Transplanted beta cell response to increased
metabolic demand. Changes in beta cell
replication and mass.
E Montaña, … , S Bonner-Weir, G C Weir
J Clin Invest. 1994;93(4):1577-1582. https://doi.org/10.1172/JCI117137.
We determined the capacity of transplanted beta cells to modify their replication and mass when stimulated by changes in metabolic demand. Five groups of Lewis rats were studied: group 1 (Tx-Px) had a 95% pancreatectomy 14 d after transplantation of 500 islets; group 2 (Px-Tx) had a 95% pancreatectomy 14 d before transplantation of 500 islets; group 3 (Tx) was transplanted with 500 islets; group 4 (Px) had a 95% pancreatectomy; and group 5 (normal) was neither transplanted nor pancreatectomized. Blood glucose was normal in Tx-Px and Tx groups at all times. Tx-Px-Tx and Tx-Px groups developed severe hyperglycemia after pancreatectomy that was corrected in Px-Tx group in 83% of rats 28 d after transplantation. Replication of transplanted beta cells increased in Tx-Px (1.15 +/- 0.12%) and Px-Tx (0.85 +/- 0.12%) groups, but not in Tx group (0.64 +/- 0.07%) compared with normal pancreatic beta cells (0.38 +/- 0.05%) (P < 0.001). Mean beta cell size increased in Tx-Px (311 +/- 14 microns2) and Px-Tx (328 +/- 13 microns2) groups compared with Tx (252 +/- 12 microns2) and normal (239 +/- 9 microns2) groups (P < 0.001). Transplanted beta cell mass increased in Tx-Px (1.87 +/- 0.51 mg) and Px-Tx (1.55 +/- 0.21 mg) groups compared with Tx group (0.78 +/- 0.17 mg) (P < 0.05). In summary, changes in […]
Research Article
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Transplanted
Beta
Cell
Response
to
Increased Metabolic Demand
Changes in Beta Cell Replicationand Mass
Eduard Montafla,SusanBonner-Weir, andGordon C. Weir
Research Division, JoslinDiabetes Center and the Department of Medicine, NewEnglandDeaconessHospitalandBrigham and Women's Hospital,HarvardMedicalSchool, Boston, Massachusetts 02215
Abstract
Wedeterminedthe capacity oftransplantedbeta cellsto mod-ify their replication and masswhen stimulated by changes in metabolic demand. Five groups of Lewis rats were studied:
group 1(Tx-Px) hada95%pancreatectomy 14d after trans-plantation of500islets;group2(Px-Tx)hada95%
pancreatec-tomy 14 d before transplantation of 500 islets;group 3 (Tx)
wastransplantedwith500 islets;group4(Px) hada95%
pan-createctomy; and group 5 (normal)was neither transplanted
norpancreatectomized.Bloodglucosewasnormal in Tx-Px and
Txgroupsatalltimes. Px-Tx and Pxgroupsdevelopedsevere hyperglycemiaafterpancreatectomythatwascorrected in
Px-Txgroupin83% ofrats28 daftertransplantation.Replication oftransplanted beta cells increased in Tx-Px (1.15±0.12%)
and Px-Tx (0.85±0.12%) groups, but not in Tx group (0.64±0.07%) compared with normal pancreatic beta cells
(0.38±0.05%)(P< 0.001).Mean betacell size increasedin Tx-Px(311±14
gm2)
and Px-Tx(328±13 Mum2)groupscom-pared withTx(252±12,tm2)and normal (239±9 Mm2)groups
(P<0.001).Transplanted beta cellmassincreased in Tx-Px (1.87±0.51 mg) and Px-Tx(1.55±0.21 mg)groupscompared
with Tx group (0.78±0.17 mg) (P < 0.05). In summary,
changesintransplanted beta cells prevented the development ofhyperglycemia in Tx-Pxrats. Transplanted beta cells
re-spondedto increased metabolic demand increasing their beta cellmass.(J. Clin. Invest. 1994.93:1577-1582.) Key words:
diabetes*islettransplantation-betacellgrowth * beta cellmass
*pancreatectomy Introduction
The firstsuccessfultransplants of islet cells in diabetic patients have been reported recently (1-4). However, restoration of
normoglycemiais oftennotachievedorhas limited duration.
