1 Steno Diabetes Center, After pancreas-kidney transplantation, it is
difficult to obtain an accurate estimate of the insulin secretion of
the pancreas graft, since several pitfalls are involved using
peripheral C-peptide and/or insulin measurements in this
determination. In this study, the individual kinetic parameters of
C-peptide and then the rates of insulin secretion were estimated by two
mathematical methods, the deconvolution method and the "combined
model" during slow (oral glucose) and fast (intravenous glucagon)
changes in insulin secretion in six successful pancreas-kidney
transplant recipients with systemic delivery of insulin (Px), six
nondiabetic kidney-transplant recipients with portal insulin secretion
(Kx), six nondiabetic controls (NS), and six C-peptide-negative
insulin-dependent diabetes mellitus patients (IDDM). Decreased
C-peptide clearance and basal and poststimulatory hyperinsulinemia were
found in both Px and Kx compared with NS
(P < 0.05). Similar glucose
responses were observed after intravenous glucagon in all groups,
whereas the responses after oral glucose were 30% higher in Px and Kx
than in NS (P < 0.05). During oral
glucose and after intravenous glucagon, both mathematical methods
resulted in significantly lower maximal and incremental insulin
secretion rates (ISR) in Px than in Kx (P < 0.05). In contrast,
calculations of incremental ISR in NS and Px induced by the two
pancreas-kidney transplantation; kidney transplantation C-peptide; kinetics; oral glucose tolerance test; glucagon test
AFTER PANCREAS TRANSPLANTATION, an accurate estimate of
insulin secretion is paramount not only for evaluation of the graft function in relation to monitoring the success of the transplantation but also for the studies of the intermediate metabolism after transplantation. Both peripheral insulin and C-peptide concentrations have been used to assess Insulin secretion can be estimated by several methods (11, 21, 23, 24,
26, 28). The commonest method has been to evaluate In the present study, Subjects
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
-cell stimuli were about the same but significantly higher in Kx
than in NS (P < 0.05). These results differed markedly from those obtained using peripheral measurements of
insulin and C-peptide alone. In conclusion, when C-peptide clearance
and insulin metabolism change, such as in pancreas-kidney transplant
recipients, accurate evaluation of insulin secretion from the graft can
be obtained only by using individual kinetics of the peptides before
calculating the ISR. This study also clearly demonstrates that insulin
secretion after pancreas transplantation is still defective.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-cell function after pancreas-kidney transplantation (2-4, 6, 7, 13, 17, 19, 25). However, it has
recently been found that there are limitations and pitfalls encountered
using these peptide levels as measures of insulin secretion after
pancreas-kidney transplantation, since they may falsely overestimate
insulin secretion compared with those of normal subjects (2-4, 6,
7). In pancreas-kidney transplant subjects, it therefore seems
advisable to determine individual kinetic parameters of C-peptide and
then calculate insulin secretion rates (ISR) to get an accurate
assessment of insulin secretion.
-cell function by
calculating ISR by deconvolution of peripheral C-peptide
concentrations, as originally proposed by Eaton et al. (11) and
confirmed by Polonsky et al. (21). With this 2-day method, individual
C-peptide kinetics are determined in each subject after a bolus
injection of biosynthetic human C-peptide while endogenous insulin
secretion is suppressed by an infusion of somatostatin. Thereafter, the
kinetic parameters are used on the day of experiment to calculate ISR
by deconvolution of peripheral C-peptide concentrations (21). Thus,
with this method, peripheral insulin measurements are not employed.
Another approach uses kinetic modeling of the concomitantly measured
concentrations of insulin and C-peptide, termed the "combined
model" (28). With this method, ISR and C-peptide and insulin
kinetics and an index of hepatic extraction of insulin are calculated
on the basis of measured profiles of the two peptides, and in contrast
to the deconvolution method, a separate day for assessing C-peptide
kinetics is not necessary (28).
