Circulating preptin levels in normal, impaired glucose tolerance, and type 2 diabetic subjects

Abstract

Background. Preptin is a novel hormone that is co-secreted with insulin and amylin from the pancreatic β-cells. Preptin increases glucose-mediated insulin secretion, while the binding of endogenous preptin by antipreptin antibodies decreases glucose-mediated insulin secretion. Thus, it appears to act as a physiological amplifier of glucose-mediated insulin secretion.

Aim. In this study, we investigate whether plasma preptin levels are different in non-diabetic subjects and patients with impaired glucose tolerance (IGT) or type 2 diabetes mellitus (T2DM).

Method. Fifty patients with T2DM, 56 subjects with IGT, and 54 sex- and age-matched normal controls participated in the study. Plasma preptin levels were measured with a radioimmunoassay. The relationships between plasma preptin levels and anthropometric and metabolic parameters were also assessed.

Results. Plasma preptin levels were higher in patients with T2DM compared to patients with IGT and controls (456±14 versus 416±13 and 398±13 ng/L, P<0.05 and P<0.01, respectively). Plasma preptin levels were lower in males than females (403±10 versus 432±10 ng/L, P<0.05). Fasting plasma preptin levels correlated positively with diastolic blood pressure (DBP) (r=0.20, P<0.01), triglyceride (TG) (r=0.24, P<0.01), total cholesterol (TC) (r=0.24, P<0.01), high-density lipoprotein cholesterol (HDL-C) (r=0.18, P<0.05), free fatty acids (FFA) (r=0.21, P<0.01), 2-h blood glucose after glucose overload (2hOGTT) (r=0.18, P<0.05), glycosylated haemoglobin (HbA₁c) (r=0.19, P<0.01), and homeostasis model assessment-insulin resistance index (HOMA_IR) (r=0.13, P<0.05) in simple regression analysis of pooled data, while in multiple stepwise regression analysis, only DBP, TG, HDL-C, and FFA were independently related with plasma preptin levels.

Conclusion. The present work suggests a potential role of preptin in the pathogenesis of T2DM.

• Metabolism
• physiological amplifier
• preptin
• T2DM

Introduction

Insulin is one of the most important hormones in regulating body metabolism. Insulin secretion and/or action are affected by several other peptides 1–3. For instance, amylin, a peptide co-secreted with insulin from pancreatic β-cells, affects both insulin secretion and sensitivity 4, 5. Pancreastatin, another peptide produced in β-cells from chromogranin-A, has also been shown to modulate insulin secretion 6.

Preptin is a peptide that consists of 34 amino acids and is derived from proinsulin-like growth factor II (pro-IGF-II) 7. It is synthesized by pancreatic β-cells and secreted together with insulin. Previous studies have demonstrated that preptin enhanced insulin secretion and that infusion of isolated pancreas with antipreptin antibodies significantly reduced glucose-mediated insulin secretion 8. These initial results obtained in animals led us to investigate plasma preptin levels in humans.

In the present study, we measured plasma preptin levels in subjects with different glucose tolerance. We also assessed the association between plasma preptin levels and body composition as well as several metabolic parameters in these subjects.

Key messages

• Preptin is a novel hormone that is co-secreted with insulin and amylin from the pancreatic β-cells.

• Preptin increases glucose-mediated insulin secretion and appears to act as a physiological amplifier of glucose-mediated insulin secretion.

• In this study, we investigate whether plasma preptin levels are different in non-diabetic subjects and patients with impaired glucose tolerance (IGT) or type 2 diabetes mellitus (T2DM).