Thecausesof the limitedsurvivalof transplanted islets in
dia-beticpatients andinlarge animals (5)arenotwellunderstood. Inadditiontorejection, nonimmunological factors could play
aroleinthesepooroutcomes.For example,impaired growth
capacity of transplanted islets could leadto adecline in beta
cellmassandcontributetograft failure.
AddresscorrespondencetoDr.Eduard Montafna, EndocrineUnit (
13-2), Hospital UniversitarideBellvitge, 08907 L'Hospitaletde
Llobre-gat,Barcelona, Spain.
Receivedfor publication 30August1993 andinrevisedform3
De-cember1993.
Amajorprobleminislettransplantationis obtaining
suffi-cient mass ofislet tissue for transplantation. A critical islet
mass must be transplanted to achieve normoglycemia, with
continuedsuccess beingdependent uponthe numberof ini-tially transplanted islets (6-8). Moreover, the islet mass
needed for islettransplantation is higherthatpredictedonthe
basis ofbetacellmeasurementsindiabetes(9).Anunknown factor in theseconsiderationsin thegrowth capacityof
trans-planted islets. Although some information about growth of
transplanted isletshasbeen obtainedoverthepast decade( 10-16), neither specific beta cell replication norbeta cell mass havebeen determined. Theimplicationsofchangesin betacell
replicationcanonly be understood when beta cellmassis also taken intoaccount.
We have shown that, in short-term successfulislet trans-plants, basal nonstimulated replication of transplanted beta cells issimilartothatofendogenouspancreaticbeta cells( 17).
The aim of this studywas todeterminethecapacity of
trans-planted beta cellsto modifytheir replication andmasswhen
stimulated by changesinmetabolicdemand. We have used the
partialpancreatectomy model ( 18-21 )tocreate asituationof increased metabolic demand in ratswith islet transplants in orderto determine thebeta cellreplicative capacity and the changes of thetransplantedbetacellmass.
Methods
Male Lewis rats (Harlan Sprague Dawley, Inc.,Indianapolis,IN) were obtained at5 wkofage. Ratsweretransplantedwith 500syngeneic islets under the kidney capsule, and/or underwent a 95%
pancreatec-tomyaccordingtotheprotocol describedbelow. Animals were bled andweighedonthedayoftransplantation,dayofpancreatectomy,and
atleastondays2, 5, 7,10, 14,18, 21, 25,and28after transplantation and/orpancreatectomy.Bloodglucose,determinedbetween 9 and 11
a.m.innonfasting conditions,wasobtainedfromthesnippedtail with
aheparinized microcapillary tube,andglucose was measured witha portableglucose meter(Accu-CheckII; Boehringer Mannheim Bio-chemicals, Indianapolis,IN). Animals were kept underconventional conditions in climatized rooms with free access to tap water and stan-dardpelleted food.
Animal groups.Fiveexperimental groupswerestudied (TableI).
Group 1 (n= 7):ratsweretransplantedand 14 d later underwent a
95%pancreatectomy(Tx-Px group); grafts andpancreaticremnants were harvested 28 d aftertransplantation. Group 2 (n= 12): a 95%
pancreatectomywasperformed and rats were transplanted 14dlater (Px-Tx group); grafts and pancreatic remnants were harvested 14(n
=6)or28 (n=6) d aftertransplantation.Group 3 (n= 13):rats were
transplanted andgraftsandpancreas were harvested14(n=6)or28 (n
=6) d later (Txgroup).Group4(n=6): rats underwent 95%
pancre-atectomy and pancreatic remnants were harvested 14 d later (Px group). Group 5 (n=6): normal animals that had their pancreases
removed(normal group). The beta cell mass of7groups of 500 islets isolatedondifferent days wasalsodetermined. Since beta cell replica-tion decreases with age(22),pancreatectomizedgroupswerematched for age at pancreatectomy(groups 1, 2, and 4) (7-8 wkold), and
J.Clin.Invest.
