Blood flow measurements in autotransplanted
pancreatic islets of the rat. Impairment of the
blood perfusion of the graft during
hyperglycemia.
S Sandler, L Jansson
J Clin Invest.
1987;
80(1)
:17-21.
https://doi.org/10.1172/JCI113044
.
No information is available on the rate of blood flow in transplanted islets. In this study, adult
rats were partially depancreatized, and islets from the excised pancreas were then isolated,
maintained for 7 d in tissue culture, and subsequently transplanted back to the animal,
beneath the renal capsule. Some rats were rendered diabetic with streptozotocin before
transplantation. A month after transplantation the blood flow of the grafts was measured by a
microsphere technique. Autotransplantation to streptozotocin-diabetic rats of approximately
500 islets did not revert the hyperglycemia, and the blood flow of these grafts was
approximately 25% of that in the normoglycemic-transplanted rats. However, in
insulin-treated diabetic rats the blood flow of the pancreatic graft was similar to that in the
normoglycemic rats. The present results suggest that the blood flow in transplanted islets is
markedly diminished by hyperglycemia and that this can be enhanced by insulin
administration.
Research ArticleFind the latest version:
Blood Flow Measurements in Autotransplanted Pancreatic Islets of the Rat
Impairment
of the Blood Perfusion of
theGraft
during HyperglycemiaStellan SandierandLeifJansson
DepartmentofMedical Cell Biology, Uppsala University, S-75123Uppsala, Sweden
Abstract
Noinformation is availableontherateof blood flowin
trans-planted islets. In this study, adult rats were partially
depan-creatized, andislets from the excisedpancreaswerethen isolated,
maintained for 7 d in tissue culture, and subsequently
trans-planted backtotheanimal,beneath the renalcapsule.Somerats
wererendereddiabetic with streptozotocin beforetransplantation.
A month after transplantation the blood flow of the graftswas
measured by a microsphere technique. Autotransplantation to
streptozotocin-diabetic ratsof - 500 islets didnot revertthe
hyperglycemia, and the blood flow of these graftswas - 25% of that inthenormoglycenic-transplanted rats.However, in in-sulin-treated diabeticratsthe bloodflowof thepancreatic graft
wassimilartothatin the norioglycemicrats.Thepresentresults
suggest that the blood flow in transplanted islets is markedly diminished byhyperglycemiaand thatthiscanbeenhanced by
insulinadministration.
Introduction
Since the firstreportin
1912
ontransplantationof isolatedpan-creaticislets(1),alargenumber ofreportshave confirmed that thisisanefficient methodof curing experimental diabetes (for a review see Sutherland [2]). Since the implanted islets have
been foundtorevertthehyperglycemiaofdiabeticanimals within
afewdaysand then maintain normoglycemia, it hasbeen
as-sumed that they are rapidly vascularized upon implantation. Although it is likely that revascularization of the grafted islets is of crucial importance for their long-term function in the
re-cipient,there isnoinformationonthe bloodflowthrough
trans-planted islets.Likewise,thepossibleeffects of the diabeticstate
onthe bloodsupplytothegrafte4isletsremainunknown. The
aim ofthe presentstudywastomeasuretherateof blood flow
inpancreaticisletstransplanted toasite beneath the left renal
capsuleinnormoglycemicand in
insilin-treated
and non-treatedstreptozotocin-diabetic recipients. For this purpose a partial
pancreatectomywasperformedinrats,and isletswereisolated fromthe excised pancreaticspecimenandautotransplantedafter
Thisworkwaspresented in partatthe 1986 Annual Meeting ofthe
European Association forthe StudyofDiabetes, Rome, Italy
(Diabe-tologia. 29:589. [Abstr.]).
Address allcorrespondenceandreprintrequeststoDr.S. Sandler, Department ofMedicalCell Biology,Biomedicum,P.O. Box 571, S-751 23Uppsala,Sweden.
Receivedfor publication 27August 1986 andinrevisedform 3 March 1987.
culture. Tohave access to normoglycemic control animals it
was nottheaim of the partialpancreatectomy toinduce hyper-glycemia. By thesame token, the autotransplantation of 500 islets didnot curethestreptozotocin-induced diabetes. 1 or3
moaftertransplantation the blood flow in the grafted islets and
inthe remainingpancreas wasmeasured by amicrosphere
tech-nique (3).Also,the insulinresponse to a glucose load was
mea-sured in theeffluent from the grafted islets and in the systemic circulation.
