0013-7227/01/$03.00/0
7
Endocrinology
Copyright © 2001 by The Endocrine Society
Leptin Administration Prevents Spontaneous
Gestational Diabetes in Heterozygous Leprdb/ⴙ Mice:
Effects on Placental Leptin and Fetal Growth*
HIROSHI YAMASHITA, JIANHUA SHAO, TATSUYA ISHIZUKA,
PATRICK J. KLEPCYK, PEGGY MUHLENKAMP, LIPING QIAO, NIGEL HOGGARD,
AND JACOB E. FRIEDMAN
Departments of Nutrition and Reproductive Biology, Case Western Reserve University School of
Medicine, Cleveland, Ohio 44106-4935; and Rowett Research Institute (N.H.), Buckburn, Aberdeen,
Scotland, United Kingdom AB21 9SB
ABSTRACT
Gestational diabetes mellitus (GDM) results from an interaction
between susceptibility genes and the diabetogenic effects of preg-
nancy. During pregnancy, mice heterozygous for the lepin receptor
(db/⫹) gain more weight, are glucose intolerant, and produce mac-
rosomic fetuses compared with wild-type (⫹/⫹) mothers, suggesting
that an alteration in leptin action may play a role in GDM and fetal
overgrowth. To investigate whether leptin administration or pair-
feeding can reduce adiposity and thereby prevent GDM and neonatal
overgrowth, we examined energy balance, glucose and insulin toler-
ance, and fetal growth in pregnant db/⫹ and ⫹/⫹ mice treated with
recombinant human leptin-IgG during late pregnancy. Leptin re-
duced food intake and adiposity in pregnant db/⫹ mice to levels
similar to pregnant ⫹/⫹ mice and significantly reduced maternal
weight gain. Maternal glucose levels were markedly lower during
glucose and insulin challenge tests in leptin-treated db/⫹ mice rel-
G ESTATIONAL DIABETES mellitus (GDM) is associ-
ated with increases in maternal and perinatal mor-
bidity, including cesarean section, neonatal hypoglycemia,
and fetal macrosomia (1, 2). Moreover, human epidemiolog-
ical and animal studies suggest that the intrauterine diabetic
environment increases risk for hypertension, obesity, and
type II diabetes in adulthood (3–5). Animal models of GDM
have generally relied on maternal streptozotocin injection to
produce diabetes during pregnancy. However, streptozoto-
cin-induced diabetes usually does not result in fetal over-
growth (6). Mice heterozygous for the leptin receptor
(Leprdb/⫹) develop spontaneous glucose intolerance during
pregnancy, and the pups from these pregnancies are mac-
rosomic compared with offspring of wild-type mothers, re-
gardless of fetal genotype (7). Studies in the db/⫹ mouse
suggest that the leptin signal is apparently attenuated re-
sulting from reduced number of molecules of the intact long
receptor isoform (8). The db/⫹ mouse has increased plasma
Received December 27, 2000.
Address all correspondence and requests for reprints to: Jacob E.
Friedman, Ph.D., University of Colorado Health Sciences Center, Section
of Neonatology-B195, 4200 East Ninth Avenue, Denver, Colorado 80262.
E-mail: jed.friedman@uchsc.edu.
* This work was supported in part by Grant DK-50272 and Perinatal
Emphasis Research Center Grant 11089 from the NICHHD, NIH (to
J.E.F.), and by a grant from the Scottish Executive Rural Affairs Depart-
ment (to N.H.).
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ative to db/⫹ and pair-fed controls. Despite reduced energy intake and
improved glucose tolerance, leptin administration did not reduce fetal
overgrowth in offspring from db/⫹ mothers. Fetal and placental leptin
levels were 1.3- to 1.5-fold higher in offspring from db/⫹ mothers and
remained unchanged with leptin administration, whereas leptin
treatment in ⫹/⫹ mothers or pair-feeding decreased placental leptin
concentration and reduced fetal birth weight. Our results provide
evidence that leptin administration during late gestation can reduce
adiposity and improve glucose tolerance in the db/⫹ mouse model of
spontaneous GDM. However, fetal and placenta leptin levels are
higher in db/⫹ mothers and are subject to reduced negative feedback
in response to leptin treatment. These data suggest that alterations
in placenta leptin may contribute to the regulation of fetal growth
independently of maternal glucose levels. (Endocrinology 142: 2888 –
2897, 2001)
leptin levels relative to ⫹/⫹ mice (7, 9), suggesting that the
receptor is not fully recessive with regard to fat mass and that
heterozygosity at the leptin receptor may play a role in sus-
ceptibility to environmental conditions favoring obesity,
such as pregnancy.
In humans and animals, plasma leptin increases early dur-
ing gestation, derived primarily from the placenta (10 –12).
Although leptin and its receptor messenger RNA (mRNA)
are expressed by the placenta (12–14), the role of increased
leptin during pregnancy in maternal-fetal metabolism and
intrauterine growth remains unclear. The leptin gene has a
placenta-specific upstream enhancer (15), implying that pla-
cental leptin is differentially regulated from leptin of adipose
origin. In the mouse, leptin protein and mRNA are colocal-
ized to the trophoblast giant cells at the maternal interface of
the placenta and to the cytotrophoblasts in close proximity
to the developing fetus (12, 16). There is no correlation be-
tween maternal leptin levels and fetal weight; however, sev-
eral studies have reported that umbilical cord blood leptin
levels are positively correlated with fetal insulin, birth
weight, ponderal index (kilograms per cm3), and length and
head circumference (17–19), suggesting a potential relation-
ship between placental leptin and fetal growth. The higher
leptin levels in umbilical veins than umbilical arteries and the
marked fall after placental delivery indicate that the placenta is
one of the major sources of leptin in the fetal circulation (20).
 LEPTIN INFLUENCES GDM AND FETAL GROWTH
Leptin normally reduces appetite and increases energy
expenditure, acting through the hypothalamus (21, 22). Lep-
tin also has direct metabolic effects on several tissues, re-
sulting in increased glucose utilization and lipolysis (23–26).
Although the effect of leptin on insulin secretion is contro-
versial, most investigators report that leptin inhibits insulin
secretion (27–29). In the mouse, serum leptin increases by 25
times on day 17 of pregnancy in the maternal circulation (7,
30). The marked increase in maternal leptin, an appetite
suppressant, suggests there is some form of maternal leptin
resistance, or perhaps there is an alternative role for maternal
leptin. Leptin also serves as a mitogen for a growing number
of cell types, including endothelial cells, hemopoietic cells,
lung epithelial cells, and pancreatic ␤-cells in vitro (31–34).
Leptin could therefore be acting as a mitogen for the placenta
in addition to stimulating growth of tissues in the developing
fetus.
Previous studies have shown that environmental factors,
including weight gain during pregnancy, maternal glucose
levels, and fetal hyperinsulinemia, can contribute to fetal
macrosomia (35–37). Pregnant women with GDM have more
severe insulin resistance and abnormal insulin secretion (im-
paired glucose tolerance) compared with weight-matched
pregnant control subjects (38 – 40). The mechanisms for in-
sulin resistance in GDM include a 30 – 40% decrease in insulin
receptor tyrosine kinase activity in skeletal muscle compared
with obese pregnant controls (41) and is exacerbated by
decreased insulin receptor substrate-1 (IRS-1) tyrosine phos-
phorylation, due in part to decreased IRS-1 expression (41).
Given these abnormalities, we hypothesized that exogenous
leptin treatment during late gestation might reduce insulin
resistance, thereby lowering maternal glucose and prevent-
ing fetal overgrowth.
Materials and Methods
Animals and experimental protocol
Male and female C57BL/KsJ-Lepr⫹/⫹ and C57BL/KsJ-Leprdb/⫹ mice
were obtained from The Jackson Laboratory (Bar Harbor, ME) at 6 weeks
of age. Mice were housed in groups of three in a temperature-, humid-
ity-, and light-controlled (lights on at 0600 h, off at 1800 h) colony room.
Mice were given ad libitum access to commercial mouse chow and water.
At 60 – 80 days of age female mice were housed individually with ⫹/⫹
males, and mating was confirmed by the presence of a copulatory plug
the next morning, designated day 0 of gestation. On day 10 of pregnancy,
a human recombinant immunoadhesin leptin fusion protein (leptin-IgG)
or vehicle was administered daily for 7 days by ip injection (1 mg/kg䡠day
in 100 ␮l sterile saline). Recombinant leptin-IgG consists of a fusion
between human leptin and the Fc region of a human IgG molecule and
has a longer half-life than native leptin (42). This protein was synthesized
and purified by Genentech, Inc. (South San Francisco, CA). This dose
was chosen because it was demonstrated to be more than twice as potent
as native leptin in reducing food intake and promoting thermogenesis
and progressive weight loss when injected into ob/ob mice (42). The body
weight and food intake of each mouse were recorded daily to the nearest
0.1 g between 0900 –1000 h. Pair-feeding began on day 1 of pregnancy
and was accomplished by measuring the food intake of ad libitum-fed
⫹/⫾ pregnant mice every 24 h and presenting this amount of food to
pair-fed db/⫹ mice. On day 18 of gestation, mice were anesthetized with
ketamine (150 mg/kg) and acepromazine (5 mg/kg), the abdominal
cavity was opened, and the fetuses were removed with the uterus. Pups
were weighed to the nearest 0.01 g and decapitated, blood was collected,
and the placenta, liver, and brain were removed, blotted, weighed, and
frozen immediately on dry ice. Maternal fat mass was obtained from
postmortem collection of the mesenteric, gonadal, retroperitoneal, and
2889
dorsal fat pads weighed to the nearest 0.001 g. All procedures performed
were approved by the Case Western Reserve University animal care and
use committee.
Measurement of serum parameters and placenta leptin
Mouse serum leptin was measured before pregnancy and on day 18
using a commercial RIA kit specific for mouse leptin (Linco Research,
Inc., St. Charles, MO). Assays were conducted in duplicate, and the
intraassay coefficient of variation was less than 5%. To confirm that the
mouse leptin RIA kit did not cross-react with human leptin, two inde-
pendent experiments were performed. Firstly, we injected 1 mg/kg
human leptin IgG into the superior vena cava of nonpregnant mice and
measured serum leptin 10 min later using the mouse-specific RIA kit.
The levels averaged 4.56 ng/ml, similar to those in the nonpregnant
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control mice. Second, we measured the same samples using a human-
specific leptin RIA kit (Linco Research, Inc.) and found levels of 201–258
ng/ml, suggesting that the human and mouse leptin kits were species
specific, and that serum leptin levels were approaching pharmacological
concentrations. Preliminary studies were also performed to determine
whether leptin was able to cross the placenta from the maternal circu-
lation. Leptin IgG was injected into maternal mice via the inferior vena
cava, and the pups were killed 15 min later. Serum leptin levels assayed
by the human leptin kit averaged 0.81 ng/ml, similar to nonleptin-
treated fetal leptin levels. This finding is in full agreement with previous
human studies showing that there is no correlation between maternal
and fetal serum leptin levels (10, 11, 43) and suggests that maternal leptin
was unable to cross the placenta. Serum glucose was measured by
colorimetric glucose oxidase assay (Sigma, St. Louis, MO). Insulin was
detected in serum using commercial RIA kits for mice (Linco Research,
Inc.). Assays were conducted in duplicate, and the intraassay coefficient
of variation was less than 5%.
Placental leptin content was measured using a mouse enzyme-linked
immunosorbent assay detection system. Placental tissues were weighed
before homogenization in 5 vol extraction buffer (100 mm NH4HCO3, 10
mm EDTA, and 10 mm EGTA, pH 9.3) and were centrifuged at 15,000 ⫻
g for 15 min. The supernatants were stored at ⫺70 C until analysis. Leptin
was measured using murine leptin standards (0.05–25 ng/ml) as de-
scribed in detail previously (12). The mouse leptin receptor OB-Rb was
measured by Western blot analysis as detailed below, using commer-
cially available antisera (Linco Research, Inc.).
Glucose and insulin tolerance tests
Glucose tolerance tests were performed before pregnancy and on
selected mice on day 17 of pregnancy. Mice were fasted for 6 h and were
injected ip with glucose (2 g/kg body wt), and blood was sampled from
the tail vein using capillary tubes at 0, 30, and 60 min after glucose
injection. The blood samples were allowed to clot on ice and centrifuged
for 20 min at 13,000 rpm at 4 C, and the serum was frozen at ⫺70 C until
assayed for glucose and insulin. An insulin tolerance test was also
performed on selected mice on day 18 of pregnancy. The mice were
fasted for 6 h and were injected ip with insulin (0.75 U/kg BW). Blood
was collected from the tail vein at 0, 15, 30, and 60 min after glucose
injection, and the serum was frozen at ⫺70 C until assayed for glucose
as described above.
Insulin-stimulated tyrosine phosphorylation of insulin
receptor (IR␤), IRS-1, and phosphoinositol trisphosphate
3-kinase (PI3-kinase; p85␣ subunit)
To determine whether leptin treatment affected skeletal muscle in-
sulin signal transduction and expression of insulin-signaling proteins,
pregnant mice were challenged by insulin in vivo using a method de-
scribed previously (7), with minor modifications. Mice were anesthe-
tized with ketamine (150 mg/kg) and acepromazine (5 mg/kg), the
abdominal cavity was opened, and the portal vein was exposed. The skin
from one hind limb was removed, and a 200-mg biopsy of the gastroc-
nemius was taken and frozen immediately in liquid nitrogen. This was
followed by injection of 500-␮l bolus of normal saline (0.9% NaCl) with
or without insulin (10 U/kg BW; Humulin R, Eli Lilly & Co., Indianap-
olis, IN) into the portal vein. Within 5 min a sample from the opposite
gastrocnemius muscle was quickly excised and frozen immediately in
2890 LEPTIN INFLUENCES GDM AND FETAL GROWTH Endo • 2001
Vol. 142 • No. 7

