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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-0019
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 6 2272-2278
Copyright © 2006 by The Endocrine Society

The Role of Endocrine Counterregulation for Estimating Insulin Sensitivity from Intravenous Glucose Tolerance Tests

Attila Brehm, Karl Thomaseth, Elisabeth Bernroider, Peter Nowotny, Werner Waldhäusl, Giovanni Pacini and Michael Roden

Division of Endocrinology and Metabolism (A.B., E.B., P.N., W.W., M.R.), Department of Internal Medicine III, Medical University of Vienna, A-1090 Vienna, Austria; Metabolic Unit (K.T., G.P.), Institute of Biomedical Engineering, I-35127 Padova, Italy; and First Medical Department (A.B., M.R.), Hanusch Hospital, A-1140 Vienna, Austria

Address all correspondence and requests for reprints to: Michael Roden, M.D., Division of Endocrinology and Metabolism, Department of Internal Medicine 3, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria, c/o 1. Medical Department, Hanusch Hospital, Heinrich Collin Strasse 30, A-1140 Vienna, Austria. E-mail: michael.roden{at}meduniwien.ac.at.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: During insulin-modified frequently sampled iv glucose tolerance tests (IM-FSIGT), which allow assessment of insulin action, plasma glucose can markedly decrease.

Objective: This study aimed to assess the counterregulatory impact of the insulin-induced fall of glucose on minimal model-derived indices of insulin sensitivity (SI) and glucose effectiveness.

Participants: Thirteen nondiabetic volunteers (seven males, six females, aged 26 ± 1 yr, body mass index 22.1 ± 0.7 kg/m2) were studied.

Design: All participants were studied in random order during IM-FSIGT (0.3 g/kg glucose; 0.03 U/kg insulin at 20 min) and during identical conditions but with a variable glucose infusion preventing a decrease of plasma glucose concentration below euglycemia (IM-FSIGT-CLAMP). Five participants additionally underwent euglycemic-hyperinsulinemic (1 mU·kg–1·min–1) clamp tests.

Results: Plasma glucose declined during IM-FSIGT to its nadir of 50 ± 3 mg/dl at 60 min in parallel to a rise (P < 0.05 vs. basal) of plasma glucagon, cortisol, epinephrine, and GH. Glucose infusion rates of 4.6 ± 0.5 mg·kg–1·min–1 between 30 and 180 min during IM-FSIGT-CLAMP prevented the decline of plasma glucose and the hypoglycemia counterregulatory hormone response. SI was approximately 68% lower during IM-FSIGT (3.40 ± 0.36 vs. IM-FSIGT-CLAMP: 10.71 ± 1.06 10–4·min–1 per µU/ml, P < 0.0001), whereas glucose effectiveness did not differ between both protocols (0.024 ± 0.002 vs. 0.021 ± 0.003 min–1, P = NS). Compared with the euglycemic hyperinsulinemic clamp test, SI expressed in identical units from IM-FSIGT was approximately 66% (P < 0.001) lower but did not differ between the euglycemic hyperinsulinemic clamp test and the IM-FSIGT-CLAMP (P = NS).

Conclusions: The transient fall of plasma glucose during IM-FSIGT results in lower estimates of SI, which can be explained by hormonal response to hypoglycemia.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE FREQUENTLY SAMPLED iv glucose tolerance test (FSIGT) is now widely used to obtain parameters of insulin sensitivity and glucose effectiveness (SG) in humans (1, 2). This approach is based on minimal model analysis of the time courses of plasma glucose and insulin concentrations in response to an iv glucose bolus. To increase the dynamics of plasma glucose and insulin in states of insufficient or absent endogenous insulin secretion, insulin-modified protocols (IM-FSIGT) were developed (3, 4, 5). These protocols introduce a second exogenously induced insulin peak by iv administration of insulin 20 min after the glucose bolus.

In insulin-sensitive humans with normal ß-cell function, plasma glucose frequently decreases below fasting concentrations during the IM-FSIGT (6, 7, 8, 9). Because any rapid fall in plasma glucose concentration results in hormonal and metabolic counterregulation, we hypothesized that such changes would also impact on minimal model-derived indices of insulin sensitivity (SI) and possibly also SG. The basis of this hypothesis is the observation of impaired SI in the posthypoglycemic state (10, 11, 12).

