Distribution
and Metabolism of Intravenously Tritiated Insulin in Rats
Injected
Philippe A. Halban, Michael Berger, and Robin E. Offord Semisynthetic [‘H] insulin was used to follow the distribution and metabolism of intravenously injected insulin in rats. Chromatographic methods were used to separate intact labeled insulin from radioactive degradation products in the plasma, liver, kidneys. and skeletal muscle. The metabolic clearance rate of the injected insulin was 23.6 ? 1 ml/min/kg and the distribution space 65 + 17 ml/kg for an injected dose of 1.5 mU/lOO g body weight. The kidneys concentrated tritium relative to the plasma by up to ninefold; the liver also concentrated radioactivity, but to a lesser degree. Radioactive degradation products were found to be of either higher or lower molecular weight than insulin. The kidneys contained predominantly low-molecularweight degradation products, accounting for 54% of the radioactivity in these organs even 5 min after injection. The liver, by contrast, contained predominantly high-molecular-weight degradation products. This material appeared in the liver before it was seen in the plasma, suggesting that the liver is responsible for its production. This suggestion was confirmed by analyzing plasma samples from rats injected intravenously with [“HI insulin following either functional hepatectomy or nephrectomy. The hepatectomized rats displayed less high-molecularweight material in the plasma and 2-3 times more intact insulin when compared to controls. By contrast, nephrectomy resulted in no significant change in the percentage of either high- or lowmolecular-weight degradation products in the plasma despite reduced insulin clearance. These data confirm the importance of the liver and kidneys in insulin metabolism. Since at least some of the high-molecular-weight-degradation products may be formed by reincorporation of [“HI phenylalanine (liberated by degradation of [“HI insulin) into newly synthesized protein, the importance of the liver in its production may be a reflection of the protein biosynthetic capacity of this organ.
A
BETTER UNDERSTANDING of the pharmacokinetics of injected insulin may lead to improved management of insulin-dependent diabetics. Whereas many studies have been directed toward the distribution, metabolism, and excretion of exogenous insulin, the majority of such studies have relied upon radioiodinated insulin preparations as tracers (see Izzo’ for review.) However carefully the iodoinsulin is prepared, it still represents a chemically altered form of the hormone2: there cannot be certainty that it will behave authentically in every situation.‘13” In order to overcome this problem, we Merabolism,Vol. 28, No. 11,
(November), 1979
have developed a semisynthetic tritiated insulin’ that has been shown to be indistinguishable from native insulin in both its biologic and immunologic activity as well as its metabolism.‘~* This labeled insulin has previously been used to study the pharmacokinetics of subcutaneously injected insulin both in rats’.” and humans.” This study describes the pharmacokinetics of intravenously injected [3H] insulin in rats. In addition to estimating the metabolic clearance rate and distribution space of the injected insulin, we have followed the distribution of the tracer in the liver, kidneys, and skeletal muscle. Previous studies have shown the importance of the liver for insulin metabolism using both in vivol?~l4 and in vitro83’5-‘7 experimental techniques. The kidney, similarly, has been shown to play an important role in insulin metabolism and clearance.‘8-20 The results described in this study serve to confirm the importance of these two organs in insulin metabolism and provide preliminary evidence as to how the two organs metabolize insulin and its degradation products.
MATERIALS
AND
METHODS
Preparation of 13H] Insulin Semisynthetic [‘HI insulin was prepared and purified as previously described.7,8In brief, the preparation involves the replacement of the Bl phenylalanine of pork insulin by [‘HI phenylalanine. followed by two purification steps to remove excess (‘H] phenylalanine and to separate intact insulin from damaged material. The purified material had a specific radioactivity of 7-10 Ci/mmole and 95%-98’S of the
From the Insiitui de Biochimie Clinique, University of Geneva, Switzerland, and the Laboratory of Molecular Biophysics, University of Oxford, United Kingdom. Received for publication August 25, 1978. Presented in part at the Twelfih Annual Meeting of the European Society for Clinical Investigation. April 1978. Rotterdam. Amsterdam. Supported by Swiss National Science Foundation Grant No. 3.774-0.76SR. Address reprint requests to Dr. Philippe A. Halban, Institut de Biochimie Clinique. Sentier de la Roseraie. 121 I Cen&e 4, Switzerland. 0 I979 by Grune & Stratton, Inc. 0026-c)495/79/2811-0004$01.00/0
1097
HALBAN.
1098
radioactivity
was associated
with
mined by gel chromatography
intact
insulin
(as deter-
tion
products,
previously
and immunoprecipitation’).
plasma
described’
samples
Male Wistar standard
rats (mean weight
Purina
chow.
[‘HI
220 g) were fed ad lib. on
insulin
was injected
pulse into a tail vein as an aqueous solution glycine, The
2.5 mg/ml
injected
bovine
serum
x
weight
(mean
+ SE, n = 31). which
mately
0.015
U insulin/kg
(BSA),
times
corresponds
body weight.
after
tubes.
Following
frozen
and stored.
sodium
taken,
of skeletal Up
min
and were
finally
used for subsequent
in a boiling
centrifuged.
