American Journal of Transplantation 2006; 6: 967–975Blackwell Munksgaard

C© 2006 The AuthorsJournal compilation C© 2006 The American Society of

Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2006.01299.x

Islets Transplanted Intraportally into the Liverare Stimulated to Insulin and Glucagon ReleaseExclusively through the Hepatic Artery

J. Laua,∗, L. Janssona and P.-O. Carlssona,b

Departments of aMedical Cell Biology and bMedicalSciences, Uppsala University, SE-751 23,Uppsala, Sweden∗Corresponding author: Joey Lau,Joey.Lau@medcellbiol.uu.se

Not much is known about the physiology of intrapor-tally transplanted islets. One reason for this is thatit is difficult to study such islets, since they are scat-tered throughout the liver. We employed a perfusiontechnique to characterize the functional properties ofsyngeneic intrahepatic 1-month-old islet grafts, andcompared them to islets transplanted beneath the kid-ney capsule, as well as native islets. The cellular com-position of the islet grafts was also examined. Glu-cose and arginine administered through the hepaticartery, but not through the portal vein, induced in-sulin release from the intraportally implanted islets.Moreover, arginine, only when administered throughthe hepatic artery, induced glucagon release from thesame islets. The first phase of glucose-stimulated in-sulin release from both islets transplanted to the liverand kidney was delayed, and less prominent whencompared to the pancreas. Intraportally transplantedislets contained fewer glucagon-positive cells thanislets transplanted to the kidney and native islets. Ourfindings demonstrate that intraportally transplantedislets respond with insulin and glucagon to secreta-gogues, but only when stimulated through the hepaticartery. Whether intrahepatic islets may sense othersubstances than glucose or arginine occurring in highconcentrations in the portal vein following intestinaluptake remains to be studied.

Key words: Engraftment, graft function, hepaticartery, islets, liver perfusion

Received 11 October 2005, revised and accepted forpublication 25 January 2006

Introduction

Pancreatic islet transplantation is a tempting strategy to

treat patients with type 1 diabetes, since if successful it

could provide a cure for the disease. The “Edmonton pro-

tocol” for islet transplantation changed the immunosup-

pressive protocol toward less b-cytotoxic agents in combi-

nation with the use of freshly isolated islets for transplan-

tation and infusion of islets from 2 to 4 donors. By these

means a 1-year insulin independence rate of 80% was ob-

tained (1, 2). However, recent long-term observations show

that also when applying this protocol there is a continuous

decline in function of islet transplants. Very few patients re-

main insulin-independent beyond 4 years after transplanta-

tion (3), which contrasts to the results for whole pancreas

transplants (4, 5). Since the histocompatibility barrier, the

underlying autoimmune disease, and the immunosuppres-

sive agents used are the same for both procedures, this

puts focus on issues that are related to the adaptation of

the implanted islets in their new microenvironment, the

liver.

The liver provides several site-specific challenges for long-

term engraftment of transplanted islets, for instance be-

cause of its inherently low oxygenation (6, 7) and its func-

tion as the major detoxification site for substances occur-

ring in the portal vein following intestinal uptake. There

are also reports on alterations in islet function after intra-

portal islet transplantation, such as a defective glucagon

response to hypoglycemia (8, 9) and a defective glucose-

stimulated insulin release in murine islets retrieved from

the liver (10). Moreover, one experimental study in rat

has indicated an impaired long-term function of intrapor-

tally transplanted islets when compared to islets implanted

to the kidney (11). In view of these combined observa-

tions, we decided to study in more detail the insulin and

glucagon release from intraportally transplanted islets in

response to different stimuli by applying a technique with

liver perfusion ex vivo in the rat. We also aimed to inves-

tigate the changes in cellular composition of intraportally

transplanted islets, and whether hormone release from in-

traportally transplanted islets is affected by the stimuli ad-

ministered through either the portal vein or hepatic artery.

Materials and Methods

AnimalsInbred male Wistar-Furth rats weighing approximately 300 g were pur-

chased from Scanbur (Sollentuna, Sweden). The animals had free access

to pelleted food and water. All experiments were approved by the animal

ethics committee for Uppsala University.

967

Lau et al.