To determine the impact of IM-FSIGT-associated hypoglycemia on calculated SI, we measured profiles of plasma glucose, insulin, free fatty acids (FFAs), and major counterregulatory hormones during a standard IM-FSIGT and during a modified IM-FSIGT protocol. This modified protocol prevented any decline of plasma glucose below euglycemia by using a variable glucose infusion (IM-FSIGT-CLAMP). A subgroup underwent additional euglycemic-hyperinsulinemic clamp [SIP(clamp)] tests to obtain an independent measure of insulin sensitivity.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Thirteen lean, healthy volunteers (seven men and six women, aged 26 ± 1 yr, body mass index 22.1 ± 0.7 kg/m2, fasting plasma glucose 90 ± 2 mg/dl, fasting plasma insulin 6 ± 1 µU/ml) without family history of diabetes mellitus participated in this study. They were not glucose intolerant, suffering from conditions related to insulin resistance, or taking any medication on a regular basis. None of the women was taking hormonal contraceptives, and all females were studied in the follicular phase of their cycle. Each subject underwent in random order an IM-FSIGT and an IM-FSIGT-CLAMP spaced by at least 5 d. A subgroup of the participants additionally underwent a euglycemic hyperinsulinemic clamp test. The protocols were approved by the local institutional ethics board, and written informed consent was obtained from all participants.

Study protocols

The participants were instructed to ingest an isocaloric diet [25 kcal/kg·d; carbohydrate/protein/fat: 55/15/30%] and refrain from physical exercise for at least 3 d before all studies (13).

IM-FSIGT and IM-FSIGT-CLAMP

After a 12-h overnight fast, each protocol started at 0800 h with insertion of two indwelling catheters (Vasofix; Braun, Melsungen, Germany) into the antecubital veins of both arms for blood sampling and infusions, respectively. During both protocols, a glucose bolus [time 0–1 min, 0.30 g/kg body weight, 33% (wt/vol)] and a bolus of regular insulin (time 20 min, 0.03 IU/kg, Actrapid; Novo Nordisk, Bagsvaerd, Denmark) were injected (14). During IM-FSIGT-CLAMP a variable adjusted (5 min intervals) iv infusion of glucose [20% (wt/vol)] was applied to prevent a fall of plasma glucose less than 100 mg/dl. During both protocols blood samples were frequently collected (9). Plasma concentrations of glucose, insulin, FFAs, glucagon, cortisol, GH, epinephrine, and norepinephrine were determined at timed intervals.

Hyperinsulinemic-euglycemic-clamp test

A subgroup of the study participants (five males, aged 25 ± 0.1 yr, body mass index 22.2 ± 1.0 kg/m2) additionally underwent a SIP(clamp) test. After an identical overnight fast, catheters were placed in antecubital veins of each arm for blood sampling and infusions, respectively. At 0700 h (–120 min), a baseline blood sample was drawn, and at 0800 h, a primed-continuous infusion of D-[6,6-2H2]glucose (–120 to –115 min: 16.7 µmol/kg per 5 mmol/liter; – 115 min to 360 min: 0.17 µmol·kg–1·min–1) (98% enriched, Cambridge Isotope Laboratories, Andover, MA) was started to determine rates of glucose turnover (15, 16). At 0955 h (–5 min), a somatostatin infusion was started (–5 to 360 min: 0.1 µg·kg–1·min–1, UCB Pharma, Vienna, Austria), and insulin was infused from 0 to 180 min and from 180 to 360 min at rates of 0.15 mU·kg–1·min–1 and 1 mU·kg–1·min–1, respectively. Plasma glucose was measured in 5-min intervals and was maintained at approximately 5.5 mmol/liter by a variable glucose infusion (20 g/dl) enriched to approximately 2% with D-[6,6-2H2]glucose (16). Blood samples for measurement of plasma insulin concentration and deuterated glucose enrichments were drawn at –180, – 15, – 5, 60, 120, 180, 210, 240, 300, and 360 min.

