Effects on Placental Leptin and Fetal

0013-7227/01/$03.00/0 Endocrinology Copyright © 2001 by The Endocrine Society

Vol. 142, No. 7 Printed in U.S.A.

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 pregnancy. During pregnancy, mice heterozygous for the lepin receptor (db/⫹) gain more weight, are glucose intolerant, and produce macrosomic 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 pairfeeding can reduce adiposity and thereby prevent GDM and neonatal overgrowth, we examined energy balance, glucose and insulin tolerance, and fetal growth in pregnant db/⫹ and ⫹/⫹ mice treated with recombinant human leptin-IgG during late pregnancy. Leptin reduced 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-

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)

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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 susceptibility to environmental conditions favoring obesity, such as pregnancy. In humans and animals, plasma leptin increases early during 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 placental leptin is differentially regulated from leptin of adipose origin. In the mouse, leptin protein and mRNA are colocalized 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 between maternal leptin levels and fetal weight; however, several 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 relationship 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).

ESTATIONAL DIABETES mellitus (GDM) is associated with increases in maternal and perinatal morbidity, including cesarean section, neonatal hypoglycemia, and fetal macrosomia (1, 2). Moreover, human epidemiological 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, streptozotocin-induced diabetes usually does not result in fetal overgrowth (6). Mice heterozygous for the leptin receptor (Leprdb/⫹) develop spontaneous glucose intolerance during pregnancy, and the pups from these pregnancies are macrosomic compared with offspring of wild-type mothers, regardless of fetal genotype (7). Studies in the db/⫹ mouse suggest that the leptin signal is apparently attenuated resulting 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: [email protected]. * 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 Department (to N.H.).

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LEPTIN INFLUENCES GDM AND FETAL GROWTH

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Leptin normally reduces appetite and increases energy expenditure, acting through the hypothalamus (21, 22). Leptin also has direct metabolic effects on several tissues, resulting in increased glucose utilization and lipolysis (23–26). Although the effect of leptin on insulin secretion is controversial, 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 (impaired glucose tolerance) compared with weight-matched pregnant control subjects (38 – 40). The mechanisms for insulin 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 phosphorylation, 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 preventing fetal overgrowth.

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.

Materials and Methods Animals and experimental protocol

Glucose and insulin tolerance tests

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-, humidity-, 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

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 independent 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 control mice. Second, we measured the same samples using a humanspecific 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 circulation. 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 nonleptintreated 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 described in detail previously (12). The mouse leptin receptor OB-Rb was measured by Western blot analysis as detailed below, using commercially available antisera (Linco Research, Inc.).

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 insulin signal transduction and expression of insulin-signaling proteins, pregnant mice were challenged by insulin in vivo using a method described previously (7), with minor modifications. Mice were anesthetized 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 gastrocnemius 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., Indianapolis, IN) into the portal vein. Within 5 min a sample from the opposite gastrocnemius muscle was quickly excised and frozen immediately in

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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 icecold 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), phenylmethylsulfonylfluoride (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 Laboratories, 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 EGTA, 0.4 mm phenylmethylsulfonylfluoride, 0.4 mm sodium vanadate, and 1% Nonidet P-40. After immunoprecipitation, the samples were 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 plus 0.1% Triton-X, followed by centrifugation at 500 ⫻ g for 1 min at 4 C; this was repeated four times. The washed precipitate was mixed with Laemmli sample buffer (50 ␮l), boiled for 5 min, and centrifuged 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 difluoride (PVDF) membrane, and the membranes were blocked with 5% nonfat dry milk. The PVDF membranes were incubated with antiphosphotyrosine antibodies (0.3 ␮g/ml; ␣-pY, Upstate Biotechnology, 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, the blots were incubated with secondary antibody linked to HRP for 1 h at room temperature. The membranes were washed and detected with enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL). Each sample was analyzed an average of three separate times involving different gels. The results are expressed as the average signal intensity (arbitrary units) expressed relative to the effect of insulin on phosphorylation in ⫹/⫹ pregnant animals. For Western blotting, muscle homogenate containing 50 –75 ␮g protein was boiled for 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 Cruz Biotechnology, Inc., Santa Cruz, CA), anti-IRS-1 (1:1000 dilution; Transduction Laboratories, Inc., Lexington, KY), or anti-p85␣ (1:2000 dilution; Upstate Biotechnology, Inc.). The results were expressed as arbitrary units compared with values obtained from the internal control protein.