liquid nitrogen. The frozen samples were pulverized in liquid nitrogen
and homogenized using a Polytron PTA 20S generator (Brinkmann
Instruments, Inc., Westbury, NY) at maximum speed for 30 sec in ice-
cold 10-fold volume of homogenization buffer [50 mm HEPES (pH 7.5),
100 mm Na2P202, 100 mm NaF, 10 mm EDTA, and 10 mm Na3VO4 plus
aprotinin (2 ␮g/ml), leupeptin (10 ␮g/ml), phenylmethylsulfonylfluo-
ride (34 ␮g/ml), and 1% Triton-X 100]. The homogenate was allowed to
incubate on ice for 30 min at 4 C, followed by centrifugation at 300,000
rpm in a 70 Ti rotor (Beckman Coulter, Inc., Fullerton, CA) at 4 C for 60
min to remove insoluble material. The supernatant was collected and
assayed for protein concentration (Bradford dye assay, Bio-Rad Labo-
ratories, Inc., Hercules, CA). For immunoprecipitation, 4 mg protein
were incubated overnight at 4 C with an antiphosphotyrosine antibody
(5 ␮g Ab/8 mg protein) in 1 ml immunoprecipitation buffer containing
2% Triton-X-100, 300 mm NaCl, 200 mm Tris-HCl, 2 mm EDTA, 2 mm
Statistical analysis
Results are presented as the mean ⫾ sem for the indicated number of
mice. Comparisons between groups were made using one-way ANOVA
and Student’s unpaired t test. Statistical significance was set at P ⬍ 0.05.
Results
Effects of pregnancy and GDM on serum parameters
Before pregnancy, fasting glucose and insulin levels were
not significantly different between ⫹/⫹ and db/⫹ mice (Ta-
ble 1); however, serum leptin levels were 25% higher in db/⫹
mice compared with those in ⫹/⫹ mice (P ⬍ 0.05). Preg-

