Surrogate measures of first-phase insulin secretion versus reference methods intravenous glucose tolerance test and hyperglycemic clamp: a systematic review and meta-analyses

Introduction

The global burden of diabetes was estimated to be 463 million people in 2019 and is steadily rising.1 The key pathomechanisms of type 2 diabetes are impaired insulin sensitivity combined with a progressive loss of pancreatic beta-cell function. Longitudinal studies have shown that beta-cell function is altered many years before the diabetes diagnosis.2 With the increasing recognition of disease heterogeneity, insulin secretion has been used as a key feature in the subclassification of pre-diabetes and diabetes.3 In one recent work, adult-onset diabetes has been classified into five clusters, each of which features different pathophysiologic phenotypes and distinct patterns of complication.4 In this approach, a reliable measurement of insulin secretion is important to differentiate insulinopenic and hyperinsulinemic diabetes endotypes. In pre-diabetes subphenotyping, adequate methods for measurement of insulin secretion are similarly important for capturing key differences between pre-diabetes subphenotypes.

In healthy individuals, insulin is secreted in a biphasic manner in response to an increase in arterial glucose concentration. The first phase lasts approximately 10 min, and the second phase reaches a plateau after 2–3 hours, provided glucose is continuously elevated, in an experimental setting.5 In type 2 diabetes, the first phase of insulin secretion is impaired or even absent and the second phase is decreased.6 The first phase of insulin secretion is frequently used in pathophysiological studies of type 2 diabetes and the prediction of type 1 diabetes.7 Homeostatic model assessment and beta cell function (HOMA-beta) is used to estimate insulin secretion for the classification of diabetes and can have importance in clinical settings for optimizing diabetes treatment.

There are numerous methods to assess first-phase secretion; of these the hyperglycemic clamp (HGC) test and intravenous glucose tolerance test (IVGTT) are referred to as the gold standards.8 Gold-standard methods are used in smaller populations for an accurate measurement of secretion, often in combination with an examination of insulin resistance in the same protocol. The HGC test allows assessment of both first-phase and second-phase insulin secretion, whereas the IVGTT is a reference test only for first-phase insulin secretion.

During the HGC test, a glucose bolus is given to quickly raise glucose levels to a fixed hyperglycemic target value, usually 125 mg/dL (6.9 mmol/L) above basal level.9 The elevated plasma glucose concentration is then maintained by continuous glucose infusion. Blood samples are taken frequently using a catheter to measure blood glucose, and the glucose infusion is adjusted as necessary. The first phase of insulin secretion is calculated from insulin levels during the first 8–10 min, and the second phase is calculated as the mean insulin value after the initial secretion peak.9

When performing an IVGTT, only one glucose bolus is given intravenously, and blood samples are collected for 3–4 hours. The most commonly used parameter is acute insulin response (AIR), which is the mean insulin concentration above basal levels during the first peak.10

The two reference methods are used to calculate the first phase of insulin secretion in similar ways and research settings. During the HGC test an amount of glucose, which is based on the participant’s level of glucose, is given to raise insulin secretion. In an IVGTT the bolus given is fixed. The difference between the reference methods is more evident in the second phase when an infusion of glucose is used in the HGC test. In both methods, the first phase is calculated from the insulin levels during the first minutes. Because both reference methods measure insulin secretion as a response to intravenous glucose administration, they cannot assess all aspects of physiological insulin secretion when food is consumed. Both reference methods are also time-consuming to perform. Therefore, several surrogate measures have been developed for broader use. Surrogate measures are used in a wide range of epidemiological studies wherein associations between insulin secretion and conditions related to diabetes are investigated. Since the oral glucose tolerance test (OGTT) is frequently used for the classification and diagnosis of diabetes and pre-diabetes, the additional blood samples taken to calculate a surrogate measure of insulin secretion are relatively inexpensive and time-efficient to collect.

