Plasminogen activator inhibitor-1 synthesis in
the human hepatoma cell line Hep G2.
Metformin inhibits the stimulating effect of
insulin.
F Anfosso, … , P Vague, I Juhan-Vague
J Clin Invest. 1993;91(5):2185-2193. https://doi.org/10.1172/JCI116445.
High plasma plasminogen activator inhibitor-1 (PAI-1) activity is associated with insulin resistance and is correlated with hyperinsulinemia. The cellular origin of plasma PAI-1 in insulin resistance is not known. The hepatoma cell line Hep G2 has been shown to synthesize PAI-1 in response to insulin. The aim of this study was to analyze the mediated response of PAI-1 and lipid synthesis in Hep G2 cells after producing an insulin-resistant state by decreasing insulin receptor numbers. The effect of metformin, a dimethyl-substituted biguanide, known to lower plasma insulin and PAI-1 levels in vivo was
concomitantly evaluated. Preincubation by an 18-h exposure of Hep G2 cells to 10(-7) M insulin aimed at reducing the number of insulin receptors, was followed by a subsequent 24-h stimulation with 10(-9) M insulin. The decrease in insulin receptors was accompanied as expected, by a reduction in [14C]acetate incorporation, an index of lipid synthesis, whereas PAI-1 secretion and PAI-1 mRNA expression were enhanced. The addition of metformin did not modify the effect of insulin on insulin receptors or [14C]acetate
incorporation. In contrast, the drug (10(-4) M) inhibited insulin-mediated PAI-1 synthesis. The results indicate that PAI-1 synthesis in presence of insulin is markedly increased in down-regulated cells, and that metformin inhibits this effect by acting at the cellular level. These in vitro data are relevant with those found […]
Research Article
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Plasminogen
Activator Inhibitor-1
Synthesis
in the Human
Hepatoma Cell Line Hep
G2
Metformin InhibitstheStimulating Effect of Insulin
F. Anfosso,N.Chomiki,M.C.Alessi,P.Vague,*and1. Juhan-Vague
Laboratory ofHematology, *DepartmentofDiabetology, Centre Hospitalier Universitaire Timone, 13385 Marseille Cedex 5, France
Abstract
High plasmaplasminogenactivator inhibitor-i (PAI-1) activ-ity is associated with insulin resistance and is correlated with
hyperinsulinemia.Thecellularoriginofplasma PAI-1in
insu-linresistance isnot known.Thehepatoma cell line Hep G2has beenshowntosynthesizePAI-1 inresponsetoinsulin. The aim of this study wastoanalyzethe insulin-mediatedresponseof
PAI-1 and lipid synthesis inHep G2 cells after producingan
insulin-resistant stateby decreasing insulinreceptornumbers.
The effect of metformin, a dimethyl-substituted biguanide,
known to lowerplasma insulin and PAI-I levels in vivowas
concomitantly evaluated. Preincubationby an18-h exposure of
HepG2cells to
1i-7
Minsulin aimedatreducingthenumber of insulin receptors, wasfollowedby asubsequent 24-h stimula-tionwith 10-9Minsulin. Thedecrease ininsulinreceptors wasaccompaniedasexpected, byareduction in
I14Clacetate
incor-poration,anindex oflipidsynthesis,whereasPAI-i secretion andPAI-i mRNAexpressionwereenhanced. The addition of metformindidnotmodify the effect of insulinoninsulin recep-tors orI14C
acetateincorporation.In contrast, thedrug(i0-4M) inhibited insulin-mediatedPAI-1synthesis.The results
in-dicate thatPAI-1synthesisinpresenceof insulin is markedly
increased indown-regulated cells,and thatmetformin inhibits thiseffect by actingatthecellularlevel. Theseinvitrodataare relevantwith those found in vivo ininsulin-resistant patients. Hep G2 cellsmay be asuitablemodel to study PAI-1regulation in response to hyperinsulinemia. (J. Clin. Invest. 1993.
91:2185-2193.)Key words:fibrinolysis*insulin*insulin
resis-tance-metformin-plasminogen activator inhibitor1
Introduction
Plasminogenactivator inhibitor-1(PAI-1)'is themajor
physio-logical inhibitor of tissue-type plasminogen activator(t-PA) ( 1). IncreasedPAIactivityisresponsible for impaired
fibrinoly-sisassociated withthromboticdiseases.ElevatedplasmaPAI- 1
concentrationshave been observed in coronary artery disease
Addressreprintrequests toDr. I.Juhan-Vague,Laboratory of Hematol-ogy,CHUTimone, 13385MarseilleCedex5,France.
Receivedfor publication6July1992 andinrevisedform 3 De-cember 1992.
I. Abbreviations used in thispaper:CM, conditionedmedia;HUVEC, human umbilical vein endothelial cells; PAI-1,plasminogenactivator inhibitor-1.
and were shownto bepredictive ofrecurrentmyocardial
in-farction(2).