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 (Table 1); however, serum leptin levels were 25% higher in db/⫹ mice compared with those in ⫹/⫹ mice (P ⬍ 0.05). Pregnancy increased fasting glucose by 24 ⫾ 6% in db/⫹ mice (P ⬍ 0.05), but had no effect on glucose levels in ⫹/⫹ mice. Fasting insulin levels increased during gestation by 2.2- to 3-fold in pregnant db/⫹ and ⫹/⫹ mice, respectively (P ⬍ 0.05). Leptin treatment reduced fasting insulin levels by 45% in pregnant ⫹/⫹ mice (P ⬍ 0.05) and by 14% in pregnant db/⫹ mice (P ⫽ NS), whereas pair-feeding pregnant db/⫹ mice to the intake of pregnant ⫹/⫹ mice throughout gestation reduced fasting insulin by 65 ⫾ 13% (P ⬍ 0.01). Serum leptin levels were measured on day 17 of pregnancy using a leptin RIA kit specific for mouse leptin. Leptin levels were increased 35- to 37-fold in pregnant ⫹/⫹ and db/⫹ mice, respectively, and were 20% higher in pregnant db/⫹ compared with pregnant ⫹/⫹ mice (P ⬍ 0.05). After 7 days of human leptin treatment, mouse serum leptin was reduced by 15 ⫾ 6% (P ⬍ 0.05) in pregnant ⫹/⫹ mice compared with vehicle ⫹/⫹ controls. However, leptin levels remained unchanged in leptin-treated db/⫹ mice compared with vehicletreated db/⫹ controls. Leptin levels were reduced by 7% in db/⫹ pair-fed mice, but this difference was not statistically significant. TABLE 1. Serum glucose, insulin, and leptin in C57BLKsJ-Lepr⫹/⫹ and Leprdb/⫹ mice

Genotyping for the Leprdb mutation We modified Horvat and Bu¨gers’s method of PCR-restriction fragment length polymorphism for identifying the db genotype in fetal tissue (44). DNA was obtained from 100 mg fetal brain digested in 100 ␮l lysis buffer [100 mm Tris HCl (pH 8.5), 5 mm EDTA, 0.2% SDS, 200 mm NaCl, and 100 ␮g protein kinase K/ml] overnight at 55 C with agitation. Five microliters of a 1:50 dilution (in water) of these lysates served as a template in a total 10-␮l PCR reaction. Five microliters of 2 ⫻ PCR premix was then added to yield final concentrations of 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 2 mm MgCl2, 150 ␮m deoxy-NTP, 0.2 ␮m of each primer (forward, 5⬘-ATGACCACTACAGATGAACCCAGTCTAC-3⬘; reverse, 5⬘-CATTCAAACCATAGTTTAGGTTTGTCT-3⬘), and 0.2 U Taq polymerase (Life Technologies, Inc., Gaithersburg, MD) Reactions were overlaid with 15 ␮l mineral oil. Amplification was carried out in Hybaid Limited Omnigene TR3 SM5 (National Labonet Co., UK) using a PCR profile of 1 cycle at 95 C for 3 min; 5 cycles of 95 C for 1 min, 60 C for 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 directly under oil 10 ␮l of 2 ⫻ digestion cocktail containing 7.0 ␮l water, 2 ␮l buffer 4 (New England Biolabs, Inc., Beverly, MA), and 1 ␮l AccI restriction enzyme (New England Biolabs, Inc.) and incubating at 37 C for 4 h. After digestion, 4 ␮l loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol) were added to each sample. Digests (20 ␮l) were analyzed in 3.5% agarose (Sigma) and 1 ⫻ TAE buffer containing ethidium bromide (0.5 ␮g/ml). Digestion with AccI yielded 85- and 24-bp fragments in ⫹/⫹ mice and 85-, 58-, 27-, and 24-bp fragments in the heterozygote db/⫹ mice.

Group

Nonpregnant db/⫹ (n ⫽ 21) ⫹/⫹ (n ⫽ 19) Pregnant db/⫹ vehicle (n ⫽ 8) db/⫹ leptin (n ⫽ 7) db/⫹ pair-fed (n ⫽ 6) ⫹/⫹ vehicle (n ⫽ 13) ⫹/⫹ leptin (n ⫽ 6)

Fasting glucose (mg/dl)

Fasting insulin (ng/ml)

Serum leptin (ng/ml)

