Original Article

|
doi: 10.1111/j.1365-2796.2011.02465.x

Type 2 diabetes mellitus interacts with obesity and common

variations in PLTP to affect plasma phospholipid transfer
protein activity
R. P. F. Dullaart1, M. Vergeer2, R. de Vries1, P. J. W. H. Kappelle1 & G. M. Dallinga-Thie2
From the 1Department of Endocrinology, University Medical Center Groningen, University of Groningen, Groningen; and 2Departments of Vascular
and Experimental Vascular Medicine, Academic Medical Center Amsterdam, Amsterdam, The Netherlands

Abstract. Dullaart RPF, Vergeer M, de Vries R, Kappelle

PJWH, Dallinga-Thie GM (University Medical Center
Groningen, University of Groningen, Groningen; and
Academic Medical Center Amsterdam, Amsterdam;
The Netherlands). Type 2 diabetes mellitus interacts
with obesity and common variations in PLTP to affect
plasma phospholipid transfer protein activity.
J Intern Med 2012; 271: 490498.
Background. Phospholipid transfer protein (PLTP) is an
emerging cardiometabolic risk marker that is important in high-density lipoprotein (HDL) and triglyceride metabolism. Plasma PLTP activity is elevated in
type 2 diabetes mellitus, whereas glucose may regulate PLTP gene transcription in vitro. Of interest, common PLTP variations that predict cardiovascular disease have been identified recently. We investigated
whether the diabetic state is able to amplify relationships between obesity and PLTP gene variations with
circulating PLTP levels.
Subjects and methods. Plasma PLTP activity (using a
phospholipid vesiclesHDL system), PLTP gene score
[number of PLTP activity-decreasing alleles based on
two tagging polymorphisms (rs378114 and rs6065904)] and waist circumference were determined in
two Dutch cohorts comprising 237 patients with type
2diabetesand78controlsubjects.
Results. Patients with diabetes were more obese
(P < 0.001 for prevalence of increased waist circumference) and had 13% higher plasma PLTP activity
(P < 0.001). PLTP gene score was not different in diabetic and control subjects (P = 0.40). PLTP activity

Introduction
Phospholipid transfer protein (PLTP) belongs to the lipid transfer lipopolysaccharide binding protein family and is expressed in several tissues and cell sys-

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2011 The Association for the Publication of the Journal of Internal Medicine

was highest in patients with diabetes with an enlarged waist and lowest in control subjects with a normal waist circumference (P < 0.001). Multiple linear
regression analysis revealed a positive interaction between diabetes status and waist circumference on
PLTP activity (b = 0.200, P = 0.005). Furthermore,
diabetes status (b = )0.485, P = 0.046) or HbA1c
(b = )0.240, P = 0.035) interacted with PLTP gene
score to affect PLTP activity.
Conclusions. Type 2 diabetes and enlarged waist circumference interact to impact on plasma PLTP activity.
Diabetes may also amplify the association between
plasma PLTP activity and common PLTP gene variations. Our findings support the hypothesis that diabetesenvironment and diabetesgene interactions
govern plasma PLTP activity.
Keywords: obesity, phospholipid transfer protein,
phospholipid transfer protein gene, type 2 diabetes
mellitus, waist circumference.
Abbreviations: anova, analysis of variance; apo, apolipoprotein; AU, arbitrary unit; BMI, body mass index;
CETP, cholesteryl ester transfer protein; CRP, highsensitivity C-reactive protein; DALI study, Diabetes
Atorvastatin Lipid Intervention study; EDTA, ethylenediaminetetraacetic acid; HbA1c, glycated haemoglobin; HDL, high-density lipoprotein; MetS, metabolic syndrome; NCEP-ATP-III, national cholesterol
education programme adult treatment panel III;
PLTP, phospholipid transfer protein; SNP, singlenucleotide polymorphism; VLDL, very low-density
lipoprotein

tems including liver, macrophages and adipose

tissue [14]. PLTP is a high-density lipoprotein (HDL)associated lipid transfer protein that is able to transfer phospholipids towards HDL during lipolysis of triglyceride-rich lipoproteins and to remodel HDL into

R. P. F. Dullaart et al.

Diabetes interacts with obesity and PLTP variation to affect PLTP

different sized (both small and large) particles [14].