Patients and methods

Subjects

Fifty patients with type 2 diabetes (T2DM) (22 men, 28 women; age 54±10 years), 56 subjects with impaired glucose tolerance (IGT) (17 men, 39 women; age 53±13 years), and 54 subjects with normal glucose tolerance (NGT) (16 men, 38 women; age 51±12 years) participated in the study. All study subjects were of Han Chinese origin and lived in the same region of the country. The NGT subjects had no family history of T2DM, no clinical evidence of any major disease, and were recruited from an unselected population who had undergone routine medical check-ups. The diagnosis of IGT and T2DM was based on oral glucose tolerance tests and World Health Organization criteria 9. Patients with type 1 diabetes and patients with macrovascular or microvascular complications, urinary tract infections, urolithiasis, liver cirrhosis, congestive heart failure, overt proteinuria, or other known major diseases were excluded. The patients with IGT and T2DM were newly diagnosed and had not been treated with oral antihyperglycaemic agents or insulin. None of the control subjects were taking any medication known to alter glucose tolerance. The study was approved by the human research ethics committee of the hospital, and informed consent was obtained from all participants.

Anthropometric parameters measured included the body mass index (BMI) and the waist-to-hip ratio (WHR). Waist and hip circumferences were measured to the nearest 0.1 cm at the narrowest point between the lowest rib and the uppermost lateral border of the right iliac crest. The hips were measured at their widest point. Blood pressure measurements were taken by trained nurses after the subjects had rested for 10 min.

Plasma biochemical parameters and preptin

Blood was drawn after an overnight fast for measurement of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), free fatty acids (FFA), glycosylated haemoglobin (HbA₁c) and glucose. Plasma was collected and kept at −80°C until assayed. The concentration of plasma preptin was determined by radioimmunoassay (RIA) (Phoenix Pharmaceuticals Inc., Belmont, CA, USA) using ¹²⁵I-labelled preptin as a tracer. The linear range of the assay was 0.1–12.8 ng/mL. The interassay and intra-assay coefficients of variation were 11% and 4.9%, respectively. Plasma insulin was measured in deproteinized serum by RIA using human insulin as standard (Linco, St Charles, MO, USA). FFA was measured in plasma with a kit (RANDOX Laboratories Ltd, Antrim, UK). Plasma glucose was assayed using the glucose oxidase method. HbA₁c was measured by isoelectric focusing. Plasma triglyceride, total cholesterol, LDL-C, and HDL-C were determined in duplicate with automated enzymatic assays (Hitachi7170A, Tokyo, Japan). The homeostasis model assessment of insulin resistance (HOMA_IR) and the homeostasis model assessment of insulin secretion (HOMA_IS) were calculated from fasting insulin and glucose levels with the following equations: HOMA_IR=insulin (µU/mL)× glucose (mmol/L)/22.5; and HOMA_IS=(20× insulin (µU/ml))/(fasting blood glucose (mmol/L)–3.5) 10. Body fat (FAT%) was calculated from the following equations: FAT%=1.2×BMI + 0.23×age – 16.2 (male), and 1.2×BMI + 0.23×age – 5.4 (female).

Statistical methods

The data are shown as the mean±standard deviation. All statistical analyses were performed using the SPSS 8.0 software (SPSS Inc., Chicago, IL, USA). Base-line characteristics of test and control subjects were compared by the one-way, Wilcoxon rank sum test, or the chi-square test. The general linear modelling function analysis was used to control for potential confounders. As the distributions of plasma insulin, HOMA_IS, and HOMA_IR values were skewed, logarithmically transformed values were used for statistical analysis. As our primary approach, we included plasma preptin concentration as continuous independent variables in the multivariable models.

The association of preptin with IGT and diabetes was examined by multivariate logistic regression analysis that contained 1) preptin, age, and gender; 2) preptin, age, gender, BMI, and WHR; 3) preptin, age, gender, BMI, WHR, and blood pressure; and 4) preptin, age, gender, BMI, WHR, blood pressure, lipid profile, FFA, blood glucose, HbA₁c, and insulin. We further divided the distribution of preptin in pooled data into tertiles and used the row mean scores difference and the Cochran-Armitage trend test to detect significant trends across increasing tertiles and to estimate the odds ratio of diabetes in each tertile using the lowest tertile as the reference category. Multivariate adjusted odds ratios are presented with 95% confidence intervals (CI).

