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Obesity has long been considered to have genetic underpinnings, but it was not until 1994 that the obesity gene (ob/ob) was positionally cloned(1). The ob/ob gene product, designated leptin, is a 16-kD adipocyte-derived protein that reflects body fat content(2). Leptin is thought to act as an afferent satiety signal from body fat to the hypothalamus, which causes a reduction in appetite and augments basal metabolic rate when fat stores are replete(3, 4). The obese (ob/ob) mouse, which is markedly hyperphagic and obese, is leptin-deficient due to a mutation in the ob gene. When given leptin, its food intake is reduced and the mice lose weight(1). Although leptin deficiency may play a role in some forms of rodent and human obesity, CNS resistance to leptin may be the more likely explanation(5–7). Leptin is now known to act through high affinity transmembrane receptors(8). High affinity binding sites for leptin have been localized in hypothalamic tissue (arcuate nucleus)(9), leptomeninges, and choroid plexus(10). Point mutations of these receptors have been associated with obesity and diabetes in select animal models(11, 12).

Both serum leptin concentrations and ob gene mRNA are elevated in adipocytes from obese humans. Moreover, there is a consistent correlation between the serum leptin concentration and the percentage of body fat, the BMI, and basal serum insulin concentrations(2, 13). Human adipocytes produce more leptin when the adipose mass increases(14). Obese patients show decreased sensitivity to the action of leptin(13, 14).

Beyond its relation with body-fat mass, little is known about the regulation or secretion of leptin. Caloric restriction reduces serum leptin concentrations and the ob mRNA level in adipose tissue, whereas refeeding increases these levels. Enhanced leptin production has been mimicked by norepinephrine, insulin, and glucocortocoids in cell culture experiments(15–17). Induction of leptin mRNA has been shown to occur in infections using the endotoxin lipopolysaccharide model in hamsters(18). Whether this is the cause for the loss of weight seen in some inflammatory processes remains to be seen. Leptin has also been shown to play a major role in fertility and the induction of puberty(19).

The role of leptin in diabetes (insulin dependent or noninsulin dependent) is not entirely clear(20). Animal experiments have shown that insulin administration can up-regulate leptin mRNA expression. Conversely, administration of recombinant leptin to normal rats significantly reduces appetite, weight, and serum glucose levels(1, 21). In humans, however, neither euglycemic hyperinsulinemia nor hyperinsulinemic hypoglycemia acutely stimulate leptin production for up to 5 h, but chronic effects of insulin have not been examined(22, 23). In patients with non-insulin-dependent diabetes, leptin concentrations differ significantly by diabetic status and are higher in female compared with male subjects(23).

Thus, the exact role of leptin in human glucose and fat metabolism is still unfolding. Little is known regarding the level of this hormone in neonates(24, 25). Pregnancy, a clinically diabetogenic process, causes a large change in body weight and fat content. Infants born to gestational or insulin-dependent diabetic mothers as well as infants who are growth-retarded in utero have metabolic changes that may impact body size, glucose, and intermediary metabolism(26).

The purpose of this study was to: 1) establish normal values for serum concentrations of leptin in neonates by gestational age, BW, and maternal diabetic status; 2) correlate various anthropometric variables of the mother and infant with cord serum leptin concentration; and 3) examine the effect of maternal antenatal steroid treatment on neonatal leptin levels.

METHODS

Cord blood samples were collected from live born infants over a 2-mo period at three major maternity hospitals in Milwaukee, WI. Blood was collected in a plain tube for serum leptin measurement and in a EDTA tube for GHB estimation. Serum was separated after the blood was clotted, and both tubes were stored at 4 °C till the time of analysis. BW; length; gestational age (early ultrasound, menstrual history or neonatal examination); maternal diabetes status; and antenatal steroid exposure were recorded. Standard growth curves(27, 28) were used to categorize infants as appropriate, large, or small for gestational age. Diagnosis of IUGR was based on accepted clinical criteria(26). BMI was calculated as BW (kg)/square of length (m)(29). Documentation was made concerning use of antenatal corticosteroids. Maternal antenatal steroid treatment was defined by exposure to at least one dose (12 mg) of betamethasone before delivery. Maternal obesity was defined by a maternal weight >90th percentile for height and a pregravid BMI >29(30). Data sheets were completed by the laboratory personnel or a research nurse at the respective hospitals. Blood samples were analyzed for leptin and GHB in a blinded fashion.

