1Department of Information Engineering, University of Padova, Padua, Italy; 2Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, Missouri; and 3San Raffaele Scientific Institute, Milan, Italy
Submitted 22 February 2005 ; accepted in final form 10 July 2005
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ABSTRACT |
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insulin resistance; oral glucose tolerance test; meal; insulin action; tracer kinetics
The purpose of this study was to address these issues. To do this, a multiple-tracer OGTT was performed in subjects with varying degrees of glucose tolerance who also underwent a labeled euglycemic hyperinsulinemic clamp. OMM SI and OMM* SI were compared against their clamp counterparts, SIclamp and SI*clamp, respectively. In addition, OMM SI, OMM* SI, and Ra ogtt were compared against the model-independent measurements provided by the multiple-tracer OGTT protocol.
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METHODS |
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Twenty-one subjects (8 females, 13 males) with varying degrees of glucose tolerance [10 normal glucose tolerance (NGT) and 11 impaired glucose tolerance (IGT); age = 41 ± 1 yr; BMI = 27 ± 1 kg/m2 in NGT and 34 ± 2 kg/m2 in IGT; body surface area (BSA) = 1.89 ± 0.06 in NGT and 2.01 ± 0.06 in IGT] underwent both a labeled euglycemic hyperinsulinemic clamp and an OGTT labeled with two glucose tracers. Experimental procedures were reviewed and approved by the Institutional Review Board at Washington University School of Medicine. All subjects provided informed consent.
Euglycemic Hyperinsulinemic Clamp
The labeled euglycemic hyperinsulinemic clamp consisted of a tracer equilibration period (from t = 0 to 120 min) and a glucose clamp period (from t = 120 to 300 min). During the tracer equilibration period, a primed continuous infusion of [2H2]glucose (Cambridge Isotope Laboratories, Andover, MA) was administered, with
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Labeled OGTT
OGTT consisted of oral administration of 75 g of glucose at time 0 (71 g unlabeled glucose, 4 g [U-13C6]glucose tracer); glucose tracer concentration (G*) was used to derive the exogenous, i.e., coming from the oral load, glucose concentration (Gogtt) as:
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It is of note that, in Ref. 2, three tracers were administered [one given with the meal: [1-13C]glucose; two given intravenously: [6,6-2H2]glucose mimicking endogenous glucose production (EGP) and [3H]glucose mimicking Ra] to assess not only the Ra of orally administered glucose, but also EGP. In the present paper, only two tracers were used (oral, [U-13C6]glucose; iv, [6,6-2H2]glucose), because the purpose here is to derive a model-independent estimate of Ra.
Blood samples were collected at 15, 0, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 120, 150, 180, 210, 240, 270, 300, and 360 min. Capillary gas chromatography-quadrupole mass-spectrometry was used to quantitate [6,6-2H2]- and [U-13C6]glucose enrichments (6, 19, 23). Plasma samples (200 µl) were deproteinized with cold acetone (200 µl), and the pentacetate derivative of glucose was formed. The derivatized sample was analyzed on an Agilent 5970 Mass Selective Detector (Palo Alto, CA) with positive chemical ionization capabilities and fitted with a DB-1 capillary column. Selected ion monitoring was used to quantify the signal intensities for the ions at mass-to-charge ratios (m/z) 331, 333, and 337. The tracer-to-tracee ratios ([6,6-2H2]- and [U-13C6]glucose enrichments) were calculated as described in Refs. 25 and 26).
Insulin Sensitivity from Clamp
Insulin sensitivity.
Insulin sensitivity (SIclamp) was calculated from plasma glucose and insulin concentrations and glucose rate of infusion as (3, 17):
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Insulin sensitivity on glucose disposal.
Insulin sensitivity on glucose disposal was calculated from [2H2]glucose and insulin plasma concentrations and from tracer GIR as (3):
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Insulin Sensitivity from Oral Minimal Models
Oral minimal model.