©P
The American Society for Clinical Investigation, Inc. 0021-9738/94/04/1577/06 $2.00Table I. Experimental Groups
Group n DayO Day 14 Day28 Day42
1 (Tx-Px) 7 Transplantation Pancreatectomy Harvest
2 (Px-Tx) 12 Pancreatectomy Transplantation Harvest(6) Harvest(6) 3 (Tx) 13 Transplantation Harvest (6) Harvest (7)
4(Px) 6 Pancreatectomy Harvest 5 (Normal) 6 Harvest
Day 0 denotes the day when thefirst experimentalprocedure wasperformedin each group. Groups 1, 2, and 4 were matched for age at pancre-atectomy and groups 3 and 5 had were matchedforage at harvestwithgroups 1 and 4, and group 2 at day 28. Figures in parentheses in groups 2and 3 show numberof animalsharvested at each timepoint.
groups 3 and 5 had the same age at harvest as groups 1 and 4. Rats were transplantedwithislets from5-7-wk-oldanimals.
Islet isolation and transplantation. The method for islet isolation and transplantation has been previously described for mice (17). Briefly, undersodium amobarbital anesthesia, pancreases were dis-tended with 6 mlofcold(40C)M-199medium(GIBCOBRL, Gaith-ersburg, MD)containing2mg/ml of collagenase(Collagenase from ClostridiumHistoliticum; Serva, Heidelberg, Germany), excised, and incubated in astationarybath at370C.Theisletswereseparated by a density gradient (Histopaque-1077; Sigma Chemical Co.,St. Louis, MO) andisolated isletswere handpickedunder astereomicroscope two or threetimes untilapopulation ofpureisletswasobtained.Only islets > 75 and < 250,umindiameterwerecollected.With this restric-tion thefinalyieldwas 200-250 islets per pancreas. Theisletswere transplanted under thekidney capsule oftheleft kidneyonthe dayof theisolation usinga200-,ul pipette tippreviously plugged with Gel-foamO(Upjohn,Kalamazoo,MI).
Pancreatectomy. Partial pancreatectomy (95%) was performed in 7-8-wk-old rats aspreviously described(18).Inbrief,animals were anesthetized with sodiumamobarbitaland 95%ofthe pancreas was removedby gentleabrasionwith cottonapplicators, being carefulto leavemajorblood vesselsintact.Thepancreaticremnant was thetissue between the commonbileduct and thefirst loop ofthe duodenum. Particularattentionwasplacedonremovingthe smallflap ofpancreas attachedtothepylorus. Nonpancreatectomized animals didnot un-dergoany shamoperation.
Graftand pancreas removal.On theday ofgraftremoval rats were injected with 5-bromo-2'deoxyuridine (BrdU)' (Sigma Chemical Co.), 100mg/kgi.p. body weight,and 6hlater thegraftwasremoved. The kidney capsule surroundingthegraftwasincisedand removed with thegraft.Usually all thegraftwas removed with thecapsule; when-everthegraftedisletswereinfiltratingthekidneycortex asecondpiece of tissuewasalso takento ensurethattheentiregraftwasharvested. ThegraftswerefixedinBouin'ssolution andprocessed forplastic em-bedding.Theweight ofthegraftwasdeterminedon abalancereading
to0.01 mg(A240; MettlerInstrumentCorp.,Hightstown,NJ)as de-scribed ( 17).Immediatelyaftergraftremoval,rats werekilledand the pancreas orthepancreaticremnantswereexcised, blotted,weighed,
and fixed inBouin's solution.Subsequently,thepancreatictissuewas
embedded inparaffin.
