Elevated 1-h post-load plasma glucose levels in subjects with normal glucose tolerance are associated with a pro-atherogenic lipid profile

Accepted Manuscript Elevated 1-h post-load plasma glucose levels in subjects with normal glucose tolerance are associated with a pro-atherogenic lipid profile Francesco Andreozzi, Gaia C. Mannino, Maria Perticone, Francesco Perticone, Giorgio Sesti PII:

S0021-9150(16)31488-5

DOI:

10.1016/j.atherosclerosis.2016.11.020

Reference:

ATH 14878

To appear in:

Atherosclerosis

Received Date: 25 June 2016 Revised Date:

25 October 2016

Accepted Date: 16 November 2016

Please cite this article as: Andreozzi F, Mannino GC, Perticone M, Perticone F, Sesti G, Elevated 1-h post-load plasma glucose levels in subjects with normal glucose tolerance are associated with a proatherogenic lipid profile, Atherosclerosis (2016), doi: 10.1016/j.atherosclerosis.2016.11.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Elevated 1-hour post-load plasma glucose levels in subjects with normal glucose tolerance are associated with a pro-atherogenic lipid profile

Francesco Andreozzi, Gaia C. Mannino, Maria Perticone, Francesco Perticone, Giorgio Sesti*

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Department of Medical and Surgical Sciences, University Magna Graecia of Catanzaro, Catanzaro, Italy

*Corresponding author: Department of Medical and Surgical Sciences, University of Catanzaro “Magna

0961 3647192. E-mail address: [email protected] (G. Sesti).

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Graecia”, Viale Europa, loc. Germaneto, I-88100, Catanzaro (CZ), Italy. Tel.: + 39 0961 3647204; Fax: +39

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Keywords: 1h-post load hyperglycemia, dyslipidemia, pre-diabetes, apolipoproteins.

Abstract

Background and aims: Evidence suggests that plasma glucose concentration ≥ 155 mg/dl at 1-h during an

oral glucose tolerance test (OGTT) (NGT 1h-high) predicts both development of type 2 diabetes (T2DM)

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and cardiovascular events, among adults with normal glucose tolerance (NGT). An atherogenic lipid profile is detectable in subjects with impaired glucose tolerance (IGT) and T2DM. Whether individuals with NGT1h-high also exhibit a pro-atherogenic lipid profile is still uncertain. Methods: The study cohort includes 1011 non-diabetic Caucasian adults participating in the CATAMERI

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study. All participants were submitted to anthropometrical evaluation before undergoing an OGTT. Subjects were categorized into NGT-1h-low (1h glucose < 155 mg/dl), NGT-1h-high, IGT, and newly diagnosed

1.

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T2DM. Lipid profile includes triglycerides, total and HDL cholesterol, apolipoprotein B (ApoB) and ApoA-

Results: 510 subjects were NGT-1h-low, 211 NGT-1h-high, 232 IGT and 58 were newly diagnosed T2DM. Triglyceride and ApoB levels were significantly higher in NGT 1h-high, IGT and T2DM subjects compared to NGT 1h-low, and HDL cholesterol was significantly lower. Triglycerides-to-HDL cholesterol ratio was significantly higher in NGT 1h-high, IGT and T2DM groups compared with NGT 1h-low individuals. The ApoB/ApoA-1 ratio was significantly higher in NGT 1h-high, IGT and T2DM groups than in the NGT 1hlow group. NGT 1h-high, IGT and T2DM subjects exhibited reduced LDL/ApoB ratio compared with NGT 1h-low. Noticeably, there were no significant differences in ApoB/ApoA-1 and LDL/ApoB ratios when comparing NGT 1h-high with IGT and T2DM. Conclusions: Individuals with NGT 1-h-high exhibited an atherogenic lipid pattern qualitatively and quantitatively similar to that observed in individuals with IGT and newly diagnosed T2DM.

