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Clinical Studies |
Steno Diabetes Center (E.C.), Gentofte, Denmark; the Department of Transplantation Surgery, Huddinge Hospital (A.T.), Huddinge, Sweden; Novo Nordisk Research Institute (Aa.V., L.S.), Bagsvaerd, Denmark; and the Department of Medical Physiology, Panum Institute (J.J.H.), the Department of Nephrology, Rigshospitalet (K.R.), and the Department of Endocrinology, Hvidovre Hospital (S.M.), University of Copenhagen, Copenhagen, Denmark
Address all correspondence and requests for reprints to: Erik Christiansen, M.D., Steno Diabetes Center, Niels Steensensvej 2, 2820 Gentofte, Denmark.
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Abstract |
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In conclusion, IPx after pancreas transplantation was characterized by 1) impaired early insulin secretion, 2) reduced ß-cell responsiveness, 3) reduced glucose uptake, 4) impaired nonoxidative glucose metabolism, and 5) impaired early inhibition of glucagon secretion.
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Introduction |
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In the fasting state, systemic glucose is derived from the release of glucose from liver and kidney (10). Postprandially, the absorption of ingested carbohydrate also contributes to systemic glucose concentration, but simultaneously, the liver extracts parts of the ingested glucose and suppresses hepatic glucose release, thereby minimizing postprandial hyperglycemia. The predominant defect causing recurrence of the diabetic state after pancreas transplantation is the failure of ß-cells to continue hypersecretion of insulin in relation to the increased demand for insulin after transplantation (8). However, it is unknown whether transition from normoglycemia to impaired glucose tolerance with mild increases in postprandial glucose concentrations is explained by an excessive rate of glucose entry into the peripheral circulation, decreased glucose utilization, or a combination of both.
To examine the causes of the impaired glucose tolerance in pancreas transplant recipients, we determined the rate of systemic glucose appearance, derived from the sum of systemic glucose release and the appearance of exogenous glucose, and the rate of glucose disappearance after an oral glucose load using a dual isotope technique. Also, the balance between the intracellular glucose metabolic pathways was explored by assessing glucose oxidation and glucose storage in the entire body using indirect calorimetry. The role of ß-cell function in the pathogenesis of impaired glucose tolerance was evaluated by estimating ß-cell responsiveness to oral glucose. To study the effect of immunosuppressive treatment on glucose metabolism, the results were compared to those of 1) recipients of pancreas transplants with normal glucose tolerance, 2) nondiabetic kidney transplants receiving identical immunosuppression, and 3) a group of normal subjects.
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Subjects and Methods |
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The study protocol was approved by the local ethics committee. Informed consent was obtained, and all participants were instructed to remain on their normal diet for at least 3 days preceding the test. Normal fasting plasma glucose concentration, normal HbA1c (4.1–6.1%), and normal hemoglobin were required for participation in the study.
Two groups of pancreas-kidney transplant recipients were investigated who required a pancreas transplant for insulin-dependent diabetes mellitus and a kidney transplant for end-stage diabetic nephropathy. Cadaveric segmental pancreas allografting was performed in all cases. One pancreas transplantation group (Px) comprised five recipients of pancreas-kidney transplants with normal glucose tolerance according to the criteria of WHO (11). A second pancreas group (IPx) consisted of four recipients of pancreas-kidney transplants with impaired glucose tolerance, also according to the WHO criteria. Based on the characteristics of the pancreas transplant recipients, two control groups with normal glucose tolerance were matched for kidney function, body mass index, age, and sex; they comprised six nondiabetic kidney transplants (Kx) and eight healthy nondiabetic subjects (Ns). Both control groups were without a family history of diabetes. The immunosuppressive treatment in the three transplanted groups, which did not differ, consisted of prednisone (5–10 mg/day), cyclosporine A (200–300 mg/day), and azathioprine (50–100 mg/day). The insulin sensitivity index and noninsulin-mediated glucose uptake (glucose effectiveness) have been assessed from an iv glucose tolerance test using the minimal model approach in all pancreas-transplanted recipients, four kidney transplant recipients, and five normal subjects as part of a recent study (12, 13). The shortcomings of this model have recently been discussed; however, none of these was observed in relation to the present estimations of insulin sensitivity indexes (14, 15). Among the transplanted groups, there were no differences in antihypertensive treatment.