The
were
samples
in 2%~ (w/v)
I g
to
of tissue
was
samples were
water bath for 2
supernatants
were
Fed rats were anesthetized the liver portal
(40 mg/kg
from
body weight).
the circulation,
A polyethylene
(internal
0.61 mm),
with a heparin
solution,
was inserted
jugular
had
which
vein was ligated
[IH]
group of animals insulin
above. After checked, aliquots
into the right jugular
by laparotomy.
of each
the total
of the columns
G50
because
than is normally fractions
from
of
found
were collected
Tritium both
SDS
in exclusion
radioactivity
plasma
and
tissue
profiles were drawn on graph paper and peak
was
measured
planimetrically
as
Peak size was expressed as percent
‘H radioactivity
eluted
from the column.
was greater than 90%~. [‘HI
tion
in plasma
samples
total
sample
radioactivity
eluting
with
Dextran
Blue, insulin.
was determined
insulin.
by the
The
insulin
were
of
The yield concentra-
by multiplying
percentage
columns
and [jH]
the
radioactivity
calibrated
with
phenylalanine.
free of radioactiv-
considered
successful.
as described
above.
Renal
sides. A control
group
by laparotomy.
liquid
[‘H]
insulin
above. After
30
and blood and tissue
above.
scintillation
as the scintillant.
for quenching intact
Behringwerke
“Instagel,”
Packard
Switzerland;
Zurich,
A.G..
Mar-
Instrument
Inter-
G50 and G75 Sephadex.
Switzerland.
Presentation of Results, Calculations, and Statistical Methods In order
to allow
for variation
injection means
different
insulin
of I x 10s dpm [‘HI tables,
in rat weight
experiments,
evaluated
by
and figures, Student’s
were expressed
insulin/l00
* SE and the statistical
and insulin
all data on plasma or
the results significance
t test
as for an
g body weight.
for
are given
as
of differences unpaired
group
as described
counter All
(Model
determina-
by the addition
of an
standard.
In order to separate
Zurich,
A.G..
were
of plasma and soluble tissue samples was
were corrected
S.A.,
Pharmacia
comparisons.
were decapitated
using “lnstagel”
national
albumin,
F.R.G.;
and
into a tail vein as described
in a Beckman
serum
burg/Lahn,
was
offon both
was sham-operated
The radioactivity
internal
weight
peaks
previously.’
of the catheter
Analytical Methods
tions
area
neck blood collected,
specimens were sampled as described
LS-230)
of
(I .5 x 80
BSA, pH 8.4.
was patent until the end of the
anesthetized
or 60 min, the animals
determined
the
described
columns
resulting
was 0.2 ml/min.
as 3 distinct
In the text.
into a tail vein as described
procedure
and veins were tied
was then injected
rate
tissue levels of ‘H or [‘HI
vena cava. A
Functional Nephrectomy
of animals
eluted
flow
The
and the liver tissue essentially
were
instead
for any given gel type. One-milliliter
to the site of insertion.
of the liver assayed for ‘H radioactivity
rats
Sephadex
and the flow tissue samples
SDS, 25 mg/ml
of lower molecular
dosage between
decapitated.
ity was the hepatectomy
Fed
molecules
Soluble
of molecules,
The
30 or 60 min, the patency
above. Only when the catheter
arteries
used
exposed.
was sham-operated
was then injected
the animals
experiment
was
pH 8.8 as the
into 0.28
had been flushed
portal blood was thus bypassed into the superior control
entry
diameter
been surgically
proximal
and the
into the portal vein. The
was inserted
previously
of
to exclude
artery
before their
catheter
other end of the catheter
injection
In order
the hepatic
diameter
which
Sephadex
Bovine by intraperitoneal
mm, external
vein,
on ti75
with 2% (w/v)
changes the properties
BSA,
as (0.8 x
Sources of Materials
vein were tied off immediately
the liver.
0.3 ml/min.
were chromatographed G75
2.5 mg/ml
columns
of I ml were collected
rate was approximately cm) developed
chromatographed
analysis.
Functional Hepatectomy pentobarbitol
Fractions
samples. The elution
muscle from the
in IO ml SDS. The homogenized
then made soluble by incubation
were sacri-
samples
and homogenized (SDS).
dose.
decapitation.
buffer.
and the
in heparinized
the plasma after
and aliquots
weighed,
dodecylsulphate
homogenized
insulin
the animals
centrifugation, Immediately
to approxi-
No hypoglycemic
and neck blood collected
of the liver. kidneys, legs were
injection,
pH 7.4.
IO’ dpm/ 100 g body
effect was observed as a result of this injected At various
in a single
in 0.2 ml of 0.2 M
albumin
dose was I .7 k 0.1
ficed by decapitation
elution
were
on G50 fine Sephadex
60 cm) using 0.2 M glycine,
Intravenous Injection and Sample Collection
BERGER, AND OFFORD
labeled
insulin
from degrada-
RESULTS
Distribution of -‘H in Plasma, Liver, Kidneys, and Skeletal Muscle Following Intravenous Injection of [3H] Insulin The radioactivity in plasma and soluble organ and skeletal muscle samples was determined at set times after intravenous injection of [‘HI insulin (Table I). Since the organs were not flushed out with a physiologic buffer before being made soluble they still contained blood and extracellular fluid. Taking the extracellular space as 20% of tissue weight, the contribution of the radioactivity in the extracellular space of the organs and muscle may be estimated, assuming that the plasma radioactivity at any time is an
DISTRIBUTION AND METABOLISM OF I.V. INSULIN
1099
Table 1. Recovery of Radioactivity
in Plasma, Liver, Kidney, and Skeletal
Following Intravenous
Muscle
Injection of [‘HI insulin Total Radwxxtvlty*
Plasm.3
Time (mm)
Idpmlmll
LIVH
Kidney
(dpmlgl
ldpmlgl
MllSCk
idpmlg)
11029
+ 897 16)
1 5
5956 2042
k 652 (8) k 175 18)
2888
+ 425 (6)
18501
t 736
(61
963 + 69
10
1551
i- 307 (3)
1056 + 114 (4)
10348
k 772
(4)
775 k 134 (4)
0.5
20 60
832 + 136 (4) 1271
k 171 15)
961 f 238 151
7524
+ 1737 (5)
873 k 166 (3)
4785
i
183
(41
456
(5)
+ 125 151 670 (21
*Results are expressed as the mean i SE normalized as for an Injection of lo5 dpmi 100 g body weight. The number of Independent observations is shown in parentheses.
