Objective
Synthetic glucocorticoids (sGCs) are administered to women threatening preterm labor. We have shown multigenerational endocrine and metabolic effects of fetal sGC exposure. We hypothesized that sGC exposure would alter the second filial generation (F2) offspring neonatal leptin peak that controls development of appetitive behavior with metabolic consequences.
Study Design
F0 nulliparous ewes were bred to a single ram. Beginning at day 103 of gestation (term 150 days), dexamethasone (DEX) ewes received 4 injections of 2 mg DEX intramuscularly, 12 hours apart. Control ewes received saline. Ewes lambed naturally. At 22 months of age, F1 offspring were mated to produce F2 offspring. At 10 months of age, F2 female offspring were placed on an ad libitum feeding challenge for 12 weeks.
Results
DEX F2 female offspring did not show a postnatal leptin peak and their plasma cortisol concentration was elevated in the first days of life. During the feeding challenge, DEX F2 offspring consumed 10% more feed and gained 20% more weight compared with control F2 offspring. At the end of the feeding challenge, DEX F2 offspring had greater adiposity compared with control F2 offspring. F2 sGC offspring showed impaired insulin secretion in response to an intravenous glucose tolerance test.
Conclusion
sGC administration to F0 mothers eliminates the neonatal leptin peak in F2 female offspring potentially by inhibition caused by elevated cortisol in the DEX F2 offspring. F2 offspring showed increased appetite, weight gain, and adiposity during an ad libitum feeding challenge accompanied by decreased insulin response to an intravenous glucose tolerance test.
The hormone leptin is produced by adipose tissue and acts on hypothalamic appetitive centers to inhibit food intake. Appropriate regulation of leptin feedback is central to the maintenance of normal body weight and composition. Dysregulation of this feedback system can lead to obesity. In neonatal rodents, leptin has a characteristic peak that occurs at postnatal days 8-21. The precise timing and duration of the leptin peak varies between studies, strains, and species. The normal leptin peak is thought to program the balance of competing activity of hypothalamic orexigenic and anorexigenic appetitive neurons and influence leptin sensitivity postnatally. The leptin peak is amplified and prolonged in offspring of obese rats.
Several factors have been implicated in the control of fetal and adult leptin production in rodents including glucocorticoids, glucose, insulin, thyroid hormones, and insulin growth factor (IGF)-1. There is controversy over the timing of the leptin peak in precocial species. We have demonstrated a leptin peak in newborn lambs that occurs between days 6 and 8 of postnatal age and is associated with increased plasma cortisol at birth.
In his classical study, Liggins and colleagues showed that glucocorticoids (GCs) prematurely accelerate fetal lung development in sheep, an observation that led to a clinical trial that showed decreased morbidity and mortality in premature babies whose mothers received synthetic GCs (sGCs). It is now routine clinical practice to treat women threatening preterm delivery before 34 weeks’ gestation with sGC. Although this treatment with sGC confers great benefit by lowering neonatal mortality and morbidity, evidence is accumulating that fetal exposure to GC levels higher than those appropriate for the current stage of fetal maturation produces intrauterine growth retardation (IUGR) in sheep, nonhuman primates, and humans. Synthetic GC administration (170 μg kg −1 day −1 of betamethasone) at 0.75 of gestation results in decreased placental endothelial nitric oxide synthase function in baboons, which could possibly explain the fetal IUGR. Synthetic GCs have also been shown to alter learning and attention in offspring.
Developmental programming can be defined as a response to a specific challenge to the mammalian organism during a critical developmental time window that alters the trajectory of development with persistent effects on offspring phenotype and predisposition to future illness. sGC alters the developmental trajectory of many fetal organ systems with the potential for adverse developmental programming effects in later life.
Similarities across species suggest a common underlying mechanism responsible for these adverse outcomes. Animal studies demonstrate that following sGC exposure, delayed offspring endocrine, renal, and metabolic effects emerge later in life with the potential to predispose to chronic disease. Increases in fetal and maternal GCs have been demonstrated to occur in response to many challenges that result in developmental programming in both precocial and altricial species.
Offspring outcomes resulting from challenges such as poor maternal nutrition can be prevented by inhibiting GC changes occurring in response to the challenge. As a result, several investigators consider exposure to excessive endogenous GC levels at critical fetal developmental periods to be a common causative factor of many, although not necessarily all, fetal programming responses to challenges during development (eg, maternal stress and poor nutrition).