Studies in mice support the notion that PLTP increases hepatic very low-density lipoprotein (VLDL)
production [5, 6]. Moreover, PLTP exchanges atocopherol between lipoproteins and is associated
with pro-inflammatory proteins in plasma [7, 8].
Although the potential role of PLTP in the development of atherosclerosis has not been unequivocally
established, available evidence from human studies
mostly supports the possibility that elevated plasma
PLTP activity is related positively to (subclinical) atherosclerosis [2, 3, 912]. Genetic variations in PLTP
that are associated with plasma PLTP activity, lipid
phenotype and obesity-related variables, as well as
cardiovascular disease, have been identified recently
[1316].
During the past few years, evidence has accumulated
to support the possibility that plasma PLTP activity is
elevated in insulin-resistant states, such as type 2
diabetes mellitus, obesity and metabolic syndrome
(MetS) [3, 4, 1722]. Circulating PLTP activity is
closely related to glucose and lipid homoeostasis.
Plasma PLTP increases in parallel with increments in
circulating free fatty acid levels and decreases in
response to acipimox administration, insulin infusion and weight loss [3, 4, 18, 19, 2327]. Of note, it
has been reported that high glucose stimulates PLTP
promoter activity in vitro, possibly via nuclear hormone receptor-dependent mechanisms [28]. Taken
together, these findings raise the hypothesis that the
diabetic state may influence relationships of obesity
and common genetic variations in PLTP with plasma
PLTP activity levels in humans.
Given an emerging role of PLTP as a possible cardiometabolic risk factor, we sought to determine
whether the diabetic state interacts with obesity to
impact on plasma PLTP activity. We also investigated
whether the presence of diabetes mellitus may amplify the observed association between plasma PLTP
activity and common PLTP gene variations. These
possibilities were tested in two cohorts, comprising
type 2 diabetic and control subjects, in which we previously identified two PLTP-tagging single-nucleotide
polymorphisms (SNPs) that are associated with plasma PLTP activity [16].
Subjects and methods
Subjects included in the present study were participants from two previous studies: the Groningen
casecontrol study, which was originally designed to

examine whether intimamedia thickness is related

to plasma PLTP activity [10]; and the Diabetes Atorvastatin Lipid Intervention (DALI) study, a doubleblind, placebo-controlled, randomized, multicentre
study, which evaluated the effect of atorvastatin on lipid metabolism, endothelial function, coagulation
and inflammatory markers [29, 30]. The protocols
were approved by the medical ethics committees of
the participating centres. All participants provided
written informed consent.
The inclusion and exclusion criteria for the Groningen and DALI studies have been described in detail
elsewhere [10, 29, 30]. In brief, the participants of the
Groningen study were recruited by advertisement in
local newspapers. Subjects with and without previously diagnosed type 2 diabetes mellitus (defined
according to World Health Organization criteria) were
included. Smokers and subjects using lipid-lowering
drugs, insulin and thiazolidones were excluded from
the Groningen cohort. Patients with diabetes participating in the DALI study had a diabetes duration of at
least 1 year, were free of clinically manifest cardiovascular disease at entry and had a glycated haemoglobin (HbA1c) level <10%. When applicable, lipidlowering drugs were withdrawn at least 8 weeks before the start of the run-in phase. Premenopausal
women were excluded. Plasma lipid inclusion criteria
were levels of fasting total cholesterol between 4.0
and 8.0 mmol L)1 and triglycerides between 1.5 and
6.0 mmol L)1. Further exclusion criteria for participation in either cohort were clinically manifest cardiac disease (history of myocardial infarction, coronary intervention or major ischaemia on an
electrocardiogram) and pregnancy. For the present
study, we included all subjects who had complete
data with respect to obesity measures and PLTP gene
score. Consequently, a total of 78 control subjects
and 67 patients with diabetes from the Groningen
cohort and 170 patients with diabetes from the DALI
cohort were included.
Blood pressure was measured using routine clinical procedures. Body mass index (BMI) was calculated as the ratio between weight and height
squared (in kg m)2). Waist circumference was measured as the smallest girth between the rib cage
and iliac crest, and waist hip ratio was measured
as the waist circumference divided by the hip circumference. The revised national cholesterol education programme adult treatment panel III (NCEPATP-III) criteria were applied for classification of
MetS, and a waist circumference >102 cm for men
and >88 cm for women [31] was used to indicate
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Diabetes interacts with obesity and PLTP variation to affect PLTP