approximate indication of the extracellular space radioactivity. Five minutes after the injection, it can be seen that the radioactivity in the liver (expressed as dpm/g wet weight of tissue) is greater than 20% of that found in the plasma (expressed as dpm/ml plasma), indicating that radioactive molecules are bound to the liver cell membranes and/or internalized. This concentration of radioactivity above that found in the extracellular space is even more pronounced for the kidneys. Between 5 and 20 min after injection the kidneys concentrate radioactivity 6-9 times over that found in the plasma. Although a moderate concentrating effect is observed in the muscle, it is less pronounced than that found in the liver. Since total muscle mass is 45% of body weight,” it can be calculated that 18% of the total injected radioactivity is either bound or internalized by skeletal muscle cells even 60 min after injection.‘2 Clearly, only a varying proportion of the total recovered ‘H in any given sample will be associated with intact, biologically active insulin. Samples from plasma, organs, and skeletal muscle were, therefore, chromatographed to separate intact insulin from labeled degradation products. Disappearance of Intact Insulin and Appearance of Labeled Degradation Products in Plasma In order to separate intact [3H] insulin from labeled degradation products, plasma samples were chromatographed on G50 Sephadex columns.R.9 Three radioactive peaks eluted from the column. The first, eluting with the void volume, consisted of insulin degradation prod-
ucts with a molecular weight higher than that of insulin itself. Intact [3H] insulin eluted with the second peak and the third peak corresponded to low-molecular-weight degradation products. The elution profile of the plasma samples was similar to that found after subcutaneous injection of [‘HI insulin.‘.” I n control experiments, aliquots of pooled fractions from each of the three peaks were subjected to immunoprecipitation using guinea pig antiporcine insulin serum. Radioactivity bound to the anti-insulin serum was precipitated by the addition of a second antibody (rabbit anti guinea-pig serum). Nonspecific binding was assessed by the use of either nonimmune guinea pig serum in place of the antiinsulin serum or by the addition of a IOO-fold excess of native porcine insulin (relative to the total binding capacity of the anti-insulin serum). Although more than 90% of the radioactivity eluting in the second, or insulin, peak was specifically immunoprecipitable, no immunopreciptable radioactivity could be detected in either the high- or the low-molecular-weight degradation peaks. Thirty seconds after injection, 92% of the radioactivity eluted as intact insulin with the remaining radioactivity distributed between the high- (3%) and the low-molecular-weight (6%) degradation products (Table 2). Prior to injection, 95%98% of the radioactivity in the labeled insulin preparation eluted with intact insulin. The percentage radioactivity associated with intact [‘HI insulin declined with time, such that after 60 min only 7% of the total plasma radioactivity still eluted in the insulin peak. Up until 20 min after injection, the predominant form of degradation products was of low molecular weight. From 30 min, however, the high-molecu-
HALBAN, BERGER. AND OFFORD
1100
Table 2. Percentage
of Radioactivity
Intact Insulin or Degradation Following Intravenous
* i
in Plasma Eluting as
Products From G5B Sephadex
Injection of [‘HI Insulin in Rats
1oooc
Time
Percentage of Radioactivity
Eluting’
After Injection
High MW
lnsukn
Low MW
(mini
Peak
Peak
Peak
0.5
3 ? 1
(6)
92 + 3
(6)
6 + 2
(6)
1
5 + 1
(7)
87 k 2
(8)
7 t 2
(7) (81
12 k 1
(81
63 + 3
(8)
26 i- 3
10
11
(5)
41+5
(5)
47
+ 4
(5)
20
1653
15)
47
(5)
36
+ 5
(5)
5
i
1
*
7
30
41
(2)
23 (2)
40
60 (2)
10 (2)
73 t 4
60
Abbreviation: MW, *Results independent
(4)
molecular
7 + 1
observations
31 (2) (5)
2123
500(
(4)
weight.
expressed as the mean
are
36 (2)
shown
+ SE with
the number
of
in parentheses.
lar-weight degradation product peak appeared as the major component. If the total radioactivity in the plasma is multiplied by the percentage of radioactivity still associated with intact insulin, the plasma concentration of intact [3H] insulin may be calculated. Figure 1 shows the disappearance with time of intact [3H] insulin from the plasma calculated by this method. The values for the metabolic clearance rate (23.6 + 1 ml/min/kg) and distribution space (85 2 17 ml/kg) were. obtained from a log-linear plot by graphing methods. At least two exponential processes were clearly distinguishable (see inset, Fig. 1) with halftimes of 2 and 20 min, respectively.