Analytical procedures

Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II; Beckman Instruments, Inc., Fullerton, CA). Plasma FFA concentration was assayed with a microfluorometric method (Wako Chem USA Inc., Richmond, VA). In vitro lipolysis was prevented by collecting blood into vials containing orlistat and rapid centrifugation of the samples (17). Plasma immunoreactive insulin, glucagon, GH, and cortisol were measured by RIAs, and plasma epinephrine and norepinephrine were determined by HPLC. Coefficients of variation for these assays have been previously reported (17, 18, 19). Gas chromatography-mass spectrometry was used for the determination of atom percent excess of 2H in glucose as previously described (15, 16). Briefly, the glucose-pentaacetate was analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with a CP-Sil5 25 m x 0.25 mm x 0.12 µm capillary column (Chrompack, Middelburg, The Netherlands) interfaced to a Hewlett-Packard 5971A mass selective detector operating in the electron impact ionization mode. Selective ion monitoring was used to determine enrichments in various molecular mass ion fragments of glucose. Deuterium (M+2) enrichments in glucose were assayed in fragments of C3-C6 with their masses of 187 and 189 (15, 16).

Calculations

IM-FSIGT and IM-FSIGT-CLAMP. Minimal model analysis of the time course of plasma glucose and insulin concentrations provided indices of SI and SG (9). The mathematical equations used to fit dynamic glucose concentrations are as follows: dG(t)/dt = –(SG + X(t)) G(t) + SGGb + uG(t)/VG and dX(t)/dt = –(P2(X(t) – S1(I(t) – Ib)), where G(t) (milligrams per deciliter) represents glucose concentration with basal value Gb and initial condition G(0) = Gb+ D/VG (glucose volume of distribution) after the bolus dose D (300 mg/kg) with normalized VG (deciliters per kilogram); I(t) (microunits per milliliter) is an assigned model input reconstructed through linear interpolation of plasma insulin concentration data. The additional glucose input uG(t) (milligrams per kilogram per minute) is the piecewise constant infusion administered during the glucose clamp-modified IM-FSIGT; X(t) (minutes–1) is the action of insulin in a remote compartment with initial condition X(0) = 0; SG (minutes–1) is SG at basal insulin that quantifies glucose disposal per se independent of dynamic insulin; SI (minutes–1 per microunit/ milliliter) is the SI that quantifies the effect of insulin in enhancing glucose disposal; P2 (minutes–1) quantifies the turnover rate of insulin action in the remote compartment. Because the basal reference concentrations of glucose and insulin, Gb and Ib, respectively, affect the estimation of SI and SG (20, 21), several different values for Gb and Ib were tested including preglucose-injection levels and final values of IM-FSIGT and IM-FSIGT-CLAMP. The different choices did not significantly affect the estimated SI and SG so that we used mean concentrations at the end of the test (130–180 min). Parameter estimates of SG, SI, P2, and VG were obtained by weighted nonlinear mixed-effects model fitting of glucose data, with weights given by the reciprocal of measurement noise variance calculated by assuming 2% error of measured glucose concentrations.

Euglycemic hyperinsulinemic clamp. Basal rate of glucose disappearance (Rdbasal), defined as the rate of glucose disappearance before the start of the clamp test, was calculated by dividing the tracer infusion rate times the tracer enrichment by the steady-state tracer enrichment of plasma glucose before the start of the clamp test and subtracting the tracer infusion rate (22). During the clamp, the rate of disappearance of glucose was calculated during the low and high insulin (Rdclamp) infusion period from Steele's equation for non-steady state (23).

Comparison of measures of SI. To compare measures of SI obtained from the FSIGT protocols and the SIP(clamp) test, the data have to be transformed to allow expressing in identical units (4, 24, 25). This was achieved by expressing SI in terms of changes in glucose clearance per unit change in the plasma insulin concentration (deciliters per minute per microunits per milliliter) (4, 24, 25).

To convert SI (minutes–1 per microunits per milliliter) from both FSIGT protocols into glucose clearance per unit change in plasma insulin (deciliters per minute per microunits per milliliter), SI was multiplied by the glucose volume of distribution (VG) expressed in deciliters (4, 24, 25).

