The PNPLA3 rs738409 G-allele associates with reduced fasting serum triglyceride and serum cholesterol in Danes with impaired glucose regulation.
ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is a common condition, associated with hepatic insulin resistance and the metabolic syndrome including hyperglycaemia and dyslipidemia. We aimed at studying the potential impact of the NAFLD-associated PNPLA3 rs738409 G-allele on NAFLD-related metabolic traits in hyperglycaemic individuals.
The rs738409 variant was genotyped in the population-based Inter99 cohort examined by an oral glucose-tolerance test, and a combined study-sample consisting of 192 twins (96 twin pairs) and a sub-set of the Inter99 population (n = 63) examined by a hyperinsulinemic euglycemic clamp (n(total) = 255). In Inter99, we analyzed associations of rs738409 with components of the WHO-defined metabolic syndrome (n = 5,847) and traits related to metabolic disease (n = 5,663). In the combined study sample we elucidated whether the rs738409 G-allele altered hepatic or peripheral insulin sensitivity. Study populations were divided into individuals with normal glucose-tolerance (NGT) and with impaired glucose regulation (IGR).
The case-control study showed no associations with components of the metabolic syndrome or the metabolic syndrome. Among 1,357 IGR individuals, the rs738409 G-allele associated with decreased fasting serum triglyceride levels (per allele effect(β) = -9.9% [-14.4%;-4.0% (95% CI)], p = 5.1×10(-5)) and fasting total cholesterol (β = -0.2 mmol/l [-0.3;-0.01 mmol/l(95% CI)], p = 1.5×10(-4)). Meta-analyses showed no impact on hepatic or peripheral insulin resistance in carriers of the rs738409 G-allele.
Our findings suggest that the G-allele of PNPLA3 rs738409 associates with reduced fasting levels of cholesterol and triglyceride in individuals with IGR.
- SourceAvailable from: Luca Valenti[Show abstract] [Hide abstract]
ABSTRACT: Evidence relating dietary patterns to obesity and related disorders such as non-alcoholic fatty liver disease (NAFLD) is limited in pediatric age. Aim of this study was to analyze the association between dietary patterns, obesity and development of severe steatosis and the metabolic syndrome in a series of children and adolescents referred for suspected NAFLD, and the interaction with the rs738409 I148M PNPLA3 polymorphism. Two hundred patients (112 females) had completed a food frequency and demographic questionnaire. Nearly 58 % were obese, and 32 % were overweight. Mild, moderate, and severe fatty liver was present in 60 (30 %), 87 (44 %), and 51 (26 %) participants, respectively. A great proportion of overweight/obese children and adolescents reported a correct dietary pattern. At multivariate ordinal regression analysis considering demographic, anthropometric, genetic, and behavioral determinants, the major determinant of steatosis severity was PNPLA3 I148M genotype (p < 0.0001), followed by older age (p = 0.017), higher waist circumference (p = 0.016), and less time spent practising physical exercise (p = 0.034). Furthermore, there was a significant interaction between PNPLA3 I148M and intake of sweetened beverages (p = 0.033) and of vegetables (p = 0.038). In conclusion, although dietary pattern was reportedly correct in at-risk overweight adolescents with NAFLD, we report a novel interaction between PNPLA3 I148M and dietary components with the severity of steatosis.Genes & Nutrition 05/2014; 9(3):392. · 3.33 Impact Factor