I
0
Fig. 1. Disappearance of intact labeled insulin from plasma following intravenous injection of fH] insulin in rats. Plasma rH] insulin concentration was determined by multiplying the total plasma radioactivity by the percentage of radioactivity still in the form of intact insulin using chromatographic methods. Each point is the mean of the number of independent observations shown in parentheses. The error bars represent the SE. The inset shows the same results plotted on a log-linear scale. The arrows indicate the time of insulin injection.
samples, the percentage of radioactivity eluting with the two degradation product peaks and the insulin peak was calculated (Table 3). The elution profiles for the two organs, skeletal muscle, and plasma differ considerably. Thus, for the skeletal muscle, only 8% of the radioactivity was accounted for by intact insulin
In order to separate intact insulin from labeled degradation products in the organs and muscle samples, aliquots of the homogenates were chromatographed by G75 Sephadex. As for the plasma
of Intact [‘HI Insulin and Radioactive Following Intravenous
60
MINUTES
TIME
Percentage Insulin and Degradation Products in Plasma, Liver, Kidney, and Skeletal Muscle Following i.v. Injection of 13H] Insulin
Table 3. Percentage
30
*
Degradation
Products in Plasma and Organs
Injection of [‘HI Insulin in Rats Percentage Radioactivity
High MW Peak
lnsukn Peak
Low MW Peak
Time (man)
Plasma
L1V.Y
Kidney
MUS&
Plasma
Liver
Kidney
MllSCle
Plasma
Liver
Kidney
MUS& NE
5
7
17
8
NE
75
29
38
NE
18
54
54
10
10
41
11
16
57
11
47
8
33
47
42
76
20
11
59
9
NE
51
9
33
NE
38
32
58
NE
60
68
76
6
67
11
13
10
0
21
11
84
33
Abbrewations:
MW,
molecular
weight;
NE,
not examined.
DISTRIBUTION AND METABOLISM OF I.V. INSULIN
10 min after injection, with 76% of that in the form of low-molecular-weight degradation products. After 60 min, when approximately 20% of the injected radioactivity was recovered in the skeletal muscle, no radioactivity was detected in the insulin position. At all time points studied, the kidney extracts contained the highest percentage of low-molecular-weight degradation products. Up to 20 min after injection, between 30% and 50% of the kidney extract radioactivity was in the form of intact insulin, but this percentage was lower than that found in the plasma at the same times. In marked contrast, the liver contained prodominantly high-molecular-weight degradation products, and the appearance of this material in the liver preceded that observed in the plasma samples. At all times, less than 30% of the liver radioactivity was in the form of intact insulin, and the percentage of radioactivity in the lowmolecular-weight degradation peak declined steadily with time, in contrast to the plasma or kidney samples. These results, and, in particular, the appearance of high-molecular-weight degradation products in the liver before appearance in the plasma, led us to speculate that the liver was the organ responsible for production of this material. It has been shown previously” that subcutaneous injection of [3H] phenylalanine results in the appearance of high-molecular-weight radioactive material in much the same way as seen following injection of the labeled insulin. Similar results were obtained when [3H] phenylalanine was injected intravenously. Five minutes after intravenous injection of [3H] phenylalanine (I &i, 8 ng), 98.5% of the radioactivity in the plasma was found in the position of small molecules following G50 Sephadex chromatography, with 1.5% eluting in the void volume (high molecular weight). After 60 min, however, 70% of the plasma radioactivity was found in the high-molecular-weight peak. The addition of 7.5 mg of native phenylalanine to the radioactively labeled amino acid resulted in only 45% of the plasma radioactivity being found in the highmolecular-weight peak. The appearance of radioactivity in the high-molecular-weight peak following injection of [‘HI phenylalanine and the observation that the amount of radioactivity eluting in this peak was reduced by isotopic dilution suggests that the high-molecular-weight
1101
product(s) are not generated by nonspecific adsorption of phenylalanine to high-molecularweight proteins, but may be the result of incorporation of the labeled amino acid into newly synthesized protein. The possibility that the high-molecularweight material consists of aggregates of insulin B-chain or of B-chain linked to higher-molecular-weight plasma proteins by disulphide bonds has also been examined. Aliquots of the highmolecular-weight peak material obtained by GSO Sephadex column chromatography of plasma samples were oxidized by performic acid. The performic-acid-oxidized products were then either rechromatographed on G50 Sephadex or, alternatively, subjected to electrophoresis on cellulose acetate.13 Neither method revealed the liberation of radioactive material displaying the physical properties of B-chain or of B-chain fragments as a result of cleavage of disulphide bridges by performic-acid oxidation (J-G. Davies and R.E. Offord, unpublished observation). It is to be expected that if [3H] phenylalanine is released from [3H] insulin at some stage in the degradation of the hormone, it would be treated in a similar fashion to the injected amino acid. Since the liver is known to be an organ that is particularly active in both protein biosynthesis and insulin degradation, it would most probably be an important site for such incorporation of [3H] phenylalanine. In order to test the hypothesis that the liver was indeed primarily responsible for production of the high-molecular-weight material found in the plasma, [3H] insulin was injected intravenously into rats following functional hepatectomy or nephrectomy. Eflects of Functional Hepatectomy or Nephrect0m.v on Insulin Clearance and Plasma G.50 Sephadex El&ion Profiles Following Intravenous Injection of 13H] Insulin Following injection of [3H] insulin into hepatectomized or control (anesthetized) rats, plasma samples were subjected to GSO Sephadex column chromatography and the percentage radioactivity eluting in the three peaks was determined as described above. Strikingly, both 30 and 60 min after injection, the percentage of radioactivity in the high-molecular-weight peak was reduced by removal of the liver from the circulation (Fig. 2). In addition, the percentage of radioactivity eluting in the insulin position