ACCEPTED MANUSCRIPT Introduction The number of people with diabetes has risen from 108 million in 1980 to 422 million in 2014 (1). It is widely recognized that adults with diabetes have a 2-3-fold increased risk of premature atherosclerosis, heart attacks and strokes, with fasting blood glucose levels and glycated hemoglobin (HbA1c) being associated with the risk of vascular disease even in non-diabetic individuals (2). Patients with type 2 diabetes (T2DM) exhibit an abnormal lipid profile, with reduced high-density lipoprotein (HDL) cholesterol, elevated

altogether conferring increased risk of cardiovascular diseases (3).

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triglyceride-rich lipoproteins and the presence of small dense low-density lipoprotein (LDL) particles,

Early recognition of subjects at risk for T2DM is crucial not only because the progression to diabetes could be largely avoidable through lifestyle changes and/or pharmacologic treatment (4,5), but also to

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prevent or delay the cardiovascular complications associated with both T2DM and prediabetes itself (6,7). Impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) are two dysglycemic disorders, also referred to as pre-diabetic categories, with high likelihood of transitioning to full blown T2DM (8).

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Dyslipidemic profiles are already detectable in pre-diabetic subjects: IFG status has been associated with higher presence of apolipoprotein B-100 (ApoB) and smaller sized HDL particles (9); whereas the IGT status has been associated with higher levels of triglycerides and triglycerides-to-HDL ratio, large very-low-density lipoprotein (VLDL) size, increased concentration of small dense LDL particles, and small HDL size (9,10). Increasing evidence suggests plasma glucose concentration ≥ 155 mg/dl (8.6 mmol/L) at 1-h during an oral glucose tolerance test (OGTT) (NGT 1h-high) predicts the development of future T2DM among

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adults with normal glucose tolerance (NGT) (11–14), and also associates with an intermediate cardiometabolic risk profile, falling between IFG and IGT (15–24). It is worth noticing that previous studies aimed at characterizing the lipid pattern in pre-diabetes conditions such as IFG and IGT have never distinguished NGT 1h-high and plain NGT (NGT 1h-low) subjects as separate categorical entities (9,25–27).

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Thus, an important portion of the population considered “not at risk” could instead exhibit atherogenic dyslipidemia, a condition in which the risk for cardiovascular disease is embedded. To address these issues, we compared the lipid profiles of 1011 individuals with different glucose

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tolerance status, including IGT and newly diagnosed T2DM, with a focus on the NGT 1h-high group. We measured both lipids routinely tested such as triglycerides, total, low-density lipoprotein (LDL) and highdensity lipoprotein cholesterol (HDL) cholesterol, and apolipoprotein B (ApoB) and ApoA-1 which, in addition to stabilizing lipoprotein structure, have a crucial role in regulating lipid metabolism. ApoB are present in very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL) and LDL, and represent the total number of atherogenic lipoproteins (28). ApoA-1 is the major apolipoprotein associated with HDL and play a protective role by removing cholesterol from peripheral cells and delivering cholesterol to the liver. Thus, ApoB and ApoA-1 exert opposing effects on atherogenic risk, and the ApoB/ApoA-1 ratio is a useful index to characterize the cardiovascular risk (29). The size of LDL particles can be estimated through the LDL/ApoB ratio, to reveal the presence of small dense LDL, whose diameter (< 25.5 nm) allows to infiltrate the subintimal space of the vascular wall

ACCEPTED MANUSCRIPT and participate in the formation of the arterial plaque (30). ApoB, LDL/ApoB and ApoB/ApoA-1 are stronger markers than conventional lipids and lipoproteins measurements alone for predicting the development of cardiovascular disease across age and ethnicity groups (31,32).

Patients and methods Study population

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The study group consisted of 1011 self-reported non-diabetic Caucasian subjects (481 men and 530 women), enrolled in the CAtanzaro MEtabolic RIsk factors Study (CATAMERIS), an observational study assessing cardiometabolic risk factors in individuals carrying at least one risk factor including overweight/obesity, hypertension, dyslipidemia, dysglycemia, and family history for type 2 diabetes (33,34).