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ABSTRACT: Metabolic syndrome is a cluster of metabolic abnormalities that identifies people at risk of diabetes and cardiovascular disease, whereas non-alcoholic fatty liver disease (NAFLD) is defined as a disorder with excess fat in the liver due to non-alcoholic causes. Two key components of metabolic syndrome, glucose and triglycerides, are overproduced by the fatty liver. The liver is therefore a key determinant of metabolic abnormalities. The prevalence of both metabolic syndrome and NAFLD increases with obesity. Other acquired causes for both disorders include excessive intake of simple sugars and physical inactivity. Both disorders predict type 2 diabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), and hepatocellular carcinoma. Because metabolic syndrome can be defined in many different ways, NAFLD might be a more direct predictor of these diseases. Half of people with NAFLD carry at least one variant (G) allele at rs738409 in the PNPLA3 gene, which is associated with high liver fat content. Steatosis in PNPLA3-associated NAFLD is not accompanied by features of metabolic syndrome. All forms of NAFLD increase the risk of NASH, cirrhosis, and hepatocellular carcinoma.The lancet. Diabetes & endocrinology. 04/2014;
- [Show abstract] [Hide abstract]
ABSTRACT: The Ile148Met (rs738409, G-allele) in the patatin-like phospholipase domain-containing protein 3 gene (PNPLA3) associates with liver fat content and may lead to loss-of-function (hydrolysis) or gain-of-function (CoA-dependent lysophosphatidic acid acyltransferase) defects. PNPLA3 is up-regulated by dietary carbohydrates, and interactions between rs738409 and carbohydrates, and sugar and ω6:ω3-polyunsaturated fatty acid (PUFA) ratio on hepatic fat accumulation have been reported. We examined interaction between rs738409 and overweight, and between rs738409 and dietary intakes (carbohydrates, sucrose and ω6:ω3-PUFA ratio), on fasting triglyceride levels. From the Malmo Diet and Cancer Study-Cardiovascular Cohort, 4,827 individuals without diabetes aged 58 ± 6 years, 2,346 with BMI ≤ 25 kg/m(2) and 2,478 with BMI > 25 kg/m(2), were included in cross-sectional analyses. Dietary data were collected by a modified diet history method. Overweight modified the association between rs738409 and fasting triglyceride levels (P interaction = 0.003). G-allele associated with lower triglycerides only among overweight individuals (P = 0.01). Nominally, significant interaction on triglyceride levels was observed between rs738409 and sucrose among normal-weight individuals (P interaction = 0.03). G-allele associated with lower triglycerides among overweight individuals in the lowest tertiles of carbohydrate and ω6:ω3-PUFA ratio (P = 0.04 and P = 0.001) and with higher triglycerides among normal-weight individuals in the highest tertile of sucrose (P = 0.001). We conclude that overweight and dietary sucrose may modify the association between rs738409 and fasting triglyceride levels.Genes & Nutrition 03/2014; 9(2):388. · 3.33 Impact Factor
The PNPLA3 rs738409 G-Allele Associates with Reduced
Fasting Serum Triglyceride and Serum Cholesterol in
Danes with Impaired Glucose Regulation
Nikolaj Thure Krarup1*, Niels Grarup1, Karina Banasik1, Martin Friedrichsen5, Kristine Færch6, Camilla
Helene Sandholt1, Torben Jørgensen2,4, Pernille Poulsen3, Daniel Rinse Witte6, Allan Vaag6,7,
Thorkild Sørensen1,8, Oluf Pedersen1,2,9, Torben Hansen1,10
1The Novo Nordisk Foundation Center for Basic Metabolic Research, Section of Metabolic Genetics, Faculty of Health Sciences, University of Copenhagen, Copenhagen,
Denmark, 2Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark, 3Novo Nordisk A/S, Bagsvaerd, Denmark, 4Research Centre for Prevention and
Health, Glostrup, Denmark, 5Department of Exercise and Sports Sciences, Copenhagen, Denmark, 6Steno Diabetes Center, Gentofte, Denmark, 7Department of Diabetes
and Metabolism, Rigshospitalet, Copenhagen, Denmark, 8Institute of Preventive Medicine, Copenhagen University Hospital, Copenhagen, Denmark, 9Faculty of Health
Sciences, University of Aarhus, Aarhus, Denmark, 10Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark
Background and Aim: Non-alcoholic fatty liver disease (NAFLD) is a common condition, associated with hepatic insulin
resistance and the metabolic syndrome including hyperglycaemia and dyslipidemia. We aimed at studying the potential
impact of the NAFLD-associated PNPLA3 rs738409 G-allele on NAFLD-related metabolic traits in hyperglycaemic individuals.
