Nonalcoholic Fatty Liver Disease
Is Prevalent in Women With Prior
Gestational Diabetes Mellitus and
Independently Associated With
Insulin Resistance and Waist
Circumference
Diabetes Care 2017;40:109–116 | DOI: 10.2337/dc16-1017
OBJECTIVE
Type 2 diabetes increases the risk of nonalcoholic fatty liver disease (NAFLD),
which is a potentiall y rever sible condition but is also associated with progressive
fibrosis and cirrhosis. Women with prior gestational diabetes mellitus (p GDM)
have a higher risk fo r NAFLD.
RESEARCH DESIGN AND METHODS
One hundred women without diabetes who had pGDM (median [interquartile
range]: age 38.6 [6.4] years; BMI 3 1.0 [6.2] kg/m
2
) and 11 healthy control subjects
without NAFLD (age 37.9 [7.8] years; BMI 28.1 [0.8] kg/m
2
) underwent a 75-g oral
glucose tolerance test (OGTT), DXA w hole- body scan, and ultrasonic evaluation of
hepatic steatosis.
RESULTS
Twenty-four (24%) women with pGDM had NAFLD on the basis of the ultrasound
scan. None had cirrhosis. Women with NAFLD had a hi gher BMI (P = 0.0002) and
waist circumference (P = 0.0003), increased insu lin resistance (P = 0.0004), and
delayed suppression of glucagon after the OGTT (P < 0.0001), but NAFLD was not
associate d with the degree of glucose intolerance (P = 0.2196). Visceral fat mass
differed amon g the three group s, with the NAFLD group ha ving the highest
amount of fat and the control subjects the lowest (P =0.0003).Bylogisticre-
gression analysis, insulin resistance (P = 0.0057) and waist circumference (P =
0.0109) were independently associated with NAFLD.
CONCLUSIONS
NAFLD was prevalent in this cohort of relatively young and nonseverely obese
women with pGDM who are considered healthy apart from their increased risk for
diabetes. Insulin res istance and a larger waist circumference were independently
associate d with the presence of NAFLD, whereas gl ucose intolerance was not.
1
Center for Diabetes Research, Gentofte Hospital,
University of Copenhagen, Hellerup, Denmark
2
Novo Nordisk Foundation Center for Basic Met-
abolic Research and Department of Biomedical
Sciences, Faculty of Health and Medical Sciences,
University of Copenhagen, Hellerup, Denmark
3
Danish Diabetes Academy, Odense University
Hospital, Odense, Denmark
4
Department of Radiology, Gentofte Hospital,
University of Copenhagen, Hellerup, Denmark
5
Department of Nuclear Medicine, Gentofte
Hospital, University of Copenhagen, Hellerup,
Denmark
6
Department of Obstetrics and Gynecology, Her-
lev Hospital, University of Copenhagen, Herlev,
Denmark
7
Department of Gynecology and Obstetrics,
Nordsjællands Hospital, University of Copenha-
gen, Hillerød, Denmark
8
Center for Pregnant Women with Diabetes, De-
partment of Endocrinology, Rigshospitalet, Uni-
versity of Copenhagen, Copenhagen, Denmark
9
Center for Pregnant Women with Diabetes,
Department of Obstetrics, Rigshospit alet, Uni-
versity of Copenhagen, Copenhagen, Denmark
Corresponding author: Tina Vilsbøll, t.vilsboll@
dadlnet.dk.
Received 10 May 2016 and accepted 10 October
2016.
Clinical trial reg. no. 2012-001371-37, clinical
trialsregister.eu.
S.F. and C.A. are co-first authors.
© 2017 by the American Diabetes Association.
Readers may use this article as long as the work
is properly cited, the use is educa tional and not
for profit, and the work is not altered. More infor-
mation is available at http://www.diabetesjournals
.org/content/license.