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EGTA, 0.4 mm phenylmethylsulfonylfluoride, 0.4 mm sodium vanadate, nancy increased fasting glucose by 24 ⫾ 6% in db/⫹ mice (P ⬍
and 1% Nonidet P-40. After immunoprecipitation, the samples were 0.05), but had no effect on glucose levels in ⫹/⫹ mice. Fast-
mixed with 50 ␮l protein-A Sepharose (10% solution) for 4 h at 4 C, and
the immunoprecipitate was washed in 1 ml immunoprecipitation buffer
ing insulin levels increased during gestation by 2.2- to 3-fold
plus 0.1% Triton-X, followed by centrifugation at 500 ⫻ g for 1 min at in pregnant db/⫹ and ⫹/⫹ mice, respectively (P ⬍ 0.05).
4 C; this was repeated four times. The washed precipitate was mixed Leptin treatment reduced fasting insulin levels by 45% in
with Laemmli sample buffer (50 ␮l), boiled for 5 min, and centrifuged pregnant ⫹/⫹ mice (P ⬍ 0.05) and by 14% in pregnant db/⫹
for 5 min at 500 ⫻ g, and the supernatant (20 ␮l) was separated on a 7%
SDS gel. Proteins were electrotransferred from the gel to polyvinylidene mice (P ⫽ NS), whereas pair-feeding pregnant db/⫹ mice to
difluoride (PVDF) membrane, and the membranes were blocked with the intake of pregnant ⫹/⫹ mice throughout gestation re-
5% nonfat dry milk. The PVDF membranes were incubated with an- duced fasting insulin by 65 ⫾ 13% (P ⬍ 0.01).
tiphosphotyrosine antibodies (0.3 ␮g/ml; ␣-pY, Upstate Biotechnology, Serum leptin levels were measured on day 17 of pregnancy
Inc., Lake Placid, NY), IR␤, IRS-1, or p85␣ in blocking buffer overnight
at 4 C, followed by extensive washing with TBS-T. After final washing, using a leptin RIA kit specific for mouse leptin. Leptin levels
the blots were incubated with secondary antibody linked to HRP for 1 h were increased 35- to 37-fold in pregnant ⫹/⫹ and db/⫹
at room temperature. The membranes were washed and detected with mice, respectively, and were 20% higher in pregnant db/⫹
enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Ar- compared with pregnant ⫹/⫹ mice (P ⬍ 0.05). After 7 days
lington Heights, IL). Each sample was analyzed an average of three
separate times involving different gels. The results are expressed as the of human leptin treatment, mouse serum leptin was reduced
average signal intensity (arbitrary units) expressed relative to the effect by 15 ⫾ 6% (P ⬍ 0.05) in pregnant ⫹/⫹ mice compared with
of insulin on phosphorylation in ⫹/⫹ pregnant animals. For Western vehicle ⫹/⫹ controls. However, leptin levels remained un-
blotting, muscle homogenate containing 50 –75 ␮g protein was boiled for changed in leptin-treated db/⫹ mice compared with vehicle-
4 min in Laemmli sample buffer, run on 7% SDS gel, transferred to PVDF
membrane, and probed with anti-IR␤ (1:1000 dilution in TBS-T; Santa treated db/⫹ controls. Leptin levels were reduced by 7% in
Cruz Biotechnology, Inc., Santa Cruz, CA), anti-IRS-1 (1:1000 dilution; db/⫹ pair-fed mice, but this difference was not statistically
Transduction Laboratories, Inc., Lexington, KY), or anti-p85␣ (1:2000 significant.
dilution; Upstate Biotechnology, Inc.). The results were expressed as
arbitrary units compared with values obtained from the internal control
protein. TABLE 1. Serum glucose, insulin, and leptin in
C57BLKsJ-Lepr⫹/⫹ and Leprdb/⫹ mice
Genotyping for the Leprdb mutation
Fasting glucose Fasting insulin Serum leptin
Group
We modified Horvat and Bügers’s method of PCR-restriction frag- (mg/dl) (ng/ml) (ng/ml)
ment length polymorphism for identifying the db genotype in fetal tissue
Nonpregnant
(44). DNA was obtained from 100 mg fetal brain digested in 100 ␮l lysis
db/⫹ (n ⫽ 21) 98.6 ⫾ 7.5 0.49 ⫾ 0.17 3.1 ⫾ 0.3
buffer [100 mm Tris HCl (pH 8.5), 5 mm EDTA, 0.2% SDS, 200 mm NaCl,
⫹/⫹ (n ⫽ 19) 106.9 ⫾ 7.5 0.30 ⫾ 0.05 2.5 ⫾ 0.2a
and 100 ␮g protein kinase K/ml] overnight at 55 C with agitation. Five
Pregnant
microliters of a 1:50 dilution (in water) of these lysates served as a
db/⫹ vehicle 122.4 ⫾ 6.3b 1.07 ⫾ 0.16b 114.3 ⫾ 7.0b,c
template in a total 10-␮l PCR reaction. Five microliters of 2 ⫻ PCR premix
(n ⫽ 8)
was then added to yield final concentrations of 20 mm Tris-HCl (pH 8.4),
db/⫹ leptin 119.3 ⫾ 6.0 0.92 ⫾ 0.28b 111.6 ⫾ 8.6b,c
50 mm KCl, 2 mm MgCl2, 150 ␮m deoxy-NTP, 0.2 ␮m of each primer
(n ⫽ 7)
(forward, 5⬘-ATGACCACTACAGATGAACCCAGTCTAC-3⬘; reverse,
db/⫹ pair-fed 107.2 ⫾ 7.2 0.37 ⫾ 0.14a 103.0 ⫾ 7.6b,c
5⬘-CATTCAAACCATAGTTTAGGTTTGTCT-3⬘), and 0.2 U Taq poly-
(n ⫽ 6)
merase (Life Technologies, Inc., Gaithersburg, MD) Reactions were over-
⫹/⫹ vehicle 109.4 ⫾ 3.3 0.91 ⫾ 0.11b 93.4 ⫾ 3.5a,b,c
laid with 15 ␮l mineral oil. Amplification was carried out in Hybaid
(n ⫽ 13)
Limited Omnigene TR3 SM5 (National Labonet Co., UK) using a PCR
⫹/⫹ leptin 111.2 ⫾ 7.6 0.50 ⫾ 0.12a 79.2 ⫾ 6.1a,b
profile of 1 cycle at 95 C for 3 min; 5 cycles of 95 C for 1 min, 60 C for
(n ⫽ 6)
1 min, and 72 C for 30 sec; and 30 cycles of 92 C for 15 sec, 50 C for 1 min,
and 72 C for 30 sec. The 10-␮l PCR reaction was digested by adding Recombinant human leptin-IgG was administered daily (1 mg/kg 䡠
directly under oil 10 ␮l of 2 ⫻ digestion cocktail containing 7.0 ␮l water, day) on days 10 –16 of pregnancy. Pair-feeding in db/⫹ mice began on
2 ␮l buffer 4 (New England Biolabs, Inc., Beverly, MA), and 1 ␮l AccI day 1 of pregnancy. Mouse and human leptin levels were assayed
restriction enzyme (New England Biolabs, Inc.) and incubating at 37 C separately on day 18 of gestation. Human leptin averaged 91.3 ⫾ 7.1
for 4 h. After digestion, 4 ␮l loading buffer (0.25% bromophenol blue, and 80.2 ⫾ 5.5 ng/ml in db/⫹ and ⫹/⫹ mice, respectively. Numbers
0.25% xylene cyanol, and 30% glycerol) were added to each sample. in parentheses indicate the total number of mice examined. Data are
Digests (20 ␮l) were analyzed in 3.5% agarose (Sigma) and 1 ⫻ TAE the mean ⫾ SE.
buffer containing ethidium bromide (0.5 ␮g/ml). Digestion with AccI a
Significantly less than db/⫹ vehicle, P ⬍ 0.05.
yielded 85- and 24-bp fragments in ⫹/⫹ mice and 85-, 58-, 27-, and 24-bp b
Significantly greater than nonpregnant control, P ⬍ 0.05.
fragments in the heterozygote db/⫹ mice. c
Significantly different than ⫹/⫹ leptin-treated group, P ⬍ 0.05.
 LEPTIN INFLUENCES GDM AND FETAL GROWTH 2891