Immunocytochemistry.Sections ofgraftandpancreaswere double stained for BrdU and for the endocrine nonbeta cells of the islets with immunoperoxidase. Immunostainingfor BrdU used a cell prolifera-tion kit (AmershamInternational, Amersham, UK). Stainingfor the endocrine nonbeta cells usedacocktail of antibodies: rabbit anti-bo-vineglucagon(finaldilution, 1:3,000;giftof Dr. M. Appel,University ofMassachusettsMedicalSchool), rabbitantisyntheticsomatostatin (final dilution, 1:300; made in our ownlaboratory),andrabbit anti-bovinepancreaticpolypeptide, finaldilution, 1:3,000;gift ofDr. R. Chance,EliLilly&Co.).
1.Abbreviation used in thispaper:BrdU,5-bromo-2'deoxyuridine.
Betacell replication, individual beta cell area, and beta cell mass. Methods usedformeasurementof beta cell replication, area and mass have beendescribed in detail ( 17). For beta cell replication, beta cells andBrdU-positivebeta cells werecountedusingmicroscope (BH-2; Olympus Corp.,LakeSuccess, NY) connected to a video camera with a black andwhite monitor.Results were expressed as the percentage of BrdU-positivebeta cells. At least 1,200 cells were counted per graft or pancreas.
The meancross sectional area of individual beta cells, a measure of betacellsize,wasdeterminedonimmunoperoxidase-stainedsections ofgraftsandisolated islets.Forbothgraftsandisolated islets the beta cellnucleion arandomfieldwerecountedand the areaofthe beta cell tissuein thatfieldmeasuredwith anelectronicplanimetryprogram (Sigma Scan;JandelScientific,Corte Madera, CA). The beta cell area was divided by the number ofbeta cell nuclei to calculate the area ofthe individualbetacells. Aspointedoutpreviously( 17), the actual num-berofbetacells washigherthan the number counted,sincenot allbeta cells were sectioned across theirnucleiand,therefore,thesize ofthe beta cellswasoverestimated.
Beta cell mass was measured bypoint countingmorphometry on immunoperoxidase-stainedsections ofgraftandendogenous pancreas. Eachsectionwascoveredsystematically usinga48-pointgridtoobtain the numberofinterceptsoverbetacells,overendocrinenonbetacells, and overothertissue.Inpancreatic sections interceptsoverexocrine tissueandovernonpancreatic tissuewerecountedseparately.The beta cellrelativevolumewascalculatedbydividingtheinterceptsoverbeta cellsbyinterceptsovertotaltissue;then the beta cell mass was esti-matedbymultiplyingbeta cellrelative volume bygraft weight. Endo-crinenonbeta cellmassandpancreaticexocrinemass wereobtainedin the same way. The beta cellmassoftheisletsatthe timeofthe trans-plantationwasdetermined in 7 groupsof500islets isolatedondifferent days. Beta cell mass wasobtained bymultiplyingtheweightofthe islets by the percentage ofbeta cell volumeasderived fromourdata(92.7%).
Statisticalanalysis. Results wereexpressedas mean and standard
errorofthe mean(x+±SEM).Forcomparisonsbetweentwogroups the
unpairedStudent'st test(twotailed)wasused andformultiple compar-isonsthe one-wayanalysisofvariance(ANOVA)wasused. A Pvalue of<0.05wasconsideredsignificant.
Results
Metabolic evolution
after transplantation
and pancreatectomyThe body weight and blood glucose at critical time points
(transplantation, pancreatectomy, andharvesting)areshown in Table II. The evolution of bloodglucose throughout the
experimentis summarizedinFig. 1.Pancreatectomy induced
severehyperglycemiainPx-Txand Px groups. In contrast, in
Table ILMetabolicCharacteristics ofExperimentalGroupsat
Different
TimePointsDayoftransplantation Dayof pancreatectomy Day ofharvesting
Body Blood Body Blood Body Blood
Group weight glucose weight glucose weight glucose g mmol/liter g mmol/liter g mmol/liter 1 (Tx+Px) 130±7 6.11±0.3 228±6 6.11±0.3 269±10 6.44±0.3 2(Px+Tx) 255±3 18.8±0.9 218±3 6.22±0.2 308±6 9.78±1.8
3 (Tx) 117±5 5.17±0.2 245±10 5.83±0.2
4(Px) 202±4 6.50±0.2 254±9 14.5±0.2
5(normal) - 279±11 6.28±0.2
Forgroups2and 3 the values showmeanbodyweightandbloodglucose of animals killed14and 28 daftertransplantation.Resultsareexpressed
asmean±SEM.
