-strated both in mammalian cells [20] and in mice afterhepatic hydrodynamic gene delivery [21]

We report here an efficient reduction in the levels ofmRNA protein and enzyme activity of hepatic PEPCK-Cin animals treated with specific shRNAs As a resultplasma glucose of diabetic mice was reduced up to 40with no associated lactic acidosis or hepatic steatosis Wedemonstrate that the inhibition of PEPCK-C gene expres-

FIG 1 pSHAG-664 induces PEPCK gene silencing in vitro

(A) pCPEPCK-C (225 Ag) was cotransfected with different

molar ratios of pSHAG-664 (10 (striped) 16 (white) and

112 (gray)) As a control for nonspecific gene silencing

pSHAG-Ff was cotransfected at a 124 ratio (black) pBlue-

script was used as stuffer As an internal transfection

control 1 Ag of pGL3 was added to all plates PEPCK-C

activity was measured 48 h after transfection and normal-

ized for luciferase activity Data are expressed as relative

PEPCK activity (PEPCK activityluciferase activity) (B)

Silencing was confirmed at the protein level using Western

blot A representative blot is shown

402

sion using shRNA also results in alterations in lipidmetabolism exemplified by reduced serum NEFA andtriglycerides (TAG) in parallel to a clear reduction of bothprecursor and mature forms of the key lipogenic tran-scription factor SREBP1c Hence liver-specific PEPCKgene silencing provides a novel therapeutic approachfor the treatment of diabetes

RESULTS

PEPCK-C Gene Silencing by Means of shRNAExpression in VitroWe generated two mammalian expression cassettes con-taining the human U6 polymerase III gene promoterdriving expression of shRNAs (pSHAG vector) comple-mentary to both mouse and rat PEPCK-C transcripts Bothsequences were designed with homology to a 29-bp stretchfrom nucleotides 482 and 664 of the cDNA (pSHAG-482and pSHAG-664 respectively) We used an equivalentcassette expressing a shRNA specific for a Photinus pyralisluciferase sequence (pSHAG-Ff) as a control

We confirmed efficient silencing of the gene forPEPCK-C in Huh-7 cells using cotransfection experimentswith a constant amount of PEPCK-C expression vector(pCPEPCK-C) and different molar ratios (16 and 112) ofthe silencing vectors (664 482 or Ff) Cotransfection ofpSHAG-Ff whatever the ratio used never altered PEPCK-C activity or protein levels In contrast as shown in Fig1 PEPCK-C activity and protein levels significantlydecreased when pSHAG-664 and pCPEPCK-C plasmidswere cotransfected The suppression of PEPCK-C wasclose to 80 as determined by enzyme activity and

MOLECULAR THERAPY Vol 13 No 2 February 2006

Copyright C The American Society of Gene Therapy

IG 3 PEPCK-C gene silencing in healthy mice Analysis of the silencing

apacity of pSHAG-664 in nondiabetic mice assessed at the mRNA enzyme

ctivity and protein levels Mice were injected in the tail vein with 10 (wv)

hysiological saline solution containing 100 Ag of either pSHAG-664 or


Western blot at both 16 and 112 molar ratios (Fig 1)whereas pSHAG-482 inhibited PEPCK-C gene expressionwith a lower efficiency (data not shown)

Hepatic Gene Silencing in Vivo Using HydrodynamicGene TransferHydrodynamic gene transfer is an efficient and conven-ient method for preferential transfection of DNA to theliver [2223] Since we aimed at down-regulating hepaticPEPCK-C hydrodynamic gene transfer seemed an appro-priate tool to achieve our objective We thereforeassessed the efficiency of shRNA silencing in vivo usinga validation model whereby luciferase gene expression inthe liver was induced by transfection of a luciferaseexpression vector (pGL3) cotransfected with pSHAG-Ff aluciferase-specific silencing vector Luciferase activity wasinhibited by about 98 in mice treated with pSHAG-Ffcompared with animals injected with reporter vectoralone or nonspecific pSHAG (Fig 2) Luciferase activitywas undetectable in kidney extracts after pGL3 injection(data not shown) This experiment demonstrates specificand efficient silencing in the liver mediated by shRNAand validates the experimental system

Specific Silencing of Endogenous PEPCK-C in HealthyAnimalsWe demonstrated silencing of endogenous PEPCK-C geneexpression in healthy animals 24 h after tail vein injectionwith pSHAG-664 plasmid We noted a reduction in PEPCK-C mRNA content independent of the nutritional status(fed or 24 h fasted) and it was statistically significant (40reduction) in fed animals (Fig 3A) Moreover hepaticPEPCK-C activity after 24 h fasting was decreased by 30compared with pSHAG-Ff (20 F 198 vs 285 F 189 mUmg protein n = 4 P b 005) (Fig 3B) We found no

SHAG-Ff plasmid Silencing of PEPCK-C was assayed 24 h later (A) Hepatic

EPCK mRNA content was analyzed from fed and 24-h fasted mice by

orthern blot and is represented as the relative amount of PEPCK-C mRNA

ith respect to GAPDH mRNA (B) Specific PEPCK activity in liver extracts from

sted animals is presented Values are expressed as the means F SE (n = 4

b 005) (C) Further confirmation of PEPCK silencing was obtained by

nalysis of the PEPCK-C protein content by Western blot Representative blots

re shown (n = 4)

FIG 2 Liver-specific RNAi-induced gene silencing using hydrodynamic gene

transfer Luciferase expression vector pGL3 (10 Ag) was co-injected with

specific (pSHAG-Ff) or nonspecific (pSHAG-664) shRNA at a 16 molar ratio

using the hydrodynamic method Luciferase activity was assayed 72 h later in

liver extracts (n = 3)



F

c

a

p

p

P

N

w

fa

P

a

a

significant reduction in PEPCK-C activity or protein inkidney compared with pSHAG-Ff or saline groups (datanot shown) In the liver and correlating with effectsobserved on mRNA content and enzymatic activityprotein levels were also reduced as determined by Westernblot analysis (Fig 3C) These animals remained euglyce-mic and euinsulinemic and we noted no lactic acidosis orchange in serum or hepatic lipids (data not shown)

Silencing of Liver PEPCK-C in Streptozotocin-TreatedAnimalsTo assess whether the inhibition of PEPCK-C in the livercould be a feasible strategy to overcome fasting hyper-

403

FIG 4 Blood glucose and glucose tolerance after silencing PEPCK-C in the

livers of diabetic animals (A) Streptozotocin-induced diabetic mice received

an intravenous injection as explained under Materials and methods with 100

Ag of either pSHAG-Ff (solid bars) or pSHAG-664 (empty bars) Glycemia at 48

h (pSHAG-Ff n = 26 and pSHAG-664 n = 25 P b 0001) and 72 h

(pSHAG-Ff n = 12 and pSHAG-664 n = 10 P = 005) in 8-h fasted animals i

presented Results are expressed as relative glycemia (B) A glucose tolerance

test was performed at 48 h after hydrodynamic gene transfer by ip injection o

a glucose bolus (1 mgg) in pSHAG-Ff (filled squares) pSHAG-664 (empty

squares) and healthy control animals (empty circles) Values are the means FSE (n = 5 P b 001)


glycemia in diabetic animal models and to study themetabolic implications of such inhibition we introducedpSHAG-664 into the liver of streptozotocin (STZ)-treatedmice At the time of injection mice were hyperglycemic(N400 mgdl) and weighed 20ndash25 g Only animals thatmaintained their weight above 20 g during the course ofthe experiment were further analyzed We used 3-mercap-topicolinic acid (3-MPA) a well-known noncompetitiveinhibitor of PEPCK-C as a positive control for the systemicinhibition of gluconeogenesis in these animals