Changes in energy intake, body weight, and fat mass
during pregnancy: effects of leptin
Before pregnancy there were no differences in food intake
or total body weight between ⫹/⫹ and db/⫹ mice (7). The
average daily food intake of pregnant db/⫹ mice was greater
by 11 ⫾ 2% compared with that of pregnant ⫹/⫹ controls
(P ⬍ 0.05, Fig. 1A). At term, pregnant db/⫹ mice had 33 ⫾ 6%
greater maternal body weight gain (P ⬍ 0.05) and 20 ⫾ 3%
greater adipose tissue mass (P ⬍ 0.05) compared with preg-
nant ⫹/⫹ mice (Fig. 1, B and C). The total body weight
(maternal ⫹ fetal mass) at term was 24 ⫾ 4% greater in db/⫹
compared with ⫹/⫹ mothers (P ⬍ 0.05; Fig. 1D).
compared with those in db/⫹ controls. Leptin treatment in
pregnant ⫹/⫹ mice decreased the average food consumed by
6% compared with that in ⫹/⫹ controls, but this difference was
not significant. Maternal weight gain in leptin-treated ⫹/⫹
mice was 13 ⫾ 6% lower (0.8 g) less at term, and maternal
adipose tissue mass was reduced by 21 ⫾ 6% (0.2 g), but was
not statistically significant (P ⫽ 0.10; P ⫽ NS) compared with
that in pregnant ⫹/⫹ controls.
Effects of leptin treatment on pregnancy-induced
glucose intolerance
Pregnant db/⫹ mice demonstrated profound glucose in-

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Leptin-treated pregnant animals continued to gain weight;
however, leptin suppressed the average food intake in db/⫹
mice by 9 ⫾ 2% (P ⬍ 0.05) during days 10 –16 of pregnancy
to levels similar to pregnant ⫹/⫹ mice. At term, maternal
body weight was 31 ⫾ 7% lower (1.6 g; P ⬍ 0.05), and maternal
adipose tissue mass was reduced by 22 ⫾ 3% (0.4 g; P ⬍ 0.05)
in leptin-treated db/⫹ mice compared with db/⫹ controls. Pair-
feeding reduced maternal weight gain at term by 54 ⫾ 11% (2.4
g; P ⬍ 0.05) and adipose tissue mass by 17 ⫾ 4% (0.3 g; P ⬍ 0.05)
tolerance during a glucose tolerance test (Fig. 2). Glucose
levels were 41 ⫾ 11% higher (P ⬍ 0.05) at 30 and 60 min
compared with those in pregnant ⫹/⫹ controls (Fig. 2) de-
spite insulin levels that were twice as high in pregnant db/⫹
mice compared with ⫹/⫹ mice during the glucose tolerance
test (P ⬍ 0.05). Leptin-treated pregnant db/⫹ mice had sig-
nificantly lower glucose levels at 30 and 60 min by 33 ⫾ 6%
and 30 ⫾ 5% (P ⬍ 0.05), respectively, and lowered insulin
secretion by 26 ⫾ 10% and 40 ⫾ 12% during the glucose

FIG. 1. Food intake, maternal weight gain, maternal adipose tissue mass, and total (maternal and fetal) body weight in db/⫹ and ⫹/⫹ mice
during pregnancy: effects of leptin treatment or pair-feeding. Recombinant human leptin-IgG (1 mg/kg BW䡠day) or vehicle was administered
daily beginning on day 10 through day 16 of pregnancy. Pair-fed db/⫹ mice had a food intake similar to that of ⫹/⫹ mice beginning on day
1 of pregnancy. Data for food intake are for days 10 –16 of gestation. Maternal weight gain was obtained by subtracting the weight of the pups
from maternal weight on day 18 of pregnancy. Maternal fat mass was obtained from postmortem collection of the mesenteric, gonadal,
retroperitoneal, and dorsal fat pads weighed to the nearest 0.001 g. *, Significantly less than db/⫹ vehicle, P ⬍ 0.05. Data are the mean ⫾
SE (n ⫽ 6 – 8 animals/group, except for ⫹/⫹ vehicle, where n ⫽ 13).
2892 LEPTIN INFLUENCES GDM AND FETAL GROWTH Endo • 2001
Vol. 142 • No. 7

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FIG. 2. Plasma glucose and insulin concentrations during an ip glu-
cose tolerance test in pregnant db/⫹ and ⫹/⫹ mice: effect of leptin
administration. Mice were fasted 6 h on day 17 of gestation and
administrated 2 g/kg BW glucose loads at time zero, and insulin and FIG. 3. Insulin tolerance test in pregnant db/⫹, ⫹/⫹, leptin-treated
glucose levels were determined before and 30 and 60 min after in- db/⫹ and ⫹/⫹, and pair-fed db/⫹ mice. Mice were fasted 6 h on day
jection. Values are the mean ⫾ SEM for 6 –10 mice/group. *, P ⬍ 0.05, 18 of pregnancy and administrated 0.75 U/kg BW regular insulin load
db/⫹ vs. ⫹/⫹ mice; ⫹P ⬍ 0.05, db/⫹ vs. db/⫹ leptin-treated; # P ⬍ at time zero, and the glucose level was determined before and 15, 30,
0.05, ⫹/⫹ vs. ⫹/⫹ leptin-treated; @, P ⬍ 0.05, db/⫹ vs. ⫹/⫹ leptin- and 60 min after injection. Results are expressed as a percentage of
treated. the blood glucose concentration before insulin injection. Values are
the mean ⫾ SEM for 6 –10 mice/group. *, P ⬍ 0.05, db/⫹ vs. ⫹/⫹ mice;
⫹, P ⬍ 0.05, db/⫹ vs. leptin-treated; @, P ⬍ 0.05, db/⫹ vs. leptin-
tolerance test, suggesting improved ␤-cell function and re- treated ⫹/⫹ mice; #, P ⬍ 0.05, pair-fed db/⫹ vs. leptin-treated db/⫹
duced insulin resistance. In pregnant ⫹/⫹ mice, leptin treat- mice.
ment significantly lowered fasting insulin by 47 ⫾ 10% (P ⬍
0.05) and decreased insulin levels during the glucose toler-
phorylation of IRS-1 and p85␣ subunit of PI-3 kinase were
ance test by 27 ⫾ 6% and 38 ⫾ 6% at 30 and 60 min (P ⬍ 0.05).
lower by 21 ⫾ 3% (P ⬍ 0.05) and 19 ⫾ 3% (P ⬍ 0.05),
However, there was no significant effect on serum glucose
respectively, in pregnant db/⫹ mice compared with that in
levels. Pair-feeding pregnant db/⫹ mice reduced glucose lev-
pregnant ⫹/⫹ mice. In leptin-treated db/⫹ mice, insulin-
els by 63 ⫾ 22% at 60 min and significantly decreased fasting
stimulated IRS-1 and p85␣ phosphorylation in skeletal mus-
insulin and insulin secretion by 50 – 60% during the glucose
cle increased by 30 ⫾ 5% and 38 ⫾ 6%, respectively, com-
tolerance test (P ⬍ 0.05; data not shown).
pared with those in pregnant db/⫹ mice (P ⬍ 0.05). Leptin
An insulin challenge test was used to acutely stimulate
treatment had no effect on the level of the insulin receptor
glucose disposal as well as reduce hepatic glucose output in
phosphorylation of the 95-kDa ␤-subunit of the insulin re-
the blood (Fig. 3). As expected, pregnant control db/⫹ mice
ceptor in skeletal muscle.
demonstrated a 45–55% higher glucose level throughout the
The effect of leptin on skeletal muscle protein expression
insulin challenge test compared with pregnant ⫹/⫹ mice
was also examined in pregnant db/⫹ and ⫹/⫹ mice (Fig. 5).
(P ⬍ 0.05). Leptin treatment in pregnant db/⫹ mice dramat-
Pregnant db/⫹ mice had a 2.5-fold higher expression of IR␤
ically improved glucose disposal in response to insulin by
compared with pregnant ⫹/⫹ mice (P ⬍ 0.05), whereas IRS-1
45–50% at all time points compared with that in db/⫹ controls
expression in pregnant db/⫹ mice was significantly lower by
(P ⬍ 0.05), whereas pair-feeding pregnant db/⫹ mice mar-
43 ⫾ 10% (P ⬍ 0.05) compared with that in pregnant ⫹/⫹ mice.
ginally improved the overall rate of glucose disposal in re-
There was no significant difference in p85␣ expression between
sponse to insulin. The glucose levels in leptin-treated ⫹/⫹
pregnant ⫹/⫹ mice and db/⫹ mice. Leptin treatment in db/⫹
mice were not significantly different compared with those in
mice increased IR␤ expression by 33 ⫾ 8% (P ⬍ 0.05), IRS-1 by
⫹/⫹ controls during the insulin challenge test.
150 ⫾ 21% (P ⬍ 0.05), and p85␣ expression by 50 ⫾ 12% (P ⬍
Effects of leptin treatment on insulin signal transduction in
0.05), respectively. Pair-feeding also increased IR␤, IRS-1, and
skeletal muscle
p85␣ expression by 53–113% (P ⬍ 0.05). Leptin treatment in
pregnant ⫹/⫹ mice had no effect on IR␤, IRS-1, or p85␣ ex-
To determine the biochemical mechanisms associated with pression, and GLUT4 levels were unchanged (data not shown)
improved insulin sensitivity in the pregnant db/⫹ mouse in db/⫹ and leptin-treated animals.
treated with leptin, several aspects of the insulin receptor
signaling system were measured in vivo in skeletal muscle
Effect of leptin treatment on the fetus and placenta
from pregnant ⫹/⫹, db/⫹, and leptin-treated db/⫹ mice (Fig.
4). There were no differences in basal tyrosine phosphory- Fetuses were delivered by cesarean section on day 18 of
lation of the insulin receptor, IRS-1, and p85␣ subunit of PI-3 gestation. The number of fetuses born to db/⫹, ⫹/⫹, and
kinase in the db/⫹ pregnant mice compared with pregnant leptin-treated dams were not different (Table 2). We geno-
⫹/⫹ mice. In response to insulin, the level of tyrosine phos- typed the fetuses for the db mutation by PCR-restriction
 LEPTIN INFLUENCES GDM AND FETAL GROWTH 2893

