Insulin sensitivity, and b-cell function in relation to hemoglobin A1C

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1 Nutrition, Metabolism & Cardiovascular Diseases (2014) 24, 27e33 Available online at journal homepage: Insulin sensitivity, and b-cell function in relation to hemoglobin A1C M.A. Marini a, S. Frontoni a, E. Succurro b, F. Arturi b, A. Sciacqua b, M.L. Hribal b, F. Perticone b, G. Sesti b, * a Department of Systems Medicine, University of Rome-Tor Vergata, Italy b Department of Medical and Surgical Sciences, University Magna Graecia of Catanzaro, Italy Received 17 November 2012; received in revised form 31 December 2012; accepted 25 January 2013 Available online 16 April 2013 KEYWORDS A1C; Insulin sensitivity; Insulin secretion Abstract Background and aims: The A1C diagnostic criterion for identifying individuals at increased risk for diabetes, introduced by the American Diabetes Association in 2010, was not defined on the basis of the principal pathophysiological abnormalities responsible for the development and progression of type 2 diabetes; we therefore wished to gain a deeper insight into the metabolic abnormalities characterizing the group of at risk individuals with an A1C value of 5.7e6.4%. Methods and results: As many as 338 non-diabetic offspring of type 2 diabetic patients were consecutively recruited. Insulin secretion was assessed using both indexes derived from oral glucose tolerance test (OGTT), and intravenous glucose tolerance test (IVGTT). Insulin sensitivity was measured by hyperinsulinemic euglycemic clamp. As compared with subjects with A1C <5.7%, individuals with A1C of 5.7e6.4% exhibited lower insulin sensitivity after adjusting for age, gender and body mass index (BMI). Insulin secretion estimated from the OGTT, did not differ between the two groups. By contrast, as compared with subjects with A1C <5.7%, the acute insulin response (AIR) during an IVGTT and both IVGTT-derived and OGTT-derived disposition indexes were reduced in individuals with A1C of 5.7e6.4% after adjusting for age, gender and BMI. As A1C increased to 5.7%, a sharp decrease in insulin sensitivity and b-cell function, measured as disposition index, was observed. Conclusions: Caucasian individuals with A1C 5.7% exhibit both core pathophysiological defects of type 2 diabetes i.e. insulin resistance and b-cell dysfunction. ª 2013 Elsevier B.V. All rights reserved. Abbreviations: AIR, acute insulin response; ADA, American Diabetes Association; AUC, area under the curve; BMI, body mass index; EASD, European Association for the Study of Diabetes; FPG, fasting plasma glucose; A1C, hemoglobin A1C; HOMA, homeostasis model assessment; IFG, impaired fasting glucose; IGT, impaired glucose tolerance; IDF, International Diabetes Federation; IVGTT, intravenous glucose tolerance test; M FFM, milligrams per minute per kilogram fat-free mass; OGTT, oral glucose tolerance test; PG, plasma glucose. * Corresponding author. Dipartimento di Scienze Mediche e Chirurgiche, Viale Europa, Catanzaro, Italy. Tel.: þ ; fax: þ address: sesti@unicz.it (G. Sesti) /$ - see front matter ª 2013 Elsevier B.V. All rights reserved.