Methods
Animals. Non-inbred maleSprague-Dawleyrats, which were3 mo old andweighed 350-400 g,wereobtained fromAnticimex AB (Sollentuna, Sweden). The animalswerehousedonepercage and had freeaccessto tap water andstandard pelletedfood (type R3; Anticimex,
SWdertilje,
Sweden)throughoutthe experiments.
Surgicaltreatment, isletisolation,and culture. The rats were anes-thetized byanintraperitoneal injection of sodium pentobarbital (Meb-umal; 40 mg/kg body wt, ACO LAkemedel, Stockholm, Sweden); if
nec-essary thisanesthesiawasmaintained by etherduringtheoperation.The animals also received 0.05 mg/kg bodyWtatropine (ACO LAkemedel) intraperitoneally before the operation. The pancreas was exposed through
atransverseincision in theupperleft part of the abdomen and theportion adjacenttothespleen and stomachwasmobilized,gently dissectedfree, and removed. Thus, the part of the pancreas in closeproximitytothe duodenumwasleftintact. By this procedure, - 0.9-1.0 g of pancreatic tissuewasremoved from eachanimal, whichcorrespondedto'
two-thirdsof the total pancreaticmass or - 1.5 gatthis age (4), before the
peritoneumandskinwereclosedbysutures.
Theexcised portion of the gland was distended bymultiple injections of Hanks'solution (Statens
hakteriologiska
Laboratorium,Stockholm, Sweden) into the parenchyma andcutinto - 30to40pieces.Thetissue piecesweredistributedto twosterilized glass vials eachcontaining25 mgof collagenase from Clostridiumhistolyticum(BoehringerMannheimGmbH, Mannheim,FederalRepublicofGermany)dissolved in 8 ml of Hanks'solution. The vialswererapidlyshaken ina370Cwaterbath for 15-20 min until the tissue haddisintegrated,asdetermined by visual inspection. The digestwassedimented three times in 20 ml of Hanks' solution(240C),andpancreaticisletswerehandpicked byusing braking
pipetteswhile viewing throughastereomicroscopeat30Xmagnification.
Toincrease thecontrastbetween the rat islets and the acinartissue,the dish with thepancreaticdigestwasplacedon adark support soaked with Hanks'solution.Bythisprocedurewe wereconsistentlyabletopick500
to700isletsfrom theexcised part of theratpancreas.
Groups of - 125islets eachwereculturedfree-floatingat370Cin
5 mlof mediumRPMI1640 (FlowLaboratories,Irvine,Ayrshire,United Kingdom) containing 100 U/ml
benzylpenicillin
(Astra LIkemedel,Sodertilje,Sweden), 0.1 mg/mlstreptomycin (GlaxoLaboratoriesLtd., Greenford, United Kingdom),and 10%calfserum
(Statens
Bakteriol-ogiskaLaboratorium)accordingtoAndersson(5).Theatmosphere
was humidified air plus5%CO2. Theculture mediumwaschanged
every secondday.2dafterthepartialpancreatectomy and while the isletswere main-tained in culture,some rats weregivenanintravenous
injection
ofstrep-tozotocin(40 mg/kgbody wt)(U-9889,lot
2408A, kindly provided
byJ.Clin. Invest.
© The American
Society
for ClinicalInvestigation,
Inc.0021-9738/87/07/0017/05
$2.00
Dr.W. E. Dulin,Upjohn Co., Kalamazoo, MI)toinduce amanifest diabetic state.
Iwkafter thepartial pancreatectomy, the animalswere
autotrans-plantedwith - 500 cultured islets each. Forthis purpose,anincision
was made in theleft renal capsuleof the animals while under ether anesthesia and theislets,collected inabraking pipette,weredeposited
beneath thecapsule. Thetransplantedstreptozotocin-diabeticratswere
subdivided intotwogroups,onetreated with subcutaneousinjections of insulin (4 IU Ultralente; Novo Research Institute,Copenhagen,
Den-mark), whereas theotherremained untreated.
