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Department of Medicine (B.A.), Lund University, Lund SE-221 84; and Department of Clinical Physiology (O.T.), Lund University, SE-20501 Malmö, Sweden
Address all correspondence and requests for reprints to: Dr. Bo Ahrén, Department of Medicine, Lund University, B11 BMC, SE-221 84 Lund, Sweden. E-mail: bo.ahren{at}med.lu.se.
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Abstract |
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Introduction |
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The curvilinear relation between insulin sensitivity and insulin secretion implies not only that insulin secretion is increased in insulin resistance but also that increased insulin sensitivity is compensated by reduced insulin secretion. This has not been explored in such a detail as the islet compensation to reduced insulin sensitivity. It has been shown, however, that athletes, who display increased insulin sensitivity, exhibit reduction in the insulin response to oral glucose (11), which is also seen in nonathletes after exercise training (12, 13). Whether this altered insulin response matches the increased insulin sensitivity has not been estimated, and, similarly, it has not been examined whether the reduced insulin response in subjects with increased insulin sensitivity reflects reduced insulin secretion alone or whether increased insulin clearance by increased hepatic extraction of insulin might contribute. In this study, we therefore examined insulin secretion and insulin sensitivity in master athletes exhibiting high insulin sensitivity. For estimation of insulin sensitivity, we used the euglycemic, hyperinsulinemic clamp technique, and for estimation of insulin secretion, we performed the glucose-dependent arginine-stimulation test (14, 15). This latter test characterizes both the baseline and maximal insulin secretion and glucose sensitivity in the ß-cells because it includes a three-step arginine challenge performed at three different glucose levels. In the present study, we also examined C-peptide, besides insulin, to examine whether compensatory changes in insulin response in subjects with increased insulin sensitivity may be entirely explained by changes in insulin secretion or whether altered hepatic extraction of insulin also contributes. Thus, C-peptide is not extracted by the liver during its first passage, whereas insulin is extracted by a considerable degree (16). The ratio of insulin to C-peptide immediately following an acute arginine challenge may therefore give an estimation of hepatic extraction of insulin.
Finally, the glucose-dependent arginine-stimulation test also discloses the 
-cell function because arginine stimulates glucagon secretion (15). In parallel to the characteristics of the ß-cells in this test, basal glucagon secretion and the glucose sensitivity of the -cells are therefore also characterized. This is of interest in view of findings that subjects with impaired glucose tolerance and diabetes exhibit hyperglucagonemia and defective suppression of glucagon, which may be a factor underlying the hyperglycemia (17, 18, 19, 20, 21, 22). However, whether the -cells are adapted to reduced insulin sensitivity is not known.
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Subjects and Methods |
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The study was undertaken in 12 male athletes, being elite sportsmen recruited from a track and field club. Six men were sprinters, and six were 800- and 1500-m runners. They were 25.8 ± 4.1 yr old (mean ± SD) with a body mass index (BMI) of 22.8 ± 2.3 kg/m2. Seven sedentary male students (age 25.0 ± 2.5 yr, BMI 22.6 ± 2.3 kg/m2) served as controls. All participating subjects were of Caucasian origin and healthy without cardiovascular diseases or impaired kidney or liver function. The ethics committee of Lund University, Sweden, approved the study. All subjects gave written informed consent before entrance in the study.
Glucose-dependent arginine stimulation test
Insulin and glucagon secretion were determined with iv arginine stimulation at three glucose levels (fasting, 14 mmol/liter and >25 mmol/liter), as previously described (15). Intravenous catheters were inserted into antecubital veins in both arms and baseline samples were taken at -5 and -2 min. A maximally stimulating dose of arginine hydrochloride (5 g) was then injected iv over 45 sec. Samples were taken at +2, +3, +4, and +5 min. Variable-rate 20% glucose infusions were then sequentially performed to raise and maintain blood glucose at 13–15 mmol/liter and above 25 mmol/liter, respectively, as determined by bedside using Accutrend (Boehringer Scandinavica AB, Bromma, Sweden). New baseline samples were taken at these blood glucose levels, wherefore arginine (5 g) was again injected and new samples taken.
Euglycemic, hyperinsulinemic clamp test
Insulin sensitivity was determined with the euglycemic, hyperinsulinemic clamp, performed according to DeFronzo et al. (14). Intravenous catheters were inserted into antecubital veins of both arms and baseline samples were taken. A primed-constant infusion of insulin (Actrapid 100 U/ml, Novo Nordisk A/S, Bagsvaerd, Denmark) with a constant infusion rate of 0.28 nmol/m2 body surface area per minute was started. After 4 min a variable rate 20% glucose infusion was added, and its infusion rate was adjusted manually throughout the clamp procedure to maintain the blood glucose level at 5.0 mmol/liter. Blood glucose levels were determined bedside every 5 min by the glucose dehydrogenase technique with a Hemocue (Hemocue, Ängelholm, Sweden), and samples for analysis of insulin and free fatty acid (FFA) levels were taken at 60 and 120 min.