-cell function was evaluated in
pancreas-kidney transplant recipients from ISR after an oral glucose load and an intravenous glucagon stimulation of the pancreatic graft,
resulting in relatively slow and rapid changes in insulin secretion,
respectively. ISR was calculated on the basis of the deconvolution
technique and the decay curve of C-peptide distribution and degradation
(11, 21) and compared with ISR based on the combined model without the
use of a bolus injection of C-peptide (28). The results obtained were
compared with those in nondiabetic kidney transplant recipients and
nondiabetic healthy controls. Because an infusion of somatostatin has
been shown to affect renal plasma flow and the glomerular filtration
rate (GFR) and thereby potentially C-peptide clearance (27), a group of
C-peptide-negative insulin-dependent diabetes mellitus (IDDM) patients
was also studied after a bolus injection of C-peptide.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Group 1. This group was made up of six successful pancreas-kidney transplant recipients with systemic delivery of insulin (Px), three of whom had segmental grafting and three whole organ grafting. Two recipients had bladder drainage and four recipients enteric drainage of the exocrine secretion.
Group 2. This group, made up of six nondiabetic kidney transplant recipients with portal insulin secretion, served as the first control group (Kx). All transplant subjects received cadaveric pancreas-kidney and/or kidney grafts. The studies were undertaken 35 ± 6 and 30 ± 8 mo posttransplantation, respectively. The immunosuppressive regimen included prednisolone (5-10 mg/day), cyclosporin A (150-300 mg/day), and azathioprine (50-75 mg/day).
Group 3. This group, made up of six nondiabetic matched controls, served as the second control group (NS).
Group 4. This group, made up of six C-peptide-negative IDDM (type 1) patients with acceptable metabolic control on a multiple insulin injection regimen and with long-standing diabetes mellitus and kidney function, evaluated by serum creatinine levels and GFR like those of the pancreas-kidney transplant and kidney transplant groups, were selected as the third control group. The IDDM group was included to control for the reduced kidney function of the transplant recipients, as assessed from GFR levels, and to evaluate the potential effect of somatostatin on C-peptide clearance during the study day of the bolus injection of C-peptide in the transplant groups. Because the IDDM group has no endogenous insulin secretion, C-peptide kinetics were studied without use of somatostatin. GFR was assessed on a separate day during an outpatient visit. Normal fasting plasma glucose, glycosylated hemoglobin (HbA1C) within the range of 4.1-6.1%, and stable serum creatinine were required by all the transplant participants before initiation of the study.
Subjects were studied as outpatients consuming an ad libitum diet, including 300 g of carbohydrate, at least 3 days before testing in the Px, Kx, and NS groups; the IDDM group kept their regular diet. There were no differences in the amount or type of antihypertensive medication in the transplant and IDDM groups. The studies were performed at Steno Diabetes Center, Copenhagen, Denmark, or at the Research Unit of the Transplant Division at Huddinge Hospital, Stockholm, Sweden, by the same investigator (EC). All analyses of blood samples were performed at the Steno Diabetes Center. The study was approved by the local committees on ethics in Copenhagen and Stockholm and was performed in accordance with the Helsinki Declaration.Study Protocols
Study 1. Assessment of C-peptide kinetics. All experiments were started between 7:00 and 8:00 AM, after the subjects had fasted overnight for 10 h. The IDDM subjects were instructed to take their bedtime insulin injection (around 10 PM) on the evening before the study and not to take their morning insulin injections. No other medications were taken by any of the participating subjects in the morning before the study. The subjects were placed at bed rest and kept supine during the study. Fasting basal blood samples were drawn from an antecubital vein kept patent with 0.9% saline, and the forearm was maintained in a heated box to ensure arterialization of the venous blood. Concentrations of plasma glucose, plasma C-peptide, plasma cyclosporin A, serum creatinine, serum insulin, and HbA1C were measured. In the contralateral antecubital vein, another 17-gauge cannula was used for the somatostatin bolus and infusion as well as the C-peptide injection. Endogenous insulin secretion was suppressed by a bolus injection of 250 µg somatostatin, with a subsequent continuous infusion of 500 µg/h somatostatin (Durascan Medical Products, Odense, Denmark) for 270 min. Ninety minutes after the start of the infusion, a 50-nmol bolus injection of biosynthetic human C-peptide (Bachem Feinchemikalien, Bubendorf, Switzerland) was administered over 30 s. Blood samples of plasma glucose, C-peptide, and insulin were collected for 270 min. Urine samples were collected from 90 to 270 min to assess urinary C-peptide excretion.
Study 2. Glucagon test (GT).