Epidemiological studieshaveindicatedthat
hyperinsuline-mia in thefastingstateandafteran oralglucoseload is
asso-ciatedwithanincreasedriskofdevelopingcoronary heart
dis-ease(3-6). Hyperinsulinemiais the
biological
marker of insu-linresistance,amultifaceted syndrome.Itischaracterizedbyadecrease in theability ofinsulin tostimulate in vivoglucose utilization and is associatedwith
hypertension,
androidobe-sity,andanabnormallipid profile includingelevated
triglycer-ide and decreased highdensity lipoproteincholesterol levels(7,
8). We have previously shown that increased plasma PAI-1
levelsarelinkedtohyperinsulinemiaandarefound in insulin-resistantstates(for reviewseereference 9).Significant correla-tionshavebeenestablishedbetweenplasmaPAI- 1 levels, fast-ingplasmainsulin, bodymassindex, android obesity,and hy-pertriglyceridemia. A direct link between PAI- 1 levels and
insulin resistancehas beenestablishedbydecreasing
hyperin-sulinemiainvivo. Diet,weightloss, andphysicaltraining
im-provetheinsulin-resistant state byreducing insulinemiaand
also lowerPAI- 1 levels. Theoral antidiabetic drug,metformin, adimethyl substitutedbiguanide,increasesperipheralglucose uptake, reducesgluconeogenesisand decreases plasma insulin and triglyceride levels (10-13). In addition, metformin
im-provesfibrinolysis in cardiovascular disease( 14). We have also
shown inapreviousstudy that, in obesenondiabeticwomen, recoveryof fibrinolyticactivityafter Metformintreatmentwas
mainly duetodecreasedPAI- 1 levels( 15).
Severalattemptshave been undertakentoexplainthelink between increasedPAI- 1 levelsandthe insulin-resistantstate.
Insulin resistance is associated withseveral celldefects
includ-ing decreased receptor numbers, alterationsininsulin receptor
tyrosinekinaseactivity leadingtoimpaired insulinaction, and
postreceptor defects (16-19). A decrease in insulin receptor numbers may besecondaryto adown-regulationof the recep-torsowingtoinsulinbindingandinternalization and may be
inducedatthecell level by acute or chronicexposureof cells to ahighconcentration of insulin(20, 21 ). InvitroPAI- 1 is
syn-thesized by several celltypesincluding endothelialcells, hepa-tocytes(22), and smooth musclecells(for reviewseereference
23).The humanhepatoma cell lineHep G2 cells have retained
severalpropertiesofhumanhepatocytes(24) and possess func-tional insulin receptors which can be down-regulated in re-sponsetochronicoracuteexposure to highconcentrationsof
insulin (25),thusleadingto insulindesensitization. We have
previously shown that insulin increased PAI- 1 synthesis by Hep G2 cells (26). The results have beenconfirmedinother
studiesusingHepG2 cells(27-29)andfreshhuman hepato-cytes(27).
Themolecular events that mediate
PAl-1
synthesisin re-sponse toinsulinarepoorly understood. Insulin exerts severalmetabolic actionsin Hep G2 cellsincludingadirecteffecton
J.Clin.Invest.
©The AmericanSociety for Clinical Investigation,Inc.
0021-9738/93/05/2185/09 $2.00
triacylglycerolsynthesis (30). In this study we have examined PAI-I synthesis and acetate incorporation into lipids after in-duction of an insulin-resistant state inHepG2cells by acute exposureto high insulin concentrations. The data have
estab-lishedthat down-regulation of insulin receptors leads to a
re-ductionin acetateincorporation and in contrast, an
up-regula-tion of PAI- 1 synthesis. Metformin in the presence of insulin, inhibited theinsulin-mediatedPAI- 1 synthesis but exerted no effect on insulin-mediated acetate incorporation indicating thatthetwo insulin actions are regulated by different
mecha-nisms.
Methods
The human hepatoma cell line Hep G2 was provided by Dr. B. Knowles, Wistar Institute ofAnatomy and Biology (Philadelphia,
PA). Fetalcalfserum, cell culture reagents, cell culture medium, and molecular biology reagentswerepurchased fromGibcoBRLLife
Bio-technologies (Cergy Pontoise, France). BSA (cell culture reagent
tested), endothelialcellgrowth supplement,andchemicalswere ob-tained fromSigma ChemicalCo.(St. Louis, MO).Plastic tissue culture flasks andplateswereobtained from Falcon Labware (Becton, Dickin-son&Co., Meylan, France).Humaninsulin(solutionforinjection,7
X lo0- M) was purchasedfromNovoIndustries (Copenhagen,DK). Monoclonal antibodies specific forPAI-l and PAI-l cDNA human probewere agift of Prof. D.Collen, Dr. P. J. Declerck, and Dr. L. Nelles (Center forThrombosisandVascularResearch, Leuven,
Bel-gium). f-actin cDNA human probe waspurchased from Clontech (Palo Alto, CA). [35S]Methionine, '25I-insulin, ['4C]aceticacid
so-dium salt, nylon N'membranes, andmultiprime labelingDNA sys-temwerepurchased fromAmersham International (Amersham, UK).
SafeEmulsieferwasfromPackard(MeridienUSA). Metforminwas a
giftfromLipha Laboratories(Lyon, France).
Cell culture
Cellsfromthe humanhepatomacell lineHep G2weregrownto con-fluencyin Ham's F12/Eagle'sMEMsupplementedwith10%
heat-in-activatedFCS,2 mML-glutamine, 100IU/mlpenicillin,and 100,ug/
ml streptomycin undera 5% CO2 atmosphere. Forspecific
experi-mentsHep G2 monolayerswerecultured on24- or 12-well culture plates. 24 hafterreaching confluence, HepG2monolayerswere cul-turedovernight in serum-starved medium supplemented with 0.5% BSAfollowed byincubationwith insulin and/or metforminat
differ-entconcentrations.Human umbilical veinendothelialcells(HUVEC)
were isolatedfrom umbilicalcordveinsaccordingtothe method of Jaffeetal.(31).Cellswereusedatconfluencyafteronepassage. For
specific experiments, confluent HUVECmonolayerswerepassedon
24-wellplates.Cellviabilitywasassessedbythe trypan bluedye exclu-siontest.Conditionedmedia (CM)wereharvested, immediately cen-trifugedat2,000gtoexclude cellulardebris,aliquoted,andstoredat -20°C.Cellsweretrypsinizedand counted.