Analyses
Blood samples were immediately centrifuged at 5 C and plasma frozen at -20 C until analysis in duplicate. Plasma insulin and glucagon concentrations were analyzed with double-antibody RIA techniques (Linco Research Inc., St. Charles, MO), using guinea pig antihuman insulin antibodies, human insulin standard, mono-125I-Tyr-labeled human insulin, guinea pig antiglucagon antibodies specific for pancreatic glucagon, 125I-labeled glucagon, and glucagon standard. Plasma C-peptide was also measured with a double-antibody RIA technique (Linco Research), using guinea pig antihuman C-peptide antibody, human C-peptide standard, and 125I-human C-peptide as tracer. Glucose was determined using the glucose oxidase technique, and FFAs were determined spectrophotometrically (Wako Chemicals, Neuss, Germany).
Calculations
The acute insulin (AIR), acute C-peptide (ACR), and acute glucagon (AGR) responses to arginine were calculated as the mean of the +2 to +5 min samples minus the mean prestimulus hormone concentration at fasting glucose (AIR1, ACR1, or AGR1) at 14 mmol/liter glucose (AIR2, ACR2, or AGR2) and at more than 25 mmol/liter glucose (AIR3, ACR3, or AGR3). The slope between AIR1 and AIR2 [slopeAIR = (AIR2 - AIR1)/
glucose] was calculated as a measure of glucose potentiation of ß-cell secretion (15). Similarly, the slope between AGR1 and AGR2 [slopeAGR = (AGR2 - AGR1)/glucose] was calculated as the glucose inhibition of -cell secretion. For estimation of insulin sensitivity, the amount of glucose infused to maintain the glucose level during the clamp study at 5.0 mmol/liter was divided by the mean insulin concentration of 60 and 120 min during the clamp (M/I). Differences in the M/I value between groups may be explained by different sensitivity to insulin in each insulin-responsive cell or to altered number of such cells. We used the term insulin sensitivity for the M/I value without knowing the relative contribution of each of these two factors. Disposition index (DI) was calculated by multiplying AIR1 by M/I or ACR1 by M/I. Hepatic extraction of insulin was quantified by dividing the insulin response (AIR1) by the C-peptide response (ACR1) at fasting glucose assuming that insulin and C-peptide are released on an equimolar basis from the basis and that insulin but not C-peptide is extracted in the liver (16).
Statistical analyses
Statistical analyses were performed with the SPSS for Windows system (SPSS, Inc., Chicago, IL). Analyses for comparison of means between the groups was performed by using Mann-Whitney U test for nonparametric comparisons. Pearson’s product moment correlation coefficients were obtained to estimate linear correlations between variables. Linear stepwise forward multiple regression was used to assess the independent effect of several variables. Means ± SE are shown if not stated otherwise.
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Results |
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Fasting plasma glucose was slightly lower in the athletes (4.2 ± 0.1 mmol/liter) than in the controls (4.7 ± 0.1 mmol/liter, P = 0.017), whereas plasma insulin levels did not differ significantly between the groups (47 ± 4 vs. 53 ± 8 pmol/liter). During the euglycemic, hyperinsulinemic clamp study, the steady-state blood glucose level was maintained equally in the two groups (athletes 4.9 ± 0.1 mmol/liter, controls 5.0 ± 0.1 mmol/liter). To achieve this, more glucose had to be infused in the athletes (92 ± 8 µmol/kg·min) than in the controls (58 ± 10 µmol/kg·min; P = 0.017) although steady-state plasma insulin during the second hour of the test was lower in the athletes (460 ± 29 pmol/liter) than in the controls (557 ± 25 pmol/liter, P = 0.028). The athletes thus exhibited more than 2-fold higher insulin sensitivity than the controls [230 ± 18 vs. 92 ± 12 (nmol glucose/kg·min)/(pmol insulin/liter); P < 0.001]. There were two subgroups of athletes because six of them were medium-distance runners (800 and 1500 m), whereas six were sprinters (100 and 200 m). Although BMI was lower in the medium-distance runners than in the sprinters (20.8 ± 1.1 SD vs. 24.7 ± 1.1 kg/m2; P < 0.001), insulin sensitivity did not differ between the groups (242 ± 20 SEM vs. 218 ± 31 nmol glucose/kg·min per picomole insulin/liter, NS).