central obesity. Venous blood was obtained after

an overnight fast.
Laboratory analyses
Venous blood was collected in ethylenediaminetetraacetic acid (EDTA)-containing tubes (1.5 mg mL)1),
which were placed on ice immediately. Plasma was
obtained by centrifugation at 1400 g for 15 min at
4 C. Plasma glucose and HbA1c were measured
shortly after blood collection. Plasma samples for
measurement of PLTP activity, lipids and apolipoproteins were stored at )80 C until analysis. In both
studies, total levels of cholesterol and triglycerides
were measured using routine enzymatic colorimetric
assays, and HDL cholesterol was measured with a direct homogeneous method [10, 29]. Non-HDL cholesterol was calculated as the difference between total
and HDL cholesterol. High-sensitivity C-reactive protein (CRP) was assayed by nephelometry (Dade Behring, Marburg, Germany) in the Groningen cohort
[22] and by enzyme immunoassay (Dako, Copenhagen, Denmark) in the DALI study [32]. Apolipoprotein
E (apoE) was measured by nephelometry (Wako,
Osaka, Japan) [30].
Plasma PLTP activity was measured with a phospholipid vesiclesHDL system, using [14C]-labelled
dipalmitoyl phosphatidylcholine as previously described [10, 32, 33]. Briefly, plasma samples (1 lL)
were incubated with [14C]-labelled phosphatidylcholine vesicles and excess pooled normal HDL for
45 min at 37 C. The method is specific for PLTP
activity; the phospholipid transfer-promoting property of cholesteryl ester transfer protein (CETP) does
not interfere with the assay. Levels of plasma PLTP
activity vary linearly with the amount of plasma
added to the incubation system. PLTP activity was related to the activity in human reference pooled plasma and was expressed in arbitrary units (AU; 100 AU
corresponds to 13.6 lmol phosphatidylcholine
transferred per mL per hour). The inter-assay coefficient of variation of the measurement of PLTP activity
is 5%.
Variation in PLTP was determined using a gene score
that is based on two common PLTP-tagging SNPs
[rs378114
(c.330)117G>A)
and
rs6065904
(c.705+256C>T)], as described in detail elsewhere
[16]. The PLTP score represents the number (from 0 to
4) of plasma PLTP activity-decreasing alleles (for
rs378114: presence of 0, 1 or 2 G alleles; for
rs6065904: presence of 0, 1 or 2 T alleles) and has
been found to inversely predict plasma PLTP activity
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in both the Groningen and the DALI cohorts [16].

A higher PLTP gene score is also linearly associated
with less PLTP mRNA expression in human liver
specimens [16].
Statistical analysis
spss 18 was used for data analysis. Results are expressed as mean SD or median (interquartile
range) unless stated otherwise. Differences in variables between diabetic and nondiabetic subjects
were determined by unpaired t-tests or MannWhitney U tests where appropriate. Differences between
diabetic and nondiabetic subjects with and without
an enlarged waist circumference were determined by
one-way analysis of variance (anova) with subsequent Bonferroni correction for multiple comparisons. Logarithmically transformed triglyceride values were used in regression analyses. Betweengroup differences in proportions were assessed by
chi-square analysis. Multivariate linear regression
analysis was applied to determine differences between diabetic and control subjects after adjustment
for age and sex. Multivariate linear regression analyses were also carried out to determine the independent contribution of variables to plasma PLTP activity. Additionally, interaction terms between diabetes
status, HbA1c, PLTP gene score and obesity variables
were calculated. To this end, a distribution of centred
to the mean was made for continuous variables by
subtracting the individual value of the variable of
interest from its group mean value. Interaction terms
were considered to be statistically significant at twosided P-values <0.1 [34]. Otherwise, the level of significance was set at two-sided P < 0.05.
Results
A total of 237 patients with diabetes and 78 control
subjects were included in the study. Patients with
diabetic were older, had higher systolic and diastolic
blood pressure and were more obese than control
subjects, but the male female ratio was similar between the groups (Table 1). Average diabetes duration
was 9 7 years. Fifty-two patients with diabetes
(22%) were current smokers (P < 0.001 compared to
control subjects, because of exclusion of smokers in
the Groningen study). Nineteen of the patients with
diabetes were treated with diet alone. Oral blood glucose-lowering drugs alone (metformin and or sulfonylurea) were used by 130 patients, whereas 43 patients used insulin alone and 45 used insulin in
combination with oral blood glucose-lowering drugs
(mainly metformin). A total of 210 (89%) patients with