We carried out tail vein injections with 100 Ag ofpSHAG-Ff or pSHAG-664 We analyzed weight andglycemia at 48 and 72 h postinjection after a short fastWe observed the greatest hypoglycemic effect 2 days aftertreatment with pSHAG-664 when fasting blood glucoseconcentration was dramatically reduced (40) comparedto control (pSHAG-Ff) animals (218 F 26 vs 364 F 33 mgdl n = 26 and n = 25 P b 0001) similar to 3-MPA-treateddiabetic mice (149 F 30 mgdl) Overt hypoglycemia(blood glucose concentration below 30 mgdl) was notobserved in any of the animals treated Three days aftertreatment glycemia in the treated group was reduced byapproximately 25 (350 F 68 vs 476 F 35 mgdl n = 10and n = 12 P = 005) (Fig 4A) These data suggest aprogressive loss of expression from the injected vectorDNA in good agreement with previously reported tran-sient expression of silencing vectors introduced byhydrodynamic gene transfer [24]

In addition we performed an intraperitoneal glucosetolerance test 48 h after injection of pSHAG expressionvector At the time of initiation of the experiment thefasting glucose concentration in treated animals (pSHAG-664) was about 40 lower than in controls (pSHAG-Ff)(111 F 30 vs 272 F 47 mgdl P b 001 n = 5) Diabeticanimals treated with pSHAG-664 showed a normalizedglucose tolerance compared to healthy mice (Fig 4B)

PEPCK-C protein content in the liver as analyzedusing Western blot was also transiently silenced corre-lating with significant enzyme activity changes (approx-imately 20 decrease) demonstrated 72 h after injection(97 F 11 vs 76 F 06 mUmg protein n = 9 and n = 7 P b

005) (Fig 5)The concentrations of TAG and free fatty acids (FFA) in

serum were significantly lower in PEPCK-silenced animals48 h after injection This reduction was also transient weobserved no differences at 72 h postinjection Accom-panying the decrease in serum FFA we observed anonsignificant increase in serum h-hydroxybutyrate(h-HBA) (Table 1) Liver glycogen content was dramati-cally decreased at 48 h whereas we observed a lesspronounced reduction 72 h after treatment In additionwe observed a significant increase in the activity oflactate dehydrogenase (LDH) 72 h after injection

To gain insight into the regulatory mechanismsinvolved in altered lipid metabolism in the livers oftreated animals we performed specific immunoblotting

MOLECULAR THERAPY Vol 13 No 2 February 2006404Copyright C The American Society of Gene Therap

s

f

against key regulatory proteins involved in lipogenesis(SREBP1c and FAS) and fatty acid h-oxidation (ACC-P) inwhole liver extracts (Fig 5) A very profound down-regulation of SREBP1c precursor (p125) and mature (p68)forms paralleled PEPCK silencing whereas we found nosignificant changes in ACC-P or FAS Densitometricanalysis of the blots demonstrated an 82 and 38reduction in p125 and p68 SREBP1c forms respectively

Green Fluorescent Protein (GFP) Expression andPEPCK-C Immunohistochemistry afterHydrodynamic Gene TransferTo assess whether the percentage of hepatocytes trans-fected using the hydrodynamic procedure correlates withsilencing efficiency of endogenous PEPCK-C we injected

y

FIG 5 PEPCK-C silencing in the liver of diabetic mice Mice received an

intravenous injection of pSHAG-Ff or pSHAG-664 (100 Ag) Liver extracts

were prepared from animals at 48 and 72 h after injection as described

under Materials and Methods PEPCK content was analyzed by Western blot

and enzymatic activity In addition the level of a number of key proteins

involved in the regulation of energy metabolism was analyzed (A)

Representative blots from independent experiments are shown (B) PEPCK

specific activity 72 h after the injection is shown Values are the means F SE

(P b 005)

TA

BLE

1

Meta

bolic

pro

file

aft

er

part

ialliv

er

PEPC

K-C

sile

nci

ng

ind

iab

eti

cm

ice

Blo

od

Seru

mLi

ver

Glu

cose

(mg

dl)

TA

G

(mg

dl)

h-H

BA

(mm

ol

L)

FFA

(mEq

L)

Lact

ate

(mg

dl)

Insu

lin

(Ag

L)

GPT

(UL

)

TA

G

(mg

g)

LDH

(mU

mg

)

Gly

(mM

g)

48

hp

SH

AG

-Ff

(meanF

SE)

364F

33

102F

16

02

5F

00

814

4F

01

1736

F79

01

1F

00

4112F

16

127

F05

nd

439

F52

PSH

AG

-664

(meanF

SE)

218F

26

65F

11

03

8F

01

408

9F

01

0

634

F60

01

3F

00

2142F

21

131

F03

nd

127

F70

72

hp

SH

AG

-Ff

(meanF

SE)

476F

35

838

F108

01

9F

00

511

F02

413

F64

nd

369

F64

133

F07

101

F00

499

F36

PSH

AG

-664

(meanF

SE)

350F

68

(P=

00

5)

894

F141

02

4F

01

113

F04

442

F99

nd

470

F89

112

F06

138

F00

305

F80

Str

ep

tozo

toci

n-in

duce

dd

iab

etic

mic

ew

ere

inje

cted

with

100Ag

ofp

SH

AG

-Ffor

pSH

AG

-664Li

ver

an

dse

rum

meta

bolit

em

easu

rem

en

tsw

ere

perf

orm

ed

at

diffe

ren

tti

mes

(48

an

d72

h)

as

desc

rib

ed

un

der

Mate

rials

an

dm

eth

od

sD

ata

are

mean

sF

SE

of

7ndash12

an

imals

exce

pt

for

blo

od

glu

cose

(n=

26

pSH

AG

-Ff

an

dn

=25

pSH

AG

-664)

nd

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dete

rmin

ed

Pb

00

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Pb

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1

Pb

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01


diabetic mice with various amounts of pEGFP Weassessed hepatic distribution of GFP and PEPCK-C pro-teins using direct (GFP) and indirect (PEPCK immunohis-tochemistry) fluorescence under confocal microscopy 48h after the injection Compared with uninjected animalsGFP expression was easily detected in liver sections andthe amount of GFP-positive cells increased with the doseof DNA injected (Figs 6A and 6B) More intense PEPCK-Cimmunostaining was localized in the periportal zonewhich is characteristic of this gene Strikingly GFP-positive hepatocytes were localized in the lower part ofthe gradient of PEPCK-C immunoreactivity (perivenousand intermediate zone) with a broader distributionapparent when a higher dose of plasmid was injected(Figs 6C and 6D) These data are consistent with gene

MOLECULAR THERAPY Vol 13 No 2 February 2006 405Copyright C The American Society of Gene Therapy

