Bioscience Reports i, 345-352 (1981)
~zinted in Great Britain
545
U p t a k e of ~ree and l i p o s o m e - e n t r a p p e d i n s u l i n by
r a t i n t e s t i n a l sacs i n v i t r o
Richard N. ROWLAND and 3ohn F. WOODLEY
Biochemistry Research Laboratory, Department of
Biological Sciences, University of Keele,
Keele, Staffordshire ST5 5BG, U.K.
(Received 31 March 1981)
Entrapment of insulin within distearoylphosphatidylcholine
and cholesterol liposomes can p r o t e c t the hormone from
d e g r a d a t i o n in an in v i t r o g u t - s a c s y s t e m and c a n
i n c r e a s e t he q u a n t i t y of i m m u n o r e a c t i v e insulin that
reaches the serosal fluid 26-fold.
95~ of t h e insulin
that reaches the serosal fluid is present within relatively
intact liposomes.
However, the presence of h y d r o l y t i c
a g e n t s , p r o b a b l y e n z y m e s , within the serosal fluid of
everted gut sacs makes it n e c e s s a r y to s e p a r a t e t h e
liposomes from the serosal fluid, by centrifugation, prior
to release of the entrapped insulin with T r i t o n X-100.
This procedure prevents hydrolysis of the released insulin
which can then be measured by radioimmune assay.
It is generally agreed that liposome-entrapped orally administered
insulin can r e d u c e blood g l u c o s e l e v e l s in d i a b e t i c r a t s ( 1 , 2 ) .
H o w e v e r , t h e c l a i m by Dapergolas and Gregoriadis (2) that insulin
reaches the circulation in i nt act liposomes has been disputed (3). We
h a v e shown p r e v i o u s l y (4) t h a t l i p o s o m e s composed of distearoylphosphatidylcholine/cholesterol r e t a i n t h e m a j o r i t y of an e n t r a p p e d
m a c r o m o l e c u l e in t h e p r e s e n c e of e x t r e m e s of pH, bile salts, and
p an cr eatic lipase and thus appear to have the qualities required of a
p r o t e c t i v e carrier for oral use.
Furt herm ore, e n t r a p m e n t of degradable horseradish peroxidase (HRP) within these liposomes increased the
quantity of enzymaticaIly active HRP t hat reached the serosal fluid of
ev er ted rat gut sacs in vitro 6.6-fold (5).
The aim of t h e p r e s e n t study was to compare the quantity of
immunoreactive insulin that reached the serosal fluid of e v e r t e d r a t
gut sacs cultured in vitro with f r ee or liposome-entrapped insulin and
to determine whether the insulin present in the serosal fluid was still
entrapped within liposomes.
M a t e r i a l s and Methods
Liposome preparation
27 mg of L-a-distearoylphosphatidylcholine (Sigma) and 3.g mg of
cholesterol ( p u r e , K o c h - L i g h t L a b s . ) w e r e d i s s o l v e d in 15 ml of
c h l o r o f o r m and r o t a r y - e v a p o r a t e d under nitrogen at 5S~
The lipid
film was resuspended at 5 g ~
by hand a g i t a t i o n , with 2.5 ml of
9
The Biochemical Society
3tt6
ROWLAND AND WOODLEY
bovine insulin B.P. (83.2 U/roll 3.176 mg of insulin/roll Weddel
Pharmaceuticals L t d , London).
A tracer Of 128I-labelled insulin (50
t~Ci/mg; R a d i o c h e m i c a l C e n t r % A m e r s h a m ) was included in some
preparations.
Unentrapped insulin was removed by c e n t r i f u g i n g the
p r e p a r a t i o n at 100 000 g (]0 min~ 20~
removing the supernatant,
and resuspending the pellet in fresh 5 mM phosphate-buffered saline
(pH 7.2). This process was repeated 4 times. It was calculated from
r a d i o a c t i v i t y measurements that the final liposome p r e p a r a t i o n s
c o n t a i n e d 0.141 • 0.003 U of i n s u l i n / m l lipid (5.3g • 0. t l lag of
insulin/mg lipid), mean • S.E. in 16 separate liposome p r e p a r a t i o n s .
'Empty' liposomes were prepared by the method described above but in
the absence of unlabelled or 12SI-labelled insulin.
