Importance of preabsorptive insulin release on oral glucose tolerance: studies in pancreatic islet transplanted rats
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Gut: first published as 10.1136/gut.21.11.1002 on 1 November 1980. Downloaded from http://gut.bmj.com/ on March 12, 2022 by guest. Protected by copyright.
Gut, 1980, 21, 1002-1009
Importance of preabsorptive insulin release on oral
glucose tolerance: studies in pancreatic islet
transplanted rats
E G SIEGEL, E R TRIMBLE, A E RENOLD, AND H R BERTHOUD
From the Institut de Biochimie Clinique and Laboratoires de Recherches Medicales, University of Geneva,
Geneva, Switzerland
SUMMARY The role of preabsorptive (cephalic phase) insulin release in oral glucose tolerance was
investigated using diabetic rats treated by intraportal transplantation of isogenic islets. This early
neurally mediated phase of insulin release has been shown to be absent in such rats. When the body
weight of transplanted rats was normalised, glucose tolerance tests (GTTs) were performed in the
unstressed state using permanent cardiac catheters. Transplanted rats had a normalised intravenous
GTT, whereas, as we have shown previously, their oral GTT remained clearly pathological. During
both tests peripheral insulin levels were decreased compared with controls. While during intravenous
GTT the onset of insulin release occurred as early in transplanted rats as in controls, during oral
GTT there was a clear delay, probably because ofthe absence of the cephalic phase. Re-establishment
of normal preabsorptive insulin levels was attempted by a small intravenous insulin injection
during this period. This resulted in a transient increase in peripheral insulin levels, which, at two
minutes after glucose ingestion, gave values similar to those found in controls at that time. This small
insulin injection caused a marked improvement of the oral GTT which was most evident after
exogenous insulin had disappeared from the blood. While the injection did not affect the 60 minute
incremental insulin area, the glucose area was decreased by 50 %, to a value not significantly different
from that of control rats. The cephalic phase of insulin release appears, therefore, to be one import-
ant factor in the control of glycaemia during food intake. Its absence plays a major role in the patho-
logical oral glucose tolerance of diabetic rats treated by intraportal islet transplantation.
It has long been known that the disposal of orally designed to measure preabsorptive (cephalic phase)
ingested glucose is controlled not only by the increase insulin release were performed, it was found that
in blood glucose concentration, but also by other this part of the early insulin response was absent in
factors such as gastrointestinal hormones and islet-transplanted rats,3 as reported previously for
neural reflexes.' Neural influences may be of par- diabetic rats treated by foetal pancreas trans-
ticular importance when transplantation of pan- plants.4 It is known that the early insulin response
creatic endocrine tissue is performed, as the trans- after both intravenous5 6 and oral4 7 8 glucose
plant may remain denervated. We have shown, for loading is an important determinant of the overall
example, that when a sufficient number of islets is glucose tolerance. Therefore, the present study was
transplanted to normalise the intravenous glucose designed to define the importance of preabsorptive
tolerance in the unstressed rat, oral glucose tolerance insulin release-or its absence-in maintaining
in the same animals remains abnormal.2 Whereas normal oral glucose tolerance in previously diabetic
after an intravenous glucose load plasma insulin rats treated by intraportal islet transplantation.
levels rose as early in transplanted rats as in controls, To this end, islet transplanted rats had oral glucose
after an oral glucose load transplanted rats displayed tolerance tests performed with and without a small
a delayed plasma insulin response. When tests injection of insulin given intravenously during the
preabsorptive period, in a dose calculated to mimic
the levels seen during cephalic phase secretion. As
Received for publication 22 July 1980 stress affects glucose tolerance in islet transplanted
1002Gut: first published as 10.1136/gut.21.11.1002 on 1 November 1980. Downloaded from http://gut.bmj.com/ on March 12, 2022 by guest. Protected by copyright.
Importance ofpreabsorptive insulin release on oral glucose tolerance 1003
rats even more than in normal animals,9 care was procedure and to drinking a glucose solution when
taken to test animals in the conscious unstressed it was presented. Tests were performed in the after-
state. noon, six to eight hours after food withdrawal.