FIG 6 Zonal expression in the liver after hydrodynamic gene delivery

promotes partial PEPCK-C silencing throughout the liver parenchyma Mice

were injected with either (A to D) 10 or 20 Ag of pEGFP or (E and F) 100 Ag o

pSHAG using the hydrodynamics procedure Direct visualization of GFP

(green) and indirect immunodetection of PEPCK (red) on fixed liver section

were analyzed using confocal microscopy (A and B) correspond to

representative fields of hepatic GFP distribution after hydrodynamic gene

transfer at either 10 or 20 Ag dose respectively Colocalization of GFP and

PEPCK signals at the indicated doses (C 10 Ag and D 20 Ag) in fixed live

sections is also shown Representative PEPCK immunostaining after trans

fection of 100 Ag pSHAG-Ff (E) or pSHAG-664 (F) is shown Origina

magnification 200


406

f

s

r

-

l

transfer to an area corresponding to the perivenous andintermediate hepatic acinus regions and are in agreementwith the metabolic zonation of the liver [25] From theseresults one might infer that the relative distribution in theliver lobule of hepatic PEPCK-C and transgene expressionusing hydrodynamic gene transfer might provide anexplanation for the partial silencing of hepatic PEPCK-Creported here Nevertheless it is important to note thatafter pSHAG-664 transfection (100 Aganimal) PEPCK-Cimmunostaining is reduced globally (Figs 6E and 6F) thatis the periportalndashperivenous intensity gradient is partiallylost suggesting that PEPCK-C is being knocked down notonly in the perivenous and intermediate zone but also inthe periportal area

Nonspecific Gene Silencing via PKRInterferonPathwaySystemic andor nonspecific suppression of transcriptiondue to dsRNA-induced PKRinterferon stress response hasbeen previously demonstrated using specific siRNAsequences and RNAi vectors [2627] To discard theinduction of this pathway in treated animals we inves-tigated the level of activation of downstream targets ofPKR ie increased phosphorylation of the eukaryoticinitiation factor 2a (eIF2a) in liver extracts [26] and theproduction of IL-12 [28]

We used poly(dIdC) a well-known dsRNA analogueas a positive control for PKR-dependent stimulation ofeIF2a phosphorylation in the liver We observed thatpoly(dIdC) injection induced 3- to 26-fold higher levelsof phosphorylated eIF2a compared to pSHAG treatmentMoreover treatments with shRNA vectors did notincrease the level of eIF2a phosphorylation comparedwith control saline-injected animals (Fig 7A) eIF2aactivation (ratio of phosphorylated over total protein)was comparable among the various pSHAG-injected(both Ff and 664 groups) and control (saline-injected)livers Furthermore the levels of IL-12 in plasma were notaltered by pSHAG gene transfer (data not shown)altogether discarding nonspecific silencing induced by aPKRinterferon-mediated response

Hydrodynamic gene transfer has also been shown toinduce transient liver damage [23] resulting in increasedserum transaminase levels immediately after injectionand a return to near normal levels by 48ndash72 h Howeverthe degree of damage induced by hydrodynamic genetransfer in the liver of STZ-administered mice has notbeen assessed to date Therefore to discard nonspecificeffects due to gene transfer we have performed bothserum transaminase (GPT) measurements and caspase 3activation analysis in whole-cell liver extracts fromtreated animals The levels of GPT were significantlyabove saline ip-injected controls (270 F 24 n = 5) 48 hafter injection and returned to control values by 72 h(Table 1) Importantly we observed no significant differ-ences between transfected groups at either time point Inaddition we investigated long-term deleterious conse-quences on hepatocyte viability using a general apoptosisinduction analysis involving the determination of cas-pase 3 activity in whole-cell liver extracts (Fig 7B) Thesedata demonstrated that gene transfer by hydrodynamicinjection did not induce apoptosis compared to saline ip-injected controls in marked contrast with galactosaminelipopolysaccharide (LPS) induction [29] used as a positivecontrol

DISCUSSION

Pharmacological intervention in diabetes focuses on aseries of targets including h-cell function (sulfonylur-eas) [30] FFA reesterification in adipose tissue (TZD)



FIG 7 PKRinterferon or apoptosis pathways are not activated upon pSHAG

hydrodynamic injection Positive control (C+) for PKRinterferon pathway

activation was obtained from liver extracts of diabetic mice injected ip with 50

Ag of a dsRNA analogue (poly(dIdC)) Negative controls (C) were saline-

injected mice eIF2a and eIF2a-P were detected by Western blot performed

with liver extracts from the various groups (A) Results are presented as the

ratio of the phosphorylated form versus total eIF2a after densitometric analysis

of the blots (n = 5) (B) Caspase 3 activity in liver extracts from healthy mice

following hydrodynamic injection of with 100 Ag of either pSHAG Ff or pSHAG

664 (n = 7) Positive control for hepatic apoptosis induction was obtained

from liver extracts of mice injected ip with 700 mgkg galactosamine and 100

mgkg LPS (n = 3) Negative controls were ip saline-injected mice (n = 3)


insulin sensitivity in the muscle (TZD) [1331] andglucose output in the liver (metformin) [1532] Despiterecognition through extensive investigation of thecritical role that PEPCK exerts in controlling gluconeo-genesis in the liver [1393334] the validation of thisenzyme as a target for liver-specific gene therapy orpharmacological intervention in diabetes has not beenextensively investigated to date Therefore we havedeveloped a therapeutic vector-based RNAi approach invivo to validate liver PEPCK as a target for diabetes genetherapy



Silencing vectors (Fig 1) together with liver-specificgene transfer (achieved using a hydrodynamic-basedprocedure) (Fig 2) provide the model to evaluate theefficacy and metabolic alterations induced by PEPCKinhibition in the liver Initially we confirmed that thesilencing vector was able to down-regulate endogenousPEPCK in healthy mice These data demonstrated anincreased capacity to silence PEPCK-C in fed (Fig 3A)versus fasted animals probably related to increased targetmRNA levels after transcription up-regulation of PEPCK-C induced by fasting [25] Nevertheless even in fastedanimals a significant reduction in PEPCK-C activity andprotein was detected after injection of pSHAG-664although the reduction of PEPCK-C mRNA was signifi-cant only in fed animals (Fig 3) This reduction was notaccompanied by changes in carbohydrate or lipid metab-olites in healthy mice as expected from a partialinhibition of the gene and in contrast to results obtainedby a complete ablation of hepatic PEPCK-C [3]

In diabetic animals treatment with pSHAG-664silencing vector also showed a significant reduction inPEPCK protein and enzyme activity that correlated witha significant reduction of blood glucose levels andimproved glucose tolerance in the absence of insulin orstimulation of insulin release by the treatment (Table 1)Such a large impact in glucose homeostasis after a partialreduction in liver PEPCK-C reinforces the importance ofthis gluconeogenic enzyme in sustaining fasting hyper-glycemia in diabetes [91435] Apart from a clearhypoglycemic effect partial silencing of liver PEPCK-Cdemonstrated several other metabolic consequencesLiver glycogen and serum FFA and TAG were signifi-cantly reduced concomitant with increased liver LDHactivity and a tendency toward increased serum h-HBAThe implications of these changes are severalfold First ofall a decrease in glycogen stores in treated animalsmight reflect a diminished glycogen synthesis fromgluconeogenic precursors in agreement with previousobservations describing a liver-specific PEPCK-C knock-out [3] Second lower plasma FFA correlating withincreased serum h-HBA levels suggests a higher rate ofFFA uptake and h-oxidation These data might bepartially explained by the maintenance of O2 consump-tion observed in perfused liver after acute inhibition ofgluconeogenesis [36] an indirect measure of the level ofh-oxidation in gluconeogenic liver Increased FFA oxida-tion could be secondary to an increase in eithermitochondrial or extramitochondrial (peroxisomal) oxi-dation Immunoblotting analysis of key targets of regu-latory pathways involved in energy metabolism such asphosphorylated ACC FAS and SREBP1c have confirmedno significant changes in the level of ACC phosphor-ylation On the other hand SREBP1c both precursor(p125) and mature (p68) forms were very significantlyreduced suggesting an inhibition of its transcription andarguing for an inhibitory effect of elevated concentra-