Methods: The rs738409 variant was genotyped in the population-based Inter99 cohort examined by an oral glucose-
tolerance test, and a combined study-sample consisting of 192 twins (96 twin pairs) and a sub-set of the Inter99 population
(n=63) examined by a hyperinsulinemic euglycemic clamp (ntotal=255). In Inter99, we analyzed associations of rs738409
with components of the WHO-defined metabolic syndrome (n=5,847) and traits related to metabolic disease (n=5,663). In
the combined study sample we elucidated whether the rs738409 G-allele altered hepatic or peripheral insulin sensitivity.
Study populations were divided into individuals with normal glucose-tolerance (NGT) and with impaired glucose regulation
Results: The case-control study showed no associations with components of the metabolic syndrome or the metabolic
syndrome. Among 1,357 IGR individuals, the rs738409 G-allele associated with decreased fasting serum triglyceride levels
(per allele effect(b)=29.9% [214.4%;24.0% (95% CI)], p=5.161025) and fasting total cholesterol (b=20.2 mmol/l
[20.3;20.01 mmol/l(95% CI)], p=1.561024). Meta-analyses showed no impact on hepatic or peripheral insulin resistance in
carriers of the rs738409 G-allele.
Conclusion: Our findings suggest that the G-allele of PNPLA3 rs738409 associates with reduced fasting levels of cholesterol
and triglyceride in individuals with IGR.
Citation: Krarup NT, Grarup N, Banasik K, Friedrichsen M, Færch K, et al. (2012) The PNPLA3 rs738409 G-Allele Associates with Reduced Fasting Serum Triglyceride
and Serum Cholesterol in Danes with Impaired Glucose Regulation. PLoS ONE 7(7): e40376. doi:10.1371/journal.pone.0040376
Editor: Ingrid A. Dahlman, Karolinska Insitutet, Sweden
Received February 8, 2012; Accepted June 4, 2012; Published July 5, 2012
Copyright: ? 2012 Krarup et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants from: the Lundbeck Foundation Centre of Applied Medical Genomics for Personalized Disease Prediction,
Prevention and Care (LuCAMP), the Danish Health Research Council, "Hepatic and adipose tissue and functions in the metabolic syndrome" (HEPADIP, http://
www.hepadip.org), which was supported by the European Commission as an integrated project under the 6th Framework Programme (LSHM-CT-2005-018734),
the Danish Diabetes Association, the Danish Council for Independent Research (Medical Sciences) and Novo Nordisk. The NNF Center for Metabolic Research is
funded by the Novo Nordisk Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: Authors NTK, KB, KF, CS, TH and OP hold stock in Novo Nordisk A/S. Author PP is employed by Novo Nordisk A/S. Authors TH and OP have
received research grants and honoraria for speaking and participating at meetings from Lundbeck A/S and Novo Nordisk A/S. Author TIAS collaborates on various
industrial obesity research as indicated on the website http://www.ipm.hosp.dk/Publications-site/tias/Disclosures.html. The study was supported by: Lundbeck A/
S, Novo Nordisk A/S, and The NNF Center for Metabolic Research is funded by the Novo Nordisk Foundation. This does not alter the authors’ adherence to all the
PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
Non-alcoholic fatty liver disease (NAFLD) is defined as the
deposition of fat in hepatocytes exceeding 5–10% of the liver-
weight, not caused by excessive alcohol consumption . The
disease is becoming increasingly prevalent and is estimated to
involve 30% of the general population in the U.S.A. . NAFLD
is often present in obese individuals and liver fat content is linearly
correlated to components of the metabolic syndrome, e.g.
increased fasting plasma glucose- and fasting serum triglyceride-
levels and increased waist circumference [3,4]. Furthermore,
accumulation of fat in the liver is associated with hepatic insulin
PLoS ONE | www.plosone.org1 July 2012 | Volume 7 | Issue 7 | e40376
resistance [5,6]. Hepatic content of triacylglycerols (TAG) is
derived from uptake of circulating albumin-bound fatty acids,
very-low density lipoprotein (VLDL) and chylomicron remnants
and increased circulating levels of glucose can act as substrate for
de novo lipogenesis and may inhibit fatty acid oxidation [7–9].
Regulation of hepatic TAG synthesis and degradation is
influenced by insulin levels and by circulating glucose levels via
the liver X-receptor (LXR) which regulates carbohydrate response
element binding protein (ChREBP), the sterol response element
binding protein-1c (SREBP-1c) and downstream enzymes involved
in fatty acid and TAG synthesis .