The most frequently used surrogate measures of insulin secretion are based on the OGTT, which is performed after an overnight fast. Subjects are given an oral load of 75 mg glucose, which triggers insulin secretion.8 Plasma insulin or C-peptide and glucose levels are measured at baseline and repeatedly for at least 120 min.

HOMA-beta is based on a fasting blood sample and assesses insulin secretion by calculating the ratio of insulin concentration to glucose concentration minus 3.5 mmol/L.8

Although there are numerous surrogate indices for the first phase of insulin secretion, no systematic review has summarized the validation studies of surrogate indices. This study investigated the diagnostic accuracy of surrogate measures by assessing the correlation between first-phase insulin secretion calculated using surrogate methods and the reference methods IVGTT and HGC. This was undertaken to help researchers decide which surrogate measure to use for larger studies.

Method

We performed a systematic review and meta-analyses according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, 2020.11 Before we started, our meta-analysis was registered at PROSPERO.

Data synthesis and analysis

In this paper, we included all surrogate measures that have been validated against first-phase insulin secretion measured using either the HGC test or IVGTT in at least three studies (figure 1). Surrogate measures validated in two studies are also presented in the meta-analysis (online supplemental figure 2). Correlation coefficients published in only one paper are presented in online supplemental table 3. No surrogate measures from meal-tolerance tests were validated in more than two studies and these are therefore only presented in online supplemental table 3.

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Figure 1

Meta-analyses of correlation coefficients between surrogate measures of insulin secretion and the reference methods intravenous glucose tolerance test (IVGTT) and the hyperglycemic clamp (HCG). CF, Cystic Fibrosis; CFRD, Cystic Fibrosis Related Diabetes; HOMA, homeostatic model assessment; IGT, impaired glucose intolerance; NGT, normal glucose tolerance; T2D, Type 2 Diabetes.

The main analysis, involved random-effects meta-analysis, because the characteristics of the participants differed between the studies.14 We also conducted fixed-effects meta-analysis as sensitivity analysis, because the sizes of the studies differed significantly and a random-effects meta-analysis may give smaller studies too much relative weight.

We used the program Comprehensive Meta-Analysis, (V.3.3 Biostat, Englewood, New Jersey, USA) which uses inverse variance weights and estimates the between-study variance τ² using the DerSimonian and Laird method.15 The correlation coefficients are transformed to Fischer’s Z before the meta-analysis. The SE of Fischer’s Z metric is determined solely by sample size rather than by the size of the correlation, to avoid larger correlations being assigned more weight. Fischer’s Z-values are then retransformed to correlation coefficients for the graphical presentation. The risk ratio for the binary outcomes was calculated with a 95% CI and a two-sided p value.

To avoid bias we converted Spearman’s ρ to Pearson’s r according to Rupinski and Dunlap’s formula r=2sin(ρ×π/6).16 When the type of correlation coefficient was not specified in two studies, we contacted the first authors. When we did not receive an answer, we assumed that it was reported as Pearson’s r. Thus, all correlation coefficient presented in this article are reported as Pearson’s r.

Although all included studies presented the relationship between the reference method and the surrogate measure in terms of first-phase insulin secretion, the first phase was defined in different ways. For example, the first phase is defined as the first 5 min by Coppack et al and the first 3 min by Philips et al; however, in most studies, it is defined as the first 10 min. The methods are described in detail for each study in online supplemental table 1. We performed a separate analysis to assess the impact of different methods of calculating first-phase insulin secretion on the correlation with the surrogate measures Insulinogenic Index 30 (IGI30) and HOMA-beta (online supplemental figure 4). We also conducted meta-analyses of subgroups (NGT, IGT, and type 2 diabetes) for surrogate measurements with at least two studies with separate correlation coefficients for each subgroup (online supplemental figure 3).