2 28 M.A. Marini et al. Introduction Hemoglobin A1C (A1C) is an integrated measure of mean glycemia over the preceding 8e12 weeks, and is largely employed for monitoring metabolic control in subjects with diabetes [1]. In 2009, an International Expert Committee including representatives of the International Diabetes Federation (IDF), the American Diabetes Association (ADA), and the European Association for the Study of Diabetes (EASD) recommended the adoption of the A1C test for the diagnosis of diabetes with a cut-off of 6.5% in addition to the criteria based on either fasting plasma glucose (FPG) 126 mg/dl or 2-h 75-g oral glucose tolerance test (OGTT) plasma glucose (2-h PG) value 200 mg/dl [2]. This cut-off was mainly derived from an appraisal of epidemiological studies evaluating the association between A1C values and risk of retinopathy, a micro-vascular complication strongly related to chronic hyperglycemia [2]. In 2010, the ADA adopted this criterion for the diagnosis of diabetes, and revised the criteria for the diagnosis of increased risk for diabetes (pre-diabetes) with the inclusion, in addition to the preexisting groups of subjects with impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT), of the group of individuals with an A1C value ranging from 5.7% to 6.4% [3]. This cut-off was mainly derived from analyzes of prospective studies evaluating the association between A1C values and the future risk of type 2 diabetes [4]. However, the A1C diagnostic criterion for identifying individuals at increased risk for diabetes was not defined on the basis of the principal pathophysiological abnormalities responsible for the development and progression of type 2 diabetes, i.e. impaired tissue sensitivity to insulin and failure of pancreatic b-cells to compensate for the enhanced insulin demand. To gain a deeper insight into the metabolic abnormalities characterizing at risk individuals with an A1C value of 5.7e6.4%, we employed the hyperinsulinemiceeuglycemic clamp technique to evaluate insulin sensitivity as well as both OGTT and intravenous glucose tolerance test (IVGTT) to achieve a detailed analysis of b-cell function in a cohort of non-diabetic offspring of type 2 diabetic subjects. Methods The study group included 338 non-diabetic offspring consecutively recruited at the Department of Systems Medicine of the University of Rome-Tor Vergata and at the Department of Medical and Surgical Sciences of the University of Catanzaro-Magna Graecia [5]. All individuals were Caucasian and had one parent with type 2 diabetes and the other with no history of type 2 diabetes and a normal response to an OGTT. No subject was taking any medication know to affect glucose metabolism. After 12-h fasting, all individuals underwent anthropometrical evaluation including body composition evaluated by bioelectrical impedance, and a 75 g OGTT was performed with 0, 30, 60, 90 and 120 min sampling for plasma glucose and insulin. All non-diabetic individuals (FPG <126 mg/dl, 2 h post-load <200 mg/dl and A1C <6.5%) were characterized according to a previously validated protocol [5,6]. Briefly, after 12-h fasting, the subjects underwent an IVGTT and a hyperinsulinemic euglycemic clamp. After baseline blood collection, a bolus of glucose (300 mg/kg in a 50% solution) was given (within 30 s) into the antecubital vein to acutely increase blood glucose levels. Samples for the measurement of blood glucose and plasma insulin were drawn at 2, 4, 6, 8, 10, 20, 30, 40, 50, and 60 min. At 60 min after the glucose bolus, a continuous insulin infusion was initiated at the rate of 40 mu/m 2 of body surface area per min, after a priming dose, in order to reach and maintain a steady state plasma insulin of 85 9 mu/ml. Plasma glucose was assessed at 5 min intervals during the 2-h clamp study by a glucose analyzer. In the study subjects, mean plasma glucose concentration during the last hour of the clamp was 92 4 mg/dl. The study was approved by Institutional Ethics Committees and informed consent was obtained from each subject in accordance with principles