The oral minimal model (OMM) (14, 15) uses the changes in plasma glucose and insulin concentrations observed after the OGTT glucose dose to derive SI and the Ra ogtt. Model equations are (4):
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Insulin sensitivity is given by (14, 15):
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Labeled OMM.
The labeled OMM, OMM*, uses exogenous glucose Gogtt, to derive SI (effect of insulin on glucose disposal) and Ra ogtt (13); it is described by
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Reference Model and Reference Model*
Both OMM and OMM* contain a parametric description of Ra ogtt, the parameters of which must be estimated from the data. An additional model-independent estimate of glucose Ra during OGTT was derived for each subject (Ra ogttref), by applying the Steele equation to the clamped tracer-to-tracee ratio TTR = [2H2]glucose/Gogtt, as explained in detail in Ref. 2:
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Ra ogttref was used to validate the estimated Ra ogtt and as known input of Reference Model (RM) and labeled Reference Model (RM*):
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Identification of RM and RM* provided reference values for OMM and OMM* parameters (see Identification), denoted, as in Eqs. 11 and 12, with a superscript "ref".
Identification
Identifiability.
OMM and OMM* were simultaneously identified on G and Gogtt data; since V and V* are not identifiable and SG and SG are not uniquely identifiable (see Ref. 15 for details), V, V* and SG, SG were fixed to the mean values obtained with RM and RM*, i.e., V = Vref, V* = V*ref, SG = SGref*, SG = SGref*, whereas parameters p2, p2, p3, p3, and 1...
7 were estimated in each individual. As in Refs. 14 and 15, the area under Ra ogtt was constrained to equal the total amount of ingested glucose, D, multiplied by the fraction that is actually absorbed, f (fixed to the mean value obtained from the Ra ogttref profiles, f = 0.87). Moreover, the availability of oral tracer measurements provided information about when Ra ogtt began to rise in each subject: if tracer concentration is zero up to time ti and is different from zero at time ti+1, then one can safely assume that Ra ogtt is zero up to ti.
Parameter estimation. All models were numerically identified by nonlinear least squares (8, 11) as implemented in SAAM II [Simulation Analysis and Modeling software (1)]. Measurement error on glucose and [U-13C6]glucose data were assumed to be independent, Gaussian, with zero mean and constant fractional standard deviation (CV = 2 and 6%, respectively).
Statistical Analysis
Data are presented as means ± SE. Two sample comparisons were done by Wilcoxon signed rank test (significance level set to 5%). Pearson's correlation was used to evaluate univariate correlation.
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RESULTS |
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Figure 1 shows the clamp mean glucose, tracer glucose, and insulin plasma concentrations. Figure 2 shows the OGTT mean glucose, exogenous glucose, and insulin plasma concentrations. The clamped TTR = [2H2]glucose/Gogtt, obtained during the OGTT, is shown in Fig. 3. It is worth noting that TTR doubles during the experiment; however, most of the variation is in the last 180 min, where the Ra is approximately zero; thus this nonconstancy will modestly affect the estimated profile. However, to minimize the nonconstancy effect of TTR (the non-steady-state error), a non-steady-state correction was employed (Eq. 10) by taking into account the derivative of TTR.
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Clamp insulin sensitivity and disposal insulin sensitivity are SIclamp = 13.66 ± 1.69, SIclamp = 8.84 ± 1.39 104 dl·kg1·min1 per µU/ml.
RM and RM*
Reference parameters estimated with RM and RM* are: SGref = 0.028 ± 0.003 min1 (CV = 15 ± 2%); Vref = 1.34 ± 0.06 dl/kg (CV = 5 ± 0.3%); p2ref = 0.0123 ± 0.0023 min1 (CV = 19 ± 3%); SIref = 8.18 ± 0.96 dl·kg1·min1 per µU/ml (CV = 11 ± 2%); SG*ref = 0.0067 ± 0.006 min1 (CV = 15 ± 3%); V*ref = 1.48 ± 0.07 dl/kg (CV = 4 ± 0.1%); p2*ref = 0.042 ± 0.004 min1 (CV = 14 ± 1%); and SI*ref = 8.00 ± 1.19 dl·kg1·min1 per µU/ml (CV = 8 ± 1%).