Experimental protocol
After an overnight fast of 10 h, a catheter was inserted into an antecubital vein to begin a primed (25-µCi) continuous \[3-3H\]glucose infusion (0.25 µCi/min) for 7 h to measure glucose turnover. After a 2-h isotope equilibration period, each subject ingested a 200-mL solution of 75 g glucose (dextrose) containing 100 µCi \[1-14C\]glucose in 3 min. The subjects remained supine throughout the experiments. From the contralateral arm, arterialized venous blood was sampled to determine concentrations of plasma glucose, insulin, C peptide, glucagon, and specific activities of 3-[3H]glucose and \[1-14C\]glucose 120, 30, 15, 10, and 5 min before and 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, 180, 210, 240, 270, and 300 min after glucose ingestion (1000 h). Indirect calorimetry was included in the last 30 min of the equilibration period and in the last 30 min of every hour after glucose ingestion to estimate the net rates of carbohydrate and lipid oxidation (16). A computerized, open circuit system was used to measure gas exchange rates through a transparent ventilated hood placed over the subject’s head (Deltatrak Metabolic Monitor, Datex, Helsinki, Finland). Urine samples were obtained before glucose ingestion, and all urine produced during the following 5 h was collected to measure glucose and nitrogen excretions. Protein oxidation was estimated from the urinary urea nitrogen excretion (1 g nitrogen = 6.25 g protein).
Analytic techniques
Plasma glucose was measured in duplicate by the glucose oxidase method on a Beckman glucose analyzer. Plasma insulin, C peptide, and glucagon were centrifuged immediately at 3000 rpm for 10 min at 4 C, and plasma was stored at -20 C until analysis. In the C peptide assay, proinsulin was removed by polyethylene glycol precipitation (17). In plasma from pancreas transplants, free insulin was determined after polyethylene glycol precipitation (18). Pancreatic plasma glucagon was assessed in tubes containing EDTA and Trasylol by RIA, using antiserum 4305 (19). All samples from one pancreas transplant recipient were measured at the same time as samples from one of the control subjects. The specific activity of \[1-14C\]glucose was determined by a scintillation method through a one-step enzymatic assay that eliminated both labeled metabolites and labeled metabolites recycled to other positions in glucose, and \[3-3H\]glucose was also determined by a scintillation method (20, 21).
Calculations
Fasting levels of plasma glucose, insulin, and C peptide were calculated as the mean of the -15 to -5 min values. The integrated responses (total or incremental above basal) of glucose, insulin, C peptide, and glucagon were calculated as the area under the curve by means of the trapezoidal rule. As early insulin secretion is of importance for glucose intolerance, the results were analyzed according to four time periods: 0–30, 0–60, 0–120, and 0–300 min after glucose ingestion. The 120 min point was used to separate the subjects with normal vs. abnormal glucose tolerance based on the WHO criteria (11). Insulin secretion rates were calculated according to the combined model of insulin and C peptide kinetics, which takes account of the altered kinetic parameters of insulin and C peptide in pancreas transplants (22). This one-compartment model has been validated and is suitable for cases of systemic insulin delivery (3, 23). The insulin secretion rates are expressed as picomoles per min/L distribution volume of C peptide (22).
ß-Cell secretion in response to changes in glucose expresses the efficacy with which changes in the plasma glucose concentration and the gut incretins stimulate insulin secretion. The correlation between the ambient glucose concentration and the insulin secretion rate during the oral glucose test was evaluated by correlation analysis. From this, an index of the ß-cell response and the slope of the regression line expressing ß-cell responsiveness to oral glucose can be obtained.
In the basal state, systemic glucose release was determined by dividing the \[3-3H\]glucose infusion rate by the steady state level of \[3-3H\]glucose specific activity in the last 30 min of the isotope equilibration period. The total systemic glucose appearance rates (comprising ingested glucose and endogenously produced glucose) and the disappearance rates of glucose were calculated in 20-min intervals from the \[3-3H\]glucose specific activity. The urinary loss of glucose was subtracted from the rate of total glucose disappearance integrated over the entire study period. The exogenous glucose appearance, expressing the glucose absorbed from the oral glucose load, was calculated from \[1-14C\]glucose specific activity using the nonsteady state equations of Cobelli and Ferrannini after correction for recycling, as previously described (24). This calculation is stable when dealing with high rates of glucose metabolism and when hyperglycemia is associated with hyperinsulinemia, as expected in this study. The systemic glucose release was calculated as the difference between the overall rate of systemic glucose appearance and the rate of exogenous glucose appearance. Splanchnic glucose uptake was calculated as the difference between the amount of oral glucose ingested and the amount reaching the systemic circulation. We assumed that all ingested glucose was absorbed or metabolized within the 5-h period. The assumptions and limitations in the use of \[3-3H\]glucose and \[1-14C\]glucose as indicators of glucose metabolism have previously been discussed (21).