of the Declaration of Helsinki. Analytical determinations A1C was measured with high performance liquid chromatography using a National Glycohemoglobin Standardization Program (NGSP) certified automated analyzer (Adams HA HbA1C analyzer, Menarini, Italy) (normal range 4.3e5.9%). Plasma insulin concentration was determined by a chemiluminescence-based assay (Immulite Ò, Siemens, Italy). Glucose, triglyceride, total and HDL-C concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). Calculation Glucose disposal (M) was calculated as the mean rate of glucose infusion measured during the last 60 min of the clamp examination (steady-state) and is expressed as milligrams per minute per kilogram fat-free mass (M FFM ) measured with the use of electrical bioimpedance. Acute insulin response (AIR) during the IVGTT was calculated as the incremental area under the curve (AUC) for insulin during the first 10 min of the IVGTT using the trapezoidal rule. Two indexes of insulin secretion were calculated from the OGTT data: the incremental area under the curve of insulin (DIns[AUC]) was divided by the incremental area under the curve of glucose (DGluc[AUC]) during the 0- to 30-min of the OGTT as a measure of early insulin secretion, or by the DGluc[AUC] during the 0- to 120-min of the OGTT as a measure of total insulin secretion [7,8]. To evaluate b-cell function, two so-called disposition indexes were calculated as follows: AIR M FFM (IVGTT-derived disposition index) or DIns(AUC 0e120)/DGluc(AUC 0e120) M FFM (OGTT-derived disposition index) [9]. Statistical analysis Continuous variables are expressed as means SD. Categorical variables were compared by c 2 test. Metabolic differences among groups were tested after adjusting for gender, age, and BMI using a general linear model with post hoc Bonferroni correction for multiple comparisons. A P value <0.05 was considered nominally significant. All

3 A1C, insulin sensitivity, and b-cell function 29 analyzes were performed using SPSS software Version 16.0 for Windows. Results A total 77 of 338 (22.8%) subjects were at increased risk for diabetes according to ADA criteria (A1C 5.7e6.4%). Table 1 shows the metabolic characteristics of the two study groups i.e. subjects with A1C <5.7%. vs. subjects with A1C of 5.7e6.4%. Significant differences between the two groups were observed with respect to age (subjects with A1C of 5.7e6.4% were older than subjects with A1C <5.7%), and BMI (subjects with A1C of 5.7e6.4% were heavier than subjects with A1C <5.7%). A tendency toward a higher prevalence of men in the group with A1C of 5.7e6.4% was also observed. Therefore, all analyzes were adjusted for age, gender, and BMI. As expected, individuals at increased risk for diabetes (A1C 5.7e6.4%) had significantly higher A1C, FPG, and 2-h post-load plasma glucose and triglycerides levels as compared with subjects with A1C <5.7% (Table 1). A significant reduction in peripheral insulin sensitivity, evaluated by the hyperinsulinemic euglycemic clamp, was observed in individuals with A1C of 5.7e6.4% as compared with subjects with A1C <5.7% (P < after adjusting for age, gender, and BMI). Total insulin secretion during the 120-min OGTT, measured as DIns(AUC)/DGluc(AUC), and early insulin secretion, estimated as DIns AUC 0e30/DGluc AUC 0e30 did not differ between the two groups of individuals. By contrast, as compared with subjects with A1C <5.7%, the AIR during an IVGTT, a direct measure of insulin secretion from b-cells, was nominally reduced in individuals with A1C of 5.7e6.4% (P < 0.02 after adjusting for age, gender, and BMI). Since insulin secretion is dependent on actual insulin sensitivity, we compared the disposition indexes, derived either from the IVGTT or from the OGTT data in the two groups of subjects. Both disposition indexes were significantly lower in individuals with A1C of 5.7e6.4% as compared with subjects with A1C <5.7% (Table 1). After further adjustment for BMI in addition to age, and gender, both the IVGTT-derived and the OGTT-derived disposition indexes were significantly reduced in individuals