On another day, the subjects in the Px, Kx, and NS groups were studied
after an overnight fast for 10 h by means of an intravenous injection
of 1 mg glucagon (Novo Nordisk, Bagsvaerd, Denmark) administered
over 30 s. Blood samples of plasma glucose, C-peptide, and
insulin were collected in the basal state (10,
5, and 0 min) and for 20 min (2, 5, 6, 8, 10, 12, and 20 min) after stimulation.
Study 3. Oral glucose tolerance test (OGTT).
On another day, the subjects in the Px, Kx, and NS groups were studied
after an overnight fast for 10 h. The -cell function was tested for
insulin secretion by means of a 4-h OGTT (75 g dextrose). Blood samples
of plasma glucose, C-peptide, and insulin were collected in the basal
state (
10,
5, and 0 min) and for 240 min (5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 120, 150 180, 210, and 240 min)
after stimulation.
Analytical Determinations
Glucose in plasma was determined in duplicate by the glucose oxidase method (Beckman Instruments, Fullerton, CA). Blood samples of serum insulin and plasma C-peptide were centrifuged immediately at 3,000 rpm for 10 min at 4°C and stored atData Analysis
The first part of the deconvolution method is estimation of the kinetic parameters of the two-compartment model for C-peptide from the decay curve after the bolus of C-peptide (21). The basis of the rate equations for the kinetic model has previously been presented (21). The parameters K1 and K2 represent the rate constants for C-peptide transport from compartment 1 to 2 and vice versa, respectively. K3 is the rate constant for the irreversible disappearance of C-peptide from compartment 1. The calculation of the two volumes, V1 and V2, and the rapid first phase (T1) and the slower second phase (T2) half-lives of C-peptide has previously been reported (21).The second part consists of calculation of the secretion rate from C-peptide concentrations measured during GT and OGTT. Although the original method (21) used a cubic spline function to smoothe the C-peptide concentration profile followed by mathematical deconvolution, we used a cubic spline function to describe the secretion rate (28), which can then be estimated from the measured C-peptide and the individual kinetic parameters for C-peptide by multiple linear regression analysis. The inherent tendency of deconvolution methods to exhibit large random deviations was avoided by choosing the knot points of the splines to be preceded and followed by three to four sampling times. The secretion rates were expressed as picomoles per minute per unit of the total distribution volume (V1 + V2) (21).
The combined model is defined by means of the rate equations for one-compartment models for insulin and C-peptide, in which kI and kC are the elimination rate constants for the two peptides (28). The prehepatic secretion rate is expressed per unit distribution volume of C-peptide (VC), and f is the fraction of the secreted insulin that is not extracted by the liver during the first passage (F) multiplied by the ratio between the distribution volumes, i.e., f = F(VC/VI), where VI is the distribution volume of insulin. This model was estimated from the measured values of insulin and C-peptide by nonlinear regression analysis where the ISR was represented by a cubic spline function.
From the ISR models, the -cell function was evaluated by calculating
the basal prehepatic ISR, the total and incremental (above basal)
amounts of prehepatic insulin during OGTT and GT, and the maximal
prehepatic ISR to both
-cell stimuli as well as time to maximum. The
contribution of the basal to total insulin secretion during the tests
was calculated by extrapolating the basal ISR during the 20 min of the
GT and 240 min of OGTT. Because we took the deconvolution method as the
"golden standard" in these experiments (21), we also calculated
the mean difference between the ISR determined by the two methods. The
integrated responses (total and incremental above basal) of insulin and
C-peptide were calculated as the area under the curve by means of the
trapezoidal rule.
Statistical Methods
Nonparametric statistical tests were employed, the Mann-Whitney rank-sum test in the analysis of unpaired data, the Wilcoxon rank-sum test in the analysis of paired data, and Spearman's ![]() |
RESULTS |
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The clinical characteristics of the subjects at baseline are given in
Table 1.
HbA1C was significantly increased
in the group of IDDM patients. Serum creatinine levels were higher in
the Px, Kx, and IDDM groups than in NS
(P < 0.05) but also higher in IDDM and Kx than in Px (P < 0.05). GFR
values were similar in Px, Kx, and IDDM and significantly lower than
the normal range for NS [80-120
ml · min1 · (1.73 m2)
1;
P < 0.05]. Plasma cyclosporin
levels were identical in the two transplant groups. Fasting
plasma glucose, plasma C-peptide, and serum insulin levels within each
group were not significantly different between the 2 study days.