1102
0
5
HALBAN,
CONTROL
80
z
z
N
30 min
1
HEPATECTOMY
NEPHRECTOMY
-I
AFTER
INJECTION
and Metabolism of Intravenously Tritiated Insulin in Rats
Injected
Philippe A. Halban, Michael Berger, and Robin E. Offord Semisynthetic [‘H] insulin was used to follow the distribution and metabolism of intravenously injected insulin in rats. Chromatographic methods were used to separate intact labeled insulin from radioactive degradation products in the plasma, liver, kidneys. and skeletal muscle. The metabolic clearance rate of the injected insulin was 23.6 ? 1 ml/min/kg and the distribution space 65 + 17 ml/kg for an injected dose of 1.5 mU/lOO g body weight. The kidneys concentrated tritium relative to the plasma by up to ninefold; the liver also concentrated radioactivity, but to a lesser degree. Radioactive degradation products were found to be of either higher or lower molecular weight than insulin. The kidneys contained predominantly low-molecularweight degradation products, accounting for 54% of the radioactivity in these organs even 5 min after injection. The liver, by contrast, contained predominantly high-molecular-weight degradation products. This material appeared in the liver before it was seen in the plasma, suggesting that the liver is responsible for its production. This suggestion was confirmed by analyzing plasma samples from rats injected intravenously with [“HI insulin following either functional hepatectomy or nephrectomy. The hepatectomized rats displayed less high-molecularweight material in the plasma and 2-3 times more intact insulin when compared to controls. By contrast, nephrectomy resulted in no significant change in the percentage of either high- or lowmolecular-weight degradation products in the plasma despite reduced insulin clearance. These data confirm the importance of the liver and kidneys in insulin metabolism. Since at least some of the high-molecular-weight-degradation products may be formed by reincorporation of [“HI phenylalanine (liberated by degradation of [“HI insulin) into newly synthesized protein, the importance of the liver in its production may be a reflection of the protein biosynthetic capacity of this organ.
A
BETTER UNDERSTANDING of the pharmacokinetics of injected insulin may lead to improved management of insulin-dependent diabetics. Whereas many studies have been directed toward the distribution, metabolism, and excretion of exogenous insulin, the majority of such studies have relied upon radioiodinated insulin preparations as tracers (see Izzo’ for review.) However carefully the iodoinsulin is prepared, it still represents a chemically altered form of the hormone2: there cannot be certainty that it will behave authentically in every situation.‘13” In order to overcome this problem, we Merabolism,Vol. 28, No. 11,
(November), 1979
have developed a semisynthetic tritiated insulin’ that has been shown to be indistinguishable from native insulin in both its biologic and immunologic activity as well as its metabolism.‘~* This labeled insulin has previously been used to study the pharmacokinetics of subcutaneously injected insulin both in rats’.” and humans.” This study describes the pharmacokinetics of intravenously injected [3H] insulin in rats. In addition to estimating the metabolic clearance rate and distribution space of the injected insulin, we have followed the distribution of the tracer in the liver, kidneys, and skeletal muscle. Previous studies have shown the importance of the liver for insulin metabolism using both in vivol?~l4 and in vitro83’5-‘7 experimental techniques. The kidney, similarly, has been shown to play an important role in insulin metabolism and clearance.‘8-20 The results described in this study serve to confirm the importance of these two organs in insulin metabolism and provide preliminary evidence as to how the two organs metabolize insulin and its degradation products.
MATERIALS
AND
METHODS
Preparation of 13H] Insulin Semisynthetic [‘HI insulin was prepared and purified as previously described.7,8In brief, the preparation involves the replacement of the Bl phenylalanine of pork insulin by [‘HI phenylalanine. followed by two purification steps to remove excess (‘H] phenylalanine and to separate intact insulin from damaged material. The purified material had a specific radioactivity of 7-10 Ci/mmole and 95%-98’S of the
From the Insiitui de Biochimie Clinique, University of Geneva, Switzerland, and the Laboratory of Molecular Biophysics, University of Oxford, United Kingdom. Received for publication August 25, 1978. Presented in part at the Twelfih Annual Meeting of the European Society for Clinical Investigation. April 1978. Rotterdam. Amsterdam. Supported by Swiss National Science Foundation Grant No. 3.774-0.76SR. Address reprint requests to Dr. Philippe A. Halban, Institut de Biochimie Clinique. Sentier de la Roseraie. 121 I Cen&e 4, Switzerland. 0 I979 by Grune & Stratton, Inc. 0026-c)495/79/2811-0004$01.00/0
1097
HALBAN.
1098
radioactivity
was associated
with
mined by gel chromatography
intact
insulin
(as deter-
tion
products,
previously
and immunoprecipitation’).
plasma
described’
samples
Male Wistar standard
rats (mean weight
Purina
chow.
[‘HI
220 g) were fed ad lib. on
insulin
was injected
pulse into a tail vein as an aqueous solution glycine, The
2.5 mg/ml
injected
bovine
serum
x
weight
(mean
+ SE, n = 31). which
mately
0.015
U insulin/kg
(BSA),
times
corresponds
body weight.
after
tubes.
Following
frozen
and stored.
sodium
taken,
of skeletal Up
min
and were
finally
used for subsequent
in a boiling
centrifuged.