407


tions of fatty acids on glucose metabolism and lipo-genesis [37] Animals treated with streptozotocin at thedoses utilized in these experiments have remarkably lowlevels of insulin (Table 1) However insulin-independentexpression of SREBP1c in liver extracts of STZ-treatedmice has been previously described [38] and is alsoapparent in the liver of mice shown here (Fig 5)Therefore glucose metabolism is sufficiently active tosustain a certain level of glucose uptake [38] that couldbe diverted from lipogenesis to glycolysis after SREBP1cinhibition in pSHAG 664 injected animals It is thereforetempting to speculate that a yet to be identified energysensing mechanism would induce FFA uptake andactivation resulting in down-regulation of SREBP1c thatin turn would inhibit TAG synthesis and release from theliver In fact the ratio of ACC-PSREBP1c in PEPCK-silenced animals is much higher suggesting an increasedflux from FFA synthesis to oxidation Conversely asignificant reduction in glycemia as observed uponPEPCK partial silencing would down-regulate SREBP1ctranscription indirectly since plasma glucose levels canaffect the levels of SREBP1c directly in the liver ofstreptozotocin-treated mice [38]

In the present report we show transient silencing usinghydrodynamic gene transfer of RNAi-inducing vectors inagreement with the reported duration of gene expressionafter hydrodynamic transfection (72ndash96 h) [24] Howeverwe cannot rule out that the transitory biological effectobserved is due to a feedback regulation responsible forsteady-state maintenance of gluconeogenesis upon stim-ulation of PEPCK transcription

We and others [2239] have shown up to 40hepatocyte transfection using this procedure althoughthe zonal distribution of hepatocyte delivery has notbeen reported to date This issue is of special importancedue to metabolic zonation of PEPCK-C (a decreasinggradient through the portocentral axis) in the liver [40]Nevertheless during fasting or diabetes the absoluteincrease in the concentration of PEPCK mRNA is similarthroughout the liver [25] Our results show extensiveimmunolocalization of PEPCK throughout the entireliver and quantitative compartmentalization of PEPCK-C in periportal hepatocytes whereas GFP expression afterhydrodynamic injection colocalizes to a discrete com-partment corresponding to a more perivenous zone (Fig6) Nevertheless the distribution and levels of transgeneexpression broaden in a dose-dependent manner as seenby the increasing number of GFP-expressing hepatocytesobtained when injecting 20 Ag versus 10 Ag of thereporter plasmid Consequently upon injection of 100Ag of therapeutic plasmids silencing of the endogenousPEPCK-C gene might achieve a broader distribution Infact direct immunohistochemistry for PEPCK-C afterhydrodynamic gene transfer of 100 Ag of pSHAG showedlower immunostaining throughout the liver parenchymawith a partial loss of the portocentral PEPCK-C gradient

408

Taking into consideration the concept of metaboliczonation in the liver and the incomplete colocalizationof the transgene and PEPCK-C one might infer that thecombination of shRNA expression vectors and hydro-dynamic gene transfer would lead to a partial silencing ofthe hepatic PEPCK-C Data presented in this articleconfirm this possibility

This study demonstrates acute effects of a partialreduction of gluconeogenesis in the diabetic liver There-fore it is not clear whether the changes observed could besustained over time in this model However preliminarydata from our group suggest that a longer lastingexpression of pSHAG-664 in diabetic dbdb mice resultsin a significant decrease in glycemia as well as weightgain both in fed and in fasted animals that wasmaintained for as long as 7 days All in all these datasupport the notion that PEPCK-C not only is a gluconeo-genic enzyme but also has an important role in cataple-rosis [5] glyceroneogenesis and the triglyceride cycleflux control [41] and its deregulation is implicated in thedevelopment of obesity and diabetes [35]

MATERIALS AND METHODS

Chemicals Polyethylenimine (PEI) was from Aldrich (PEI 25000 Da Cat

No 40872-7 Steinheim Germany) Media sera and antibiotics were

obtained from Life Technologies Inc (Grand Island NY USA) Poly(dIdC)

was purchased from Amersham Biosciences Corp (Piscataway NJ USA)

and 3-MPA from Toronto Research Chemicals Inc (North York ON

Canada) Galactosamine and LPS from Escherichia coli 0111B4 were from

Sigma (St Louis MO USA)

Plasmids pEGFP was purchased from Clontech (Palo Alto CA USA) and

contains an early cytomegalovirus promoter and an enhanced green

fluorescent protein The firefly (P pyralis) luciferase reporter vector

(pGL3) was obtained from Promega (Madison WI USA) The cDNA for

rat cytosolic PEPCK-C was kindly provided by Dr Richard W Hanson (Case

Western Reserve University Cleveland OH USA) and it was cloned into

the BamHIndashBglII site of a pCAGGS vector (pCPEPCK) which allows high

levels of transgene expression [42] Short-hairpin RNA expression vectors

pSHAG-Ff and pSHAG-1 were a kind gift from Dr Greg Hannon (Cold

Spring Harbor Laboratory Cold Spring Harbor NY USA) pSHAG-1 contains

the U6 promoter region from 265 to +1 a cloning site for short-hairpin

RNAs (BamHIndashBseRI) and a U6 terminator sequence pSHAG-Ff contains the

U6 promoter followed by a short-hairpin RNA directed against P pyralis

luciferase [2043] Two shRNA sets of oligonucleotides targeted against rat and

mouse PEPCK-C mRNA were designed utilizing a published algorithm [2043]

available at httpkatahdincshlorg 9331siRNAhtmlshrna The first

shRNA targeted a sequence that starts at nucleotide 482 from the start site

of translation (5V-CATGCTGGCCACCACATAGGGCGAGTCTGAAGCTTGA-

GACTCGTCCTATGTGGTGGCCGGCGTGTGGTTTTTT-3V and 5V-GAT-

CAAAAAACGGTGAGCCATACTCAGCCAATGCGCCAGATCAAGCTT-

CACCTGGCGCACTGGCTGAGCATGGCCCACG-3V) The second targeted a

sequence that starts at nucleotide 664 from the start site of translation

(5V-AGGAGATGATCTCTCTGCGGTCCGGGAGAAGCTTGTTCCGGATCG-

CAGGGAGATTATCTCCTTCGGTTTTTT-3V and 5V-GATCAAAAAACCGAAG-

GAGATAATCTCCCTGCGATCCGGAACAAGCTTCTCCTGGACCGCAGA-

GAGATCATCTCCTTCG-3V) Each pair of primers was annealed and cloned

into BamHIndashBseRI of pSHAG-1 The plasmids obtained were named pSHAG-

482 and pSHAG-664 respectively

Plasmid DNA was prepared using Endo-Free (Sigma) or Machereyndash

Nagel (Dqren Germany) Maxi Prep kit and contained no detectable

bacterial genomic DNA or RNA contamination by DNA gel electro-




phoresis Plasmid DNA preparations had less than 20 open circular or

linear DNA

Cell culture For in vitro assays the human hepatoma cell line Huh-7 was

maintained in DMEM supplemented with 5 mM glutamine 100 unitsml

penicillin 01 mgml streptomycin and 10 fetal bovine serum Cells

were transfected at 30ndash50 confluence using PEI in 10-cm diameter

plates

Animal care and treatment Male ICR (CD1) mice purchased from Harlan

Interfarma IBERICA SL (Spain) were maintained under a constant 12-h

lightndashdark cycle and fed a standard rodent chow and water ad libitum All

animal protocols were approved by the Ethics Committee at the

University of Barcelona

Mice weighing 22ndash25 g were made diabetic with a single ip injection

of 200 mgkg streptozotocin in 100 mM citriccitrate buffer pH 45 One

week later glycemia was assessed after a 6-h fast Only those mice that

had concentrations of blood glucose over 400 mgdl were used in this

study

Hydrodynamic gene transfer was as described by Liu et al [22] Only 5

of 35 animals injected with shRNA-664 did not respond to hydrodynamic

gene delivery in terms of decreased postinjection glycemia probably due

to the variability intrinsic to this procedure [22] and to noted problems

during injection Therefore only those animals that responded to the

injection were subsequently analyzed

3-MPA was injected into diabetic animals as described elsewhere [44]

