1Consiglio Nazionale delle Ricerche Institute of Biomedical Engineering, Padua; 2Department of Internal Medicine and Consiglio Nazionale delle Ricerche Institute of Clinical Physiology at the University of Pisa, Pisa, Italy; and 3Department of Medicine M (Endocrinology and Diabetes), University Hospital, Aarhus, Denmark
Submitted 19 April 2004 ; accepted in final form 25 October 2004
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ABSTRACT |
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insulin secretion; insulin sensitivity; glucose tolerance; mathematical models
To assess -cell function in a state approximating free living, we have employed a 24-h, multiple-meal test with modeling analysis to quantify
-cell function and the euglycemic insulin clamp to measure insulin sensitivity. We also evaluated the role of gastrointestinal incretin hormones in insulin secretion.
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SUBJECTS AND METHODS |
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Subjects.
Data for the assessment of insulin secretion and insulin sensitivity were taken from a previous study (16). Twenty-six FDR and 17 subjects without any history of type 2 diabetes (control subjects) participated in the study. FDR and control subjects were closely matched for sex, age, and body mass index (BMI), were healthy, and had a normal oral glucose tolerance test according to the American Diabetes Association criteria (Table 1). Within the offspring group, six subjects had one parent with type 2 diabetes, 13 had one parent and two or more known second-degree relatives with type 2 diabetes, and seven had two FDR relatives (both parents). The 26 relatives were from 22 unrelated families; if more than two offspring were available in a family, a maximum of two were randomly selected to participate. The control group was recruited by advertising from healthy volunteers without any family history of diabetes. Subjects in the control group were all unrelated, healthy, and of Caucasian origin and were taking no medication. Participants were asked to consume a weight-maintaining diet containing 250 g of carbohydrate for 3 days before all examinations, and none was engaged in heavy physical exercise during this period. A questionnaire (18) dealing with daily physical activity patterns during work and leisure did not show differences between the two groups. All subjects underwent a 24-h triple-meal test and a hyperinsulinemic euglycemic glucose clamp on separate days, as briefly described below. Written informed consent was obtained from all subjects, and the protocol was approved by the Ethics Committee of the County of Aarhus. Additional details on the subjects characteristics and experimental procedures can be found in Ref. 16. Part of the results in the control group have been reported previously (13).
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Euglycemic hyperinsulinemic clamp.
A 2.5-h euglycemic hyperinsulinemic glucose clamp was performed in all subjects in the morning on the day after the 24-h test. The insulin infusion rate was 1 mU·min1·kg1 (6 pmol·min1·kg1), and plasma glucose was clamped at 5 mM. Insulin-stimulated glucose uptake (M value) was calculated as the average glucose infusion rate between times 120 and 150 min, and was expressed in micromoles per minute per kilogram of lean body mass, as measured by bioelectric impedance. Plasma glucose, insulin, C-peptide, FFA, GIP, and GLP-1 concentrations were measured as previously reported (16). The assay for GIP and GLP-1 available at the time of measurement allowed determination of total hormone concentration only.
Modeling Analysis
The -cell model used in the present study, describing the relationship between insulin secretion and glucose concentration, has been previously illustrated in detail (13, 14). Insulin secretion, S(t), consists of two components:
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In the present context, the term potentiation refers to the fact that insulin secretion during the 24-h test can be higher or lower than that predicted by a simple dose-response model. In this sense, P(t) expresses a relative potentiation of insulin secretion, which encompasses physiological mechanisms that have a different origin and are known in the literature by different names. Among these mechanisms, the most prominent are the glucose-mediated potentiation, discussed, e.g., by Cerasi (5), and the potentiating effects of the gastrointestinal hormones (6).
The second insulin secretion component represents a dynamic dependence of insulin secretion on the rate of change of glucose concentration, expressed as
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Statistical Analysis
All data are presented as means ± SE. The Mann-Whitney U-test was used to compare group differences. Associations were tested by Spearman correlation, unless otherwise stated. Multiple regression analysis was carried out using standard techniques.
