Small elevations of glucose concentration
redirect and amplify the synthesis of guanosine
5'-triphosphate in rat islets.
S A Metz, … , M E Rabaglia, A Kowluru
J Clin Invest.
1993;
92(2)
:872-882.
https://doi.org/10.1172/JCI116662
.
Recent studies suggest a permissive requirement for guanosine 5'-triphosphate (GTP) in
insulin release, based on the use of GTP synthesis inhibitors (such as myocophenolic acid)
acting at inosine monophosphate (IMP) dehydrogenase; herein, we examine the glucose
dependency of GTP synthesis. Mycophenolic acid inhibited insulin secretion equally well
after islet culture at 7.8 or 11.1 mM glucose (51% inhibition) but its effect was dramatically
attenuated when provided at < or = 6.4 mM glucose (13% inhibition; P < 0.001). These
observations were explicable by a stimulation of islet GTP synthesis derived from IMP
since, at high glucose: (a) total GTP content was augmented; (b) a greater decrement in
GTP (1.75 vs. 1.05 pmol/islet) was induced by mycophenolic acid; and (c) a smaller "pool"
of residual GTP persisted after drug treatment. Glucose also accelerated GTP synthesis
from exogenous guanine ("salvage" pathway) and increased content of a pyrimidine, uridine
5'-triphosphate (UTP), suggesting that glucose augments production of a common
regulatory intermediate (probably 5-phosphoribosyl-1-pyrophosphate). Pathway-specific
radiolabeling studies confirmed that glucose tripled both salvage and de novo synthesis of
nucleotides. We conclude that steep changes in the biosynthesis of cytosolic pools of GTP
occur at modest changes in glucose concentrations, a finding which may have relevance to
the adaptive (patho) physiologic responses of islets to changes in ambient glucose levels.
Research Article
Find the latest version:
http://jci.me/116662/pdf
Small Elevations
of Glucose Concentration
Redirect
and
Amplify
the
Synthesis
of
Guanosine 5'-Triphosphate
in Rat
Islets
Stewart A. Metz, MelissaMeredith, Mary E. Rabaglia, andAnjaneyulu Kowluru
Department ofMedicine andDivisionofEndocrinology, UniversityofWisconsin, Madison, Wisconsin53792; andMiddleton Memorial Veterans Medical Center, Madison, Wisconsin53705
Abstract
Recent
studies
suggest a permissiverequirement
for guanosine5'-triphosphate (GTP) in insulin
release, based on the use ofGTP synthesis inhibitors (such
asmycophenolic
acid) acting atinosine
monophosphate (IMP)dehydrogenase;
herein, weex-amine
the glucose dependency of GTP synthesis.Mycopheno-lic acid inhibited
insulin secretion equally well after islet cul-ture at7.8
or 11.1 mM glucose (51 % inhibition) but its effect wasdramatically
attenuated when provided at <6.4 mM glu-cose(13% inhibition;
P <0.001).
These observations wereexplicable by
astimulation of islet GTP synthesis
derived from IMPsince,
athigh
glucose: (a) total GTP content wasaug-mented;
(b)
agreaterdecrement
inGTP (1.75
vs. 1.05 pmol/islet)
wasinduced
bymycophenolic acid;
and(c)
a smaller"pool" of residual GTP persisted after
drugtreatment.
Glucosealso
accelerated GTP synthesis from
exogenousguanine
("sal-vage"
pathway) and increased
contentof
apyrimidine,
uridine5'-triphosphate
(UTP),
suggesting that glucose
augmentspro-duction
of
a common regulatoryintermediate (probably
5-phosphoribosyl-1-pyrophosphate).
Pathway-specific
radiola-beling studies confirmed that glucose tripled both salvage and
de novosynthesis of nucleotides. We
conclude that steepchanges
in thebiosynthesis of cytosolic pools of GTP
occur atmodest changes in glucose concentrations,
afinding which
mayhave relevance
to theadaptive (
patho) physiologic
responsesof
islets
tochanges in ambient glucose levels.
(J.
Clin. Invest.
1993.
92:872-882.)
Key words:
(Pancreatic)
islet
*nucleotide.
purine
*adenosine
triphosphate
*guanine
Introduction
We
recently
reported
that
exocytotic
insulin secretion
from
isolated, intact
ratpancreatic
islets
depends
uponanadequate
islet
contentof
guanosine 5'-triphosphate
(GTP)'
(
1).
Portions ofthisworkwerepresentedatthe52nd Annual
Meeting
oftheAmerican Diabetes
Association,
SanAntonio,
TX,
June21,
1992(1992. Diabetes.41[Suppl.] :3a);the MidwestSection oftheAmerican
Federation for Clinical Research, Chicago, IL, November
6,
1992(1992.Clin. Res.
40:734a[Abstr.]);
and the 28th AnnualMeeting
of theEuropean AssociationfortheStudy
ofDiabetes, Prague,
Czechoslo-vakia, September8, 1992(1992.
Diabetologia.
35:[Suppl.]
Al16).
AddresscorrespondencetoDr.Stewart A.
Metz,
Division of Endo-crinology,University
ofWisconsinMedicalSchool(1H4/554
ClinicalScienceCenter),600
Highland
Avenue,Madison,
WI53792. Receivedforpublication
IDecember1992and inrevisedform
24 March 1993.1.Abbreviations usedinthispaper:CTP,
cytidine 5'-triphosphate; Gln,
L-glutamine;GTP,guanosine 5'-triphosphate; HGPRTase,hypoxan-The Journal
of ClinicalInvestigation,
Inc. Volume92, August 1993,
872-882The
conclusion
was based on the use of four selective inhibi-torsof inosine
monophosphate
(IMP)dehydrogenase
(EC1.1.1.205),
arate-limiting
step in the de novo synthetic path-wayfor purine
nucleotides. Two of these drugs, mycophenolicacid (MPA)
andmizoribine,
when provided during an 18-hculture
period in
RPMI 1640 (a purine-free medium), wereshown
todrastically curtail
islet content of GTP and,pari
passu,the subsequent insulin secretory
responses to glucoseand other
secretagogues. MPA or mizoribine also caused a more modestinhibition
of ATP content (by about 40%) anddramatic elevations of
apyrimidine,
uridine5'-triphosphate
(UTP), presumably due
toredirection of
aprecursor common toboth
pyrimidine
and
purine synthesis,
such as5-phosphori-bosyl-
1-pyrophosphate
(PRPP).
However,GTP
wasimpli-cated
asthe regulatory factor since:
(a)provision ofexogenous
adenine reversed changes in
ATP and UTP,while
leavingunal-tered the decrements in GTP
andin insulin
release; and (b)provision of
exogenous
guanine, by fueling the "salvage" path-wayfor purine synthesis,
restored both GTP and secretionnearly
tonormal
levels. We concludedtherefore
thatGTP
was apermissive
factor required for exocytotic
insulin release (1 ). Inthose
studies, pancreatic
islets
werecultured
overnight
in
1.