Before the test, an extension tube was attached to the
Methods cardiac catheter, 50-100 U heparin was given per
rat, and 20 to 30 minutes later the tests were begun.
After two basal blood samples glucose was injected
ANIMALS via the cardiac catheter or presented in a petri
Highly inbred male Wistar-Lewis rats obtained dish for oral consumption. Zero time for the oral
from the Charles-River Breeding Laboratories, test was when the rat spontaneously began to drink
Wilmington, Mass., were used. After food had been the glucose solution, and for the intravenous test
withdrawn for 12 to 16 hours, diabetes was induced when glucose was injected. After intravenous
in rats weighing 200-250 g (8-10 weeks old) by the glucose loading, the catheter was flushed through
intravenous injection of streptozotocin (50 mg/kg) several times with saline to remove any traces of
freshly dissolved in acidified saline (pH 4.5). Control glucose. The glucose loads and blood-sampling
rats received an intravenous injection of acidified times for each test are as follows:
saline. Islet transplantation or sham operation was IVGTT Glucose load 1 g/kg (40 % glucose solution).
performed four to five weeks later. Islets were Sampling times: basal (twice), 2, 5, 10, 20, 40, and
isolated by a modification of the collagenase tech- 60 minutes.
nique.10 After hand-picking, the islets were suspended OGTT Glucose load lg/kg and 2 g/kg (400%glucose
in modified Krebs-Ringer bicarbonate (KRB) solution). Sampling times: basal (twice), 2, 5, 10, 20,
buffer containing Na+ 145.6, K+ 6-0, Ca++ 2.0, 40, 60, 90, and 120 minutes.
Mg++ 2-4, Cl- 127.8, P04- - 1.2, S04- - 2.4,
HCO3- 24-6 mEq/l, 5.5 mM glucose, and 1 % bovine
serum albumin. An aliquot was taken from each ORAL GTT (lg/kg) WITH INSULIN INJECTION
batch of islets for measurement of insulin content. A The procedure was similar to the above except that
known number of islets was then placed in a sili- an intravenous injection of Actrapid insulin (Novo)
conised glass syringe containing 0-2-0-3 ml KRB was given during the first minute (55-60 s) after the
buffer. The recipient rat was anaesthetised with rat had started to take the glucose. The dose of
sodium pentobarbital. The abdomen was opened insulin was calculated to mimic the concentration
through a midline incision and the portal vein found in peripheral veins during cephalic phase
exposed. A 23-gauge needle, connected by a fine release. These calculations were made taking into
catheter to the syringe, was inserted into the portal account the known disappearance rate of intra-
vein and the islets were slowly injected. After the venously injected insulin in rats12 and adjusted
injection, the syringe, catheter, and needle were empirically after pilot experiments in normal rats
repeatedly flushed into a Petri dish and the remaining under basal conditions. Each blood sample was
islets were counted. 200,u1. The total blood withdrawn was therefore
small (1 -6-2 ml) and it was replaced at the end of
each test. The time interval between tests was three
GLUCOSE TOLERANCE TESTS to five days.
Thirteen to 15 weeks after transplantation or sham
operation of non-diabetic control rats, permanent
cardiac catheters were implanted via the jugular PANCREAS AND ISLET EXTRACTION FOR
vein according to the method of Steffens.1" These DETERMINATION OF INSULIN CONTENT
catheters were filled with a 60-70 % polyvinyl- Immediately after death the pancreata were re-
pyrrolidone (PVP) solution containing heparin moved, weighed, and stored at -20°C. Insulin was
500 U/ml and penicillin 20 000 U/ml; the mixture extracted by a modification of the Kenny technique,'3
was aspirated every second day and the whole using acidified ethanol (0.7 M HCI/ethanol, 1:3
catheter was flushed out with heparinised saline and v/v). Acidified ethanol was also used for the extrac-
then refilled with the PVP/heparin/saline solution. tion of islet insulin from the isolated islets.