Simple and multiple regression analyses were used to examine the association between plasma concentrations of preptin and the values of other biomarkers. All of the statistical analyses were two-sided, and a P-value < 0.05 was considered significant.

Results

The clinical characteristics of our subjects are shown in Table I. Diabetic subjects had higher fasting glucose, 2-h blood glucose after glucose overload (2hOGTT glucose), WHR, HOMA_IR, HOMA_IS, and HbA₁c levels than IGT and control subjects (P < 0.05 or P < 0.01). Their systolic blood pressure (SBP) and TG were also higher than the controls’ (P < 0.05 and P < 0.01). IGT subjects had higher BMI, FFA, 2hOGTT glucose, 2hOGTT insulin (2hIns), FFA, and HbA₁c than the controls (P < 0.05 and P < 0.01). Plasma preptin levels were found higher in patients with T2DM compared with IGT and controls (P < 0.05 and P < 0.01, Table I). There were significant differences between male and female subjects in plasma preptin levels (403±10 versus 432±10 ng/L, P < 0.05). Fasting plasma preptin correlated positively with diastolic blood pressure (DBP) (r = 0.20, P < 0.01), TG (r = 0.24, P < 0.01), TC (r = 0.24, P < 0.01), HDL-C (r = 0.18, P < 0.05), FFA (r = 0.21, P < 0.01), 2hOGTT glucose (r = 0.18, P < 0.05), HbA₁c (r = 0.19, P < 0.01), and HOMA_IR (r = 0.13, P < 0.05) in simple regression analysis of pooled data, while in multiple stepwise regression analysis, only DBP, TG, HDL-C, and FFA were independently related factors with plasma preptin levels (Table II). The multiple regression equation was: Y_preptin=147.959 + 1.876×DBP + 17.827×TG + 47.869×HDL-L + 59.964×FFA.

Table I.  Clinical characteristics and preptin levels of study subjects. Data are mean±standard deviation or frequency (percentage).

Factor	NGT	IGT	T2DM
n	54	56	50
Age (years)	51±12	53±13	54±10
Weight (kg)	58±11	62±10	61±10
BMI (kg/m^2)	23.4±3.8	25.0±3.6^a	24.4±3.3
WHR	0.86±0.05	0.87±0.06	0.90±0.06^b,d
FAT (%)	31.2±7.2	33.7±6.6	30.6±7.8^c
SBP (mmHg)	121±18	125±14	127±16^a
DBP (mmHg)	76±8	78±8	78±10
TG (mmol/L)	1.38±0.89	1.73±0.83	2.06±1.26^b
TC (mmol/L)	4.47±0.94	4.54±1.04	4.79±1.01
HDL-C (mmol/L)	1.21±0.28	1.21±0.52	1.18±0.31
LDL-C (mmol/L)	2.64±0.69	2.67±0.69	2.73±0.79
FFA (µmol/L)	0.60±0.26	0.73±0.35^a	0.69±0.22
FBG (mmol/L)	5.4±0.4	5.7±0.5	9.3±3.7^b,d
2hOGTT (mmol/L)	6.3±1.0	9.1±0.9^b	18.4±6.8^b,d
FIns (mU/L)	9.6±5.0	9.49±4.4	11.2±7.2
2hIns (mU/L)	41.6±3.1	81.5±8.6^b	44.3±4.1^d
HbA1c (%)	5.5±0.4	6.0±0.5^b	8.7±2.5^b,d
HOMAIR	2.3±0.2	2.4±0.2	4.5±0.3^b,d
HOMAIS	108±8.4	100±9.3	60±8.6^b,d
Preptin (ng/L)	398±13	416±13	456±14^b,c

^a P < 0.05 compared with NGT.

^b P < 0.01 compared with NGT.

^c P < 0.05 compared with IGT group.

^d P < 0.01 compared with IGT group.