Signe Foghsgaard,
1–3
Camilla Andreasen,
1
Louise Vedtofte,
1
Emilie S. Andersen,
1
Emilie Bahne,
1
Charlotte Strandber g,
4
Thora Buhl,
5
Jens J. Ho lst,
2
Jens A. Svare,
6
Tine D. Clausen,
7
Elisabeth R. Mathiesen,
8
Peter Damm,
9
Lise L. Gluud,
1
Filip K. Knop,
1,2
and Tina Vilsbøll
1
Diabetes Care Volume 40, January 2017 109
PATHOP HYSIOLOGY/COMP LICATIONS
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Nonalcoholic fatty liver disease (NAFLD)
is hepatic accumulation of triglycerides
in the absence of excessive alcohol con-
sumption (1). NAFLD is the most com-
mon liver abnormality i n t he Western
world, with a prevalenc e of 20–33% in
the general European population of
adults and 43–70% in patients with
type 2 diabetes (T2D) (2) . NAFLD spans
from simple steatosis to the more
aggressive form nonalcoholic steato-
hepatitis (NASH) that may progress to
fibrosis, cirrhosis, and end-stage liver
failure (3). Visceral, as opposed to sub-
cutaneous, adipose tissue is especially
unhealthy because of its metabolically
active nature and because it releases
free fatty acids directly int o the portal
venous system (4–6). The majority of
patients with mild to moderate NAFLD
without NASH are asymptomatic, and in
up to 70% of these patients, no abnor-
malities in plasma liver enzyme levels
are observed (3,4,7). NAFLD is associ-
ated with hyperglucagonemia, visceral
adiposity, obesity, insulin resistance,
and T2D, the latter three of which are
features of the metabolic syn drome
(3,8,9). Of no te, b oth NAFLD and T2D
are associated with a marked ly in-
creased risk of cardiovascular disease
(3,5,7). Liver biopsy is the gold standard
for diagnosing NAFLD and necessary for
the diagnosis of NASH, but imaging
methods are increasingly being ac-
cepted as nonin vasive alternatives be-
cause the invasive nature of biopsy has
several important disadvantages (10).
Gestational diabetes mellitus (GDM)
is glucose intolerance first detected
during pregnancy and affects 2–6% of
pregnant European women (11). In the
majority of women, normal glucose tol-
erance (NGT) is reestablished after de-
livery (12,13). Nevertheless, women
with prior GDM (pGDM ) may progress
to overt T2D, with a long-term risk o f
up to 70% (14), and even if NGT is main-
tained, these women are more prone to
the metabolic syndrome than those who
had NGT during pregnancy (15). Addition-
ally, women with pGDM have a twofold
risk of NAFLD compared with women
without a history of GDM, even when ad-
justed for BMI (16). Because of their in-
creased risk, women without diabetes but
with pGDM represent a valuable target
group for investigating the early meta-
bolic changes that precede T2D. In this
study, we investigated the presence of
NAFLD and its association with glucose
intolerance, insulin resistance, and fat dis-
tribution in a cohort of overweight or
obese women without diabetes but with
pGDM (16).
RESEARCH DESIGN AND METHODS
Study Design
The study included baseline data from
women who were recruited for an inves-
tigator-initiated, randomized, placebo-
controlled, double-blind intervention trial
carried out in women without diabetes
but with pGDM before the intervention
was initiated (17). The protocol was ap-
proved by the Danish Data Protection
Agency (01714 GEH-2012-024) on 4 June
2012, the Danish Medicines Agency
(EudraCT no. 2012-001371-37) on 10
July 2012, and the Scientific-Ethical Com-
mittee of the Capital Region of Denmark
(H-2-2012-073) on 13 July 2012 . The
study was carried out under the surveil-
lance of the Good Clinical Practice unit
(Copenhagen, Denmark) and conducted
in accordance with the Declaration of
Helsinki.
Outcomes
The primary outcome was the presence
of ultrasound-detectable NAFLD in a
cohort of wo men with pGDM.
