0021-972X/98/$03.00/0
Journal of Clinical Endocrinology and Metabolism
Copyright © 1998 by The Endocrine Society
Vol. 83, No. 4
Printed in U.S.A.
Effect of Glucose on Production and Release
of Proinsulin Conversion Products by Cultured
Human Islets*
YASMEENI ZAMBRE, ZHIDONG LING, XUE HOU, ANDRE FORIERS,
BAS VAN DEN BOGAERT, CHRIS VAN SCHRAVENDIJK, AND DANIEL PIPELEERS
Diabetes Research Center and the Department of Pharmaceutical and Biochemical Analysis (B.V.D.B),
Vrije Universiteit Brussel, Brussels, Belgium
ABSTRACT
Isolated human islets were examined for the rates of conversion
and release of newly formed (pro)insulin-like peptides. The rate of
proinsulin (PI) conversion was 2-fold slower in human b-cells (t1/2 5
50 min) than in rat b-cells (t1/2 5 25 min). During the first hour
following labeling of newly synthesized proteins, PI represented the
main newly formed hormonal peptide in the medium; its release was
stimulated 2-fold over the basal level by 20 mmol/L glucose. During
the second hour, newly synthesized hormone was mainly released as
insulin, with 10- to 20-fold higher rates at 20 mmol/L glucose. Prolonged preculture of the islets at 20 mmol/L glucose did not delay PI
conversion, but markedly increased the release of newly formed PI,
des31,32-PI, and insulin at both low and high glucose levels. Our data
P
ROINSULIN (PI) conversion requires the activity of two
endoproteases, PC1 (PC3) and PC2, and one exopeptidase, carboxypeptidase H (1–3). These enzymes are evolutionary conserved, but their regulation and mode of action differ
among species (4, 5) and experimental models (6–10). Although
a mutation in carboxypeptidase H was recently observed in
hyperproinsulinemic fa/fa mice (11), it is still unknown whether
an imbalance between prohormone synthesis and enzyme activities contributes to the increased plasma levels of PI and
des31,32-PI that are often observed in noninsulin-dependent diabetes (12). The possibility should also be considered that the
elevated glucose levels in diabetes influence the biosynthetic
and enzyme activities in islet b-cells (13). In the present study,
we have investigated the effects of acutely and chronically
elevated glucose on the rate of PI conversion and the release of
PI-like products in human b-cell preparations.
Materials and Methods
Islet cells
Human islets were isolated within the framework of a collaborative
program on islet cell transplantation in diabetes (14). Isolated human
Received May 19, 1997. Revision received October 29, 1997. Accepted
December 15, 1997.
Address all correspondence and requests for reprints to: Prof. D.
Pipeleers, Department of Metabolism and Endocrinology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail:
[email protected].
* This work was supported by grants from the Flemish Community
(GOA 92/97–1807), the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek (F.G.W.O. 3.0132.91), and BIOMED (BMH 1-CT 92–
0805).
demonstrate that 1) the release of PI provides an extracellular index
for the hormone biosynthetic activity of human b-cells; 2) an acute rise
in glucose exerts a stronger amplification of the release of converted
hormone than in that of nonconverted hormone; and 3) prolonged
exposure to high glucose levels results in an elevated basal release of
converted and nonconverted PI; this elevation is not associated with
a delay in PI conversion, but is attributed to the hyperactivated state
of the human b-cell population, which was recently found to be responsible for an elevation in basal rates of hormone synthesis. These
in vitro observations on human b-cells provide a possible explanation
for the altered circulating (pro)insulin levels measured in nondiabetic
and noninsulin-dependent diabetic subjects. (J Clin Endocrinol
Metab 83: 1234 –1238, 1998)
islets were cultured for 3–7 days in Ham’s F-10 (Life Technologies,
Paisley, Scotland) containing 0.5% BSA (Boehringer Mannheim, Mannheim, Germany) at 6.7 or 20 mmol/L glucose (15). After culture, preparations consisted of 70% endocrine cells (15). Rat islet isolation and
b-cell purification were carried out as previously described (16); b-cells
were cultured for 20 h in Ham’s F-10 medium before labeling (17).