Briefly 3-MPA was administered in a 1 (wv) starch suspension to 2-h

fasted mice An initial dose of 100 mgkg followed 3 h later by a second

dose of 25 mgkg was administered by intraperitoneal injection Blood

glucose was analyzed 5 h later

GalactosamineLPS has been shown to produce extensive hepatocel-

lular apoptosis in mice [45] and was used as a positive control Control

mice (20ndash25 g) were injected ip with 700 mgkg galactosamine and 100

mgkg LPS in 200 Al of saline Negative control animals were injected with

an equivalent volume of saline Animals were killed 6 h after

Animals were killed after ketaminendashxylazine anesthesia or CO2

inhalation and liver and kidney were dissected and snap frozen in liquid

nitrogen Tissues were stored at 808C until analysis Blood was taken by

heart puncture and serum was obtained by centrifugation at 2500 rpm at

48C for 15 min

Confocal microscopy Four percent buffered paraformaldehyde-fixed

tissue was cut into 50-Am sections using a Leica VT M1000 slicing blade

microtome GFP was detected in sections using a spectral confocal

microscope (Leica TCS-SL) PEPCK-C was immunostained using indirect

immunofluorescence with a sheep anti-PEPCK-C primary antibody

(kindly provided by Dr Daryl Granner Vanderbilt University) at a

11000 dilution followed by a donkey (1200 dilution) anti-sheep anti-

body conjugated to Alexa Fluor 546 (Molecular Probes Europe BV

Leiden The Netherlands)

Enzyme activity assays Liver extracts were obtained using a Polytron in

appropriate lysis buffer PEPCK activity was measured spectrophotometri-

cally by coupling the conversion of phosphoenolpyruvate to oxaloacetate

by PEPCK to the subsequent conversion to malate by malate dehydrogen-

ase as described previously [46] Activity was expressed as mUnitsmg

protein in the supernatant Caspase 3 activity assay was performed using a

fluorometric assay essentially as described [47]

Western blot Western blot was performed with 50 Ag of cell protein

extract from cultured cells or 20 Ag from liver or kidney extracts in RIPA

buffer Proteins were separated in 10 SDSndashPAGE and transferred to an

Immobilon membrane (Millipore Corp Bedford MA USA)

Sheep anti-PEPCK-C antiserum was used at a 120000 dilution

Antibodies against eIF2a-P Ser51 (Oncogene Research Products San

Diego CA USA) and ACC-P (Ser79) (Upstate Biotechnology Lake

Placid NY USA) were used at a 11000 dilution FAS antibody (Santa

Cruz Biotechnology Santa Cruz CA USA) was used at 1500 All

membranes were normalized using monoclonal anti-a-tubulin (14000)

(Sigma) or anti-MAPK (12000) antibodies (New England Biolabs Inc

Hitchin UK)



RNA isolation and Northern blot Total RNA was isolated using Ultraspec

RNA (Biotecx Houston TX USA) The PEPCK-C probe used was a BamHIndash

BglII fragment (15 kb) from the rat cDNA Loading differences were

normalized using a GAPDH-specific probe

Analytical procedures Blood glucose levels were measured using a

Glucocard Memory 2 apparatus (A Menarini Inc Florence Italy) Blood

was collected from the tail tip Unless indicated otherwise animals were

fasted for 8 h prior to blood and specimen collection

The concentration of FFA in serum was measured using a NEFA C kit

(Wako Pure Chemical Industries Osaka Japan) Serum triglycerides

lactate and h-hydroxybutyrate were quantified using a colorimetric kits

(Sigma) Some measurements of metabolites were performed by the

Clinical Biochemistry Service from the Veterinary Hospital in Bellaterra

Spain Serum insulin and IL-12 were determined using mouse insulin

(healthy animals) and ultrasensitive mouse insulin (diabetic animals)

ELISAs (Mercodia AB Uppsala Sweden) and a mouse IL-12 ELISA (Bender

MedSystems San Bruno CA USA) respectively

To determine hepatic glycogen content livers were homogenized in

400 mM aceticacetate buffer pH 48 and boiled for 15 min The

homogenates were centrifuged for 5 min at 6000g The supernatant was

digested with 1 unit of a-amiloglucosidase from Leuconostoc (Sigma) and

the glucose produced was quantified using a glucose oxidase kit (Sigma)

The hepatic TAG content was quantified using a TAG kit (Sigma) from 3 M

KOH 65 ethanol extracts based on the method of Salmon and Flatt for

liver saponification

LDH activity was measured from liver extracts (50 mM Tris 01

Triton X-100 25 mM DTT) using a LDH kit (Roche Indianapolis IN

USA)

Transaminase (GPT) levels in serum were quantified using a Reflotron

system (Roche)

Statistics Results are expressed as the means F standard error Statistical

analysis was always performed by one-way analysis of variance and

StudentTs t test A P b 005 was considered significant

ACKNOWLEDGMENTS

The authors are indebted to Dr Richard W Hanson for helpful discussions and

reviewing the manuscript A G Gomez-Valades and A Vidal-Alabro were

supported by fellowships awarded from FPU the Ministerio de Educacion y

Ciencia (Spain) and FI DURSI Generalitat de Catalunya respectively This

study was supported by grants from the Ministerio de Ciencia y Tecnologıa

(Spain) (SAF02-02964 and BFI03-02539) and the Fundacio Marato de TV3

(031633) We also thank the Research Support Services from the Biology Unit of

Bellvitge University of Barcelona for their technical assistance

RECEIVED FOR PUBLICATION MARCH 10 2005 REVISED AUGUST 31 2005

ACCEPTED AUGUST 31 2005

REFERENCES1 Landau B R Wahren J Chandramouli V Schumann W C Ekberg K and Kalhan

S C (1996) Contributions of gluconeogenesis to glucose production in the fasted

state J Clin Invest 98 378 ndash 385

2 Katz J and Tayek J A (1998) Gluconeogenesis and the Cori cycle in 12- 20- and

40-h-fasted humans Am J Physiol 275 E537 ndash E542

3 She P Shiota M Shelton K D Chalkley R Postic C and Magnuson M A

(2000) Phosphoenolpyruvate carboxykinase is necessary for the integration of hepatic

energy metabolism Mol Cell Biol 20 6508 ndash 6517

4 Curthoys N P and Gstraunthaler G (2001) Mechanism of increased renal gene

expression during metabolic acidosis Am J Physiol Renal Physiol 281 F381 ndash F390