SI, SI, and Ra ogtt
Oral minimal models SI, SI are estimated with good precisions. Mean values are SI = 8.08 ± 0.89 (CV = 6 ± 1%) and SI = 8.17 ± 1.59 104 dl·kg1·min1 per µU/ml, (CV = 3 ± 1%). The model-reconstructed Ra ogtt (Fig. 4) is in good agreement with the model-independent Ra ogttref obtained using the tracer-to-tracee clamp technique. SI and SI were virtually identical to, respectively, SIref and SI*ref, obtained using Ra ogttref as a known input: 8.08 ± 0.89 vs. 8.18 ± 0.96 (P = 0.72) and 8.17 ± 1.59 vs. 8.00 ± 1.19 (P = 0.79).
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Oral models and clamp measurements of insulin sensitivity were well correlated (Fig. 5) : r = 0.81, P < 0.001 for SI vs. SI*clamp and r = 0.70, P < 0.001 for SI vs. SI*clamp. SI was lower than SIclamp by 34%: 8.08 vs. 13.66 104 dl·kg1·min1 per µU/ml (P = 0.0002), whereas SI was similar to SI*clamp: 8.17 vs. 8.84 104 dl·kg1·min1 per µU/ml (P = 0.52).
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DISCUSSION |
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Oral minimal-model insulin sensitivity indexes SI and SI are well correlated with their counterparts obtained with the euglycemic hyperinsulinemic clamp technique, SIclamp and SI*clamp, respectively r = 0.81 and r = 0.70. However, OMM SI was significantly lower than SIclamp, whereas OMM* SI was virtually identical to SI*clamp.
The good correlation shown between OMM and clamp SI is similar to that observed when SI of the IVGTT minimal model is compared against that of the clamp technique (20). An analogous trend toward underestimating SI was also present when IVGTT SI was compared with glucose clamp SI. However, as discussed in detail (21), values/comparison of IVGTT and clamp depend on how the IVGTT and clamp are performed (i.e., standard insulin- or tolbutamide-boosted IVGTT; and low-, medium-, or high-dose hyperinsulinemia during the clamp). Similarly, for OMM SI and SIclamp to be equivalent a number of conditions must be met; the most important are that the minimal-model single-pool description of glucose is adequate; insulin action on the combination of glucose utilization and production increases linearly with insulin concentration across the insulin range experienced during OGTT and clamp; and insulin sensitivity is independent from the route of insulin delivery, i.e., portal vs. peripheral. As regards the first condition, the OGTT dynamic milieu is well described by single-compartment glucose kinetics, at variance with IVGTT, so we tend to exclude an undermodeling effect on OGTT SI. As regards the second condition, in the current study plasma insulin levels during the clamp were designed to mimic the mean plasma insulin levels observed during an OGTT (4050 µU/ml), which are within the linear portion of the insulin vs. glucose disposal curve. In fact, although the steady-state relationship between glucose disposal and insulin concentration is approximately linear in the range of 10100 µU/ml, the relationship between endogenous glucose production and insulin concentration can be safely assumed linear only up to 4050 µU/ml. Thus these considerations speak in favor of having met the linear assumption, but one has to keep in mind that the time courses of insulin concentration during an OGTT and a glucose clamp are very different (i.e., constant in the clamp, vs. bell shaped during the OGTT). Finally, in comparing SI with SIclamp, one assumes that the peripheral and portal routes of insulin delivery are equally effective in inhibiting endogenous glucose production. To the best of our knowledge, the only study addressing this issue is that of Steil et al. (22). In that study, paired insulin-modified IVGTTs were performed in dogs infused with insulin (portal vs. peripheral) while circulating insulin levels were matched. Parameter estimates of SI under those two different routes of insulin infusion did not differ significantly. Unfortunately no data are available on the contribution of portal insulin to the assessment of SI during OGTT or meal. However, one has to consider the very different physiological milieu seen by the liver during clamp and OGTT, i.e., a constant insulin level of 4050 µU/ml vs. a bell-shaped insulin time course with a maximum of approximately three times the peripheral concentration, i.e., 240 µU/ml (see also below).