Results

Our initial search resulted in 12,555 titles; and 33 articles were used for the final analysis (online supplemental figure 1). Eight of the studies presented no baseline participant characteristics. The authors of these articles were contacted to retrieve information about age, sex, BMI and glycemic status; however, after a month no reply had been received from seven of these authors. Therefore, we compared the studies with missing data to the studies with complete information in a separate analysis. This is shown in online supplemental figure 5, and there was no significant difference between studies with missing data as compared with studies with complete data for HOMA-beta and IGI(30). In addition to the included articles, at one center a separate analysis was undertaken using data from two populations; 1 of 309 participants who underwent both IVGTT and OGTT and 75 participants who underwent both HGC and OGTT.20

The characteristics of the included studies are presented in online supplemental table 1, and the formulae of the included indices are shown in table 1. This systematic review presents surrogate measures that have been compared with first-phase insulin secretion reference methods in at least three published articles (figure 1). Surrogate measures that have been compared with first-phase insulin secretion reference methods, in two studies are presented in online supplemental figure 2. Surrogate measures that have been compared with first-phase insulin secretion reference methods in only one paper are listed in online supplemental table 2.

Comparison of surrogate and reference measures of first-phase insulin

We found at least three validation studies for the surrogate measures HOMA-beta, IGI(30) Insulin30, Insulin 120, AUC30Insulin/AUC30Glucose, Stumvoll’s first-phase secretion, Stumvoll’s second-phase secretion, BIGTT0-60-120, BIGTT0-30-60, Kadowaki, AUC120Insulin/AUC120Glucose and Beta-index (figure 1). All surrogate measures validated in at least three studies are calculated from an OGTT, except HOMA-beta. The surrogate measures with the strongest correlation to reference methods were Kadowaki (0.67 (95% CI 0.61 to 0.73)) and Stumvoll’s first-phase secretion (0.65 (95% CI 0.58 to 0.71)) (figure 2). These measures were only validated in three respectively seven studies. All five surrogate measures with the highest correlation are based on the first 30 min of an OGTT. In addition, Stumvoll’s second phase is based on the first 30 min of an OGTT (table 1), and was compared with the first phase calculated using a reference method. HOMA-beta and IGI(30) were the surrogate measures validated in most studies, (17 respectively 13 studies) and their pooled correlations to reference methods are 0.48 (95% CI 0.40 to 0.56) and 0.61 (95% CI 0.54 to 0.68) respectively (figure 1A).

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Figure 2

Summary of all meta-analyses of this systematic review. This figure summarizes the results of the 12 meta-analyses reported in figure 1A–L. In this figure, one row shows the results of one meta-analysis of figure 1; for example, the row “HOMA-beta” describes the results from the meta-analysis of figure 1A. The rows of this figure are ordered by the strengths of the pooled correlations, for example, the surrogate measure “Kadowaki” has the highest pooled correlation (0.672) to the gold standard examinations and the surrogate measure “Insulin 120” has the lowest pooled correlation (0.318) according to the results reported in figure 1A–L. Pooling all meta-analyses together shows that the correlations of the different surrogate measures to the gold standard examination differ substantially from each other with a heterogeneity I² of 95%. AUC, area under the curve; BIGTT, oral glucose tolerance test-based estimate of insulin secretion; HOMA, homeostatic model assessment; IGI, Insulinogenic Index.

All meta-analyses included in figure 1 are summarized with their correlations and CIs in figure 2. The figure shows that the surrogate measure Kadowaki has the highest pooled correlation to the first phase of insulin secretion according to gold standard examinations and the surrogate measure “Insulin 120” has the lowest pooled correlation. Figure 2 shows that the correlations of the different surrogate measures to the gold standard examination differ greatly from each other which results in a heterogeneity I² of 95%.

Discussion

We performed a systematic review in order to find the most appropriate surrogate method for measuring first-phase insulin secretion, with a view to this being used in epidemiological studies. The surrogate measures with the highest correlation to the reference methods are Kadowaki and Stumvoll’s first-phase secretion. However, the most frequently validated surrogate measures are IGI(30) and HOMA-beta.