PAI-I
antigen assayPAI- Iantigen(PAI-IAg) assay in CMwasperformed usinganELISA asdescribed elsewhere ( 32). Resultswereexpressed intermsof PAI- 1 Ag per106cells.
RNAextraction and Northern blot
analysis
TotalRNA wasextractedaccordingtoChomczynski et al. (33) and fractionatedon1.2% agarosegelcontaining20%formaldehyde.RNA wastransferred to nylon N+ membranes. Hybridization conditions andprocessingof the filterswerecarriedout asdescribed by Medcalfet al.(34). ThePAI-l cDNA probewas a436-bpfragment ofhuman
PAI-l cDNA.The humanf3-actinprobewas acomplete 2-kb human
fl-actin
cDNAandwasusedascontrol. ThePAI- 1 cDNA and 3-actincDNAprobeswerelabeledwith [a-32P]dCTPusingthe random
prim-ing technique(35).
['4C]Acetate incorporation
Serum-starved Hep G2 monolayers were incubated with['4C]acetic
acid sodium salt(['4C]acetate 1 gCi/ml,spact 56.2 mCi/mM) in presenceof insulin and/or metformin. At the end ofthe incubation the
conditionedmediawerediscarded,themonolayers were rinsed three times with cold PBS and the cells were incubated on ice with a lysing
buffer (Tris HCI0.05 M, pH 8.0, NaCl 0.15 M, NP-40 I%, PMSF 1 mM, andaprotinin 1 gg/ml). The cell extracts were centrifuged to
eliminatecell debris andanaliquot (50,g)wasusedforquantifyingthe
proteincontent.Incorporation of ['4C]acetatein the lipid phase ofthe
cell extractswasassayedasdescribedby Folchetal.(36).Analiquot
(150
p1)
of thelipidphase was mixed with 10ml of Safe Emulsifier and counted inaf:counter.Residual binding of '25I-insulin to Hep G2 cells
Residualinsulinbindingwasperformedonserum-starved cell mono-layersin the presence of'25I-insulinasfollows:after a 24-h incubation with insulin and/or metformin, cells were acid-washed three times at
4°Casdescribed by Garveyetal. (18) to remove bound insulin, fol-lowed by three rinses in PBS. Cellswerethenincubated for3 hat4°Cin MEMHam's F12 medium 0.5% BSAcontaining0.2 ng'25I-insulinper well. Free hormone was removed with five rinses of cold PBS and the cells wereagainacid-washed with 250ulof MEM, pH 4.0, todissociate receptor-boundinsulin. Theradioactivityrecovered in theacid-washed
eluatewascounted.Nonspecific binding determined withtheaddition of200 ng of unlabeledinsulinwas<5%oftotalcountsbound.
Down-regulation ofHep
G2cells
Down-regulationof Hep G2 cell receptorswasperformedasdescribed byBlakeetal.(25):serum-starvedconfluentHepG2cells were
pre-in-cubated withahigh concentrationof insulin(l0- M)foraprolonged
time ( 18h),cooledonicetoavoid receptorrecycling,acid-washed in MEM,pH4.0,toremoveboundinsulin,andthen washed withPBS,
andfinallywith culturemedium,eachwashing being performedthree times. Whenrequired,cellswerepreincubated with10- Minsulin for differenttimes andsubsequentlytreatedasdescribed above.
Insulin stimulation
of
Hep
G2cells
Control cells.Serum-starvedHep G2cells wereincubatedfor24 hwith doses of insulinrangingbetween 10-10andI0-7M.Unstimulated cells were used ascontrols. When required control cells were incubated with
l0-7orI0-9 M insulin fordifferenttimes.PAl-1secretion,PAI-lRNA
expressionand['4C]acetateincorporation into lipidswereassayedas above.
Down-regulatedcells. Downregulationwasperformedasdescribed above; cellsweresubsequentlystimulatedwith10-9Minsulin for 24 h. Controlunstimulatedcellswerekeptin serum-starved mediumduring
thetwo steps of this procedure. Control down-regulated cellswere
down-regulated by l0-7Minsulinfor 18h, acid-washed,andkept in serum-starved medium forafurther 24-hincubation.
Measurements ofinsulinremainingin the conditionedmediawere
performedduetoinsulindegradation.Weassayed insulin concentra-tionsduringthetwoincubationperiods:(a) when10-9Minsulinwas addedtoHep G2 cells,theinsulincontent inthe mediumdecreased 12 hafter thebeginningof the incubation andwasnotdetectable after 18 h;(b)whenlO-7Minsulinwasaddedtothecells, the insulin remain-ing in the medium afteran18-hincubation representsonethird of the initialcontent.Residual insulinwaseliminated by acid washes (25).
Effect
of metformin
on insulin-mediatedPAI-I
synthesis
and
['4C
acetateincorporation
Theeffects of metforminoninsulinstimulationwerestudied by incu-batingserum starved control Hep G2 cells for24 hwith insulin in presence of different concentrations of metformin ranging between
106 and I0-3M.Thetime-dependenteffect of metforminon insulin-mediatedPAI- Isecretionwasstudiedby incubating HepG2cells with
l0-4Mmetformin forupto24 h.