98.6 ⫾ 7.5 106.9 ⫾ 7.5

0.49 ⫾ 0.17 0.30 ⫾ 0.05

3.1 ⫾ 0.3 2.5 ⫾ 0.2a

122.4 ⫾ 6.3b

1.07 ⫾ 0.16b

114.3 ⫾ 7.0b,c

119.3 ⫾ 6.0

0.92 ⫾ 0.28b

111.6 ⫾ 8.6b,c

107.2 ⫾ 7.2

0.37 ⫾ 0.14a

103.0 ⫾ 7.6b,c

109.4 ⫾ 3.3

0.91 ⫾ 0.11b

93.4 ⫾ 3.5a,b,c

111.2 ⫾ 7.6

0.50 ⫾ 0.12a

79.2 ⫾ 6.1a,b

Recombinant human leptin-IgG was administered daily (1 mg/kg 䡠 day) on days 10 –16 of pregnancy. Pair-feeding in db/⫹ mice began on day 1 of pregnancy. Mouse and human leptin levels were assayed separately on day 18 of gestation. Human leptin averaged 91.3 ⫾ 7.1 and 80.2 ⫾ 5.5 ng/ml in db/⫹ and ⫹/⫹ mice, respectively. Numbers in parentheses indicate the total number of mice examined. Data are the mean ⫾ SE. a Significantly less than db/⫹ vehicle, P ⬍ 0.05. b Significantly greater than nonpregnant control, P ⬍ 0.05. c Significantly different than ⫹/⫹ leptin-treated group, P ⬍ 0.05.

LEPTIN INFLUENCES GDM AND FETAL GROWTH 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 pregnant ⫹/⫹ 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). 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. Pairfeeding 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)

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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 intolerance 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) despite 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 significantly 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).

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FIG. 2. Plasma glucose and insulin concentrations during an ip glucose 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 glucose levels were determined before and 30 and 60 min after injection. Values are the mean ⫾ SEM for 6 –10 mice/group. *, P ⬍ 0.05, db/⫹ vs. ⫹/⫹ mice; ⫹P ⬍ 0.05, db/⫹ vs. db/⫹ leptin-treated; # P ⬍ 0.05, ⫹/⫹ vs. ⫹/⫹ leptin-treated; @, P ⬍ 0.05, db/⫹ vs. ⫹/⫹ leptintreated.

tolerance test, suggesting improved ␤-cell function and reduced insulin resistance. In pregnant ⫹/⫹ mice, leptin treatment significantly lowered fasting insulin by 47 ⫾ 10% (P ⬍ 0.05) and decreased insulin levels during the glucose tolerance test by 27 ⫾ 6% and 38 ⫾ 6% at 30 and 60 min (P ⬍ 0.05). However, there was no significant effect on serum glucose levels. Pair-feeding pregnant db/⫹ mice reduced glucose levels by 63 ⫾ 22% at 60 min and significantly decreased fasting insulin and insulin secretion by 50 – 60% during the glucose tolerance test (P ⬍ 0.05; data not shown). An insulin challenge test was used to acutely stimulate glucose disposal as well as reduce hepatic glucose output in the blood (Fig. 3). As expected, pregnant control db/⫹ mice demonstrated a 45–55% higher glucose level throughout the insulin challenge test compared with pregnant ⫹/⫹ mice (P ⬍ 0.05). Leptin treatment in pregnant db/⫹ mice dramatically improved glucose disposal in response to insulin by 45–50% at all time points compared with that in db/⫹ controls (P ⬍ 0.05), whereas pair-feeding pregnant db/⫹ mice marginally improved the overall rate of glucose disposal in response to insulin. The glucose levels in leptin-treated ⫹/⫹ mice were not significantly different compared with those in ⫹/⫹ controls during the insulin challenge test. Effects of leptin treatment on insulin signal transduction in skeletal muscle

To determine the biochemical mechanisms associated with improved insulin sensitivity in the pregnant db/⫹ mouse treated with leptin, several aspects of the insulin receptor signaling system were measured in vivo in skeletal muscle from pregnant ⫹/⫹, db/⫹, and leptin-treated db/⫹ mice (Fig. 4). There were no differences in basal tyrosine phosphorylation of the insulin receptor, IRS-1, and p85␣ subunit of PI-3 kinase in the db/⫹ pregnant mice compared with pregnant ⫹/⫹ mice. In response to insulin, the level of tyrosine phos-

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FIG. 3. Insulin tolerance test in pregnant db/⫹, ⫹/⫹, leptin-treated db/⫹ and ⫹/⫹, and pair-fed db/⫹ mice. Mice were fasted 6 h on day 18 of pregnancy and administrated 0.75 U/kg BW regular insulin load at time zero, and the glucose level was determined before and 15, 30, and 60 min after injection. Results are expressed as a percentage of 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. leptintreated ⫹/⫹ mice; #, P ⬍ 0.05, pair-fed db/⫹ vs. leptin-treated db/⫹ mice.