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Kinetics of C-Peptide by Deconvolution Method
Baseline data are presented in Tables 1 and 2. Fasting hyperinsulinemia was observed in Kx and Px but not in NS (P < 0.05). Fasting plasma C-peptide levels were increased in Kx and Px, unlike in NS and IDDM (P < 0.05), and in Kx compared with Px (P < 0.05). Before the somatostatin infusion was started, IDDM subjects had fasting insulinemia comparable to that of NS. One IDDM subject, who had high insulin binding antibodies (40% binding capacity to the tracer), has been excluded from the insulin data shown.
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The profiles of plasma glucose and serum insulin and the decay curve of C-peptide after the bolus injection of C-peptide in the four groups are shown in Fig. 1. Plasma glucose declined initially in all groups after initiation of somatostatin and increased thereafter in NS from 3.6 ± 0.2 to 5.9 ± 0.4 mM, in IDDM from 8.1 ± 1.1 to 10.4 ± 1.4 mM, in Kx from 3.5 ± 0.5 to 4.6 ± 0.7 mM, and in Px from 3.7 ± 0.6 to 6.6 ± 1.1 mM, respectively (P < 0.05). Meanwhile, the fasting serum insulin levels were suppressed to levels around detection limit (5 pM) of the insulin assay in the Px, Kx, and NS groups. Likewise, fasting C-peptide decreased to levels around detection limit (<100 pM) before injection of C-peptide. Plasma C-peptide increased to similar peak levels in all groups after the bolus injection of C-peptide (Fig. 1). Urine C-peptide excretion was similar in all groups, constituting 7.6 ± 1.6% in Kx, 6.2 ± 1.1% in Px, 6.4 ± 1.3% in NS, and 7.0 ± 1.2% in IDDM of the amount of C-peptide injected. K1 was higher in Px compared with NS (P < 0.05), and K3 was higher in Px and Kx than in NS (P < 0.05, Table 2). No differences was seen in K1, K2, and K3 between Px and Kx, and K2 was similar in the Px, Ks, and NS groups. K1, K2, and K3 were higher in IDDM than in the other groups (P < 0.05). Distribution volumes (V, ml/kg) and the short half-life (T1) were similar in all groups, whereas the long half-life (T2) was significantly increased in IDDM, Kx and Px compared with NS (P < 0.05).
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OGTT
Plasma glucose, C-peptide, and serum insulin profiles. Plasma glucose, C-peptide, and serum insulin levels determined by OGTT are reported in Fig. 2 and Table 3. The plasma glucose response was 30% higher in the two transplant groups than in normal subjects (P < 0.05). However, glucose tolerance was normal in the two transplant groups, according to WHO criteria. The total insulin and C-peptide responses were significantly higher than in NS (P < 0.05), and the total C-peptide response was higher in Kx than in Px (P < 0.05). The corresponding incremental insulin response was twice as high in Kx as in NS (P < 0.05), and the incremental C-peptide response was threefold higher than in NS (P < 0.05). In contrast, the incremental C-peptide responses in Px recipients were not significantly different from those in NS but were 55% lower than that those of Kx (P < 0.05). The incremental insulin response was 44% higher in Px than in NS (P < 0.05) and 20% lower than in Kx (P < 0.05).
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ISR by deconvolution model. The mean ISR profile during OGTT in Px, Kx, and NS is depicted in Fig. 3A. Basal ISR values were similar in Px and NS, whereas basal ISR was twofold higher in Kx. Both the total amount of insulin secreted and the increment above basal secretion were ~50% higher in Kx than in Px and NS (P < 0.05). Maximal ISR was also 50% higher in Kx than in Px (P < 0.05), and maximal ISR was 30% lower in Px than NS. Maximal ISR occurred significantly later in Px and Kx than in NS (P < 0.05). There were no differences in basal-to-total ISR between groups. The correlation between basal ISR and total ISR was r = 0.93 (P < 0.00001), between basal ISR and the increment in ISR was r = 0.80 (P = 0.0002), and between basal ISR and maximal ISR was r = 0.65 (P = 0.0048; n = 18).