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Subjects, aged 21–70 years (mean ± SD 46.9 ± 13), were excluded if they had a history of cardiovascular disease, chronic gastrointestinal diseases, chronic pancreatitis, history of any malignant disease, history of alcohol or drug abuse, liver or kidney failure and treatments able to modulate glucose metabolism, including

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lipid-lowering and hypoglycemic agents. All participants were submitted to anthropometrical evaluation before undergoing an oral glucose tolerance test (OGTT): weight, height, body mass index (BMI), and waist circumference were measured, and body composition was evaluated by bioelectrical impedance. After a 12-h fast, a 75 g OGTT was performed with 0, 30, 60, and 120 min sampling for plasma glucose and insulin. The study was approved by Institutional Ethics Committees of University "Magna Graecia" of Catanzaro. Written informed consent was obtained from each subject in accordance with principles of the Declaration of

Laboratory determinations

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Helsinki.

Glucose, triglycerides, total and high density lipoprotein (HDL) cholesterol concentrations were

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determined by enzymatic methods (Roche®, Basel, Switzerland). ApoA-1 and ApoB levels were quantified by means of immunonephelometry (Siemens Healthcare Diagnostics Inc®, Marburg, Germany). Plasma insulin concentration was measured with a chemiluminescence-based assay (Immulite®, Siemens Healthcare

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Diagnostics Inc, Marburg, Germany).

Calculations

Subjects were classified into three groups according to glucose tolerance status as having normal glucose tolerance (NGT) when fasting plasma glucose (FPG) was < 7.0 mmol/L (100 mg/dl) and 2-h postload < 7.8 mmol/L (140 mg/dl), IGT when FPG was < 7.0 mmol/L (126 mg/dl) and 2-h post-load was 7.811.0 mmol/L (140-199 mg/dl) and diabetes when FPG was ≥ 7.0 mmol/L (126 mg/dl) and/or 2-h plasma glucose ≥ 11.1 mmol/L (200 mg/dl). Individuals classified as NGT were subsequently divided into two groups (NGT 1h-low and NGT 1h-high) based upon their 1-h plasma glucose concentration, below or above 155 mg/dl (8.6 mmol/L), respectively.

ACCEPTED MANUSCRIPT Non-HDL cholesterol was calculated as the difference between total cholesterol (TC) and HDL. Clinical indicators of lipids metabolism were directly computed from the laboratory results (ApoB/ApoA-1 and LDL/ApoB ratios). Insulin sensitivity was evaluated using the Matsuda index (insulin sensitivity index [ISI]) calculated as follows: 10,000/square root of [fasting glucose (mmol/L) × fasting insulin (mU/L)] × [mean glucose × mean insulin during OGTT]. The homeostasis model assessment (HOMA) index was calculated as fasting

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insulin × fasting glucose/22.5 (35).

Statistical analysis

The results for continuous variables are given as means ± SD. Anthropometric and metabolic

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differences between groups were tested after adjusting for age, gender and waist circumference using a general linear model. The χ2 test was used for categorical variables. Variables with skewed distribution (i.e.

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triglycerides, fasting, 30 min, 90 min, 1-h and 2-h insulin, and HOMA index) were log transformed to meet the normality assumption for statistical purposes. A multiple linear regression analysis was used to examine the relation of 1-h glucose during an OGTT to each of the lipoproteins and apolipoproteins (dependent variable) in a model also including age, gender, waist circumference, and fasting glucose levels. Bonferroni post hoc correction for multiple comparisons was applied and a p value < 0.05 was considered statistically significant. All analyses were performed using the statistical package SPSS 22.0 for Windows (SPSS,

Results

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IBM®, Chicago, IL).

Cardiometabolic characteristics

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Anthropometric and biochemical characteristics of the study cohort, according to glucose tolerance classification are summarized in Table 1. A total of 1011 subjects were evaluated, of which 721 (71%) had normal glucose tolerance (NGT), 232 (23%) had impaired glucose tolerance (IGT) and 58 (6%) had newly