Liposomal
latency
A l i q u o t s of l i p o s o m e s containing both unlabelled and l~SI-labelled
insulin were counted for total radioactivity. The p e r c e n t a g e of t h e
t o t a l r a d i o a c t i v i t y that was present in a 100 000-g (10 min, 20~
supernatant was d e t e r m i n e d by y c o u n t i n g bot h b e f o r e and a f t e r
incubation of liposomes with Triton X-100 (2.5% v/v; 58~
30 rain).
T h e s e e x p e r i m e n t s s how e d t h a t 94.g • 1.3% ( m e a n • S . E . , 21
e x p e r i m e n t s ) of the liposome-entrapped 12SI-labelled insulin could be
released by t r e a t m e n t With Triton X-100.
The TCA-solubility of the
insulin was u n a f f e c t e d (<2%).
A l i q u o t s of liposomes containing unlabelled insulin were incubated
with Triton X-I00, and the quantity of insulin present in solution was
d e t e r m i n e d by r a d i o i m m u n e as s ay ( R . I . A . ; R a d i o c h e m i c a I Centr%
Amersham).
A standard curve was constructed with insulin that had
b e e n i n c u b a t e d at 58~
for 30 min with Triton X-100 (2.5% v/ v).
Assuming a release of 94.g% of the entrapped insulin, R.I.A. of the
l i p o s o m e s t r e a t e d w i t h T r i t o n X-100 gave an e n t r a p m e n t value of
0.140 • 0.010 U of insulin/mg lipid ( 5 . ] 5 • 0.39 pg of i n s u l i n / r a g
lipid), mean • S . E , 10 separate liposome preparations. This figure is
t h e s a m e as t h e e n t r a p m e n t v a l u e d e t e r m i n e d
by y c o u n t i n g .
E x p e r i m e n t s with f r ee insulin showed that the activity of insulin, as
measured by R.I.A. in the presence of Triton X-100~ was u n a f f e c t e d by
the liposome preparation procedure, by incubation at 5g~ for 30 rain,
or by the presence of empty liposomes.
Everted gut sac culture
The preparation and method of culture of e v e r t e d sacs of adult rat
i n t e s t i n e ha ve been d e s c r i b e d ( 6 ) .
Briefly, everted sacs are
i n d i v i d u a l l y i n c u b a t e d in 10 ml of o x y g e n a t e d TC 199 m e d i u m
(Wellcome) containing 10% inactivated calf serum (Wellcome) and 1
mM ATP a t 37~
E a c h e v e r t e d sac is 3-4 cm long (40-60 mg
protein) and contains = 0.5 ml of oxygenated medium. Gut sacs were
cultured for 1 h in medium containing (a) g0 mU of free insulin/ml
(3.05 pg of insulin/ml) (b) gO mU of f r e e i n s u l i n / m l plus e m p t y
liposomes at 580 lag of lipid/ml, or (c) gO mU of insulin/ml entrapped
within liposomes at 580 lag of lipid/ml.
Measurement
of immunoreactive
insulin in the serosal fluid
M e t h o d 1.
In preliminary experiments the serosal fluid from each
sac was collected after 1 h, and the volume was measured. Standard
UPTAKE
OF
LIPOSOMES
BY RAT GUT
3it7
c u r v e s f o r t h e R . I . A . w e r e p r e p a r e d ( a ) with insulin in m e d i u m at
room t e m p e r a t u r e and (b) w i t h m e d i u m c o n t a i n i n g i n s u l i n t h a t h a d
been incubated
w i t h T r i t o n X - 1 0 0 (2.5~
v/v, 5g~
30 r a i n ) .
I m m u n o r e a c t i v e insulin p r e s e n t in t h e serosal fluid was d e t e r m i n e d by
R.I.A., both b e f o r e and a f t e r t r e a t m e n t with T r i t o n X-100 (2.5% v / v ,
58~
30 min) by use of the a p p r o p r i a t e s t a n d a r d curve.
M e t h o d 2.
In l a t e r e x p e r i m e n t s the serosal fluid f r o m 5 or 6 sacs
was pooled, the v o l u m e m e a s u r e d , and c e n t r i f u g e d a t 100 000 g ( 1 0
rain, 2 0 ~
The s u p e r n a t a n t was r e m o v e d and r e t a i n e d .
The pellet
was r e s u s p e n d e d in f r e s h m e d i u m and r e c e n t r i f u g e d .