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ISR and insulin and C-peptide kinetics by combined model. The mean ISR profile during OGTT in Px, Kx, and NS is shown in Fig. 3B and Table 4. The mean elimination rate constant of insulin, kI, did not differ significantly among the three groups, although it tended to be higher in Px. The mean elimination rate constant of C-peptide, kC, was significantly lower in the two transplant groups, corresponding to a longer half-life of C-peptide in the two transplant groups than in NS. The analysis according to the combined model also provided estimates of f = F(VC/VI), which were significantly higher in Px than in NS and Kx. Compared with the deconvolution model results, the basal ISR was higher in Px and Kx than in NS (P < 0.05), whereas there were no significant differences between basal ISR in Kx and Px. The total amounts of insulin secreted were 2.2- and 2.8-fold higher in Px and Kx than NS, respectively (P < 0.05). The incremental amount above basal insulin secretion was higher in Kx than in Px and NS (P < 0.05), but similar in Px and NS. The time to maximal ISR was significantly higher in Px and Kx than in NS, but the maximal insulin rate was increased only in Kx vs. NS. The correlation coefficient between basal ISR and total ISR was r = 0.93 (P < 0.00001), and the basal ISR and incremental ISR correlation was r = 0.75 (P < 0.0008), whereas the basal ISR and maximal ISR correlation coefficient was r = 0.47 (P < 0.0661), and the basal ISR correlated highly with the time to the maximal ISR (r = 0.83, P = 0.0001; n = 18).
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Comparison between ISR estimated by deconvolution method and combined model during OGTT. The mean difference between the calculated ISR determined by the two methods is shown in Fig. 3C. The combined model gave the highest rates during the first 15 min (P = 0.05 at 5 min), whereas the deconvolution method gave the highest mean rates thereafter, without reaching statistical significance. The profiles in the separate groups in Fig. 3, A and B, showed that the combined model gave significant higher secretion in the Px group in the early part of the test and significantly lower secretion in the NS group from 45 to 150 min than did the deconvolution method, but there were only small differences in the Kx group.
GT
Plasma glucose, C-peptide, and serum insulin profiles. Plasma glucose, C-peptide, and serum insulin levels determined by GT are reported in Fig. 4 and Table 3. The glucose response to intravenous glucagon resulted in higher glucose concentrations at the end of the 20-min study period in Kx (8.9 ± 0.7 mM) than in NS (7.0 ± 0.5 mM) and Px (7.6 ± 0.6 mM), (P < 0.05). The total insulin response in Kx was 3-fold higher than in NS and 2.5-fold higher than in Px, respectively (P < 0.05), and 2-fold higher in Px than NS (P < 0.05). The total C-peptide response was 2.5- and 1.5-fold higher in Kx than in NS and Px, respectively (P < 0.05). In contrast, only incremental insulin and C-peptide responses were higher in Kx than in NS (P < 0.05).
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ISR by deconvolution model. The ISR profile during GT in Px, Kx, and NS is demonstrated in Fig. 5A and Table 3. Basal ISR values were similar in Px and NS but twofold higher in Kx (P < 0.05, Table 4). Total and incremental amounts of insulin secreted were significantly higher in Kx than in Px and NS (P < 0.05), which had not statistically different responses. Maximal ISR values were significantly lower in Px than in Kx (P < 0.05), but the difference between Px and NS failed to reach statistical significance. No differences were observed in the basal-to-total ISR or in time-to-maximal ISR between groups.
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ISR and insulin and C-peptide kinetics by combined model. The ISR profile during GT is demonstrated in Fig. 5B and Tables 3 and 4. Kinetic parameters were not significantly different compared with the OGTT day. No differences between groups were observed in the calculated basal ISR, whereas the total and incremental amounts of insulin secreted were significantly lower in Px than in Kx and NS (P < 0.05) and lower in NS than in Kx (P < 0.05). Although maximal ISR was lower in Px than in Kx and NS, these differences were not significant. The time-to-maximal ISR was similar in the three groups.