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diagnosed T2DM. NGT subjects were divided into two groups using a 1-hour post-load plasma glucose cutoff point of 155 mg/dl during OGTT: 510 subjects with 1-h post-load plasma glucose < 155 mg/dl (NGT 1h-low) and 211 individuals with 1-h post-load plasma glucose ≥ 155mg/dl (NGT 1h-high). Gender, age and BMI distributions were unevenly scattered among the four groups: NGT 1h-high and T2DM harbored more men than women, while subjects with NGT 1h-low were younger and exhibited lower waist circumference as compared with subjects having IGT or T2DM. Since, these three parameters are associated with lipid profiles, all subsequent analyses were adjusted for age, gender and waist circumference. After adjusting for age, gender, and waist circumference, NGT 1h-high, IGT and newly diagnosed T2DM groups exhibited significantly higher fasting and 2-h post-load glucose and insulin levels as compared with NGT 1h-low group. Additionally, NGT 1h-high, IGT and newly diagnosed T2DM groups exhibited higher values of HOMA index of hepatic insulin resistance and lower values of Matsuda index of insulin sensitivity even after adjusting for age gender, and waist circumference.

ACCEPTED MANUSCRIPT As compared with the NGT 1h-high group, IGT and newly diagnosed T2DM groups exhibited higher values of fasting, 1h and 2-h post-load glucose, fasting and 2h post-load insulin, HOMA index and lower values of Matsuda index of insulin sensitivity even after adjusting for age gender, and waist circumference (Table 1).

Lipid profiles

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Serum triglycerides and ApoB levels were significantly higher in NGT 1h-high, IGT and T2DM subjects compared to NGT 1h-low, and HDL cholesterol was significantly lower. Triglycerides-to-HDL cholesterol ratio was significantly higher in NGT 1h-high, IGT and T2DM groups as compared with NGT 1h-low individuals. No significant differences were observed among the four groups for total cholesterol,

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LDL and non-HDL cholesterol levels, and ApoA-1 levels after adjusting for age, gender, and waist circumference (Table 2). Nevertheless, the ApoB/ApoA-1 ratio was significantly higher in NGT 1h-high, IGT and T2DM groups than in the NGT 1h-low group. In addition, NGT 1h-high, IGT and T2DM subjects

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exhibited reduced LDL/ApoB ratio compared with NGT 1h-low. Noticeably, there were no significant differences in ApoB/ApoA-1 and LDL/ApoB ratios when comparing NGT 1h-high with IGT and T2DM (Table 2).

To estimate whether 1-h glucose levels during an OGTT was an independent contributor to lipoproteins and apolipoproteins, a multivariable regression analysis was performed in a model also including age, gender, waist circumference, and fasting glucose levels (Table 3). 1-h glucose levels during an

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OGTT were independently associated with HDL, triglycerides, ApoB, ApoB/ApoA-1 ratio, and LDL/ApoB ratio after adjustments for other confounders including age, gender, waist circumference, and fasting glucose

Discussion

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levels (Table 3).

Increasing evidence suggests that 1-h post load plasma glucose value ≥ 155 mg/dl (8.6 mmol/L) in

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individuals with NGT is associated with an increased risk of progression to diabetes (11–14). Moreover, these individuals exhibit increased risk of subclinical organ damage such as common carotid artery thickness (10), left ventricular hypertrophy (11), vascular stiffness (14), and left ventricular diastolic dysfunction (13), all independent predictors of cardiovascular events. Remarkably, two longitudinal studies have shown that 1hour post-load glucose levels ≥ 161 mg/dl and ≥ 155 mg/dl, respectively, predict cardiovascular mortality and all-cause mortality (36,37). A major contributor to the increased risk of cardiovascular disease amongst individuals with dysglycemic conditions is dyslipidemia, which includes abnormalities in all lipoproteins (9). These observations, coupled with the availability of a carefully characterized cohort of nondiabetic individuals participating in the CATAMERI study, have provided the rationale for addressing the question of whether individuals with NGT, whose 1-hour post-load plasma glucose is ≥ 155 mg/dl (NGT 1h high), are

ACCEPTED MANUSCRIPT characterized by an atherogenic lipid profile. In the present cross-sectional study, we provided evidence that individuals with NGT 1-h high exhibited an atherogenic lipid pattern with increased fasting triglycerides, higher triglycerides-to-HDL cholesterol ratio, ApoB levels, ApoB/ApoA-1 ratio, and reduced HDL cholesterol and LDL/ApoB ratio. Notably, the derangement of lipid pattern was qualitatively and quantitatively similar to that observed in individuals with IGT and newly diagnosed T2DM. Furthermore, a stronger association was observed between 1-h and 2-h glucose levels and lipoproteins and apolipoproteins

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concentrations as compared with fasting glucose levels. Taken together, these results support the notion that individuals with NGT 1h-high may represent a dysglycemic condition characterized by a pronounced derangement of lipoproteins and apolipoproteins.