Islet isolation, culture and transplantationPancreatic islets were isolated from Wistar-Furth rats by collagenase diges-

tion (12), and cultured in groups of 150 islets for 3–4 days in 5 mL of culture

medium consisting of RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supple-

mented with L-glutamine (Sigma-Aldrich) and 10% (vol/vol) fetal calf serum

(Sigma-Aldrich). Culture medium was changed every second day.

At transplantation, groups of 200 islets were packed in a butterfly needle

(25G) and infused via the portal vein into the liver, or packed in a brak-

ing pipette and implanted beneath the left renal capsule, of syngeneic

pentobarbital-anesthetized (60 mg/kg i.p.; Apoteket, Goteborg, Sweden)

Wistar-Furth rats. The transplanted islet volume was estimated to as a mean

2.53 lL by measurements of islet volume packed in the braking pipette be-

fore transplantation.

Perfusions of graft-bearing liversEx vivo perfusion of graft-bearing livers was performed 1 month posttrans-

plantation. The animals were anesthetized with pentobarbital (see above),

and placed on a heated operating table. The abdominal cavity of each animal

was opened. Ligatures were placed around the inferior vena cava immedi-

ately below the liver, whereas the portal vein and hepatic artery were cannu-

lated with polyethylene catheters (Figure 1A). The catheters were fixed with

ligatures and connected to separate infusion pumps. The inferior vena cava

was then incised cranially to the liver and the liver perfusion was started.

The liver was dissected, placed in a funnel, and kept at a constant tempera-

ture (37◦C) and humidity throughout the experiments. The liver preparation

was perfused without recirculation at 3.5 mL/min and 1.5 mL/min through

the portal vein and hepatic artery, respectively, with a continuously gassed

(95% O2: 5% CO2) bicarbonate buffer (13) supplemented with 10 mmol/L

Hepes (Sigma-Aldrich) and 2 mg/mL each of dextran T70 (Pharmacia,

Uppsala, Sweden) and bovine serum albumin (Fraction V; Miles, Slough,

UK).

Each graft-bearing liver was allocated to one of the three different exper-

imental groups with regard to stimulation with D-glucose and L-arginine:

(i) stimuli provided through both the portal vein and the hepatic artery; (ii)

stimuli provided only through the hepatic artery or (iii) stimuli provided only

through the portal vein. The blood vessel through which stimuli were not

provided in group 2 (portal vein) and 3 (hepatic artery) was perfused with

a medium containing low glucose (2.8 mmol/L) during the whole experi-

Figure 1: Schematic drawings ofthe perfusion procedures. Schematic

drawing of the preparation used for per-

fusion of the islet graft-bearing livers

(A). Experimental protocol on the per-

fusion procedure (B). Experimental pro-

tocol on the perfusion procedure when

measuring insulin and glucagon extrac-

tion rates in the liver (C).

ment. For the blood vessel(s) through which stimuli were to be provided,

the experiments started with a 10-min equilibration period with the perfu-

sion medium containing 2.8 mmol/L D-glucose, which was followed by a

20-min stimulation period with high-glucose medium (16.7 mmol/L

D-glucose). Thereafter, a 10-min reset period with low-glucose medium

(2.8 mmol/L D-glucose) was followed by a 10-min stimulation period with

10 mmol/L L-arginine + 5.5 mmol/L D-glucose added to the perfusion

medium. The perfusions were completed with a 20-min period with low-

glucose medium (2.8 mmol/L D-glucose; Figure 1B). The effluent medium

from the preparations was collected on ice at minutes 9–15, 17, 20,

25, 30, 35, 40–45, 47, 50, 55, 60 and 70 in tubes containing aprotinine

(7.4% of the final sampling volume, 10 000 KIU/mL; Bayer, Leverkusen,

Germany).

Perfusions of pancreas-duodenumNontransplanted control animals were anesthetized with pentobarbital. The

pancreas and duodenum were removed from the animals as previously

described (14,15). The pancreas-duodenum preparations were perfused at

1.5 mL/min without recirculation applying the same perfusion protocol with

high glucose and arginine stimuli and collection of samples as used for the

perfusion of graft-bearing livers (see above).

Perfusions of graft-bearing kidneysOne month posttransplantation, animals with an islet graft implanted be-

neath their left renal capsule were anesthetized with pentobarbital. The

graft-bearing kidneys were then removed from the animals as previously

described (16). The kidney preparations were perfused at 3 mL/min with-

out recirculation applying the same perfusion protocol with high glucose

and arginine stimuli and collection of samples as used for the perfusion of

graft-bearing livers and control pancreata given above.