Recent investigations have examined whether common genetic
variants associate with NAFLD [11–13]. These studies identified
the G-allele of rs738409 in PNPLA3, changing the amino-acid
isoleucine to methionine at location 148 (I148M) to associate with
NAFLD on a genome-wide significant level (p-value ,561028).
Interestingly, although association with hepatic lipid accumulation
was validated, the variant did not affect VLDL-, LDL-, HDL-,
total-cholesterol levels, insulin resistance or circulating glucose
We aimed at examining the effect of the rs738409 on traits
related to the metabolic syndrome in individuals in the population
based Inter99 study. Moreover, as lipogenesis is influenced by
availability of glucose [7–9] and PNPLA3 expression is influenced
by glucose-levels  we aimed to examine whether the effect of
PNPLA3 rs738409 on metabolic traits is influenced by hypergly-
Ethnicity and ethical statement
All participants were Danes by self-report and written informed
consent was obtained from all individuals before participation.
The studies were approved by the regional Ethical Committee of
Copenhagen and were conducted in accordance with the
principles of the Helsinki Declaration.
A case-control study of the metabolic syndrome defined
according to the 1999 WHO criteria  was performed in the
Inter99 population (n=5,847) where genotype was available. The
Inter99 population is a randomised multi-factorial lifestyle
intervention study for prevention of ischemic heart disease
(ClinicalTrials.gov ID-no: NCT00289237). Control individuals
(n=1,691) were defined as not having any of the components
comprised in the WHO-defined criteria of the metabolic
syndrome. Case-individuals with a HOMA-IR value in the highest
quartile of the population distribution were defined as having
insulin resistance (n=1,497). Cases with dyslipidemia (n=1,526)
were defined as having serum TAG .1.7 mM and/or HDL-
cholesterol ,0.9 mM for men and ,1 mM for women or if they
were treated with lipid-lowering agents. Cases with hypertension
(n=2,383) were defined as having a systolic blood pressure above
140 mmHg and/or diastolic blood pressure above 90 mmHg or if
they were treated with antihypertensive medication. Obese cases
(n=2,637) were defined either by a BMI above 30 kg/m2or a
waist-to-hip ratio above 0.9 for men and above 0.85 for women.
Cases with albuminuria (n=170) were defined as having an
albumin/creatinine ratio above 30 mg/g. Individuals can belong
to more than one group.
Analysis of quantitative metabolic traits related to NAFLD was
also performed in the population-based Inter99 cohort of
individuals of Danish nationality (n=5,663). The cohort was
stratified in individuals with normal glucose-tolerance (NGT,
n=4,306) and individuals with impaired glucose regulation (IGR,
n=1,357) according to WHO-criteria . Individuals with
known type 2 diabetes (T2D) receiving oral antidiabetic or insulin
treatment (n=118) and individuals receiving lipid-lowering
treatment (n=68) were excluded from the analyses of quantitative
metabolic traits. Individuals with IGR included individuals with 1)
IFG (n=459), 2) IGT (n=644) and 3) newly-diagnosed untreated
T2D (n=254). All participants were examined by a standardised
questionnaire and interview, physical examination and blood
sampling before and during a standardized 75g OGTT.
In vivo hepatic and peripheral insulin sensitivity was measured by
euglycemic hyperinsulinemic clamps using tritiated glucose in two
separate study populations: 1) 98 young (22–31 years) and elderly
(57–66 years) monozygotic and same-sex dizygotic twin pairs
(YOND), including 149 twins with NGT, 23 twins with IFG, 21
with IGT and 3 with previously undiagnosed diabetes [20–22] and
2) a sub-set of the Inter99 population (n=63) including 18
participants with NGT, 17 with isolated IFG and 28 with isolated
IGT . Meta-analyses included a study sample of 183 NGT and
68 IGR individuals in total.
Biochemical and anthropometric measures
In the Inter99 study population, height and body weight were
measured in light indoor clothing without shoes. BMI was
calculated as weight (kg)/(height [m])2. All blood samples were
obtained after a 12 hour overnight fast and Inter99 participants
underwent an OGTT. Insulin sensitivity was estimated by the
homeostasis model assessment of insulin resistance (HOMA-IR;
(fasting plasma glucose [mmol/l] 6fasting serum insulin [pmol/
l])/22.5) . Levels of LDL cholesterol were calculated as
described earlier .