By this means catheters could be used for six to eight
weeks. Animals were housed individually in special ASSAYS
cages, handled frequently, and all tests were carried Plasma and urinary glucose concentrations were
out in their home cages. The tests were started measured by a glucose oxidase method.'4 Immuno-
one to two weeks after catheter implantation when reactive insulin was measured according to Herbert
the rats had returned to a normal rate of weight gain. et al.1" using rat insulin standard and guinea-pig
The rats were habituated to the blood-sampling antipork insulin serum.2wX r| Gut: first published as 10.1136/gut.21.11.1002 on 1 November 1980. Downloaded from http://gut.bmj.com/ on March 12, 2022 by guest. Protected by copyright.
1004 Siegel, Trimble, Renold, and Berthoud
MATERIALS contained in the transplanted islets corresponded to
Streptozotocin was a gift from Dr W J Dulin, about 62 % of the content of one normal rat pancreas.
Upjohn Company, Kalamazoo, Michigan. Colla-
genase (type IV) was obtained from Serva GmbH, DEVELOPMENT OF BODY WEIGHT
Heidelberg, West Germany. Rat insulin standard The body weight of transplanted rats and of diabetic
was obtained from Dr J Schlichtkrull, Novo and non-diabetic controls is shown in Fig. 1.
Research Institute, Bagsvaerd, Denmark. Guinea-pig Animals injected with streptozotocin stopped gain-
anti-insulin serum was a gift from Dr H H Schone, ing weight. They, in fact, lost about 10 g over the
Farbwerke Hoechst, Frankfurt, West Germany. period of investigation. Not all diabetic controls
Insulin Actrapid was obtained from Novo Industri, survived for the whole period, however, so that the
Copenhagen, Denmark. value shown for six surviving animals corresponds
to a positive selection. After four weeks of diabetes
PRESENTATION OF RESULTS AND (zero time, Fig. 1) the mean weight of the recipient
STATISTICAL METHODS rats was about 75 g less than that of the age-matched
Results are given as mean ± SEM unless stated non-diabetic controls at the time of transplantation.
otherwise. The glucose and insulin areas for the Rapid weight gain occurred after transplantation, so
various glucose tolerance tests were calculated by that from the eighth week onwards no significant
integrating the incremental glucose and insulin difference was observed between transplanted rats
concentrations above basal for each animal. Stu- and non-diabetic controls. Transplanted rats gained
dent's two-tailed unpaired t test was used to evaluate 223 + 21 g and non-diabetic controls 164±14
differences between transplanted rats and controls. (pGut: first published as 10.1136/gut.21.11.1002 on 1 November 1980. Downloaded from http://gut.bmj.com/ on March 12, 2022 by guest. Protected by copyright.
Importance of preabsorptive insulin release on oral glucose tolerance 1005
. Islet transplanted rats (n = 5) (n =5) and was normalised by transplantation (see
- Nondiabetic controls (n = 5) below). Non-diabetic controls had plasma glucose
35
values of 6-9 ±0.6 mmol/l (n= 5) before sham opera-
tion. At death the insulin content of the pancreas in
O 30- transplanted rats was 0 50+0 05% of non-diabetic
E controls and that of diabetic controls was 0.46+
F= 25-
0-22%.
Q)
0
O 20
GLUCOSE TOLERANCE TESTS
Permanent cardiac catheters for use in glucose
tolerance tests were implanted after the weight of the
k
k=2.5320.08 t 0 transplanted rats had caught up with that of the age-
9s matched controls (13th to 15th week, Fig. 1).