1 mM(200 mg/dl)
of
glucose, since this usually is the
mosteffective concentration
for cultured islets
tomaintain
insulin
contentand secretion
(2,
3). However,
wewereintrigued
by
anabsence
of
reportsof
the
induction
ofhyperglycemia
in normal
animals
orhumans
treated with
high concentrations
of these
drugs
(e.g.,
MPA,
provided
asits
pro-drug RS-61443,
refer-ences 4
and
5).
Therefore,
wequestioned
whether the
ambient
glucose
concentration
might
modulate the islet
contentof
pu-rine
nucleotides and, if
so,whether
glucose
might
alter
the
rela-tive
predominance of
the
pathways
involved in the
synthesis
of
GTP. The
currentstudies (the first
to ourknowledge
which
address
purine
nucleotide metabolic
pathways
in
peptide-se-creting endocrine cells)
provide
datathat
supportboth
possibili-ties and
suggest thatglucose
alterspurine
nucleotide
metabo-lism in both
quantitative
and
qualitative
fashions. The
narrowrange
of relevant
glucose
concentrations
overwhich such
re-sponsescan
be
discerned
in
pancreatic
islets suggests that these
events could have(patho)physiologic
relevancetosomeof
theislet's
nucleotide-dependent
adaptive
responsestohyperglyce-mia, such
asinsulin
secretion (1),
RNAand
protein synthesis
(6),
and DNAsynthesis (7).
Methods
Materials. Mycophenolic acid,nucleotidestandards,monobasic
am-moniumphosphate (forHPLC mobile
phase)
andguanine
werepur-thine-guanine
phosphoribosyltransferase;
IMP, inosinemonophos-phate; ITP, inosine
5'-triphosphate;
MPA,mycophenolic acid; NTP,
nucleoside
triphosphate;
PRPP,5-phosphoribosyl-1-pyrophosphate;
chased fromSigmaChemicalCo.,(St. Louis, MO). Mizoribine,also referredto asbredinin(4-carbamoyl-I --D-ribofuranosylimidazolium-5-olate;references8-10)was agenerousgiftof Dr. N. Kazmatani (To-kyo Women's MedicalCollege,Tokyo).The diluents used for making stocksolutions of thesedrugswereethanol(formycophenolicacid),
water(formizoribine),andMe2SO (for guanine), respectively;control tubesalways contained an equal amountof the relevant diluent as
experimental tubes. [3H(G)]hypoxanthine (17 Ci/mmol) and
[
'4C(U)Iglycine
(1 10.5mCi/mmol)
werepurchased
from NewEn-glandNuclear(Boston, MA)orAmershamCorp.(ArlingtonHeights,
IL). HPLC columns were purchased from AlltechAssociates, Inc.
(Deerfield, IL); HPLC water was purchased from Fisher Scientific (Itasca,IL).RPMI 1640 mediumwaspurchasedfrom Gibco (Grand Island, NY)asglucose-free powdertowhich the desiredamountof
D-glucosewasaddedtoachieveglucoseconcentrations of 1.7to 11.1 mM in theovernightculturemedium. L-glutamine (Gln,2.05 mM)
waspresent inthe culturemediumtoprovide adequatesubstrate for the denovosynthesisofpurinenucleotides(11-14);whereindicated, glycinecontentwasselectively modifiedusingthe Select-Amine Kit from Gibco.UndialyzedFBSwasfrom Gibco. In all studiescomparing
theeffects oftwodifferentglucoseconcentrationsduringthe culture
period,
theosmolarity
of themediumcontaining
the lowerglucoseconcentrationwasbroughttothatof the
higher
concentrationthroughthe
provision
of additionalNaCl.Isolationand treatmentofpancreatic isletsfor studies ofinsulin
release.Intactpancreatic islets (usually500-600islets perstudy)were
isolated from adult male Sprague-Dawley rats (Sasco-King, Inc., Omaha, NE) using collagenase digestionandseparation from acinar tissue and debrisonficoll
gradients,
followed by hand-pickingunderstereomicroscopic
control,aspreviously
described( 1, 15-17). Isletswereculturedovernight (X18 h)in RPMI 1640 medium(containing
10%FBS, 100U/ml
penicillin,
100Ag/ml
ofstreptomycin,and vari-ableglucoseconcentrations). Generally speaking,
each secretionex-periment
contained 4-8experimental conditions,
eachcomprised
ofmultiple (3to8)replicatedeterminations(10isletseach).The incuba-tion medium forsubsequent insulin release studies
(carried
outthenextday)wasKRB, pH7.4,containing0.5% BSA and 3.3or16.7 mM
glucose,andgassedwith 95%02/5%
CO2.
Static, batch-type incuba-tionswerecarriedoutfor45minsso as toinclude contributions from both firstand secondphasesecretion,
sinceourpreviousstudies( 1)indicated that MPA inhibits bothphasesofglucose-inducedinsulin release. The basal glucoseconcentration foracute incubations (i.e.,
after the
overnight
cultureperiod)was3.3mM;theglucoseconcentra-tion usedtostimulate islets in allincubationswaskeptconstant at16.7 mM. MPA(orothertestagents)waspresentduringnotonly the
cul-tureperiodbut alsowasincludedinisletpicking, wash,or
preincuba-tion steps the
following
day,in ordertoavoidattenuation of itseffectsovertime. However, drugswereexcludedfromtheincubation period,
since theireffectswere notrapidly dissipatedafter removal (1).
Insulin contentandfractionalrelease.After incubations to assess
insulinrelease,the tubescontaining islets,stillrestingontheir
polyeth-ylenefilters ( 15), wereplaced in 15-mlconical centrifuge tubes
con-taining
1 mlof acid alcohol(comprisedof77%absolute ethanol, 22% water, and 1% concentratedHCl; vol / vol), sonicated and leftover-nightat4°C.After 20 h, one ml of phosphate-buffered saline and 30
Al
of SN NaOHwereaddedtoraise the pH to 8.0, and the contents were vortexedafter removing the glass tube but leaving behind the mesh-filterso as toretain any islet tissue which might remain adherent to the filter. Tubes werecentrifuged at 4°C for 5 min (X2,000 rpm); the supernatantwasremoved, diluted (1:10)in RIA buffer (PBS) to a final dilution of1:20,and assayed.Insulin content of media or of islets was measured by RIA as
de-scribed(l, 15-17).
Determinationofnucleotide content of islets. Islets were cultured overnight (asdescribed aboveandinreference 1 ) except that exactly 200islets foreachcondition were cultured in individual dishes
contain-ing
testsubstances,
sothatisletscouldberapidly
extracted thenextday
withoutthe needfor
time-consuming
counting
andaliquoting,
during
which thenucleosidetriphosphate (NTP)contentsmightchange. A
typical experimentcontained 12 determinationscomprisedof
2,400
islets/study;threetofourexperimentalconditions(eachintriplicateorquadruplicate)werestudiedin each experiment. After the culture
pe-riod,isletsweretreatedaspreviously described in detail(1) andwere
extracted in trichloroacetic acid (TCA), containing sufficient internal standardcytidine 5'-triphosphate (CTP)andinosine5'-triphosphate (ITP)toyieldafinal concentration of0.8 nmol/ 100 d. Isletswerethen
lightly vortexed,sonicated(2X20s)onice,and the supernatantwas
obtained after centrifugation, allasdescribed(1).Supernatantswere
extracted(X4) using0.75 ml ether(toremovetheTCA); the ether phasewasthendiscarded. Samplesweredirectly analyzed by HPLCor
frozen at -20'C for futureanalysiswithintwoweeks;therewasno
difference in results between thetwohandlingprocedures.