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Diabetes interacts with obesity and PLTP variation to affect PLTP

Table 1 Clinical characteristics, plasma lipids, phospholipid transfer protein (PLTP) activity and variation in PLTP in 237 type 2 diabetic and 78 control subjects
Type 2 diabetic

Control

subjects (n = 237)

subjects (n = 78)

Age (years)

59 8

56 9

Sex (m f)

129 108

44 34

Systolic blood pressure (mmHg)

151 21

130 18

<0.001

<0.001

Diastolic blood pressure (mmHg)

87 9

82 10

<0.001

<0.001

BMI (kg m)2)

30.2 5.1

26.0 4.1

<0.001

<0.001

Waist circumference (cm)

105 14

90 13

<0.001

<0.001

Enlarged waist

174 (73%)

18 (23%)

<0.001

P-value

P-value*

0.003
0.87

Waist hip ratio

0.98 0.10

0.90 0.08

<0.001

<0.001

Glucose (mmol L)1)

10.0 3.1

5.7 1.0

<0.001

<0.001

HbA1c (%)

7.9 1.3

5.4 0.4

<0.001

<0.001

CRP (mg L)1)

2.77 (1.295.43)

1.36 (0.512.61)

<0.001

<0.001

Total cholesterol (mmol L)1)

5.80 0.96

5.70 1.03

0.43

0.86

Non-HDL cholesterol (mmol L)1)

4.70 0.99

4.22 1.11

<0.001

0.004

HDL cholesterol (mmol L )

1.11 0.31

1.48 0.42

<0.001

<0.001

Triglycerides (mmol L)1)

2.20 (1.762.97)

1.26 (0.892.02)

<0.001

<0.001

ApoE (g L)1)

0.041 0.011

0.040 0.010

PLTP activity (AU)

107 17

PLTP gene score

)1

0.027

0.021

95 11

<0.001

<0.001

0: n = 16

0: n = 1

0.40

1: n = 59

1: n = 20

2: n = 92

2: n = 30

3: n = 51

3: n = 21

4: n = 19

4: n = 6

BMI, body mass index; CRP, high-sensitivity C-reactive protein; HDL, high-density lipoprotein; SNPs, single-nucleotide
polymorphisms.
Data are given as mean (SD), median (interquartile range) or number (%).
*P-values for difference between type 2 diabetic and control subjects adjusted for age, sex and smoking. PLTP gene score: derived
from two tagging SNPs (rs378114 and rs6065904) associated with plasma PLTP activity.

diabetes fulfilled the criteria for MetS compared to 21

control subjects (27%, P < 0.001).
As shown in Table 1, systolic and diastolic blood pressure, BMI, waist circumference, waist hip ratio, fasting plasma glucose, HbA1c and CRP levels were higher in patients with diabetes than in control subjects.
Plasma total cholesterol was similar between diabetic
and control subjects. Non-HDL cholesterol, triglycerides and apoE levels were higher, whereas HDL cholesterol was lower in patients with diabetes (Table 1).
All these differences remained significant after
adjustment for age, sex and smoking. Plasma PLTP
activity was on average 13% higher in patients with
diabetes; this difference remained significant after