Tostudy theeffect ofmetforminoninsulin stimulation in
down-re-gulatedcells, l0-4 M metforminwasadded eithertoI0-7 M insulin in
the 18-hpreincubationstep, or to 10-9 M insulin in the stimulation
step.
Whenrequired, Hep G2 cells were stimulated for 24 hby 10-' M PMA(37) in absence or presence of10-' Mmetformin.
HUVEC were stimulated for 24 hbyIL-10 IU/ml (38)in pres-ence or abspres-ence ofl0-4Mmetformin.
Statistical analysis
Data areexpressed as mean±SE. Thesignificance ofdifferenceswas examinedusingunpairedStudent's t test. P values<0.05were
consid-eredsignificant.
Results
Effects
of
insulin on PAI-Isynthesis
andacetate incorporationTheeffectsof insulinon PAI- 1synthesisandacetate incorpora-tionwerestudied in control ordown-regulatedcells.
Control cells. Serum-starvedHepG2 cellswereculturedfor
up to 24 h withincreasingdoses(1O-10to lO-'M)of insulin. Aspreviouslyshown(26)ourresults confirm that insulin
in-ducedadose-dependentincrease inPAI-I secretion upto
4.8-fold(Fig. 1A).The maximal effect occurredwith 1O-8M
insu-lin(469±39.3 instimulatedcells versus 10 1±8.3ng/106 cells
in control cells, P< 0.01). Insulin also induced changes in
PAl-1 mRNAexpression.Thesteady-statelevelof bothforms
ofmature PAl-I mRNA (3.2 and 2.3 kb) wasincreased by insulin stimulation. Nochangeinthe 3-actingeneexpression
wasobserved (Fig. 1, inset).
Insulin induced adose-dependent increase from 10-10 to
I0-7Min [
`4C]acetate
incorporationintoHepG2cells upto2.2-fold ( 1.52±0.12vs.0.69±0.09nM/mgprotein[P<0.051) when thecellswerestimulated by i0-7Minsulin.
Kinetic studies indicate that insulin at
l0-'
and l0-9 M, induced a time-dependent increase in PAI-l secretion and['4C]acetate incorporation into Hep G2 cells with a 4-h lag-phase(Fig. 1B).
Down-regulated cells. Serum-starved Hep G2 cells were down-regulated byI0 -7 Minsulin for 18 h, thenstimulatedfor
24 h with 10-9 M insulin. PAI-I synthesis and ['4C]acetate
incorporationwereevaluatedatthe endofthestimulationstep.
Results in Fig. 2A indicated that in down-regulated cells, a
stimulation with 10-9 M insulin led to an increasein PAI- 1
secretion by 1.4-foldoverthe levelinducedby thesame stimula-tionofcontrolcells(597±54vs.408±39ng/1O6cells), and by
5.9-fold over the level of the unstimulated control cells (597±54 vs. 101±8.3 ng/106 cells). It should be notedthat PAI- 1 secretion levels from down-regulated cells kept in serum-starvedmedium foranadditional24hafteracid wash
werenotsignificantlydifferentfromthoseobserved with con-trol cells (132±15.9vs. 101±8.3 ng/ 106 cells,Fig.2A).
Stimu-lationmediatedby l0-9 M insulinofdown-regulated Hep G2 cellsinduced changesin PAI- 1 mRNA expression. This proce-dure resulted in thestimulation ofbothforms ofmature PAI- I mRNA over the expression mediated by l0-9 M insulin in controlcells(Fig. 2, inset). No change in
0-actin
mRNA ex-pression wasobserved.Incontrast toPAI-1 synthesis, the stimulation with I 0 -9 M
insulin in down-regulated cells, resulted in a net decrease in
_ = | 2.3KB PAI-1
H
0 500 0
%0
0 H
0 0
-5300
01
H
as 100
500
-H
H 0 001
01
-' 100, Im
W-ssW
A B C D 3
a A&IN
0
go
1.5 4 $A
10.
0.5
0
go
~0 "4 r
O -0u o, 0
u of a
U
0 2 4 6 8 12 18 24 TIME (hours)
Figure 1. Dose- andtime-dependent effects of insulin onPAI-l se-cretion ( ) and [ '4C]acetateincorporation(x) in Hep G2 cells. (A) Dose-dependenteffects ofa24-hinsulinstimulationon PAI- 1 secretion (*) and['4C] acetateincorporation(x). Results represent means±SEoffiveexperimentsperformedintriplicate: *P < 0.05, * *P <0.01. Inset:Effects of increasing doses of insulin on PAI- I and
f-actinmRNAexpression in Hep G2 cells. Lane A, control cells; laneB, 10-0Minsulin;laneC, 10-9 M insulin; lane D, 10-8 M
in-sulin;lane E, I0O- M insulin. (B)Time-dependenteffects ofinsulin onPAl-1 secretion(.)and['4C]acetateincorporation (x). Hep G2 cellswerestimulated up to 24 h by 10-9M(-) or 10-7M (...)
insulin.Results represent means of two experiments performed in
duplicate.
['4C]acetate incorporation (Fig. 2B): itresultedina2.4-fold
decrease compared with the level obtained withl0-9M insulin in control cells (0.5±0.2 vs. 1.18±0.09 nM/mg protein [P
<0.05])anda 1.4-folddecrease comparedwithunstimulated
control cells (0.51±0.04 vs. 0.69±0.09 nM/mg protein [P
<0.05]).Down-regulatedcellskept forafurther24-h
incuba-tion in serum-starved medium expressed[14C] acetate
incorpo-ration identical to control unstimulated cells (0.76±0.1 vs.
