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Original Studies |
Medizinische Klinik, Abteilung für Endokrinologie, Stoffwechsel und Pathobiochemie, Eberhard Karls Universität, 72076 Tubingen, Germany
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Abstract |
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Introduction |
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In subjects with impaired glucose tolerance (IGT), a condition preceding type 2 diabetes, abnormalities of insulin secretion have been demonstrated (8, 9). As a particular aspect of the ß-cell dysfunction of IGT, a defect in proinsulin processing resulting in disproportionately elevated amounts of proinsulin in the secretory granule has recently been proposed (6). This was based on an increased plasma PI/I ratio observed in subjects with IGT after acute stimulation of insulin secretion with arginine. We recently proposed a modified hyperglycemic clamp in which three secretagogues [glucose, glucagon-like peptide-1 (GLP-1), and arginine] were administered in an additive fashion (10). This secretion test resulted in very high insulin secretion rates (C peptide levels in the 10 nmol/L range) in normal subjects. In subjects with IGT we found various phases of insulin secretion to be significantly reduced (10). If the secretion defect in IGT is in a substantial way secondary to abnormalities in proinsulin processing, one would expect a clear increase in the PI/I ratio during maximal stimulation of insulin secretion in these subjects.
In the present study, therefore, we analyzed circulating proinsulin concentrations and the PI/I ratio in plasma in 11 subjects with IGT and 11 normal glucose-tolerant (NGT) controls during a squarewave hyperglycemic clamp (10 mmol/L, 0–120 min), additional GLP-1 (120–180 min), and an arginine bolus. The specific aim of this study was to determine how a maximal stimulation of insulin secretion influenced the PI/I ratio, especially in response to acute stimuli, and how this compared with normal controls.
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Subjects and Methods |
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We studied 11 healthy NGT volunteers and 11 subjects with IGT of German origin. The NGT subjects were selected on the basis of age, waist/hip ratio, body mass index, and sex to pair-match the IGT subjects. The family histories of the subjects were not suggestive of MODY diabetes. They did not take any medication known to affect glucose tolerance, insulin sensitivity, or insulin secretion. A 75-g oral glucose tolerance test was performed to classify subjects according to WHO criteria (11). Insulin secretion data for the subjects have been previously reported (10).
Clamp protocols
The study protocol was approved by the ethics committee of the University of Tubingen. Before the study, informed written consent was obtained from all participants. All subjects underwent a modified hyperglycemic clamp (10 mmol/L) with administration of GLP-1 and arginine. Subjects had been instructed to maintain their usual diet before the study. After an overnight fast, at around 0800 h a hand vein was cannulated retrogradely and kept in a thermoregulated box at 55 C to obtain arterialized blood samples. At the same time, an antecubital vein was cannulated for infusions. After baseline samples had been obtained, a modified hyperglycemic clamp was performed as previously described (10). An iv bolus of 20% glucose over 1 min was given to instantaneously raise blood glucose to 10 mmol/L [bolus dose (mg) = body weight (kg) x desired increase in blood glucose (mg/dL) x 1.5]. Subsequently, a glucose infusion was adjusted to maintain blood glucose at 10 mmol/L. After 120 min, GLP-1 [human GLP-1-(7–36) amide, Poly Peptide, Wolfenbhttel, Germany] was given as a primed-continuous infusion (0.6 pmol/kg; 1.5 pmol/kg·min) during the next 80 min (12). At 180 min, a bolus of 5 g arginine hydrochloride (Pharmacia & Upjohn, Inc., Erlangen Germany) was injected over 45 s, and the GLP-1 infusion was continued. GLP-1 from the same batch was used in all subjects.
Plasma glucose was determined at bedside with a HemoCue blood glucose photometer (HemoCue AB, Aengelholm, Sweden) at 5-min intervals. Samples for insulin (Microparticle Enzyme Immunoassay, Abbott Laboratories, Tokyo, Japan; coefficient of variation, 2.5–6%) and proinsulin (enzyme immunoassay, IBL, Hamburg, Germany) determinations were taken at -30, -15, 0, 2.5, 5, 7.5, 10, 20, 40, 60, 80, 100, 120, 125, 130, 140, 150, 160, 170, 180, 182.5, 185, 187.5, 190, and 200 min. According to the manufacturer’s information, the proinsulin essay has 0% cross-reactivity with human insulin and C peptide. The insulin assay has 0% cross-reactivity with proinsulin. The proinsulin assay has less than 2% cross-reactivity with 32,33 proinsulin split products and 50–75% cross-reactivity with 65,66 proinsulin split products. The 32,33 split products are those that appear in plasma to a relevant extent.