The clinical examinations of the YOND cohort (n=192) and
the Inter99 subset (n=63) have previously been described in detail
[20,21,23]. Individuals without genotype information were
excluded (n=4). In brief, peripheral insulin sensitivity was in both
populations examined by a 2 hour euglycemic-hyperinsulinemic
clamp (40 mU m22min21). A primed constant continuous
infusion of [323H]-tritiated glucose (bolus 22 mCi, 0.22 mCi
min21) was initiated at 0 min and continued throughout the
clinical investigation (basal period [120 min] and clamp period
[120 min]). Steady-state was defined as the last 30 min of the basal
and insulin-stimulated periods . Peripheral insulin sensitivity
was calculated as the rate of glucose disappearance during insulin
stimulation (Rd clamp) using the non-steady-state equation .
Hepatic insulin resistance was estimated as basal endogenous
glucose production multiplied by fasting serum insulin concentra-
For all the included study samples, genotyping of the PNPLA3
rs738409 was performed using KASPar SNP Genotyping
(KBioscience, Hoddesdon, UK). Genotyping success rate was
96.7% and the error rate was 0.92% as estimated from 1,187
duplicate samples. Genotype distribution obeyed Hardy-Weinberg
equilibrium in the analysed study samples (p.0.05).
In the case-control study of the metabolic syndrome and related
traits, we used logistic regression to examine differences in
genotypes assuming an additive model adjusting for age and
gender. The analysis consisted of pair-wise analysis of genotype
frequencies between metabolic syndrome related traits and control
individuals (with none of the characteristics of metabolic
derangement). We assumed an additive genetic model, based on
The PNPLA3 rs738409 and Fasting Lipid Levels
PLoS ONE | www.plosone.org2 July 2012 | Volume 7 | Issue 7 | e40376
earlier findings of the effect of the variant. In the Inter99 study
population, a general linear model was used to test anthropomet-
ric, OGTT-derived and biochemical traits. All analyses were
adjusted for sex, age and BMI. Serum TAG levels, HOMA-IR
and estimates of hepatic and peripheral insulin sensitivity were
logarithmically transformed before analysis. Interaction analyses
between genotype and glucose tolerance status (divided into two
groups; NGT and IGR), obesity status (BMI below 25, BMI from
25 to 30 and BMI over 30) and OGTT-derived glucose levels
(quantitative outcomes) on fasting TAG and total cholesterol levels
were performed using linear regression with a multiplicative
interaction term. Statistical analyses were performed using RGui,
version 2.7.2 (available at http://www.r-project.org).
In the YOND study sample and the Inter99 subset, statistical
tests were performed in SAS (version 9.1, SAS Institute, Cary, NC)
also using the linear regression model adjusting for age, sex, and
BMI. All response variables were log-transformed before analysis.
Effect sizes of log-transformed variables (with b .0.05) were
calculated as 1006eb-1. Fixed-effect meta-analyses were per-
formed to increase power to detect an association with estimates of
hepatic (Hepatic IR basal) and peripheral insulin resistance (Rd
clamp). We used effect size estimates and standard errors derived
from analyses of the mentioned traits where the effect of the
genetic variant was estimated as the effect per allele. Weight of
studies was estimated using inverse variance assuming fixed effects.
Heterogeneity was measured by Q-statistics. Multiple regression
analyses using the proc mixed procedure in SAS allowed for
adjustment of twin pair and zygosity status and other contributing
A p-value below 0.05 was considered significant. No correction
for multiple testing (Bonferroni) was performed. Power analysis
was based on 1000 simulations. We used empirical variance of the
observed traits in the Inter99 cohort to simulate phenotypes from a
normal distribution. Statistical power analysis showed approxi-
mately 50% statistical power to detect an association with
measures of insulin resistance in the small combined study-sample
(ntotal=251) if the true allelic effect size is 10% on the level of a
given trait. Conversely, power estimates on the quantitative trait
analysis in Inter99 revealed more robust statistical power (.80%)
to detect association assuming a minor allele frequency of 22%
and an allele-dependent effect size of 8% on the level of a given
Case- control study of the metabolic syndrome
A case-control study showed no associations with components of
the metabolic syndrome or the metabolic syndrome (Table 1).