There are several plausible pathophysiological mechanisms explaining the association between 1-

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hour post-load plasma glucose ≥ 155 mg/dl and the atherogenic lipid pattern herein reported. There is evidence that individuals with NGT 1h-high are characterized by skeletal muscle insulin resistance as assessed by euglycemic hyperinsulinemic clamp studies and OGTT-derived indexes of insulin sensitivity,

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and reduced insulin clearance, thus resulting in sustained hyperinsulinemia after an oral glucose load (14,15,23,38,39). Impaired insulin sensitivity in skeletal muscle may lead to diversion of ingested carbohydrate from muscle glycogen synthesis to the liver. This change, in combination with elevated plasma insulin levels, leads to increased hepatic de novo lipogenesis, resulting in the atherogenic lipid pattern characterized by increased triglyceride concentrations, reduced HDL levels, and increased hepatic triglyceride synthesis, which was observed in individuals with NGT 1h-high (40). On the other hand,

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individuals with NGT 1h-high are also characterized by hepatic insulin resistance (18). Since insulin directly inhibits hepatic VLDL production (41), and stimulates ApoB degradation in hepatocytes, it is conceivable that an impaired capability of insulin to promote these processes may lead to the increase in plasma concentrations of triglycerides and ApoB observed in individuals with NGT 1h-high (42). In addition, post-

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load hyperglycemia and hyperinsulinemia observed in individuals with NGT 1h-high may induce de novo lipogenesis in the liver by increasing expression of carbohydrate responsiveness element-binding protein (ChREBP) and sterol regulatory element-binding protein (SREBP)-1c, respectively (42). There is also

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evidence that individuals with NGT 1h-high exhibit higher dietary intake of both saturated fatty acids and fructose (43). Dietary saturated fatty acids are a major source of substrate for hepatic triacylglycerol synthesis and fructose is able to quickly enter the glycolytic pathway and to provide acetyl-CoA for de novo lipogenesis in the liver. Individuals with NGT 1h-high also exhibit low HDL cholesterol, and a low LDL cholesterol per apolipoprotein B (LDL/ApoB) ratio, reflecting an increase in small LDL particles. It has been shown that in conditions of insulin resistance, an increase in large VLDL promotes cholesterol estertriglyceride exchange between VLDL and LDL, resulting in LDL particles with more triglycerides and fewer cholesterol esters (44,45). These LDL particles enriched in triglycerides are a good substrate for hepatic lipase, and the ensuing triglyceride hydrolysis leads to the formation of small dense LDL particles, which are more atherogenic. The increased pool of triglyceride-rich large VLDL and small LDL promotes triglyceride exchange with HDL particles via lecithin-cholesterol acyltransferase and cholesterol ester transfer protein,

ACCEPTED MANUSCRIPT resulting in an increase of HDL catabolism, since HDL-rich particles are very good substrates for hepatic lipase. The current study has a number of strengths, including the large sample size encompassing both men and women, the detailed anthropometric and metabolic variables data collected according to a standardized protocol, the centralization of biochemical analyses comprising conventional lipid and lipoproteins measurement, the assay of metabolites in fresh blood samples rather than in stored samples that may lead to

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their degradation, the exclusion of subjects with pharmacological treatments affecting glucose and lipid metabolism.