m 15 Tests were started one week after this operation
when the rats had regained their preoperative
20 -
weight. The mean basal plasma glucose values for
mean SEM all the tests described below (calculated from the
b 15 pcO.05 mean of six values for each animal) were 68 ±0.2
.C
~~~~p010 308±17 E
Insulin areas 121+19 < :0 005 304±32 8-
Incremental glucose areas during oral GTT (60 min)
.5
6-
Without insulin injection 307 +63 0-10 76±7 coc
Incremental insulin areas during oral GTT (60 min) 2-
Without insulin injection 128 +20 080 >0-10
With insulin injection 131+28 >0.30 190+49 0
0 20 40 60 90 120
Incremental insulin areas during first 5 min of oral GTT minutes
Without insulin injection 2.6+1-2 0 10 203±2.8 conscious unstressed rats during oral GTT (1 g/kg). The
curves compare the oral glucose tolerance of the same
The areas were calculated by integrating the insulin or glucose values animals as Fig. 2. Tests were started (0 min) when
above basal for each individual rat. Statistical comparison was by the rat spontaneously began to take the glucose. Early
Student's unpaired t test when transplanted rats were compared with blood sampling was at two, five and 10 minutes. The
controls and by the paired, when the rats with insulin injection were
compared with themselves without insulin injection. Values are insert shows the first S min of the insulin curves on an
mean ± SEM, n= S, for both transplanted rats and controls. expanded scale, 1 ng insulin= 24 jsU.Gut: first published as 10.1136/gut.21.11.1002 on 1 November 1980. Downloaded from http://gut.bmj.com/ on March 12, 2022 by guest. Protected by copyright.
1006 Siegel, Trimble, Renold, and Berthoud
Islet transplanted rats: planted and control rats both showed a rapid insulin
o-o non injected (n = 5 ) response with maximal insulin concentrations
injected with 24 mU/kg kisuin iv achieved at two minutes. The insulin concentrations,
at 1 min (n = 5) however, were significantly lower in transplanted
15- rats during the first 20 minutes, and the incremental
( moan t SEM ) insulin areas were smaller in transplanted rats than
**
0 pGut: first published as 10.1136/gut.21.11.1002 on 1 November 1980. Downloaded from http://gut.bmj.com/ on March 12, 2022 by guest. Protected by copyright. Importance ofpreabsorptive insulin release on oral glucose tolerance 1007 exhibited a significant increase of plasma insulin insulin levels was a decrease at 10 minutes compared above their own basal level (mean increase 2.5 ±0.6 with the tolerance test without insulin injection. mg/ml, P
Gut: first published as 10.1136/gut.21.11.1002 on 1 November 1980. Downloaded from http://gut.bmj.com/ on March 12, 2022 by guest. Protected by copyright. 1008 Siegel, Trimble, Renold, and Berthoiid rats than in controls during both oral and intra- phenomenon remains to be established. It seems venous glucose tolerance. Caution should, however, unlikely, however, that their secretion would be be taken in extrapolating from peripheral plasma abnormal in transplanted rats with intact innervation insulin levels to the amount secreted. In normal rats, of the gastrointestinal tract and normalised body 50% of secreted insulin can be extracted during one weight. passage through the liver,'617 whereas no such data While decreased insulin secretory capacity not exist for rats with islets transplanted to the liver related to cephalic phase may have contributed to where regions of low and high insulin concentrations the pathological 1 g/kg oral GTT in islet-trans- are found within the organ. The insulin areas of planted rats, results from the 2 g/kg test demon- controls during an oral GTT were comparable strate that the transplanted rats had not reached the with those during an intravenous GTT (Table). limit of their insulin secretory capacity during the The insulin areas of transplanted rats were reduced 1 g/kg test. The principal observation reported here by a similar amount during both tests. For the intra- is that an artificially reestablished 'cephalic phase venous GTT, the insulin output was sufficient to insulin response' resulted in remarkably improved normalise the K-values, whereas the oral GTT oral glucose tolerance most clearly seen some time remained clearly abnormal. While during the intra- after exogenous insulin has disappeared from the venous GTT the onset of insulin release occurred as blood. A preabsorptive increase of 2.5 ng/ml in early in transplanted rats as in controls, during the peripheral plasma insulin in controls two minutes oral GTT transplanted rats showed a clear delay in after oral glucose may seem rather