phorylation of IRS-1 and p85␣ subunit of PI-3 kinase were lower by 21 ⫾ 3% (P ⬍ 0.05) and 19 ⫾ 3% (P ⬍ 0.05), respectively, in pregnant db/⫹ mice compared with that in pregnant ⫹/⫹ mice. In leptin-treated db/⫹ mice, insulinstimulated IRS-1 and p85␣ phosphorylation in skeletal muscle increased by 30 ⫾ 5% and 38 ⫾ 6%, respectively, compared with those in pregnant db/⫹ mice (P ⬍ 0.05). Leptin treatment had no effect on the level of the insulin receptor phosphorylation of the 95-kDa ␤-subunit of the insulin receptor in skeletal muscle. The effect of leptin on skeletal muscle protein expression was also examined in pregnant db/⫹ and ⫹/⫹ mice (Fig. 5). Pregnant db/⫹ mice had a 2.5-fold higher expression of IR␤ compared with pregnant ⫹/⫹ mice (P ⬍ 0.05), whereas IRS-1 expression in pregnant db/⫹ mice was significantly lower by 43 ⫾ 10% (P ⬍ 0.05) compared with that in pregnant ⫹/⫹ mice. There was no significant difference in p85␣ expression between pregnant ⫹/⫹ mice and db/⫹ mice. Leptin treatment in db/⫹ mice increased IR␤ expression by 33 ⫾ 8% (P ⬍ 0.05), IRS-1 by 150 ⫾ 21% (P ⬍ 0.05), and p85␣ expression by 50 ⫾ 12% (P ⬍ 0.05), respectively. Pair-feeding also increased IR␤, IRS-1, and p85␣ expression by 53–113% (P ⬍ 0.05). Leptin treatment in pregnant ⫹/⫹ mice had no effect on IR␤, IRS-1, or p85␣ expression, and GLUT4 levels were unchanged (data not shown) in db/⫹ and leptin-treated animals. Effect of leptin treatment on the fetus and placenta

Fetuses were delivered by cesarean section on day 18 of gestation. The number of fetuses born to db/⫹, ⫹/⫹, and leptin-treated dams were not different (Table 2). We genotyped the fetuses for the db mutation by PCR-restriction


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FIG. 4. Effect of insulin on tyrosine phosphorylation of IR␤ (A), IRS-1 (B), 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 immunoprecipitated with antiphosphotyrosine antibody and immunoblotted with anti-IR␤, IRS-1, and PI3-kinase (p85␣). The tyrosine-phosphorylated bands corresponding to these proteins were analyzed by scanning densitometry. The bar graph shows quantification of the autoradiograms of these experiments using six mice per group. The values are expressed as arbitrary units relative to pregnant ⫹/⫹ mice, assigning a value of 100 to the insulin-stimulated 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 significant 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 ⫹/⫹ control offspring (P ⬍ 0.05). Pair-feeding db/⫹ mothers to the food intake from ⫹/⫹ mothers throughout pregnancy significantly 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 pregnancies 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

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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 Mother

db/⫹ vehicle (n ⫽ 58) db/⫹ leptin (n ⫽ 46) db/⫹ pair-fed (n ⫽ 42) ⫹/⫹ vehicle (n ⫽ 81) ⫹/⫹ leptin (n ⫽ 42)

Litter size

Birth wt (g)

Placenta (mg)

7.3 ⫾ 0.4 6.6 ⫾ 0.7 7.0 ⫾ 0.6 6.2 ⫾ 0.3 7.0 ⫾ 0.5

1.14 ⫾ 0.02 a 1.15 ⫾ 0.02 a a 1.07 ⫾ 0.02 1.09 ⫾ 0.01 a 1.03 ⫾ 0.02

] ]

]]

89.2 ⫾ 2.1 91.2 ⫾ 2.2 88.3 ⫾ 2.2 90.2 ⫾ 2.1 a 84.4 ⫾ 2.3

]

Placental leptin (ng/g wet wt)

74.6 ⫾ 5.0 a 76.4 ⫾ 4.2 a a 53.6 ⫾ 3.4 58.8 ⫾ 4.8 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 human 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 gestation (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-


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 resistance of pregnancy. One of the main reasons for the effectiveness of peripherally administered human leptin in the db/⫹ mouse may be the relatively higher and sustained halflife 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, peptide 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 appetite, but significantly reduced insulin resistance in pregnant db/⫹ mice, in part through an improvement in skeletal muscle 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-associated 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 adipose tissue mass and lower insulin levels than db/⫹ mice during gestation, which may have contributed to the inability to detect small differences in absolute weight loss and insulin sensitivity in response to leptin administration. Leptin also down-regulated the endogenous leptin expression levels in ⫹/⫹ mice, which may have contributed to the reduced sensitivity. Leptin treatment was, however, successful 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 leptin 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 independently 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 wildtype 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

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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 detailed 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 evidence, 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, suggesting 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 circulation 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 levels in response to glucose tolerance tests are important modifiable variables for infant birth weight and future obesity (36, 65). Our findings suggest that pair-feeding throughout gestation was more effective than short-term leptin treatment at reducing fetal overgrowth in db/⫹ mice. Unlike leptin treatment, 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 decreasing 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 effective means of reducing the abnormal glucose tolerance

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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 retardation (67). In addition, high insulin concentrations in umbilical 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 placental 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 ability to influence fetal growth could have important implications for susceptibility to adult disease and will be an important 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.

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