Insulin secretion as judged by insulin levels
Figure 1
shows the insulin levels in the glucose-dependent arginine-stimulation test. It is seen that plasma insulin levels were lower in the athletes than in the controls, both under basal conditions, after raising the glucose levels to 14 and more than 25 mmol/liter and after the iv administration of arginine. Figure 2 and Table 1 show the calculated insulin responses to arginine at the three glucose levels. It is seen that the insulin responses were significantly lower in the athletes than in the controls using all variables, i.e. AIR1 (by 65%, P < 0.001), AIR2 (by 48%, P = 0.010), AIR3 (by 60%, P < 0.001), and slopeAIR (by 32%, P = 0.023) (Table 1). The variables reflecting insulin secretion were all inversely related to the insulin sensitivity, as illustrated in Fig. 3 for AIR1. A hyperbolic regression showed a higher coefficiency (r = -0.86, P < 0.001) than a linear regression (r = -0.69, P = 0.001).
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). These results show that insulin secretion, when measured using insulin levels, is reduced in subjects with the increased insulin sensitivity and the reduction almost perfectly matches the increased insulin sensitivity yielding an unchanged DI. No estimate of insulin secretion based on insulin data was significantly different between the two subgroups of athletes. Insulin secretion as judged by C-peptide levels
The C-peptide response to glucose and arginine during the glucose-dependent arginine-stimulation test showed a similar pattern as the insulin responses in the glucose-dependent arginine stimulation test (Fig. 1). In fact, AIRs and ACRs correlated linearly to each other (r = 0.78, P < 0.001). Thus, plasma C-peptide levels, as insulin levels, were lower in the athletes than in the controls, both under basal conditions, after raising the glucose levels to 14 and more than 25 mmol/liter, and after the iv administration of arginine. The calculated ACRs were all found to be lower in the athletes than in the controls (ACR1 by 16%, P = 0.034), ACR2 (by 46%, P = 0.003), ACR3 (by 60%, P = 0.001), and slopeCpeptide (by 70%, P = 0.011) (Table 1). Also the C-peptide responses to arginine were inversely related to insulin sensitivity, as were the insulin responses, although the relation between C-peptide secretion and insulin sensitivity described a linear relation, rather than a hyperbolic relation (Fig. 3). However, in contrast to the unchanged DI when calculated using AIR1 as a measure for insulin secretion, DI was higher in athletes than in controls when ACR1 was used (184 ± 19.4 vs. 76.4 ± 11.6 µmol glucose/kg·min; P < 0.001). This inconsistency may be explained by differences in hepatic extraction of insulin between the groups and therefore would indicate that ß-cell secretion is not perfectly reduced to match the increased insulin sensitivity. No estimate of insulin secretion based on C-peptide data was significantly different between the two subgroups of athletes.
Insulin clearance
Insulin is partially degraded during its first passage in the liver, whereas C-peptide is not (16). To examine whether this process is altered in the athletes, we formed an AIR1/CPR1 ratio, which reflects the relative extraction of the insulin molecule in the liver in relation to insulin secretion (Table 1). The results show that in the controls, hepatic extraction of insulin was 53.6% ± 4.7% whereas in the athletes, insulin extraction was significantly higher (81.1% ± 1.8%, P < 0.001). In fact, there was a linear relation between hepatic insulin extraction and insulin sensitivity (r = 0.61, P = 0.005; Fig. 3). This was also supported by the lower insulin levels in the athletes during the clamp study, in spite of the infusion of the same amount of insulin in the two groups. This shows that insulin extraction is up-regulated under baseline conditions when insulin sensitivity is increased. This will then, together with the down-regulation of insulin response to arginine, result in an unchanged DI. Hence, the up-regulation of hepatic insulin extraction explains why there is dissociation between the results in DI when levels of insulin vs. levels of C-peptide are used. Insulin clearance was significantly higher in the medium-distance runners than in the sprinters (85.5% ± 1.6% vs. 76.8% ± 2.1%; P = 0.010).
Glucagon secretion
Figure 1 and Table 1 show that, whereas baseline glucagon levels did not differ between the groups, all three AGRs were significantly lower in the athletes than in the controls. In contrast, slopeAGR did not differ between the groups. This shows that increased insulin sensitivity is associated with reduced glucagon secretion but not with altered glucose sensitivity in the -cells. No estimate of glucagon secretion was significantly different between the two subgroups of athletes.