HPLC wascarriedout asdescribed (1) usingaLiChrosorb-NH2
anion exchange column (Alltech Associates, Inc.) withoutaguard
col-umn.The detectorwassetat254 nM and 0.02to0.005 AUFS. Buffer A
was0.5 mM NH4H2PO4, pH 2.65; buffer Bwas0.65 MNH4H2PO4, pH2.65. Theexactproportionsof A and B and thepHwerevariedas
described in(1) usingasolvent programmer (Model 660; Waters In-struments,Inc., Milford, MA) in ordertomaximize separations, which
wereaffected by changes in columnsor newbatches of solvent,and,
especially, byevenminorchanges in pH. Separations were achieved by isocratic elutionat1.0-1.3 ml/min. 100 d each of sample(comprising
the extract of - 70 islets) was injected in duplicate; results were
corrected for the meanrecovery oftwoexogenousnucleotides(ITP
andCTP, whichwereusedasinternal standards after preliminary stud-ies ascertained that theywere notdetectable endogenously in islet
ex-tracts). A standard curvewasroutinelyruneachday, covering the relevant range (0.2-1.0 nmol) of ITP, CTP, GTP, ATP, and UTP. Valuesfor therecoveries of internal standards (ITP, CTP) have been reported, as have been the coefficients of variation for the overall preci-sion of themeasurements( 1). Values for basal islet nucleotidecontent
weresufficiently constant (inter-experiment CVs of 6.8, 7.2, and 10.8% for basal ATP, GTP, and UTP content, respectively) as to permit meaningful comparisons of the effect of experimental perturbations
notonly within, but also between, studies.
Labeling ofpurinenucleotidesviathe de novo orsalvagepathways. In order todetermine whether the de novo or salvage pathways contrib-uted tothe total GTPorATP content, islets were labeled with 10
ACi/ml
of[
14C(U)]glycine
(which
donates carbonsatposition
4 and 5, andnitrogen at position 7, of the newly-synthesized purine ring)orwith 2 ,uCi/ml of
[3H(G)]hypoxanthine
(asubstrate for HGPRTase)overanidentical 18-h period. The added mass of hypoxanthinewas
kept low ('- 120 nM), and labeled glycine replaced an equal mass of unlabeled glycine in the RPMI 1640 media, so as not to perturb the conditions under which ATP/GTP had been assessed by mass in the preceding studies; in addition, all other experimental conditions re-mainedunchanged. After 18 h, islets were extracted and analyzed by HPLC asindicated (1) except that an additional rapid wash in 1 ml KRBbuffercontaining 0.5% BSA was added in order to remove more completely anyunincorporated radiolabeled compounds adherent to theislets. Eluant fractions were collected every 30 s (glycine label) or every 18s(hypoxanthine label) using a fraction collector (Multi-rac 21 1;LKBInstruments, Bromma, Sweden) and were matched against theexacttimes ofelution of GTP and ATP using the concomitant UV
chromatograms.Samples were counted for 5 min in 4 mls of scintilla-tion cocktail.
Data presentationand statistical analysis. Absolute insulin secre-tion is expressed as ,uU/ 10 islets-45min (mean±SEM) for static
insulin released fractional release = . . r
initial insulin
content]
where the denominator equals the insulin content remaining in islets at the endof the incubation period plus that insulin released during the incubation period. Fractional release is expressed as percent of initial insulincontent released per 45 mins.
Nucleotide content is expressed as pmol/islet; specific activity is expressed as dpm/ pmol. Increments or decrements in nucleotide con-tent are expressed as the concon-tent ofeach experimental tube (minus) the meancontent of the control tubes in that study; alternatively, experi-mental values are expressed in terms of percent of their mean control values (the glucose concentration used to define the "control" response for each experiment is indicated in the text or figure legends). Data are expressed asmean±SEM,with (n) representing the number of obser-vations in a representativestudy, or the number of separate experi-ments, as indicated. Statistical analyses were by paired or nonpairedt
tests, and ANOVA, asappropriate.
Results
Dependency on glucose concentration ofthe inhibition by MPA
of
glucose-induced insulin release. Glucose concentrations
from 50-200 mg/dl (2.8-1
1.1mM)
werechosen for
theover-night culture period. 25 ,ug/ml of
MPAwasused in
allstudies
since it induces
amaximal inhibition of
GTP
synthesis (1).
It wasconfirmed
( 1) that
anovernight
exposure to MPA inhibitssubsequent insulin release induced by 16.7
mMglucose;
how-ever,the
inhibitory effect of
MPA wasinfluenced dramatically
by the
glucose
concentration
presentduring
the preceding 18-hculture
period (Fig.
1). Islets cultured
at7.8
mMglucose
were atleast
assensitive
to MPAas wereislets cultured
at 11.1
mMglucose, but
sensitivity
to MPAdeclined steeply
atlower
glu-coseconcentrations, being
half-maximal
at6.4
mMglucose,
and
essentially
absent
at4.4
mMglucose (Fig. 1).
Inpaired
analysis of
data accrued
within
the
sameexperiments,
inhibi-tion
wasalways
greaterwhen
MPA wasprovided
at7.8-1
1.1 mMglucose (-51±6%) than
at<6.4
mM(-13±6%;
P<
0.001 for six
paired glucose-concentration comparisons
within five
separateexperiments,
asdepicted
by individual
symbols in the inset
toFig.
1).
In ourcumulative
experience
using
MPA(including
data in reference
1),
there
wasnoover-lap in the
rangeof inhibition by MPA
athigher
glucose levels
(range: 34
to86%;
n =34
separateexperiments)
and that
atlower
glucose level (range: 0
to34%;
n =6;
P <0.001
by
nonpaired
ttest). Such
comparisons
underestimate the
differ-ence at
higher
vs.lower
glucose
concentrations,
since preexpo-sure to MPA at4.4
mMglucose actually
augmented
subse-quentinsulin release in
twoexperiments;
these values aretreated
as "zeroinhibition"
for statistical purposes. It is notlikely
thatthe
findings with
MPA represent aaberration
spe-cific
tothat
pharmacologic probe,
sinceasecond,
selectivein-hibitor of inosine
monophosphate dehydrogenase (75
,tg/ml
mizoribine) did
notinhibit
subsequent
insulin
release at allwhen
provided
at4.4
mMglucose (data
notshown),
in
contra-distinction
to aninhibition of
47%(absolute release)
andof
52%
(fractional
release),
seenafter 11.1
mMglucose.