0.69±0.09).
-w
H
4)
0 0
-ll
H
p4
500
300
100
a
0
-A
,1 1.0
0
) 0.5.
U
4,1
Hi
f:..w.g
...f.: 3.BI-,_ ow iP as f3 ACTIN
A B C D
A
*
*
B
AlL1
A BIn
C DFigure2.Effects of i0-9M insulin stimulationfor 24 honPAI-I se-cretion(A), ['4C]acetate incorporation (B),and PAl-I andf3-actin mRNAexpression (inset)incontrol anddown-regulated HepG2 cells. LaneA, control unstimulatedcells;laneB, control cells
stimu-latedby l0-9Minsulin;laneC, down-regulatedcells washed and
keptinserum-starved medium for24h;laneD, down-regulatedcells
washedandstimulated for24 hby l0-9M insulin. Results inpanels AandBrepresentmeans±SEof fiveexperiments performedin
tripli-cate:*P<0.05; **P<0.01;***P<0.001.
investigated. Hep G2 cells were preincubated for different
times,upto 18h,withI0-7M insulin and after acidwash, they
werestimulated for24 h with 10-9Minsulin. Kinetic studies
(Fig. 3)reveal thatPAI-I secretionbegantograduallydecrease in down-regulated cellsduringthe first4 hofpreincubation
andwasthenprogressivelyenhancedwhen thepreincubation
timewasincreased from4 to 18h. In contrast toPAI-
secre-tion, a4 hpreincubationwith
10-'
Minsulinwaseffective indecreasing[
"4C]acetate
incorporation, and this level remained unchanged irrespective of the time used for the preincubation.Effects ofmetformin
on insulin-mediated PAI-1synthesis
and
acetateincorporation
Control cells.Theeffectsofincreasing doses (10-6to
10-3
M) of metforminonPAI-I synthesis mediated by IO1 Minsulin during a 24-h stimulation of control cells were first investi-gated. Metformin exerted a dose-dependent inhibition on the insulin-mediated PAI-1 secretion for doses ranging betweenlO-'
andl0-'
M(Fig. 4A). When cells were stimulated for increasing times with10-'
M insulin in presence oflO-' M metformin, the inhibitory effect of the drug was observed as soon as the stimulating effect of insulin was detectable, i.e., after a 4-h incubation. From the 8th h, a complete inhibition of insulin-mediatedPAI-l synthesis was obtained (Fig.4B).Inthe presence ofl0-4Mmetformin, the stimulating effect of
10-10
to l0-7 M insulin was abolished, PAI-I levels being identical to unstimulated control cell levels (Fig. 5). The same results were obtained withl0-'
M metformin (data not shown). Metformin alone had noeffecton PAI-1constitutive secretion(Table I)orPAI-1 mRNAexpression (Fig. 6, inset, lane A). The inhibitory effect of metformin oninsulin-me-diated PAI-1 synthesis wasalsoobserved at the mRNA level. Metformin inhibited the insulin-mediated increase in both 3.2-and 2.3-kb transcriptsof PAI-1 mRNA. Nochange in thef
actin geneexpressioncould be observedafterinsulin or insulin plusmetforminstimulation (Fig. 6, inset, lane B).
Theeffects of10 -4 Mmetforminon [ 4C]acetate incorpora-tionmediated by I0 10Mto l0-7Minsulinwerestudied. As illustrated inFig.5, incubation ofHep G2 cellswith
l0-'
Mmetforminhad nosignificant effectoninsulin-mediated[
14C]-acetateincorporation irrespective oftheinsulin concentration
studied. Therefore, in Hep G2 cells, metformin was able to suppress theinsulin-mediatedPAI-1synthesisbutnotthe
insu-lin-mediatedacetateincorporation into lipids.
Down-regulated cells. The effects of metformin on
insulin-*a 5002
H
o
400.
%0 0 M
tn 300
--T
200_-14
of 100~
I I I I I
0 2 4 6 8 12
a
0
I4.
-1.5 *f f
- 1.0 * s
_
0.5 o
H0
18
DOWN-REGULATION TIME (hours)
Figure3. Effects of thedown-regulationtimeonPAI-Isecretion(. ) and[4C]acetateincorporation (x ). Hep G2cellswere down-regu-lated forincreasing times, by l0-' Minsulin,and after acidwashes, stimulated for24 hby lO-9Minsulin. Resultsrepresentmeansof
threeexperiments performedinduplicate.
2188 Anfossoetal.
k.
....***-**--mediated PAI-I synthesisand acetateincorporationin
down-regulated cells were then investigated.Inthefirst set of experi- 0
ments Hep G2cellswere incubated for 18 h with IO Minsu- T q .
linin presenceof I04- Mmetformin,andafter washing,were |I stimulated by lO9Minsulin. Results inFig. 6A indicated that ° 50 '| addition of metformin in the preincubation procedure corm- 8
pletely reversed the overproduction of PAl-1
synthesis
me-Tadiated
by
10-
Minsulin indown-regulated
cells,
the levels of c 300 /PAI- I secretion being identical to unstimulated control cell O T x.o
levels (P < 0.001I). In a further set ofexperiments IOM
metformin was added to
10-'
M insulin in the stimulation H 100 -| <Wr u.*.*-**0-**'*. 0 XIC ~~~~~~~~~U
step. Results indicated that metformin inhibited the insulin- V
mediated PAl-1
synthesis,
toasimilarextent tothat obtained * I * *-10 -9 -8 -7
INSULIN (L0og K)
A Figure5. Effects of10-4M metforminonPAI-I secretionand ['4C]acetateincorporation mediated by increasing doses of insulin in
500 Hep G2 cells. PAI-l secretion after stimulation with insulin alone
H Soo0
0_(e-*)
orwithinsulin plus10-4
M metformin ( -.0).