The p e l l e t was
r e s u s p e n d e d to I ml and the insulin in the original s u p e r n a t a n t a n d the
r e s u s p e n d e d p e l l e t was d e t e r m i n e d by R.I.A., b o t h b e f o r e a n d a f t e r
i n c u b a t i o n with T r i t o n X-100. In both m e t h o d s the sacs w e r e dissolved
in 1.0 M N a O H a n d a s s a y e d f o r p r o t e i n ( 7 ) .
The quantity
of
immunoreactive
insulin p r e s e n t in the serosal fluid was e x p r e s s e d as
launits/mg tissue protein.
Determination of the extent of hydrolysis of 125I-labelled insulin
(1) I n t h e m e d i u m .
Medium c o n t a i n i n g ( a ) f r e e i n s u l i n a t 4 x
lO -4, 4 X lO -3, g x 10 -2, 4 x 10 -1, or t+ U / m l and a 'spike' of
1251-1abelled insulin (2.0 nCi/ml; l IJU; t~O pg); (b) free insulin at g x
I0 -2 U/ml, a spike of 1251-1abelled insulin, and empty liposomes at 580
lag lipid/ml; (c) 8 x lO -2 U insulin/ml and a spike of 12Sl-labelled
insul.in e n t r a p p e d within l i p o s o m e s at 580 lag of lipid/ml was i n c u b a t e d
a t 37~ for 1 h in the p r e s e n c e or a b s e n c e of e v e r t e d gut sacs filled
w i t h plain m e d i u m .
(2) I n t h e s e r o s a l f l u i d .
E v e r t e d gut sacs w e r e p r e p a r e d i n the
n o r m a l m a n n e r e x c e p t t h a t t h e y w e r e filled with 0.5 ml of the m e d i a
d e s c r i b e d a b o v e in (1) and w e r e i n c u b a t e d in plain m e d i u m at 37~
f o r 1 h.
The p e r c e n t a g e of the t o t a l r a d i o a c t i v i t y in the m e d i u m or serosal
fluid t h a t was T C A - s o l u b l e was d e t e r m i n e d at t h e b e g i n n i n g of t h e
experiment
a n d a f t e r I h of c u l t u r e .
The liposome-entrapped
125I-labelled insulin w a s r e l e a s e d by t r e a t m e n t
with Triton X-100
(2~
v/v; 5g~
30 rain) prior to d e t e r m i n a t i o n of the p e r c e n t a g e of
TCA-soluble radioactivity.
Results
and Discussion
Measurement
Method 1
of
the immunoreactive
insulin
in the serosal
fluid:
T a b l e i s h o w s t h a t t h e a m o u n t of i m m u n o r e a c t i v e insulin t h a t
r e a c h e d the serosal . f l u i d w a s v e r y s m a l l .
Furthermore,
although
e n t r a p m e n t of insulin within l i p o s o m e s i n c r e a s e d the level of insulin in
the serosal fluid 2-fold, it was not i n c r e a s e d f u r t h e r by t r e a t m e n t of
t h e serosal fluid with T r i t o n X-100.
This result implied t h a t all the
d e t e c t a b l e insulin was f r e e in solution a n d n o t l i p o s o m e - e n t r a p p e d ,
T h e s e results w e r e in c o n t r a s t to our previous work with H R P , w h e r e
it was shown t h a t l i p o s o m e - e n t r a p m e n t i n c r e a s e d the q u a n t i t y of H R P
that
reached
the serosal fluid 6.6-fold and that 95% was still
l i p o s o m e - e n t r a p p e d ( 6 ) . The serosal fluid f r o m gut sacs c u l t u r e d with
f r e e a n d w i t h l i p o s o m e - e n t r a p p e d insulin c o n t a i n e d 6.9 x 10-e% and
1.21 x 10-5% r e s p e c t i v e l y of t h e t o t a l i n s u l i n ( 0 . 8 U) o r i g i n a l l y
348
ROWLAND AND WOODLEY
Table i. Immunoreactive insulin present in the serosal fluid
of gut sacs cultured for i h with free or liposome-entrapped
insulin: Method 1
~U insulin/mg tissue protein
Before Triton
After Triton
Free insulin a
0.043 • 0.006
0.055 + 0.014
Free insulin +
'empty' liposomes b
0.048 • 0.007
0.049 + 0.023
Liposome-entrapped
insulin c
0.i01 • 0.015
0.097 + 0.018
Gut sacs were
either free or
37~
Insulin
i n c u b a t i o n of
represents the
c u l t u r e d in medium
liposome-entrapped
was determined by
the serosal fluid
mean -+ S.E. of all,
containing 80 mU of i n s u l i n / m l
(580 ~g of lipid/ml) for 1 h at
R.I.A. both b e f o r e and after
with Triton X-100.