thefollowup.Onerat,however, developed transientmoderate
hyperglycemia (blood glucose, 10.2 mM/liter). Transplanta-tion in alreadypancreatectomizedrats(Px-Txgroup) restored euglycemia (blood glucose,<7.2mM/liter;meanplus2SDof bloodglucoseinnormalgroup)in 7of 12 rats 14 d after Tx and in5 of 6 rats 28 d after transplantation.
Betacellreplication
Transplantedbeta cells. AsshowninFig. 2,beta cellreplication
was increased in transplanted islets from Tx-Px group
(1.15±0.12%) compared with pancreatic islets from normal
group(0.38±0.05%) and with transplanted beta cells from Px-Tx group (0.56±0.09%) and Tx group (0.64±0.07%) (P <0.001). Replicationwas notstatistically differentin Px-Tx,
Tx,and normalgroups.However, inPx-Tx groups,whenday 28 and 42 graftswereconsidered separately, replicationwas
increased at day 28 (0.81±0.12%) compared with day 42
(0.33±0.04%)andwith normalpancreatic islets (P<0.001).
Replication wassimilar inTx group at days 14and 28 after
transplantation.
Endogenouspancreas. Asshown inFig. 3, inTx-Px group
beta cellreplication in the pancreatic remnantwasincreased (1.01±0.25%)comparedwithPx-Tx group(0.49±0.11%),Tx group (0.40±0.11%), Px group (0.65±0.18%), and normal group(0.38±0.05%)(P <0.05).Whenexperimental animals
wereanalyzed individually, itwas apparent that replication in
transplantedbeta cells and inpancreaticbeta cellswassimilar
0-0
E
0
0
a
0
8
25-20
-15
-
10-5.
----0--- Tx-Px
-U-- Px-Tx
0 Tx
----.--- Px
0 7 1 4 21 28
Day
35 42
Figure 1. Evolution of fed blood glucose in experimentalgroups. Groupsaredescribed in TableI.Valuesaremean±SEM.
and the presence of apositivecorrelation between replication inboth locationswasestablished (r=0.55,P=0.003). How-ever, in Px-Tx group replicationwas increased on day28 in
transplantedbetacells(0.81±0.12%)but not inpancreaticbeta cells (0.55±0.18%). The discrepancy between replication in
endogenous andtransplantedislets in this group could reflect the different exposure tohyperglycemia,< 14d intransplanted
islets and > 21 d in the endogenous islets ( 17);.
Betacellarea
Asshown inFig. 4, the individualcrosssectionalareaofbeta cells in isolated islets was 239±10 4Mm2. Beta cell size was in-creased intransplantedislets from Tx-Px group(311±141Im2)
and Px-Tx group (328±13 1Im2) comparedwith
nonpancre-atectomizedgroup(Txgroup: 252±12 Rm2)orwith beta cell
areainisolated islets (P=0.001).
Betacellmass
Transplanted islets.Asshown inFig. 5,transplanted betacell mass increased in Tx-Px (1.81±0.51 mg) and Px-Tx
(1.55±0.21 mg) rats compared with nonpancreatectomized animals (Txgroup:0.78±0.17 mg) (P< 0.05). In Tx group oneoutlying value (higherthan meanplus 2.5 SD) was
ex-1,4 *
_ 1,2 1,0 m 0,8
0,6-Ch
0,40,I2r
Normal Tx-Px Px-Tx Tx
Pancreas
Figure 2.Betacellreplication innormalpancreas and in transplanted
islets.Replicationis expressedas percentageof BrdU-positivebeta
cells. Namesonthex-axiscorrespond togroups shown in TableI. Valuesaremean±SEM.*P <0.001 betweenTx-Px group and all
b-%
mla
In0
0
C.)