SI from the SIP(clamp) test [SIP(clamp) in deciliters per minute microunits per milliliter] was calculated as the steady-state ratio of the increment in glucose disposal (i.e. {Delta}Rd in milligrams per minute) to the increment in plasma insulin concentration ({Delta}I in microunits per milliliter), normalized to the ambient plasma glucose concentration (G; milligrams per deciliter) during the high insulin infusion period of the clamp; i.e. SIP(clamp) = {Delta}Rd/({Delta}I/G).

{Delta}Rd was calculated as the difference between Rd before the clamp and Rd during the final 30 min of the 1 mU·kg–1·min–1 insulin infusion period of the clamp (i.e. {Delta}Rd = Rdclamp – Rdbasal). Likewise, {Delta}I was calculated as the difference between fasting plasma insulin concentration and the steady-state plasma insulin concentration during the final 30 min of the high insulin period of the clamp (4, 24, 25).

Statistical analysis

Data are presented as means ± SE. Results from both IM-FSIGT protocols were compared with the two-tailed paired Student’s t test. One-way ANOVA with post hoc Tukey correction for multiple comparisons was used to compare SI from the euglycemic hyperinsulinemic clamp test and both IM-FSIGT protocols. Linear correlations are Pearson product-moment correlations. SPSS 11.0 software (SPSS Inc., Chicago, IL) was used. P < 0.05 was considered to indicate statistically significant differences.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
IM-FSIGT and IM-FSIGT-CLAMP

Glucose, insulin, and FFA concentrations. Fasting plasma glucose and insulin did not differ between both protocols (Fig. 1Go, A and B). Plasma glucose concentrations peaked at 3 min after the glucose bolus during both protocols (IM-FSIGT: 262 ± 17 vs. IM-FSIGT-CLAMP: 254 ± 15 mg/dl, P = NS) and then followed a similar pattern of decrease until 35 min (Fig. 1AGo). After 35 min, plasma glucose continued to decrease during IM-FSIGT to its nadir of 50 ± 3 mg/dl at 60 min and thereafter rose to 87 ± 3 mg/dl at 180 min. During IM-FSIGT-CLAMP, the variable glucose infusion (Fig. 1DGo) prevented the fall of plasma glucose below the normoglycemic range (mean plasma glucose 30–180 min: 104 ± 3 mg/dl) (Fig. 1AGo).


Figure 1
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  		FIG. 1. Time course of plasma concentrations of glucose (A), insulin (B), and FFAs (C) during IM-FSIGT (open circles) and IM-FSIGT-CLAMP (filled circles). During IM-FSIGT-CLAMP the decline of plasma glucose concentrations below euglycemic levels was prevented by a frequently adjusted variable glucose infusion (D). *, P < 0.05; {dagger}, P < 0.01; and {ddagger}, P < 0.005 for IM-FSIGT vs. IM-FSIGT-CLAMP.

 
Plasma insulin concentration peaked at 4 min (IM-FSIGT: 51 ± 8 vs. IM-FSIGT-CLAMP: 47 ± 6 µU/ml, P = NS) and upon the exogenous insulin injection at 22 min (IM-FSIGT: 569 ± 95 vs. IM-FSIGT-CLAMP: 494 ± 50 µU/ml, P = NS) (Fig. 1BGo). After its peak, plasma insulin decreased comparably until 70 min on both study days and was then lower (mean 70–180 min) during IM-FSIGT (10 ± 2 vs. IM-FSIGT-CLAMP: 5 ± 1 µU/ml, P < 0.005).

Fasting plasma concentrations of FFAs were comparable on both study days and similarly decreased after the glucose bolus on both study days to a nadir of approximately 10% of basal at 50 min (Fig. 1CGo). From 50 min on, FFAs increased during both protocols but were approximately 2-fold higher during IM-FSIGT (50–180 min: 242 ± 32 vs. IM-FSIGT-CLAMP: 121 ± 27 µmol/liter, P < 0.05).