adjustment for age, sex, smoking and study cohort

(P < 0.001). The distribution of PLTP gene score was
not different in diabetic compared to control subjects
(Table 1; median gene score (interquartile range): 2
(13) in both diabetic and control subjects).
As shown in Fig. 1, plasma PLTP activity was higher
in patients with diabetes with an enlarged waist
(n = 174), compared to patients with diabetes with a
normal waist circumference (n = 63), control
subjects with an enlarged waist (n = 18) and control
subjects with a normal waist circumference (n = 60)
(anova, P < 0.001). These differences remained significant after adjustment for age, sex, smoking and
study cohort (anova, P < 0.001), after additional
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R. P. F. Dullaart et al.

Diabetes interacts with obesity and PLTP variation to affect PLTP

P < 0.001
P = 0.021

120

PLTP activity (AU)

P < 0.001

100

80
0

Fig. 1 Plasma phospholipid transfer protein activity according to diabetes status and enlarged waist circumference.
Data are means SEM. (a) Nondiabetic subjects with normal
waist (n = 60); (b) nondiabetic subjects with enlarged waist
(n = 18); (c) type 2 diabetic subjects with normal
waist (n = 63); (d) type 2 diabetic subjects with enlarged
waist (n = 174). anova, P < 0.001.

adjustment for PLTP gene score (anova, P < 0.001)

and also after excluding the 88 patients with diabetes
using insulin (n = 227, anova, P < 0.001).
Multiple linear regression analysis was carried out to
determine the extent to which plasma PLTP activity
was governed by PLTP gene score, diabetes status
and obesity. In initial models that included either
BMI or waist circumference, it was found that plasma
PLTP activity was related to BMI (b = 0.179,
P = 0.002) or waist circumference (b = 0.255,
P < 0.001), independently of the presence of diabetes
(P < 0.001, data not shown). When BMI and waist circumference were included together, PLTP activity
was found to be determined by waist circumference
alone (b = 0.258, P = 0.002) and not by BMI
(b = 0.01, P = 0.95). Therefore, waist circumference
was selected as the primary obesity variable for evaluating diabetesobesity interactions to impact on plasma PLTP. As shown in Table 2, plasma PLTP activity
was independently and inversely related to PLTP gene
score and positively related to diabetes status and enlarged waist circumference after adjustment for age,
sex, smoking, CRP and study cohort (model 1). We
next examined whether the presence of diabetes
modified the relationship between plasma PLTP
activity and enlarged waist circumference. As shown
in model 2, there was a positive interaction between
diabetes status and waist circumference on PLTP
activity. When HbA1c, non-HDL cholesterol, triglycerides and apoE levels were added to the models, PLTP
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activity remained independently related to enlarged

waist (model 3), and there was still an interaction between diabetes status and waist circumference on
PLTP activity (model 4). When patients with diabetes
using insulin were excluded, the interaction between
diabetes status and waist circumference on PLTP
activity also remained significant (b = 0.213, P =
0.013 and b = 0.180, P = 0.038 without and with
additional adjustment for HbA1c, non-HDL cholesterol, triglycerides and apoE, respectively; data not
shown). In further analyses, there was also an interaction between diabetes status and waist hip ratio
on PLTP activity (model 2: b = 0.219, P < 0.001; model 4: b = 0.215, P = 0.001, data not shown).
Finally, we determined whether diabetes status and
the degree of chronic hyperglycaemia, as reflected by
the HbA1c level, interacted with PLTP gene score to affect plasma PLTP activity (Table 3). Remarkably, diabetes status showed an interaction with PLTP gene
score on PLTP activity after adjustment for age, sex,
smoking, CRP, study cohort and enlarged waist circumference (model 1), as well as after additional
adjustment for non-HDL cholesterol, triglycerides
and apoE (model P < 0.001 compared to control subjects, because of exclusion of smokers in the Groningen study3). Thus, the presence of diabetes amplified
the association between plasma PLTP activity and the
number of PLTP activity-decreasing alleles. This
interaction was essentially unaltered after exclusion
of patients with diabetes using insulin (model 1:
b = )0.449, P = 0.085; model 3: b = )0.418, P =
0.098, data not shown). Likewise, interactions
between the HbA1c level and the PLTP gene score on
PLTP activity were observed in analysis without (model 2; graphically depicted in Fig. 2) and with (model 4)
additional adjustment for non-HDL cholesterol, triglycerides and apoE.
Discussion
In the present study, we have demonstrated for the
first time that the presence of diabetes strengthens
the relationship between plasma PLTP activity and
enlarged waist circumference independently of the
influence of common PLTP variations. Furthermore,
our results suggest that the association between
plasma PLTP activity and variations in PLTP is modified by the diabetic state, meaning that plasma PLTP
activity is more strongly affected in individuals with
diabetes mellitus for a given PLTP gene score. Such
an interaction was also observed when using the
HbA1c level as a measure of the degree of chronic hyperglycaemia. Taken together, our findings support