FFA
Baseline FFA was not significantly different between athletes and controls. Similarly, the suppression of FFA levels during the clamp or after raising the glucose level to 14 or more than 25 mmol/liter in the glucose-dependent arginine stimulation test was not different between the two groups (Table 2).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A main outcome of this study is, however, that the unaltered DI in the subjects with increased insulin sensitivity is not entirely explained by a down-regulation of insulin secretion from the ß-cells. Thus, when we used C-peptide instead of insulin to calculate insulin secretion, we found that DI was increased. Hence, the reduction in ß-cell secretion in response to increased insulin sensitivity is not as large as the reduction in the insulin response. This is explained by increased insulin clearance in the subjects with increased insulin sensitivity. The increased insulin clearance was evident by the increased ratio of the insulin vs. the C-peptide response to arginine and by the lower insulin levels in the athletes at steady-state during the euglycemic, hyperinsulinemic clamp study in spite of infusion of the same amount of insulin. The site and mechanism of this increased insulin clearance cannot be established from this study. Although increased insulin clearance in the athletes may be explained both by increased peripheral insulin clearance and increased hepatic extraction, it is most likely that increased hepatic extraction of insulin contributes, considering that it was observed in samples taken already during the first minutes after the arginine bolus. Hence, besides a reduction in insulin secretion, there is also an increase in insulin clearance under conditions of increased insulin sensitivity, and these two processes in combination result in the perfect regulation of DI. Therefore, insulin clearance processes also seem to contribute to the adaptation of insulin availability to increased insulin sensitivity. It has previously been demonstrated that insulin resistance in high-fat-fed dogs is associated with reduced insulin clearance, which contributes to the increased insulin compensation to insulin resistance (24). In fact, we found that insulin clearance is linearly related to insulin sensitivity and therefore an important contributor to the adaptation to changes in insulin sensitivity. The mechanism explaining this regulation needs to be established.
We also found that glucagon secretion was reduced in the athletes, suggesting that increased insulin sensitivity is associated with down-regulation of secretion not only from the ß-cells but also from the -cells. This is of clinical interest, in view of previous findings that subjects with impaired glucose tolerance and type 2 diabetes have hyperglucagonemia or reduced suppression of glucagon secretion, which may contribute to the hyperglycemia (17, 18, 19, 20, 21, 22). It may thus be speculated that insulin sensitivity is involved in the regulation of glucagon secretion such that insulin resistance is associated with hyperglucagonemia and increased insulin sensitivity is associated with down-regulation of glucagon secretion. Because hyperglucagonemia is a potential target for treatment of type 2 diabetes (25), it would be of interest to establish the mechanism underlying this regulation. One possibility is that - and ß-cells are regulated in parallel through changes in insulin sensitivity, which may be due to autonomic nerves (26). Another possibility is, however, that the -cell sensitivity to insulin parallels the whole-body insulin sensitivity determined by the euglycemic, hyperinsulinemic clamp: Insulin resistance will therefore be accompanied by hyperglucagonemia and, conversely, increased insulin sensitivity will be accompanied by exaggeration of the inhibitory action of insulin to inhibit glucagon secretion. This would be executed through insulin receptors on the -cells (27, 28, 29).
In this study, we examined elite athletes being either medium-distance runners (800 or 1500 m) or sprinters (100 or 200 m). There were only six athletes in each group, making it difficult to rely on findings of differences or no differences between these two groups. Nevertheless, the medium-distance runners had significantly lower BMI and higher insulin clearance, whereas measures of insulin sensitivity and insulin or glucagon secretion were not significantly different between the groups. Studies in more subjects, however, are required to allow conclusions on differences between these two subgroups.
In conclusion, we show that increased insulin sensitivity in master athletes is associated with reduced insulin response to stimulation resulting in unchanged DI. The reduced insulin response is explained both by reduced insulin secretion and increased insulin clearance, most likely because of increased hepatic extraction of insulin. In addition, the increased insulin sensitivity is associated with reduced glucagon secretion. Therefore, both islet hormone secretion and insulin clearance seem to be regulated by the ambient insulin sensitivity.
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Acknowledgments |
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Footnotes |
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Abbreviations: ACR, Acute C-peptide response; AGR, acute glucagon response; AIR, acute insulin response; BMI, body mass index; DI, disposition index; FFA, free fatty acid; M/I value, amount of glucose infused to maintain the glucose level during the clamp study divided by the mean insulin concentration.
Received October 7, 2002.
Accepted December 11, 2002.
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). Lines represent the hyperbolic (insulin) or linear (C-peptide and hepatic extraction) relation in the entire study group. The r values are the regression coefficients, P values the degree of significance.