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Insulin and Proinsulin Dynamics Progressively Deteriorate From Within the Normal Range Toward Impaired Glucose Tolerance

Abstract Context Slight elevations in plasma glucose (PG) manifest in advance of diabetes onset, but abnormalities in immunoreactive insulin (IRI), proinsulin (Pro), and adiponectin dynamics during this stage remain poorly understood. Objective The objective of this work is to investigate whether IRI and Pro dynamics become abnormal as glucose tolerance deteriorates from within the normal range toward impaired glucose tolerance (IGT), as well as the relationship between PG, and these dynamics and serum adiponectin levels. Design A cross-sectional study was designed. Setting This study took place at Jichi Medical University in Japan. Participants and Measurements PG, IRI, and Pro levels were determined in 1311 young Japanese individuals (age < 40 years) with normal or IGT before and at 30, 60, and 120 minutes during a 75-g oral glucose tolerance test. Participants were assigned to 4 groups according to glucose tolerance, and then background factors, adiponectin levels, insulin sensitivity (SI), and insulin secretion (β) indexes were determined. Results PG levels as well as IRI and Pro levels 60 and 120 minutes after glucose-loading increased incrementally with deteriorating glucose tolerance. All measures of β and the SI measure index of insulin sensitivity (ISI)-Matsuda decreased incrementally. Serum adiponectin levels were not significantly different among the glucose tolerance groups, but were independently and negatively correlated with fasting glucose. Conclusions Early β decreased and postloading Pro levels became excessive in a progressive manner as glucose tolerance deteriorated from within the normal range toward IGT.

Glucose intolerance develops as a consequence of β-cell dysfunction and/or insulin resistance. Glucose tolerance is classified as normal glucose tolerance (NGT), impaired glucose tolerance (IGT), impaired fasting glucose (IFG), and diabetes mellitus (DM) according to plasma glucose (PG) levels during an oral glucose tolerance test (OGTT) [1]. Impaired glucose-stimulated insulin secretion (GSIS) is an indicator of β-cell dysfunction in the prediabetic categories of IGT and IFG [2,3]. High triglyceride (TG) levels, hypertension, and high body mass index (BMI) are also risk factors for prediabetes and diabetes, even in individuals with NGT [4]. We previously revealed that the disposition index, which is the product of insulin sensitivity (SI) and insulin secretion (β), decreases with increasing BMI in young Japanese individuals with NGT, and that β-cell dysfunction can manifest with increasing BMI even when glucose tolerance is normal [5]. However, in a population of mainly NGT individuals, whether SI and β deteriorate with worsening glucose tolerance has been poorly elucidated.
Slight elevations in PG manifest in advance of glucose intolerance [6], but the understanding of abnormalities in immunoreactive insulin (IRI) and proinsulin (Pro) dynamics in this stage remains insufficient. A recent study in Chinese individuals with NGT or IGT (but not IFG or DM) showed a delayed peak in Pro level with deteriorated glucose tolerance despite no abnormalities in insulin dynamic patterns, even in those with NGT following a 75-g OGTT [7]. However, that study had a small sample size and did not seem to sufficiently characterize the relationship between glucose tolerance and insulin and Pro dynamics during OGTT. Differences in Pro assay procedures and background factors, including age and ethnicity, can also affect this relationship.
Adiponectin is an insulin-sensitizing hormone secreted by adipocytes. High circulating levels of adiponectin reduce the risk of prediabetes and diabetes [8,9]. Serum adiponectin levels are low in individuals with visceral fat accumulation, insulin resistance, and diabetes [10,11]. Hypoadiponectinemia was also found to be related to impaired β and diabetes onset in the participants, including those with prediabetes [12,13]. It is unclear, however, whether serum adiponectin levels are related to PG, β, and SI in people with NGT.
In the present study, we used data from a large number of young Japanese individuals to test the hypothesis regarding whether insulin and Pro dynamics progressively become abnormal as glucose tolerance deteriorates from within the normal range toward IGT. We also investigated the relationship of serum adiponectin levels to PG, β, SI, and other parameters in this population.

A. Diagnosis of Glucose Tolerance
The criteria of the Japan Diabetes Society define NGT as a fasting PG of less than 110 mg/ dL and a 120-minute value of less than 140 mg/dL, IGT as a fasting PG of less than 110 mg/ dL and a 120-minute value greater than or equal to 140 mg/dL to less than 200 mg/dL, IFG as a fasting PG of greater than or equal to 110 mg/dL to less than 126 mg/dL, and DM as a fasting PG of greater than or equal to 126 mg/dL or a 120-minute value of greater than or equal to 200 mg/dL during a 75-g OGTT [1].