Insulin and glucagon extractionSeparate control experiments were performed to estimate the immediate

hepatic uptake of insulin and glucagon. Livers from control, nontransplanted

animals were prepared for organ perfusion, as described above. Insulin (26

or 52 ng/mL; Actrapid®, 100 IU/mL; Novo Nordisk, Bagsværd, Denmark)

or glucagon (72 ng/mL; Glucagon Novo Nordisk®, 1 mg/mL; Novo Nordisk)

was then added for 1-min pulses to the medium perfusing the hepatic artery

in these preparations (n = 4 animals for each hormone), while the perfusion

968 American Journal of Transplantation 2006; 6: 967–975

Perfusion of Islet Graft-Bearing Livers

medium contained a low glucose (2.8 mmol/L), a high glucose concentration

(16.7 mmol/L) or arginine (Figure 1C). In each case, the perfusion medium

had been unchanged for 10 min prior to insulin or glucagon infusion. Follow-

ing each infusion, the contents of the perfusion medium were unchanged

for at least another 10 min, while the effluent from the incised caval vein

was collected on ice in tubes prefilled with aprotinin (7.4% of the final sam-

pling volume, 10 000 KIU/mL). Moreover, there was a 10-min wash-out

period between stimulation with high glucose and arginine. The insulin and

glucagon concentrations used for these control experiments were chosen

to mimic the physiological concentrations of insulin and glucagon in the

liver.

Hormone measurementsThe effluents from the different perfusions of pancreata or graft-bearing

organs were analyzed for insulin concentrations by a rat insulin ELISA

(Mercodia, Uppsala, Sweden). Glucagon measurements in the samples

were carried out using rat glucagon RIA kits (Linco Research, St. Louis,

MO). The mass insulin and glucagon responses to stimuli were calculated

by planimetry (17).

In order to detect glucagon concentrations in the effluents from the livers

containing an islet graft, the samples were concentrated with Centricon

YM-3 tubes (membrane cutoff 3 000 Da; Millipore, Billerica, MA) approxi-

mately 5–6 times. The exact magnitude of concentration was determined

for each sample by weighing it before and after centrifugation.

Insulin and glucagon concentrations in samples obtained from the con-

trol experiments where insulin and glucagon were infused into the hepatic

Figure 2: Insulin release in effluent medium collected from perfused native Wistar-Furth pancreas (A), islet graft-bearing kidneys(B) and islet graft-bearing livers of Wistar-Furth rats (C, D and E). Each graft-bearing liver was allocated to one of three different

experimental groups, where stimuli to insulin release were provided through both the portal vein and the hepatic artery (experimental

group 1; C) only through the hepatic artery; (experimental group 2; D), or only through the portal vein (experimental group 3; E). After

10 min equilibration period with a perfusion medium containing 2.8 mmol/L D-glucose, insulin secretion was stimulated by a 20-min period

with 16.7 mmol/L D-glucose followed by a 10 min reset period with 2.8 mmol/L D-glucose. Thereafter, insulin secretion was stimulated

with a perfusion medium containing 10 mmol/L L-arginine + 5.5 mmol/L D-glucose followed by 20 min with 2.8 mmol/L D-glucose. The

blood vessel through which stimuli were not provided in the perfusion of graft-bearing livers, i.e. portal vein in group 2 and hepatic artery

in group 3, was perfused with low-glucose medium (2.8 mmol/L D-glucose). All values are given as means ± SEM for 5–7 animals in each

group.

artery were analyzed by a human insulin ELISA (Mercodia) and a glucagon

RIA kit (Linco Research), respectively.

Morphological studiesSeparate graft-bearing kidneys/livers and control pancreata were removed,

fixed in 10% (vol/vol) formalin, embedded in paraffin and sectioned at 5 lm.

To determine the percentage of b-cells and a-cells in the different sam-

ples, sections of native or transplanted islets were stained with insulin

(ICN Biomedicals, Aurora, OH) and glucagon (Novo Nordisk). These sec-

tions were then examined using a point-counting method (18), where the

number of intersections overlapping insulin- and glucagon-positive cells, re-

spectively, was counted in a light microscope. A total of 121–726 points

were counted in each pancreas or islet graft. The volume of the islets in

the native pancreata was evaluated as previously described (19). Briefly,

each pancreata of 9 separate animals was weighed, cut into 45–48 pieces

and placed between object slides. Following visualization of the islets by a

freeze-thawing technique (20), the percentage of islet volume was deter-

mined by point-counting (18). A total of 5000–5700 points were counted in

each pancreas. The total islet volume in a Wistar-Furth pancreas could then

be estimated to 12.4 ± 0.6 lL.