In our attempt to find a surrogate measure for the first phase of insulin secretion, we found that indices with the highest correlations to the reference methods are all based on the first 30 min of the OGTT, as is for the case of, for example, Kadowaki, Stumvoll’s first-phase secretion and IGI(30). Kadowaki is the surrogate measure with the highest correlation, but it is not as frequently used and was only validated in three studies. Like Stumvoll’s first-phase secretion, one of the surrogate measures with the highest correlation, it is based on the increment of insulin and the level of glucose after 30 min during an OGTT. Both Kadowaki and Stumvoll’s first-phase secretion are less frequently used, and fewer studies and participants validate these measures compared with IGI(30), which is similar, and had only a slightly lower pooled correlation to the gold standard reference measure.

Insulin secretion is measured mainly in research and there is no cut-off to separate healthy and diseased individuals. Although it is not used as a diagnostic tool in clinical settings an accurate measurement of insulin secretion using surrogate measures is important in order to classify individuals into different levels of diabetes development and help patients with type 2 diabetes in different clusters access more specific treatment and obtain a better understanding of the pathophysiology.

As expected, the surrogate measure with the lowest correlation to the first phase of insulin secretion is Insulin120, which is measured later in the OGTT. There are substantial differences between the reference methods and surrogate measures. With the gold standard examinations, the first phase of insulin secretion is measured during minutes after the intravenous glucose bolus. Such a direct examination of the first phase is not possible, neither with indices based on the OGTT or with those based on fasting samples. Therefore, this systematic review can only examine the association between surrogate indices and the direct measurement of the first phase. We find correlation coefficients that are moderate at the most.

However, surrogate indices after the OGTT may reflect a more physiological insulin responses. Moreover, OGTT-based surrogate measures are influenced by additional factors that do not contribute to the response to intravenous glucose. For example, the incretin effect amounts to a twofold to threefold increase in secreted insulin in response to oral glucose as compared with intravenous glucose administration.25 The measurements based on the OGTT could therefore provide a good estimate of physiological insulin secretion and still only having a modest correlation to the IVGTT. This applies also to measures based on a meal tolerance test; unfortunately, we had too little data to compare those in our analyses. The meal tolerance test not only mimics the uptake of glucose but also takes into account the variety of nutrients in a normal meal. The correlation to the reference method, IVGTT, could be low and the surrogate index may still be a closer estimate of physiological insulin secretion.

In type 2 diabetes the incretin effect is reduced compared with NGT individuals.26 This could result in substantially lower insulin secretion when measured during an OGTT. Other confounding factors are variation in gastric emptying and intestinal absorption of glucose. Variation in gastric emptying has been shown to account for 35% of the variance in peak blood glucose levels at the end of an OGTT for both healthy people and patients with type 2 diabetes. However, most of the surrogate measures involving the OGTT use the insulin-to-glucose ratio, and therefore the impact of gastric emptying should be reduced.

HOMA-beta was the surrogate measure that was validated in the most studies and is an index that captures another aspect of insulin secretion. Unlike the surrogate measure involving the OGTT, it is based on a single fasting blood sample and is not specifically intended to measure the first phase of insulin secretion. It is used as a non-specific measure of insulin secretion, without any information about an acute stimulation of beta cells and can thus be considered a measure of steady-state insulin secretion during fasting. In a recent cluster analysis aimed at reclassifying adult-onset diabetes, insulin secretion was estimated using HOMA2-beta, a computer-estimated version of HOMA, using either insulin or C-peptide in a fasting state, both of which are available in many laboratories and can be used in routine healthcare (27–31). In this systematic review, it is evident that HOMA-beta has a consistently good correlation to first-phase insulin secretion even when calculated using insulin. In addition, this correlation had the lowest heterogeneity across the analyzed studies. This encourages the use of HOMA-beta as an estimate of insulin secretion in a clinical setting, even though it is a measure of steady-state insulin secretion and should not be interpreted as a measure of the first phase.