Each value
b5, or c14 experiments.
p r e s e n t in the l0 ml of m e d i u m / m g tissue p r o t e i n . T h e s e values w e r e
22 x and 80 x l o w e r t h a n t h e c o r r e s p o n d i n g
values for free and
liposome-entrapped
HRP.
T h i s s u g g e s t s t h a t insulin was far m o r e
s u s c e p t i b l e to d e g r a d a t i o n in the g u t - s a c s y s t e m t h a n H R P , a n d w a s
not p r o t e c t e d by l i p o s o m e e n t r a p m e n t .
Hydrolysis of 125I-labelled insulin
(1) In the medium. Table 2 shows that in the absence of everted
gut sacs virtually no hydrolysis of f r e e 125I-labelled insulin o c c u r r e d in
t h e m e d i u m during 1 h of incubation.
H o w e v e r , the p r e s e n c e of gut
sacs r e s u l t e d in the d e g r a d a t i o n of m o r e t h a n 90% of the f r e e insulin
(80 m U / m l + spike of 12SI-labelled insulin), as m e a s u r e d by solubility
in TCA. As the c o n c e n t r a t i o n of insulin was i n c r e a s e d , the p e r c e n t a g e
t h a t was hydrolysed was reduced.
Similar results h a v e been r e p o r t e d
for i n t e s t i n a l e p i t h e l i a l cells in v i t r o ( 8 ) .
T h e p r e s e n c e of e m p t y
l i p o s o m e s had no e f f e c t on t h e d e g r a d a t i o n of f r e e insulin.
The
d e g r a d a t i o n of f r e e i n s u l i n in t h e m e d i u m e i t h e r by b r u s h - b o r d e r
e n z y m e s ( 9 ) a n d / o r by a d s o r b e d p a n c r e a t i c
e n z y m e s ( 1 0 ) would
explain why only a m i n u t e a m o u n t of f r e e insulin r e a c h e d the s e r o s a l
fluid.
H o w e v e r , e s s e n t i a l l y no d e g r a d a t i o n
of l i p o s o m e - e n t r a p p e d
insulin o c c u r r e d in the m e d i u m during 1 h of incubation with e v e r t e d
gut sacs (Table 2).
These results suggested that the liposomee n t r a p p e d insulin was d e g r a d e d e i t h e r in the e p i t h e l i a l cells or in t h e
serosal fluid a f t e r t r a n s l o c a t i o n .
(2) In the serosal
fluid.
When m e d i u m c o n t a i n i n g a spike of
f r e e 125I-labelled insulin and up to 80 m U / m l of unlabelled insulin was
placed inside gut sacs and i n c u b a t e d for l h at 37~
m o r e t h a n 97%
of the insulin was h y d r o l y s e d ( T a b l e 3 ) .
T h e p r e s e n c e of e m p t y
l i p o s o m e s i n s i d e the sac had no e f f e c t on the p e r c e n t a g e of insulin
t h a t was hydrolysed.
H o w e v e r , i n c r e a s i n g the c o n c e n t r a t i o n of f r e e
unlabelled insulin inside the sac did r e d u c e the e x t e n t of d e g r a d a t i o n .
When m e d i u m c o n t a i n i n g l i p o s o m e - e n t r a p p e d l~-SI-labelled insulin and 80
UPTAKE
OF
LIPOSOMES
BY RAT GUT
3Lt9
Table 2. Hydrolysis of free and liposome-entrapped insulin
in medium in the presence and absence of gut sacs
Unlabelled
insulin
% of TCA-soluble
radioactivity
(Ulml)5
tO
t60
t~o + sacs
Free insulin
4
4
8
4
4
x
x
x
x
10 -4
10 -3
10 -2
10 -!