with A1C of 5.7e6.4% (P < , and P Z 0.004, respectively). Stratification according to gender did not change results. Thus, men with A1C of 5.7e6.4% had lower insulinstimulated glucose disposal ( mg/min Kg FFM) as compared with men with A1C <5.7% ( mg/ min Kg FFM; P < after adjusting for age) as well as reduced IVGTT-derived disposition index ( vs for men with A1C <5.7% and 5.7e6.4%, respectively; P Z after adjusting for age). Accordingly, women with A1C of 5.7e6.4% had lower insulinstimulated glucose disposal ( mg/min Kg FFM) as compared with women with A1C <5.7% ( mg/ min Kg FFM; P < after adjusting for age) as well as reduced IVGTT-derived disposition index ( vs for women with A1C <5.7% and 5.7e6.4%, respectively; P Z after adjusting for age). Next, we stratified subjects with A1C <5.7% and those with A1C of 5.7e6.4% into two groups based on glucose tolerance during the OGTT: i) Normal glucose tolerant Table 1 Anthropometric and metabolic characteristics of the study subjects stratified according to A1C value. Variables Subjects with A1C <5.7% Subjects with A1C 5.7e6.4% P n (Male/Female) 261 (91/170) 77 (35/42) 0.09 Age (yr) <0.0001* BMI (kg/m 2 ) <0.0001** A1C (%) < Fasting glucose (mg/dl ) < h glucose (mg/dl) < Total cholesterol (mg/dl) HDL (mg/dl) Triglycerides (mg/dl) SBP (mmhg) DBP (mmhg) DInsAUC 0e30/DGluAUC 0e DInsAUC 0e120/DGluAUC 0e AIR (mu/ml min) Insulin-stimulated glucose disposal < (mg/min Kg FFM) Disposition index (AIR M FFM ) < Disposition index (DIns[AUC 0e120]/DGluc[AUC 0e120] M FFM ) IFG and/or IGT (%) 48 (18.4%) 41 (53.2%) < Data are means SD. Comparisons between the two groups were performed using a general linear model. P values refer to results after analyzes with adjustment for age, gender, and BMI. *P values refer to results after analyzes with adjustment for gender. **P values refer to results after analyzes with adjustment for age and gender. Categorical variables were compared by c 2 test. IFG Z Impaired fasting glucose; IGT Z Impaired glucose tolerance; AIR Z acute insulin response during IVGTT; SBP Z systolic blood pressure; DBP Z diastolic blood pressure.

4 30 M.A. Marini et al. (NGT) having fasting plasma glucose (FPG) < 100 mg/dl and 2-h plasma glucose <140 mg/dl; and ii) IFG and/or IGT having FPG Z 100e125 mg/dl or 2 h plasma glucose Z 140e199 mg/dl, and compared the metabolic characteristics of the four groups of individuals (Table 2). In the group of subjects having A1C <5.7%, IFG/IGT individuals exhibited significantly lower insulin-stimulated glucose disposal and IVGTT-derived disposition index as compared with NGT subjects. These differences remained significant after adjusting for age, gender, and BMI using a general linear model with post hoc Bonferroni correction for multiple comparisons. Accordingly, NGT subjects with A1C of 5.7e6.4% exhibited significantly lower insulin-stimulated glucose disposal and IVGTT-derived disposition index as compared with NGT subjects with A1C <5.7% even after adjusting for age, gender, and BMI. Then, all subjects were divided into categories based on 0.1% differences in A1C levels, and insulin sensitivity, OGTT-derived insulin secretion indexes, AIR, and disposition indexes were related to A1C values. Whole-body insulin sensitivity, measured with the hyperinsulinemic euglycemic clamp, remained unchanged up to an A1C value of 5.6%. However, as A1C increased to a value 5.7%, a sharp decrease in insulin sensitivity was observed (Fig. 1A). Insulin secretion during an OGTT, as measured by DIns(AUC 0e120)/DGluc(AUC 0e120) (Fig. 1B) and DInsAUC 0e30/DGluAUC 0e30 (Supplementary Fig. 1) indexes, did not change significantly up to A1C >6%. First phase insulin secretion measured as AIR during an IVGTT was nominally reduced as A1C increased to a value 5.9% (Supplementary Fig. 3). By contrast, the IVGTT-derived disposition index remained unchanged up to an A1C value of 5.6%, but it decreased as A1C increased to a value 5.7% (Fig. 1C). Discussion ADA has recently revised criteria for the diagnosis of type 2 diabetes and the categories at increased risk for