Statistical analysisAll values are given as means ± SEM. Multiple comparisons for normally

distributed data were performed using ANOVA and the Bonferroni post-hoc

test, whereas nonparametric values were compared using nonparametric

ANOVA and Dunn’s test. For all comparisons, a p-value < 0.05 was consid-

ered to be statistically significant.

American Journal of Transplantation 2006; 6: 967–975 969

Lau et al.

Results

Insulin releaseA biphasic insulin response to high glucose (16.7 mmol/L;

Figure 2A) was consistently seen from perfused pancre-

ata. A rapid and prominent first peak was followed by a

sustained second phase of insulin release. The incremen-

tal value for the first phase was in average of 150% higher

than the mean plateau values during the second phase.

Similarly to the response to high glucose, two prominent

peaks of insulin release were discerned when arginine was

added to the perfusate. From perfused graft-bearing kid-

neys, there was a first and second phase of insulin release,

as from the control pancreata, in response to 16.7 mmol/L

D-glucose (Figure 2B). However, the first phase of insulin

release from these renal subcapsular grafts was delayed

when compared to the control pancreata with a mean incre-

mental value of 3 min instead of 1 min after introduction of

the high glucose in the perfusion medium (p < 0.05, non-

parametric ANOVA). Moreover, the incremental value for

the first phase of insulin release from these grafts was only

approximately 50% higher than the insulin release values

during the second phase, which is a weaker first-phase re-

sponse than that seen in the pancreata (p < 0.05, ANOVA).

The renal subcapsular grafts responded to arginine with a

prominent first peak followed by a smaller second peak of

insulin release.

In the first experimental group with perfusion of graft-

bearing livers, stimuli to insulin secretion were provided

simultaneously through both the hepatic artery and the

portal vein. Introduction of a high-glucose concentration

(16.7 mmol/L) in the perfusion medium elicited insulin re-

lease (Figure 2C), but as for the renal subcapsular grafts,

the response was delayed with an incremental value oc-

curring 2 min later when compared to control pancre-

ata (p < 0.05, nonparametric ANOVA). Moreover, the

first peak of insulin release from the graft-bearing livers

could barely be separated from the second phase of in-

sulin release, and constituted, as for the renal subcapsu-

lar grafts, a weaker first-phase response than that seen in

the pancreata (p < 0.05, ANOVA). However, a prominent

insulin release response from the intraportally implanted

islets was recorded when the islets were challenged with

arginine.

Also when stimuli to insulin release (high glucose or argi-

nine) were provided only through the hepatic artery into

graft-bearing livers (experimental group 2), there was a pro-

nounced insulin secretion (Figure 2D). The insulin release

in this setting was similar in magnitude to that seen when

stimuli to insulin release were provided through both the

hepatic artery and portal vein. The first phase of insulin

release in response to high glucose tended to be slightly

more prominent than when both blood vessels were per-

fused with high glucose stimuli, but only ∼20% higher than

the plateau values during the second phase.

Figure 3: Glucagon response (as area under curve, AUC) to10 mmol/L L-arginine + 5.5 mmol/L D-glucose from nativeislets, and 1-month-old renal subcapsular or intraportal isletgrafts consisting of 200 islets. All values are given as means

± SEM for 2–8 animals in each group. ∗Denotes p < 0.05 when

compared to native islets, and †p < 0.05 when compared to renal

subcapsular islet grafts. All comparisons were made using ANOVA

and Bonferroni’s post hoc test.

No insulin response at all could be detected when stimuli

to insulin release were administered only through the por-

tal vein (experimental group 3; Figure 2E). In this setting,

the insulin release remained at basal levels throughout the

whole perfusion.

Glucagon releaseWhen the control pancreata were perfused, there was a

rapid and transient peak of glucagon release (mean incre-

mental value 904 ± 403 pg/min) during the first 3 min

of perfusion with the high glucose (16.7 mmol/L) perfu-

sion medium. Moreover, when the perfusion medium con-

tained arginine, a distinct and sustained glucagon release

was elicited from the preparations (Figure 3). Glucagon

concentrations in the effluents during other parts of the

perfusion were below the detection level of the assay

(20 pg/mL).