FIG. 4. Effect of insulin on tyrosine

phosphorylation of IR␤ (A), IRS-1 (B),

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and PI 3-kinase (p85␣; C) in skeletal
muscle from pregnant ⫹/⫹, db/⫹, and
leptin-treated db/⫹ mice. Selected mice
were fasted overnight and anesthetized
before obtaining a muscle biopsy from a
hind limb on day 18 of pregnancy. This
was followed by insulin injection (10
U/kg BW) via the portal vein, and after
5 min a second muscle sample (Insulin
⫹) was obtained from opposite side hind
limb. The proteins were isolated, and
aliquots of the supernatant were immu-
noprecipitated with antiphosphoty-
rosine antibody and immunoblotted
with anti-IR␤, IRS-1, and PI3-kinase
(p85␣). The tyrosine-phosphorylated
bands corresponding to these proteins
were analyzed by scanning densitome-
try. The bar graph shows quantification
of the autoradiograms of these experi-
ments using six mice per group. The
values are expressed as arbitrary units
relative to pregnant ⫹/⫹ mice, assign-
ing a value of 100 to the insulin-stim-
ulated result. Values are the mean ⫾
SEM for 6 –10 mice/group. *, P ⬍ 0.05,
⫹/⫹ vs. db/⫹ mice; ⫹, P ⬍ 0.05, db/⫹ vs.
leptin-treated db/⫹ mice.

fragment length polymorphism, and there were no signifi-
cant differences between ⫹/⫹ and db/⫹ fetuses from the
same litter in terms of birth weight, liver size, or placenta size
(data not shown). We therefore grouped the data from each
litter together for analysis. The birth weight of pups from
db/⫹ control mothers was significantly heavier by 6.1 ⫾ 1.7%
compared with that of pups from ⫹/⫹ mothers (P ⬍ 0.05).
Placenta weights were similar in fetuses from ⫹/⫹ and db/⫹
mothers. Leptin treatment in db/⫹ mothers had no significant
effect on fetal birth weight or placenta weight. However,
leptin treatment of ⫹/⫹ mothers decreased fetal birth
weight and placenta weight in fetuses by 5.5 ⫾ 1.8% and
6.7 ⫾ 1.2%, respectively, compared with those of ⫹/⫹ con-
trol offspring (P ⬍ 0.05). Pair-feeding db/⫹ mothers to the
food intake from ⫹/⫹ mothers throughout pregnancy sig-
nificantly decreased birth weight by 7.2 ⫾ 1.8%, with no
change in placenta weight.
Placenta leptin was 26.9 ⫾ 9.3% higher (P ⬍ 0.05) in preg-
nancies from db/⫹ compared with ⫹/⫹ mothers. Leptin
treatment had no effect on placental leptin in db/⫹ mice,
whereas in ⫹/⫹ mothers, leptin treatment reduced placental
leptin by 17.6 ⫾ 5.4% (P ⬍ 0.05). Pair-feeding db/⫹ mice
during gestation reduced placental leptin by 28.2 ⫾ 4.6%
compared with that in db/⫹ controls (P ⬍ 0.05). Expression
2894 LEPTIN INFLUENCES GDM AND FETAL GROWTH Endo • 2001
Vol. 142 • No. 7

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FIG. 5. IR␤, IRS-1, and PI3-kinase (p85␣ subunit) in skeletal muscle from pregnant ⫹/⫹, db/⫹, leptin-treated db/⫹ and ⫹/⫹ mice, and db/⫹
mice pair-fed to ⫹/⫹ mice throughout gestation. Mice were anesthetized on day 18 of pregnancy, and a muscle biopsy was obtained from the
hind limb. Equivalent amounts of protein were subjected to SDS-PAGE and Western blotted with anti-IR␤, IRS-1, and anti-PI 3-kinase (85-kDa
subunit). The values are the mean ⫾ SE of the scanning densitometry values expressed in arbitrary units compared with the values obtained
in an internal control sample.

TABLE 2. Average fetal birth weight, litter size, placenta weight, and leptin content

Placental leptin
Mother Litter size Birth wt (g) Placenta (mg)
(ng/g wet wt)
db/⫹ vehicle (n ⫽ 58) 7.3 ⫾ 0.4 1.14 ⫾ 0.02 89.2 ⫾ 2.1 74.6 ⫾ 5.0
db/⫹ leptin (n ⫽ 46)
db/⫹ pair-fed (n ⫽ 42)
⫹/⫹ vehicle (n ⫽ 81)
6.6 ⫾ 0.7
7.0 ⫾ 0.6
6.2 ⫾ 0.3
1.15 ⫾ 0.02 a
1.07 ⫾ 0.02
1.09 ⫾ 0.01 a
a

]
a
]] 91.2 ⫾ 2.2
88.3 ⫾ 2.2
90.2 ⫾ 2.1
76.4 ⫾ 4.2 a
53.6 ⫾ 3.4
58.8 ⫾ 4.8 a
]
a
a
]]
⫹/⫹ leptin (n ⫽ 42) 7.0 ⫾ 0.5 1.03 ⫾ 0.02 ] 84.4 ⫾ 2.3 ]
a
48.4 ⫾ 3.2 ]
Fetal mice were delivered by cesarean section on day 18 and genotyped using PCR. No differences were noted in birth weight between fetal
genotypes; therefore, the results have been pooled. Numbers in parentheses indicate the total number of pups pups examined. Data are the
mean ⫾ SE.
a
P ⬍ 0.05.