small. However, their insulin response. the increase in portal vein insulin concentrations at The lack of early insulin response during oral that time is almost certainly much greater, a finding GTT can be attributed, at least in part, to a missing that has been demonstrated in dogs during the first cephalic phase of insulin release.3' This phase of five minutes of an oral GTT.26 In man, portal insulin release occurs before the absorption of insulin levels are twice as high as in the periphery nutrients,34 22 is a reflex that can be triggered by in the basal state and may be as much as 10 times food-related stimuli in the mouth, and results in greater when insulin release is stimulated.27 In stimulation of the P-cells probably via efferent addition, it has been shown that the disappearance fibres of the vagus nerve.42223 Physiological import- rate of intraportally injected insulin is greater than ance of cephalic phase insulin secretion in oral that of insulin injected into the periphery.28 There- glucose tolerance has been shown in experiments fore, an insulin injection calculated to mimic peri- where the reflex has been by-passed in otherwise pheral insulin levels at two minutes probably intact rats with permanent gastric catheters. Fifteen does not overestimate the amount of insulin actually and 20 minutes after intragastric administration of secreted from the pancreas during the preabsorptive glucose, plasma glucose levels were higher and phase. In addition, insulin secreted into the portal plasma insulin levels were lower than when the circulation is more effective in improving the meta- same load was given orally.4 Higher plasma glucose bolic state of pancreas-transplanted rats than is concentrations were also found after intragastric insulin secreted into the periphery.29 Nevertheless, administration of a mixed meal.'9 After abolition the peripheral injection of a small amount of insulin of the reflex by vagotomy21 otherwise intact rats caused striking changes in overall oral glucose showed much higher plasma glucose levels during tolerance of islet-transplanted rats. This result an oral glucose tolerance test than did normal suggests an important physiological role for the controls.4 Cephalic phase insulin release can be neurally-mediated cephalic phase insulin response. abolished in dogs by chemical vagotomy that is, After intravenous glucose administration, the atropinisation.23 In man atropinisation has been liver accounts for about 10% of the glucose meta- shown to cause deterioration of oral glucose toler- bolised30 as opposed to 60% after oral administra- ance while intravenous glucose tolerance remained tion of glucose.30 31 In general, the function of intact.24 It is not surprising, therefore, that islet- cephalic phase responses is to prepare the gastro- transplanted rats, with presumably vagotomised intestinal tract to digest and absorb food and to P-cells and no cephalic phase insulin release,234 prepare the viscera to metabolise and store nut- should exhibit abnormal oral glucose tolerance, rients.22 The primary physiological role of cephalic even when intravenous glucose tolerance has been phase insulin secretion may thus be to prime the normalised. Whether, in addition, gastrointestinal liver for the uptake of glucose absorbed from the hormones such as gastric inhibitory polypeptide intestines. Pathological oral glucose tolerance may, hormones such as gastric inhibitory polypeptidel therefore, occur when cephalic phase insulin secre- and gastrin,2' which play a role in the insulin res- tion is absent and the liver is not prepared for this ponse to oral glucose, may have contributed to this task.
Gut: first published as 10.1136/gut.21.11.1002 on 1 November 1980. Downloaded from http://gut.bmj.com/ on March 12, 2022 by guest. Protected by copyright.
Importance ofpreabsorptive insulin release on oral glucose tolerance 1009
The authors are grateful to Mrs Liza Cavillier, '6Karakash C, Assimacopoulos-Jeannet F., Jeanrenaud
Mrs Danielle Nappey, and Miss Dominique Rutsch- B. An anomaly of insulin removal in perfused livers of
mann for their excellent technical help. This work obese-hyperglycemic (ob/ob) mice. J Clin Invest 1976;
was supported by grant nos. 3.774.0.76, 3.154.0.77, 57:1117-24.
3.487.0.79 of the Swiss National Science Foundation 1Mondon CE, Olefsky JM, Dolkas CB, Reaven GM.
Removal of insulin by perfused rat liver: effect of con-
and NIH grant no. ROI AM 25220-01. centration. Metabolism 1975; 24: 153-60.
18Hommel H, Fischer U, Retzlaff K, Knofler H. The
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