Also from perfused graft-bearing kidneys, there was

a glucagon response during perfusion with arginine

(Figure 3). Glucagon concentrations in the effluents dur-

ing other parts of the perfusion were below the detection

level of the assay.

When graft-bearing livers were perfused, we could at first

not detect glucagon concentrations in the effluents, even

in response to arginine, in any of the three experimental

groups. However, following concentration of our samples

5–6 times, there was a rapid and transient peak of glucagon

release in response to a high (16.7 mmol/L) glucose con-

centration (mean incremental value 28.9 ± 2.5 and 35.3 ±8.3 pg/min for experimental groups 1 and 2, respectively)

970 American Journal of Transplantation 2006; 6: 967–975

Perfusion of Islet Graft-Bearing Livers

in the effluents of our liver preparations, as in the efflu-

ents from the control pancreata. Likewise, glucagon re-

lease was increased when the perfusion medium was sup-

plemented with arginine (Figure 3). Quite in contrast, there

was no glucagon release at all in response to these stim-

uli in experimental group 3, i.e. when high glucose and

arginine were supplemented to the medium perfusing the

portal vein.

Insulin and glucagon extraction rates in liverIrrespective of whether insulin was administered into

the hepatic artery during perfusion with low glucose

(2.8 mmol/L), high glucose (16.7 mmol/L) or arginine

supplemented medium, 43.6 ± 3.8% (n = 4 animals) of

infused insulin was extracted in the liver. In comparison,

the glucagon extraction rate in the liver was 67.5 ± 5.1%

(n = 4).

Cellular composition of transplanted isletsIn the pancreas of Wistar-Furth rats, 75–80% of all

islet cells were b-cells (Figure 4A,B) and ∼20% a-cells

(Figure 5A, B). Islets transplanted beneath the renal cap-

sule had a decreased fraction of b-cells compared to control

islets (Figure 4A, C), whereas for intraportally transplanted

islets there was only a tendency to a decrease in b-cell

number (Figure 4A, D). In contrast, the fraction of a-cells

was markedly decreased in the intraportally transplanted

islets (Figure 5A, D), but not in the islets grafted to the kid-

ney (Figure 5A, C). Moreover, when comparing the fraction

of a-cells at the different implantation sites, intraportally

transplanted islets had much less glucagon-positive cells

than islets transplanted beneath the renal capsule.

Figure 4: Comparisonbetween native andtransplanted islets. (A)

Percentage insulin-positive

cells in native Wistar-Furth

rat islets, and 1-month-old

syngeneic renal subcap-

sular or intraportal islet

grafts. All values are given

as means ± SEM for

4–5 experiments in each

group. ∗Denotes p < 0.05

when compared to native

pancreatic islets as eval-

uated by nonparametric

ANOVA and Dunn’s test.

(B–D) Micrographs of a

native rat islet (B), renal

subcapsular islet graft (C)

and intraportal islet graft

(D) stained for insulin.

Scale bar 10 lm in B and

D, and 20 lm in C.

Discussion

When pancreatic islets are implanted intraportally they dis-

perse throughout the liver and become lodged deep in dis-

tal tributaries of the portal vein. It has therefore been en-

visaged that islet endocrine cells are chronically exposed

to substances occurring in the portal vein. The present

study shows that only nutrients present in the hepatic

artery could elicit insulin and glucagon release from trans-

planted islets 1 month posttransplantation, whereas nutri-

ents administered through the portal vein had no effects on

islet hormonal release. The latter finding suggests that not

even the endocrine cells located in the islet periphery were

reached by stimuli in the portal vein. We did not investigate

to what extent other substances, e.g. immunosuppressive

drugs, occurring in high concentrations in the portal vein fol-

lowing intestinal uptake could affect islet cells through the

portal vein route. Since both glucose and arginine have a

very high capacity for diffusion in tissues, we think that our

findings are also applicable to other substances following

engraftment. It is possible that the high concentrations of

immunosuppressive drugs occurring in portal blood exert

detrimental effects on islets cells in the immediate post-

transplantation period (21, 22).