of the leptin receptor OB-Rb was unchanged in the placenta
from db/⫹ mice or by leptin treatment (data not shown).
Because of poor recovery of fetal blood, we were unable to
assay fetal leptin in leptin-treated mice. However, we were
able to assay leptin levels in pooled samples from fetuses
from ⫹/⫹ and db/⫹ pregnancies. The leptin levels averaged
0.81 ⫾ 0.03 and 1.21 ⫾ 0.02 ng/ml in pups from ⫹/⫹ and
db/⫹ mothers, respectively (P ⬍ 0.05).
Discussion
The present study was designed to investigate the effects
of exogenous leptin administration on insulin sensitivity and
fetal overgrowth in the db/⫹ model of GDM. In the mouse,
serum leptin levels increased approximately 30-fold during
pregnancy and were 20% higher in pregnant db/⫹ compared
with pregnant ⫹/⫹ mice. The murine placenta, unlike hu-
man placenta, expresses large amounts of the circulating
OB-Re (short form) of the leptin receptor. Leptin binding
capacity in the serum rises about 40-fold by day 18 of ges-
tation (30), and this may contribute to the large increase in
serum leptin in pregnant mice as well as the leptin resistance
of pregnancy. Despite leptin resistance, however, exogenous
human leptin administration lessened maternal weight gain
and improved glucose tolerance in the db/⫹ mouse. Admin-
 LEPTIN INFLUENCES GDM AND FETAL GROWTH
istration of human recombinant leptin-IgG increased human
serum leptin levels to about 250 ng/ml, suggesting that large
daily injections were required to overcome the leptin resis-
tance of pregnancy. One of the main reasons for the effec-
tiveness of peripherally administered human leptin in the
db/⫹ mouse may be the relatively higher and sustained half-
life of the leptin immunoadhesion compared with native
leptin (42). High levels of leptin have been shown to reduce
fat content in heterozygous Zucker diabetic fatty (fa/⫹) rats
by blocking intracellular FFA esterification and by enhancing
intracellular oxidation of lipids (46, 47). More recently, pep-
tide analogs of leptin have been produced that decrease
blood glucose and body weight gain, but not food intake, in
db/db mice lacking the long form of the leptin receptor (48),
suggesting an important role for the leptin receptor short
form in some of the metabolic actions of leptin.
Leptin administration had only marginal effects on appe-
tite, but significantly reduced insulin resistance in pregnant
db/⫹ mice, in part through an improvement in skeletal mus-
cle insulin signal transduction at the level of IRS-1. Several
studies have shown that leptin increases insulin sensitivity
(23, 24, 49 –51), and leptin directly stimulates IRS-1-associ-
ated PI-3 kinase activity, although less than insulin alone
(52). Our results suggest that high doses of exogenous leptin
were slightly more effective at mobilizing lipid stores and
slowing maternal weight gain in pregnant db/⫹ compared
with ⫹/⫹ mice. The basis for this apparent difference in
responsiveness to leptin between pregnant ⫹/⫹ and db/⫹
mice is not clear. Pregnant control ⫹/⫹ mice had less adi-
pose tissue mass and lower insulin levels than db/⫹ mice
during gestation, which may have contributed to the inabil-
ity to detect small differences in absolute weight loss and
insulin sensitivity in response to leptin administration. Lep-
tin also down-regulated the endogenous leptin expression
levels in ⫹/⫹ mice, which may have contributed to the
reduced sensitivity. Leptin treatment was, however, success-
ful at improving glucose-stimulated insulin secretion during
a glucose tolerance test in pregnant ⫹/⫹ mice, an effect
reported by others in islets from normal animals (27, 28, 53,
54) and in islets from leptin-treated heterozygous Zucker
diabetic fa/⫹ rats (55), suggesting a positive effect on the
pancreatic ␤-cell.
The effects of leptin on fetal and placental growth have not
been investigated previously. Previous studies have found
that the leptin receptor mediates autocrine regulation of lep-
tin mRNA expression in a tissue-specific manner (56, 57).
Leptin administration reduces leptin synthesis in adipose
tissue, whereas in skeletal muscle it induces the protein in-
dependently of differences in fat mass or insulin levels. In
vitro studies indicate that placental leptin mRNA and protein
secretion increase in response to retinoic acid, cAMP, and
protein kinase C (58, 59). In the present work placental leptin
protein was reduced with leptin administration in the wild-
type mouse, but not in the db/⫹ pregnant animal. As our
studies were carried out in vivo, there could be a number of
causes of the reduced leptin levels, including a change in one
or more of the above-named intracellular mediators. Leptin
administration in db/⫹ mothers did not affect placenta or
maternal leptin protein levels or reduce fetal growth. The
lack of effect of leptin treatment on placenta leptin levels in
2895
db/⫹ mice suggests that the leptin receptor may play a role
in regulating its own expression by leptin in the placenta.
However, changes in leptin clearance, binding proteins,
and/or other hormones cannot be completely ruled out as
additional factors contributing to increased leptin levels in
pregnant db/⫹ mice.
In ob/ob mice, which lack leptin, leptin administration
throughout 19 days of gestation limited maternal weight
gain, while allowing a normal pregnancy to proceed (60).
Moreover, leptin administration for only 0.5 day postcoitus
in ob/ob mice also restored fertility and allowed ob/ob females
to sustain the pregnancy. These results indicate that leptin is
not required beyond day 0.5 for gestation; however, no de-
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tailed results for fetal or placental weight were reported. The
observation that ob/ob offspring from heterozygous (ob/⫹)
matings have reduced brain weight and decreased DNA
content, which is restored by leptin treatment postnatally
(61), suggests a role for leptin in the regulation of fetal brain
development.
Most of the leptin produced by the placenta is released into
the maternal circulation (62), but there is some recent evi-
dence, at least from human placenta studies, that a higher
proportion of leptin is released into the fetal circulation (63).
The fact that all pregnancies are associated with maternal
leptin resistance suggests that fetal macrosomia would more
likely be associated with changes in placental or fetal leptin
expression. The factors that increase fetal leptin levels in
macrosomia are not known. In the rodent there is very little
or no fetal adipose tissue; thus, the macrosomia may be a
function of increased placental production, whereas in other
animal models fetal leptin correlates with adipose tissue
mass (64). We found that leptin administered to pregnant
⫹/⫹ mice was undetectable in the fetal circulation, suggest-
ing that maternally derived leptin (e.g. of adipose origin)
does not contribute to fetal leptin levels. Because of the
smaller numbers, we were unable to assay fetal leptin in all
of the leptin-treated mice. However, we were able to detect
leptin, albeit in low levels, in the fetal circulation from ⫹/⫹
and db/⫹ pregnancies, and there was a 1.5-fold increase in
fetal and placental leptin level in db/⫹ compared with ⫹/⫹
offspring. Increased leptin in the placenta and fetal circula-
tion in db/⫹ mice are consistent with the hypothesis that
changes in placental leptin expression may play a significant
role in the regulation of fetal growth.
Weight gain during pregnancy and maternal glucose lev-
els in response to glucose tolerance tests are important mod-
ifiable variables for infant birth weight and future obesity (36,
65). Our findings suggest that pair-feeding throughout ges-