Participants and Recruitment
One hundred five women without dia-
betes but with pGDM were recruited to
the main study (17), and of these, 100
underwent B-mode ultrasonographic
evaluation of the liver for the presence
or absence of NAFLD. The trial allowed
inclusion o f all ethnicities, but only 4 of
the 100 women were non-Caucasian
(one African and three Asian). We did
not include women with known liver dis-
ease (based on patient history and bio-
chemical and ultrasonic assessment),
increased liver enzyme s, or ongoing al-
cohol abuse. Four women presented
with levels of liver enzymes above normal
limits (but below three times the normal
limit). These women were examined
with concern for hepatitis B and C with
negative results. Eleven healthy women
without ultrasound-detectable NAFLD,
pGDM, or glucose intolerance were in-
cluded as the control group (17). The eval-
uated groups were 1) women without
pGDM and without NAFLD (control), 2)
womenwithpGDMwithoutNAFLD
(nonNAFLD pGDM), and 3)women
with pGDM and NAFLD (NAFLD pGDM).
After signed consent, a screening visit
was completed wherein fasting blood
samples for creatinine, sodium, potassium,
alanine transaminase (ALT), aspartate
transaminase (AST), alkaline phosphatase,
albumin, hemoglobin, fasting plasma glu-
cose, and glycated hemoglobin A
1c
(HbA
1c
)
levels were collected to verify that the par-
ticipant fulfilled all the inclusion criteria
and none of the exclusion criteria (17).
Medical history was recorded, and a full
physical examination was performed.
Procedure
On the experimental days, participants
came in the morning after a 10-h fast
and underwent a number of examina-
tions ( 17).
Ultrasonography
Real-time B-mode ultrasonography of
the liver was p erformed with a hig h-
end ultrasound scanner (Logiq E9; Gen-
eral Electric, Milwaukee, WI), using a
convex probe (2.5–6 MHz). All exami-
nations we re performed by the same
specialized radiologist (C.S.).
Transient Elastography
To rule out hepatic fibrosis, vibration-
controlled transient elastography (FibroScan;
Echosens, Paris, France) was performed
by one of two trained investigators (S. F. ,
L.V.). All transient elastography results with
an interquartile range (IQR) ,30 % of the
median value and a success rate of at
least 60% were analyzed. A median of
hepatic elasticity .8 kPa implied hepatic
fibrosis (18).
DXA
Body composition and fat distribution
measures were acquired using Lunar
iDXA (GE Healthcare, Chicago, IL) and ana-
lyzed with the accompa nying software
enCORE version 13.6, where visceral fat
mass was computed by subtracting subcu-
taneous fat mass from total abdominal fat
mass in the predefined android region (19).
Oral Glucose Tolerance Test
A 4-h 75-g oral glucose tolerance test
(OGTT) was performed to define glucose
tolerance status and to rule out diabe-
tes. Women were categorized as having
NGT or prediabetes according 2006
World Health Organization criteria (20).
Whole-body insulin resistance was calcu-
lated according to the Matsuda index (21),
and hepatic insulin resistance was evalu-
ated with the computerized HOMA2 for
insulin resistance (HOMA2
IR
) (22). The in-
sulinogenic index was calculated as the
110 NAFLD Is Prevalent in Women With Prior GDM Diabetes Care Volume 40, January 2017
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ratio between the total area under the
curve(tAUC)ofseruminsulinandplasma
glucose during the OGTT (23). The disposi-
tion index (insulinogenic index / HOMA2
IR
)
wasusedasanadjustedmeasureofb-cell
function (23). Presence of the metabolic
syndrome was assessed by joint scientific
statement criteria (24).
Biochemical Markers
Liver function was evaluated by biochem-
ical markers (ALT, AST, and g-glutamyl
transferase[GGT]).WomenwithALTand
AST levels three times above normal limi ts
were excluded according to protocol (17).