Hypoglycemia counterregulatory hormones. Fasting plasma concentrations of glucagon, cortisol, and GH were comparable on both study days (Fig. 2Go). Plasma concentrations of glucagon, cortisol, and GH increased after 30 min during IM-FSIGT and were temporarily higher (P < 0.05), compared with IM-FSIGT-CLAMP (Fig. 2Go, A–C). Fasting plasma concentrations of epinephrine and norepinephrine also did not differ between IM-FSIGT and IM-FSIGT-CLAMP (epinephrine: 71 ± 40 vs. 117 ± 64 pmol/liter, norepinephrine: 1.15 ± 0.21 vs. 1.29 ± 0.12 nmol/liter, for both P = NS). Plasma epinephrine increased (P < 0.05 vs. basal) during IM-FSIGT between 70 and 90 min (768 ± 341 vs. 83 ± 57 pmol/liter, P < 0.05), during which plasma norepinephrine concentrations did not change during IM-FSIGT-CLAMP (Fig. 2DGo).


Figure 2
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  		FIG. 2. Time course of plasma concentrations of glucagon (A), cortisol (B), GH (C), and epinephrine (D) during IM-FSIGT (open symbols) and IM-FSIGT-CLAMP (filled symbols). {dagger}, P < 0.05 for IM-FSIGT vs. IM-FSIGT-CLAMP.

 
Minimal model parameters of SI and SG. VG did not differ between both FSIGT protocols (IM-FSIGT: 1.79 ± 0.10 dl/kg vs. IM-FSIGT-CLAMP: 1.93 ± 0.08, P = NS).

SG did not differ between both protocols (IM-FSIGT: 0.024 ± 0.002 vs. IM-FSIGT-CLAMP: 0.021 ± 0.003 min–1, P = NS), whereas SI from IM-FSIGT was approximately 68% lower, compared with SI from IM-FSIGT-CLAMP (3.40 ± 0.35 vs. 10.71 ± 1.06 10–4·min–1 per µU/ml, P < 0.00001) (Fig. 3AGo). Correlation analysis revealed no significant relationship between SI from IM-FSIGT and IM-FSIGT-CLAMP (r = 0.277, P = NS). The difference between SI from IM-FSIGT-CLAMP and IM-FSIGT was positively correlated with the mean glucose infusion rate (R = 0.564, P < 0.05) and the area under the curve of the glucose infusion rate during IM-FSIGT-CLAMP (R = 0.560, P < 0.05, Fig. 3BGo).


Figure 3
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  		FIG. 3. A, Plot of indices of SI (10–4 min–1/µU·ml) from IM-FSIGT and IM-FSIGT-CLAMP (n = 13). Solid lines connect SI data points from each individual. {ddagger}, P < 0.0001 for IM-FSIGT vs. IM-FSIGT-CLAMP. B, Relationship between the SI difference ({Delta}SI) from IM-FSIGT and IM-FSIGT-CLAMP with the area under the glucose infusion curve (AUC) required, preventing hypoglycemia during IM-FSIGT-CLAMP. The solid line represents the linear regression line, and the dotted lines represent the mean 95% confidence interval.

 
Euglycemic hyperinsulinemic clamp

Fasting plasma insulin concentration before the clamp was 6 ± 1 µU/ml in the five participants who additionally underwent the euglycemic hyperinsulinemic clamp test. During the steady-state period of the 0.15 and 1 mU·kg–1·min–1 insulin infusion period, mean plasma glucose and insulin concentrations were 96 ± 1 and 96 ± 2 mg/dl and 14 ± 2 and 72 µU/ml, respectively. Rdbasal was 1.76 ± 0.09 mg·kg–1·min–1 before the clamp test, rate of disappearance of glucose during the low insulin infusion period was 4.88 ± 1.11 mg·kg–1·min–1 during the final 30 min of the 0.15 mU·kg–1·min–1 insulin infusion period, and Rdclamp was 13.44 ± 0.28 mg·kg–1·min–1 during the final 30 min of the 1 mU·kg–1·min–1 insulin infusion period of the euglycemic hyperinsulinemic clamp.

SI from IM-FSIGT, IM-FSIGT-CLAMP, and the clamp test

When SI of all protocols was expressed in identical units, SI from IM-FSIGT was approximately 66% (P < 0.001) lower than SI obtained from the euglycemic hyperinsulinemic clamp, whereas SI obtained from IM-FSIGT-CLAMP was comparable (P = NS) with that obtained with the euglycemic hyperinsulinemic clamp test (IM-FSIGT: 0.041 ± 0.009 vs. IM-FSIGT-CLAMP: 0.096 ± 0.012 vs. hyperinsulinemic clamp test: 0.122 ± 0.007 dl/min per µU/ml). Correlation analysis revealed no significant relationship between SI from IM-FSIGT and the euglycemic hyperinsulinemic clamp test (R = 0.216, P = NS) (Fig. 4Go). SI from IM-FSIGT-CLAMP positively correlated with SI from the euglycemic hyperinsulinemic clamp test (R = 0.878, P < 0.05) (Fig. 4Go).