0
co
1,4
-1,2
-1,0-' 0,8-0,6
- 0,4-0,2
-I
A
Normal Tx-Px Px-Tx
Pancreas
a
Tx Px
Figure 3. Beta cellreplicationinendogenouspancreaticbeta cells. Replication isexpressedaspercentageofBrdU-positivebeta cells. Names onthex-axiscorrespond to groups shown in Table I. Values aremean±SEM. *P<0.001 between Tx-Px group and all other groups.
2,8
-2,4
-E a 0)o0 m
2,0- 1,6- 1,2- 0,8-
0,4-*
*
Isolated Islets
/
/
Tx-Px Px-Tx Tx
Figure 5. Transplanted beta cell mass. Groups on the x-axis corre-spond tothosedescribedin Table I. Isolatedisletsshowtheinitially transplanted beta cell mass. Values are mean±SEM.*P< 0.05, be-tween Tx-Pxand Px-Tx groups and Tx group.
cluded. Beta cellmassinTxgroupgraftswasreducedto 74%of the initially transplanted beta cell mass (1.05±0.07 mg, P
=NS).
Endogenouspancreas. Beta cellmass was notsignificantly differentamong the remnantsofthe threepancreatectomized
groups orbetween thetwononpancreatectomized groups, de-spite the different protocols for transplantation (Table III). Beta cell mass in pancreatic remnants of Tx-Px and Px-Tx groupsincreasedto23 and 19%, respectively,ofthe totalbeta
cell mass in normalpancreases. In Pxgroup the value
repre-sented 42% ofthe beta cell mass in normalpancreases. Beta cell mass in Tx group wassimilar to thatofnormal rats.
To compare the beta cell volume in the pancreas of all five groups beta cell mass was expressed per gramofpancreas
(Ta-bleIII). Beta cell relative volume was increased in Pxgroup
comparedwith all other groups.
Endocrine nonbeta
cells and
exocrinepancreas
massDifferences in endocrine nonbeta cellmassoftransplanted is-lets among groups didnotreachstatisticalsignificance(results
cN
E
S
m)
co S
a)
0
400-
300-
200-
100-0
*
7
/
Isolated Islets
*
/-r
Tx-Px Px-Tx Tx
Figure 4.Areaof individual beta cells from isolated andtransplanted islets.Groupsonthex-axiscorrespondtothosedescribedin Table I.Isolated islets show beta cellarea onthetransplantationday.Values
aremean±SEM.*P<0.001betweenTx-Px andPx-Tx groups and isolatedislets andTxgroups.
notshown). Endocrine nonbeta cell mass inTx-Pxand Px-Tx groups was - 10% that of normal pancreas. No differences
wereseen between Tx and normal groups.
Theexocrine tissue showed a significant regeneration in pancreatectomized groups (Table III), attaining 15-17% ofthe exocrinemassinthe normal group instead ofthe 5% remaining after surgery. The mass ofexocrinetissue in the pancreatic
remnant was similar in all three pancreatectomized groups,
confirmingthat theextentof pancreatectomywassimilar in all groups.
Discussion
Betacells transplantedinto normal rats increased their replica-tion and size whena95% pancreatectomy wasperformed
(Tx-Pxgroup), and total beta cell mass almost tripled. Similar re-sultswereobtained when islets were transplanted into already
pancreatectomized rats (Px-Tx group). However, although
normoglycemiawasmaintainedin Tx-Px rats after pancreatec-tomy, it took severaldaystorestoreit in Px-Tx group and in
some casesthiswas notachieved.Beta cell response to pancre-atectomy in thegrafted isletswassimilartothatof isletsin the
endogenouspancreas, indicatingthat normal growthcapacity
was preserved in beta cells after transplantation.