The probability score of steatosis was cal-
culated according to the recently validated
fatty liver index (FLI), cate gorizing the
women into three group s: G1 with
FLI #30 (very-low risk of steatosis); G2
with FLI .30 and ,60 (intermediate risk
of steatosis); and G3 with FLI $60 (high
risk of steatosis) (25).
Alcohol Consumption
Habits were ev aluated through a vali-
dated questionnaire, the Alcohol Use
Disorder Identification Test (AUDIT), to
rule out excessive alcoho l consumption
(score ,8) (26).
Analysis
Pla sma glucos e (27), glucagon (28 ,29),
and insulin and C-peptide (30) levels
were analyzed as previously described.
Statistics
Data are tabulated as median and IQR
or numbe r and percent. Fasting values
of glucose, insulin, C-pep tide, and glu-
cagon were calculated as the mean
of 215, 210, and 0 min. The AUC was
calculated by the trapezoidal rule and
expressed as either the tAUC or the in-
cremen tal AUC ( iAUC) for all 240 min,
unless otherwise stated. The statistical
analyses were performed using GraphPad
Prism version 6.0 (GraphPad Software, La
Jolla, CA) and RStudio version 0.98.1083
(RStudio, Boston, MA) software. Bartlett
test was used to assess for normal distri-
bution. Comparisons among the three
groups were performed using Kruskall-
Wallis test with Dunn correction for
multiple comparisons for continuous var-
iables. Differences of categorical vari-
ables were analyzed using x
2
test. Logistic
regression analysis of the significant vari-
ables in the univariate regression anal-
ysis were used to identify clinically
relevant determinants associated with
the presence of NAFLD. Differences
resulting in P , 0.05 were considered
significant.
RESULTS
Baseline Characteristics
Anthropometric and metabolic character-
istics of the control (n = 11), nonNAFLD
pGDM (n =76),andNAFLDpGDM(n =
24) groups are listed in Table 1. The three
groups were similar with regard to age,
waist-to-hip ratio, heart rate, HbA
1c
,
android-to-gynoid fat ratio, AUDIT scores,
and AST-to-ALT ratio as well as with regard
to use of hormonal contraception, polycys-
tic ovarian syndrome, number of pregnan-
cies, and time since last pregnancy. BMI
was highest in the NAFLD pGDM group.
Systolic and diastolic blood pressure was
lowest in the control group. Total choles-
terol and LDL were similar among groups,
but HDL was lower in the NAFLD pGDM
group, and VLDL and triglycerides were
lower in the control group. DXA scan re-
vealed different amounts of visceral fat in
the three groups, with the control group
being the leanest and having the lowest
total fat mass. GGT was similar and within
the normal range in all three groups,
whereas ALT was higher in the NAFLD
pGDM group. Four women in this group
had values that exceeded the upper limit
of normal (35 units/L) but less than three
times this. AST was higher in the NAFLD
pGDM group than in the nonNAFLD pGDM
group, but all three groups were well
within the normal range. FLI was higher
in the NAFLD pGDM group, with 88% of
women categorized as G3 (high risk of
steatosis) (Table 1). No differences were
observed with respect to ethnicity.
Glucose Tolerance and Indices of
Insulin Resistance
Data fro m the OGTT are listed in Table 2;
the dynamic responses of glucose, gluca-
gon, insulin, and C-peptide are illustrated
in Fig. 1A–D. Fasting plasma glucose, the
2-h value, and the peak value and tAUC
for plasma glucose excursions were lower
in the control group. No differences be-
tween the pGDM groups were observed,
and no differences with respect to preva-
lence of prediabetes were found. Al-
though there was a numerical tendency,
fasting plasma glucagon did not differ sig-
nificantly among groups, but during the
first 45 min after glucose ingestion, both
tAUC and iAUC were higher in the NAFLD
pGDM group. Fasting serum insulin was
higher in NAFLD pGDM, but no difference
between nonNAFLD pGDM and control
was found. The tAUC for serum insulin dif-
fered among the three groups, with the
largest being in the NAFLD pGDM group
and the smallest being in the control
group. C-peptide followed the same pat-
tern, with higher fasting serum C-peptide
in the NAFLD pGDM group and increasing
tAUC for serum C-peptide across the three
groups, the largest being in the NAFLD
pGDM group. The insulinogenic index
was higher in t he NAFLD pGDM group,
whereas the disposition index was sim-
ilar in all three groups. The Matsuda
index was lowest in the NAFLD pGDM
group and highest in the control group.