Measurements
ofbloodflows.
The method formeasuring pancreaticandislet blood flow byusingnon-radioactive microsphereshas been described indetail (3, 4).Briefly,thetransplantedrats wereanesthetized intraperitoneally with 130
mg/kg
bodywtsodiumthiobutabarbital (In-actin; Byk Gulden, Konstanz, Federal Republic of Germany) and hep-arinized. Polyethylene catheterswereinserted into the left ventricle of the heart via therightcommon carotid artery and into the abdominalaortavia theleft femoral artery.After5 min, non-radioactive micro-spheres (NewEngland Nuclear, Boston,MA)withadiameter of 10.2±0.6
Am
(mean±SD)
thatweresuspended
in 0.3 ml of 0.9% saline and 0.002%Tween80(Sigma ChemicalCo., St.Louis, MO)wereinjected through
theintracardiac catheter. The catheterwasimmediately flushed with 0.3 mlof saline. This solutioncontained - 1.0-1.5 X
101
microspheres.Simultaneously, the catheter in the abdominalaorta wasusedtoobtain
anarterial blood reference sample by using a peristaltic pump (Minipuls II; Gilson,Villiers-le-Bel,France)set at awithdrawalrateof 0.6ml/min
for 90s.The accuracy of thisrate wasconfirmed in each experiment by weighing the sample. When theanimals had beenkilled, thegraft-bearing kidney, thepancreatic remnant, and adrenal glandswereremoved. The kidneywas fixed in Bouin's solution, and thepancreaticand adrenal tissuewasblotted,weighed,andtreatedfor visualization of the isletsby usingafreeze-thawingtechnique (4).
Afterthepancreatic preparationshad beenthawed, theirmicrosphere
content wasdeterminedwith the aid ofastereomicroscope(M3;Wild Heerbrugg, Heerbrugg,
Switzerland)
that had both dark andbright field illumination. Themicrosphere contentof the adrenal glandswas esti-matedsimilarly, whereas that ofthe reference blood sample was calculated bytransferringthesampleto2.5-cmmicrofiber filters (GF/A; Whatman Ltd., London, UnitedKingdom)withaporesizeof 0.2tm
and then counting themicrospheresilluminated by transmittedlightwhileviewingthrougha stereomicroscope. Beforeembedding,the isletgraftwas lo-calized as adistinct whitish spot andcut out withan - 4-mm wide margin. The fixed isletgraftwithsurrounding kidney tissuewas subse-quently processed forlight microscopy by serial sectioning into 7-am thick sections, whichwerestained withhematoxylin and eosin. The total
areasof theisletgraftswere measuredin theconsecutive sections by
using a computer system formorphometry (MOP-Videoplan; Zeiss, SvenskaAB,Stockholm, Sweden), and the totalgraftvolume in each animalwascalculatedasthe total areatimes the section thickness. The
microspherecontent oftheentiregraftwas then counted byusing a light
microscope;The darkmicrospheres were easily recognized at low power magnification (X 100) (Fig. 1) and their identity was then confirmed at higher magnification (X 400) (Fig. 2). Because of their physical properties,
themicrosphereswere notcutwith the microtome knife; thus, they were present in only one section and could therefore be counted only once.
Insomeinitial experiments the freeze-thawing technique was adopted for visualization of the islets in the pancreas (4) on preparations from thegraft-bearingpart ofthe kidney, but with this procedure the islet cells couldnotbedistinguished from the kidney cells.
Theblood
flow
ofthetransplantedislets and ofthe pancreatic remnant was calculated according to the formula: QOZ8=Qe
XNorgiNref,
whereQO08
isthe organ blood flow(milliliters
perminute);
Q.,>
is therateof withdrawal of the reference blood sample (milliliters per minute);N0rg
is the number of microspheres present in the organ; and N fis the number of microspheres present in the reference sample. The blood flow values basedonthemicrosphere content in the adrenal glands were used to confirm that the microspheres were adequately mixed in the circulation.Adifference of< 10%
(expressed
asmilliliters per minute times gram)Figure 1.Light micrographofanislet graftbeneath the renalcapsule inanormoglycemicratkilled 1moafterautotransplantation.Normal
kidneyparenchymacanbeseenadjacenttothegraft.Three
micro-spherescanbe seenwithin thegraft
(arrows).