TableIII.Endocrine andExocrine MassinPancreas
andPancreatic Remnants
Exocrine Relative beta
Group Beta cell Nonbeta cell pancreas cell volume
mg mg mg
1 (Tx-Px) 1.08±0.26 0.15±0.05 152±12 6.95±1.70 2 (Px-Tx) 0.87±0.25 0.13±0.04 171±10 4.83±1.30 3(Tx) 5.09±0.59 1.29±0.16 913±30 5.55±0.58
4(Px) 1.99±0.75 0.22±0.05 150±14 12.3±3.84*
5(normal) 4.68±0.65 1.48±0.26 977±30 4.81±0.65
Relative beta cell volume is beta cellmassper gram of pancreas.
The increase inreplication and size oftransplanted beta cells afterpancreatectomyin Tx-Pxratsresulted in increased betacellmass.Theseverehyperglycemiathat followed pancre-atectomyinPxandPx-Tx groups wasprevented in Tx-Pxrats
by the transplantation of 500 islets. Thus, transplanted beta cellswereableto compensatefor the increased metabolic de-mand placeduponthemby the surgical reduction of
endoge-nous betacell mass. Itisnot clear, however, how beta cells sensed this increased metabolic demand. Theconceptof
adap-tive beta cellproliferation has been usedtosuggestthat func-tional demandisamechanism of control of beta cell replica-tion(23).Glucose isawell-known stimulus forbeta cell
replica-tion both in vitro (22, 24) and in vivo(25),andhyperglycemia
ispresentinmostoftheconditions in whichadaptivebetacell
proliferationoccurs.However,hyperglycemiawasnotdetected inTx-Pxgroup.Theresults resemble theisletgrowthdescribed
in othersituationsofincreasedmetabolicdemand where
hyper-glycemiaisnotdetected, suchas40 and60%pancreatectomies
(20, 21 )orpregnancy(26). Subtleelevationsin blood glucose could have beenresponsiblefor increased beta cellreplication
inTx-Px rats.Moreover, inadditiontoglucose, other
media-torsmay also havecontributedtoincrease beta cell
prolifera-tion. Theidentificationofthese mediators couldopenthe possi-bilityfor interventionstomodulate thegrowthoftransplanted
betacells.
When isletsweretransplanted into already pancreatecto-mized rats (Px-Tx group) beta cell replication and size in-creased and beta cellmassdoubled 14d aftertransplantation.
However, although transplanted and endogenous beta cell
massweresimilarinTx-PxandPx-Tx groups,normoglycemia
wassustained inTx-Px ratsthroughout the followupwhile its restorationwasdelayed, and insomecase even notachieved,in
Px-Tx rats. Since pancreatectomy in Tx-Px group was
per-formed 14daftertransplantation, onecould wonder whether
an increase ingrafted beta cell massduring thatperiod was
responsiblefor the maintenanceofnormoglycemiaafter pan-createctomy. Thequestionwasaddressedbythemeasurement
ofbetacell massinTx group 14 daftertransplantation. Beta
cellmassinthisgroup wasfoundtobe 74% ofthat initially
transplanted, rulingoutthepossibilityofanincreasedbetacell
massinTx-Px rats atthe timeofpancreatectomy.The reduc-tionconfirmsourpreviousresults inmiceshowingthat
trans-plantation of isletsinto normal recipients results inadecreased beta cellmassin thegraftaftertransplantation( 17).