These results
might
beexplicable
if
MPAwere to exertaglucose-dependent
effect
oninsulin
contentof islets.
Asprevi-ously
observed(1),
MPAdid,
infact,
reduceinsulin
contentafter culture in
high glucose (by 20±4%)
whereas afterexpo-0~
._
0-0) 0.
0)
-UL)
01)
01)
U1)
0
C.)
0
C
.0
-c
C
70r
60
50
F
40 F
0
0 X
0
A~~~x~
/ 80
40
<6.4mM 7.8-11.1mM
/(X11
(140-200/VS mg/di) m/d1)
30
F
20 H
2.8
4.4
5.6
6.4
7.8
(50) (80) (100)(115) (140)
II.1mM (200mg/dl)
[Glucose] During Culture Period
Figure1.Glucose dependency ofMPA inhibition of insulin secretion.
The glucose concentration duringthepreceding18-h culture period
isexpressedontheabscissa and the inhibitionby MPAofthe
subse-quentincremental insulinsecretoryresponsetoamaximalglucose
( 16.7 mM) stimulus is presentedontheordinate.Each symbol
com-prises themeanof multiple (3-8) determinations carriedoutafter
cultureatthe indicatedglucose concentrationon asingle day, with
eachsymbol representingasingle such experiment. Identical symbols
indicatefindings withinasingle experiment.Thehorizontal lines in themainpanel indicate themeanresponseateach glucose
concen-tration. Statistical analysis (by pairedttest) is presented in the inset, inwhichadividing point of 6.4 mM glucosewasusedtoanalyze
re-sponsestohighervs.lowerglucose concentrations during culture. Pairedresponseswithin thesamestudyareconnectedby solid lines.
sureofisletstolowerglucose levels,MPAreduced insulin
con-tentby only7±3%(n= 5experiments;P<0.01;TableI).In
fact,at2.8mMglucose,MPAactually increasedinsulin
con-tent(Table I). However,theeffects of MPAonexocytotic
re-leasewereindependentof theseeffects of MPAoninsulin
con-tent, since fractional insulin release (insulin release/insulin content)wasalsoreduced much lessbyMPAduringculture of
isletsatlowerglucoselevels (- 14±7%;P = ns vs. noMPA) than after cultureat7.8-1 1.1 mM glucose (-44±5%;n = 5
experiments;P<0.0001vs. noMPA;P<0.01vs.inhibitionby
MPAatlowerglucose levels;TableI).
Effects ofglucoseonGTP and A TP content, and their
de-pendency on MPA-inhibitablepools. In orderto explain the
glucose dependency of MPA effectsonsecretion,the
nucleo-tidecontentofisletswasanalyzed usingasimilar
experimental
paradigm. In addition, we estimated the proportion of thesteady-state GTP (and ATP) content that was derived
ulti-matelyfromIMP, by taking advantageof the facts thatMPA is
Table
I.Insulin
Content(ApU/10
islets) of
PancreaticIslets,
and Fractional Insulin Release(%o
* 45min),
in the Presence and Absenceof
PriorExposure (X18 h) to MPA at Lower vs. Higher Glucose Concentrations * t4.4-6.4mMglucose$ 7.8-1 1.1 mMglucose P
A. Insulin
content*
(-) MPA 10,914±700 8,009±1,016 <0.05
t
t
P<0.05 P<0.01
(+) MPA
10,079±450
6,282±628 <0.01B.Fractional insulinrelease
(-)MPA 3.35±0.91 6.05±0.54 <0.05
t
t
NS P<0.000I
(+)MPA 3.11±0.49 3.37±0.35 NS
* Dataare mean
(±SEM)
for five separateexperimentscomprised
ofsixsetsofpaired
dataatdifferentglucose
concentrations(as
indicated in legendtoFig. 1). *In anadditionalexperiment,MPAreducedinsulincontentby
30%at11.1 mMglucose (control: 8,359±756,
n =7;
MPA:5,872±394,n=7,P<0.01)whereasinsulincontent
actually
roseby
20% in thepresenceof MPAat2.8 mMglucose
(control: 10,459±498,n=8; MPA: 12,504±808,n=8;P<0.025).The controlinsulin secretoryresponseto
glucose
wastoolow for the effect of MPAtobeinterpretable
inthis experiment.
a
selective inhibitor of GTP
synthesis
atthe level
of
IMPdehy-drogenase (
18-22) with
nodirect effects
on enzymes of the"salvage" (or
other) pathways
(20, 21, 23, 24).
The
contribu-tion of IMP-dependent pools
ateach glucose level
wasdefined
asthe decrement
inGTP induced by
amaximally-effective
concentration
of
MPA(25
#g/ml;
reference 1), whereas the
contribution of
IMP-independent pathways
tosteady-state
GTP
content wasestimated
asthe
amountof GTP resistant
toblockade
by
MPA.Additionally,
the increment in GTP
con-centration induced by
751uM
of
guanine, provided
exoge-nously,
wastaken
as a measureof
netsalvage pathway
activity
(i.e.,
reflecting hypoxanthine-guanine
phosphoribosyltransfer-ase
activity).
Culturing
islets
at 11.1 mMglucose
reproducibly
increased
their
contentof
both GTP
(by 24
to33%) and
ATP(by 13
to20%) above values
seen at4.4 mMglucose; UTP also
rose(seebelow)
(Figs. 2-5). Therefore,
total NTP contentalso
in-creased
by 33%
from
13.77±0.79
to18.38±1.24
pmol/islet
(df
1
1;
P<0.01 ). These
effects
were notexplicable
by
anonspe-cific (e.g.,
osmotic)
effect of hexoses, since osmotic controls
were present in all studies (see Methods); furthermore, theaddition
to 4.4 mM glucoseof
6.7 mM 3-0-methylglucose(which is
transported similarly to glucose but which is notme-tabolized; reference
25) actually
slightly
inhibited
(by 5, 6, and10%,
respectively)
contentof
GTP, ATP, and UTP (data notshown). Furthermore, the
NTP contentof islets declined if
the glucose level was reduced from 4.4 to 1.7 mM glucose (TableII);
it fell
to acomparable degree when 3.0 mM of mannohep-tulose(which
blocks thephosphorylation
andfurther
metabo-lism
of
glucose; reference 26) was added to 4.4 mM glucoseduring
culture
(TableII).
Additionally,
MPAreduced GTP or ATP content more at 11.1
mM glucose than at 4.4 mM glucose (both P < 0.001;Figs.
2, 3, and 5). Thus, despite starting from higher levels at1
1.1
mMglucose,
MPAreduced
GTP
contentby
a greater amountand
tolower absolute levels
athigh glucose (Figs.