tation was more effective than short-term leptin treatment at
reducing fetal overgrowth in db/⫹ mice. Unlike leptin treat-
ment, caloric restriction significantly decreased placental
leptin levels during pregnancy. Pair-feeding may have also
limited maternal-fetal nutrient transfer throughout the entire
pregnancy, and this may be another important factor, in
addition to decreased placental leptin, responsible for de-
creasing fetal growth in offspring from pair-fed db/⫹ mice.
In summary, our data demonstrate that 7 days of leptin
treatment in the db/⫹ model reduces adiposity and improves
insulin sensitivity, suggesting it may potentially be an ef-
fective means of reducing the abnormal glucose tolerance
2896 LEPTIN INFLUENCES GDM AND FETAL GROWTH
associated with GDM. Our results also suggest a role for fetal
and placental leptin expression in the regulation of fetal
growth, independent of maternal glucose. Placental leptin
levels are increased in human diabetic pregnancies (66) and
decreased in pregnancies complicated by fetal growth retar-
dation (67). In addition, high insulin concentrations in um-
bilical cord blood are associated with higher concentrations
of leptin in cord blood and placenta. A role for leptin in
stimulating fetal pancreatic development has been suggested
(34, 68), which could result in early insulin production and
stimulate an increase in fetal growth. Alternatively, fetal
hyperinsulinemia could stimulate increased fetal and pla-
cental leptin, which, in turn, could contribute to increased
fetal growth in tissues expressing the leptin receptor. Studies
are currently underway in our laboratory to determine
whether maternal leptin administration alters insulin and the
␤-cell gene expression profile in neonatal mice. Leptin’s abil-
ity to influence fetal growth could have important implica-
tions for susceptibility to adult disease and will be an im-
portant area for future research.
Acknowledgments
We gratefully acknowledge Dr. Austin Gurney (Genentech, Inc.,
South San Francisco, CA) for providing recombinant leptin, and Jennifer
Crabtree (Rowett Research Institute) for determination of placental
leptin.
References
1. Schwartz R, Teramo KA 2000 Effects of diabetic pregnancy on the fetus and
newborn. Semin Perinatol 24:120 –135
2. Ferber A 2000 Maternal complications of fetal macrosomia. Clin Obstet Gy-
necol 43:335–333
3. Baker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS
1993 Fetal nutrition and cardiovascular disease in adult life. Lancet
341:938 –941
4. Pettitt DJ, Aleck KA, Baird HR, Carraher JM, Bennett PH, Knowler WC 1988
Congenital susceptibility to NIDDM. Role of the intrauterine environment.
Diabetes 37:622– 628
5. Holemans K, Aerts L, Van Assche FA 1991 Evidence for an insulin resistance
in the adult offspring of pregnant streptozotocin-diabetic rats. Diabetologia
34:81– 85
6. Gewolb IH, Barrett C, Wilson C, Warshaw JB 1982 Delay in pulmonary
glycogen degradation in fetuses of streptozotocin diabetic rats. Pediatr Res
16:869 – 873
7. Ishizuka T, Klepcyk P, Liu S, Panko L, Liu S, Gibbs EM, Friedman JE 1999
Effects of over expression of human GLUT4 gene on maternal diabetes and
fetal growth in spontaneous gestational diabetic C57BLKS/J Leprdb/⫹ mice.
Diabetes 48:1061–1069
8. Lee GH, Proenca R, Montez JM, Carrol KM, Darvishzadeth JG, Lee JI,
Friedman JM 1996 Abnormal splicing of the leptin receptor in diabetic mice.
Nature 379:632– 645
9. Chung Wk, Belfi D, Chua M, Wiley J, Mackintosh R, Nicolson M, Boozer
CN, Leibel RL 1998 Heterozygosity for Lepob or Leprdb affects body compo-
sition and leptin homeostasis in adult mice. Am J Physiol 274:43:R985–R990
10. Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nish-
imura H, Yoshimasa Y, Tanaka I, Mori T, Nakao K 1997 Non-adipose tissue
production of leptin: leptin as a novel placenta-derived hormone in humans.
Nat Med 3:1029 –1033
11. Highman T, Friedman JE, Huston L, Wong W, Catalano P 1998 Longitudinal
changes in maternal serum leptin concentrations, body composition and rest-
ing metabolic rate in pregnancy. Am J Obstet Gynecol 178:1010 –1015
12. Hoggard N, Hunter L, Lea RG, Trayhurn P, Mercer JG 2000 Ontogeny of the
expression of leptin and its receptor in the murine fetus and placenta. Br J. Nutr
83:317–326
13. Henson MC, Swan KF, O’Neil J 1998 Expression of placental leptin and leptin
receptor transcripts in early pregnancy and at term. Obstet Gynecol
92:1020 –1028
14. Bodner J, Ebenbichler CF, Wolf HJ, Muller-Holzner E, Stanzl U, Gander R,
Huter O, Patsch JR 1999 Leptin receptor in human term placenta: in situ
hybridization and immunohistochemical localization. Placenta 20:677– 682
15. Bi S, Gavrilova O, Gong DW, Mason MM, Reitman M 1997 Identification of
a placental enhancer for the human leptin gene. J Biol Chem 272:30583–30588
Endo • 2001
Vol. 142 • No. 7
16. Ashworth CJ, Hoggard N, Thomas L, Mercer JG, Wallace JM, Lea RG 2000
Placental leptin. Rev Reprod 5:18 –24
17. Tamura T, Golddenberg RL, Johnston KE, Cliver SP 1998 Serum leptin
concentration during pregnancy and their relationship to fetal growth. Obstet
Gynecol 91:389 –395
18. Ong KK, Ahmed ML, Sherriff A, Woods KA, WattsA, Golding J, Dunger DB
1999 Cord blood leptin is associated with size at birth and predicts infancy
weight gain in humans. ALSPAC study team. Avon longitudinal study of
pregnancy and childhood. J Clin Endocrinol Metab 84:1145–1148
19. Lepercq J, Lahlou N, Timsit J, Girard G, Haugel-de Mouzon S 1999 Mac-
rosomia revisited: ponderal index and leptin delineate subtypes of fetal over-
growth. Am J Obstet Gynecol 181:621– 625
20. Yura S, Sagawa N, Mise H, Masuzaki H, Ogawa Y, Nakao K 1999 A positive
umbilical venous-arterial difference of leptin level and its rapid decline after
birth. Am J Obstet Gynecol 178:926 –930
21. Campfield LA, Smith FJ, Guiser Y, Devos R, Burn P 1995 Recombination
mouse OB protein; evidence for a peripheral signal linking adiposity and
Downloaded from https://academic.oup.com/endo/article/142/7/2888/2989199 by guest on 23 October 2022
central neural networks. Science 269:546 –549
22. Vaisse C, Halaas JL, Horvath CM, Darnell Jr JE, Stoffel M, Friedman JM 1996
Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but
not db/db mice. Nat Genet 14:95–97
23. Sivitz WI, Walsh SA, Morgan DA, Thomas ML, Haynes WG 1997 Effect of
leptin on insulin sensitivity in normal rats. Endocrinology 138:3395–3401
24. Brazilai N, Wang J, Massilon D, Vuguin P, Hawkin M, Rossetti L 1997 Leptin
selective decrease visceral adiposity and enhances insulin action. J Clin Invest
100:3105–3110
25. Brison JM, Phuyal JL, Swan V, Caterson ID 1999 Leptin has acute effects on
glucose and lipid metabolism in both lean and gold thioglucose-obese mice.
Am J Physiol 277:E417–E422
26. Kamohara S, Burceline R, Halaas JL, Friedman JM, Charron MJ 1997 Acute
stimulation of glucose metabolism in mice by leptin treatment. Nature
389:374 –377
27. Poitout V, Rouault C, Guerre-Millo M, Briaud I, Reach G 1998 Inhibition of
insulin secretion by leptin in normal rodent islets of Langerhans. Endocrinol-
ogy 139:822– 826
28. Ookuma M, Ookuma K, York DA 1998 Effect of leptin on insulin secretion
from isolated rat pancreatic islets. Diabetes 47:219 –223
29. Seufert J, Kieffer TJ, Leech CA, Holz GG, Moritz W, Ricordi C, Habener JF
1999 Leptin suppression of insulin secretion and gene expression in human
pancreatic islets: implications for the development of adipogenic diabetes
mellitus. J Clin Endocrinol Metab 84:670 – 676
30. Gavrilova O, Barr V, Marcus-Samuels B, Reitman M 1997 Hyperleptinemia
of pregnancy associated with the appearance of a circulating form of the leptin
receptor. J Biol Chem 272:30546 –30551