The net rates of glucose oxidation and lipid oxidation were calculated from indirect calorimetry measurements of CO2 production (VCO2), O2 consumption (VO2), and urinary nitrogen excretion. The constants used to calculate glucose, lipid, and protein oxidation from gas exchange data have been reported previously (16, 25). Nonoxidative glucose metabolism, representing mainly glycogen storage, was calculated as the difference between the glucose oxidation rate and the total rate of glucose disposal.
Statistical analysis
The data in the figures and text are presented as the mean \ SEM. Results from the four groups were compared by the Kruskall-Wallis test, and when significant differences (P < 0.05) were found, they were further evaluated by the Wilcoxon test or the Mann-Whitney rank sum test. The present results and conclusions should be viewed in light of the low number of subjects in the two pancreas-transplanted groups, which increases the risk of statistical type 2 errors and reduced statistical power.
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Results |
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and 2 and Figs. 1 and 2)The mean fasting plasma glucose concentration tended to be higher in the IPx group than in the three other groups, and HbA1C was significantly higher in IPx patients than in normal controls. After the ingestion of glucose, plasma glucose increased rapidly and leveled off between 50–100 min in IPx at a significantly higher mean peak concentration of 12 mmol/L, whereas the mean peak glucose concentration was 8 mmol/L at 50 min in Ns, 9.6 mmol/L at 80 min in Kx, and 8.3 mmol/L at 60 min in Px, respectively (P > 0.05 among Ns, Kx, and Px). The total area under the curve of glucose was between 1.4- and 1.6-fold higher in IPx, and the incremental glucose response was between 2.5- and 3.7-fold higher in IPx than in the other groups (IPx, 1068 \ 325; Px, 289 \ 49; Kx, 435 \ 82; Ns, 293 \ 52 mmol/L·5 h; P < 0.05).
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After 120 min, the incremental plasma insulin and C peptide responses
increased significantly in the transplant recipients compared to those
in normal subjects (P < 0.05).
The kinetic parameters and physiological equivalents estimated from the
combined model are shown in Table 3. The basal insulin
secretion rates were elevated 2- and 1.6-fold in Kx and Px compared to
those in the normal subjects and pancreas recipients with impaired
glucose tolerance (Fig. 2). In IPx, the insulin secretion rates were
markedly lower during the initial 120 min and remained increased
throughout the test compared to those in normal subjects (Fig. 2). Both
the total (Table 2) and the incremental amounts of insulin secreted
were significantly reduced in IPx compared to the other three groups
(IPx, 5,936 \ 1,540; Px, 10,534 \ 3,447; Kx, 11,629
\ 1,135; Ns, 7,527 \ 1,027 pmol/L·5 h; P <
0.05). In kidney recipients and pancreas transplant patients with
normal glucose tolerance, the total amount of insulin secreted was
significantly increased compared to that in normal subjects
(P < 0.05), but there was no statistical difference
between Kx and Px. The glucose-stimulated insulin secretion above
baseline, expressed as a percentage of the integrated basal insulin
secretion over the 5-h period, was not significantly different in IPx
compared to all other groups (IPx, 34 \ 6%; Px, 40 \ 8%;
Kx, 38 \ 5%; Ns, 40 \ 5%).
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Fasting hyperglucagonemia was observed in the three transplanted groups
(Table 1 and Fig. 1), and glucagon secretion was relatively less
suppressed in IPx during the initial 120 min (IPx, 14 \ 5%; Px,
23 \ 4%; Kx, 21 \ 5%; Ns, 32 \ 5%;
P < 0.05) and remained suppressed from 120–300 min
(IPx, 35 \ 6%; Px, 3 \ 1%; Kx, 8 \ 3%; Ns, 2
\ 1%; P < 0.05). Despite the slow initial decrease
in glucagon secretion and the slow increase in insulin secretion in
IPx, similar molar ratios of mean insulin to mean glucagon
concentration were seen in the four groups (P >
0.05).
Rates of total glucose appearance, exogenous glucose appearance,
systemic glucose release, and total glucose disappearance (Tables 1 and 4 and Figs. 3 and 4)
In all four groups, the rate of exogenous glucose appearance
peaked within 60 min after glucose ingestion and then declined slowly
toward basal values. There was no significant difference in the pattern
of glucose absorption between the groups at any sample time. Of the
75 g glucose ingested, the amount reaching the systemic
circulation did not differ significantly among the groups of subjects
(IPx, 56.2 \ 1.4; Px, 50.2 \ 2.0; Kx, 56.2 \ 4.0; Ns,
46.0 \ 3.2 g; P > 0.05). Thus, splanchnic
glucose uptake (amount of glucose metabolized in the splanchnic bed)
corresponded to 28 \ 2% in IPx, 33 \ 3% in Px, 25
\1 2% in Kx, and 39 \ 4% in Ns (P > 0.05).
