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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 Article

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(2)

Blood Flow Measurements in Autotransplanted Pancreatic Islets of the Rat

Impairment

of the Blood Perfusion of

the

Graft

during Hyperglycemia

Stellan 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 isolated

pan-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-treated

streptozotocin-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(BoehringerMannheim

GmbH, 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).The

atmosphere

was humidified air plus5%CO2. Theculture mediumwas

changed

every secondday.

2dafterthepartialpancreatectomy and while the isletswere main-tained in culture,some rats weregivenanintravenous

injection

of

strep-tozotocin(40 mg/kgbody wt)(U-9889,lot

2408A, kindly provided

by

J.Clin. Invest.

© The American

Society

for Clinical

Investigation,

Inc.

0021-9738/87/07/0017/05

$2.00

(3)

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 pancreatic

andislet 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 abdominal

aortavia theleft femoral artery.After5 min, non-radioactive micro-spheres (NewEngland Nuclear, Boston,MA)withadiameter of 10.2±0.6

Am

(mean±SD)

thatwere

suspended

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 0.2

tm

and then counting themicrospheresilluminated by transmittedlightwhileviewing

througha 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

X

NorgiNref,

where

QO08

isthe organ blood flow

(milliliters

per

minute);

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 animals

metthisrequirement. Insomeof the organs the number of observed

microsphereswasfairlylow(- 25),butwehavepreviouslyshown that evensuchasmall number ofmicrosphereswillgiveareliable blood flow estimate(6).

Insulinresponseto aglucose injection.

Immediately

after the

micro-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 killed

by

cervical dislocation. Serum insulin concentrations in the

samples

weremeasured

byradioimmunoassay(RIA) (7) by

using

mouse

crystalline

insulin

(Novo)

asstandard and

251-insulin (Novo)

as tracer.

Measurements

ofbody weight

andserum

glucose

concentrations. The

rats wereweighedbefore theexperiment,onthedayof

transplantation,

andbeforeinjectionsofmicrospheres.Bloodsamplesofnon-fasted an-imalsweretakenwithoutanesthesia from the tail veins

before

the

ex-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

Analyzer

2;

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);

and

insulin-treated

streptozotocin-diabeticrats

killed

1moafter

transplan-tation

(IDl).

Statistical

analyses.

The resultsareexpressedasmeans±SEM.

Groups

of datawerecompared bymeansof Student'sunpairedttest.

Results

General

characteristics

of

the

transplanted

rats.Beforethestart

of

the

experiments

there

wasno

difference

in

body weight

be-1.Abbreviationsusedinthis

paper:

Dl,

streptozotocin-diabetic

ratskilled 1 moaftertransplantation;ID1,insulin-treated

streptozotocin-diabetic

ratskilled 1 moaftertransplantation; NI, normoglycemicratskilled 1 mo aftertransplantation; N3, normoglycemicrats killed 3 mo after

(4)

,

*

*

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 of

transplantation),

the streptozotocin-treated animals (experi-mental groups Dl and

IDl)

had lost weight as compared with the Nl group

(Table

I). 1 mo

after transplantation

the weight of IDl rats was

similar

to that of Nl rats, whereas the body weight of

Dl

rats was - 30% lower than that of Nl rats. N3

rats(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-treated

animals

(Table

I).

The

insulin

treatment promptly

normalized

the

hyperglycemia

and theanimals remained normoglycemic

during

the month of insulin administration (datanotshown), until the day of

killing

(Table I). It should be emphasized that

neither did

partial

pancreatectomy

induce

overt

hyperglycemia

or

impaired glucose

tolerance

(data

notshown), nordid

auto-transplantation of

500

islets

reversethe

diabetes

of the

strepto-zotocin-treated animals.

When theanimalswere

killed,

theserum

insulin

concentrations

in the blood samples taken through the

abdominal arterial

catheterappearedtobehigherin N3 than in

Nl rats,althoughthis differencewas notstatistically

significant

(P>0.05).

Pancreatic

and islet

bloodflows

in

the 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).