Since nutrients in the vicinity of the transplanted islets,

i.e. administered through the portal vein, did not affect

islet hormonal release, it is likely that nutrient sensing

of the islet a- and b-cells in the islet transplants occurs

only through the newly formed vascularity. There have

previously been contradictory reports whether intraportally

transplanted islets become revascularized by tributaries

American Journal of Transplantation 2006; 6: 967–975 971

Lau et al.

Figure 5: Comparisonbetween native andtransplanted islets. (A)

Percentage glucagon-

positive cells in native

Wistar-Furth rat islets, and

1-month-old syngeneic

renal subcapsular or intra-

portal islet grafts. All values

are given as means ± SEM

for 4–7 experiments in

each group. ∗Denotes p <

0.05 when compared to

native pancreatic islets,

and †p < 0.05 when com-

pared to renal subcapsular

islet grafts. All compar-

isons were evaluated by

nonparametric ANOVA and

Dunn’s test. (B–D) Micro-

graphs of a native rat islet

(B), renal subcapsular islet

graft (C) and intraportal

islet graft (D) stained for

glucagon. Scale bar 10 lm

in B, and 20 lm in C–D.

both from the portal vein and the hepatic artery (23), or

mainly by tributaries from the hepatic artery (24). Our data

are consistent with the latter report.

There are some previous studies concerning perfusion

of graft-bearing livers (25–27). Those only concluded that

a biphasic insulin release from intraportally transplanted

islets does occur in response to glucose, but did not com-

pare the response either to native islets or islets implanted

to other organs. In the present study, both islets implanted

to the kidney and intraportally into the liver had a delayed

and decreased first phase of insulin release in response to

glucose when compared to native islets. The decreased

first-phase response, compared to the native pancreas,

may be related to a lower islet mass transplanted and ul-

timately engrafted in the liver. Although we corrected the

first-phase response for the number of islets transplanted,

it is unknown how many islet survive in the ectopic environ-

ment, and the lower first-phase response may simply rep-

resent a lower functional islet mass. It may also reflect dis-

turbances in the secretory machinery of the transplanted

b-cells, as previously indicated in in vitro studies of islets re-

trieved from the renal subcapsular (28) and intraportal site

(10). Alternatively, it may reflect a vascular dysfunction of

the transplanted islets due to their lower vascular density

(29, 30) and blood perfusion (6). In line with this, we have

recently observed that improvements in the blood perfu-

sion of islet renal subcapsular grafts are associated with an

enhanced first phase of glucose-stimulated insulin release

(31).

Since the liver is a major target organ for insulin, some of

the insulin secreted by intraportally transplanted islets is

metabolized within the organ before entering the systemic

circulation. This makes the comparison of the response of

insulin from intraportally transplanted islets to that of islets

implanted at other implantation sites difficult. In order to

estimate the fraction of insulin metabolized in the liver al-

ready during the first passage, we injected insulin into the

hepatic artery of control liver preparations during perfusion.

We found that approximately 55% of injected insulin could

be retrieved in the effluents from the caval vein, irrespec-

tive of whether insulin was injected during perfusion with

buffer containing the low or high glucose concentration or

arginine. These estimations are also consistent with previ-

ous observations on the insulin extraction rate in liver (32–

34). If compensating for the immediate hepatic uptake of

some of the secreted insulin from intraportally transplanted

islets with this factor when performing the comparison to

insulin release from islets in the renal subcapsular site,

these two implantation sites seemed to have a similar in-

sulin response (cf. Figure 6A, B). Following measurements

of islet mass in a Wistar-Furth pancreas, as well as of the

transplanted islet mass, it could also be estimated that the

insulin response from the islet transplants was 26–47% of

the expected for an optimal graft with this number of islets.

However, the insulin response from the transplanted islets

in our perfusion experiments seemed still quite prominent,

which contrasts to previous findings on retrieved intrapor-

tally transplanted islets investigated in vitro. Such islets

showed a much lower insulin content and insulin release

972 American Journal of Transplantation 2006; 6: 967–975

Perfusion of Islet Graft-Bearing Livers

Figure 6: Insulin mass response (as area under curve, AUC) to 16.7 mmol/L D-glucose (A) and 10 mmol/L L-arginine + 5.5 mmol/LD-glucose (B), and glucagon mass response to 10 mmol/L L-arginine + 5.5 mmol/L D-glucose (C), for native Wistar-Furth ratislets and 1-month-old syngeneic renal subcapsular or intraportal islet grafts. For intraportal islet grafts, nutrient stimuli were