Nevertheless, some limitations should be acknowledged in the interpretation of our results. First, each lipid variable has been measured once and small day-to-day changes would be expected. A second

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limitation of our study is that participants underwent a single 75 g OGTT to assess glucose tolerance. Although such an approach is common in both clinical practice and epidemiological studies, the intraindividual variability of 1-h and 2-h post-challenge glucose cannot be taken into account and this may have

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introduced some imprecision in the classification of recruited individuals into glucose tolerance groups. Furthermore, our cohort comprises outpatients recruited at a referral university hospital, representing individuals at risk for T2DM, and, therefore, our results may not necessarily be extendible to the general population. Next, a surrogate measure of hepatic insulin resistance was used because clamp studies combined with tracer techniques are not feasible in large scale studies. Additionally, all participants to the present study were Caucasians, and whether the present findings can also be extended to non-Caucasian

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ethnic groups remains to be established. Finally, the cross-sectional design of the study reflects only an association of 1-hour post-load plasma glucose with atherogenic lipid profile, and precludes us to draw any conclusion about causal relationships between this dysglycemic condition and incident cardiovascular disease. Therefore, our findings should be considered hypothesis generating and requiring confirmation by

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further prospective studies assessing the impact of 1-hour post-load plasma glucose ≥ 155 mg/dl in predicting cardiovascular events.

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Conflict of interest

The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript. The founding source took no part in the conceptualization and realization of this project, or the writing of the present manuscript.

Financial support Società Italiana di Diabetologia - SID (Fondazione Diabete Ricerca FO.DI.RI. - MSD scholarship 2014 and 2015) to Gaia Chiara Mannino.

ACCEPTED MANUSCRIPT Author contributions G.S. designed the study, analyzed, interpreted the data and wrote the manuscript; F.A. acquired, analyzed, and interpreted the data, and wrote the manuscript; G.C.M. analyzed and interpreted the data and reviewed the manuscript; M.P. acquired the data and reviewed the manuscript; F.P. acquired the data and

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reviewed the manuscript.

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35. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28(7):412–419. 36. Strandberg TE, Pienimäki T, Strandberg AY, Salomaa VV, Pitkälä KH, Tilvis RS, Miettinen TA. Onehour glucose, mortality, and risk of diabetes: a 44-year prospective study in men. Arch Intern Med 2011; 171(10):941–943.

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37. Bergman M, Chetrit A, Roth J, Dankner R. One-hour post-load plasma glucose level during the OGTT predicts mortality: observations from the Israel Study of Glucose Intolerance, Obesity and Hypertension. Diabet Med J Br Diabet Assoc 2016. doi:10.1111/dme.13116. 38. Marini MA, Succurro E, Frontoni S, Mastroianni S, Arturi F, Sciacqua A, Lauro R, Hribal ML, Perticone F, Sesti G. Insulin sensitivity, β-cell function, and incretin effect in individuals with elevated 1-hour postload plasma glucose levels. Diabetes Care 2012; 35(4):868–872.

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39. Marini MA, Frontoni S, Succurro E, Arturi F, Fiorentino TV, Sciacqua A, Hribal ML, Perticone F, Sesti G. Decreased insulin clearance in individuals with elevated 1-h post-load plasma glucose levels. PloS One 2013; 8(10):e77440.

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40. Petersen KF, Dufour S, Savage DB, Bilz S, Solomon G, Yonemitsu S, Cline GW, Befroy D, Zemany L, Kahn BB, Papademetris X, Rothman DL, Shulman GI. The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome. Proc Natl Acad Sci U S A 2007; 104(31):12587–12594. 41. Malmström R, Packard CJ, Caslake M, Bedford D, Stewart P, Yki-Järvinen H, Shepherd J, Taskinen MR. Effects of insulin and acipimox on VLDL1 and VLDL2 apolipoprotein B production in normal subjects. Diabetes 1998; 47(5):779–787. 42. Vergès B. Pathophysiology of diabetic dyslipidaemia: where are we? Diabetologia 2015; 58(5):886–899. 43. Sciacqua A, Perticone M, Falbo T, Grillo N, Tassone EJ, Sinopoli F, Lo Russo C, Succurro E, Andreozzi F, Sesti G, Perticone F. Dietary patterns and 1-h post-load glucose in essential hypertension. Nutr Metab Cardiovasc Dis NMCD 2014; 24(5):547–553. 44. Garvey WT, Kwon S, Zheng D, Shaughnessy S, Wallace P, Hutto A, Pugh K, Jenkins AJ, Klein RL, Liao Y. Effects of insulin resistance and type 2 diabetes on lipoprotein subclass particle size and concentration determined by nuclear magnetic resonance. Diabetes 2003; 52(2):453–462.