R. P. F. Dullaart et al.

Diabetes interacts with obesity and PLTP variation to affect PLTP

Table 2 Multiple linear regression models showing independent relationships between plasma phospholipid transfer protein (PLTP)
activity and PLTP gene score, diabetes status and waist circumference, and interaction between diabetes status and enlarged
waist in 237 patients with type 2 diabetes and 78 control subjects
Model 1

Model 2
P-value

b
Age

)0.120

Sex (men vs. women)

Model 3
P-value

0.019

)0.098

Model 4
P-value

0.057

)0.10

P-value

0.053

)0.087

0.094

0.21

)0.117

0.043

)0.073

0.162

)0.137

0.015

)0.067

Smoking (yes no)

0.127

0.022

0.123

0.025

0.128

0.021

Ln CRP

0.035

0.54

0.01

0.94

0.001

0.99

)0.022

0.71

0.002

0.98

DALI vs. Groningen cohort

)0.152

0.026

)0.129

0.058

)0.011

0.90

PLTP gene score

<0.001

0.122

0.028

)0.280

<0.001

)0.284

<0.001

)0.265

)0.271

<0.001

Diabetes mellitus (yes no)

0.155

0.021

0.173

0.010

0.101

0.17

0.113

0.125

Enlarged waist circumference (yes no)

0.208

0.001

0.076

0.32

0.176

0.006

0.070

0.38

0.200

0.005

0.162

0.028

Diabeteswaist circumference interaction

HbA1c

0.174

0.040

0.183

0.030

)0.021

0.74

)0.026

0.68

Ln triglycerides

0.059

0.44

0.039

0.61

ApoE

0.184

0.004

0.189

0.003

Non-HDL cholesterol

CRP, high-sensitivity C-reactive protein; DALI, Diabetes Atorvastatin Lipid Intervention; HDL, high-density lipoprotein; SNPs,
single-nucleotide polymorphisms.
PLTP gene score: derived from two tagging SNPs (rs378114 and rs6065904) associated with plasma PLTP activity. b: standardized regression coefficient. Logarithmically transformed values of triglycerides and C-reactive protein (CRP) levels are used in
the analyses.
All models are adjusted for age, sex, smoking, CRP and study cohort (DALI cohort, Groningen cohort).
Other statistical determinants of PLTP activity included in the multivariate linear regression analyses:
Model 1: diabetes status, enlarged waist circumference, PLTP gene score;
Model 2: diabetes status, enlarged waist circumference, PLTP gene score and interaction between waist circumference and diabetes status;
Model 3: diabetes status, enlarged waist circumference, PLTP gene score, glycated haemoglobin (HbA1c), non-high-density lipoprotein (HDL) cholesterol, ln triglycerides and apolipoprotein E (apoE);
Model 4: diabetes status, enlarged waist circumference, PLTP gene score, HbA1c, non-HDL cholesterol, ln triglycerides, apoE
and interaction between waist circumference and diabetes status.