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RESULTS |
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Figure 1 shows the mean 24-h profiles of plasma glucose, insulin, and C-peptide concentrations. Mean 24-h glucose was slightly but significantly higher in FDR than in control subjects (Table 1). Mean 24-h total plasma GIP (28 ± 3 vs. 34 ± 2 pM in control subjects and FDR, respectively) and GLP-1 concentrations (18 ± 1 vs. 17 ± 1 pM) were similar in the two groups. Mean 24-h FFA levels were not different between control and FDR subjects (0.42 ± 0.02 vs. 0.45 ± 0.02 mM). Fasting (0.54 ± 0.05 vs. 0.52 ± 0.05 mM) and nocturnal (0.55 ± 0.02 vs. 0.57 ± 0.03 mM) levels were also not different.
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Figure 2 shows the 24-h insulin secretion profiles. Total insulin secretion during the whole day was 40% higher in FDR than in control subjects, as was the mean nocturnal insulin secretion rate (Table 2).
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The model-derived -cell dose-response function is shown in Fig. 3, and the
-cell function parameters ISR5, glucose sensitivity, and kd are given in Table 2. Daily and nocturnal insulin secretion, the
-cell response at 5 mM glucose, and the early secretion phase (represented by the kd) were increased in FDR.
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M was 30% lower in FDR than in control subjects (Table 2). In control subjects, M was reciprocally related to the insulin secretion rate at 5 mM (
= 0.74, P = 0.003). This relationship could be described by the power function ISR5 = 13,050/M1.23; an index of compensation for insulin resistance was thus calculated in each subject as ISR5·M1.23. The correlation between ISR5 and M in FDR was not significant (
= 0.13, P = 0.53); in addition, the data fell to the left of those of the control subjects (Fig. 5), and the calculated compensation index was significantly lower (10.8 ± 1.0 vs. 13.4 ± 0.6 arbitrary units, P < 0.05), indicating impaired
-cell compensation for insulin resistance. In the pooled data from the whole study group, the compensation index was positively related to the potentiation factor value measured during the first 2 h (
= 0.42, P < 0.005).
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Regulation of Basal Secretory Tone
The mechanisms regulating the increase with insulin resistance of the basal secretory tone, as assessed by nocturnal insulin output, were different in the two groups. In control subjects, the increase in the basal secretory tone with insulin resistance was mostly mediated by upregulation of -cell function by augmentation of ISR5, with a marginal role of glucose levels. In fact, in control subjects nocturnal insulin output was linearly related to ISR5 without a statistically significant independent contribution of mean nocturnal glucose (partial r for ISR5 = 0.78, P < 0.0005; partial r for nocturnal glucose = 0.43, P = 0.096). In contrast, in FDR the increase of the basal secretory tone involved the development of mild basal hyperglycemia: nocturnal insulin output was linearly related to both ISR5 and nocturnal glucose (partial rs of 0.68 and 0.74, respectively, P < 0.0001 for both; overall r = 0.83, P < 0.0001). This significant dependence of nocturnal secretion on glucose rather than on ISR5 alone in FDR was not due to the presence of severely resistant subjects in the group, in which adaptation of ISR5 to insulin resistance was insufficient (Fig. 5, leftmost subjects); in the FDR subgroup with M within the normal range (n = 10), nocturnal secretion was still strongly related to nocturnal glucose (partial rs of 0.75, P < 0.02, and 0.94, P < 0.0002, for ISR5 and glucose, respectively; overall r = 0.95, P < 0.0001).
This indicates that, although in control subjects the upregulation of the -cell dose response with insulin resistance (Fig. 5) is complete and preserves normal glucose levels, in FDR the upregulation is incomplete, and mild basal hyperglycemia develops.
Gastrointestinal Hormones and Potentiation
Plasma GIP concentrations and the potentiation factor were generally correlated with each other on a minute-by-minute basis (i.e., at corresponding sampling times during the 24-h measurement period) in each subject. The correlation was statistically significant in 50% of the individual cases in both groups. To obtain an average figure of the degree of correlation, the pooled data of each group (control or FDR) were analyzed by multiple linear regression by using the potentiation factor values at all time points of all subjects of each group as independent variable, GIP concentrations as dependent variable, and distinct slope coefficients for each subject. The correlation was highly significant (control subjects, r = 0.49, P < 0.0001; FDR, r = 0.38, P < 0.0001). Correlation with GLP-1 was somewhat weaker; plasma GLP-1 concentrations were directly related to the potentiation factor in 20% of control subjects and 35% of FDR.
The GIP potentiation index tended to be decreased in FDR (5.7 ± 1.1 vs. 10.7 ± 1.7 nM1, P = 0.056). In the whole study group, this index was positively related to the potentiation factor measured during the first 2 h of the test ( = 0.44, P < 0.003). This suggests that impaired potentiation and reduced sensitivity to GIP in FDR are related phenomena.