NGT = normal glucose tolerance; IGT = impaired glucose tolerance; T2DM = type 2 diabetes mellitus; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; WHR = waist-to-hip ratio; FAT (%) = the percentage of fat in vivo; FBG = fasting blood glucose; 2hOGTT = 2-h blood glucose after glucose overload; HOMA_IR=homeostasis model assessment-insulin resistance index; HOMA_IS=homeostasis model assessment-β-cell insulin secretion index; HbA₁c = glycosylated haemoglobin; FFA = free fatty acids; TG = triglyceride; TC = total cholesterol; HDL = high-density lipoprotein; LDL = low-density lipoprotein; FIns = fasting insulin; 2hIns = 2-h insulin.

Table II.  Linear and multiple regression analysis of variables associated with plasma preptin levels in subjects studied.

	Simple		Multiple
Variable	Estimate	P-value	Estimate	P-value
Age	−0.039	0.619	——	——
BMI	0.094	0.228	——	——
WHR	−0.061	0.437	——	——
FAT%	0.119	0.128	——	——
Systolic BP	0.027	0.731	——	——
Diastolic BP	0.197	0.007	1.876	0.024
Fasting glucose	0.022	0.118	——	——
2hOGTT glucose	0.180	0.023	——	——
Insulin	0.087	0.265	——	——
2hIns	−0.007	0.933	——	——
HOMAIR	0.130	0.045	——	——
HOMAIS	−0.027	0.73	——	——
HbA1c	0.189	0.008	——	——
Total cholesterol	0.241	0.002	——	——
Triglyceride	0.244	0.001	17.827	0.022
HDL-C	0.191	0.014	47.869	0.009
LDL-C	0.119	0.127	——	——
FFA	0.212	0.004	59.964	0.023

In multiple linear stepwise regression analysis, values included for analysis were age, gender, body mass index (BMI), waist-to-hip ratio (WHR), systolic blood pressure, diastolic blood pressure, fasting glucose, 2-h blood glucose after glucose overload (2hOGTT glucose), glycosylated haemoglobin (HbA₁c), insulin, homeostasis model assessment of insulin resistance (HOMA_IR), homeostasis model assessment-β-cell insulin secretion index (HOMA_IS), free fatty acid (FFA), total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride, and FAT% (the percentage of fat in vivo), 2-hinsulin (2hIns).

The plasma preptin levels were significantly correlated with T2DM even after controlling for anthropometric variables, blood pressure, lipid profile, age, and gender (Table III). Increasing levels of preptin showed a significant linear trend and were independently associated with T2DM, especially when concentrations were analysed both by row mean scores differ and Cochran-Armitage trend test (Table IV).

Table III.  Association of plasma preptin with IGT and T2DM in fully adjusted models. Results of multivariate logistic regression analysis are presented as the odds ratio (OR) in type 2 diabetes mellitus (T2DM), and impaired glucose tolerance (IGT) status decrease in plasma preptin.

	IGT			T2DM
Model adjusted	OR	95% CI	P	OR	95% CI	P
Plasma preptin
Age	1.004	0.999–1.009	0.082	1.007	1.003–1.012	0.002
Age, BP	1.004	0.999–1.009	0.085	1.008	1.003–1.013	0.001
Age, BP, BMI, WHR	1.005	1.000–1.010	0.069	1.009	1.004–1.015	0.000
Age, BP, BMI, WHR, FAT%	1.005	1.000–1.010	0.066	1.011	1.005–1.016	0.000
Age, BP, BMI, WHR, FAT%, lipid profile	1.004	0.998–1.009	0.195	1.009	1.003–1.015	0.003

BMI = body mass index; WHR = waist-to-hip; BP = blood pressure; FAT% = the percentage of fat in vivo; lipid profile = including total cholesterol, triglyceride, low- and high-density lipoprotein cholesterol; NGT = normal glucose tolerance;IGT = impaired glucose tolerance;DM = diabetes mellitus

Table IV.  Row mean scores differ and Cochran-Armitage trend analysis of the impact of plasma preptin level on type 2 diabetes mellitus. Values shown are cut-offs of plasma preptin levels of all subjects. Adjusted for age, sex, body mass index, waist-to-hip ratio, systolic blood pressure, diastolic blood pressure, total cholesterol, triglyceride, and low- and high-density lipoprotein cholesterol.