diabetes, already including IFG and IGT, recommending the use of A1C assay as another diagnostic test option in addition to FPG or 2-h PG glucose values. The ADA criteria identify as individuals with high risk for future diabetes those with A1C ranging from 5.7 to 6.4% [3]. In the present study, we provide evidence that Caucasian individuals with A1C of 5.7e6.4% already exhibit abnormalities of the two main pathophysiological defects responsible for the development of type 2 diabetes, i.e. insulin resistance and b-cell dysfunction. We found that, as compared with subjects with A1C <5.7%, individuals with A1C of 5.7e6.4% exhibited a significant decrease ( 31%) in insulin sensitivity evaluated by Table 2 Anthropometric and metabolic characteristics of the study subjects stratified according to A1C value and glucose tolerance (NGT, IFG and/or IGT). Variables A1C <5.7% A1C 5.7e6.4% P NGT IFG and/or IGT NGT IFG and/or IGT n (%) 213 (63.0) 48 (14.2) 36 (10.7) 41 (12.1) Male/Female 65/148 26/22 14/22 21/ Age (yr) 34 10zzz*xxx xx <0.0001# BMI (kg/m 2 ) **xxx < A1C (%) ***xxx ***xxx < Fasting glucose (mg/dl) 85 8zzzxxx 98 11***xx 89 7xxx < h glucose (mg/dl) zzzxxx ***xxx xxx < Total cholesterol (mg/dl) HDL (mg/dl) 54 14* Triglycerides (mg/dl) x SBP (mmhg) *x DBP (mmhg) DInsAUC 0e30/DGluAUC 0e DInsAUC 0e120/DGluAUC 0e AIR (mu/ml min) Insulin-stimulated glucose disposal (mg/min Kg FFM) z**xxx xx < Disposition index (AIR M FFM ) z**xxx x < Disposition index (DIns[AUC 0e120]/DGluc[AUC 0e120] M FFM ) xxx x x Data are means SD. Comparisons between the four groups were performed using a general linear model with post hoc Bonferroni correction for multiple comparisons. P values refer to results after analyzes with adjustment for age, gender, and BMI. #P values refer to results after analyzes with adjustment for gender. Categorical variables were compared by c 2 test. NGT Z normal glucose tolerance; IFG Z Impaired fasting glucose; IGT Z Impaired glucose tolerance; AIR Z acute insulin response during IVGTT; BMI Z body mass index; SBP Z systolic blood pressure; DBP Z diastolic blood pressure. zp < 0.05; zzp < 0.01; zzzp < compared with subjects with A1C <5.7% and IFG/IGT after adjustment for age, and gender. *P < 0.05; **P < 0.01; ***P < compared with subjects with A1C 5.7e6.4% and NGT after adjustment for age, and gender. xp < 0.05; xxp < 0.01; xxxp < compared with subjects with A1C 5.7e6.4% and and IFG/IGT after adjustment for age, and gender.

5 A1C, insulin sensitivity, and b-cell function 31 Figure 1 Relationship between A1C and insulin-stimulated glucose disposal (A), DIns(AUC 0e120)/DGluc(AUC 0e120) (B), and OGTT-derived disposition index (C). Number of subjects according to A1C categories: A1C of 4.9 Z 44; A1C of 5 Z 24; A1C of 5.1 Z 31, A1C of 5.2 Z 30; A1C of 5.3 Z 33; A1C of 5.4 Z 25; A1C of 5.5 Z 41; A1C of 5.6 Z 33; A1C of 5.7 Z 24; A1C of 5.8 Z 10; A1C of 5.9 Z 15; and A1C of 6.0 Z 23. NS Z Not significant. the hyperinsulinemic euglycemic clamp, and a nominally significant reduction ( 13%) in first-phase insulin secretion assessed by IVGTT. Since the b-cell response to an increment in glucose levels is modulated by the severity of underlying insulin resistance, accounting for differences in insulin sensitivity is a critical point when evaluating b-cell function. Thus, adjusting insulin secretion, assessed by OGTT or IVGTT, for the degree of insulin sensitivity using the disposition index may be a better measure of b-cell function [9]. Using this approach, we observed that individuals with A1C of 5.7e6.4% exhibited a significantly reduction of both IVGTT-derived ( 38%) and OGTT-derived ( 22%) disposition indexes as compared with subjects with A1C <5.7% after adjusting for age, gender, and BMI indicating the presence of b-cell dysfunction. Although measurements of FPG, 2-h post-load glucose, and A1C are likely to reflect diverse pathophysiological feature of glucose metabolism, we observed that both individuals with IFG/IGT and A1C <5.7% and individuals with NGT and A1C value of 5.7e6.4% exhibited impairment in insulin sensitivity and