The relationship between gastrointestinal hormones and insulin secretion was specific for the potentiation factor. We did not find significant associations with other -cell function parameters. In particular, neither GIP nor GLP-1 was related to fasting insulin secretion, total insulin output, or insulin secretion at 5 mM glucose. Furthermore, the potentiation factor during the first 2 h was not related to the mean nocturnal or fasting FFA levels either in the whole data set or in the individual groups.
Determinants of Glucose Tolerance
By multivariate analysis of the whole data set, glucose tolerance, as expressed by the mean 24-h glucose level, was independently related (after logarithmic transformation of variables) to the potentiation factor (partial r = 0.57, P = 0.006), the compensation index (r = 0.65, P < 0.0001), and glucose sensitivity (r = 0.38, P = 0.015) (with a total explained variance of 63%), whereas mean GIP and GLP-1 concentrations did not reach statistical significance as independent predictors. M, which in univariate analysis was correlated to glucose tolerance (r = 0.64, P < 0001), did not reach statistical significance in multivariate analysis.
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DISCUSSION |
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In the present study, we selected normotolerant FDR, rigorously matched for age, sex, and BMI to a control group of normotolerant subjects with negative family history of type 2 diabetes. To characterize -cell function under normal living conditions, we analyzed data from a 24-h triple-meal test by use of a mathematical model. The model retains the known constitutive characteristics of
-cell secretory function: the ability to respond to glucose in a dose-response fashion, the ability to respond rapidly to a rapid glucose increase (represented by the rate component), and the ability to potentiate the secretory response during sustained hyperglycemia or after release of incretin hormones. This model has proved effective in previous applications (1, 7, 9, 10, 13, 14). Although the
-cell exhibits such a complex behavior that all models are necessarily approximate,
-cell models are nevertheless an indispensable tool for the analysis of meal tests, as empirical
-cell function indexes do not bear a clear relation to
-cell physiology.
The novelty of this work is thus the quantitative analysis of -cell function by means of a mathematical model under conditions approximating normal living. The use of a 24-h test is particularly relevant for
-cell function, because shorter tests do not fully disclose the complexity of
-cell responses (13). Furthermore, the analysis of the dynamic characteristics of insulin secretion, the investigation of the mechanisms of
-cell adaptation to insulin resistance, and the examination of the role of the incretin hormones reveal aspects of
-cell function that cannot be evaluated with more traditional intravenous tests.
First, this work shows that the ability to upregulate tonic -cell function in response to insulin resistance is compromised in FDR (as shown by loss of correlation with insulin sensitivity, impaired compensation index, development of mild hyperglycemia to sustain basal insulin secretion). This defect has been previously identified in part by use of intravenous glucose (2); the current study detects this defect under normal living conditions, describes its characteristics more precisely, and shows that it plays a role in determining glucose tolerance. In agreement with previous work (9), we have also found that the
-cell function parameter that best represents adaptation to insulin resistance is the basal secretory tone, whereas other
-cell function parameters are less sensitive, or not at all insensitive, to insulin resistance. In addition, we show for the first time that the basal secretory tone is regulated differently in control and FDR subjects. In the presence of insulin resistance, control subjects are able to increase the basal secretory tone solely by modulating the
-cell dose response (ISR5 upregulation) so that basal glycemia remains constant, whereas in FDR subjects the ISR5 upregulation is inappropriate, and mild hyperglycemia develops.
Second, we have found that glucose sensitivity is not compromised in FDR (Table 2 and Fig. 3); this indicates that the glucose-sensing ability of -cells is intact in FDR at this stage of the condition, i.e., when subjects are young and glucose tolerance is still normal. This result extends to the normal living condition the observation made by Byrne et al. (4), who found a slightly steeper increase in insulin secretion rates in FDR than in control subjects in response to graded intravenous glucose infusions. Furthermore, in contrast to tonic insulin secretion, glucose sensitivity was largely unrelated to insulin sensitivity, as we have previously reported in other conditions (10). This finding clearly implies that insulin sensitivity is functionally linked with some
-cell responses but not others.