	Quartiles of preptin
Factor	Q1	Q2	Q3	Q4	Chi-square	P
Cut-off preptin (ng/L)	<350	350–450	450–550	≥550	9.4178^a	0.0021^a
No. of NGT/IGT/DM	19/16/3	21/21/29	14/15/9	0/4/9	3.0785^b	0.0021^b

^a Row mean scores differ test.

^b Cochran-Armitage trend test.; NGT = normal glucose tolerance; IGT = impaired glucose tolerance; DM = diabetes mellitus.

Discussion

Type 2 diabetes mellitus (T2DM) is a genetically heterogeneous disorder involving resistance of peripheral tissues to insulin and reduced insulin secretion from pancreatic β-cells 11, 12. Despite extensive studies, the underlying mechanism of these defects remains elusive. Preptin is a recently isolated 34-amino acid peptide hormone that is co-secreted with insulin and amylin from the pancreatic β-cells 1, 8. Preptin corresponds to Asp69-Leu102 of pro-IGF-II. Recent data have shown that preptin may have relevance to glucose-mediated insulin secretion 8, 13. Buchanan et al. demonstrated that preptin increased glucose-stimulated insulin secretion from glucose-stimulated βTC6-F7 cells in a concentration-dependent and saturable manner 8. Preptin infusion into the isolated, perfused rat pancreas increased the second phase of glucose-mediated insulin secretion by 30%, while antipreptin immunoglobulin infusion decreased the first and second phases of insulin secretion by 29% and 26%, respectively 8. These findings suggest that preptin may be an amplifier of glucose-mediated insulin secretion. In the present study, we have determined plasma levels of preptin in patients with T2DM and IGT and in non-diabetic controls.

Fasting preptin levels were significantly higher in women compared to men.

The results of the current study also showed that fasting plasma preptin concentrations were significantly higher in patients with T2DM compared to patients with IGT or non-diabetic controls. Elevated preptin level in patients with T2DM might be the result of increased secretion and/or decreased preptin metabolism. This needs to be clarified by further studies.

Furthermore, we found that fasting plasma preptin was positively correlated with DBP, TG, TC, HDL-C, FFA, 2hOGTT glucose, HbA₁c, and HOMA_IR in simple regression analysis of pooled data, while in multiple stepwise regression analysis, only DBP, TG, HDL-C, and FFA were independently related factors with plasma preptin levels. On the other hand, plasma preptin did not correlate with BMI and other biomarkers, such as insulin and HOMA_IS. These results suggested a potential link between preptin and glucose-lipid metabolism and insulin resistance, but not with insulin secretion. Our finding is not in agreement with a recent report in which preptin was found to be correlated with glucose-mediated insulin secretion in perfused rat pancreas 8. Species differences and differences in study design may contribute to this disparity.

Row mean scores differ and Cochran-Armitage trend test showed that plasma preptin concentration was significantly associated with type 2 diabetes. This suggests that preptin may play a role in the pathogenesis of insulin resistance and T2DM.

Some limitations of this study need to be considered. The cross-sectional design limits our ability to infer a causal relationship between increased plasma preptin level and T2DM. Our analyses are based on single measurements of blood preptin, which may not reflect the relationship over time. It would be interesting to measure serial changes of plasma preptin levels in prediabetic and diabetic subjects to further clarify the role of preptin in the pathogenesis of T2DM.

In conclusion, our results of elevated plasma preptin levels in newly diagnosed patients with T2DM suggested that preptin may play a role in the pathogenesis of T2DM. However, this needs to be clarified in further studies.

Acknowledgements

This work was supported by research grants from the National Natural Science Foundation of China (30771037), Chongqing Municipal Education Commission (KJ 050304), and the National Institutes of Health (R01-DK 58895 to G.B.).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. GangYi Yang and Ling Li contributed equally to this project.