Similarly, HOMA2
IR
was highest in the
NAFLD pGDM group.
Logistic Regression Analysis
Univariate logistic regression analysis of
women with pGDM showed several de-
terminants to be significantly associated
with the presence of NAFLD (Table 3).
The following variables did not reach
significance: glucose tolerance status,
age, waist-to-hip ratio, HbA
1c
,2-hplasma
glucose during OGTT, fasting plasma gluca-
gon, family disposition to diabetes, use of
hormonal contraceptive, total cholesterol,
LDL cholesterol, AUDIT score, and pres-
ence of the metabolic syndrome. All signif-
icant univariable associations contributed
in the multivariable analysis with back-
ward elimination, showing that waist cir-
cumference (P = 0.011) and Matsuda index
(P = 0.006) were independent determi-
nants associated with NAFLD (Table 3).
CONCLUSIONS
We show that NAFLD is present in women
withou t diabetes but with pGDM. Our
thorough examin ation of glucose dynam-
ics and metabolism showed no association
with the degree of glucose intolerance,
whereas increasing insulin resistance and
larger waist circumference were indepen-
dently associated with the presence of
NAFLD. When comparing the NAFLD
pGDM group with the control group, we
found that body composition was signifi-
cantly more android, with a larger waist
circumference, more visceral fat mass,
and greater total fat mass. We found no
differences in fasting levels of plasma glu-
cagon, but the suppression of plasma glu-
cagon after oral glucose ingestion was
significantly delayed, and the initial sup-
pression was reduced in women with
NAFLD and pGDM.
care.d iabetesjournals.org Foghsgaard and Associates 111
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A limitation to this study is the use of
ultrasound to determine the presence
or absence of NAF LD. Ultrasonography
does not detect mild steatosis (5–33%
fat infiltration) and is operator depen-
dent (5). We ac commodated the latt er
by letting one specialized radiologist
(C.S.) perform and describe all examina-
tions. Liver biopsy remains the gold
standard for grading and staging NAFLD
(10), but for ethical and practical rea-
sons, liver biopsy was not performed.
Thus, we may not have detected the
presence o f mild cases of NAFLD in this
cohort, but whether detection of these
stages of NAFLD has a clinical signifi-
cance is debatable. Two studies have
shown that simple steatosis does not
necessarily progress to NASH and more
severe liver damage (2,6). The me dian
FLI in the nonNAFLD pGDM group indi-
cates that a large percentage of these
women have m ild steatosis. Indeed,
only 13 of 76 women had an FLI ,30
(25). To our knowledge, only two previ-
ous studies have investigated the p rev-
alence of NAFLD in women with pGDM
(16,31). The fi rst study was cross-
sectional a nd showed a highe r preva-
lence of ultrasound-detectable NAFLD
among European women with pGDM
(38%) compared with the current find-
ings (24%) (16). The difference in reported
prevalence does not seem to be ex-
plained by d ifferences in age, BMI,
body composition, fat percent, years
since index pregnancy, ethnicity, means
of detecting NAFLD, or size of study co-