b-cell function assessed by the disposition indexes. This observation suggests that IFG/IGT, and A1C-based diagnosis of pre-diabetes may capture at risk individuals with common underlying abnormalities in glucose metabolism. Interestingly, insulin sensitivity and b-cell function remained unchanged with the increase in A1C up to a value of 5.6%; thereafter, both insulin sensitivity and b-cell function steeply decreased with increasing A1C levels 5.7%. Consistent with the present data, a recent report including subjects of Mexican American descent who were part of the San Antonio Veterans Administration Genetic Epidemiology Study [10] has shown that insulin sensitivity (measured with an OGTT-derived index) remained unchanged up to a value of 5.5%, then decreasing, as A1C levels increased to 5.5%. However, b-cell function, measured with an OGTT-derived insulin secretion/insulin resistance disposition index, did not change significantly up to an A1C value of 5.7%, thereafter decreasing, as A1C levels increased to >5.7%. It is possible that the different A1C cut-point identifying subjects with reduced insulin sensitivity between the two studies may be due to differences in age or ethnicity since the Mexican Americans were older and had higher risk for type 2 diabetes as compared with the Caucasians participating to the present study. A recent systematic review of prospective studies that have examined the relationship of A1C to future diabetes incidence has reported that individuals with an A1C between 5.5 and 6.0% have an increased risk of diabetes with 5-year incidences ranging from 9 to 25% [4]. In consideration of the

6 32 M.A. Marini et al. expected increased use of A1C as a screening test to identify individuals with type 2 diabetes and pre-diabetic conditions [11,12], the findings of the present study showing that individuals with A1C of 5.7e6.4% already display abnormalities of insulin sensitivity and b-cell function may be clinically relevant in order to identify high-risk subjects who will benefit from lifestyle interventions [13e16] or even pharmaceutical treatment [16e20]. The current study has several strengths including the use of the gold standard hyperinsulinemic euglycemic clamp for assessment of insulin sensitivity, the measurement of insulin secretion by both OGTT and IVGTT, the collection of data by trained staff, following a standardized protocol [5], the use of a rigorously standardized A1C measurement, the exclusion of individuals with conditions that affect red cell turnover, such as anemia and major blood loss, the strict quality control of data collection and centralized laboratory analyzes. Nevertheless, the present study has some potential limitations. A first limitation is the availability of only single measures of each test including A1C, OGTT, IVGTT, euglycemic clamp study. A second limitation is the cross-sectional design, making causal interpretations of associations between underlying pathophysiological defects, i.e. insulin resistance and b-cell dysfunction and risk of type 2 diabetes difficult. Furthermore, the observed differences in insulin sensitivity and b- cell function may be, in part, due to differences in age and BMI between the groups with different A1C levels; however, all measures of insulin action and b-cell function were adjusted for these variables. Moreover, it can also be argued that the results of the current study might have been affected by the presence of a family history of type 2 diabetes. Nonetheless, type 2 diabetes is multifactorial disease arising from a complex interplay between environmental factors and genetic susceptibility, and many individuals who develop type 2 diabetes have a family history of diabetes. Additionally, it should be mentioned that insulin secretion and b-cell function indexes offer only an estimate of the underlying complex physiological phenomena; hence it is possible that where no differences were found, it was because of the lack of sensitivity and sophistication of the applied methods and, likewise, where differences were found, they could