B. Participants
The participants were 1311 medical students of Jichi Medical University who were younger than 40 years and had NGT or IGT (but not IFG or DM) among the about 1400 medical students who underwent a 75-g OGTT from December 2002 to April 2015. The present study was approved by the ethics committee of Jichi Medical University (EKI 09-45). The participants were fully informed of the purpose of the study and gave written consent to participate. The participants were assigned into 4 groups according to their glucose tolerance (OGTT 120-minute value; PG120) (NGT0: PG120 < 100, n = 864; NGT1: 100 ≤ PG120 < 120, n = 331; NGT2: 120 ≤ PG120 < 140, n = 87, IGT: 140 ≤ PG120 < 200, n = 29, in mg/dL). Background factors, PG, insulin, SI, β, Pro, and adiponectin were determined.

C. Measurements and Calculation of Indices
PG was determined using a glucose oxidase assay, and insulin using an immunoradiometric assay for IRI (Insulin RIA Beads II; Yamasa), as described previously [5]. The manufacturer claims that there is little cross-reactivity with Pro in the immunoradiometric assay for IRI. Proinsulin was determined as intact Pro with the Intact-Proinsulin Assay (MLT Research, Ltd), a chemiluminescent immunoassay procedure, as described previously [14]. Interassay and intra-assay variability for insulin and intact Pro were less than 5% and 10%, respectively. Samples for insulin and intact-Pro analysis were frozen until immunoassays were performed at about 6-month intervals.
Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL), and adiponectin levels of the participants were determined using serum collected under fasting conditions. Total adiponectin concentrations were determined using an enzyme-linked immunosorbent assay kit (Otsuka Pharmaceutical). Sitting heart rate and blood pressure were measured after the participants had been at rest for 5 minutes.

D. Statistical Analysis
JMP version 5.1 (SAS Institute Inc) was used to conduct the statistical analyses. Most variables lacked a normal distribution. Values are shown as median (25th-75th percentile). The Wilcoxon or chi-square test was used to test for differences among the 4 groups. Because serum adiponectin levels are susceptible to the effects of sex [21], the participants were classified into the 4 groups according to glucose tolerance described earlier and further classified according to sex when evaluating adiponectin.
Multiple regression analysis was used to calculate regressions of the background factors for PG120. When conducting multiple regression analysis, factors potentially contributing to PG120 other than PG, insulin, SI, and β-that is, age, sex, BMI, blood pressure, heart rate, lipids, and family history of diabetes-were selected as explanatory factors. At this time, pairs of variables with a variation inflation factor greater than 2 (systolic and diastolic blood pressure) were considered to have multicollinearity, and the variable with a lower coefficient of correlation with PG120 (systolic blood pressure) was excluded.
PG, insulin, Pro, SI, β, and P/I were considered to constitute glucose-insulin-Pro profiles, and their correlation with serum adiponectin levels was tested using the Spearman rank correlation for bivariate analysis. Factors for which a significant correlation was identified were analyzed with multiple regression analysis after adjustment for background factors (age, sex, BMI, diastolic blood pressure, heart rate, lipids, family history of diabetes). In all statistical tests, a P value less than .05 was taken to indicate statistical significance.

A. Characteristics of Participants' Background Factors According to Glucose Tolerance
The participants' background factors according to glucose tolerance are shown in Table 1. The participants were young, and no significant differences in age, BMI, high-density lipoprotein cholesterol, or TG were seen among the glucose tolerance groups. Blood pressure and TC increased from NGT0 to NGT2, but tended to decrease in IGT. As glucose tolerance deteriorated, the proportion of men decreased (NGT0: 79%, NGT1: 73%, NGT2: 69%, IGT: 59%), heart rate increased (NGT0: 62 beats per minute [bpm], NGT1: 64 bpm, NGT2: 67 bpm, IGT: 69 bpm), and the proportion of participants with a family history of diabetes (in a first-or second-degree relative) increased (NGT0: 32%, NGT1: 39%, NGT2: 46%, IGT: 59%). No significant difference among the glucose tolerance groups was seen in the proportion with a family history of diabetes in a first-degree relative.
The SI and β values of the participants according to glucose tolerance are shown in Fig. 2. The SI measure ISI-Matsuda (Fig. 2B) and all β measures (Fig. 2D-2F) incrementally decreased with deteriorating glucose tolerance. Few significant differences in 1/HOMA-IR ( Fig. 2A) and 1/FIRI (Fig. 2C) were seen among the glucose tolerance groups.

C. Relationship Between Background Factors and 120-Minute Plasma Glucose Value
The results of multiple regression analysis of the background factors for PG120 are shown in Table 2. Multiple regression analysis revealed negative correlations of PG120 with the proportion of men and TG, and positive correlations with BMI, heart rate, TC, and family history of diabetes. The association between the 4 groups is linear.