Endoge-nousbeta cellmassin thepancreas remnant wasalso similar in
Tx-Px and Px-Tx rats. Theextent ofpancreatectomies was
equivalent inboth groups, asconfirmed bytheamountof
exo-crine tissuefoundatthe end of followup.Therefore, factors other than the absolute beta cell mass contributed to
differ-encesin blood glucose in thesetwogroups.Theprevious
obser-vation that maintenance of normoglycemia may require a
lower beta cellmassthan its restoration (27)agreeswithour currentresults in thatasimilarbeta cellmasswasableto
sus-tainnormoglycemiainTx-Pxratsbutnot torestoreitin Px-Tx rats. Hyperglycemia, which is associated with insulin
resis-tance, could have contributed to thedifferences in beta cell
massrequirement.Inaddition, vascularizationcould have also
playedaroleinthedifferent evolution.It hasbeenshown that
transplanted isletsarefullyvascularized 10dafter
transplanta-tion (28, 29), and therefore islets transplanted intoTx-Pxrats werewellvascularizedatthetimeofpancreatectomy. In
con-trast,the isletstransplanted intoPx-Tx ratswereconfronted with hyperglycemiaat thetime of transplantation, when they haddeficient vascularization. Impaired vascularization could
reduce beta cell capacity to sense and respond to increased metabolic demand, and therefore decrease the functional beta cellmass. In asituation suchourexperimentalgroups, where onlyamarginal isletmass wastransplanted(30,31), vascular-ization of islets could have beencrucialforanappropriate re-sponse tochanges in metabolic conditions. Therefore, the de-layed but eventual achievement of normoglycemia inmost rats
ofPx-Txgroup may have resulted from the combination of progressive increase in beta cellmassafter transplantationand restoration of islet vascularization.
Betacellreplicationandbetacellmass werealso increased in the pancreaticremnantofTx-PxandPx-Tx groups.
Replica-tion in transplanted and endogenous beta cells showedagood correlationin all threetransplantedgroups,suggestingthat the responsiveness of transplantedbetacellswasnormal. Theone
exception was Px-Tx group 14 d aftertransplantation when beta cell replicationwasincreased in thegraftbutnotinthe
pancreaticremnant.Thediscrepancycould be duetodifferent
exposure timesto severe hyperglycemia: >21 dfor
endoge-nous pancreaticbeta cells and < 14 d for transplanted beta cells. We havepreviouslyreported that the increased beta cell replication accompanying hyperglycemia didnotpersistwhen
exposure to severehyperglycemiabecamechronic ( 17).
Simi-larly, replicationwas notsignificantlyincreased inPx ratsina
situationofchronicsevere hyperglycemia, suggesting againa
limitation ofbetacellreplication.
Both beta cells and exocrine pancreas underwent
signifi-cantregenerationafter pancreatectomy. Beta cellmassand
exo-crine massincreasedto asimilar extentin Tx-Pxand Px-Tx groups asshownby thepreservation oftheratio between beta cellmassandpancreaticmass.Theincrease in exocrine tissue inPx group wasequivalenttothat in the other
pancreatecto-mizedgroups,but betacellmassincreasedmoremarkedly in this nontransplanted group. Perhaps the presence of
trans-planted beta cells restrained the increase ofendogenousbeta cellmassinTx-PxandPx-Tx groups.
The capacity of transplanted beta cells to respond to
changes inmetabolicdemand and thesimilaritybetweentheir replicative responseand thatofendogenousnontransplanted beta cells haveimportant implicationsfor islettransplantation.
In clinical transplantation, different factors maymodify the metabolic demand placed on transplanted beta cells. For
in-stance, episodes ofacute rejection decrease the transplanted beta cellmassandhigherdosesofimmunosuppressivedrugs
canincreaseinsulin resistance and impair beta cell functionor
islet cellDNAreplication (32, 33).Itis thereforeencouraging to find that transplanted beta cells are able to respond effec-tivelyto severereductions inbeta cell mass. Moreover,ifthe
transplantationmethod could be optimized itmay bepossible
to obtaina successful result with asmaller numberof islets,
thusalleviatingtheislet shortagethatlimitscurrent
transplan-tationefforts.
Acknowledgments
Wethank J. Hollister, C. J. Cahill, and R. S. Schlesinger for expert technicalassistance.
Dia-betes Foundation.E.Montafiawasthe recipient ofapostdoctoralgrant
from the Ministry of Education and Science ofSpain.
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