infused through both hepatic artery and portal vein. Measured values in the effluents are shown in solid bars. Hatched bars for the islet

transplants denote recalculated values from those shown in solid bars when correcting for differences in islet mass to the pancreas and

also for the immediate hormone uptake by the liver parenchyma in the graft-bearing liver perfusions. The calculations were based on the

assumption of an islet mass of 12.4 lL in the pancreas of Wistar-Furth rats, a transplanted islet mass of 2.53 lL and immediate extraction

rates of 43.6% and 67.5% for insulin and glucagon secreted from the intraportal islet grafts, respectively. All values are given as means ±SEM.

than islets isolated from the pancreas, and also had a dis-

turbed glucose oxidation rate (10). It is possible that this

discrepancy may be explained by that the employed perfu-

sion technique enabled us to study the insulin release from

all transplanted b-cells in the liver, and that it is rather small

or fragmented islets that remain functional after transplan-

tation instead of larger, easily retrievable islets.

The reason for the decreased b-cell fraction in the renal

subcapsular islet grafts is unknown (35). It may, however,

be envisaged that the preferential location of rodent b-cells

to the islet core may predispose them more than other islet

cells to hypoxic injury in the immediate posttransplantation

period (35,36).

As also previously described (8, 9, 37), both renal subcap-

sular islet transplants and intraportal islet grafts responded

similarly to native islets with glucagon release in response

to arginine stimulation. Consistent with previous studies

(38, 39), we observed a high clearance of glucagon by

the liver (65–70%), which underlines the importance of

the liver as a major target organ for glucagon. The lower

size of the glucagon response from the intraportally trans-

planted islets compared to the renal subcapsular grafts

seemed mainly to be explained by this high-clearance rate

(Figure 6C). However, the intraportal islet grafts also con-

tained a decreased fraction of a-cells, both when compared

to native islets and islets implanted to the renal subcapsu-

lar site. Since intraportally transplanted islets do not seem

to sense nutrient stimuli in portal blood, but only those oc-

curring in the hepatic artery, downregulation of glucagon

production induced by higher concentrations of islet hor-

mones such as glucagon in portal than in systemic blood is

unlikely to provide an explanation. Instead, the decrease in

a-cells may be due to the factors associated with the hep-

atic environment per se, or be a result of the intraportal

transplantation procedure.

We consistently recorded a transient peak of glucagon re-

lease in both the effluents of graft-bearing livers and con-

trol pancreata in response to high glucose in the perfusate.

The reason for this seemingly paradoxal glucagon release is

obscure, but a previous study has suggested that a pulse

of glucagon secretion from a-cells may occur after high-

glucose stimulation (40). We could not detect a similar re-

sponse from the renal subcapsular grafts, which may be

explained by the detection limit of the assay (20 pg/mL),

since these samples were not concentrated.

We did not record an increased glucagon release from

either native islets in the control pancreata or the trans-

planted islets during organ perfusion with buffer containing

a low-glucose concentration (2.8 mmol/L). These results

are consistent with previous data on perfused rat pancre-

ata (41, 42) and renal subcapsular grafts (37), and may be

explained by that glucagon release following switching to

low glucose is delayed for at least 10 min in several set-

tings, including pancreas perfusions in rat (9, 43, 44).

The present findings that intraportally transplanted islets

respond with insulin and glucagon release to secreta-

gogues only when stimulated through the hepatic artery

mean that the high concentrations of nutrients in the por-

tal vein following intestinal uptake do not reach such islets.

Further studies on changes in islet physiology following in-

traportal islet transplantation may provide us with impor-

tant clues to the site-specific challenges faced by these

islets, and ultimately strategies on how to counter-act

these to improve islet graft function.

Acknowledgments

The technical assistance of Birgitta Bodin, Astrid Nordin and Eva Tornelius

is gratefully acknowledged.

American Journal of Transplantation 2006; 6: 967–975 973

Lau et al.

This study was supported by grants from the Juvenile Diabetes Re-

search Foundation, the Swedish Research Council (72XD-15043, 72X-109),

EFSD/Novo Nordisk Research Program, the Swedish Diabetes Foundation,

the Swedish Juvenile Diabetes Fund, the Ake Wiberg Foundation, the Aner

Foundation and the Family Ernfors Fund.

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