the hypothesis that diabetesenvironment and diabetesgene interactions are involved in the regulation
of circulating PLTP activity.
In this study, plasma PLTP activity was more strongly
related to waist circumference than to BMI. Of note,
both BMI and waist circumference are correlated with
visceral abdominal as well as with subcutaneous
abdominal fat tissue [35]. Therefore, these obesity
measures do not fully discriminate between relationships between plasma PLTP activity and either obesity per se or a centrally distributed adiposity pattern.
Of further note, PLTP is expressed in visceral as well
as in subcutaneous adipose tissue, although PLTP
mRNA has been found to be related positively to BMI
in subcutaneous but not in visceral adipose tissue [3,
36]. Nonetheless, a more centrally distributed obesity

pattern may be regarded as a major driving force leading to increasing free fatty acid delivery to the liver
and hence to disturbances in hepatic lipid homoeostasis [37], which are likely to include elevations in
plasma PLTP activity [22]. We did not specifically assess visceral and subcutaneous fat areas. Yet, we
consider the present finding that the diabetic state
interacted positively with waist circumference to affect PLTP activity robust, as this interaction was independent of plasma CRP, which is known to be elevated in (central) obesity [38], and was replicated
using the waist hip ratio as a measure of obesity.
The present observation that the diabetic state and,
in alternative analyses, the HbA1c level interact with
the PLTP gene score to modulate circulating PLTP
activity clearly supports the hypothesis that the ex 2011 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2012, 271; 490498

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Diabetes interacts with obesity and PLTP variation to affect PLTP

Table 3 Multiple linear regression models showing relationships between plasma phospholipid transfer protein (PLTP) activity and
PLTP gene score, diabetes status and HbA1c, and interactions between PLTP gene score and diabetes status and HbA1c in 78
nondiabetic patients and 237 type 2 diabetic subjects
Model 1

Model 2

P-value

Age

)0.125

0.014

)0.130

Sex (men vs. women)

Model 3
P-value

Model 4

P-value

0.027

)0.106

0.042

)0.106

P-value
0.041

0.22

)0.068

0.21

)0.072

0.167

)0.075

0.130

)0.066

Smoking (yes no)

0.126

0.022

0.127

0.020

0.122

0.027

0.125

Ln CRP

0.047

0.41

0.025

0.66

0.021

0.71

0.007

0.90

0.026

)0.064

0.83

)0.080

0.28

)0.004

0.96

)0.272

)0.263

<0.001

DALI vs. Groningen cohort

)0.152

PLTP gene score

0.153

0.49

Diabetes mellitus (yes no)

0.370

0.004

HbA1c
DiabetesPLTP gene score interaction

)0.485

0.046

0.209

0.001

HbA1cPLTP gene score interaction

Enlarged waist circumference (yes no)

<0.001

0.104

0.64

0.101

0.14

0.356

0.006

0.393

0.002

)0.240

0.035

0.196

0.001

)0.423

0.084

0.192

0.002

0.024

0.104

0.16

0.355

0.008

)0.203

0.078

0.182

0.004

)0.006

0.92

)0.015

0.81

Ln triglycerides

0.064

0.41

0.059

0.44

ApoE

0.178

0.005

0.177

0.005

Non-HDL cholesterol

DALI, Diabetes Atorvastatin Lipid Intervention; HDL, high-density lipoprotein; SNPs, single-nucleotide polymorphisms.
PLTP gene score: derived from two tagging SNPs (rs378114 and rs6065904) associated with plasma PLTP activity. b: standardized regression coefficient. Logarithmically transformed values of triglycerides and C-reactive protein (CRP) levels are used in
the analyses.
All models are adjusted for age, sex, smoking, CRP and study cohort (DALI cohort, Groningen cohort).
Other statistical determinants of PLTP activity included in the multivariate linear regression analyses:
Model 1: PLTP gene score, diabetes status, enlarged waist circumference and interaction between PLTP gene score and diabetes
status.
Model 2: PLTP gene score, diabetes status, HbA1c, enlarged waist circumference and interaction of PLTP gene score with HbA1c.
Model 3: PLTP gene score, diabetes status, enlarged waist circumference, non-HDL cholesterol, ln triglycerides, apolipoprotein
E (apoE) and interaction between PLTP gene score and diabetes status.
Model 4: PLTP gene score, diabetes status, glycated haemoglobin (HbA1c), enlarged waist circumference, non-high-density lipoprotein (HDL) cholesterol, ln triglycerides, apoE and interaction between PLTP gene score and HbA1c.