Leptin was measured by an 125I-labeled leptin RIA (Linco Research, St. Louis, MO)(31). The coefficient of variation of this method is 3.5% (intraassay) and 2.4% (interassay). GHB was measured using affinity chromatography (Isolab, Inc., Akron, OH)(32). The GHB assay measures all glycohemoglobins; importantly, fetal Hb does not interfere with this method(33). The study was approved by the respective Internal Review Boards of the three hospitals.

Statistical methods. Data were analyzed using SAS(6, 12) for the PC. Means were compared using the t test(34). Adjustments for multiple comparisons were made using Fisher's least significant difference method. Adjusted means were obtained using analysis of covariance, and differencts were compared using Fisher's least significant difference t test(35). Statistical significance was set at <0.05 when the overall experiment error rate was <0.05.

The relationship between BW, BMI, and leptin concentration was determined using univariate and multivariate regression analysis techniques. Data were assessed using a quadratic term in the regression model(Yij = β0 +β1Xi +β2Xi2 +εij), where Yij is the leptin level, β0 is the intercept, and β1 and β2 are the parameter estimates for the linear and quadratic components, andεij is the error term for the model used. Model assumptions were checked graphically using residual plots. If nonhomogeneous error variances were encountered, then weighted least squares regression analysis was used. The weights used in the regression model were inverse of the square of the dependent variable. Model assumptions were again checked for the weighted regression model. Both linear and quadratic effects were tested in the models. A nonsignificant value of β2 indicated that a quadratic term did not improve the fit of the data.

Multivariate analysis was done using a weighted multiple regression model to determine variables associated with leptin levels. Linear and quadratic components were considered for BW, BMI, and GHB. Forward stepwise regression was used to find the “best” model to predict leptin concentrations(35, 36).

RESULTS

Leptin was detected in the cord blood of preterm infants as small as 24 wk of gestation. Cord leptin concentration increased with increasing BW, BMI, and gestational age (Figs. 1 and 2,Table 1). The relationship between cord blood leptin and BW was quadratic (Fig. 1) but linear for BMI (Fig. 2). There were significant differences in cord serum leptin concentration between infants with different characteristics (Table 1). Analysis of AGA infants (after excluding infants whose mothers received antenatal steroids, infants of diabetic mothers and adjusting for BWs) did not find any gender differences. These leptin levels were (mean ± SD) 3.23 ± 2.76 ng/mL in female infants and 2.98 ± 2.96 ng/mL in male infants(p = 0.15). Intrauterine growth retardation was associated with a significantly higher cord serum leptin concentration compared with BW matched AGA infants (p = 0.03). Maternal obesity had no effect on fetal leptin levels (Table 3). Antenatal steroid exposure was associated with higher cord serum leptin concentration (1.9 ± 5.34versus 6.4 ± 10.01) (p = 0.006)(Fig. 3). The smaller twin had an insignificantly greater leptin level compared with the larger twin (Table 2).

Figure 1
figure 1

Regression of leptin level vs BW. Regression analysis shows a quadratic relationship between cord serum leptin concentration (ng/mL) and BW (kg) (excluding infants of diabetic mothers and those who received steroids) (R2=0.41) (p = 0.002).

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Figure 2
figure 2

Regression of leptin level vs BMI. Regression analysis shows a linear relationship between cord serum leptin concentration(ng/mL) and BMI (R2 = 0.2) (p = <0.001).

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Table 1 Results of cord serum leptin levels in different type of infants (mean ± SD) (excluding infants whose mothers received steroids)
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Table 3 Effect of maternal obesity and intrauterine growth retardation on neonatal cord serum leptin concentration (BW adjusted data)
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Figure 3
figure 3

Effect of antenatal steroids on cord serum leptin concentration. Preterm infants (n = 47) whose mothers did not receive antenatal steroids (·) had a mean cord serum leptin of 1.9 ± 0.79 (SEM) compared 6.4 ± 2.42 (SEM) for infants (n = 17) exposed to steroids (▪) antenatally (p = 0.006) (BW-adjusted data).