Quantitative trait analysis of traits related to metabolic
Among 4,306 individuals with NGT, the rs738409 G-allele was
associated with fasting plasma glucose levels (b=20.4% [20.7%;
20.01% (95% CI)], p=0.04). Among 1,357 individuals with IGR,
the minor, methionine-coding G-allele was significantly associated
with decreased fasting levels of both serum TAG (per allele effect
(b) [95% CI] =29.2% [214.4%;24.0%], p=5.161025) and
total serum cholesterol(b=20.2 mmol/l
p=1.561024) (Table 2). In an interaction analysis between
genotype and glucose-tolerance status on the two associating lipid
traits, we found statistically significant interactions between the
rs738409 G-allele and glucose tolerance status on TAG and
cholesterol (p=261024and p=861025, respectively Table 2). In
individuals with IGR, no other associations were observed
between the rs738409 genotype and metabolic disease-associated
Interaction of genotype with quantitative glucose traits and BMI
was subsequently analyzed (Table S1). Interaction was observed
between genotype and 2-hour OGTT plasma glucose values for
fasting serum TAG levels (p=0.009, Table S1). No interaction
with BMI was observed.
Meta-analysis of hepatic and peripheral insulin resistance
To investigate whether the NAFLD risk rs738409 G-allele was
associated with altered insulin resistance in either the liver or
peripheral tissues, we performed a meta-analysis based on
estimates of hepatic and peripheral insulin resistance obtained
from a euglycaemic hyperinsulinaemic clamp examination in
NGT (n=183) and IGR (n=68) individuals (Supplemental
Figure S1). For NGT individuals, a nominal increase in peripheral
insulin sensitivity in rs738409 G-allele carriers measured by
p=0.04) was observed. No association between the rs738409
genotype and peripheral insulin sensitivity was found in IGR
individuals. Furthermore, hepatic insulin resistance was not
associated with the rs738409 genotype in either NGT or IGR
This study reports an association of the PNPLA3 rs738409
NAFLD risk G-allele with decreased fasting levels of serum TAG
and serum cholesterol in individuals with IGR but not among
individuals with NGT. Analysis of interaction between genotype
and glucose tolerance status or OGTT derived 2 hour glucose
levels revealed significant interactions. The rs738409 G-allele
conferred reduced serum TAG and total cholesterol levels among
individuals with elevated glucose levels. In a population-based
sample of 5,847 Danes we found no association with the metabolic
syndrome. In NGT individuals, we observed a nominal significant
association with decreased fasting plasma glucose levels. Further-
more, the rs738409 G-allele nominally associated with increased
peripheral insulin sensitivity, estimated by a euglycemic hyperin-
sulinemic clamp. Our findings indicate that the effect of the
rs738409 G-allele on fasting lipid levels is unmasked in a state of
Changes in fasting circulating levels of TAG are believed to be
conferred endogenously by changes in levels of circulating VLDL,
stemming from hepatic secretion. Interestingly, recent findings of
an association of GCKR variation with increased VLDL particle
concentrations [28,29] suggests that enhanced glycolytic flux leads
to increased levels of de novo TAG and cholesterol synthesis.