2and
5). Furthermore,
during
MPA treatment,glucose
nolonger
increased GTP (or ATP)
content;rather, the residual
a) c
0
a)
(I)
0
4-0
2
E
0
I--0
[Glucose]
u*'MPA,25,ug/mI
4mM 4.4mM 11.1 mM II.ImM
_+ -+
Figure2. Glucose dependency of GTP contentof islets and its
inhi-bitibilitybyMPA.Barsareexpressed as%of a common reference value (4.4 mM glucose alone). Data are expressed as mean(±SEM) for six determinations each, fromtwoseparateexperiments showing identical results. The rise in GTPat11.1 mM glucose is statistically
0)1
0
Z
120-4;
(10.21t0.6O.
pmol/islet) 00
0_ (6)
CD
E 080
(- 40_
'4-0
a
20
[Gl ucose) 4.4mM
MPA,25,ug/ml
-4.4mM
+
A= -5.24t0.45
pmol/islet; p<O.O0I (-31±2%) (6) 11 mm T+V
Figure3. GlucosedependencyofATP contentofislets andits inhibiti-bility byMPA.Barsareexpressedas %ofacommonreferencevalue (4.4 mM glucose alone). Data areexpressedasmean(±SEM) for six determinations each, fromtwoseparate experimentsshowing
identi-calresults. The 20%risein ATPathigh glucosewas notstatistically significantwithin thisstudy,butwas seenineveryexperiment.
GTP
content was nowlower
(P
<0.05)
athigh
glucose
after MPA(cf. Figs.
2
and
5).
The increment in both
purine
and
pyrimidine
nucleotides
induced
by glucose
suggeststhat
anelevation in theambient
glucose
concentration
might
increase the
contentof
aninter-mediate
required
in
both
pathways (possibly PRPP;
seebe-low). To
testthis
possibility further,
exogenous
guanine
wasprovided
in order
tofuel
the
"salvage" pathway by
direct
cou-pling
to PRPP.When 75
,uM
guanine
wasprovided
at4.4 mMglucose
(in
the absence
of MPA),
GTP
content rosesignifi-cantly (by 33%,
orby
0.65±0.06
pmol/islet)
to levels now01) c
-o
o 0 08E
4- S. CX 4-:[Glucose]
MPA,25,ug/ml
similar
tothose
seen at11.1
mM glucose alone (Fig. 5), whereas,in
contrast, no significant further rise in GTP (Fig. 5) or ATPwas seenwhen guanine
wasprovided
at 11.1
mM glu-cose(Fig. 5).
However, when guanine (75,uM)
was providedin
the presenceof
MPA, islet content of GTP rose 65% more,and reached higher final levels (2.24±0.13
vs. 2.99±0.22; P<
0.05)
atthe
higher
glucoseconcentration despite again
start-ing from
alower absolute level
(P <0.05) after
MPAtreatment
(Fig. 5). Likewise, guanine increased
ATPcontent at 1 1. 1 mMglucose only in
the presenceof
MPA; ATP rose from7.32±0.33
to 11.5±0.16 pmol/islet
(P <0.01
). These findings supportthe
formulation that
aregulatory
precursor(presum-ably PRPP) does indeed rise with high
glucose levels but isshunted
into
IMP-dependent
pathways; however, when thispreferred pathway(s) is
renderedunavailable
due to the blockimposed
by MPA, themetabolite
becomes free to fuel thegua-nine-induced
"salvage"synthesis of
GTP as well (cf. references27
and28).
Effects
ofglucose, MPA, and/or guanine on UTP content.
Theformulation
aboveis
supported by measurements of UTP. MPAhas
been observed
toinduce
arise in
cellular UTP con-tent( 1,
21, 29)
attributable
to anincrease
in the availability of
PRPP2
( 1,27, 28,
30, 31 ) which is
then shuntedinto
thepyrimi-dine
synthetic
pathway.
Therefore,
wequantitated
UTPduring
the
currentstudies
to assessindirectly whether glucose
in-creasesavailability of
precursors
for
NTPsynthesis.
Areduc-tion in glucose
concentration (from
4.4
to 1.7mM)
orthe
addition of
mannoheptulose
to4.4
mMglucose reduced
UTPin parallel
tothe changes in GTP
and ATP (Table II).Recipro-cally,
increasing
glucose
from
4.4 to 11.1 mM glucose nearly
doubled UTP
(Fig.
4; Table III).
Inthe
presenceof MPA, UTP
levels
rose bysimilar
amounts at both glucoseconcentrations
2. PRPP content reflects changes in both its synthesis and consump-tion. MPA disinhibits PRPP synthesis (11, 31 ) by reducing purine
nucleotide synthesis.Inaddition,IMP,which rises (23, 28) proximal to theMPA-inducedblock at IMP dehydrogenase (32), can be degraded
toinosine (33, 34) whichinislets(35) as in other tissues (4, 28, 31 ) is
degradedtoribose-l-phosphate (36). Ribokinase action converts ri-bose-
I-phosphate
toribose-5-phosphate,which could increase synthe-sisofPRPP(33, 36),which,in turn,is utilized for UTP synthesis.(+253+ 16%) ;(6)
...
...* X X@@
*@@@@ * @* * * * * * * * * @ @ @ @ *@*... *-*@
-*@--s*
*** * * **.... ** * * * * * @ @ @ . ... ... ... ...... ... ... ... .@.@.*.*.*. * ... ... *-**@ * @ @* @@@ @ - * * *@@-@. *@@** * * @ @ . . *@@@@ *@@*** *@@@@ * * @ @ @ * **@-@@@@@@-*@ @ @ -* @ @@ @ @ *@@@@
* s ss . . *s s s s *.s.s.s.s.* * * * * * . **@-X-X-X@*@@* *
* * - * * . ***@@@---@-- *@ @ @ * I I.lmM + + +2.70±0.22 pmol /islet; p=ns
Figure4.Glucosedependencyof UTPcontentof islets and its stimulationbyMPA. Barsare
ex-pressedas %ofacommonreference value
(4.4
mMglucose alone). Dataare
expressed
as mean(±SEM)for six determinations
each,
fromtwoCultured in
- 4.4mM I
glucose
but
again reached
ahigher
final
levelat 11.1
mMglucose
(P
<
0.001; Fig.
4; and Table
III,
Experiment
Ivs.2); conversely,
exogenous
guanine (which
consumes PRPP in thesalvage
pathway), reduced
the UTP levelsachieved
inthe presence of MPA(Table
III) towards control values (no
MPA) (Table III).
Thus,
provision of
guanine largely reversed the effects of
MPA onUTP,
as itdid on GTP and ATP content(reference 1; Figs.
2and
3). To
summarize,
glucose and
MPAboth
increase,
andguanine decreases, UTP (probably
due tochanges
in both PRPPsynthesis and
consumption;
seeDiscussion).
Effect of
guanine
oninsulin
release.
The
effect
oninsulinrelease
of
guanine
supplementation of
the culture
medium
wasalso
examined
(Table
IV). By
itself, guanine (30-100
,qM)
hadlittle
effect
onsubsequent secretion after
culture,
whether
pro-vided
at 4.4 or 11.1
mMglucose (Table IV).