The discussion above assumes that OMM parameters accurately describe the manner in which changes in glucose and insulin concentrations regulate endogenous glucose production and disposal rates. We found that insulin action on glucose disposal estimated by OMM* SI, was virtually identical to that obtained during the clamp, SI*clamp. This may indicate that the glucose disposal component of OMM is more correctly described than the glucose production component. Thus a possible explanation for the 34% underestimation of OMM SI (compared with SIclamp) might be an inadequate description of the control of glucose and insulin on endogenous glucose production by OMM. This potential inaccuracy may also explain the physiologically implausible finding that SI was greater than SI in 7 of 21 subjects studied here. This paradoxical result has been observed in a large percentage of IVGTT studies (10, 12, 24). In the present OGTT study, the phenomenon has been mitigated, but it is still present in 33% of the subjects.
As in previously reported meal studies (13, 14), the multiple-tracer OGTT was used to reconstruct a model-independent estimate of the appearance rate of ingested glucose, Ra ogttref. Thanks to Ra ogttref, we have addressed several questions about OMM: is its prediction of Ra ogtt reliable? Is there any compensation between Ra ogtt and disposal/production model parameters? Figure 4 confirms our previous meal results: OMM reliably predicted the "true" Ra ogttref. In addition, when that true input Ra ogttref was used to identify either RM or RM*, identical values of SI and SI were obtained. This suggests that values for V, SG, f, and V*, SG for OMM and OMM* identification can be fixed to population averages without introducing appreciable bias in the estimation of SI. However, it is worth noting that when RM* instead of OMM* was used, the correlation between SI and SI*clamp increased from r = 0.70 to 0.83, whereas, if RM instead of OMM is used, correlation between SI and SIclamp does not change (r = 0.81 with OMM; r = 0.80 with RM). Ra estimate can also be affected by the choice of reference V, SG, V*, and SG: from a theoretical sensitivity analysis it emerged that the sensitivity of Ra estimate to V and V* is 1 in each breakpoint i, thus any error in V or V* results in a percentage equal error in the calculated Ra: luckily, neither V nor V* varies too much in the population [(Vref V)/Vref = 4 ± 4%; (V*ref V*)/V*ref = 5 ± 6%]. Conversely, the sensitivity of Ra parameters to SG and SG, different in each breakpoint, is lower than 0.64 for t < 240 min, whereas it increases until 3.8 in the last part of the experiment; thus, e.g., a 50% error in SG produces an error <32% in the first 5
i, whereas the percentage error can reach 190% for
6 and
7, which, however, are very close to zero.
In conclusion, we have presented good evidence of the ability of OMM and OMM* to assess both the net effect of insulin action on glucose utilization and endogenous production, SI, and the effect of insulin on glucose utilization only, SI, from a labeled OGTT by comparing SI and SI with their corresponding gold standard euglycemic hyperinsulinemic clamp values. Future studies should address the physiological explanation for the difference between SI and SIclamp values and to assess whether OGTT and meal models of glucose kinetics provide equivalent estimates of insulin action. Despite their limitations, our findings support the reliability of the OMM and OMM* tools for quantifying insulin action in clinical studies using a standardized oral glucose tolerance test. The potential to simultaneously assess -cell responsivity to glucose during an OGTT adds significant value to the proposed new oral minimal model method. This will require measurements of plasma C-peptide concentration and the use of the minimal model for insulin secretion and kinetics (6, 23) and will provide the ability to express
-cell function in relation to insulin sensitivity [e.g., the glucose disposition index (4, 18)]. Studies are in progress that will validate the use of fewer blood samples during an OGTT to estimate insulin sensitivity and
-cell responsivity parameters, advances that will make this approach more useful and applicable to large clinical trials (16).
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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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