Functional studies of PNPLA3 I148M-substituted cells reveal a
plausible mechanistic explanation for the decreased lipid levels
observed in IGR G-allele carriers of rs738409. PNPLA3 encodes
adiponutrin, a member of the calcium-independent phospholipase
A2 family, having triacylglycerol hydrolase activity and possibly
acylglycerol transacylase activity . The enzyme is regulated by
insulin levels and in animal models the mRNA levels of PNPLA3
are low in the fasting state but rise significantly during
carbohydrate feeding . Furthermore, a study of in vitro assays
of wild-type PNPLA3 and I148M-substituted PNPLA3 revealed a
marked reduction in hydrolysis of intracellular stores of TAGs in
hepatocytes . We suggest that IGR rs738409 G-allele carriers
have an increased glycolytic flux and generation of precursors for
synthesis of lipid molecules, e.g. TAGs and cholesterol. Individuals
with decreased hepatic lipolysis conferred by the I148M variant
have a decreased hepatic release of TAG and cholesterol to the
The PNPLA3 rs738409 and Fasting Lipid Levels
PLoS ONE | www.plosone.org3 July 2012 | Volume 7 | Issue 7 | e40376
Table 1. Genotype and allele frequency of rs738409 among individuals with traits related to the metabolic syndrome in Inter99.
One or more
OR (95% CI)
The table presents a case-control study of the association of the rs738409 with the metabolic syndrome. It includes 5,847 individuals from the Inter99 cohort. Individuals may have more than one trait. The metabolic syndrome was
defined according to the 1999 WHO criteria. Control individuals (n=1,691) were defined as not having any of the components comprised in the WHO-defined criteria of the metabolic syndrome. Case-individuals with IGR were
defined as mentioned in the main text. Individuals with a HOMA-IR value in the highest quartile of the population distribution were defined as having insulin resistance (n=1,497). Cases with dyslipidemia (n=1,526) were defined
as having serum TAG .1.7 mM and/or HDL-cholesterol ,0.9 mM for men and ,1 mM for women or treated with lipid-lowering agents. Cases with hypertension (n=2,383) were defined as having a systolic blood pressure above
140 mmHg and/or diastolic blood pressure above 90 mmHg or treated with antihypertensive medication. Obese cases (n=2,637) were defined either by a BMI above 30 kg/m2or a waist-to-hip ratio above 0.9 for men and above
0.85 for women. Cases with albuminuria (n=170) were defined as having an albumin/creatinine ratio above 30 mg/g. Values are means 6 SD. Traits were defined as according to the 1999 WHO-defined metabolic syndrome
components. Individuals may have more than one trait. P-values were adjusted for age, sex and BMI.
The PNPLA3 rs738409 and Fasting Lipid Levels
PLoS ONE | www.plosone.org4 July 2012 | Volume 7 | Issue 7 | e40376
Table 2. Quantitative traits among individuals from the Inter99 cohort according to PNPLA3 rs738409 genotype.
Normal glucose tolerance (n=4,306)
N (men/women) 2559
Age (years) 4568 4568 4568
BMI (kg/m2) 25.564.125.564.1 24.963.6
Waist (cm) 84612 84612 83611
5.461 5.461 5.461.10.0002
HDL (mmol/l) 1.560.41.460.4 1.560.4
LDL (mmol/l)3.461 3.560.9 3.5610.04
VLDL (mmol/l) 0.560.30.660.30.560.3
Impaired glucose regulation (n=1,357)
Age (years)4967 4968 4967
5563556636 64635 3%
Waist (cm) 93613 93614 97616
HDL (mmol/l) 1.460.41.460.41.360.40.005
LDL (mmol/l)3.861 3.661.13.760.9
VLDL (mmol/l) 0.860.40.760.4 0.860.4
Numbers are mean 6 SD. Analyses were made using an additive model. P-values were adjusted for age, sex and BMI. Individuals from the Inter99 receiving oral
antidiabetic or lipid-lowering treatment (n=186) were excluded. Effects are changes in units unless calculated on log-transformed traits (change in percent).
The PNPLA3 rs738409 and Fasting Lipid Levels
PLoS ONE | www.plosone.org5 July 2012 | Volume 7 | Issue 7 | e40376
circulation and they may have a relatively benign form of elevated
intrahepatocellular concentration of TAG, which does not
associate with the metabolic syndrome.