Pulse-chase experiments
Human islets (1–5 3 105 cells/tube) or rat b-cells (1–3 3 105 b-cells/
tube) were labeled for 30 min at 10 mmol/L glucose (known to activate
all b-cells) with 16.7 Ci/mmol [3H]tyrosine (SA, 48 Ci/mmol; Amersham, Aylesbury, UK), washed in cold Earle’s HEPES buffer with 1
mmol/L unlabeled tyrosine, resuspended (3– 6 3 105 rat b-cells or 0.3–
2 3 106 human islet cells/mL) in chase medium (Ham’s F-10, 0.5% BSA,
2 mmol/L glutamine, 2 mmol/L Ca21, 50 mmol/L isobutylmethylxanthine with 2.5 or 20 mmol/L glucose, and 1 mmol/L unlabeled tyrosine,
gassed with 5% CO2-95% O2), and incubated in a shaking water bath at
37 C. After a 60- or 120-min chase period, tubes were centrifuged,
supernatants were stored at 220 C for later analysis, and pellets were
extracted in 500 mL 2 mol/L acetic acid with 0.25% BSA. One sample was
extracted after the 30-min labeling period to determine the composition
at chase time zero.
High performance liquid chromatography (HPLC)
Islet cell extracts and media were analyzed by reverse phase HPLC
(18), using a Nucleosil 300 –5C4 (5-mm C4 column; 250 3 4 mm; Macherey
Nagel, Düren, Germany) connected to an LKB 2150 pump (Bromma,
Sweden) and a 2152 HPLC controller. Elution buffer consisted of acetonitrile (ACN) and 0.1% trifluroacetic acid in Milli-Q water (Millipore
Corp., Bedford, MA). After equilibration at 28% ACN, elution (at 1
mL/min, 0.5 mL/fraction) was started by increasing ACN over a linear
gradient during 60 min to 29.6%. The column was washed with 80%
ACN before the next run. Collected fractions were mixed with 7.5 mL
scintillation liquid (Ultima Gold, Packard, Downers Grove, IL), and
counted (Packard Tri-Carb). Elution times were determined with human
and rat insulin standards from Novo Nordisk (Copenhagen, Denmark),
1234
HUMAN PROINSULIN CONVERSION INTERMEDIATES
human PI from Sigma Chemical Co. (St. Louis, MO), human (h)
des31,32-PI (lot A52-AWK-117), des64,65-hPI (lot A52-AWK-100), split
32,33 hPI (lot A52–9UL-141), and split 65,66 hPI (lot A52-AUK-94) from
Eli Lilly Research Laboratories (Indianapolis, IN). Purified rat b-cell
extracts and media were separated with a similar ACN gradient, running from 28.3–34% at 45 C (18). Elution times of the two rat PI forms
were determined by injection of b-cell extract samples at the end of the
30-min labeling period. Comparable profiles were found for samples
that were first immunoprecipitated with insulin antiserum and for those
that were not, allowing cell extracts and media to be analyzed without
pretreatment. In some human islet cell extracts the PI peak exhibited a
shoulder between standard positions of intact PI and split 65,66 PI
(asterisk in Fig. 1B). This shoulder could be immunoprecipitated by
polyclonal anti-(pro)insulin serum, by monoclonal antibody S2 to the
intact AC junction of human PI, but not by monoclonal antibody S53 to
the BC junction (19, 20). Therefore, it is considered a des31,32-PI-related
truncation product and was added to the des31,32-PI peak in the analysis.
Although degradation of newly formed PI and insulin was not studied,
HPLC analysis of the islet cell extracts was not indicative of the presence
of additional peaks that would correspond to degraded hormone.
Calculations
b-Cell number in human islet samples was calculated from the DNA
content and the percentage of insulin-positive cells in immunocytochemistry (16). Partly overlapping peaks in human samples were resolved by fitting a multipeak signal model to the data using the Levenberg-Marquardt algorithm for nonlinear regression on Matlab 4.0 (The
Math Works, Natick, MA). The required fitting flexibility was obtained
by exponentially modifying a tailing peak model, i.e. the Fraser-Suzuki
function (21). Profiles from rat samples were analyzed for insulin and
1235
PI; in subsequent analysis, peaks of insulin I and II and of PI I and II were
counted separately and/or in combination. For each time point, the cell
extract and the corresponding medium were eluted; the radioactivity
was counted per peak, then for all peaks together, and then expressed
per 1000 b-cells. The radioactivity per peak was finally calculated as a
percentage of the total (pro)insulin-like radioactivity. The rate of PI
conversion (t1/2) was calculated as the time required for the conversion
of 50% of the newly formed PI. Data are expressed as individual values
or as the mean 6 sem. Statistical significance was calculated by twotailed paired Student’s t test.