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45. Malmström R, Packard CJ, Caslake M, Bedford D, Stewart P, Yki-Järvinen H, Shepherd J, Taskinen MR. Defective regulation of triglyceride metabolism by insulin in the liver in NIDDM. Diabetologia 1997; 40(4):454–462.

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Table 1. Anthropometric and metabolic characteristics of the study subjects stratified according to glucose tolerance. Variables

IGT

T2DM

p

211 (134/77)

232 (111/121)

58 (40/18)

<0.0001

43±13

48±11 e

52±12 e,g

54±10 e,g

<0.0001 a

BMI (kg/m2)

28.9±6.2

29.4±5.6

31.3±6.4 e,h

31.9±6.3 e,g

<0.0001 b

Waist circumference (cm)

98.3±14.3

100.8±13.5

105.4±15.2 e,h

105.6±13.4 d,g

<0.0001 b

32±8

31±7

38±8 e,g

34±8 e,g

<0.0001 b

88.7±8.8

94.3±9.8 e

98.1±11.6 e,g

116.5±18.2 e,h

<0.0001

1-h glucose (mg/dl)

117.1±23.1

179.1±21.3 e

187.2±33.2 e,g

237.9±37.9 e,h

<0.0001

2-h glucose (mg/dl)

102.6±19.1

114.6±18.8 e

161.3±15.3 e,h

231.3±34.3 e,h

<0.0001

Fasting insulin (µU/ml)

12.2±8.45

14.2±10.9 d

15.8±10.2 e

21.1±24.2 e,g

<0.0001

1-h insulin (µU/ml)

91.5±66.1

153.3±1.9 e

120.2±95.2 d,h

104.3±62.7 h

<0.0001

2-h insulin (µU/ml)

66.5±54.2

103.4±100.2 e

155.3±137.2 e,h

139.6±85.8 e,g

<0.0001

HOMA-IR index

2.71±1.91

3.31±2.51 c

3.81±2.29 e

5.98±6.18 e,h

<0.0001

59.43±32.31 e

51.32±31.87 e

39.21±26.04 e,f

<0.0001

86.86±49.53

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Matsuda Insulin Sensitivity index (mg x L2 x mmol-1 x mU-1 x min-1)

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Fasting glucose (mg/dl)

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Fat mass (%)

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Age (yr)

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510 (196/314)

EP

Number (male/female)

NGT 1-h glucose <155 mg/dl 1-h glucose ≥155 mg/dl

Data are means ± SD. Fasting glucose, fasting, 30 min, 1-h and 2-h insulin, and HOMA index were log transformed for statistical analysis, but values in the table represent back transformation to the original scale. Comparisons between the four groups were performed using a general linear model with post hoc Bonferroni correction for multiple comparisons. Categorical variables were compared by χ2 test. p values refer to results after analyses with adjustment for age, gender, and waist circumference. a

p values refer to results after analyses with adjustment for gender. bp values refer to results after analyses with adjustment for age and gender. cp <

0.05 vs. NGT with 1-h glucose < 155 mg/dl; dp < 0.001 vs. NGT with 1-h glucose < 155 mg/dl; ep < 0.0001 vs. NGT with 1-h glucose < 155 mg/dl.

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p < 0.05 vs. NGT with 1-h glucose ≥ 155 mg/dl; gp < 0.003 vs. NGT with 1-h glucose ≥ 155 mg/dl; hp < 0.0001 vs. NGT with 1-h glucose ≥ 155

mg/dl. BMI, body mass index; HOMA-IR, homeostatic model assessment; IGT, impaired glucose tolerance; NGT, normal glucose tolerance; T2DM, type 2

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diabetes mellitus.