Likewise, after glucose ingestion, the patterns of systemic glucose
release did not differ significantly among the four groups, although it
tended to be more suppressed in IPx in the last part of the study.
During the entire 5-h period, systemic glucose release was suppressed
by 46 \ 6% in IPx, 58 \ 5% in Px, 44 \ 7% in Kx,
and 37 \ 5% in Ns (P > 0.05). Consequently, the
rates of total glucose appearance in the systemic circulation during
the entire study were comparable in all groups. In contrast, during the
first 120 min after glucose ingestion, when the IPx group demonstrated
a reduced insulin secretion, the total rate of glucose disappearance
was, at the most, 40% reduced compared to that in subjects with normal
glucose tolerance. Glucose disappearance rates increased during the
remaining 180 min in IPx to rates similar to those in the
glucose-tolerant subjects. The glucose clearance rate failed to
increase in IPx during the first 120 min compared to that in the
glucose-tolerant subjects (IPx, 200 \ 22; Px, 491 \ 51; Kx,
345 \ 59; Ns, 445 \ 45 mL/kg·120 min; P
< 0.05), and the mean glucose clearance rate during the 5-h study was
also significantly reduced in IPx compared to that in the
glucose-tolerant subjects (IPx, 2.24 \ 0.27; Px, 4.12
\ 0.36; Kx, 3.00 \ 0.38; Ns, 3.32 \ 0.20 mL/kg·min;
P < 0.05; Fig. 4). Thus, during the first 120 min, the
difference between glucose appearance and disappearance was 2.6- to
4.5-fold higher in IPx than in the glucose-tolerant subjects, whereas
during the last 180 min, the increase in glucose disappearance ensured
a reverse relationship to reestablish basal glucose concentrations
(P < 0.05).
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)The rates of basal nonoxidative glucose uptake were similar between groups, whereas after glucose ingestion, the increase in the rates of nonoxidative glucose uptake was 35–45% lower in IPx than in the other groups (IPx, 0.55 \ 0.14; Px, 1.24 \ 0.27; Kx, 0.91 \ 0.22; Ns, 0.82 \ 0.15 mg/kg·min; P < 0.05). The basal rates of glucose oxidation were similar and the increments in glucose oxidation after glucose ingestion and suppression of lipid oxidation were not significantly different between the groups.
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Discussion |
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After pancreas transplantation, the kinetics of insulin and C peptide differ from those in normal subjects, which might lead to overestimation of insulin secretion if the insulin and C peptide levels are used as an index of insulin secretion (2, 3, 28). Insulin is secreted into the peripheral circulation, reducing first pass hepatic insulin extraction. Likewise, C peptide measurements may be misleading as an index of insulin secretion, as pancreas-kidney-transplanted recipients have only one kidney, and therefore a reduced C peptide clearance rate (3, 4). In the current study we used the combined model approach to estimate individual C peptide and insulin kinetics and prehepatic insulin secretion rates (22). The hyperglycemia in recipients with impaired glucose tolerance was primarily due to diminished early insulin secretion, approximately 50% that in the glucose-tolerant transplant recipients. The overall amount of insulin secreted, taking the degree of hyperglycemia and insulin resistance into consideration, was also reduced. Further, ß-cell responsiveness was reduced approximately 70% in glucose-intolerant recipients compared to that in the groups with normal glucose tolerance. As the transplanted ß-cell masses in the two pancreas-transplanted groups receiving a segmental pancreas graft presumably were similar, the reduction in responsiveness in the glucose-intolerant recipients probably reflects a loss of viable ß-cells, although a dysfunction of the individual ß-cells due to perioperative ischemia or rejection damage cannot be ruled out (8).