have been much greater with better methods. Moreover, although the present results are remarkably similar to those reported in Mexican Americans, they are only based on Caucasian subjects, and generalizing them to other ethnic groups must be done with caution because differences between ethnic groups in insulin resistance have been reported. In addition, because there is evidence that these ethnicities have higher A1C levels at any given level of FPG as compared with their Caucasian counterparts even after adjustment for confounding factors affecting glycemia, our results may not be generalizable to Hispanics, American Indians, Blacks, and Asian Americans [21]. Finally, it can also be argued that our results might have been influenced by the strong genetic predisposition to type 2 diabetes since all the individuals were offspring of type 2 diabetes patients. In conclusion, the results of the present study demonstrate that Caucasian individuals with A1C >5.7% exhibit both core pathophysiological defects of type 2 diabetes i.e. insulin resistance and b-cell dysfunction. These findings, together with the increased relative risk of incident type 2 diabetes in individuals with A1C ranging from 5.5 to 6.0% favor using an A1C value ranging from 5.7% to 6.4% to identify individuals at increased risk for diabetes in analogy to subjects with IFG or IGT as recommended by ADA. Acknowledgments The study was supported in part by the Ministry of University grant MERIT RBNE08NKH7 to G.S. Appendix A. Supplementary data Supplementary data related to this article can be found at References [1] Nathan DM, Kuenen J, Borg R, Zheng H, Schoenfeld D, Heine RJ, A1c-Derived Average Glucose Study Group. Translating the A1C assay into estimated average glucose values. Diabetes Care 2008;31:1473e8. [2] International Expert Committee. International Expert Committee report on the role of the A1C assay in the diagnosis of diabetes. Diabetes Care 2009;32:1327e34. [3] American Diabetes Association. Standards of medical care in diabetes Diabetes Care 2012;35(Suppl. 1):S11e63. [4] Zhang X, Gregg EW, Williamson DF, Barker LE, Thomas W, Bullard KM, et al. A1C level and future risk of diabetes: a systematic review. Diabetes Care 2010;33:1665e73. [5] Laakso M, Zilinskaite J, Hansen T, Boesgaard TW, Vänttinen M, Stancáková A, et al. Insulin sensitivity, insulin release and GLP-1 levels in subjects with IFG and/or IGT in the EUGENE2 study. Diabetologia 2008;51:502e11. [6] Succurro E, Andreozzi F, Marini MA, Lauro R, Hribal ML, Perticone F, et al. Low plasma insulin-like growth factor-1 levels are associated with reduced insulin sensitivity and increased insulin secretion in nondiabetic subjects. Nutr Metab Cardiovasc Dis 2009;19:713e /j.numecd [7] Stancakova A, Javorsky M, Kuulasmaa T, Haffner SM, Kuusisto J, Laakso M. Changes in insulin sensitivity and insulin release in relation to glycemia and glucose tolerance in 6,414 Finnish men. Diabetes 2009;58:1212e21. [8] Abdul-Ghani MA, Jenkinson CP, Richardson DK, Tripathy D, DeFronzo RA. Insulin secretion and action in subjects with impaired fasting glucose and impaired glucose tolerance: results from the Veterans Administration Genetic Epidemiology Study. Diabetes 2006;55:1430e5. [9] Kahn SE. The importance of b-cell failure in the development and progression of type 2 diabetes. J Clin Endocrinol Metab 2001;86:4047e58. [10] Kanat M, Winnier D, Norton L, Arar N, Jenkinson C, Defronzo RA, et al. The relationship between b-cell function and glycated hemoglobin: results from the veterans administration genetic epidemiology study. Diabetes Care 2001;34:1006e10. [11] Marini MA, Succurro E, Arturi F, Ruffo MF, Andreozzi F, Sciacqua A, et al. Comparison of A1C, fasting and 2-h postload plasma glucose criteria to diagnose diabetes in Italian Caucasians. Nutr Metab Cardiovasc Dis 2012;22:561e [12] Marini MA, Succurro E, Castaldo E, Cufone S, Arturi F, Sciacqua A, et al. Cardiometabolic risk profiles and carotid atherosclerosis in individuals with prediabetes identified by

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