Statistical analysis
Unless otherwise stated data are given as the mean ± SEM. Three PI/I ratios were defined at the troughs in response to the three acute stimuli (glucose, GLP-1, and arginine): PI/IGluc, mean of 2.5 and 5 min values; PI/IGLP 125 min value; and PI/IGLP+Arg, mean of 182.5 and 185 min values. These time points were chosen because the lowest absolute PI/I value after an acute stimulus is the least affected by the differences in elimination kinetics between insulin and proinsulin.
Insulin sensitivity was assessed as an insulin sensitivity index, calculated by dividing the average glucose infusion rate during the last 40 min of the hyperglycemic clamp by the average plasma insulin concentration during the same interval (13). Comparisons of a single parameter between NGT and IGT subjects were made using unpaired Student’s t test (two-tailed). PI/I ratios within one group (IGT or NGT) were compared with Dunnett’s method of comparison with control (by definition PI/IGluc). Comparison of the change of the PI/I ratio across the phases (time) between IGT and NGT was made using multivariate ANOVA with repeated measures design (factors time and group). P < 0.05 was considered to be statistically significant. The statistical software package JMP (SAS Institute, Inc., Cary, NC) was used.
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Results |
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)During the first phase of the hyperglycemic clamp, insulin concentrations increased from about 50 pmol/L at baseline to a peak value of 397 ± 119 pmol/L in NGT and 144 ± 20 pmol/L in IGT (P = 0.02). After reaching a trough at about 20 min insulin increased progressively to 436 ± 138 pmol/L in NGT and to 219 ± 28 pmol/L in IGT (P = 0.13) at 120 min. In response to the GLP-1 priming dose, insulin concentrations rose sharply to 1815 ± 371 pmol/L at 125 min in NGT and to 695 ± 89 pmol/L in IGT (P = 0.008). Subsequently, insulin concentrations increased progressively to 5430 ± 1091 pmol/L in NGT and 2142 ± 350 pmol/L in IGT (P = 0.002) at 180 min. In response to the arginine bolus insulin concentrations once again rose to a peak of 8663 ± 1430 pmol/L in NGT and 3983 ± 375 pmol/L in IGT (P = 0.005) at 182.5 min.
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and 2)During the hyperglycemic clamp, proinsulin concentrations increased progressively from about 2 pmol/L at baseline to 12 ± 2 pmol/L in NGT and 8 ± 1 pmol/L in IGT (P = 0.08). In response to the GLP-1 priming dose, proinsulin concentrations rose sharply to 21 ± 3 pmol/L in NGT and 11 ± 2 pmol/L in IGT (P = 0.02) at 125 min. Subsequently, proinsulin increased progressively to 76 ± 18 pmol/L in NGT and 35 ± 5 pmol/L in IGT (P = 0.04) at 180 min. In response to the arginine bolus, proinsulin concentrations once again rose to a peak value of 102 ± 24 pmol/L in NGT and 47 ± 6 pmol/L in IGT (P = 0.04) at 182.5 min.
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). Subsequently, the PI/I ratios
increased progressively to 3.7 ± 0.6% in NGT and 4.0 ±
0.5% in IGT (P = 0.72) at 120 min. In response to the
GLP-1 priming dose, the PI/I ratios decreased sharply to a
trough value at 140 min of 1.2 ± 0.2% in NGT and 1.4 ±
0.2% in IGT, which was not significantly different (P
= 0.43). The PI/I ratio subsequently increased to 1.7 ± 0.3% in
NGT and 2.0 ± 0.3% in IGT (P = 0.61) at 180 min.
In response to the arginine bolus it decreased to a trough value at
182.5 min of 1.3 ± 0.2% in NGT, which again was not
significantly different in IGT (1.2 ± 0.1, P =
0.93).