hort and might instead be attributed to
chance. An equal and important limita-
tion of this study is the relatively small
sample size, but despite this, we were
able to demonstr ate clinically rele vant
differences among the groups. The sec-
ond study is a recent subgroup analysis
Table 1—Anthropometric, metabolic, and ultrasonic characteristics and indices of insulin resistance and b-cell function
Group P value
Control (A) nonNAFLD pGDM (B) NAFLD pGDM (C) A – BA– CB– C
Number of participants 11 76 24
Age (years) 37.9 (7.8) 39.0 (5.6) 36.9 (5.6) .0.0999 .0.0999 0.2945
BMI (kg/m
2
) 28.1 (0.8) 29.9 (4.7) 34.6 (4.7) 0.3629 0.0003 0.0002
Waist circumference (cm) 98.0 (14.0) 101 (16) 109 (17) 0.2009 0.0001 0.0003
Waist-to-hip ratio 0.9 (0.0) 0.9 (0.0) 0.9 (0.1) .0.9999 .0.9999 .0.9999
Systolic blood pressure (mmHg) 116 (14) 127 (15) 128 (13) 0.0166 0.0500 .0.9999
Diastolic blood pressure (mmHg) 76 (11) 80 (14) 82 (7) 0.0439 0.0268 .0.9999
Heart rate (beats/min) 72 (12) 68 (14) 74 (11) .0.9999 .0.9999 0.4093
HbA
1c
(mmol/m ol) 31.0 (4.5) 33.0 (5.0) 34.0 (7.5) 0.3349 0.0732 0.5721
Metabolic syndrome 1 (9) 35 (46) 15 (63) 0.0131 0.0131 0.0131
Use of hormonal contraception 5 (45) 47 (62) 15 (63) 0.8889 0.8889 .0.9999
Pregnancies 2.0 (1.5) 2.0 (1.5) 2.0 (0.0) .0.9999 .0.9999 .0.9999
Time since pregnancy (years) 4.5 (4.5) 4.8 (4.2) 4.5 (2.6) .0.9999 .0.9999 .0.9999
Polycystic ovarian syndrome * 4 (5.3) 4 (17) * * 0.0726
Total chole sterol (mmol/L) 4.4 (0.9) 4.7 (1.2) 5.0 (0.9) 0.7034 0.1106 0.3280
HDL cholesterol (mmol/L) 1.4 (0.6) 1.2 (0.3) 1.1 (0.4) 0.0813 0.0022 0.0081
LDL cholest erol (mmol/L) 2.8 (1.0) 3.2 (1.2) 3.3 (0.7) 0.7108 0.1624 0.5165
VLDL cholesterol (mmol/L) 0.3 (0.1) 0.5 (0.2) 0.6 (0.5) 0.0266 0.0030 0.3600
Triglycerides (mmol/L) 0.7 (0.1) 1.0 (0.6) 1.3 (1.0) 0.0144 0.0006 0.1640
Visceral fat (g) 375 (113) 908 (771) 1,469 (896) 0.0094 ,0.0001 0.0003
Android-to-g ynoid fat ratio 1.0 (0.1) 1.1 (0.2) 1.1 (0.2) .0.9999 .0.9999 .0.9999
Fat mass (%) 39.5 (1.0) 43.7 (7.5) 46.4 (6.9) 0.0253 0.0012 0.1846
AUDIT score 3.0 (3.0) 3.0 (2.0) 2.0 (1.0) .0.9999 .0.9999 .0.9999
GGT (units/L) 16.5 (5.8) 18.0 (10.0) 20 .0 (8.3) .0.9999 .0.9999 0.8332
ALT (units/L) 21.0 (7.0) 22.0 (9.5) 27.5 (6.5) .0.9999 0.0591 0.0037
AST (units/L) 27.0 (9.5) 25.0 (6.0) 27.0 (8.5) .0.9999 .0.9999 0.0232
AST-to-ALT ratio 1.2 (0.8) 1.1 (0.3) 1.0 (0.5) .0.9999 0.1762 0.2315
FLI 36.7 (18.8) 50.4 (41.9) 85.2 (20.2) 0.1929 ,0.0001 0.0001
E-median (kPa) 4.7 (1.7) 3.9 (1.1) 5.5 (2.1) 0.3205 0.7638 0.0002
Insulinogenic index 0.3 (0.1) 0.3 (0.2) 0.7 (0.5) 0.3521 0.0010 0.0019
HOMA2
IR
1.2 (0.6) 1.5 (0.8) 2.4 (1.2) 0.4226 ,0.0001 ,0.0001
Disposition index 0.2 (0.1) 0.2 (0.2) 0.3 (0.4) .0.9999 0.5779 0.0981
Matsuda index 5.2 (1.7) 2.8 (1.9) 1.5 (1.2) 0.0155 ,0.0001 0.0004
Data are median (IQR) for continuous variables and n (%) fo r categorical variables. Continuous variables were analyzed using the Kruskall-Wallis
test with Dunn correction for multiple comparisons. Differences of categorical variables were analyzed by x
2
test with Bonferroni test for
multiple comparisons. P , 0.05 was c onsidered significant. Disposition index, insulinogenic index/HOMA2