Figure 4
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  		FIG. 4. Relationship between SI from the IM-FSIGT (ordinate, open circles) and IM-FSIGT-CLAMP (ordinate, closed circles) with SI from the SIP(clamp) test (abscissa) expressed in identical units (i.e. change in glucose clearance per change in plasma insulin concentration, deciliters per minute per microunits per milliliter, calculation described in Subjects and Methods).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study demonstrates that plasma glucose decreases into the hypoglycemic range in young nondiabetic nonobese humans during a standard IM-FSIGT as previously noted by others (4, 7, 8, 9, 13, 14, 25). This decrease of plasma glucose concentrations to a nadir of approximately 50 mg/dl during IM-FSIGT is associated with a transient increase in plasma concentrations of glucagon, cortisol, GH, and epinephrine, indicating hypoglycemia counterregulation. This endocrine response is absent when a variable glucose infusion during IM-FSIGT-CLAMP prevents any decrease of plasma glucose concentration below normoglycemic levels.

The insulin bolus applied in both protocols resulted in peak plasma concentrations of insulin that are comparable with values reported by other investigators (3, 4, 14) and led to nadirs of plasma glucose during IM-FSIGT that are also comparable with that of previous reports in nondiabetic humans (3, 7, 25). However, searching the literature, we were unable to find a previous study on the role of the major hypoglycemia counterregulatory hormones during IM-FSIGT. SG did not differ between IM-FSIGT and IM-FSIGT-CLAMP, indicating that this minimal model parameter is robust, whether or not hypoglycemia occurs during IM-FSIGT. This is in agreement with the fact that SG characterizes the early glucose decay during FSIGT (26), i.e. the time interval before the counterregulatory hormone response. In agreement with this view, previous FSIGT studies found at least a trend for lower SG when plasma concentrations of counterregulatory hormones such as epinephrine were already increased before the time point of glucose injection (27, 28).

Of note, the minimal model-derived parameter of SI was approximately 68% lower from IM-FSIGT, compared with IM-FSIGT-CLAMP. This difference may be explainable by several factors.

Hypoglycemia counterregulation as observed during IM-FSIGT results in a rapid stimulation of endogenous glucose production, inhibition of peripheral glucose uptake, and stimulation of lipolysis (29, 30, 31). In the present study, we did not directly quantify endogenous glucose production, whole-body glucose uptake, or lipolysis, but our data provide strong indirect evidence for the activation of these metabolic processes during IM-FSIGT in our study.

When plasma glucose concentration reached its nadir of approximately 50 mg/dl during IM-FSIGT in our study, glucose infusion rates required to prevent a fall of glycemia below euglycemic levels were approximately 8 mg·kg–1·min–1 during IM-FSIGT-CLAMP. Thus, the glucose infusion rates during IM-FSIGT-CLAMP provide a raw estimate of the glucose amount needed to prevent a decline of plasma glucose concentration into the hypoglycemic range during IM-FSIGT.

In this context it is of interest that a previous study assessed the time course of endogenous glucose production during IM-FSIGT in nondiabetic humans (28). Although the nadir of plasma glucose in this previous study was approximately 75 mg/dl, endogenous glucose production rebounded from nearly complete suppression to approximately 1.5-fold of basal. Unfortunately, plasma concentrations of counterregulatory hormones were not reported in that study (28). The minimal model was originally designed for the regular iv glucose tolerance test without any additional insulin application. In this case, even in insulin-sensitive subjects, glucose rarely reaches nadirs in the hypoglycemic range (9). In the mathematical representation, endogenous glucose production is represented as a constant term times the difference between basal glucose and the actual glucose concentration, and this may therefore result in an underestimation of endogenous glucose production when hypoglycemic counterregulation occurs during IM-FSIGT.