Diabetes status could in theory influence the correlation between the reference and surrogate measure, but this is not supported by our data on type 2 diabetes and pre-diabetes as compared with that on NGT. Only a few studies reported stratified correlations for pre-diabetes. On the other hand, there is a low heterogeneity of correlation coefficients in pre-diabetes across the examined studies. In this meta-analysis it is shown that IGI(30), Stumvoll’s first-phase secretion and HOMA-beta all have consistent correlation coefficients to reference methods for pre-diabetes. It is especially important to accurately capture variation in insulin secretion for pre-diabetes because early dysfunction can be present without a profound change in glucose levels.27

The high heterogeneity in the majority of our meta-analyses can partially be explained by different methods of measuring and calculating insulin secretion in the reference methods. Insulin secretion expressed as AIR10min (insulin secretion during the first 10 min calculated as the AUC) was the most common reference. There are other possible reasons for the high heterogeneity in our analyses, including differences in age, sex, BMI, diabetes duration, fasting glucose, Hemoglobin A1c (HbA1c), treatment and technical differences. We could not stratify our analyses on these factors, since the included studies did not report correlations by these groups. There was also a large variation in intra-individual reproducibility among indices as regards measuring insulin secretion. Intra-individual variability is generally higher for insulin-based indices than for C-peptide-based measurements,28 which is also reflected by a high heterogeneity across surrogate measures of insulin resistance.29 All of the studies in our meta-analysis used the same insulin measurement assays for both surrogate and reference measurements, which argues against increased variation originating from a technical variation across different immunoassays.30 A further source of variation with insulin-derived measures could be a considerably shorter plasma half-life of insulin as compared with C-peptide combined with a physiologically pulsatile insulin secretion.24

This review of the correlation between reference and surrogate measures of first-phase insulin secretion is the largest to have been conducted; it includes comparative work and presents all available data. The screening of articles and data extraction was performed by two researchers separately to reduce inclusion bias.

One weakness of our meta-analyses is the small number of studies that compare surrogate and reference methods. While there was a larger number of studies available for HOMA-beta and IGI(30) (17 and 13, respectively), there were too few articles available for many other indices to conduct a meta-analysis. We decided not to include Embase database in our search since the database has a focus on pharmacovigilance; thus, there may have been validation studies that we did not include in our meta-analysis. In line with the Cochrane handbook we decided that the first step of the literature search was done by one author only.12 This is a possible limitation because of the risk of missing potential studies. There are other methodological limitations regarding the search strategy, the information about the breakdown for each database and the number of duplicates was not saved. This is of course an obstacle for verification of the method.

Another limitation is the different ways of calculating insulin secretion during its first phase. It would be useful for epidemiological studies to know which surrogate measure to use for different glycemic groups, for example, IGT and NGT. Because the first phase is blunted early in the natural history of type 2 diabetes it can be measured and used as a prognostic index, and it would therefore be interesting to restrict the analysis to participants with pre-diabetes. However, too few studies investigated subgroups with specific clinical characteristics to allow a conclusion to be drawn. The statistical analysis plan was not predefined in detail, which we consider in retrospect a limitation.

In this systematic review, validation studies that compare surrogate and reference measures of first-phase insulin secretion are summarized. The indices with the highest correlation to reference methods are Kadowaki and Stumvoll’s first-phase secretion. This shows that measurements of insulin and glucose during the first 30 min of an OGTT are sufficient for a reliable estimate of insulin secretion. But the correlations between surrogate measures and the reference examination for first-phase insulin secretion are modest at the most, which is explained by the reference method being an intravenous test. However, in epidemiological studies, only surrogate measures are affordable and may even present a more physiological measure. The most validated surrogate measures are HOMA-beta based on one fasting blood sample, and IGI(30) measured 30 min after an OGTT; the first being an estimate of basal insulin secretion and the latter a measure of early secretion after an oral glucose bolus.