Results
PI conversion
The rate of PI conversion, expressed as the half-time of
disappearance of the intact prohormone, was 2-fold slower
in human islets than in rat b-cells (Table 1). A similar difference was noticed when the rates of insulin appearance
were determined (data not shown). The rate of PI disappearance was not influenced by the glucose concentration in
the chase medium. At the end of a 60-min chase at 2.5
mmol/L glucose, 60% of PI was converted. The major conversion products were insulin and des31,32-PI; only 6% appeared as other intermediates (Table 2). Comparable data
were obtained at the end of a 60-min chase at 20 mmol/L
glucose (not shown).
After 120-min chase at 2.5 mmol/L glucose, 87% of newly
formed PI was converted; compared to the 60-min chase, a
larger fraction of converted PI was now recovered as insulin
(84% vs. 58%), and a smaller fraction was recovered as
des31,32-PI (15% vs. 36%; Table 2). After a chase at 20 mmol/L
glucose, the same amount (87%) of PI was converted, but
resulted in a higher proportion of insulin (91% vs. 84% of
converted products) and a lower proportion of des31,32-PI
(9% vs. 15%) than for a 2.5 mmol/L glucose chase (Table 2).
The conversion rate was not decreased in islets cultured with
20 mmol/L glucose (Table 1).
TABLE 1. Rates of proinsulin conversion in human islets and rat
islet b-cells
FIG. 1. Reverse phase HPLC (rp-HPLC) of human PI, insulin, and PI
conversion intermediates. Standards (1, insulin; 2, split 32,33 PI; 3,
des31,32-PI; 4, PI; 5, split 65,66 PI; 6, des64,65-PI) were separated as
described in Materials and Methods (A). Human islets were pulse
chased and then analyzed for radioactivity in the cellular extracts (B)
and media (C) after 60-min chase at 20 mmol/L glucose.
Species
Age (yr)
Gender
Human islets
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
58
47
27
19
27
48
M
M
F
M
M
F
BMI
(kg/m2)
23.1
19.0
20.8
22.9
32.7
Proinsulin disappearance
t1/2 (min)
Chase glucose conc.
2.5 mmol/L
20 mmol/L
42
44
45 (37)
41 (35)
60 (43)
70
41
45
41 (37)
41 (37)
60 (46)
77
Mean
Rat b-cells
PI-I
PI-II
50 6 5
51 6 6
23 6 3
32 6 2
22 6 3
32 6 2
Overall
25 6 3
24 6 3
Data are the mean 6 SEM; human islets were cultured for 3–7 days,
and rat b-cells (n 5 3) were cultured for 20 h at 6.7 mmol/L glucose;
values obtained after high glucose culture are indicated in parentheses. The overall t1/2 of PI disappearance for rat b-cells was calculated
after pooling the peaks of the two (pro)insulin isoforms.
1236
JCE & M • 1998
Vol 83 • No 4
ZAMBRE ET AL.
TABLE 2. Effect of chase time and chase glucose concentration on conversion of newly formed human proinsulin
Culture glucose
(mmol/L)
Pulse chase
Chase time
(min)
Chase glucose
(mmol/L)
60
120
2.5
2.5
20
6.7
6.7
Conversion products
(% of converted proinsulin)
Proinsulin
conversion
(%)
Insulin
Des 31,32 PI
Others
60 6 5
87 6 2
87 6 2
58 6 4
84 6 2a
91 6 2
36 6 4
15 6 2b
962
661
261
1 6 0.4
Data presented are the mean 6 SEM of five or six experiments. Statistical significance of differences with chase at 20 mmol/L glucose was
calculated by paired Student’s t test.
a
P , 0.05.
b
P , 0.02.