The
were
samples
in 2%~ (w/v)
I g
to
of tissue
was
samples were
water bath for 2
supernatants
were
Fed rats were anesthetized the liver portal
(40 mg/kg
from
body weight).
the circulation,
A polyethylene
(internal
0.61 mm),
with a heparin
solution,
was inserted
jugular
had
which
vein was ligated
[IH]
group of animals insulin
above. After checked, aliquots
into the right jugular
by laparotomy.
of each
the total
of the columns
G50
because
than is normally fractions
from
of
found
were collected
Tritium both
SDS
in exclusion
radioactivity
plasma
and
tissue
profiles were drawn on graph paper and peak
was
measured
planimetrically
as
Peak size was expressed as percent
‘H radioactivity
eluted
from the column.
was greater than 90%~. [‘HI
tion
in plasma
samples
total
sample
radioactivity
eluting
with
Dextran
Blue, insulin.
was determined
insulin.
by the
The
insulin
were
of
The yield concentra-
by multiplying
percentage
columns
and [jH]
the
radioactivity
calibrated
with
phenylalanine.
free of radioactiv-
considered
successful.
as described
above.
Renal
sides. A control
group
by laparotomy.
liquid
[‘H]
insulin
above. After
30
and blood and tissue
above.
scintillation
as the scintillant.
for quenching intact
Behringwerke
“Instagel,”
Packard
Switzerland;
Zurich,
A.G..
Mar-
Instrument
Inter-
G50 and G75 Sephadex.
Switzerland.
Presentation of Results, Calculations, and Statistical Methods In order
to allow
for variation
injection means
different
insulin
of I x 10s dpm [‘HI tables,
in rat weight
experiments,
evaluated
by
and figures, Student’s
were expressed
insulin/l00
* SE and the statistical
and insulin
all data on plasma or
the results significance
t test
as for an
g body weight.
for
are given
as
of differences unpaired
group
as described
counter All
(Model
determina-
by the addition
of an
standard.
In order to separate
Zurich,
A.G..
were
of plasma and soluble tissue samples was
were corrected
S.A.,
Pharmacia
comparisons.
were decapitated
using “lnstagel”
national
albumin,
F.R.G.;
and
into a tail vein as described
in a Beckman
serum
burg/Lahn,
was
offon both
was sham-operated
The radioactivity
internal
weight
peaks
previously.’
of the catheter
Analytical Methods
tions
area
neck blood collected,
specimens were sampled as described
LS-230)
of
(I .5 x 80
BSA, pH 8.4.
was patent until the end of the
anesthetized
or 60 min, the animals
determined
the
described
columns
resulting
was 0.2 ml/min.
as 3 distinct
In the text.
into a tail vein as described
procedure
and veins were tied
was then injected
rate
tissue levels of ‘H or [‘HI
vena cava. A
Functional Nephrectomy
of animals
eluted
flow
The
and the liver tissue essentially
were
instead
for any given gel type. One-milliliter
to the site of insertion.
of the liver assayed for ‘H radioactivity
rats
Sephadex
and the flow tissue samples
SDS, 25 mg/ml
of lower molecular
dosage between
decapitated.
ity was the hepatectomy
Fed
molecules
Soluble
of molecules,
The
30 or 60 min, the patency
above. Only when the catheter
arteries
used
exposed.
was sham-operated
was then injected
the animals
experiment
was
pH 8.8 as the
into 0.28
had been flushed
portal blood was thus bypassed into the superior control
entry
diameter
been surgically
proximal
and the
into the portal vein. The
was inserted
previously
of
to exclude
artery
before their
catheter
other end of the catheter
injection
In order
the hepatic
diameter
which
Sephadex
Bovine by intraperitoneal
mm, external
vein,
on ti75
with 2% (w/v)
changes the properties
BSA,
as (0.8 x
Sources of Materials
vein were tied off immediately
the liver.
0.3 ml/min.
were chromatographed G75
2.5 mg/ml
columns
of I ml were collected
rate was approximately cm) developed
chromatographed
analysis.
Functional Hepatectomy pentobarbitol
Fractions
samples. The elution
muscle from the
in IO ml SDS. The homogenized
then made soluble by incubation
were sacri-
samples
and homogenized (SDS).
dose.
decapitation.
buffer.
and the
in heparinized
the plasma after
and aliquots
weighed,
dodecylsulphate
homogenized
insulin
the animals
centrifugation, Immediately
to approxi-
No hypoglycemic
and neck blood collected
of the liver. kidneys, legs were
injection,
pH 7.4.
IO’ dpm/ 100 g body
effect was observed as a result of this injected At various
in a single
in 0.2 ml of 0.2 M
albumin
dose was I .7 k 0.1
ficed by decapitation
elution
were
on G50 fine Sephadex
60 cm) using 0.2 M glycine,
Intravenous Injection and Sample Collection
BERGER, AND OFFORD
labeled
insulin
from degrada-
RESULTS
Distribution of -‘H in Plasma, Liver, Kidneys, and Skeletal Muscle Following Intravenous Injection of [3H] Insulin The radioactivity in plasma and soluble organ and skeletal muscle samples was determined at set times after intravenous injection of [‘HI insulin (Table I). Since the organs were not flushed out with a physiologic buffer before being made soluble they still contained blood and extracellular fluid. Taking the extracellular space as 20% of tissue weight, the contribution of the radioactivity in the extracellular space of the organs and muscle may be estimated, assuming that the plasma radioactivity at any time is an
DISTRIBUTION AND METABOLISM OF I.V. INSULIN
1099
Table 1. Recovery of Radioactivity
in Plasma, Liver, Kidney, and Skeletal
Following Intravenous
Muscle
Injection of [‘HI insulin Total Radwxxtvlty*
Plasm.3
Time (mm)
Idpmlmll
LIVH
Kidney
(dpmlgl
ldpmlgl
MllSCk
idpmlg)
11029
+ 897 16)
1 5
5956 2042
k 652 (8) k 175 18)
2888
+ 425 (6)
18501
t 736
(61
963 + 69
10
1551
i- 307 (3)
1056 + 114 (4)
10348
k 772
(4)
775 k 134 (4)
0.5
20 60
832 + 136 (4) 1271
k 171 15)
961 f 238 151
7524
+ 1737 (5)
873 k 166 (3)
4785
i
183
(41
456
(5)
+ 125 151 670 (21
*Results are expressed as the mean i SE normalized as for an Injection of lo5 dpmi 100 g body weight. The number of Independent observations is shown in parentheses.