5 Owen O E Kalhan S C and Hanson R W (2002) The key role of anaplerosis and

cataplerosis for citric acid cycle function J Biol Chem 277 30409 ndash 30412

6 Hanson R W and Reshef L (2003) Glyceroneogenesis revisited Biochimie 85

1199 ndash 1205

7 Croset M Rajas F Zitoun C Hurot J M Montano S and Mithieux G

(2001) Rat small intestine is an insulin-sensitive gluconeogenic organ Diabetes 50

740 ndash 746

8 DeFronzo R A and Ferrannini E (1991) Insulin resistance a multifaceted syndrome

responsible for NIDDM obesity hypertension dyslipidemia and atherosclerotic

cardiovascular disease Diabetes Care 14 173 ndash 194

9 Consoli A Nurjhan N Capani F and Gerich J (1989) Predominant role of

409


gluconeogenesis in increased hepatic glucose production in NIDDM Diabetes 38

550 ndash 557

10 Hanson R W and Reshef L (1997) Regulation of phosphoenolpyruvate carbo-

xykinase (GTP) gene expression Annu Rev Biochem 66 581 ndash 611

11 Bailey C J (1992) Biguanides and NIDDM Diabetes Care 15 755 ndash 772

12 Goldstein B J (2000) Rosiglitazone Int J Clin Pract 54 333 ndash 337

13 Schoonjans K and Auwerx J (2000) Thiazolidinediones an update Lancet 355

1008 ndash 1010

14 Hundal R S et al (2000) Mechanism by which metformin reduces glucose

production in type 2 diabetes Diabetes 49 2063 ndash 2069

15 Zhou G et al (2001) Role of AMP-activated protein kinase in mechanism of

metformin action J Clin Invest 108 1167 ndash 1174

16 Lefebvre A M et al (1998) Activation of the peroxisome proliferator-activated

receptor gamma promotes the development of colon tumors in C57BL6J-APCMin+

mice Nat Med 4 1053 ndash 1057

17 Sarraf P et al (1998) Differentiation and reversal of malignant changes in colon

cancer through PPARgamma Nat Med 4 1046 ndash 1052

18 Saez E et al (1998) Activators of the nuclear receptor PPARgamma enhance colon

polyp formation Nat Med 4 1058 ndash 1061

19 Stumvoll M Nurjhan N Perriello G Dailey G and Gerich J E (1995) Metabolic

effects of metformin in non-insulin-dependent diabetes mellitus N Engl J Med 333

550 ndash 554

20 Paddison P J Caudy A A Bernstein E Hannon G J and Conklin D S (2002)

Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

Genes Dev 16 948 ndash 958

21 McCaffrey A P Meuse L Pham T T Conklin D S Hannon G J and Kay M A

(2002) RNA interference in adult mice Nature 418 38 ndash 39

22 Liu F Song Y and Liu D (1999) Hydrodynamics-based transfection in animals by

systemic administration of plasmid DNA Gene Ther 6 1258 ndash 1266

23 Zhang G Budker V and Wolff J A (1999) High levels of foreign gene expression in

hepatocytes after tail vein injections of naked plasmid DNA Hum Gene Ther 10

1735 ndash 1737

24 Kobayashi N et al (2004) Vector-based in vivo RNA interference dose- and

time-dependent suppression of transgene expression J Pharmacol Exp Ther 308

688 ndash 693

25 Ruijter J M Gieling R G Markman M M Hagoort J and Lamers W H (2004)

Stereological measurement of porto-central gradients in gene expression in mouse

liver Hepatology 39 343 ndash 352

26 Sledz C A Holko M de Veer M J Silverman R H and Williams B R

(2003) Activation of the interferon system by short-interfering RNAs Nat Cell Biol

5 834 ndash 839

27 Bridge A J Pebernard S Ducraux A Nicoulaz A L and Iggo R (2003)

Induction of an interferon response by RNAi vectors in mammalian cells Nat Genet

34 263 ndash 264

28 Pruett S B Fan R and Zheng Q (2003) Acute ethanol administration profoundly

alters poly IC-induced cytokine expression in mice by a mechanism that is not

dependent on corticosterone Life Sci 72 1825 ndash 1839

29 Martin E J and Forkert P G (2004) Evidence that 11-dichloroethylene induces

apoptotic cell death in murine liver J Pharmacol Exp Ther 310 33 ndash 42

410

30 Korytkowski M T (2004) Sulfonylurea treatment of type 2 diabetes mellitus focus on

glimepiride Pharmacotherapy 24 606 ndash 620

31 Miyazaki Y et al (2001) Effect of rosiglitazone on glucose and non-esterified fatty

acid metabolism in Type II diabetic patients Diabetologia 44 2210 ndash 2219

32 Kirpichnikov D McFarlane S I and Sowers J R (2002) Metformin an update Ann

Intern Med 137 25 ndash 33

33 Valera A Pujol A Pelegrin M and Bosch F (1994) Transgenic mice overexpressing

phosphoenolpyruvate carboxykinase develop non-insulin-dependent diabetes mellitus

Proc Natl Acad Sci USA 91 9151 ndash 9154

34 Sun Y et al (2002) Phosphoenolpyruvate carboxykinase overexpression selectively

attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice J Biol

Chem 277 23301 ndash 23307

35 Beale E G Hammer R E Antoine B and Forest C (2004) Disregulated

glyceroneogenesis PCK1 as a candidate diabetes and obesity gene Trends Endocrinol

Metab 15 129 ndash 135

36 Jomain-Baum M Schramm V L and Hanson R W (1976) Mechanism of

3-mercaptopicolinic acid inhibition of hepatic phosphoenolpyruvate carboxykinase

(GTP) J Biol Chem 251 37 ndash 44

37 Kawaguchi T Osatomi K Yamashita H Kabashima T and Uyeda K (2002)

Mechanism for fatty acid sparing effect on glucose-induced transcription regulation

of carbohydrate-responsive element-binding protein by AMP-activated protein kinase

J Biol Chem 277 3829 ndash 3835

38 Matsuzaka T et al (2004) Insulin-independent induction of sterol regulatory

element-binding protein-1c expression in the livers of streptozotocin-treated mice

Diabetes 53 560 ndash 569

39 Zeini M et al (2005) Assessment of a dual regulatory role for NO in liver

regeneration after partial hepatectomy protection against apoptosis and retardation

of hepatocyte proliferation FASEB J 19 995 ndash 997

40 Jungermann K and Kietzmann T (1996) Zonation of parenchymal and non-

parenchymal metabolism in liver Annu Rev Nutr 16 179 ndash 203

41 Reshef L et al (2003) Glyceroneogenesis and the triglyceridefatty acid cycle J Biol

Chem 278 30413 ndash 30416

42 Niwa H Yamamura K and Miyazaki J (1991) Efficient selection for high-expression

transfectants with a novel eukaryotic vector Gene 108 193 ndash 199

43 Paddison P J Caudy A A and Hannon G J (2002) Stable suppression of gene

expression by RNAi in mammalian cells Proc Natl Acad Sci USA 99 1443 ndash 1448

44 Simon C Herling A W Preibisch G and Burger H J (2000) Upregulation of

hepatic glucose 6-phosphatase gene expression in rats treated with an inhibitor of

glucose-6-phosphate translocase Arch Biochem Biophys 373 418 ndash 428

45 Gujral J S Knight T R Farhood A Bajt M L and Jaeschke H (2002) Mode of cell

death after acetaminophen overdose in mice apoptosis or oncotic necrosis Toxicol

Sci 67 322 ndash 328

46 Petrescu I Bojan O Saied M Barzu O Schmidt F and Kuhnle H F (1979)

Determination of phosphoenolpyruvate carboxykinase activity with deoxyguanosine

5V-diphosphate as nucleotide substrate Anal Biochem 96 279 ndash 281

47 Herrera B et al (2001) Activation of caspases occurs downstream from radical

oxygen species production Bcl-xL down-regulation and early cytochrome C release in

apoptosis induced by transforming growth factor beta in rat fetal hepatocytes

Hepatology 34 548 ndash 556




drugs for the treatment of NIDDM biguanides (egmetformin) and thiazolidinediones (eg rosiglitazone)[1112] induces an increase in insulin sensitivity inperipheral tissues and the liver The antihyperglycemicaction of thiazolidinediones is a direct result of activationof the nuclear receptor peroxisome proliferator-activatedreceptor-g (PPAR-g) [13] whereas metformin has beenlinked directly to inhibition of liver gluconeogenesis[1415] However these drugs activate upstream regula-tors (eg PPAR-g or AMP-K) that are involved in a broadrange of physiological functions [1316ndash19]