* ['4C]]Acetate
incorporation
afterstimulationwith insulin alone%D _(x-x)orwith insulin plus l0-4M metformin(x... x). Results
I-I representmeans±SEof five
experiments
performed
intriplicate.a
300by addition of metformininthepreincubationstep. The sup-<4x T ** * * pressive effect of metformin on the insulin-mediated
PAM-1
"4 -x 1overproductionresulted fromaninhibition of theexpressionof
loo 2
I
I I
u
[0^1
,,PAL-1
mRNA
(Fig.
6,
inset,
laneC),
withoutchange
in
the100
*3-actingeneexpression.
Theeffect of10-4Mmetforminonincorporationof[
14C]-acetateintolipidswastestedusingthesameconditionsof
cul-A B C D U F ture. Results in Fig. 6 B indicate that the addition of metformin toinsulin didnotmodifytheinsulin-mediatedacetate
incorpo-B ration in down-regulated cells irrespective of the time that the
, 500 drugwasadded.
'-4
* |
Specificity
Adof metformin effects for
insulin-mediated
PAL-1
o
* synthesis
"4 / To testwhethermetformin could inhibitPAI- 1 synthesis
me-diated by other agents knowntoregulatePAI- I synthesis,we
~'300
_
o-testedtheeffect ofmetforminon PMAstimulation. Additionto
"4
~
~
~~6000Table
I.Effects of Metformin onPAl-I SynthesisH
0 _ /. * . - . . . .
Of
101
0 2 4 6 8 12 18 24
TIM (hours)
Figure 4. Dose- and time-dependent effects of metformin on insulin-mediated PAI-Isecretion by Hep G2 cells. (A) Dose-dependent ef-fectsofa24-hinsulin stimulation withincreasing doses of metformin (10-6toI0-3M) for 24 h. Lane A, control cells; laneB,control cells stimulated bylI0- Minsulin; lane C, control cells stimulated by l0 Minsulin-plus 10-6Mmetformin; lane D, -plusI0-5Mmetformin; laneE,-plus I0-4M metformin; laneF,-plusl0-3Mmetformin. Results represent means±SE of three experiments performed in trip-licate: *P<0.05;**P<0.01.(B) Time-dependent effects of metfor-minoninsulin-mediatedPAI-1 synthesis. Hep G2 cells were incu-bated with l0-' Minsulinin the absence ( )or inpresence
(... )ofl0-' Mmetformin for upto24h. Results representmeans oftwoexperiments performed induplicate.
Cells Stimulatingagents Metformin PAl-1Ag
ng/lo6cells
Hep G2 100.6±18.3
0-3M 127.9±15.3
10-4M 113.3±17.2
lo-5M 107.5±8.9
10-6M 105.8±12.4
PMA10-7M 1446.6± 186.3
PMAl0-7M 10-4M 1451.6±201.4
Insulin 10-9M 408.4±39.3
Insulin l0-9M l0-4M 110.8±17.4
HUVEC 908.3± 10.2
IL-1 10IU/mrl 3537.5±408.4
IL-1 10IU/ml 10-4M 3462.5±393.7
Metforminwasaddedaloneorin combination with the different
w
500.._
r-~0
0 H
01,
H
300-100
-a
0
_
d14 -H,I 1.0 _
0
0V
e m ~~~~~~3.2KB
* " 3 * 2 RB
~~~PAI
-1 2~2.3KBl-*
*j*"S~~~~
ACTIN1 2 1 2 1 2 3
A B C
A
-ii-~~~~~~~~~~7
B
A B C
Figure 6. Effectsof10' MmetforminonPAI-I secretion(A),
['4C]acetateincorporation(B), PAI-I andf3-actinmRNAexpression (inset) mediated by
0'-9
Minsulinincontrolanddown-regulatedHepG2 cells.LaneA,controlcells:(black bar)lane1,without met-formin; (graybar)lane2, withIO-4Mmetformin.LaneB, control
cellsstimulatedby 10-' Minsulin:(openbar)lane1,without met-formin; (graybar)lane2,with l0-' Mmetformin. LaneC,
down-regulatedcellsstimulatedby
10-'
Minsulin:(open bar)lane1,withoutmetformin; (graybar)lane2,with l0-' Mmetformin
added in thepreincubationstep;(dotted bars)lane3, with
10-'
Mmetforminadded in thestimulationstep. Results represent means±SE of fourexperiments performedintriplicate.
of l0-4 M metformin to l0-7 M PMA did not modify the PMA-induced PAI-1 accumulation in a 24-hCM (Table I). The effect of metformin on IL-l-induced PAI-1 secretionby
HUVECwasalso tested. HUVECwereincubatedfor24 hwith
IL-1 (10UI/ml)inpresenceandabsence of metformin l0-'
M). ResultsinTableIindicated that,atthis dose, Metformin didnotmodifythe IL-l-induced PAI-l secretion.