Incontrol

experiments

the

identity

of the apparent

grafts

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

renal

vein, before

any

glucose

had been

infused,

it was

found

thatthe portal and renal vein serum con-centrations of insulinwerehigherinN3 thanin Nl rats(Table III). Dl rats showed reduced insulin concentrations bothinthe

portal

and renal vein as compared with Nl rats. In Dl rats,

glucose

administration did not resultinasignificantly increased

serum

insulin

concentration either

in the

portal

or the renal venous blood. N3 rats also

failed

to

increase their

seruminsulin

concentration

in

either vein

in responseto

glucose.

N1 rats

re-sponded

with elevatedseruminsulin levels bothintheportal (P

<0.001) and renal

vein

(P<

0.01)

within 2 min after glucose

injection

ascompared

with time

zero.

Before

the

glucose

load, IDl rats

exhibited

elevated insulin concentrationsinthe renal

TableLCharacterization

of

RatsSubmittedtoAutotransplantation

Nlrats 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±15

Weight 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 blood

samplesfromatail 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,

(5)

Table II.Pancreaticand Islet BloodFlow and

Graft

VolumeinRatsSubmitted

toIslet 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

the

microsphere technique

itwasfoundpossible inthis

investigation

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 volume

of

an

islet

graft consisting of

500

islets

would then be - 500 nl.

Themeasured

graft

volumes in

this study

were 313-470 nI in

different

groups

of

rats,

which is

in good accordance with the

theoretical estimate.

The observed blood

flow

rates inthe

grafts

in the

normoglycemic

and

insulin-treated diabetic

rats corre-sponded toa

single islet

blood

flow of

- 35

nl/islet

Xmin for anislet volumeof1 nl.Theblood flow rates found in this study

arethus in excellent agreement

with

values reported

for

asingle

islet

blood

flow

in the pancreas of normal rats (20 nl/min) (8). There was no

difference

in thegraft blood flow values in the

normoglycemic

rats 1 and 3 mo after transplantation, which suggests that the

vascularization of

the

grafts

wasalready

com-pleted

after

1 mo. In line with this observation Griffithet al.

(9)

found ina

morphologic

studythat rat isletstransplanted

intra-hepatically

were

fully

vascularized after14d.

Moreover,

Brown et al.

(10)

observed in the

light microscope

that a

prominent

vascular supply

appeared

a

few days after transplantation

of fetal ratpancreas to a site beneath the renal capsule.

Although the

blood

flow in the

grafts

of the non-insulin-treated

diabetic

ratsshowed

large variations,

itwasmuchreduced

compared

with that in the control group

(Ni

rats).This may

indicate

that the

diabetic

state

impaired

the vascularization of the

grafted islets, especially

ifthe insulin

production

was

insuf-ficient

to reversethe

hyperglycemia.

This notion is

supported

by the

finding

that

insulin-treated

ratshadablood flowinthe

transplanted

islets thatwassimilartothatin the

normoglycemic

rats.The

possibility

cannot beruled out,

however,

thatitwas

the more

normal insulin

concentrations orthe

production

of

angiogenic factors,

rather than the

normoglycemia

per se, that promoted the

development of

higher

blood

flow

rates in the

insulin-treated diabetic animals.

The

findings

nevertheless sug-gest that

intensive insulin

treatmentmay be beneficial for the

vascularization

of

grafted isolated islets.

The

difference

in

insulin concentrations

in vivo inserum

from the

portal

and

left

renal vein after

glucose injection

indicates

Table 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±0.32

10 (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

(6)

that thegrafts of the insulin-treated rats secreted insulin, whereas those

of

the

manifest diabetic

rats

did

not function significantly. Thenormoglycemic rats tested 1 mo after transplantation

ex-hibited

increases in serum insulin concentrations both in the portal and renal

vein.

It

is 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 elevated

concen-trations of insulin

were notedin both veins before glucose

ad-ministration. This

observation wasunexpected and might reflect a

combined effect

from the regenerating pancreas and the trans-plantedislets.

The

pancreatic

remnant

weighed

900 mg in all four groups

of

rats,

which

shows that the

pancreatic

glands had almost

dou-bled

their weight after

the

partial

pancreatectomy. Bonner-Weir et al.