In the current study, we used a dual isotope technique to determine whether the impaired glucose tolerance is due to lower glucose disposal or faster glucose appearance caused by the increased exogenous glucose absorption, a reduced first pass hepatic uptake of absorbed glucose, or impaired suppression of glucose output. Total whole body glucose uptake after glucose ingestion was reduced by 40% in the glucose-intolerant pancreas recipients. The increase in glucose disappearance after oral glucose administration lagged 30–60 min behind that of the glucose-tolerant subjects. This was also illustrated by a 35% reduction in the glucose clearance rate in the glucose-intolerant pancreas transplant group compared to that in glucose-tolerant subjects. Both insulin sensitivity and glucose effectiveness were markedly reduced in the glucose-intolerant pancreas transplant recipients, as previously reported (13). These indexes of glucose tolerance reflect effects on hepatic and extrahepatic glucose uptake, and they should be judged to the prevailing systemic insulin and glucose levels (12). To what extent the impairment of these components contributes to the reduced glucose uptake and tolerance is not directly discernible from the present study. It is, however, most likely, that insulin secretion, especially early insulin secretion, is the most important factor, as insulin resistance was not statistically significantly between the glucose-intolerant recipients and their pancreas transplant counterparts. The reduced glucose effectiveness is largely compensated by the elevated glucose concentrations in the glucose-intolerant pancreas recipients, rendering the reduction in noninsulin-mediated glucose uptake less important. In comparison, glucose-tolerant transplanted subjects responded with an augmented insulin secretion sufficient to maintain normal glucose tolerance despite insulin resistance, which also has been demonstrated using other investigational designs (1, 3, 5, 6, 29, 30). These findings are supported by insulin resistance and insulin secretion being inversely correlated to maintain normal glucose tolerance in healthy subjects (31).
The hyperglycemia of the glucose-intolerant pancreas recipients was not caused by differences in total glucose appearance or exogenous glucose appearance rates. The splanchnic glucose uptake was approximately 70% of the glucose administered and was similar in the various groups, which suggests that delayed gastric emptying, as often observed in diabetic subjects with long term diabetes mellitus, did not affect the postprandial glycemic response. Although systemic glucose release tended to be more suppressed at the end of the study in the glucose-intolerant pancreas recipients, systemic glucose release during the entire study period was not different between glucose-tolerant and glucose-intolerant subjects. This may be explained by a higher insulin/glucagon ratio in the group with impaired glucose tolerance at the end of the study. In addition, hyperglycemia regulates its own metabolism by increasing glucose uptake and reducing systemic glucose release (32), which also could have contributed to the suppression of systemic glucose release in glucose-intolerant pancreas recipients. Another possibility, however, is that hepatic tissue has adapted appropriately to the prevailing portal insulin concentrations in the transplanted recipients, as recently demonstrated in glucose-tolerant pancreas recipients, who displayed a normal dose-response relationship between insulin and systemic glucose release when glucose concentrations were matched (9).
The cause of the intracellular impairment in glucose utilization seen in glucose-intolerant pancreas transplants immunosuppressed by steroids and cyclosporine is currently unknown, but could hypothetically be due to receptor and/or postreceptor defects in insulin action (33). Thus, defects in the number of insulin receptors and affinity through down-regulation caused by hyperinsulinemia as well as postreceptor defects occurring distal to insulin receptor binding and, recently, reduced levels of Glut4, the major insulin-sensitive glucose transporter, and reduced activation of glycogen synthase have been reported in glucose-tolerant pancreas transplants recipients (9, 34, 35). Hyperinsulinemia and hyperglycemia per se both stimulate glucose disposal by oxidative and nonoxidative metabolism (36). Although the overall glucose oxidation rate tended to be lower in the glucose-intolerant pancreas transplants, which could be due to the lower insulin secretion, the ability of insulin, rather than glucose, to stimulate oxidative glucose uptake was not significantly impaired (37, 38). Using the hyperinsulinemic-euglycemic clamp technique, reduced glucose uptake is primarily due to impairment of the nonoxidative glucose metabolism, whereas under hyperglycemic clamp conditions glucose uptake was normal, but associated with 4-fold increased insulin levels (1, 7, 9). In the present study, nonoxidative glucose metabolism was significantly reduced in glucose-intolerant pancreas transplant recipients, but was normal in the glucose-tolerant recipients. As immunosuppressive treatment was comparable in the transplanted groups, the effects of these drugs on glucose uptake could not be the precipitating cause of the lower glucose disposal in the glucose-intolerant pancreas recipients. More likely, if peripheral insulin levels are relatively decreased, glucose disposal and nonoxidative metabolism become impaired.
In summary, insufficient early insulin secretion due to reduced ß-cell responsiveness of the graft combined with insulin resistance contributed markedly to the reduced glucose uptake observed in recipients of pancreas transplants with impaired glucose tolerance. A delayed suppression of glucagon secretion may contribute to hyperglycemia in glucose-intolerant pancreas transplant recipients. Glucose-tolerant pancreas or kidney transplant recipients with adequate ß-cell function have a normal metabolic handling of orally administered glucose.
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Acknowledgments |
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Footnotes |
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Received November 22, 1996.
Revised January 22, 1997.
Revised April 10, 1997.
Accepted April 18, 1997.
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References |
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