2.87
2.38
2.50
2.70
2.35
•
•
+
•
•
0.01
0.01
0.02
0.01
0.01
3.31
3.16
2.70
2.76
2.44
•
i
•
•
•
0.02
0.01
0.02
0.02
0.02
98.90
96.33
93.90
17.39
7.60
+
•
+
•
+
4.54
4.58
0.50
3.44
0.94
Free insulin +
'empty' liposomes
8 x 10 -2
2.43 + 0.01
2.49 • 0.01
87.53 • 3.16
Liposome-entrapped
insulin
8 x 10 -2
2.50 -+ 0.01
3.61 • 0 . 0 5
3.74 • 0.06
Medium containing free or liposome-entrapped 1251-1abelled insulin
(2.0 nCi/ml, i ~U, 2.0 pg) and various concentrations of unlabelled insulin was incubated for 1 h at 37~
in the p r e s e n c e or
absence of everted gut sacs.
The • of the total radioactivity in
the medium that was TCA-soluble was determined at the start of the
experiment
(t O ) and after 60 m i n (t60).
Liposome-entrapped
1251-1abelled
i n s u l i n was r e l e a s e d by T r i t o n X-100 prior to
determination of the • of TCA-soluble radioactivity.
Each value
represents the mean • S.E. of 3 experiments.
Liposome concentration = 580 ~g of lipid/ml.
mU of unlabelled insulin/ml was placed inside gut sacs, i n c u b a t e d for 1
h at 37~
and t h e n t r e a t e d with T r i t o n X-100, 97% of t h e i n s u l i n
was d e g r a d e d ( T a b l e 3 ) .
H o w e v e r , if the sac c o n t e n t s were cent r i f u g e d (100 000 g, 10 rain, 20~
prior to t r e a t m e n t
with Triton
X - 1 0 0 , a w h i t e l i p o s o m a l p e l l e t was visible.
The s u p e r n a t a n t was
removed~ t h e p e l l e t was r e s u s p e n d e d in f r e s h m e d i u m , and b o t h
s a m p l e s w e r e i n c u b a t e d with T r i t o n X-100.
Under these conditions
g6.5 + 2.3%* of the r e c o v e r e d 125I-labelled insulin was present in the
p e l l e t and only 3.6 -+ 0.7%* of this was TCA-soluble. 97.4 -+ 1.9%* of
the r a d i o a c t i v i t y in the s u p e r n a t a n t was TCA-soluble.
(*Mean + S.E.,
4 experiments. )
T h e s e r e s u l t s i n d i c a t e d t h a t l i p o s o m e - e n t r a p p e d insulin was not
being degraded during c u l t u r e , but was being hydrolysed in the serosal
fluid a f t e r its r e l e a s e from liposomes by T r i t o n X-100. The n a t u r e oi
the ' h y d r o l y t i c agents' p r e s e n t in the serosal fluid is unknown, but the
demonstration
t h a t t h e y r e m a i n e d in t h e s u p e r n a t a n t f o l l o w i n g
c e n t r i f u g a t i o n showed they were in solution.
Furthermore, the fact
t h a t insulin breakdown could be r e d u c e d by increasing its c o n c e n t r a t i o n
suggested t h a t t h e h y d r o l y t i c s y s t e m c o u l d be s a t u r a t e d and was
350
ROWLAND AND WOODLEY
Table 3. Hydrolysis of free and liposome-entrapped insulin
incubated for 1 h inside everted rat gut sacs
Unlabelled
insulin
U/ml
% of TCA-soluble
radioactivity
in the serosal fluid
to
4
4
8
4
4
Free insulin
x
x
x
x
10 -4
10 -3
10 -2
i0 -!
2.87
2.38
2.50
2.70
2.35
•
•
•
•
•
t60
0.01
0.01
0.02
0.01
0.01
106.00
97.01
98.73
69.10
20.43
•
•
•
•
•
3.63
3.92
0.64
4.36
2.33
Free insulin +
'empty' liposomes
8 x 10 -2
2.43 • 0.01
98.42 + 3.11
Liposome-entrapped
insulin
8 x 10 -2
2.50 • 0.01
97.54 • 1.61
Everted rat gut sacs (3-4 cm long) were filled w i t h = 0.5 ml
medium containing free or liposome-entrapped 1251-1abelled insulin
(2.0 nCi/ml~ 1 U ~ 2.0 pg) and various concentrations of unlabelled
insulin and incubated at 37~
for 1 h in plain medium.
The • of
the total radioactivity p r e s e n t inside the gut sacs that was
TCA-soluble was determined at the start of the experiment (t O ) and
after 1 h of culture (t60).