Hematoxylin-eosin.X 125.
between theglandswastakentoindicate sufficient
mixing
andall animalsmetthisrequirement. Insomeof the organs the number of observed
microsphereswasfairlylow(- 25),butwehavepreviouslyshown that evensuchasmall number ofmicrosphereswillgiveareliable blood flow estimate(6).
Insulinresponseto aglucose injection.
Immediately
after themicro-sphereinjection,theabdomenwasopenedand catheterswereinserted
bydirect puncture into theportalvein and left renal vein. I mlofa30%
(wt/vol)glucose solutionwassubsequentlyinfused into the left femoral vein. Blood samples
(0.2
ml)weretaken before theglucoseinfusion and after2, 5,and 10 min, and the animalswere then killedby
cervical dislocation. Serum insulin concentrations in thesamples
weremeasuredbyradioimmunoassay(RIA) (7) by
using
mousecrystalline
insulin(Novo)
asstandard and
251-insulin (Novo)
as tracer.Measurements
ofbody weight
andserumglucose
concentrations. Therats wereweighedbefore theexperiment,onthedayof
transplantation,
andbeforeinjectionsofmicrospheres.Bloodsamplesofnon-fasted an-imalsweretakenwithoutanesthesia from the tail veins
before
theex-periments,thenatweeklyintervals,andfinallybefore injection of
mi-crospheres.Thislatterbloodsample, however,wasc6llectedthroughan arterial catheter(seeabove)whileanimalswereunder anesthesia. The serumglucoseconcentrations in thesampleswereassayed bya
semiau-tomatic
glucose-oxidase
method(Glucose
Analyzer2;
Beckmam Instru-ments,Inc.,Fullerton,CA).Experimentalgroups. Thedesignof thisstudyyieldedfour
experi-mental groups,namely:normoglycemicratskilled1moafter
transplan-tation (N1);' normoglycemicratskilled 3moafter transplantation (N3); streptozotocin-diabeticratskilled1 moafter transplantation
(Dl);
andinsulin-treated
streptozotocin-diabeticratskilled
1moaftertransplan-tation
(IDl).
Statistical
analyses.
The resultsareexpressedasmeans±SEM.Groups
of datawerecompared bymeansof Student'sunpairedttest.
Results
General
characteristicsof
thetransplanted
rats.Beforethestartof
theexperiments
there
wasnodifference
inbody weight
be-1.Abbreviationsusedinthis
paper:
Dl,
streptozotocin-diabetic
ratskilled 1 moaftertransplantation;ID1,insulin-treatedstreptozotocin-diabetic
ratskilled 1 moaftertransplantation; NI, normoglycemicratskilled 1 mo aftertransplantation; N3, normoglycemicrats killed 3 mo after
,
*
*
V
4**.
.
/*es}^*
sE
.*
gXw
o,
AL^A;s
5*
ki *
*M
^
M ,
g.4
H
(0
~(~
Figure 2. Two of thesame microspheresas in Fig. I observed in higher magnification. Hematoxylin-eosin.X 500.
tweenthe
experimental
groups of rats (data not shown). How-ever, I wkafter the partial pancreatectomy (i.e., on the day oftransplantation),
the streptozotocin-treated animals (experi-mental groups Dl andIDl)
had lost weight as compared with the Nl group(Table
I). 1 moafter transplantation
the weight of IDl rats wassimilar
to that of Nl rats, whereas the body weight ofDl
rats was - 30% lower than that of Nl rats. N3rats(6-mo old) had further increased their body weight as com-pared with Nl rats (4-mo old). The weight of the pancreatic remnant at killing was - 900 mg in all the experimental groups
(Table I). The serum glucose concentrations at the time of trans-plantation were markedly
increased
in the streptozotocin-treatedanimals
(TableI).
Theinsulin
treatment promptlynormalized
thehyperglycemia
and theanimals remained normoglycemicduring
the month of insulin administration (datanotshown), until the day ofkilling
(Table I). It should be emphasized thatneither did
partial
pancreatectomyinduce
overthyperglycemia
or
impaired glucose
tolerance(data
notshown), nordidauto-transplantation of
500islets
reversethediabetes
of thestrepto-zotocin-treated animals.