D. Serum Adiponectin Levels According to Glucose Tolerance and Sex
Serum adiponectin levels according to glucose tolerance in all participants, women and men, are shown in Fig. 3. Serum adiponectin levels in the participants with NGT were significantly higher in women than in men (P < .0001 for NGT0, P < .0001 for NGT1, P < .001 for NGT2). Serum adiponectin levels in the participants with IGT tended to be higher in women than in men (P = .058). Serum adiponectin levels in all participants, women and men, were not significantly different among the glucose tolerance groups.

E. Relationship Between Serum Adiponectin Levels and Glucose-Insulin-Proinsulin Profiles
The results of the Spearman rank correlation analysis of serum adiponectin levels to glucoseinsulin-Pro profiles and multiple regression analysis following adjustment for background factors are shown in Table 3. In the Spearman rank correlation analysis, serum adiponectin levels were negatively correlated with some PG, IRI, Pro, and SI measures in OGTT, but not with measures of β. Serum adiponectin levels were independently and negatively correlated with PG0 in the multiple regression analysis. However, the other measures of PG, IRI, Pro, P/I, and SI, as well as β, were not independently correlated with serum adiponectin levels.

Discussion
Slight elevations in PG in NGT and prediabetes have been reported to be associated with the long-term risk of diabetes onset [6,22,23]. Elucidating the relationship between slight Figure 1. Glucose, insulin, proinsulin, and the proinsulin/insulin ratio according to glucose tolerance. Median bar graphs are shown for A, glucose; B, insulin; C, proinsulin; and D, the proinsulin/insulin ratio according to glucose tolerance. FIRI, fasting IRI; IGT, impaired glucose tolerance; IRI, immunoreactive insulin; NGT, normal glucose tolerance; NGT0, PG120 < 100; NGT1, 100 ≤ PG120 < 120; NGT2, 120 ≤ PG120 < 140, in mg/dL; PG, plasma glucose; P/I, proinsulin/insulin ratio; Pro, proinsulin. P values for the variables were determined by paired Wilcoxon test. *P is less than .05. elevations in PG and β-cell function in NGT and prediabetes is therefore important to clarify pathogenic factors for progression from NGT to diabetes. However, it is not well known whether slight elevations in PG are correlated with β-cell function or markers related to β-cell function in individuals with NGT. The present study showed that in a large sample of young Japanese participants, early β progressively decreases, and postloading Pro levels become excessive as glucose tolerance deteriorates from within the normal range toward IGT. Serum adiponectin levels were inversely correlated with fasting glucose. Chinese individuals with NGT or IGT showed no abnormalities in insulin dynamic patterns across the NGT categories of glucose tolerance, but the peak of Pro level was delayed [7]. In the present study, even participants with NGT had delayed and/or excessive Insulin sensitivity (SI) and β according to glucose tolerance. Median bar graphs are shown for A to C, SI indices, and D to F, β indices, according to glucose tolerance. Abbreviations are the same as those in Fig. 1 FIRI, fasting IRI; IGT, impaired glucose tolerance; IRI, immunoreactive insulin; NGT, normal glucose tolerance; NGT0, PG120 < 100; NGT1, 100 ≤ PG120 < 120; NGT2, 120 ≤ PG120 < 140, in mg/dL. P values for the variables were determined by paired Wilcoxon test. *P is less than .05.  Abbreviations: FIRI, fasting immunoreactive insulin; HOMA-IR, homeostasis model assessment of insulin resistance; IRI, immunoreactive insulin; ISI, index of insulin sensitivity; PG, plasma glucose; P/I, proinsulin/insulin ratio; Pro, proinsulin. Median bar graphs are shown for serum adiponectin levels according to glucose tolerance and sex. Abbreviations are the same as those in Fig. 1 IGT, impaired glucose tolerance; NGT, normal glucose tolerance; NGT0, PG120 < 100; NGT1, 100 ≤ PG120 < 120; NGT2, 120 ≤ PG120 < 140, in mg/dL. No differences in any comparison were significant. P values for the variables were determined by paired Wilcoxon test.
insulin and Pro levels as glucose tolerance deteriorated, but the peak of the Pro level was not delayed (Fig. 1C). In the previously mentioned study [7], those with NGT (OGTT 120min value < 120 mg/dL; equivalent to NGT0 or NGT1 in the present study) had a Pro peak at 60 minutes after glucose loading, but intact Pro levels in the present study continued to increase through 120 minutes after glucose loading, even in the NGT0 and NGT1 categories (Fig. 1C). Differences in the Pro assay procedures, age, or sample size may explain this disparity. In the present study, SI was used to evaluate systemic and hepatic insulin sensitivity, and β was used to evaluate GSIS. ISI-Matsuda and all β measures decreased with deteriorating glucose tolerance. It cannot be concluded from the earlier mentioned study [7] that β abnormalities did not manifest over the glucose tolerance categories of NGT because that study did not evaluate SI and β in detail, as in the present study.