(1

1)observed

similarly

that thepancreatic remnant had more thandoubled in

weight

8-10 wk

after

partial

pancreatec-tomy. The

pancreatic regeneration

was

accompanied

by higher total

pancreatic

blood

flows

than have

previously

been observed in normal rats with intact glands (3, 12). Also, the islet blood

flow

in the pancreases

of

the

normoglycemic

rats was much increased and represented a fraction

of

not < 20-30% of the

total

pancreatic blood flow.

The

mechanism underlying

the markedlyincreased islet blood

flow

in the regenerating pancreas

is

at present unclear. It could be assumed that an increase in

insulin secretion is

parallelied

by

increased islet

blood

flow

(compared

with

the

6-mo-old

rats).

On

the other hand, we have

previously

shown that there is in some situations a dissociation

between islet

blood

flow

and

insulin

release

in vivo

(13). Finally,

note

in this context

that the

vascularization

and the blood

flow

rates

of

thetransplanted islets may have been

influenced

by the

regenerating

pancreas. However,

it

appears less

likely

that such

influence

would account

for

the observed

differences

in the blood

flow of

the

transplanted islets

between

the

experimental

groups

in

the present

investigation.

In

conclusion, this

study has shown

that

transplanted

islets

are

vascularized within

amonth

after

implantation and that the

blood

flow

rates

in

these

islets in

normoglycemic

rats are

com-parable

tothose in

islets located

in

the

pancreas. The

vascular-ization ofgrafted islets

seems to be

diminished

by

hyperglycemia;

however,

this

can be

normalized

by

insulin

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.

References

1.Ballinger, W. F., and P. E. Lacy. 1972. Transplantation of intact pancreatic islets inrats.Surgery (St. Louis). 72:175-186.

2. Sutherland,D.E. R. 1981. Pancreas and islet transplantation I. Experimentalstudies.Diabetologia.20:161-185.

3. Jansson, L., and C. Hellerstrom. 1983.Stimulation by glucose of theblood flow to the pancreatic islets of the rat.Diabetologia. 25:45-50.

4.Jansson, L., and C. Hellerstrom. 1981.Arapidmethod of visu-alizing the pancreatic islets for studies of islet capillary blood flow using non-radioactivemicrospheres.ActaPhysiol. Scand. 113:371-374.

5. Andersson,A. 1978. Isolatedmousepancreaticislets in culture: effects ofserumand different culture mediaonthe insulinproduction of the islets.Diabetologia. 14:397-404.

6. Jansson, L. 1985. Pancreatic islet blood flow withspecialreference

totheeffects of glucose administration.ActaUniv. Ups. 521:1-38. 7.Heding, L. G. 1972. Determination of total serum insulin (IRI) ininsulin-treated patients. Diabetologia.8:260-266.

8.Lifson, N., C. V. Lassa, and P. K. Dixit. 1985. Relation between blood flowandmorphologyinislet organ ofratpancreas.Am.J.Physiol.

249:E43-E48.

9.Griffith,R.C.,D.W.Sharp,B. K.Hartman,W. F.Ballinger,and P.E.Lacy. 1977.Amorphologic studyofintrahepatic portalvein islet

isografts.

Diabetes.26:201-214.

10. Brown, J.,I. G. Molnar, W. Clark, and Y. Mullen. 1974. Control ofexperimentaldiabetes mellitus inratsbytransplantationof fetal

pan-creases.Science(Wash. DC). 184:1377-1379.

11. Bonner-Weir, S., D. F. Trent, and G. C. Weir. 1983. Partial pancreatectomy in theratandsubsequent defect inglucose-induced in-sulin release. J. Clin. Invest. 71:1544-1554.

12. Jansson, L., and S. Sandler. 1985. Pancreatic islet circulation in relationtothediabetogenic action ofstreptozotocin in therat.

Endo-crinology. 116:896-900.

13. Jansson, L. 1985. Dissociation between pancreatic islet blood flow and insulin release in therat. ActaPhysiol.Scand. 124:223-228.

References

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