Importance of Obtaining Independent Measures of Insulin Secretion and Insulin Sensitivity During the Same Test: Results with the Botnia clamp

Subjects were classified into different stages of glucose tolerance according to the new World Health Organization (WHO) criteria (14). Informed consent was obtained from all subjects, and the studies were approved by the local ethics committees. Subject characteristics are given in Table 1.

For this protocol, 13 subjects (11 men, 2 women; 5 with normal glucose tolerance [NGT], 8 with type 2 diabetes or impaired glucose tolerance [IGT]) participated in two Botnia clamps performed at a 1-week interval.

The study cohort for this protocol included nine subjects (five men, four women; four with NGT, five with type 2 diabetes or IGT) who participated in random order in a Botnia clamp and a euglycemic, hyperinsulinemic clamp at a 1-week interval.

We also applied the Botnia clamp to 41 glucose-tolerant subjects, 25 of whom had a first-degree relative with type 2 diabetes.

The Botnia clamp was designed to obtain independent measures of insulin secretion and S_I during the same test (Figure 1). In brief, 0.3 g/kg body wt of a 20% glucose solution was given at time 0. Blood samples for the measurement of plasma glucose and serum insulin were obtained at −10, 0, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 120, and 180 min. The incremental trapezoidal area during the first 10 min was called first-phase insulin response (FPIR). The FPIR was also measured as the sum of three insulin concentrations at 2, 4, and 6 min after the glucose challenge. The incremental insulin secretion during the last 50 min was called the second-phase insulin secretion. After 60 min, a priming dose of insulin was given followed by an infusion (45 mU/m²) of short-acting human insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) for 120 min, thereby achieving steady-state insulin concentrations of 88.5 ± 5.4 and 92.5 ± 5.4 mIU/l during the last 60 min of clamp 1 and clamp 2, respectively. Blood samples for the measurement of plasma glucose were obtained at 5-min intervals throughout the clamp. A variable infusion of 20% glucose was started to maintain the plasma glucose concentration at 5.5 mmol/l. The mean coefficient of variation (CV) of glucose during the clamps was 6%. S_I (M-value) was calculated from the glucose infusion rates during the last 60 min of the euglycemic clamp and as the S_I index (M/I_clamp; mg glucose · kg⁻¹ body wt · min⁻¹:mU/l insulin) as the ratio of glucose infusion rates during the last 60 min and the mean steady-state insulin levels during the last 60 min of the clamp ([M/I] · 100) (6). The DI is a measure of insulin secretion adjusted for insulin sensitivity and was calculated from the product of the FPIR and the M-value (3). In a different group of nine subjects, the Botnia clamp and a euglycemic-hyperinsulinemic clamp without prior glucose infusion was performed to study the potential influence of the glucose bolus on the M-value.

Subjects with and without family history of diabetes (protocol 3) also participated in an OGTT by ingesting 75 g of glucose in a volume of 300 ml (Glucodyn; Leiras, Turku, Finland) after a 12-h overnight fast. Samples for the measurement of glucose and insulin were drawn at −10, 0, 30, 60, and 120 min.

Body height, weight, and fat-free mass (FFM) were recorded. The FFM was measured with a bioelectrical impedance technique using a two-terminal portable impedance analyzer (BIA 101, RJL; Akern, Copenhagen, Denmark) (15).

Plasma glucose was measured in duplicate by a glucose oxidase method using a Beckman Glucose Analyzer II (Beckman Instruments, Fullerton, CA). Plasma insulin concentrations were measured with a double antibody enzyme-linked immunosorbent assay (Dako, Cambridgeshire, U.K.) with an intra-assay CV of 7.5% and an interassay CV of 7%.

Data are expressed as means ± SEM or SD. Statistical analyses included Mann Whitney test and Spearman rank correlation and were carried out using the NCSS statistical software (Number Cruncher Statistical System, version 6). Data for insulin were log transformed for normality. Variability and the repeatability of measures of S_I and insulin secretion were assessed using the CV and the recently proposed measure, the discriminant ratio (DR) (16). For the CV, the following formula was used:

The DR was measured using the following formula:

in which MS_B and MS_w are the between- and within-subject error terms derived from a standard repeated measure of ANOVA and k is the number of replicate tests in each subject (16).

The first study examined reproducibility of measures of insulin secretion and S_I obtained from the Botnia clamp. Glucose and insulin values during the repeat Botnia clamps were comparable in subjects with normal (Fig. 2A and B) and abnormal glucose tolerance (Fig. 2C and D). The rates of insulin-stimulated glucose uptake did not differ between the two tests, whether measured as the M-value (NGT: 10.4 ± 1.2 and 11.3 ± 1.2 mg · FFM kg⁻¹ · min⁻¹; type 2 diabetes/IGT: 6.3 ± 0.9 and 6.7 ± 0.9 mg · FFM kg⁻¹ · min⁻¹) or as the S_I index M/I_clamp (NGT: 10.2 ± 1.3 and 10.7 ± 1.4 mg · FFM kg⁻¹ · min⁻¹/mU/l⁻¹; type 2 diabetes/IGT: 6.3 ± 1.5 and 6.7 ± 1.5 mg · FFM kg⁻¹ · min⁻¹/mU/l⁻¹), thereby yielding a mean CV for repeat M-values from the Botnia clamps of 9.0% and a correlation coefficient of r = 0.983, P < 10⁻⁵. The variability and reproducibility of the M-value were also assessed by Altman-Bland plots for the repeat M-values (Fig. 3A), and showed a uniform variability across the range of values from NGT to diabetes. The DR for M-value from repeat clamps was 1.81 (CI 1.11–2.78). In contrast, the CV for mean fasting insulin concentrations from three samples on 2 different clamp days was 20%, and therefore the S_I measured as HOMA obtained from the fasting glucose and insulin on the day of the clamp had a CV of 19%.