Because of the theoretical potential of blocking HGOby means of a specific inhibition of a key enzymatic stepin the pathway we have aimed at down-regulatinghepatic PEPCK-C To accomplish this goal we havedeveloped specific short-hairpin RNA (shRNA) reagentsto knock down PEPCK-C and achieved organ specificityusing liver-directed transfection Silencing of endogenousgenes mediated by RNA interference is a conservedbiological process that can be used to induce experimen-tal sequence-specific gene silencing with either smallinterference duplexes (siRNA) or shRNA expression vec-tors This kind of vector drives shRNA expression from ahuman U6 promoter and their efficacy has been demon-strated both in mammalian cells [20] and in mice afterhepatic hydrodynamic gene delivery [21]

We report here an efficient reduction in the levels ofmRNA protein and enzyme activity of hepatic PEPCK-Cin animals treated with specific shRNAs As a resultplasma glucose of diabetic mice was reduced up to 40with no associated lactic acidosis or hepatic steatosis Wedemonstrate that the inhibition of PEPCK-C gene expres-

FIG 1 pSHAG-664 induces PEPCK gene silencing in vitro

(A) pCPEPCK-C (225 Ag) was cotransfected with different

molar ratios of pSHAG-664 (10 (striped) 16 (white) and

112 (gray)) As a control for nonspecific gene silencing

pSHAG-Ff was cotransfected at a 124 ratio (black) pBlue-

script was used as stuffer As an internal transfection

control 1 Ag of pGL3 was added to all plates PEPCK-C

activity was measured 48 h after transfection and normal-

ized for luciferase activity Data are expressed as relative

PEPCK activity (PEPCK activityluciferase activity) (B)

Silencing was confirmed at the protein level using Western

blot A representative blot is shown

402

sion using shRNA also results in alterations in lipidmetabolism exemplified by reduced serum NEFA andtriglycerides (TAG) in parallel to a clear reduction of bothprecursor and mature forms of the key lipogenic tran-scription factor SREBP1c Hence liver-specific PEPCKgene silencing provides a novel therapeutic approachfor the treatment of diabetes

RESULTS

PEPCK-C Gene Silencing by Means of shRNAExpression in VitroWe generated two mammalian expression cassettes con-taining the human U6 polymerase III gene promoterdriving expression of shRNAs (pSHAG vector) comple-mentary to both mouse and rat PEPCK-C transcripts Bothsequences were designed with homology to a 29-bp stretchfrom nucleotides 482 and 664 of the cDNA (pSHAG-482and pSHAG-664 respectively) We used an equivalentcassette expressing a shRNA specific for a Photinus pyralisluciferase sequence (pSHAG-Ff) as a control

We confirmed efficient silencing of the gene forPEPCK-C in Huh-7 cells using cotransfection experimentswith a constant amount of PEPCK-C expression vector(pCPEPCK-C) and different molar ratios (16 and 112) ofthe silencing vectors (664 482 or Ff) Cotransfection ofpSHAG-Ff whatever the ratio used never altered PEPCK-C activity or protein levels In contrast as shown in Fig1 PEPCK-C activity and protein levels significantlydecreased when pSHAG-664 and pCPEPCK-C plasmidswere cotransfected The suppression of PEPCK-C wasclose to 80 as determined by enzyme activity and


















re shown (n = 4)








F

c

a

p

p

P

N

w

fa

P

a

a



403




















s

f



y









(P b 005)

TA

BLE

1

Meta

bolic

pro

file

aft

er

part

ialliv

er

PEPC

K-C

sile

nci

ng

ind

iab

eti

cm

ice

Blo

od

Seru

mLi

ver

Glu

cose

(mg

dl)

TA

G

(mg

dl)

h-H

BA

(mm

ol

L)

FFA

(mEq

L)

Lact

ate

(mg

dl)

Insu

lin

(Ag

L)

GPT

(UL

)

TA

G

(mg

g)

LDH

(mU

mg

)

Gly

(mM

g)

48

hp

SH

AG

-Ff

(meanF

SE)

364F

33

102F

16

02

5F

00

814

4F

01

1736

F79

01

1F

00

4112F

16

127

F05

nd

439

F52

PSH

AG

-664

(meanF

SE)

218F

26

65F

11

03

8F

01

408

9F

01

0

634

F60

01

3F

00

2142F

21

131

F03

nd

127

F70

72

hp

SH

AG

-Ff

(meanF

SE)

476F

35

838

F108

01

9F

00

511

F02

413

F64

nd

369

F64

133

F07

101

F00

499

F36

PSH

AG

-664

(meanF

SE)

350F

68

(P=

00

5)

894

F141

02

4F

01

113

F04

442

F99

nd

470

F89

112

F06

138

F00

305

F80

Str

ep

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n-in

duce

dd

iab

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mic

ew

ere

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cted

with

100Ag

ofp

SH

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-Ffor

pSH

AG

-664Li

ver

an

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meta

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easu

rem

en

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ere

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(48

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desc

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der

Mate

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an

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eth

od

sD

ata

are

mean

sF

SE

of

7ndash12

an

imals

exce

pt

for

blo

od

glu

cose

(n=

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pSH

AG

-Ff

an

dn

=25

pSH

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-664)

nd

n

ot

dete

rmin

ed

Pb

00

5

Pb

00

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Pb

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01















magnification 200


406

f

s

r

-

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DISCUSSION






















407





408














































linear DNA





plates










study





















48C for 15 min




























Hitchin UK)






























USA)


system (Roche)




ACKNOWLEDGMENTS
























1199 ndash 1205



740 ndash 746





409



550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416



































re shown (n = 4)








F

c

a

p

p

P

N

w

fa

P

a

a



403




















s

f



y









(P b 005)

TA

BLE

1

Meta

bolic

pro

file

aft

er

part

ialliv

er

PEPC

K-C

sile

nci

ng

ind

iab

eti

cm

ice

Blo

od

Seru

mLi

ver

Glu

cose

(mg

dl)

TA

G

(mg

dl)

h-H

BA

(mm

ol

L)

FFA

(mEq

L)

Lact

ate

(mg

dl)

Insu

lin

(Ag

L)

GPT

(UL

)

TA

G

(mg

g)

LDH

(mU

mg

)

Gly

(mM

g)

48

hp

SH

AG

-Ff

(meanF

SE)

364F

33

102F

16

02

5F

00

814

4F

01

1736

F79

01

1F

00

4112F

16

127

F05

nd

439

F52

PSH

AG

-664

(meanF

SE)