approximate indication of the extracellular space radioactivity. Five minutes after the injection, it can be seen that the radioactivity in the liver (expressed as dpm/g wet weight of tissue) is greater than 20% of that found in the plasma (expressed as dpm/ml plasma), indicating that radioactive molecules are bound to the liver cell membranes and/or internalized. This concentration of radioactivity above that found in the extracellular space is even more pronounced for the kidneys. Between 5 and 20 min after injection the kidneys concentrate radioactivity 6-9 times over that found in the plasma. Although a moderate concentrating effect is observed in the muscle, it is less pronounced than that found in the liver. Since total muscle mass is 45% of body weight,” it can be calculated that 18% of the total injected radioactivity is either bound or internalized by skeletal muscle cells even 60 min after injection.‘2 Clearly, only a varying proportion of the total recovered ‘H in any given sample will be associated with intact, biologically active insulin. Samples from plasma, organs, and skeletal muscle were, therefore, chromatographed to separate intact insulin from labeled degradation products. Disappearance of Intact Insulin and Appearance of Labeled Degradation Products in Plasma In order to separate intact [3H] insulin from labeled degradation products, plasma samples were chromatographed on G50 Sephadex columns.R.9 Three radioactive peaks eluted from the column. The first, eluting with the void volume, consisted of insulin degradation prod-
ucts with a molecular weight higher than that of insulin itself. Intact [3H] insulin eluted with the second peak and the third peak corresponded to low-molecular-weight degradation products. The elution profile of the plasma samples was similar to that found after subcutaneous injection of [‘HI insulin.‘.” I n control experiments, aliquots of pooled fractions from each of the three peaks were subjected to immunoprecipitation using guinea pig antiporcine insulin serum. Radioactivity bound to the anti-insulin serum was precipitated by the addition of a second antibody (rabbit anti guinea-pig serum). Nonspecific binding was assessed by the use of either nonimmune guinea pig serum in place of the antiinsulin serum or by the addition of a IOO-fold excess of native porcine insulin (relative to the total binding capacity of the anti-insulin serum). Although more than 90% of the radioactivity eluting in the second, or insulin, peak was specifically immunoprecipitable, no immunopreciptable radioactivity could be detected in either the high- or the low-molecular-weight degradation peaks. Thirty seconds after injection, 92% of the radioactivity eluted as intact insulin with the remaining radioactivity distributed between the high- (3%) and the low-molecular-weight (6%) degradation products (Table 2). Prior to injection, 95%98% of the radioactivity in the labeled insulin preparation eluted with intact insulin. The percentage radioactivity associated with intact [‘HI insulin declined with time, such that after 60 min only 7% of the total plasma radioactivity still eluted in the insulin peak. Up until 20 min after injection, the predominant form of degradation products was of low molecular weight. From 30 min, however, the high-molecu-
HALBAN, BERGER. AND OFFORD
1100
Table 2. Percentage
of Radioactivity
Intact Insulin or Degradation Following Intravenous
* i
in Plasma Eluting as
Products From G5B Sephadex
Injection of [‘HI Insulin in Rats
1oooc
Time
Percentage of Radioactivity
Eluting’
After Injection
High MW
lnsukn
Low MW
(mini
Peak
Peak
Peak
0.5
3 ? 1
(6)
92 + 3
(6)
6 + 2
(6)
1
5 + 1
(7)
87 k 2
(8)
7 t 2
(7) (81
12 k 1
(81
63 + 3
(8)
26 i- 3
10
11
(5)
41+5
(5)
47
+ 4
(5)
20
1653
15)
47
(5)
36
+ 5
(5)
5
i
1
*
7
30
41
(2)
23 (2)
40
60 (2)
10 (2)
73 t 4
60
Abbreviation: MW, *Results independent
(4)
molecular
7 + 1
observations
31 (2) (5)
2123
500(
(4)
weight.
expressed as the mean
are
36 (2)
shown
+ SE with
the number
of
in parentheses.
lar-weight degradation product peak appeared as the major component. If the total radioactivity in the plasma is multiplied by the percentage of radioactivity still associated with intact insulin, the plasma concentration of intact [3H] insulin may be calculated. Figure 1 shows the disappearance with time of intact [3H] insulin from the plasma calculated by this method. The values for the metabolic clearance rate (23.6 + 1 ml/min/kg) and distribution space (85 2 17 ml/kg) were. obtained from a log-linear plot by graphing methods. At least two exponential processes were clearly distinguishable (see inset, Fig. 1) with halftimes of 2 and 20 min, respectively.