In contrast,in the
presenceof MPA,
guanine increased
(i.e., restored)
insulin
re-lease in
proportion
tothedegree
of
inhibition of GTP
contentand
insulin release caused by MPA, i.e.,
to alarge degree
at 11.1
mMglucose and
minimally, if
atall,
at 4.4 mM glucose(Table
IV). Thus,
guanine,
via the salvage pathway,
reversesthe
defi-ciency of
pools of GTP
involved
in
exocytosis,
which had been
inhibited by MPA.
Table II.
Effect*
ofGlucose Concentration and Mannoheptuloseon NTPContents
of
PancreaticIslets After
anOvernight CultureCondition GTP ATP UTP TotalNTP
A. Control (4.4mM
glucose) 2.10±0.07 7.83±0.24 1.42±0.05 11.36±0.33
B. 4.4 mMglucose
+3mM 1.71±0.11 6.35±0.31 1.74±0.11 9.11±0.48 mannoheptulose (-19%) (-19%) (-17%) (-19%)
C. 1.7mMglucose 1.74±0.11 6.76±0.41 1.09±0.07 9.60±0.57
(-17%) (-14%) (-23%) (-15%)
*Values represent mean(±SEM)ofthree determinationseach.Comparisonof
experimental conditions(B)or (C)vs.their respective controls(A)were
statisti-callysignificant(P<0.05orgreater) foreachnucleotide.
Figure 5. Glucose dependency of the GTP content ofislets,
in thepresenceandabsence of MPA (25
,g/
ml)orguanine(75 ,M). High glucose (1
1.1
mM)
increased GTPcomparedto4.4mMglucose (open bars); exogenously-provided
gua-nine by itself (cross-hatched bars) increased GTP
signifi-cantly onlyatthe lower glucose concentration. Effects of MPAareshown(stippled bars)asis the addition of
guanine
toMPA(solidbars).Valuesare mean(±SEM)for 2-3
de-terminations each.
Radiolabeling of denovo orsalvage GTP synthesis (Table V). These results together suggested that glucoseaugmentsflux into MPA-sensitive (i.e.,
IMP-derived)
pool(s) of GTP.How-ever,since IMPcanbe derived via several pathways,we
ascer-taineddirectly whether denovoand salvage pathways
contrib-ute toGTP and ATP synthesis in islets. Purine nucleotide
pools
were labeled using ['4C]glycine (which is incorporated into
nucleotides specifically via the de novo pathway) or [3H
I-hypoxanthine (an index of salvage pathway activity), under conditions
(see "Methods")
essentially identicaltothose usedto ascertain the bulkcontent (mass) of ATP/GTPin islets.
Glucose
progressively augmented the labeling of both nucleo-tidesusing eitherprecursor(Fig. 6). High glucose (11.1 mM)increased theareasunder thecurvefor the GTP peakto208 and 331% of values at4.4 and 1.7 mM glucose, respectively (labeled glycine) and to 190 and 279% (labeled hypoxan-thine); corresponding values for ATPwere215 and 307%
(la-beled glycine) and 174 and 353% (la(la-beledhypoxanthine)
(Ta-ble V and Fig. 6). Similar changeswere seen in the specific activities (dpm/pmol) of GTPorATP (Table V).Discussion
Choice and relevance of experimental model. These studies
weredesignedtodetermine whether islet GTPcontent,which hasapermissive effect in exocytotic insulin release (1), is
modi-fied by the ambient glucose level. The experimental paradigm is basedonthe specific inhibition by MPA (or
mizoribine)
of IMP-dependent GTP synthesis, as assessed by steady-state GTPcontent (thebiologically
relevant parameter; reference 1).These drugs (8-10, 18-20) block the conversion of IMPtoxanthosine monophosphate (XMP), possibly also reinforcing thiseffect by
inhibiting
conversionof XMPtoguanine nucleo-tides( 19-21 ), but withoutanydirect effectsonothernucleo-tidepathways (20-24). RPMI 1640wasselectedasthe culture
medium since it is purine-free (thus permitting manipulation
ofsalvage pathway activity) and has anadequate
Table III. Effect ofGlucose Concentration During Culture, in the Presence and Absence
of
MPA(25tg/ml)
and
Guanine (75,1M),
on UTP ContentofPancreatic IsletsCondition UTP,pmol/islet*
Experiment 1. MPA absent
a. 4.4 mMglucose b. 1 1.1 mM glucose
c. 4.4 mMglucose+guanine d. 1 1.1 mMglucose+guanine
1.92±0.05 3.43±0.19J
2.12±0.08 2.90±0.40
A = 1.51 pmol/islet: df 4; P<.01
A = 0.78 pmol/islet: df 4;n =ns Experiment 2. MPA present throughout
a. 4.4mMglucose+MPA b. 1 1.1 mMglucose+MPA
c. 4.4 mMglucose+MPA+guanine
d. 1 1.1 mMglucose+MPA+guanine
4.59±0.36 6.02±0.14 2.60±0.
191]
4.31±0.17§JA = 1.43; df 3; P < .05
A = 1.71;df3;P<.01
*Values are the mean
(±SEM).
t P<0.02vs.guanine-freevalues. § P<0.005 vs.guanine-freevalues.cofactor for
synthesis of
PRPPand
of
purine nucleotides ( 12,
37-40).
10%undialyzed FBS was provided as a potential
source(33,
41,
42)
of small
amountsof
hypoxanthine,
in order
tomimic
the
presenceof
the low
amountsof hypoxanthine
circulating
in blood (43-45). This
amountof hypoxanthine
supports abasal
production
of salvage
pathway-derived
nu-cleotides and thereby provides
aphysiologic restraint
onthe de novopathway (41 )
without
which the visualization of
a regula-toryrole
for
PRPP concentration in de
novopathway
activity
might
be
obfuscated
(33).
Additionally,
a narrowrangeof
po-tentially
relevant glucose concentrations
wasstudied.
How-ever, aswith
anyin
vitro
study, extrapolation of these findings
tothe
situation in vivo should be made with caution.
Effects of
MPA
onGTP
contentand insulin release.
Inourprevious
studies
at 1 1.1 mMglucose,
asaturating
concentra-tion
of
MPA
(25 ,ug/ml)
reduced GTP
by
-80% and
glucose-induced insulin release by 54±2%. The
currentstudies confirm
these
observations
atnearly-normal glucose
levels
(7.8 mM)
during the antecedent overnight culture period.
A central newobservation
wasthat
the
inhibition of insulin
releaseinduced
by
MPA(or
mizoribine) declined steeply
atonly
slightly
lowerglucose levels,
being half-maximal
at 6.4 mM glucose andvir-tually absent
at4.4 mM.