218F

26

65F

11

03

8F

01

408

9F

01

0

634

F60

01

3F

00

2142F

21

131

F03

nd

127

F70

72

hp

SH

AG

-Ff

(meanF

SE)

476F

35

838

F108

01

9F

00

511

F02

413

F64

nd

369

F64

133

F07

101

F00

499

F36

PSH

AG

-664

(meanF

SE)

350F

68

(P=

00

5)

894

F141

02

4F

01

113

F04

442

F99

nd

470

F89

112

F06

138

F00

305

F80

Str

ep

tozo

toci

n-in

duce

dd

iab

etic

mic

ew

ere

inje

cted

with

100Ag

ofp

SH

AG

-Ffor

pSH

AG

-664Li

ver

an

dse

rum

meta

bolit

em

easu

rem

en

tsw

ere

perf

orm

ed

at

diffe

ren

tti

mes

(48

an

d72

h)

as

desc

rib

ed

un

der

Mate

rials

an

dm

eth

od

sD

ata

are

mean

sF

SE

of

7ndash12

an

imals

exce

pt

for

blo

od

glu

cose

(n=

26

pSH

AG

-Ff

an

dn

=25

pSH

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-664)

nd

n

ot

dete

rmin

ed

Pb

00

5

Pb

00

1

Pb

00

01















magnification 200


406

f

s

r

-

l





DISCUSSION






















407





408














































linear DNA





plates










study





















48C for 15 min




























Hitchin UK)






























USA)


system (Roche)




ACKNOWLEDGMENTS
























1199 ndash 1205



740 ndash 746





409



550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416







































s

f



y









(P b 005)

TA

BLE

1

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iab

eti

cm

ice

Blo

od

Seru

mLi

ver

Glu

cose

(mg

dl)

TA

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(mg

dl)

h-H

BA

(mm

ol

L)

FFA

(mEq

L)

Lact

ate

(mg

dl)

Insu

lin

(Ag

L)

GPT

(UL

)

TA

G

(mg

g)

LDH

(mU

mg

)

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(mM

g)

48

hp

SH

AG

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(meanF

SE)

364F

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102F

16

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4F

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1736

F79

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4112F

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127

F05

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F52

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AG

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(meanF

SE)

218F

26

65F

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8F

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408

9F

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634

F60

01

3F

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2142F

21

131

F03

nd

127

F70

72

hp

SH

AG

-Ff

(meanF

SE)

476F

35

838

F108

01

9F

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511

F02

413

F64

nd

369

F64

133

F07

101

F00

499

F36

PSH

AG

-664

(meanF

SE)

350F

68

(P=

00

5)

894

F141

02

4F

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113

F04

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F99

nd

470

F89

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F06

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305

F80

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ep

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toci

n-in

duce

dd

iab

etic

mic

ew

ere

inje

cted

with

100Ag

ofp

SH

AG

-Ffor

pSH

AG

-664Li

ver

an

dse

rum

meta

bolit

em

easu

rem

en

tsw

ere

perf

orm

ed

at

diffe

ren

tti

mes

(48

an

d72

h)

as

desc

rib

ed

un

der

Mate

rials

an

dm

eth

od

sD

ata

are

mean

sF

SE

of

7ndash12

an

imals

exce

pt

for

blo

od

glu

cose

(n=

26

pSH

AG

-Ff

an

dn

=25

pSH

AG

-664)

nd

n

ot

dete

rmin

ed

Pb

00

5

Pb

00

1

Pb

00

01















magnification 200


406

f

s

r

-

l





DISCUSSION






















407





408














































linear DNA





plates










study





















48C for 15 min




























Hitchin UK)






























USA)


system (Roche)




ACKNOWLEDGMENTS
























1199 ndash 1205



740 ndash 746





409



550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416




























(P b 005)

TA

BLE

1

Meta

bolic

pro

file

aft

er

part

ialliv

er

PEPC

K-C

sile

nci

ng

ind

iab

eti

cm

ice

Blo

od

Seru

mLi

ver

Glu

cose

(mg

dl)

TA

G

(mg

dl)

h-H

BA

(mm

ol

L)

FFA

(mEq

L)

Lact

ate

(mg

dl)

Insu

lin

(Ag

L)

GPT

(UL

)

TA

G

(mg

g)

LDH

(mU

mg

)

Gly

(mM

g)

48

hp

SH

AG

-Ff

(meanF

SE)

364F

33

102F

16

02

5F

00

814

4F

01

1736

F79

01

1F

00

4112F

16

127

F05

nd

439

F52

PSH

AG

-664

(meanF

SE)

218F

26

65F

11

03

8F

01

408

9F

01

0

634

F60

01

3F

00

2142F

21

131

F03

nd

127

F70

72

hp

SH

AG

-Ff

(meanF

SE)

476F

35

838

F108

01

9F

00

511

F02

413

F64

nd

369

F64

133

F07

101

F00

499

F36

PSH

AG

-664

(meanF

SE)

350F

68

(P=

00

5)

894

F141

02

4F

01

113

F04

442

F99

nd

470

F89

112

F06

138

F00

305

F80

Str

ep

tozo

toci

n-in

duce

dd

iab

etic

mic

ew

ere

inje

cted

with

100Ag

ofp

SH

AG

-Ffor

pSH

AG

-664Li

ver

an

dse

rum

meta

bolit

em

easu

rem

en

tsw

ere

perf

orm

ed

at

diffe

ren

tti

mes

(48

an

d72

h)

as

desc

rib

ed

un

der

Mate

rials

an

dm

eth

od

sD

ata

are

mean

sF

SE

of

7ndash12

an

imals

exce

pt

for

blo

od

glu

cose

(n=

26

pSH

AG

-Ff

an

dn

=25

pSH

AG

-664)

nd

n

ot

dete

rmin

ed

Pb

00

5

Pb

00

1

Pb

00

01















magnification 200


406

f

s

r

-

l





DISCUSSION






















407





408














































linear DNA





plates










study





















48C for 15 min




























Hitchin UK)






























USA)


system (Roche)




ACKNOWLEDGMENTS
























1199 ndash 1205



740 ndash 746





409



550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416































magnification 200


406

f

s

r

-

l





DISCUSSION






















407





408














































linear DNA





plates










study





















48C for 15 min




























Hitchin UK)






























USA)


system (Roche)




ACKNOWLEDGMENTS
























1199 ndash 1205



740 ndash 746





409



550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416






































407





408














































linear DNA





plates










study





















48C for 15 min




























Hitchin UK)






























USA)


system (Roche)




ACKNOWLEDGMENTS
























1199 ndash 1205



740 ndash 746





409



550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416
























408














































linear DNA





plates










study





















48C for 15 min




























Hitchin UK)






























USA)


system (Roche)




ACKNOWLEDGMENTS
























1199 ndash 1205



740 ndash 746





409



550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416






















linear DNA





plates










study





















48C for 15 min




























Hitchin UK)






























USA)


system (Roche)




ACKNOWLEDGMENTS
























1199 ndash 1205



740 ndash 746





409



550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416






















550 ndash 557






1008 ndash 1010














550 ndash 554










1735 ndash 1737



688 ndash 693






5 834 ndash 839



34 263 ndash 264






410












Chem 277 23301 ndash 23307




















Chem 278 30413 ndash 30416




















overcoming diabetes-induced hyperglycemia through inhibition of hepatic phosphoenolpyruvate...

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