Comparison between ISR estimated by deconvolution method and combined model during GT. The mean difference between the calculated ISR determined by the two methods during GT is shown in Fig. 5C. The combined model gave the highest rates during the first 5 min (P < 0.001 at 5 min), whereas the deconvolution method gave the highest mean rates thereafter (P < 0.05 at 8, 10 and 12 min). There was also a significantly lower basal secretion with the combined model relative to the deconvolution method. Figure 5, A and B, shows that the profile for the NS group obtained from the combined model peaked earlier than the deconvolution profile (at 4 min).
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DISCUSSION |
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In the present study, ISR was estimated in pancreas transplant
recipients by two mathematical methods, the deconvolution technique and
the combined model, during slow and fast changes in insulin secretion.
Compared with that in nondiabetic kidney transplant recipients,
incremental ISR in pancreas-kidney transplant recipients was absolutely
reduced using both approaches during oral glucose load as well as after
intravenous glucagon. In contrast, calculations of incremental ISR in
normal subjects and pancreas-kidneytransplant recipients induced by the
two -cell stimuli were about the same.
After pancreas-kidney transplantation, it is important to obtain an
accurate estimate of the insulin secretion of the pancreas graft. The
present study clearly illustrated the problems of using peripheral
measurements of insulin and C-peptide in the interpretation of -cell
function in pancreas-kidney transplant recipients. The total insulin
and C-peptide responses in pancreas-kidney transplant recipients to
oral glucose both indicated higher insulin secretion than in normal
subjects, whereas incremental C-peptide responses did not. In fact, the
insulin data merely reflect the effect of systemic delivery of insulin
with no first-pass hepatic uptake of insulin, and the C-peptide data
are influenced by the reduced C-peptide metabolic clearance rate. This
is also evident when peripheral insulin and C-peptide responses are
compared between the pancreas transplant recipients and the nondiabetic
kidney transplant recipients. No statistical difference in total
insulin response was demonstrated, whereas the C-peptide response was reduced with ~50% in the pancreas transplant recipients. However, when the kinetics of the peptides are taken into consideration in
calculation of the actual ISR, no major differences in ISR were
demonstrated between pancreas transplant recipients and normal subjects. Both the total amount of insulin secreted and the incremental insulin secretion were ~50% higher in the kidney transplant
recipients than in the pancreas transplant recipients and normal
subjects. The same pattern was demonstrated when analyzing the peptide
responses to intravenous glucagon, in which the peripheral peptide
measurements in pancreas-kidney transplant recipients indicated
improved responses compared with normal subjects, but the actual ISR
values were similar. Both peripheral peptides and insulin secretion
were reduced in pancreas-kidney transplant recipients compared with
kidney transplant recipients.
In the basal state, the increased ISR seen in kidney transplant recipients might be the result of the immunosuppressive treatment inducing insulin resistance, whereas the peripheral hyperinsulinemia in pancreas-kidney transplant recipients, caused by a combination of systemic insulin delivery and immunosuppressive induced insulin resistance, did not to the same extent result in higher basal ISR, as previously demonstrated (2, 3, 6-8). This was demonstrated by both mathematical methods, but especially using the deconvolution model.
The incremental ISR to oral glucose was higher or similar,
respectively, in the kidney and pancreas-kidney transplant groups than
in normal subjects, but these responses have to be related to the
glycemic responses and insulin resistance. By doing so, an impairment
in ISR in both transplant groups was obvious, independent of the method
used to evaluate insulin secretion, since the hyperglycemic responses
were increased by 30% in the transplant groups. The hyperglycemic
response and impairment in insulin secretion in pancreas-kidney
transplant recipients have been reported in other study designs (2, 3,
6, 19). The predominant causes of this impairment have previously been
discussed in detail, the main components being the number of
functioning -cells, reduced by the amount transplanted (segmental
vs. whole pancreas graft), and/or by the number and severity of
rejection episodes (2, 3, 6, 8, 19). We and others (8, 9, 15) have previously shown a markedly reduced insulin sensitivity in these recipients. A close relationship between insulin secretory capacity and
insulin resistance has been shown in subjects with normal glucose
tolerance (8). Therefore the present data also suggest that the
transplanted
-cells have not fully adapted to the concomitant insulin resistance. A direct effect of the immunosuppressive treatment on the
-cells has also been suggested as a mechanism inducing impaired
-cell function and insulin sensitivity as seen in our nondiabetic kidney transplant recipients (18, 30). Also, as previously
reported, basal insulin secretion constituted significantly more of the
total amount of insulin secreted in pancreas transplant recipients than
in the control groups calculated with the combined model, whereas a
similar, though not a significant difference, was seen with the
deconvolution method (2, 3, 6, 8).