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, in NGT the PI/IGluc (1.4 ± 0.5%),
PI/IGLP (1.3 ± 0.2%), and
PI/IGLP+Arg (1.3 ± 0.2%) were not
significantly different from one another. In IGT, in contrast, the
PI/IGLP (1.9 ± 0.3%; P <
0.05 vs. NGT) and PI/IGLP+Arg
(1.4 ± 0.2%; P < 0.01 vs. NGT) were
significantly lower than PI/IGluc (3.4 ±
0.6%, by Dunnett’s method). Although PI/IGluc
was significantly different between IGT and NGT, this was not the case
for PI/IGLP (P = 0.21) or
PI/IGLP+Arg (P = 0.90). The
change was also significantly different between NGT and
IGT(P = 0.003, by multivariate ANOVA, time by
group). |
Discussion |
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Most strikingly, the PI/I ratio in response to the acute arginine stimulus was not significantly different between the two groups. In response to a strong acute stimulation of insulin secretion, the PI/I ratio is least influenced by the differences in elimination kinetics between insulin and proinsulin. The PI/I ratio immediately after the acute stimulus most adequately reflects the PI/I ratio in the ß-cell and can be used as an index for the effectivity of the conversion of proinsulin to insulin. Thus, our data not only refute the hypothesis that maximal stimulation of insulin secretion exhausts the processing of proinsulin to insulin in IGT, but suggest that this process is actually normalized. Our finding of no difference between IGT and NGT during maximal stimulation is somewhat at variance with a study in women with IGT that reported a significantly higher PI/I ratio in response to arginine at 14 and 25 mmol/L glucose (6). In that study, however, insulin secretion was not stimulated to a comparable degree, as evident from absolute insulin and proinsulin concentrations of only one third of those in the present study.
The data suggest that the primary lesion resulting in the secretory defect of our IGT subjects does not necessarily involve abnormal proinsulin processing. If this had been the case, more proinsulin per mol insulin should have been cosecreted, resulting in an even greater difference between the two groups during maximal stimulation. It is difficult to explain why the obvious defect present during the first phase should be alleviated by more vigorous stimulation. It is possible that the activity of the specific endopeptidases and exopeptidases responsible for the enzymatic conversion of proinsulin to insulin increased overproportionately in IGT during maximal stimulation. This could indicate that a defect in the rate of conversion of proinsulin to insulin is overcome when the activity of the ß-cell is strongly stimulated.
It is important to note that we used a specific selection and somewhat arbitrary sequence of secretagogues. It is possible that, for example, repetitive arginine injections (basally, during hyperglycemia, and during hyperglycemia and GLP-1) would have produced a different picture.
Interestingly, with prolonged hyperglycemia the significant difference in the PI/I ratio between IGT and NGT had disappeared. This may not necessarily indicate normalization of proinsulin processing by the prolonged glucose stimulus; it could be explained by the slower elimination kinetics of proinsulin (compared with insulin), which at the higher absolute proinsulin levels in the NGT group would result in a faster increase in the PI/I ratio compared with IGT. Such influences of elimination kinetics would be minimal after the acute stimuli (GLP-1 and arginine bolus) during the later phases of the clamp.
Another interpretation of our results would be that GLP-1 beneficially affected proinsulin processing in IGT subjects. Any effect specific for GLP-1 is unlikely, however, because the decline in the PI/I ratio after the start of the infusion was parallel to that in NGT, reflecting similar PI/I ratios in prestored granules. Moreover, the time needed for processing proinsulin to insulin is longer than 5–10 min, after which time a putative GLP-1 effect was reached. Overall, the question of whether GLP-1 improves proinsulin processing cannot be answered by the present design.
There is also the possibility that split products of proinsulin (mainly the 32,33 split) that were not detected by our assay had accumulated after the strong stimulation of insulin secretion. Conceivably, in subjects with IGT these split products could have accumulated to a greater extent than in NGT subjects. A greater build-up of split products in IGT would, in contrast to the central contention of this report, indicate a defect in the processing of proinsulin to insulin downstream from the initial endopeptidases. Nevertheless, assuming this speculative scenario, the data would still allow us to conclude that the initial steps of proinsulin processing are not impaired in subjects with IGT.
Finally, the striking discrepancy between basal and postarginine PI/I ratios in IGT vs. NGT subjects deserves a comment. Although the unstimulated PI/I ratios are limited by the above-explained differences in elimination kinetics, there is an undisputed hypersecretion of proinsulin in the IGT group. This is probably one feature of ß-cell dysfunction in IGT during physiological conditions. Our data obtained by a highly artificial experimental set-up, however, suggest that any defect in proinsulin conversion involved can be overcome by maximal stimulation. This is also supported by the crossing of the proinsulin curves with prolonged stimulation and points at a functional nature of the hyperproinsulinemia.
In summary, maximal stimulation of insulin secretion in subjects with IGT results in a significant decrease in the PI/ratio in plasma that was not different from that in normal controls. This strongly argues against the hypothesis that defective processing of proinsulin to insulin represents a major component of the ß-cell dysfunction in IGT.
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Acknowledgments |
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Footnotes |
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1 This work was supported in part by a Fortune Grant
(Project 1284100 to A.F. and M.S.) and a grant from the European
Community (QLRT-1999–00674). 
Received June 8, 2000.
Revised September 6, 2000.
Revised November 28, 2000.
Accepted December 1, 2000.
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References |
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