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Table 2 Analysis of cord serum leptin concentration in twin pairs and its relation to BW, and BMI
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GHB (%) analysis was performed in the first 97 infants enrolled in the study. Term AGA infants born to nondiabetic mothers had lower GHB levels, 3.01± 0.28%, compared with infants born to diabetic mothers, 3.49 ± 0.6% (p = 0.01). GHB was linearly related to leptin concentration(p = 0.03) (Table 4). There was no statistically significant effect of gestational age, BW, BMI, maternal obesity, or steroid treatment on GHB in all other groups of infants.

Table 4 Univariate weighted regression analysis for BW, BMI, and GHB in predicting leptin level (excluding infants of diabetic mothers and those treated with steroids
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In the univariate regression model, BW was a better predictor of leptin concentration than was BMI (Table 4). Multiple weighted regression analysis (excluding infants exposed to antenatal steroids) examined relationships between infant type (AGA, LGA, and IUGR), maternal obesity, diabetes status, BW, BMI, and leptin concentration. Only BW (p = 0.002) showed a significant relationship with leptin concentration(R2 = 0.41).

DISCUSSION

In adults, children, and all normal animals studied to date, serum leptin concentrations correlate with adiposity. This report examines whether a similar relationship is apparent at birth and moreover whether maternal obesity, diabetes, or antenatal steroid treatment is related to neonatal cord leptin concentrations. Our study has shown a direct quadratic relation of serum leptin to BW, and it is linear for BMI. The latter linear relationship may be explained by the fact that in the growth-retarded human fetus there could be sparing of head and length growth (asymmetric IUGR) that leads to a disproportionate change in BMI in relation to BW(37). Analysis of cord blood leptin in 16 pairs of twins revealed no significant differences in BW, BMI, and leptin concentration (Table 2). This could be due to asymmetric growth retardation in the smaller twin due to intrauterine stress and not due to genetic factors as shown in adults(38).

Neonates with IUGR had elevated cord leptin (Table 1 and 3). The etiology of intrauterine growth retardation is multifactorial and includes both maternal and fetal factors. Major causes of IUGR include pregnancy-induced hypertension, intrauterine infections, placental dysfunction, drugs, and genetic factors. Pregnancy-induced hypertension, the commonest cause of IUGR, is associated with increased maternal inflammatory mediators such as tumor necrosis factor-α and IL-12(39, 40). Because inflammation increases leptin expression in cell culture experiments(18) and leptin receptors are closely linked to IL-6 receptors(41, 42), these factors might enhance leptin production. It is possible that insulin resistance and hyperinsulinemia in infants with IUGR is responsible for the elevated cord leptin concentration. Insulin resistance and hyperinsulinemia correlate with leptin concentrations in normal and diabetic adults(43, 44). Moreover, studies in cell cultures suggest that insulin promotes adipocyte leptin production. Because IUGR infants have a smaller, dysfunctional placenta, leptin levels may be inversely correlated with placental weight(24). Finally, elevated concentrations of endogenous corticosteroid hormones, present in some infants with IUGR(45, 46), may contribute to the elevated leptin concentrations. Thus the intrauterine growth-retarded fetus is known to be under a state of stress with high endogenous corticosteroid production, which may augment leptin synthesis(47–50). Intrauterine infections such as syphilis have also been shown to be associated with elevated serum cortisol(47).

Neonatal cord serum leptin levels were elevated in infants whose mothers received antenatal steroids (Fig. 3). The wide variation of leptin concentrations in control and treated infants can be explained by the diverse range of gestational age, BWs, and dosage of steroid used, along with the time lag (12 h to 2 wk) between administration and delivery of the infant. The ability of steroids to increase leptin has been demonstrated in cell culture experiments(48), animal experiments(18), and in adult healthy women(49). That antenatal steroid treatment is associated with increased neonatal leptin suggests that a similar cellular response mechanism may exist in both the fetus and adult. Whether this elevated leptin is persistent or involved in the increased adiposity or catabolism associated with prenatal steroids remains to be determined. There is a possibility that leptin may be secreted periodically during fetal development, and our one measurement at birth may not reflect cumulative secretion of this protein. However, leptin levels in an individual are somewhat stable over a 24-h period, except for a slight circadian rhythm of small amplitude(50).

In summary, neonatal cord serum concentrations of leptin correlate with BW and BMI. IUGR infants and infants whose mothers received antenatal steroids had an increased cord leptin concentration. Leptin, which alters pituitary function and affects numerous extraneuronal tissues, may be responsible for the regulation of body mass of the human fetus and neonate.