IR
; E-median, median of hepatic elastici ty
as measured by vibrant-controlled transient elastography; Matsuda index, 10,000 /!(fasting glucose 3 fasting insulin) 3 (mean glucose 3 mean
insulin). *Polycystic ovarian syndrome was an exclusion criterion for control subjects.
112 NAFLD Is Prevalent in Women With Prior GDM Diabetes Care Volume 40, January 2017
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of the longitudinal Coronary Artery Risk
Development in Young Adults cohort
(31). The subgroup comprised black and
white Americans with (n = 124) and with-
out (n = 991) self-reported GDM. The
women underwent computed tomogra-
phy quantification 25 years after entry
into the study. The study found a preva-
lenceof14%inthepGDMgroupand5.8%
in the non-GDM group. The strong as-
soc iation between NAFLD and GDM was
due to t he development of diabetes in
the GDM group. In the current cohort,
the 2-h plasma glucose value did not
predict NAFLD in the multivariate anal-
ysis, which would have been expected if
the association between NAFLD and
GDM was only due to t he development
of diabetes. Furthermore, we are the
first, to our knowledge, to carry out a
4-hOGTTinwomenwithpGDMand
NAFLD. An OGTT allows for the c alcu la-
tion of the degree of peripheral insulin
resistance by the Matsuda index and
determines the dynamic response of
glucose, insulin, C-peptide, and gluca-
gon by calculating the AUC during the
4-h glucose challenge. This provides a
much more detailed image of the dy-
namics in glucose metabolism of these
women than previously described and a
chance to describe the impact of gluca-
gondynamicsduringanOGTTinthe
presence of NAFLD. The association be-
tween NAFLD and insulin resistan ce is
well established in the general popula-
tion, and the current findings support
this association (8).
In a previous study from our group,
fasting plasma glucagon levels were
higher in patient s with biops y-pro ven
NAFLD than in those with T2D and
no evidence of NAFLD and in healthy
control subjects (9). In the present co-
hort, fasting plasma glucagon was not
associated with NAFLD, but the initial
suppression of glucagon during the
OGTT in the NAFLD pGDM group was
significantly delayed. This phenomenon
could reflect hepatic insulin resistance
(9) or hepatic gl ucagon resistance (32),
and these findings suggest an important
role of NAFLD in the regulat ion of post-
absorptive glucagon secretion.
During the screen ing visit, a thorough
med ical history was taken, including a
record of recent an d present use of hep-
atotoxic and lipogenic medicatio n, and
ALT, AST, and GGT levels were mea-
sured. Similarly to other studies (33),
we found that w omen with NAFLD
have higher plasma levels of ALT, AST,
and GGT, although these were all within
the normal range. These liver enzymes
are markers of liver dam age, and several
studies have found ALT and GGT, even
within the norm al range, to predict di-
abetes (34). In the current study, none
of the liver enzymes were significantly
associated w ith the presence of NAFLD,
which is partially in line with Forbes
et al. (16), who found that ALT, but not
AST and GGT, is associated with NAFLD.