When theanimalswerekilled,
theseruminsulin
concentrations
in the blood samples taken through theabdominal arterial
catheterappearedtobehigherin N3 than inNl rats,althoughthis differencewas notstatistically
significant
(P>0.05).Pancreatic
and islet
bloodflows
inthe autotransplanted
rats.Theblood flow in the pancreatic remnant wasthesameinall fourexperimentalgroups(Table II).Afewislets couldstill be observed in the frozen-thawedpreparations of thepancreasin the streptozotocin-treatedratsand thisallowed for thecalculation ofaprofounddiminuition of the islet blood flow. Incontrast,
theislet blood flowwashigh in the pancreaticremnantofNl rats; and when expressed aspercentage of the totalpancreatic blood flow in these animals. This latter figure was further in-creased in N3rats.
Inall animals, irrespective of theirtreatment,distinct islet grafts could be identified andno significantdifferences in the volume of the grafts were found (Table II).The bloodflowper
islet-graft volume was the same in the two normoglycemicgroups of rats and in IDl rats(Table II). However, the blood flow of thegrafted-islet mass in Dl rats wasreducedto <25% that in Nl rats. Note that actual measurements ofsingle islet blood flow were not possible in the grafted islets since individual isletscould notbedistinguished as separate structures in the sectioned grafts
(Fig.
1).
Incontrolexperiments
theidentity
of the apparentgrafts
has been confirmed as being mainly composed of insulin-positive cellsby immunocytochemical staining. However, itwas not fea-sible touseimmunocytochemical stained sections when counting the dark microspheres.
Insulin secretion in vivo. When the serum insulin
concen-trations were measured in vivo through catheters insertedinthe
portal vein and left
renalvein, before
anyglucose
had beeninfused,
it wasfound
thatthe portal and renal vein serum con-centrations of insulinwerehigherinN3 thanin Nl rats(Table III). Dl rats showed reduced insulin concentrations bothintheportal
and renal vein as compared with Nl rats. In Dl rats,glucose
administration did not resultinasignificantly increasedserum
insulin
concentration either
in theportal
or the renal venous blood. N3 rats alsofailed
toincrease their
seruminsulinconcentration
ineither vein
in responsetoglucose.
N1 ratsre-sponded
with elevatedseruminsulin levels bothintheportal (P<0.001) and renal
vein
(P<0.01)
within 2 min after glucoseinjection
ascomparedwith time
zero.Before
theglucose
load, IDl ratsexhibited
elevated insulin concentrationsinthe renalTableLCharacterization
of
RatsSubmittedtoAutotransplantationNlrats N3rats Di rats IDirats
n 8 7 6 6
Bodyweightattransplantation(g) 389±17 392±8 306±6* 333±12*
Bodyweightatkilling(g) 467±18 536±11
332±107#
446±15Weight of pancreaticremnant(mg) 964±44 984±23 934±59 869±46 Serumglucoseattransplantation(mM) 8.6±1.2 7.6±0.2 38.7±0.2'
24.5±1.21
Serumglucoseatkilling (mM) 10.8±0.3 12.4±0.7* 34.9±1.9§ 11.8±2.7 Serum insulinatkilling(ng/ml) 1.58±0.30 4.98±2.0 1.12±0.33 1.10±0.06
Apartialpancreatectomy wasperformedonanesthetizedratsandisletswereisolated fromtheexcisedspecimenandcultured. After 1wk,groups
of 500 cultured isletsweretransplantedback tothesameanimal,beneaththerenalcapsule,underbrief ether anesthesia. Some animalsweregiven streptozotocin2 dafter thepartialpancreatectomytoinduce diabetes.Theserumglucoseconcentrationat
transplantation
wasmeasured in bloodsamplesfromatail vein in non-anesthetizedrats,whereas thesamplesforserumglucoseandseruminsulin before
killing
werecollected froman arterial catheter inanesthetizedrats.Valuesaremeans±SEMforn,number ofrats. * $,andIdenote P<0.01,P<0.05,andP<0.001,
Table II.Pancreaticand Islet BloodFlow and
Graft
VolumeinRatsSubmittedtoIslet Autotransplantation Measured byaMicrosphere Technique
N irats N3 rats D rats ID rats
n 8 7 6 6
Pancreatic blood flow (ml/minxgpancreas) 1.28±0.24 1.08±0.21 1.24±0.19 1.14±0.11 Islet blood flow
(,Ml/min
Xgpancreas) 245±41 335±66 12±3* 13+5* Islet blood flow (percent ofpancreaticbloodflow) 19.3±2.6 32±2.5* 0.8±0.2* 1.3±0.5$Volume of graft(nl) 446±98 376±71 313±81 470±96
Graft blood flow(nl/nl graftxmin) 34.9±9.9 41.5±16 8.2±2.6§ 30.3±13
Thedifferent groups ofratshad been treatedasdescribed in Table I.Organ blood flowsweremeasured inanesthetized animals byusinga micro-sphere technique. The graft volumewascalculated with the aid ofacomputer system formorphometry. Valuesaremeans±SEM for n, number of rats. * $, and § denoteP <0.001,P <0.01, andP <0.05,respectively,ascompared with Nl rats(unpaired Student'sttest).