Vicini et al. (28) demonstrated that even small increases of plasma epinephrine brought by means of a low-dose epinephrine infusion during FSIGT result in a delayed suppression of endogenous glucose production and a reduction of whole-body glucose clearance. In agreement with increased lipolysis in response to hypoglycemia and hypoglycemic counterregulation in our study (31), the rebound of plasma FFA concentration was more pronounced during IM-FSIGT, compared with IM-FSIGT-CLAMP. Comparable increases in plasma FFA concentrations have been shown to reduce whole-body glucose uptake by approximately 15% (32).

The relevance of hypoglycemia counterregulation accounting for the differences in SI between the IM-FSIGT and IM-FSIGT-CLAMP protocols is underlined by the positive correlation between the area under the glucose infusion curve during IM-FSIGT-CLAMP and the difference in minimal model derived insulin sensitivity between both protocols. This relationship demonstrates that when hypoglycemia can be prevented by only small amounts of infused glucose during IM-FSIGT-CLAMP, SI will not dramatically differ between both protocols. In turn, when high glucose infusion rates are required to prevent hypoglycemia during IM-FSIGT-CLAMP, SI differs considerably between both FSIGT protocols. This relationship demonstrates that the amount of infused glucose required to prevent hypoglycemia during IM-FSIGT-CLAMP determines the underestimation of SI during FSIGT-CLAMP. This relationship could also explain why we could not detect a correlation between SI as determined with the two IM-FSIGT protocols.

To provide for an independent measure of SI, we additionally compared insulin sensitivity from both FSIGT protocols with SI from the euglycemic hyperinsulinemic clamp test expressed in identical units (4, 24). Although the number of subjects in this subgroup was rather small, the comparison of SI from the clamp test with parameters of SI from both IM-FSIGT protocols allows one to draw some conclusions. This comparison demonstrates that IM-FSIGT associated with a decrease of plasma glucose into the hypoglycemic range results in lower estimates of SI, compared with both IM-FSIGT-CLAMP and the hyperinsulinemic euglycemic clamp test.

Although we found a positive correlation between SI from the IM-FSIGT-CLAMP and hyperinsulinemic clamp test, these data do not validate the IM-FSIGT-CLAMP as a new technique to determine SI due to the small number of participants. However, these data demonstrate that when hypoglycemia and thus counterregulatory hormone response is prevented, the minimal model is able to describe the physiological situation and parameter estimation yields SI values similar to those evaluated with the euglycemic hyperinsulinemic clamp test.

In conclusion, our results demonstrate that decreases of plasma glucose down to the hypoglycemic range during IM-FSIGT result in endocrine counterregulation associated with an underestimation of minimal model-derived parameters of SI. To avoid hypoglycemia and underestimation of SI during IM-FSIGT protocols in nondiabetic subjects, the administered insulin dose should be as small as possible, and the plasma glucose profiles should be carefully checked for values in the hypoglycemic range.


   Acknowledgments
 
We gratefully acknowledge O. Lentner for technical assistance and the staff of the Endocrine Laboratory.


   Footnotes
 
This work was supported in part by grants from the Austrian Science Foundation (FWF, P15656) (to M.R.), the European Foundation for the Study of Diabetes (Novo Nordisk type 2 diabetes grant), and the Herzfelder Family Trust. This study is also part of an ongoing cooperation between the Division of Endocrinology and Metabolism of the 3rd Department of Internal Medicine of the Medical University of Vienna and the Metabolic Unit of Institute of Biomedical Engineering (formerly Institute of Systems Science and Biomedical Engineering).

First Published Online April 4, 2006

Abbreviations: FFA, Free fatty acid; FSIGT, frequently sampled iv glucose tolerance test; IM-FSIGT, insulin modified frequently sampled iv glucose tolerance test; IM-FSIGT-CLAMP, IM-FSIGT combined with a variable glucose infusion preventing any decrease of plasma glucose concentration below euglycemia; Rdbasal, basal rate of glucose disappearance; Rdclamp, rate of disappearance of glucose during the low and high insulin infusion period; SG, glucose effectiveness; SI, insulin sensitivity index; SIP(clamp), euglycemic-hyperinsulinemic clamp; VG, glucose volume of distribution.

Received January 5, 2006.

Accepted March 28, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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