If our nonNAFLD pGDM group had been
without mild stages of NAFLD, we might
also have found an association between
ALT an d NAFLD, but this remains specu -
lative. On the basis of the small and non-
significant difference in AUDIT scores,
we were able to rule out alcohol-induced
liver damage.
The majority of women with pGDM in
this study had abnormal glucose toler-
ance. Of note, we found no difference in
theprevalenceofprediabetesamong
the groups. This i s in contrast to previ-
ous studies, which have demonstrated a
higher prevalence of prediabetes in pa-
tients with NAFLD (5) and a strong cor-
relation between T2D and NAFLD (3).
Significantly higher fasting concentra-
tions of C-peptide observ ed in the
NAFLD pGDM group suggest that these
patients’ b-cells are still capable of ade-
quately increasing insu lin secretion at
this time point , resulting in a nondiabetic
glucose tolerance. This may explain why
no difference in the prevalence of predi-
abetes was found between the pGDM
groups and probably accounts for the
similar disposition index. We found
that the women with NA FLD had in-
creased perip heral and hepatic insulin
resistance. Hepatic insulin resistance up-
regulates lipogenic mechanisms in the
Table 2—Glucose data
Group P value
Control (A) nonNAFLD pGDM (B) NAFLD pGDM (C) A – BA– CB– C
Glucose baseline (mmol/L) 4.9 (0.5) 5.4 (0.5) 5.4 (0.8) 0.0095 0.0108 .0.9999
Glucose peak (mmol/L) 8.2 (1.6) 10.0 (2.1) 10.7 (1.6) 0.0003 0.0002 .0.9999
2-h plasma glucose (mmol/L) 7.0 (0.4) 8.0 (2.1) 8.1 (2.1) 0.0031 0.0003 0.4103
Glucose tAUC (mmol/L 3 min) 1,433 (178) 1,668 (245) 1,764 (356) 0.0004 ,0.0001 0.2196
Glucose iAUC (mmol/L 3 min) 203 (66) 389 (226) 466 (215) 0.0056 0.0004 0.3000
Prediabetes * 46 (61) 17 (71) * * 0.5031
Insulin baseline (pmol/L) 54.2 (29.5) 78.6 (42.7) 127 (59) 0.3029 ,0.0001 ,0.0001
Insulin tAUC (nmol/L 3 min) 49.1 (21.3) 73.7 (57.3) 157 (101) 0.0308 ,0.0001 0.0009
Insulin iAUC (mmol/L 3 min) 34.4 (14.9) 57.0 (48.4) 122 (93) 0.0111 ,0.0001 0.0043
C-peptide baseline (pmol/L) 372 (143) 503 (237) 664 (199) 0.1586 0.0001 0.0005
C-peptide tAUC (nmol/L 3 min) 241 (138) 352 (122) 468 (168) ,0.0001 ,0.0001 0.0164
C-peptide iAUC (nmol/L 3 min) 163 (79) 223 (92) 265 (136) ,0.0001 ,0.0001 0.1757
Glucagon baseline (pmol/L) 5.0 (3.8) 6.0 (3.1) 6.8 (2.2) .0.9999 0.3305 0.3027
Glucagon tAUC (0–45) (pmol/L 3 min) 178 (132) 258 (112) 1,038 (386) 0.5892 ,0.0001 ,0.0001
Glucagon iAUC (0–45) (pmol/L 3 min) 266.3 (49.0) 210.0 (76.0) 730 (389) 0.0875 ,0.0001 ,0.0001
Data are median (IQR) for continuous variables and n (%) for categorical variables. Continuous variables were analyzed using the Kruskall-Wallis t est
with Dunn correction for multiple comparisons. Differences of categorical variables were analyzed by x
2
test with Bonferroni test for multiple
comparisons. P , 0.05 was considered significan t. *Prediabetes was an exclusion criterion for control subjects.
care.d iabetesjournals.org Foghsgaard and Associates 113
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