These
studies
suggestthat
even mod-estelevations in ambient glucose
concentrations sensitize islets
toinhibition by structurally
dissimilar,
selective inhibitors of
IMPdehydrogenase. These
findings imply
thathigh
glucose acts as abiologic
"switch"
toshunt nucleotide
precursorspref-erentially
into IMP-dependent pathways, rendering islet GTP
content moresusceptible
toinhibition by the blockers. One
might question whether
the markedly bluntedinhibition
by MPAof
insulin
secretion
seenafter islet culture
atlower glu-coselevels could be attributed
tothe
relatively
modest
differ-ences
in
the reduction of GTP
achieved by
MPA(inhibition of
GTP after
1 1.1 mMglucose
=80%; inhibition of
GTP
after
4.4
mMglucose
=63%). However, this does indeed
seem tobe the
case,since this effect of 25
,tg/ml
MPA at4.4
mMglucose
is
Table
IV.Effects
of
MPA(25
tg/ml)
inthe Absence and
Presenceof
Guanine, During
Overnight
Cultureat4.4or11.1mM
Glucose,
onSubsequent
Insulin Secretion*Incremental insulin response,
AsU
* 45 minI. Cultureat4.4 mMglucose
a. Control(5)
b. MPA(5)
c. MPA+75
gM guanine (5)
d. 75
gM
guanine
alone(5)
343±32]
275±30]364±12 226±28
-20% A =
89±12
II. Cultureat11.1 mMglucose
a. Control(5)
b. MPA(5)
c. MPA+75,M
guanine (4)
d. 75uM
guanine
alone(3)
433±28 212±16 479±38 554±56
-51% A=267±39.
*Numbers in
parantheses
arethenumbers of observations. Dataaremean±SEM.
[3H(G)]
Hypoxanthine
C
%
a. aMinutes
nearly
superimposable
onthe effect of
amuch lower
concen-tration (0.5ptg/ml)
ofMPAprovided
at11.1
mMglucose
(GTP:-60±2%;
insulinsecretion;
-12%)
inourprevious
stud-ies(
1).
Inother words,
MPA isonly
1/ 50th
aspotent onexocytotic secretion
atlow ascompared
tohigher glucose
con-centrations. This is because insulin release isnotinhibited
until GTP content falls(by
>80%)
to alevel of<0.6-0.8
pmol/is-let(
1),
butfalls
offprecipitously
thereafter.
Thus,
the
lesser effect of MPA on insulin release afterculture
inlower
glucoseconcentrations reflects
adifferent
origin (and
therein
alesserinhibitability
by MPA) ofthe GTP
content.However,
not only GTPderived
via IMPbut also that derived via the
guaninesalvage pathway
is
capable
of
supporting secretion,
since
exoge-nousguanine restores basal- and
glucose-stimulated
GTP
lev-els inMPA-treated
islets(current
data) and,
concomitantly,
reverses
the
effect of MPA
toinhibit insulin secretion
(current
data andreference 1).
Thus,
these studies
provide
noevidence of afunctional compartmentation
of(MPA-sensitive
vs.MPA-insensitive)
cytosolic GTP
poolswith regard
toexocytosis.
Effects of
glucose
onnucleotide
content.Increasing
glucose
levels modestly but consistently augmented
totalislet
GTP content(by
40-50%,
between
1.7and
11.1
mMglucose).
These
findings
arein accord with studies of Zunkler
etal. (46),
Hoenig
and
Matschinsky (47),
and Meglasson
etal.
(48)
whonoted
small changes in GTP
content(or
GTP/GDP ratio)
within 30-150 min
of
marked
increases
in the glucose
concen-tration
towhich pancreatic islets
wereexposed.
Glucose alsoslightly
(but
again consistently)
increased
ATPlevels and
dra-matically
increased
UTPlevels. The mostparsimonious
expla-nation for these findings is
that glucoseincreases
theavailabil-ity of PRPP,
theprecursor common
to allthree purine
orpy-rimidine nucleotides. Since
theS05 (PRPP) for
the de novopurine synthetic pathway is higher than the
Km(PRPP) in
thepyrimidine synthetic
and purinesalvage pathways
( 1 1, 12,31,
32, 37, 38, 49),
andsince
PRPPavailability
isprobably
regula-tory for
all three ( 1 1, 41,50,
5 1),
anincrement
in PRPPmight
Figure
6.Glucosedepen-dency
ofpurine
nucleotidelabeling
with[3H(G)]-hypoxanthine (A)
or[
'4C(U)Jglycine
(B).
The firstpeak corresponds
toATP and the second
peak,
toGTP.Valuescomprisethe meanof 3-6
determinations
ateach
glucose
concentration fromtwoindependent
exper-> imentsandareuncorrected
a for
recoveries,
whichaver-aged
96%.Eachinjection
comprised
thenucleotidesextractedfromthe
equivalent
of33
(for hypoxanthine
la-bel)
or67(for glycine label)
ratislets.Resultsat1.7 mM
glucose
areexpressed
by
(o
- --o),
at4.4 mMglu-cose
by
(A
A),
andat1
1.1
mMglucose by
(0 0).
indeed
beexpected
tobeutilized
by
theUTP
pathway
andby
HGPRTase
first(36, 41),
followed
by
the de novosynthetic
pathway. Additionally,
it isenergetically
reasonable(
11)
thatsalvage pathways
would bepreferred (41, 52)
atlower levels
of fuelavailability (27, 36), since,
unlike
the de novo3pathway,
itdoes
notdirectly
consumeATP. Effects
ofglucose
onNTP
contentwere seennot
only
uponincreasing glucose
concentra-tion,
butalso,
upondecreasing
itor uponimpeding glucose
phosphorylation through
the
use ofmannoheptulose.
The
latter
findings,
inconjunction
with thelack
ofastimulatory
effect
of3-O-methylglucose,
demonstrate
that theglucose
in-creases
NTP
contentonly
after itsmetabolism, perhaps
toribose-5-phosphate (the
precursorof
PRPP).
The conclusion that
glucose
preferentially
shunts GTP
pro-duction from
IMP-independent pathways
toIMP-dependent
pathways
issupported
by
four
findings:
First, MPA,
aselective
inhibitor
ofIMP
dehydrogenase,
reduced GTP
content more athigher glucose
levels.
Second,
MPA
prevented
theglucose-in-duced increment
inGTP
(and ATP). Third,
theresidual GTP
remaining
after MPAexposure4
wasconsistently
smaller
athigher glucose
concentrations.
Lastly,
exogenousprovision
ofguanine (to
fuel
thesalvage
pathways)
stimulated
agreater rise inGTP
athigher glucose levels,
butonly
when fluxfrom IMP
to
GTP
wasprevented
by
treatmentwithMPA.
In contrast, the3. In additional
studies,
wehave documented thatattheglucoselevelsstudied, hypoxanthine salvage
contributesmore tothemassofGTP and ATP in islets than does the denovosynthetic pathway(Meredith,
M.,
M.Rabaglia,
andS.Metz,
manuscriptinpreparation).
4. This
pool
ofGTPmight
reflect severalpotentialsourcesofGTP derived viatransphosphorylationorsubstrate(GDP)levelphosphory-lations
(including
nucleoside diphosphokinase,succinatethiokinase,
andphosphoenolpyruvate carboxykinase) aswellassomesalvage of
endogenously-produced
guanine. Note that nucleosidediphosphoki-nase
(reference
46 andKowluru, A.,andS.Metz, unpublishedTableV.Effects
of
Glucose ConcentrationDuring Overnite Culture on theIncorporation*ofLabeledPrecursorsintoGTP and ATPt
Absolute areas under Specific activity, dpm/pmol the peaks, totaldpmt
GTP ATP GTP ATP
A.