Association of the rs738409 G-allele with decreased fasting
levels of TAG has only been observed in smaller study populations
(n=144) of individuals selected for NAFLD and steatohepatitis of
Japanese, Indian, Malayan and Chinese ancestry [31,32] as well as
in 516 American non-Hispanic individuals . All individuals
had severe NAFLD. Subsequent analyses in larger cohorts
(n=19,840) who were not ascertained for fatty liver disease (and
were not stratified on the degree of glucose intolerance) did not
reveal association with lipid levels . Another study has shown
an interaction between the rs738409 G-allele and a reduction in
apoB-containing lipoproteins in 23,274 individuals from eight
independent West-Eurasian study populations and one population
from Utah, U.S.A .
In NGT individuals, careful interpretation must be taken in
relation to the nominal association with decreased levels of fasting
plasma glucose for the risk G-allele. The nominal significant
difference between genotype groups for fasting plasma glucose in
NGT individuals is only significant when adjusting for BMI, and
likely a chance finding.
In smaller study samples, the rs738409 G-allele has been
associated with decreased insulin resistance measured by HOMA-
IR [14,17,35]. In a meta-analysis of relatively small but carefully
phenotyped study samples, we find increased insulin-stimulated
glucose disposal in NGT individuals as estimated by the
hyperinsulinaemic euglycaemic clamp. However, careful appraisal
of this finding must be adopted as the association is not corrected
for multiple testing.
The present study points to an association of the common
rs738409 minor G-allele of PNPLA3 with decreased levels of
fasting serum TAG and total serum cholesterol in individuals with
impaired glucose tolerance. Future studies should stratify study
participants according to glucose-tolerance status (i.e. normogly-
caemia and hyperglycaemia) or include information on 2-hour
OGTT plasma glucose levels to assess the effect of the PNPLA3
rs738409 G-allele on lipid levels.
sures. Meta-analyses of 251 individuals of YOND (n=188) and
Inter99 sub-set (n=63) stratified into individuals with normal
Meta-analyses of insulin resistance mea-
glucose-tolerance (NGT, n=165 in YOND; n=18 in I99 sub-set)
or impaired glucose regulation (IGR, n=23 in YOND; n=45 in
I99 sub-set). Effect sizes for the G-allele are in percentages and
standard errors were obtained from analyses done separately in the
study samples. The values were combined using the inverse
variance method. Black squares are effects in single studies
according to weight in the meta-analysis. Black diamonds are the
combined change in either hepatic insulin resistance (Basal
Hepatic IR) or peripheral insulin resistance (Rd clamp). A shows
hepatic insulin resistance(IR) in NGT individuals (Combined effect
size [95% CI] =213.3% [228.6 to 2.1%], p=0.09), B shows
peripheral insulin resistance in NGT individuals (Combined effect
size [95% CI] =9.7% [0.05% to 18.8%], p=0.04), C shows
hepatic insulin resistance in IGR individuals (Combined effect size
[95% CI] =3.3% [212.5% to 19.1%], p=0.7), C shows
peripheral insulin resistance in IGR individuals (Combined effect
size [95% CI] =1.0% [27% to 9%], p=0.8).
interaction variables. The table shows p-values and effect
estimates for interaction of glucose-tolerance, glucose-levels or
BMI with genotype on triglyceride and total cholesterol levels.
Effect estimates are percentage change in levels of fasting serum
triglyceride or changes in millimoles per liter for total cholesterol
levels. The strongest interaction is seen between glucose tolerance
and genotype on both lipid traits. Interaction is also seen between
genotype and levels of 2-hour glucose after an OGTT. All p-values
are adjusted for age and gender. Glucose-related p-values are
additionally adjusted for BMI, and BMI was adjusted for glucose-
tolerance. The BMI variable is categorized into lean (BMI,25),
overweight (BMI=25–30) and obese individuals (BMI.30).
Interaction analyses of rs738409 genotype and
The authors wish to thank Annemette Forman, Inge-Lise Wantzin and
Marianne Stendal for technical assistance. A. L. Nielsen, G. Lademann,
and M.M.H. Kristensen have been appreciated for their assistance of
management and data-handling.
Conceived and designed the experiments: NTK TS OP TH. Performed
the experiments: TJ AV PP DRW KF. Analyzed the data: NK NG KB
CHS MF KF. Contributed reagents/materials/analysis tools: KF MF AV
TS OP TH. Wrote the paper: NTK. Conceived and performed study
sampling: KF PP AV TJ.
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