Third, we found that potentiation of glucose-induced insulin release, which is one of the determinants of glucose tolerance (9, 10), was impaired in FDR. In addition, this defect was found to be related to the defect in compensation for insulin resistance. Whether these dynamic abnormalities are indicative of a common cellular defect is not known, although it is tempting to speculate that acute potentiation (the potentiation peak at breakfast) and chronic potentiation (adaptation to insulin resistance) may be two aspects of the same phenomenon.
Fourth, although we have observed a minute-by-minute relationship between the potentiation factor and GIP concentrations in FDR, as previously reported in control subjects (13) and expected on the basis of GIP physiology, the slope of this relationship was considerably reduced in FDR, albeit with borderline significance. This is suggestive of a decreased sensitivity to GIP in FDR. Furthermore, the association between the potentiation factor during the first 2 h of the test and the sensitivity to GIP suggests that the relative insensitivity to GIP in FDR and the decreased potentiation response may be part of the same -cell defect. Of note is that a decreased
-cell sensitivity to GIP in FDR has been previously observed with intravenous administration of glucose and GIP (15).
Finally, kd, which accounts for the prompt initial secretory response during a meal and is likely linked to first-phase insulin secretion (14), was significantly increased in FDR. The model-derived kd has limited precision (13). However, an increased initial response is confirmed by the calculation of the average insulinogenic index (average ratio of insulin and glucose increments 30 min after ingestion of each of the three meals). This index tended to be increased in FDR (140 ± 15 vs. 101 ± 7 pM/mM, P = 0.06 vs. control subjects). Most of the reports favor the view that first-phase insulin secretion is impaired in FDR (8, 11, 20). In our study group, compensation for insulin resistance may have masked this phenomenon [although we, like others (2), did not observe a relationship between first-phase secretion markers and insulin sensitivity]; alternatively, this discrepancy may originate from the intrinsic difference between insulin secretion during a meal and the acute insulin response to intravenous glucose, as already pointed out (10).
The presence of subtle -cell function abnormalities associated with insulin resistance but only minimal hyperglycemia (
0.25 mM over 24 h; Table 1) and the relationship between the parameters of
-cell function and mean 24-h glucose levels (extensively discussed in Ref. 10) resubmit the dilemma of whether the
-cell defects are the cause or consequence, through glucotoxicity (17), of hyperglycemia. This cannot be decided from this cross-sectional study. However, it should be noted that the glucotoxicity vicious circle can start only if
-cell function is defective ab origine, because otherwise the
-cell would be capable of maintaining normal glucose tolerance in the presence of insulin resistance. Thus the finding that FDR subjects present abnormalities in the adaptation of the
-cell to insulin resistance is interesting, as this is the kind of defect that may trigger the glucotoxicity vicious circle.
Taken together, our results indicate that -cell dysfunction in FDR involves late rather than early insulin release during meals and in particular the mechanisms of potentiation and compensation for insulin resistance. These abnormalities are suggestive of a common
-cell defect, which can tentatively be ascribed to some step in the so-called amplifying (or KATP-independent) pathway of insulin secretion [as opposed to the triggering or KATP-dependent pathway (3, 12, 19)]. Although we do not have cellular markers to support this conclusion, current understanding of the
-cell machinery is compatible with this hypothesis. For instance, cAMP, which belongs to the GIP signaling cascade, is reputed to interact with the amplifying pathway (3, 12, 19). The meal-induced potentiation observed in this study may also originate from prolonged exposure to glucose, similarly to what occurs during a hyperglycemic clamp. The glucose-induced potentiation seen with prolonged hyperglycemia is usually referred to as time-dependent potentiation (TDP), which is also thought to be a component of the amplifying pathway, although it is poorly understood (19). The cellular basis for the phenomenon of compensation for insulin resistance is unknown. However, the same mechanisms underlying acute TDP may also be involved in the compensation for insulin resistance, as the latter can be viewed as a long-term form of the former. It has been proposed that phospholipase C and protein kinase C are involved in this process (21); although their role is controversial, these factors are also reputed to be involved in the amplifying pathway (12).
In summary, in our group of insulin-resistant first-degree relatives of diabetic subjects, we have established that -cell defects are present under normal living conditions. These defects consist of a cluster of subtle, interrelated abnormalities, involving potentiation,
-cell sensitivity to GIP, and compensation for insulin resistance, and are suggestive of a common defect in the amplifying pathway of insulin secretion. In contrast, early insulin release, as a marker of the triggering pathway, does not appear to be impaired. These
-cell defects are related to glucose tolerance.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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