Other studies have found that in people with prediabetes or diabetes, Pro levels increase as glucose tolerance deteriorates, and that high Pro levels precede diabetes onset [24][25][26]. Those studies, however, did not evaluate insulin and Pro dynamics in different glucosetolerance categories within NGT. Fasting Pro and the P/I ratio did not essentially differ across the NGT categories and IGT in the present study ( Fig. 1C and 1D). Postloading insulin and Pro levels became delayed and/or excessive as the NGT categories approached IGT ( Fig. 1B and 1C). P/I120 values from NGT1 to IGT were lower than that of NGT0 (Fig. 1D), whose PG120 level had returned to baseline (Fig. 1A). This is consistent with the results that P/I120 values were lower in IFG/IGT and diabetic participants than those in NGT [26]. These findings suggest that β exceeds Pro secretion in response to sustained postloading elevated glycemia in different NGT categories or in IGT, which seems to be a plausible response [27].
We also investigated the relationship of various clinical background factors to glucose tolerance (OGTT 120-minute value; PG120). As glucose tolerance deteriorated, the proportion of men decreased, and the proportion of participants with a family history of diabetes and resting heart rate increased. Multivariable analysis showed these factors to be independently correlated with PG120. Studies have associated sex and a family history of diabetes with glucose intolerance in participants, including those with IGT [28,29]. In those studies, IGT was more prevalent in women and those with a family history of diabetes, consistent with our findings. One possible explanation for the preponderance of women with IGT is assumed to be lower body mass and skeletal muscle mass in female participants [30]. A recent genome-wide assessment of the relationship between resting heart rate and the risk of diabetes onset found resting heart rate to be strongly associated with the onset of type 2 diabetes [31]. Consistent with that study, increased resting heart rate was associated with deteriorated glucose tolerance in the present study. Elevated resting heart rate in young Japanese individuals with NGT could therefore be a risk factor for future glucose intolerance.
We also investigated whether serum adiponectin levels were associated with glucoseinsulin-Pro profiles in our mainly NGT participants. As in previous studies, serum adiponectin levels were higher in women, although serum adiponectin levels across the glucose-tolerance categories showed no differences in all participants or in women or men. In bivariate analysis with glucose-insulin-Pro profiles and multivariable analysis adjusted for background factors, serum adiponectin levels were independently and negatively correlated with fasting glucose. This correlation between serum adiponectin levels and glucose-insulin-Pro profiles has often been examined in people with prediabetes or diabetes, but less often in those with NGT [32][33][34]. A study in a non-DM population found low serum adiponectin levels to be associated with IFG [33]. Consistent with that study, our findings suggest that low serum adiponectin levels could be associated with moderately elevated fasting glucose in non-IFG individuals consisting mainly of those with NGT. Other studies have associated hypoadiponectinemia with impaired β [12,13], but the present study found no association between serum adiponectin levels and impaired β in the stages of NGT or IGT.
Several limitations must be noted. Owing to the cross-sectional nature of this study, a direct causal relationship of β-cell function and Pro dynamics affecting glucose tolerance was not proven. However, the strength of the present study is that β-cell function and Pro dynamics were examined using a large sample of participants with common characteristics (mainly NGT and young). Abnormalities in insulin and Pro dynamics have been associated with aging [35], but no age differences were observed in this study across the different glucose-tolerance categories. Because we measured peripheral venous levels of insulin and Pro and the half-lives of those in serum are different, serum levels may not accurately reflect secretion rates. However, comparison of those levels at the same time during OGTT could allow us to estimate secretion abnormalities [17][18][19][20]. Because measurements were sparse and restricted within 120 minutes after glucose loading, the true insulin and Pro peak by differences in glucose tolerance may not be captured. Some of the participants in the present study could possibly experience glucose intolerance and develop diabetes in the future, and a comparative study between this study and disease onset may be of potential interest.
In conclusion, all measures of β and ISI-Matsuda decreased with deteriorating glucose tolerance, even within individuals with NGT. Delayed and/or excessive Pro and insulin levels and a decrease in the P/I ratio with deteriorating glucose tolerance are plausible responses to postloading elevated glycemia. The proportion of women, proportion of participants with a family history of diabetes, and elevated resting heart rate were associated with deteriorated glucose tolerance (ie, high PG120), and low serum adiponectin levels were associated with elevated fasting glucose. Insulin and Pro dynamics progressively became abnormal as glucose tolerance deteriorated incrementally from within the normal range toward IGT.