The fast insulin secretory responses after intravenous glucagon demonstrated a higher similarity between the groups than did the slower changes seen after OGTT and were comparable to previous reports (5, 17). Thus insulin secretory responses to glucagon were similar in normal subjects and pancreas-kidney transplant recipients despite different peripheral levels of insulin and C-peptide.
Several pitfalls are involved using peripheral C-peptide and/or
insulin measurements in the evaluation of insulin secretion, and the
present results demonstrate that the seemingly higher insulin secretion
in pancreas-kidney transplant recipients and nondiabetic kidney
transplant recipients is explained by different mechanisms (22). The
systemic delivery of insulin in pancreas-kidney transplant recipients
avoids first-pass hepatic extraction unlike portal insulin delivery in
non-pancreas-kidney transplant subjects and therefore makes peripheral
insulin measurements unsuited for this evaluation (2-4, 6, 7).
Instead, C-peptide has been used as a marker of insulin secretion after
pancreas-kidney grafting (2-4, 6, 7, 13, 17, 19, 25). C-peptide is
cosecreted from the -cells in equimolar amounts with insulin, and
hepatic extraction of C-peptide is negligible and its metabolic
clearance rate constant over the physiological range of concentrations
(20). However, pancreas-kidney transplant recipients still have
slightly reduced kidney function, since they receive only one kidney,
and kidney function may further be adversely affected by cyclosporin (2, 3, 25). Accordingly, the kinetics of C-peptide differ between
pancreas-kidney transplant recipients and normal subjects, resulting in
an increased half-life of C-peptide (2, 3, 6-8), as demonstrated
in the present study as well and independent of the model approach. The
prolonged half-life results in overestimation of insulin secretion in
the transplant recipients. Furthermore, during non-steady-state
conditions the peripheral concentration of C-peptide does not change in
proportion to its secretion rate, because C-peptide is distributed
outside the plasma compartment and because of the long half-life of
C-peptide (16). Last, the faster the changes in insulin secretion, the
less accurate is the estimate of ISR directly from C-peptide
concentrations (20). Therefore C-peptide levels may be representative
only in the basal state of ISR and comparable only among subjects with
normal kidney function. For these reasons, it can be stated that
insulin secretion cannot be accurately assessed from peripheral plasma
insulin or C-peptide concentrations and that the peptide
comparisons between pancreas transplant recipients with systemic or
portal insulin delivery will not be valid unless mathematical modeling
based on the individual kinetics of the peptides is used for reliable assessment of insulin secretion.
As previously mentioned, ISR can be obtained by various methods (11, 21, 23, 24, 26, 28). The insulin secretion model described by Rudenski et al. (24) is inapplicable, since it uses only insulin measurements and does not take account of the lack of first-pass hepatic insulin extraction in pancreas-kidney transplant recipients with peripheral insulin secretion. A simplified version of the deconvolution method has previously been published (26). Being based on standardized kinetic parameters, it eliminates the separate experimental day for assessment of C-peptide kinetics (26). However, this model cannot be used in subjects with impaired kidney function, such as pancreas-kidney transplant recipients (26). At present, it therefore seems that only the two models employed in the present study can account for the various physiological and anatomical circumstances in pancreas-kidney recipients and kidney transplant recipients. The deconvolution method has previously been applied to pancreas-kidney transplant recipients demonstrating a reduced clearance of C-peptide (2), but it is time consuming because of the separate day reserved for estimation of the kinetics of C-peptide and is also expensive because of the costs of human biosynthetic C-peptide and somatostatin (21). The combined model has previously been applied in a study of subjects with altered hepatic extraction of insulin and peripheral hyperinsulinemia and also after pancreas-kidney transplantation (3, 6-8, 29).