Although repeat Botnia clamps yielded similar FPIRs regardless of glucose tolerance (152 ± 81 vs. 186 ± 81 mIU · l⁻¹ · 10 min⁻¹; NS) and a strong correlation was observed between FPIRs performed on 2 different days (r = 0.923, P < 0.005), the CV for the repeat tests was 27% in subjects with NGT and 12% in subjects with IGT, thereby yielding a combined CV in NGT/IGT subjects of 23%. The FPIR measured as a sum of the three insulin values at 2, 4, and 6 min also yielded a CV of 25% in NGT/IGT subjects. The CV for FPIR in subjects with diabetes was not included, as all subjects lacked the FPIR on repeat studies. The Altman-Bland plots (Fig. 3B) show that the variability is to some extent proportional to the magnitude of the FPIR. Similarly, the CV for second-phase insulin secretion in subjects with NGT/IGT was 22%. The DR for the repeat FPIR was 1.09 and for the second-phase insulin response was 1.45.

The M-values obtained from the Botnia and euglycemic clamps (7.23 ± 1.14 and 7.79 ± 1.14 mg · FFM kg⁻¹ · min⁻¹; NS) were comparable and correlated strongly (r = 0.953; P < 0.005).

As previously described (12), FH+ subjects had lower insulin-stimulated glucose uptake compared with FH− subjects (5.6 ± 0.4 vs. 7.5 ± 0.6 mg · kg⁻¹ · min⁻¹; P = 0.02), whereas no difference was observed in the FPIRs between the two groups (258 ± 37 vs. 285 ± 59 mIU · 1⁻¹ · 10 min⁻¹; NS). However, when β-cell function was expressed as a DI, FH+ subjects showed a significant reduction in their β-cell function (1,284 ± 181 vs. 1985 ± 288 mUl · mg · kg⁻¹ · min⁻¹; P = 0.04) (Figure 4).

The present study validated a means to assess S_I and β-cell function from the same test, the Botnia clamp. S_I measured with the Botnia clamp was highly reproducible, with a CV of 9.0%, and was independent of subjects’ glucose tolerance. It may be argued that the glucose bolus given at the start of the study affected the subsequent S_I measured during the clamp. However, this did not occur, as the M-value from the Botnia clamp was similar to and correlated strongly with the M-value obtained from the euglycemic clamp without prior glucose bolus. All subjects with IGT and type 2 diabetes reached the desired steady-state plasma glucose level during the last 60 min of the Botnia clamp.

In addition to the CV, we also estimated the DR (16), which takes into account the within- and intersubject variations. Indeed, the S_I obtained from the Botnia clamp showed a low CV, high DR, and low variability across a wide range of glucose tolerance.

In contrast, a slightly higher variation was observed in the FPIR values between the tests. This finding was in agreement with previous reports of up to 50% variation in FPIR, even in subjects with NGT (17–19). Given that the mean of three fasting insulin concentrations demonstrated considerable variation, it is not surprising that the glucose-stimulated insulin values showed an even greater variation. This may have represented a biological day-to-day variation rather than a technical one. Also, standard formulas for estimating CV usually underestimate CV at the lower range (16). Of note, all patients with type 2 diabetes lacked the FPIR on repeat studies.

The study of quantitative traits such as S_I and insulin secretion has been proposed as one way to reduce phenotypic heterogeneity in genetic studies. Therefore, there is an urgent need for reliable tests of S_I and β-cell function for phenotyping purposes in genetic studies. The Botnia clamp provides a simple and reproducible tool for such purposes. In fact, the length of the clamp is the same as the FSIGT test, and the number of insulin samples can be reduced to five or six (0, 4, 6, 8, and 10 min and two samples at 150 and 180 min for checking the insulin levels during infusion). It also has the advantage that tracers can be added for estimating endogenous glucose production (EGP), although it remains to be shown whether the preceding glucose bolus affected the EGP during the Botnia clamp. Another advantage is that it can be combined with indirect calorimetry to obtain estimates of intracellular substrate partitioning.

We also applied the Botnia clamp to subjects with and without a family history of diabetes. As expected, the FH+ subjects were insulin resistant, whereas the unadjusted FPIR was similar in subjects with and without a family history of diabetes. However, when the degree of insulin resistance was taken into account (DI), the FH+ subjects demonstrated impaired β-cell function. Several studies have shown that first-degree relatives of patients with type 2 diabetes are insulin resistant (20–22), but few studies have shown the presence of a defect in insulin secretion if the subjects have had NGT (22–25). This is not surprising given the strong relation between insulin secretion and S_I; if both S_I and insulin secretion are impaired, there should be some degree of impaired glucose tolerance (2).

In conclusion, the Botnia clamp provides reliable and independent measures of S_I and β-cell function during the same test. As illustrated above, knowledge of the degree of S_I is important when presenting data on insulin secretion.

A computer program for the Botnia clamp is available on request, www.endo.mas.lu.se.

This study was supported by grants from the Sigrid Juselius Foundation, the Folkhälsan Research Foundation, the Juvenile Diabetes Foundation−Wallenberg, the Academy of Finland, the Swedish Medical Research Council, the Finnish Diabetic Research Foundation, the Swedish Diabetic Research Foundation, EEC GIFT, and the Novo Nordisk Foundation.

The skillful assistance by the Botnia Research group is gratefully acknowledged.