Liposome-entrapped
1251-1abelled
insulin was released by Triton X-100 prior to determination of the
• of TCA-soluble radioactivity.
Each value represents the mean i
S.E. of 3 e x p e r i m e n t s .
L i p o s o m e c o n c e n t r a t i o n = 580 ~g of
lipid/ml.
therefore probably enzymic.
The exocytosis of lysosomal enzymes
from gut ceils could lead to a buildup of proteolytic enzymes within a
closed sac.
T h e s e r es ul t s explain the failure to d e t e c t insulin in the serosal
fluid by Method 1. Consequently a second method, involving removal
of t h e l i p o s o m e s from the hydrolytic agents in the serosal fluid, by
centrifugation, was employed.
Measurement
Method 2
of the immunoreactive
insulin
in the serosal
fluid:
The q u a n t i t y of insulin in the supernatant (100 000 g, l0 min,
20~
of the serosal fluid from sacs cultured with free or liposomeentrapped insulin, both before and a f t e r t r e a t m e n t with Triton X-100
(Table #), was very similar to the amount of insulin found in t h e
w h o l e s e r o s a l f l u i d ( T a b l e 1).
T h i s is not s u r p r i s i n g , as t h e
supernatant would contain the hydrolytic agents present in the whole
s e r o s a l f lu i d.
However, the resuspended 100 000-g pellets from the
s er o s al fluid of gut sacs c u l t u r e d with f r e e or l i p o s o m e - e n t r a p p e d
insulin all c o n t a i n e d =0.2 IJU of i n s u l i n / m g t i s s u e p r o t e i n in the
UPTAKE
OF
LIPOSOMES
BY
RAT
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352
ROWLAND AND WOODLEY
absence oi Triton X-100 (Table 4). These values are 2-5 times higher
than the values measured in the whole serosal fluid (Table 1). It is
possible that some f r ee insulin reached the serosal fluid and bound to
components of the medium, to particles oi gut tissue, such as sloughed
c e l l s , or to e m p t y or d i s r u p t e d liposomes, and was p r o t e c t e d from
degradation.
Such bound insulin m ay p e l l e t on c e n t r i f u g a t i o n ,
dissociate in fresh medium, and then be d e t e c t e d by the R.I.A.
Triton-X-100 t r e a t m e n t of the 100 000-g (10 min, 20~
pellets
f r o m t h e s e r o s a l fluid of gut sacs cultured with liposome-entrapped
insulin r e s u l t e d in a 6 0 - f o l d i n c r e a s e in t h e q u a n t i t y of insulin
m e a s u r e d by R.I.A. ( T a b l e 4) .
The control experiments with free
insulin, with or without empty liposomes, show ed t h a t only a v e r y
small p e r c e n t a g e ( < 5 % ) of this i n c r e a s e coul d be a t t r i b u t e d to
Triton-induced release of insulin irom tissue or medium components or
irom empty or disrupted liposomes. These experiments also rule out
the possibility t h a t t h e i n c r e a s e was due to t h e f o r m a t i o n of a
p h o s p h o l i p i d - h o r m o n e c o m p l e x which could only i n t e r a c t e i f i c i e n t l y
with anti-insulin antibodies in the presence of d e t e r g e n t .
Therefore
t h e m a j o r i t y oi t h e i n c r e a s e was due to the rupture oi relatively
intact l i p o s o m e s by T r i t o n X-100, l e a d i n g to t h e r e l e a s e of t h e
entrapped insulin. Table 4 shows that liposomal e n t r a p m e n t of insulin
increased the total quantity of immunoreactive insulin that reached the
serosal fluid 26-fold, compared with f r e e insulin, and that 95% of this
insulin was liposome-entrapped.
In c o n c l u s i o n , l i p o s o m e - e n t r a p m e n t can p r o t e c t insulin i r o m
degradation and can transport the intact hormone to the serosal side
oi an e v e r t e d gut sac.
The present study showed that =600 laU of
immunoreactive insulin reached the serosal fluid of a 3- to 4-cm (40to 6 0 - m g of p r o t e i n ) gut sac in one hour. Extrapolation of these
results to the whole rat intestine would suggest a translocation of "-24
mU of insulin/h to the blood stream.
Acknowledgements
Th e f i n a n c i a l support of the Medical Research Council is gratefully acknowledged.
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4.
5.
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