Residual binding
of
'25I-insulin
onHep G2 cellsChanges in insulinresponsiveness inthe presence of metfor-min could result from differences in cellsurfaceinsulin
ing. We studied the expression of insulin receptors by the bind-ing of tracer amounts (0.2 ng) of
'251I-insulin
on Hep G2 cellsafter incubation withinsulin and/ormetformin. Results are presented inFig. 7. Stimulation ofHep G2 cells with insulin
10 10to I0-7Mfor24 hresulted in a dose-dependent loss of cell surface receptors compared tounstimulatedcontrol cells taken as 100%.Stimulation ofcellsby 10-9Minsulin, led to a 40% decrease andstimulationby I0-7Minsulinled to a 81% decrease in residualinsulin binding.
The effect ofinsulin stimulation on down-regulated cells resulted, as expected, in a more marked decrease of residual insulinbinding: 10-9Minsulin added to down-regulated cells induced a decreaseof70% compared to unstimulated control cellstaken as 100%.
Addition of
l0-4
M metformin to unstimulated control cellsandtoinsulin-stimulatedcontrol cells had no significanteffectonthe
'251-insulin
bindingwhen compared to cells prein-cubated withoutthe drug. In the down-regulationprocedure, 10 -4 Mmetforminexerted nosignificanteffect on the125I
resid-ualinsulinbinding irrespective ofthetime atwhichthe drug was added to insulin, i.e., during the preincubation or the stim-ulation step. These results showed that insulin stimstim-ulation in-ducedaloss incellsurfacereceptors andaddition ofmetformin displayed nosignificant effectontheexpression of insulin re-ceptors.Discussion
Resistance to insulin-mediated glucose disposal is a well-known phenomenon associated with obesity (7). The syn-dromeof insulinresistanceischaracterized by a compensatory
hyperinsulinemia, hypertriglyceridemia, low plasma high
den-sity lipoproteincholesterolconcentration,andhighblood
pres-sure.Thesefeatures usuallycluster in the sameindividualand have beenidentified as increasingtherisk ofcoronary heart
9 100
*r
.94 0
,r40
4J
HO
0 5
'0
H
H -.
0
-10 -9 -8 -7
INSULIN (Log M)
Figure7. '251I-insulinresidualbindingincontrol
(.)
anddown-regu-latedHep G2 cells(x)stimulatedby increasingconcentrations of insulin.Bindingin control unstimulated cellswastakenas100%:(-)
withoutmetformin; (...)with l1-0 Mmetformin(addedin the
preincubationstep fordown-regulated cells); (---)with l0-' M metformin added in the stimulation step.
disease (8). High plasmaPAI-I activityhas been shown ina
cohort ofyoung survivors ofmyocardial infarction to bean
independent risk marker for reinfarction (39) and isassociated with insulin resistance(9). Clear relationshipshave been ob-served among insulinemia, serum triglycerides, and PAI- 1
(40). Interventional measuresincluding weightlossand
met-formin administration result inareduction ofboth insulin resis-tanceandplasma PAI-1 levels ( 15 ).
Hepatic insulin resistance is found in obesityandin
non-insulin-dependent diabeticsubjects (41 ). Cellular sensitivityto
insulin is dependentonthe level offunctional insulinreceptors onthe cellsurface,andawell-established inverse relationship exists between ambient insulin levels and the number of cell surface insulin receptors(18, 20,21). Ourpurpose was to re-produce a hepaticinsulin-resistant statebydecreasing insulin
receptor numbers in the human hepatoma cell lineHepG2, which isknown tosynthesizePAI- 1inresponse toinsulin (26). Hep G2 cellreceptors weredown-regulated byanexposure to a
high concentration of insulin. It has been shown that exposure of Hep G2 cellstoIO-7 Minsulin for 18 hwasefficienttoallow internalization of the insulinreceptor(25) and toselectively
and markedly down-regulate the steady-state level of its
mRNA(42, 43 ). Ourresultsfromtheresidual'251-insulintrace amountbindingasshown inFig.7 areinagreementwith these data.Theresidualbindingisinversely proportionaltoboth the time and concentration ofinsulin to which the cells are
ex-posed.
Insulin is a potentstimulator offatty acid synthesis and
lipogenesis(30). Physiological concentrations of insulin have been showntoincrease the cellularcontentoftriacylglycerolin hepatocytes fromnormal anddiabeticrats(44, 45). We first confirmed that insulin stimulated PAI-1 synthesis in a dose-andtime-dependentmanner.Themaximaleffectwasobtained foraconcentration of insulin similartothatfound in theportal
vein afterameal(46).Wealsoconfirmedthat thestimulation
of [
"4C]acetate
incorporationintolipidextractbyinsulinwasdose andtime dependent.Nevertheless,the cellswereless
sen-sitivetotheinsulin stimulationasfaraslipid synthesisis con-cerned.
Comparisonsbetweeninsulin residualbindingandacetate incorporation indicated that down-regulated cells expressed lowinsulin receptor numbersand consequently, ['4C]acetate incorporation inresponse to aninsulin stimulationwas mark-edly reduced. These dataare inagreement with the develop-mentofaninsulin-resistantstateinducedby the down-regula-tion of the insulin receptors,leadingto areduced responsive-nessofthe cells toinsulin.In contrast totheabove-mentioned data, down-regulation of Hep G2 cells did not induce a de-crease in the insulin-mediated PAI-1 synthesis in spiteofthe
lossof surfacereceptors.On thecontrary, PAI- 1 secretionwas
increased above the secretion level observed in stimulated non-down-regulated cells. Kinetic analysis showed that the overexpression ofinsulin-mediated PAI- 1 synthesisin down-regulated cells was observedfordown-regulationtimeslonger than 4 h.This effectwasdependentonthetime of down-regula-tion. Thisoverproduction ofPAI-1 resulted froman insulin-mediatedstimulation ofPAI- 1 mRNA steady state levels. The
mechanism leading to this increased synthesis remains un-known.