butnotinthe portal veinascompared with NI rats. 2 min after glucose administration the insulin-treatedratshadhigher insulin concentrations in the renal thanintheportal vein (P<0.05).
Discussion
Using
themicrosphere technique
itwasfoundpossible inthisinvestigation
to measurethe blood flow of transplanted islets in rats.Since
the mean volume of a single islet in situ in the rat pancreas hasbeen calculated to -1nI(8), the expected volumeof
anislet
graft consisting of
500islets
would then be - 500 nl.Themeasured
graft
volumes inthis study
were 313-470 nI indifferent
groupsof
rats,which is
in good accordance with thetheoretical estimate.
The observed bloodflow
rates inthegrafts
in thenormoglycemic
andinsulin-treated diabetic
rats corre-sponded toasingle islet
bloodflow of
- 35nl/islet
Xmin for anislet volumeof1 nl.Theblood flow rates found in this studyarethus in excellent agreement
with
values reportedfor
asingleislet
bloodflow
in the pancreas of normal rats (20 nl/min) (8). There was nodifference
in thegraft blood flow values in thenormoglycemic
rats 1 and 3 mo after transplantation, which suggests that thevascularization of
thegrafts
wasalreadycom-pleted
after
1 mo. In line with this observation Griffithet al.(9)
found inamorphologic
studythat rat isletstransplantedintra-hepatically
werefully
vascularized after14d.Moreover,
Brown et al.(10)
observed in thelight microscope
that aprominent
vascular supplyappeared
afew days after transplantation
of fetal ratpancreas to a site beneath the renal capsule.Although the
blood
flow in thegrafts
of the non-insulin-treateddiabetic
ratsshowedlarge variations,
itwasmuchreducedcompared
with that in the control group(Ni
rats).This mayindicate
that thediabetic
stateimpaired
the vascularization of thegrafted islets, especially
ifthe insulinproduction
wasinsuf-ficient
to reversethehyperglycemia.
This notion issupported
by thefinding
thatinsulin-treated
ratshadablood flowinthetransplanted
islets thatwassimilartothatin thenormoglycemic
rats.The
possibility
cannot beruled out,however,
thatitwasthe more
normal insulin
concentrations ortheproduction
ofangiogenic factors,
rather than thenormoglycemia
per se, that promoted thedevelopment of
higher
bloodflow
rates in theinsulin-treated diabetic animals.
Thefindings
nevertheless sug-gest thatintensive insulin
treatmentmay be beneficial for thevascularization
of
grafted isolated islets.