TABLE 3. Effect of glucose concentration on the release of newly formed hormone
Culture glucose
(mmol)
Pulse chase
Chase time
(min)
Medium composition [% of total (pro)insulin-like material]
Chase glucose
(mmol/L)
PI
Des 31,32 PI
INS
INS/PI
6.7
60
2.5
20
2.2 6 0.6
6.1 6 2.5
0.4 6 0.1
2.3 6 0.9
0.2 6 0.1
2.3 6 0.5a
0.1 6 0.01
0.6 6 0.2b
6.7
120
2.5
20
2.7 6 0.7c
4.7 6 1.1b
0.6 6 0.2
3.0 6 0.9b
0.5 6 0.1d
12.5 6 0.1a,d
0.2 6 0.03c
2.8 6 0.4a,d
Data presented are the mean 6 SEM of five or six experiments. Total (pro)insulin-like material was measured in cells plus medium. Significance
was determined by paired Student’s t test.
a
P , 0.02 vs. corresponding values at 2.5 mmol/L glucose.
b
P , 0.05 vs. corresponding values at 2.5 mmol/L glucose.
c
P , 0.05 vs. corresponding values after 60 min of chase.
d
P , 0.02 vs. corresponding values after 60 min of chase.
Release of newly synthesized (pro)insulin
At the end of a 60-min chase at 2.5 mmol/L glucose, 2.7 6
0.7% (n 5 6) of newly formed hormone was discharged into
the medium, mainly (;80%) as PI (Table 3). A higher proportion (10.7 6 3.60%; n 5 6) was recovered after a 20
mmol/L glucose chase (Table 3); this increase occurred for all
peptides, but was most pronounced for insulin (12-fold elevation), intermediate for des31,32-PI (6-fold), and lowest for
PI (3-fold; Table 3).
A longer chase period resulted in a greater release of newly
synthesized hormone. After 120 min at 2.5 mmol/L glucose,
3.8 6 1.0% (n 5 5) of newly formed hormone was recovered
in the medium, again with PI as the predominant peptide
(;70%; Table 3). At 20 mmol/L glucose, 20.2 6 4.8% (n 5 5)
of newly formed hormone was released; stimulation was
again highest for insulin (25-fold elevation), intermediate for
des31,32-PI (5-fold), and lowest for PI (2-fold). Glucose stimulation thus reverts the relative proportions of newly synthesized insulin and PI in the medium: insulin now represents 65% of the newly formed peptides in the medium, and
PI comprises only 24%. As a result of these variations, the
medium ratio of newly formed insulin over PI increases with
the length of chase and with the glucose concentration in the
chase medium (Table 3).
In the three experiments in which human islets were also
cultured at 20 mmol/L glucose, the release of newly synthesized hormone was markedly increased during the 120min chase period (Table 4). More than 20% of newly synthesized hormone was released during this time period
regardless of the presence of a low (2.5 mmol/L) or a high
(20 mmol/L) glucose concentration. This release was thus
exceptionally high at low glucose concentration (10- to 40-
fold higher than in 6.7 mmol/L glucose-cultured islet cells)
and was only marginally amplified at high glucose concentration (,2-fold increased vs. 3- to 10-fold in 6.7 mmol/L
glucose-cultured islet cells; Table 4). With both low and high
glucose chases, insulin was the predominant form in the
released newly synthesized hormone, which led to a high
medium ratio of insulin over PI (Table 4).
Discussion
The present study demonstrates that the conversion of PI
is 2-fold slower in human b-cells than in rat b-cells, at least
under the selected in vitro conditions. In the rat, this rate is
mainly derived from the more abundant PI-I, but human PI
conversion is also slower than that of rat PI-II. This idea
appeared when data were compared from previous studies
on human and rat islet preparations (4, 5). It is unknown
whether methodological differences, such as pancreas procurement and islet cell isolation, or differences in cellular
composition of the tested rat and human preparations might
influence their respective conversion rates. Although the prevailing glucose concentration did not influence the rate of
conversion, we cannot exclude that other in vivo parameters,
which are absent in vitro, maintain similar conversion rates
in both species. We have not yet investigated whether part
of the newly formed (pro)insulin products undergoes degradation under the present experimental conditions; if such
a process did occur, it was not detected by our HPLC
analysis.