Insulinresistanceis usually expressedintermsof reduced
glucose
transport (8, 47) associated with otherpost receptordefects
(48, 49)
including
a loss oftyrosine kinase activitywhich isimportant for transducing the biological effects of
in-sulin(50-52). Down-regulated Hep G2 cells expressed
multi-functional defects includingan increased proportion of tyro-sine kinaseincompetent insulinreceptorsleadingto adefect of insulin-receptor complex internalization (53). Althoughthere
is a general agreement that insulin receptor tyrosine kinase
activity mediatesmanyof the actions of insulin, the
stimula-tion of thisenzyme may notbeanecessarystepfor all insulin signaling pathways. Some studiesnowsuggestthata
non-tyro-sine kinase-dependent pathwaymay also exist (54, 55). The
discrepancy between the regulations of lipid and PAI-1 synthe-sis inourmodelmaybe relevant formultiple signal transduc-tionpathwaysthat wouldexplainthebiological actions of insu-lin.Nevertheless,adirectrelationshipbetween PAI-1synthesis andnon-tyrosine-dependent pathwaysremainstobeproven.
Metformin has been shown in vivotolower both basal
PAI-1 levels in obese nondiabeticwomen( 15) andpostvenous
oc-clusion PAI-1 levels intype IIdiabetic patients (56). We have
previouslyshown that the decrease infastinginsulinemiawas
associated withadecrease in plasma PAI-1 ( 15). Ourpresent studyindicates that metformin intherapeuticdoses (57)exerts
adirect effect onPAI- 1 synthesisat the cellular levelby sup-pressing the insulin stimulating effect both in control and down-regulatedcells.Metformin addedtoinsulin in down-reg-ulated cells inhibitedtheoverproductionof PAI-1synthesisby
adirect effectonPAI-1 mRNAexpression.Indeeditsaddition
to 10-7 Minsulin in thepreincubationsteprendered
down-re-gulated HepG2cellsunresponsiveto afurtherstimulationby
10-9 Minsulin,inspite ofitseliminationfromthemedium by
washings. However, metformin did not display any activity
eitheron thesteady-statelevelof PAI-I synthesisor onbasal
['4C]acetate incorporation and residual
'21I-insulin
binding. The resultson'251I-insulin
bindingin thepresence ofmetfor-min are in agreement with previous reports indicatingthat metformin modified neithertheinsulinbindingnorthe
affin-ityof insulinreceptors(58). The actiononlipogenesisby
met-forminremains unclear. Melinet al.(59) reporteda
restora-tion oflipogenesis bymetformin inratinsulin-resistant hepa-tocytes but such differences between our respective results
could beexplained bytheuseof differentcells and protocols.
Theintracellularsite of action of metforminpresently
re-mains unknown. Usually metforminrestoresthebiological
ef-fect of insulinbyenhancingthe substrate uptake(58). In con-trast,inourstudy,metformin decreased theparadoxical effect
of insulin on PAI-1 synthesis to normal levels. Our results clearly indicatethat metformin may modify some aspects of theaction of insulin in Hep G2 cells butnot byincreasingthe
binding
of insulintoitsreceptor.Similarly metforminhas beenshown tohave noeffecton insulin-mediated amino butyrate incorporation in rat hepatomacells whereas it enhanced the insulin-stimulated glucose incorporation into glycogen (60).
The data thus suggestthat several effects ofinsulin are
me-diatedbyseveral postreceptor mechanisms and that only some
oftheseweremodified by metformin. Metforminexerts several postreceptoreffects (61, 62) includinganaction on the
restora-tion oftyrosinekinaseactivity(63),animprovementof
glu-cosetransport, andpotentiation ofGLUT 4translocation (64, 65).This latteraction is unlikelyin our study because Hep G2 cells donotexpress GLUT4transporter (66).
In conclusion,thepresentworkdemonstratesthe ability of
insulintoinducean upregulationof PAI-I indown-regulated
insulin-resistant patients PAI-I plasmalevels are largely in-creased (9) whereas insulin receptor numbers are dein-creasedon
insulintarget cells (48). The results also indicate that
metfor-minacts at a cellular level by inhibiting the insulin-mediated PAI-1 synthesis. This effect could be responsible for the de-creased PAI- 1 plasma levels observed in vivo after metformin
therapy ( 15). Althoughextrapolation to clinical circumstances
should bedone with caution, our results are consistent with the hypothesis that Hep G2 cells which mimic humanhepatocytes (24) behave as insulin-resistant cells in terms of PAI- 1
syn-thesis.
Acknowledgments
Theauthorswouldliketothank Mrs. B. Bonardo for herskillfull
tech-nical assistance.TheyaregratefultoDr. P. Andre and Dr. N.
Weirn-sperger(LiphaInstitute) for their adviceonMetforminuseduringthis work,toDr. E.vanObberghen(INSERM U 145, Nice) for his criticism inreading this manuscript,andtoDr. R.Medcalf(Laboratoryof
He-matology, CHUVLausanne,Switzerland) for hisadvice in performing RNA assays. The researchwas supported bygrants from INSERM
(CRE 900501), Universite d'Aix-Marseille II, and ConseilRegional
Provence-COte d'Azur.
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