The
difference
ininsulin concentrations
in vivo inserumfrom the
portal
andleft
renal vein afterglucose injection
indicatesTable III. SerumInsulin Concentrations in the Portal and Left Renal Vein in Rats Submitted toAutotransplantation before and2.5and10minafteranIntravenous Glucose Injection
N rats N3rats D rats IDIrats
n 8 6 6 6
Seruminsulin(ng/ml)
Portalvein
0 (min) 1.70±0.20 9.06±2.52* 0.36±0.08* 1.40±0.31
2(min) 3.73±0.40 10.0±3.07$ 0.45±0.08§ 1.72±0.53*
5(min) 3.46±0.48 9.08±2.89t 0.47±0.10§ 1.39±0.45*
10 (min) 3.74±0.52 11.6±3.97t 0.40±0.10§ 1.40±0.36* Renalvein
0 (min) 1.38±0.24 6.38±2.23t 0.47±0.11* 2.79±0.60*
2(min) 2.95±0.40 7.53±2.63 0.75±0.22§ 3.14±0.32
5(min) 2.68±0.24
7.97±2.42*
0.55±0.13* 2.39±0.3210 (min) 2.45±0.46 8.04±2.42* 0.58±0.13* 3.52±0.98
Thedifferent groups of rats had been treated as described in TableI.Glucose(1 ml 30% glucose wt/vol) was infused into the left femoral vein in anesthetizedratsandblood samples were taken through catheters inserted in the portal vein and left renal vein. Insulin concentrations were mea-suredby RIA. Values are means±SEM for n, number of rats. *, andIdenote P<0.05, P <0.01, and P < 0.001, respectively, as compared
that thegrafts of the insulin-treated rats secreted insulin, whereas those
of
themanifest diabetic
ratsdid
not function significantly. Thenormoglycemic rats tested 1 mo after transplantationex-hibited
increases in serum insulin concentrations both in the portal and renalvein.
Itis possible,
however, that this increase in the renal vein was due to insulin secretion from the pancreas. In the 6-mo-old normoglycemic rats, highly elevatedconcen-trations of insulin
were notedin both veins before glucosead-ministration. This
observation wasunexpected and might reflect acombined effect
from the regenerating pancreas and the trans-plantedislets.The
pancreatic
remnantweighed
900 mg in all four groupsof
rats,which
shows that thepancreatic
glands had almostdou-bled
their weight after
thepartial
pancreatectomy. Bonner-Weir et al.(1
1)observedsimilarly
that thepancreatic remnant had more thandoubled inweight
8-10 wkafter
partialpancreatec-tomy. The
pancreatic regeneration
wasaccompanied
by higher totalpancreatic
bloodflows
than havepreviously
been observed in normal rats with intact glands (3, 12). Also, the islet bloodflow
in the pancreasesof
thenormoglycemic
rats was much increased and represented a fractionof
not < 20-30% of thetotal
pancreatic blood flow.
Themechanism underlying
the markedlyincreased islet bloodflow
in the regenerating pancreasis
at present unclear. It could be assumed that an increase ininsulin secretion is
parallelied
byincreased islet
bloodflow
(compared
with
the6-mo-old
rats).On
the other hand, we havepreviously
shown that there is in some situations a dissociationbetween islet
bloodflow
andinsulin
releasein vivo
(13). Finally,
notein this context
that thevascularization
and the bloodflow
ratesof
thetransplanted islets may have beeninfluenced
by theregenerating
pancreas. However,it
appears lesslikely
that suchinfluence
would accountfor
the observeddifferences
in the bloodflow of
thetransplanted islets
betweenthe
experimental
groupsin
the presentinvestigation.
In
conclusion, this
study has shownthat
transplantedislets
arevascularized within
amonthafter
implantation and that the
bloodflow
ratesin
theseislets in
normoglycemic
rats arecom-parable
tothose inislets located
inthe
pancreas. Thevascular-ization ofgrafted islets
seems to bediminished
byhyperglycemia;
however,this
can benormalized
byinsulin
treatment.Acknowledgments
WethankProfessor ClaesHellerstromand Dr. Arne Andersson for valu-ableadviceduringthecourseof this work. We aregratefulto
Anna-Britta Andersson, Birgitta Bodin, Eva Forsbeck, and Astrid Nordin for theirexcellent technical assistance, and to Agneta Snellman for careful preparation of the manuscript.
This work was supported in part by grants 12X-109, 12X-8273,
17X-7501, and 12P-7680 from the Swedish Medical Research Council, and by the Swedish Diabetes Association, the Juvenile Diabetes Foundation International, theSwedish Society of Medicine, the Nordic Insulin Fund, theC.Groschinsky Memorial Fund, the Ernfors Family Fund, Syskonen Svenssons Fond, and the Ake Wiberg Foundation.
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