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Table 2. Lipid profile of the study subjects stratified according to glucose tolerance. Variables

NGT 1-h glucose < 155 mg/dl 1-h glucose ≥ 155 mg/dl

IGT

T2DM

p

205.3±37.4

198.9±38.4 d

0.220

134.3±32.8

135.6±32.6

0.174

b

0.002

154.6±37.3

0.293

199.4±39.2

206.2±32.9

LDL cholesterol (mg/dl)

127.5±34.1

135.2±30.5

HDL cholesterol (mg/dl)

53.9±14.4

49.3±13.8

Non-HDL cholesterol (mg/dl)

145.9±39.8

156.9±34.1

Triglycerides (mg/dl)

110.3±66.3

129.3±65.9

Triglycerides-to-HDL cholesterol ratio

2.38±2.32

3.03±2.21 a

3.27±2.67 b

Apolipoprotein AI (mg/dl)

1.46±0.25

1.42±0.24

1.45±0.26

1.36±0.22

0.190

Apolipoprotein B (mg/dl)

0.92±0.24

1.01±0.23 a

1.01±0.24 a

1.02±0.22 a

0.05

Apolipoprotein B/Apolipoprotein A1 ratio

0.65±0.21

0.73±0.22 a

0.72±0.22 a

0.77±0.24 a

0.02

LDL cholesterol/Apolipoprotein B ratio

140.8±36.6

135.3±15.6 a

134.4±21.5 a

127.8±17.8 b

0.003

a

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50.1±13.9

a

155.3±38.3

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Total cholesterol (mg/dl)

140.7±81.5

c

44.2±12.4

b

<0.0001

c,d

<0.0001

155.1±75.8 4.06±2.81

Data are means ± SD. Triglycerides were log transformed for statistical analysis, but values in the table represent back transformation to the original scale. Comparisons between the four groups were performed using a general linear model with post hoc Bonferroni correction for multiple p < 0.05 vs. NGT with 1-h glucose < 155 mg/dl; bp < 0.001 vs. NGT with 1-h glucose < 155 mg/dl; cp < 0.0001 vs. NGT with 1-h glucose < 155

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a

EP

comparisons. p values refer to results after analyses with adjustment for age, gender, and waist circumference. mg/dl. dp < 0.05 vs. NGT with 1-h glucose ≥ 155 mg/dl.

HDL, high density lipoprotein; IGT, impaired glucose tolerance; LDL, low density lipoprotein; NGT, normal glucose tolerance; T2DM, type 2 diabetes mellitus.

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Table 3. Multiple regression analysis with individual lipoproteins and apolipoproteins as the dependent variable and age, gender, waist circumference, fasting and 1 h post-load glucose levels as independent variables.

RI PT

1-h post load glucose Dependent variable

p

Total cholesterol (mg/dl)

- 0.030 (±0.031)

0.439

LDL cholesterol (mg/dl)

-0.001 (±0.028)

0.984

- 0.104 (±0.011)

0.003

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β(±SE)

Non-HDL cholesterol (mg/dl)

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HDL cholesterol (mg/dl)

0.009 (±0.032)

0.815

0.114 (±0.001)

0.003

0.103 (±0.002)

0.008

- 0.025 (±0.001)

0.500

Apolipoprotein B (mg/dl)

0.086 (±0.001)

0.020

Apolipoprotein B/Apolipoprotein A-I ratio

0.092(±0.001)

0.014

- 0.077 (±0.026)

0.050

Triglycerides (mg/dl)

Triglycerides-to-HDL cholesterol ratio

EP

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Apolipoprotein A-I (mg/dl)

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LDL cholesterol/Apolipoprotein B ratio

p values refer to results after analyses with adjustment for age, gender, waist circumference and fasting plasma glucose. Triglycerides were log transformed for statistical analysis, but values in the table represent back transformation to the original scale. Effect sizes (β and SE) per study groups and corresponding p values after post hoc Bonferroni correction for multiple comparisons are shown. HDL, high density lipoprotein; LDL, low density lipoprotein.

ACCEPTED MANUSCRIPT Highlights Diabetes and pre-diabetes at diagnosis already possess atherogenic dyslipidemia

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Fasting, 1-h and 2-h glycemia strongly modulate individual’s lipid pattern

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NGT-1h-high ≥155 mg/dl have a worse cardio-metabolic profile than plain NGT

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NGT subjects may disguise an increased risk of cardiovascular disease

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