Conversion of human PI results in accumulation of mainly
des31,32-PI and insulin. The amount of newly formed
des31,32-PI decreases with the time of processing, in particular
at elevated (20 mmol/L) glucose levels, whereas the amount
HUMAN PROINSULIN CONVERSION INTERMEDIATES
TABLE 4. Effect of prolonged exposure to high glucose on the
subsequent release of newly formed hormone
Culture Pulse chase,
glucose chase glucose
(mmol/L)
(mmol/L)
6.7
20
Medium composition
[% of total (pro)insulin-like material]
PI
Des 31,32 PI
INS/PI
INS
2.5
20
1.7 6 0.4
3.5 6 0.7
0.3 6 0.0
1.8 6 0.9
0.3 6 0.1 0.2 6 0.1
8.4 6 2.1 2.5 6 0.7
2.5
20
5.8 6 2.0
8.1 6 3.5
9.4 6 4.0
14.5 6 5.8
23.0 6 5.1 5.5 6 2.4
32.1 6 9.6 5.4 6 1.7
Data presented are the mean 6 SEM of three experiments. Total
(pro)insulin-like material was measured in cells plus medium after
120 min of chase.
of newly formed insulin increases. The endopeptidase PC2
thus seems to mediate a glucose-dependent rate-limiting step
in the formation of insulin; this finding might represent the
functional consequence of the glucose-stimulated PC2 expression and biosynthesis that have been described in recent
reports (13, 22, 23). A fraction of the newly formed PI and
des31,32-PI is released within 60 min after formation of the
hormone. This fraction does not increase during the second
hour of chase. Release of PI is substantial (2–3%) at low
glucose (2.5 mmol/L) and doubles (to 5– 6%) at high glucose
(20 mmol/L). That of des31,32-PI is low (0.5%) at low glucose
(2.5 mmol/L) and increases 5-fold (to 2–3%) at high glucose
(20 mmol/L). These data suggest that circulating PI and
des31,32-PI levels will vary with the prevailing rates of both
hormone synthesis and hormone release. The more marked
stimulation of plasma des31,32-PI than of plasma PI observed
during oral glucose tests thus seems to be the result not only
of differences in respective plasma half-lives (24), but also of
the stronger glucose stimulation on the release of newly
formed des31,32-PI.
The fraction of newly formed hormone that is released as
insulin increases with time and is greatly amplified by glucose (10- to 20-fold). As for rat b-cells, this glucose-regulated
release of newly synthesized insulin probably results from a
glucose-induced recruitment of b-cells into secretory activity
(25). In the three experiments in which human b-cells were
also examined after chronic exposure to high glucose levels
(20 mmol/L), this glucose-dependent amplification was no
longer observed. In fact, this condition exhibited a high fractional release of newly formed insulin at low glucose levels
(2.5 mmol/L); this finding is consistent with the observation
that b-cells exhibit a prolonged activation after culture at
high glucose, even when subsequent glucose levels are low
(15, 26). It is now shown that these b-cells present a faster,
rather than a slower, rate of PI conversion, which indicates
that the rise in medium PI is not the result of a delay in its
conversion but instead is an expression of the elevated biosynthetic activity of these cells. These data are also in agreement with the recent finding that PC2 (gene) expression is
increased after chronically elevated glucose levels (13).
Our data support the idea that increased ratios of circulating PI over insulin, as observed in noninsulin-dependent
diabetes, are not necessarily attributable to inadequate PI
processing, but can result from the increased biosynthetic
and secretory activities that occur under hyperglycemic conditions (27). Prolonged exposure to high glucose does not
decrease the rate of PI conversion, but increases the release
1237
of newly formed PI-like peptides in the medium by more
than 7-fold. This release is no longer dependent on the glucose concentration during the incubation, as might occur in
hyperactivated cells (15); further studies are needed to identify the underlying secretory pathway as constitutive(-like)
or regulated. It will also be necessary to analyze the release
of preformed (pro)hormone in parallel. Only after this analysis will it become clear to what extent the presently described observations might be responsible for increased PI/
insulin ratios in patients with or without noninsulindependent diabetes (28).
Acknowledgments
We thank the technical personnel at the Central Unit of b Cell Transplant for providing the human islet preparations, and R. De Proft and
A. De Loof for DNA measurements. Décio Eizirik is thanked for advice
during preparation of the manuscript. PI conversion intermediate standards were kindly provided by Lilly Research Laboratories (Dr. H.
Schmitt).
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