['4C(U)]glycine
label (de novosynthesis)i. 1.7 mM glucose 0.45±0.02 0.78±0.01 57±3 359±8
ii. 4.4 mM glucose 0.70±0.04 1.15±0.10 90±6 512±38
iii. 11.1mMglucose 1.22±0.09 2.06±0.06 187±14 1103±36
B. [3H(G)]hypoxanthinelabel
(salvage pathway)
i. 1.7 mM glucose 66±3.18§ 78±4.26
3,645+30
16,172±569ii. 4.4 mMglucose 70±3.61§ 124±0.88 5,374±231 32,817±2491
iii. 11.1 mMglucose 132±9.56 219±2.17
10,186+679
57,096±1195*Values are expressed as specific activity(dpm/pmol)or areas under the peaks; mean
(±SEM)
for 2-6determinationseach from twoindepen-dentexperiments. IAllcomparisonsfor ATP or GTP between different glucose levels are statistically significant (P < 0.05 or greater level of
significance)exceptforthesingle comparisonindicated
by
I(hypoxanthine labeling of GTP at 1.7 vs. 4.4 mM glucose). *200islets per condi-tion were culturedoverniteattheindicatedglucoseconcentrations inthe presence of 10ACi/ml
['4C(U)]glycine
or 2MCi/ml
[3H(G)]hypoxanthine,
and then were extracted. Analiquotof the supernatant (equivalent to the content of 66 islets for glycine labeling and of33 islets for hypoxanthine labeling)wasanalyzed by HPLC and expressed as total counts under thecorrespondingcurves for data analysis.
exogenous
provision
of
guanine
did
notcause anincrementin
GTP
contentathigh glucose
levels in the absence of MPA(i.e.,
where
feedback inhibition of the salvage pathway by
endoge-nousnucleotides
wasmaximally
operative);
this
indicates,
in
addition, that glucose
probably
does
notdirectly
stimulate
gua-nine
uptake
orsalvage
pathway
activity.
Theseobservations
indirectly
supportthe
formulation suggested
above that
glu-cose
might
increase PRPP levels
which,
in the absence ofMPA,
is funneled preferentially by glucose,
first intopyrimidine
(UTP)
synthesis,
and then into the
hypoxanthine
salvage and
de
novopathways. Rigorous study
of the fluxesthrough
the
biochemical pathways involved,
and
direct
measurementsof
PRPP
availability,
will be
required
tosubstantiate
orrefute
these
tentative
conclusions,
since itcannot be assumedthat
physiologic
changes in
glucose
metabolism
wouldnecessarily
increase
PRPPsynthesis. However,
this
formulation isrein-forced
by
the studies of Pilz
etal.
using
humanlymphocytes
(27), in which
they observed
that
increasing
extracellular
glu-cose
concentrations
augmented
intracellularribose-5-phos-phate and
PRPPconcentrations, promoted
denovopurine
nu-cleotide
synthesis,
and
rendered
the nucleotidecontentof thecells
moresusceptible
toblockade
by
inhibitors of de novosynthetic pathways.
These
investigators
noted that inhibitors ofde
novoGTP
synthesis
further augment
PRPPavailability2;
ifthis
occurs inislets,
it would
explain
the additive effects
ofhigh
glucose and
MPA onUTP
contentof islets.
Relationship between
GTP
contentand
insulin secretion.
In ourstudies,
modest increments
in GTP contentinducedby
guanine
at4.4 mMglucose (to
levels identicaltothoseseenat1
1.1
mMglucose alone;
Fig. 5)
had little effect
onsubsequent
insulin
release,
suggesting
that GTP levels
must beclose
tosaturating
for
secretion
atphysiologic glucose
concentrations.
Infact,
areduction of
upto60% in GTP
contentinduced
by
submaximal
concentrations of
MPA(up
to0.1-1
,g/ml)
at1
1.1
mMglucose (
1)
donotdetectably
impair subsequent
se-cretion.
However,
whenGTP
contentis inhibited more(see
above)
thereis
asteepdecline
insubsequent
insulinsecretory
responses
(and, reciprocally,
aconsiderable
rise in both GTPand
insulin
release when
exogenous guanine isprovided).
Thus, under physiologic conditions, glucose availability
may never becomelimiting for
the GTP involved in insulinsecre-tion
(leading
ustodefine its
role as"permissive"
only; refer-ence1).
However,
similar
studies
need to be carried out withregard
toother glucose-dependent effects
ofpurine
nucleo-tides; for
example, purine nucleotides
are utilized for RNAsynthesis
(6) and
DNAsynthesis
(7),which
in turn are neededfor islet
protein synthesis
and beta cell proliferation.
The
rise
in GTP (and in insulin secretion)
induced bygua-nine, and the fall in both
parameters induced by MPA, were ingeneral, inversely proportional
tothe starting
contentof
purinenucleotides,
which
arefeedback inhibitors of
salvage ( 11 ) andde
novo(
11,
31, 55 )
synthetic
pathways
as well asofthe
synthe-sis
of their
common precursor PRPP(1
1, 24, 41, 55-57).Thus,
ourstudies overall
arequite compatible with
datafrom
other
cells,
which
suggestthat
the cellular
contentof
NTPis
modulated by
twointeractive
variables-the negative feedback
control of nucleotides
upontheir
ownsynthesis
(at several
en-zymatic
levels;
references
11,
24, 31, 55-58) and positive
regu-latory control by
PRPP(27,
38, 41, 56).
Possible site(s) of
action
ofglucose
on
nucleotide synthesis.
We
hypothesized
that
glucose shunts substrates such
as PRPPinto
IMP;however,
it was unknown whether the twomajor
pathways potentially yielding
IMP(the de
novoand
salvage
pathways)
actually
arepresentin
islets.
Therefore,
weassessed
their
presence inislets
via, respectively,
the
incorporation
of
[
I4C]glycine
or[3H hypoxanthine
into
nucleotides. Our
stud-ies indicate
thatboth
pathways
cangenerate
ATPand GTP in
islets and
thatglucose
augmentsboth their
activities.
The
effect
of
glucose
is
even moreimpressive
when oneconsiders
thatglucose inhibits
the
uptake
of
glycine
into islets
(59).
Such
labeling studies
cannot beused
toquantitate precisely
the
rela-tive
contributions3
of
the twopathways
to theabundance
and/or ATP,
compared
toits effect
ontheir total
mass, sug-geststhat these
twopathways
may,in
fact,
be
selectively
acti-vated
by glucose.
This
finding
canbe
utilized
profitably
in
fu-ture
studies
to assess moresensitively
any acuteeffects of
glu-cose on
ATP
orGTP
synthesis.
Acknowledaments
Thetechnical assistance ofThom
Rabaglia
and Kenneth Finnaregrate-fully acknowledged.
These studieswerefundedin part
by
the VeteransAdministration,
andthe National Institutes of Health
(grant
DK37312).
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