31. Bouloumie A, Drexler HC, Lafontan M, Busse R 1998 Leptin, the product of
Ob gene, promotes angiogenesis. Circ Res 83:1059 –1066
32. Gainsford T, Willson TA, Metcalf D, Handman E, McFarlane C, Ng A, Nicola
NA, Alexander WS, Hilton DJ 1996 Leptin can induce proliferation, differ-
entiation, and functional activation of hemopoietic cells. Proc Natl Acad Sci
USA 93:14564 –14568
33. Tsuchiya T, Shimizu H, Horie T, Mori M 1999 Expression of leptin receptor
in lung: leptin as a growth factor. Eur J Pharmacol 365:273–279
34. Islam MS, Morton NM, Hansson A, Emilsson V 1999 Rat insulinoma-derived
pancreatic beta cells express a functional leptin receptor that mediates a pro-
liferative response. Biochem Biohophys Res Commun 238:851– 855
35. Kalkhoff DK 1991 Impact of maternal fuel and nutritional state on fetal
growth. Diabetes [Suppl 2] 40:61– 65
36. Galtier-Dereure F, Boegner C, Bringer J 2000 Obesity and pregnancy: com-
plications and cost. Am J Clin Nutr [Suppl 5] 71:1242S–1248S
37. Frienkel N 1980 Banting lecture: Of pregnancy and progeny. Diabetes
29:1023–1035
38. Kuhl C 1991 Insulin secretion and insulin resistance in pregnancy and GDM.
Implications for diagnosis and management. Diabetes [Suppl 2] 40:18 –24
39. Catalano PM, Huston L, Amini SB, Kalhan SC 1999 Longitudinal change in
glucose metabolism during pregnancy in obese women with normal glucose
tolerance and gestational diabetes mellitus. Am J Obstet Gynecol 180:903–916
40. Buchanan TA, Metzger BE, Freinkel N, Bergman RN 1990 Insulin sensitivity
and beta cell responsiveness to glucose during late pregnancy in lean and
moderately obese women with normal glucose tolerance or mild gestational
diabetes. Am J Obstet Gynecol 162:1008 –1014
41. Friedman JE, Ishizuka T, Huston L, Highman T, Shao JH, Catalano P 1999
Impaired tyrosine kinase activity and insulin receptor substrate-1 expression
in obese women with gestational diabetes mellitus. Diabetes 48:1807–1814
42. Luoh SM, Di Marco F, Levin N, Armanini M, Xie MH, Nelson C, Bennet GL,
Willams M, Spencer SA, Gurney A, Sauvage FJ 1997 Cloning and charac-
terization of human leptin receptor using a biologically active leptin immu-
noadhesin. J Mol Endocrinol 18:77– 85
43. Schubring C, Kiess W, Englaro P, Rascher W, Blum W 1996 Leptin concen-
trations in amniotic fluid, venous and arterial cord blood: high leptin synthesis
in the fetus and inverse correlation with placental weight. Eur J Pediatr
155:830 – 834
44. Horvat S, Bügers L 1999 Polymerase chain reaction-restriction fragment length
 LEPTIN INFLUENCES GDM AND FETAL GROWTH
polymorphism (PCR-RFLP) assay for the mouse leptin receptor (Leprdb) mu-
tation. Lab Anim 33:380 –384
45. Bjorbaek C, Uotani S, da Silva B, Flier JS 1997 Divergent signaling capacities
of the long and short isoforms of the leptin receptor. J Biol Chem 272:32686 –32695
46. Zhour YT, Shimabukuro M, Koyama K, Lee Y, Wang MY, Trie F, Newgard
CB, Unger RH 1997 Induction by leptin of uncoupling protein-2 and enzymes
of fatty acid oxidation. Proc Natl Acad Sci USA 94:6386 – 6390
47. Shimabukuro M, Koyama K, Chen G, Wang MY, Trieu F, Lee Y, Newgard
CB, Unger RH 1997 Direct antidiabetic effect of leptin through triglyceride
depletion of tissues. Proc Natl Acad Sci USA 94:4637– 4641
48. Grasso P, White DW, Tartaglia LA, Leinung MC, Lee DW1999 Inhibitory
effects of leptin-related synthetic peptide 116 –130 on food intake and body
weight gain in female C57BL/6J ob/ob mice may not be mediated by peptide
activation of the long isoform of the leptin receptor. Diabetes 48:2204 –2202
49. Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL 1999 Leptin
reverses insulin resistance and diabetes mellitus in mice with congenital lip-
odystrophy. Nature 401:73–76
50. Buettner R, Newgard CB, Rhodes CJ, O’Doherty RM 2000 Correction of
diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin
resistance by moderate hyperleptinemia. Am J Physiol 278:E563–E569
51. Masuzaki H, Ogawa Y, Aizawa-Abe M, Hosoda K, Suga J, Ebihara K, Satoh
N, Iwai H, Inoue G, Nishimura H, Yoshimasa Y, Nakao K 1999 Glucose
metabolism and insulin sensitivity in transgenic mice overexpressing leptin
with lethal yellow agouti mutation: usefulness of leptin for the treatment of
obesity-associated diabetes. Diabetes 48:1615–1622
52. Kim YB, Uotani Sh, Pierroz DD, Flier JS, Kahn BB 2000 In vivo administration
of leptin activates signal transduction directly in insulin-sensitive tissues:
overlapping but distinct pathways from insulin. Endocrinology 141:2328 –2339
53. Emilsson V, Liu YL, Cawthorne MA, Morton NM, Davenport M 1997 Ex-
pression of the functional leptin receptor mRNA in pancreatic islets and direct
inhibitory action of leptin on insulin secretion. Diabetes 46:313–316
54. Roduit R, Thorens B 1997 Inhibition of glucose-induced insulin secretion by
long-term preexposure of pancreatic islets to leptin. FEBS Lett 415:179 –182
55. Zhou YT, Shimabukuro M, Lee Y, Koyama K, Trieu F, Unger R 1997 Leptin
normalizes the impaired response of proinsulin mRNA to long chain fatty
acids in heterozygous Zucker diabetic fatty rats. J Biol Chem 272:25648 –25651
56. Zhang Y, Olbort M, Schwarzer K, Nuesslein-Hildesheim B, Nicolson M,
Murphy E, Kowalski TJ, Schmidt I, Leibel RL 1997 The leptin receptor
2897
mediates apparent autocrine regulation of leptin gene expression. Biochem
Biophys Res Commun 240:492– 495
57. Wang J, Liu R, Liu L, Chowdhury R, Barzilai N, Tan J, Rossetti L 1999 The
effect of leptin on Lep expression is tissue-specific and nutritionally regulated.
Nat Med 5:895– 859
58. Guibourdenche J, Tarrade A, Laurendeau I, Rochette-Egly C, Chambon P,
Vidaud M, Evain-Brion D 2000 Retinoids stimulate leptin synthesis and se-
cretion in human syncytiotrophoblast. J Clin Endocrinol Metab 85:2550 –2555
59. Yura S, Sagawa N, Ogawa Y, Masuzaki H, Mise H, Matsumoto T, Ebihara
K, Fujii S, Nakao K 1998 Augmentation of leptin synthesis and secretion
through activation of protein kinases A and C in cultured human trophoblastic
cells. J Clin Endocrinol Metab 83:3609 –3615
60. Mounzih K, Qiu J, Ewart-Toland A, Chehab FF 1998 Leptin is not necessary
for gestation and parturition but regulates maternal nutrition via a leptin
resistance state. Endocrinology 139:5259 –5262
61. Ahima RS, Bjorbaek C, Osei S, Flier JS 1999 Regulation of neuronal and glial
proteins by leptin: implications for brain development. Endocrinology
Downloaded from https://academic.oup.com/endo/article/142/7/2888/2989199 by guest on 23 October 2022
140:2755–2762
62. Linnemann K Malek A, Sager R, Blum WF, Schneider H, Fusch C 2000 Leptin
production and release in the dually in vitro perfused human placenta. J Clin
Endocrinol Metab 85:4298 – 4301
63. Hoggard N, Crabtree J, Allstaff S, Abramovich DR, Haggarty P 2001 Leptin
secretion to both the maternal and fetal circulation in the ex vivo perfused
human term placenta. Placenta 22:347–352
64. Chen X, Lin J, Hausman DB, Martin RJ, Dean RG, Hausman GJ 2000 Al-
terations in fetal adipose tissue leptin expression correlate with the develop-
ment of adipose tissue. Biol Neonate 78:41– 47
65. Kjos SL, Buchanan TA 1999 Gestational diabetes mellitus. N Engl J Med
341:1749 –1756
66. Lepercq J, Cauzac M, Lahlou N, Timsit J, Girard J, Auwerx J, Hauguel-de
Mouzon H 1998 Overexpression of placental leptin in diabetic pregnancy.
Diabetes 47:847– 850
67. Lea RG, Howe D, Hannah LT, Bonneau O, Hunter L, Hoggard N 2000
Placental leptin in normal, diabetic and fetal growth-retarded pregnancies. Mol
Hum Reprod 8:763–769
68. Islam MS, Sjoholm A, Emilsson V 2000 Fetal pancreatic islets express func-
tional leptin receptors and leptin stimulates proliferation of fetal islet cells. Int
J Obesity 24:1246 –1253