tent to which genetic variation in PLTP affects circulating PLTP activity levels may be amplified by the degree of chronic hyperglycaemia in vivo. The PLTP gene
score used here is strongly associated with hepatic
mRNA expression [16]. Earlier studies have demonstrated that high glucose stimulates PLTP promoter
activity in vitro [28]. Accordingly, PLTP mRNA expression is modulated by nuclear receptors, such as the
peroxisome proliferator-activated receptor, the liver
X-receptor and the farnesoid X-receptor [28, 39, 40].
In the present study, we did not aim to delineate the
mechanisms whereby exposure to hyperglycaemia
may modify PLTP expression.
Several other aspects of our study should be addressed. In view of the possibility that PLTP activity is
higher in cigarette smokers [41], and given the exclu496

2011 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2012, 271; 490498

sion of smokers in the Groningen cohort, we took

account of smoking status in the multivariable
analyses. Indeed, current smoking was found to be
associated with higher plasma PLTP levels in this
study. Furthermore, the notion that PLTP has
pro-inflammatory properties [8, 42] provides an additional argument to evaluate whether or not interactions between diabetes and obesity on circulating
PLTP activity are confounded by enhanced chronic
subclinical inflammation. We also included the plasma level of apoE, which has been considered to be
involved in activating PLTP [30, 43] and in stimulating the ability of PLTP to remodel HDL particles [44]. A
strong correlation between plasma PLTP activity and
apoE was observed that was independent of plasma
apoB-containing lipoproteins and obesity. Of note,
interactions between diabetes status and obesity and

R. P. F. Dullaart et al.

Diabetes interacts with obesity and PLTP variation to affect PLTP

actions are involved in the regulation of plasma

activity of PLTP, an emerging cardiometabolic risk
factor. The present observations impact on the supposition that improving metabolic control and weight
reduction may coordinately and perhaps even synergistically affect lipoprotein metabolism via PLTP
regulation.

130

110

Conflict of interest statement

100

No conflicts of interest were declared.

y

9.30
12
.20
8.10
9.2
0
6.80
8.0
0
5.60
6.7
0
4.50
5.5
0

or

90

ca

%)

2
3
gene
score

c(

1
PLTP

A1

teg

80

Hb

PLTP activity (AU)

120

Fig. 2 Graphical presentation of the interaction between the

HbA1c level (presented in categories) and PLTP gene score
[number of phospholipid transfer protein (PLTP) activitydecreasing alleles] on plasma PLTP activity (data derived
from Table 3, model 2; interaction term: b = )0.240, P =
0.035).

between diabetes status and PLTP gene score to affect

plasma PLTP activity were not essentially influenced
by inclusion of apoE in the multivariable analysis.
The current findings are based on an analysis of two
cohorts combined. In the Groningen cohort, insulintreated patients with diabetes were excluded [10],
whereas a considerable number of DALI participants
used insulin [29]. Furthermore, mild to moderate hypertriglyceridaemia was a selection criterion for the
DALI study [29], which probably explains the high
prevalence of enlarged waist circumference among
patients with diabetes. For these reasons, we took account of the origin of the study cohort in all multivariable analyses. In this regard, it is relevant that all
interactions remained essentially unaltered after
exclusion of insulin-treated patients. Moreover, the
analyses concerning PLTP gene variation that we
used here are based on a recently described PLTP
gene score [16]. This approach was chosen in the
expectation that statistical power to detect diabetes
statusPLTP gene and HbA1cPLTP interactions was
better compared to that using each PLTP gene variation separately. Finally, it should be appreciated that
the cross-sectional design is a limitation of the present study.
In conclusion, our findings support the hypothesis
that diabetesenvironment and diabetesgene inter-

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Correspondence: R. P. F. Dullaart, MD, PhD, Department of Endocrinology, University Medical Centre Groningen, PO Box 30001,
9700 RB Groningen, The Netherlands.
(fax: +31503619392; e-mail: r.p.f.dullaart@int.umcg.nl).

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