I
0
Fig. 1. Disappearance of intact labeled insulin from plasma following intravenous injection of fH] insulin in rats. Plasma rH] insulin concentration was determined by multiplying the total plasma radioactivity by the percentage of radioactivity still in the form of intact insulin using chromatographic methods. Each point is the mean of the number of independent observations shown in parentheses. The error bars represent the SE. The inset shows the same results plotted on a log-linear scale. The arrows indicate the time of insulin injection.
samples, the percentage of radioactivity eluting with the two degradation product peaks and the insulin peak was calculated (Table 3). The elution profiles for the two organs, skeletal muscle, and plasma differ considerably. Thus, for the skeletal muscle, only 8% of the radioactivity was accounted for by intact insulin
In order to separate intact insulin from labeled degradation products in the organs and muscle samples, aliquots of the homogenates were chromatographed by G75 Sephadex. As for the plasma
of Intact [‘HI Insulin and Radioactive Following Intravenous
60
MINUTES
TIME
Percentage Insulin and Degradation Products in Plasma, Liver, Kidney, and Skeletal Muscle Following i.v. Injection of 13H] Insulin
Table 3. Percentage
30
*
Degradation
Products in Plasma and Organs
Injection of [‘HI Insulin in Rats Percentage Radioactivity
High MW Peak
lnsukn Peak
Low MW Peak
Time (man)
Plasma
L1V.Y
Kidney
MUS&
Plasma
Liver
Kidney
MllSCle
Plasma
Liver
Kidney
MUS& NE
5
7
17
8
NE
75
29
38
NE
18
54
54
10
10
41
11
16
57
11
47
8
33
47
42
76
20
11
59
9
NE
51
9
33
NE
38
32
58
NE
60
68
76
6
67
11
13
10
0
21
11
84
33
Abbrewations:
MW,
molecular
weight;
NE,
not examined.
DISTRIBUTION AND METABOLISM OF I.V. INSULIN
10 min after injection, with 76% of that in the form of low-molecular-weight degradation products. After 60 min, when approximately 20% of the injected radioactivity was recovered in the skeletal muscle, no radioactivity was detected in the insulin position. At all time points studied, the kidney extracts contained the highest percentage of low-molecular-weight degradation products. Up to 20 min after injection, between 30% and 50% of the kidney extract radioactivity was in the form of intact insulin, but this percentage was lower than that found in the plasma at the same times. In marked contrast, the liver contained prodominantly high-molecular-weight degradation products, and the appearance of this material in the liver preceded that observed in the plasma samples. At all times, less than 30% of the liver radioactivity was in the form of intact insulin, and the percentage of radioactivity in the lowmolecular-weight degradation peak declined steadily with time, in contrast to the plasma or kidney samples. These results, and, in particular, the appearance of high-molecular-weight degradation products in the liver before appearance in the plasma, led us to speculate that the liver was the organ responsible for production of this material. It has been shown previously” that subcutaneous injection of [3H] phenylalanine results in the appearance of high-molecular-weight radioactive material in much the same way as seen following injection of the labeled insulin. Similar results were obtained when [3H] phenylalanine was injected intravenously. Five minutes after intravenous injection of [3H] phenylalanine (I &i, 8 ng), 98.5% of the radioactivity in the plasma was found in the position of small molecules following G50 Sephadex chromatography, with 1.5% eluting in the void volume (high molecular weight). After 60 min, however, 70% of the plasma radioactivity was found in the high-molecular-weight peak. The addition of 7.5 mg of native phenylalanine to the radioactively labeled amino acid resulted in only 45% of the plasma radioactivity being found in the highmolecular-weight peak. The appearance of radioactivity in the high-molecular-weight peak following injection of [‘HI phenylalanine and the observation that the amount of radioactivity eluting in this peak was reduced by isotopic dilution suggests that the high-molecular-weight
1101
product(s) are not generated by nonspecific adsorption of phenylalanine to high-molecularweight proteins, but may be the result of incorporation of the labeled amino acid into newly synthesized protein. The possibility that the high-molecularweight material consists of aggregates of insulin B-chain or of B-chain linked to higher-molecular-weight plasma proteins by disulphide bonds has also been examined. Aliquots of the highmolecular-weight peak material obtained by GSO Sephadex column chromatography of plasma samples were oxidized by performic acid. The performic-acid-oxidized products were then either rechromatographed on G50 Sephadex or, alternatively, subjected to electrophoresis on cellulose acetate.13 Neither method revealed the liberation of radioactive material displaying the physical properties of B-chain or of B-chain fragments as a result of cleavage of disulphide bridges by performic-acid oxidation (J-G. Davies and R.E. Offord, unpublished observation). It is to be expected that if [3H] phenylalanine is released from [3H] insulin at some stage in the degradation of the hormone, it would be treated in a similar fashion to the injected amino acid. Since the liver is known to be an organ that is particularly active in both protein biosynthesis and insulin degradation, it would most probably be an important site for such incorporation of [3H] phenylalanine. In order to test the hypothesis that the liver was indeed primarily responsible for production of the high-molecular-weight material found in the plasma, [3H] insulin was injected intravenously into rats following functional hepatectomy or nephrectomy. Eflects of Functional Hepatectomy or Nephrect0m.v on Insulin Clearance and Plasma G.50 Sephadex El&ion Profiles Following Intravenous Injection of 13H] Insulin Following injection of [3H] insulin into hepatectomized or control (anesthetized) rats, plasma samples were subjected to GSO Sephadex column chromatography and the percentage radioactivity eluting in the three peaks was determined as described above. Strikingly, both 30 and 60 min after injection, the percentage of radioactivity in the high-molecular-weight peak was reduced by removal of the liver from the circulation (Fig. 2). In addition, the percentage of radioactivity eluting in the insulin position
1102
0
5
HALBAN,
CONTROL
80
z
z
N
30 min
1
HEPATECTOMY
NEPHRECTOMY
-I
AFTER
INJECTION