When the estimated ISR values from the two mathematical methods are compared, the insulin secretion profiles for the OGTT showed steeper increases when calculated from the combined model, and the secretion rates for the normal subjects and kidney transplant subjects decayed more rapidly using the combined model than using the deconvolution approach. On the much shorter time scale of the GT study, the profiles from the combined model also showed faster increases and subsequent decays than those based on deconvolution. Higher peak values were observed in all groups using the combined model. In relation to the overall mean differences between the two methods, there were some differences in the subgroups. Thus the combined model seemed to overestimate ISR in the early poststimulatory phase of insulin secretion and to underestimate ISR in the later phase of insulin secretion compared with the deconvolution method, this being most pronounced during fast changes in insulin secretion. These findings are probably explained by the fact that the estimated ISR in the combined model are based on the single compartment approximation for C-peptide rather than the two-compartment model on which the deconvolution method is based. Therefore the dynamic differences between the mean secretion profiles obtained with the two methods are probably due to the different C-peptide kinetic modeling. In agreement with this explanation it is also apparent that the dynamic differences between the two methods was more pronounced in the short GT than in the OGTT.
From this study alone, one cannot determine which of the two methods gives the most correct and precise results. The deconvolution method should in theory give more accurate secretion rates than the combined model, since it is based on the two-compartment model for C-peptide, which seems to be required for an adequate description of the C-peptide kinetics after intravenous bolus administration (26). It has been validated and used extensively in many clinical studies; however, the kinetic parameters obtained from this test may be influenced by the continuous infusion of somatostatin, which, of course, is not infused during the OGTT or GT. This might lead to some bias in the estimation of the secretion rates. The combined model was designed with approximate one-compartment models for C-peptide and insulin, but a validation study in dogs showed that it gave accurate estimates of portally infused insulin and C-peptide rates resembling responses to OGTT (28). These findings have later been reiterated with insulin and C-peptide infusions in humans (14). On the basis of the present data, the two methods yield relatively large dynamic differences in the GT, and it therefore seems very likely that the deconvolution method gave the most reliable estimates of the fast changes in insulin secretory responses. This conclusion, however, will be dependent on the accuracy of the C-peptide assay and on the assumption that somatostatin does not influence C-peptide kinetics. The latter seems to be confirmed by the findings of similar GFR and C-peptide decay curves in the transplant groups and the C-peptide-negative IDDM group, which did not receive somatostatin; these findings, however, contrast with previous reports (27). The combined model may give sufficiently accurate results in many situations with slow changes in insulin secretion, i.e., OGTT, and might be the method of choice when the additional efforts required to determine individual C-peptide kinetics are not warranted.
In conclusion, several limitations and pitfalls exist in the evaluation
of insulin secretion after pancreas-kidney transplantation with
systemic delivery of insulin. In general, the use of the stimulated
peripheral insulin and/or C-peptide measurements as an index of
-cell function cannot be recommended. We suggest that, to facilitate
the interpretation of peripheral measurements of these peptides, at
least the individual kinetics of C-peptide should be estimated in
pancreas-kidney transplant recipients followed by calculation of ISR by
deconvolution. Alternatively, the combined model can by employed
especially in situations with slow changes in insulin secretion. The
present study has clearly demonstrated that insulin secretion after
pancreas transplantation is not entirely normalized.
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ACKNOWLEDGEMENTS |
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The technical assistance of Annemette Forman, Bente Mottlau, Lene Åboe, and Jane Falk, Steno Diabetes Center, is gratefully acknowledged. The authors thank Bo Feldt-Rasmussen, MD, PhD, Dept. of Nephrology, Rigshospitalet, University of Copenhagen, Denmark, and Gunnar Tydèn, MD, PhD, Dept. of Transplantation Surgery, Huddinge Hospital, Sweden, for recruiting participants to the study. We thank Zoe and Francis Walsh for revising the manuscript.
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FOOTNOTES |
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This study was supported by grants from the Danish Medical Research Council, the Nordisk Insulin Foundation, Novo Nordisk Pharmaceuticals, Denmark, the Mogens and Jenny Vissing Grant, the Danish Society of Internal Medicine, and the Danish Diabetes Association.
Address for reprint requests: E. Christiansen, Steno Diabetes Center, Niels Steensensvej 2, 2820 Gentofte, Denmark.
Received 29 August 1997; accepted in final form 8 January 1998.
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