Most multigenerational studies on the effects of fetal exposure to GCs have been conducted in rodents: altricial species with a very different profile of fetal and neonatal development to precocial species such as humans and sheep. However, in sheep we have shown that sGCs result in effects on both F1 and F2 female offspring with reduced birthweight and postnatal growth, increased plasma glucose, decreased plasma insulin during an intravenous glucose tolerance test, and increased basal and reduced stimulated hypothalamic pituitary adrenal axis function.
In view of the relationship we have demonstrated of the normal leptin peak in newborn lambs to the plasma cortisol concentration, we hypothesized that exposure of female fetal lambs (F1 generation) to doses of sGCs well within the range administered clinically to their mothers (F0 generation) at 27 weeks’ human gestation equivalent would have effects on F2 female offspring appetite drive, metabolism, endocrine function, and weight changes when allowed ad libitum food access. To test this hypothesis, we administered either dexamethasone (DEX) or vehicle to F0 pregnant ewes at 103 and 104 days’ gestation (term 150 days), maintained all the F1 female generation (both DEX and control offspring) on a similar diet, and determined F2 female offspring outcomes at approximately 1 year of age.
Materials and Methods
Care and use of animals
All procedures were approved by the University of Wyoming Animal Care and Use Committee. A description of animal generation and housing has previously been published. Briefly, 22 founder generation (F0) nulliparous Rambouillet X Columbia cross-bred ewes bred to a single ram were used to produce the first filial (F1) and second filial (F2) generation of ewe lambs. After natural mating, F0 ewes were fed in accordance with National Research Council (NRC) maintenance requirements.
On day 103 and 104 of gestation, ewes weighed 73.6 ± 4.0 kg (mean ± SEM, n = 22) body weight and were randomly assigned to 1 of 2 treatment groups: DEX ewes (n = 10) received 4 injections of 2 mg of DEX (intramuscularly; Vedco, St. Joseph, MO) 12 hours apart, a dose equal to approximately 60 μg/kg −1 per day −1 . Control ewes (n = 12) received equivalent volumes of saline intramuscularly.
Ewes were allowed to lamb naturally. After lambing, all F0 ewes were given free choice access to alfalfa hay. Prior to 2 weeks of age, F1 female lambs (n = 10 DEX and 12 control F1) were tail docked as per Federation of Animal Science Societies recommendations. F1 ewe lambs were given free access to a standard commercially available creep feed (Lamb Creep B30 w/Bovatec; Ranch-Way Feeds, Ft. Collins, CO) from birth to weaning at 4 months of age and placed in an outdoor housing facility with shelter and ad libitum water. These F1 ewe lambs were maintained in accordance with NRC recommendations requirements for replacement ewes with a diet that consisted of alfalfa and corn with ad libitum access to a trace mineral salt block. Diets were adjusted up for weight gain every month.
At maturity nulliparous control and DEX F1 ewes exhibited similar body weights (65.1 ± 3.5 and 64.7 ± 3.7 kg, respectively) and were naturally mated to a single ram of similar breeding. There were 3 F2 control twin (3 male-female twin sets) and 5 F2 control singleton ewe lambs. In the F2 DEX group, there were 4 twin (1 female twin set and 3 male-female twin sets) and 4 singleton ewe lambs. Jugular venous blood was obtained from F2 ewe lambs (n = 8, control and 9 DEX; approximately 6 mL of blood) at birth and daily at 6:00 am from postnatal day 1 to 7 and days 9 and 11.
F2 lambs were fed in a similar fashion as their F1 mothers, and postnatal body weights were recorded monthly until 10 months of age when lambs were placed on a 12 week feeding challenge using previously published procedures. Briefly, F2 lambs were adapted from a hay and grain diet to the experimental ration at maintenance levels over a 10 day acclimation period. The experimental diet was a highly palatable, pelleted diet (containing 71.05% total digestible nutrients, 1.08 Mcal/kg net energy for gain, 1.64 Mcal/kg net energy for maintenance, 88.2% dry matter, 13.5% crude protein, and 4.05% fat on an as-fed basis (ADM; Alliance Nutrition, Quincy, IL). At the end of this acclimation period, lambs were weighed and removed from feed and water for 12 hours and evaluated by dual-energy x-ray absorptiometry (DEXA) to determine body composition (fat and lean tissue). The lambs were then returned to the experimental ration, which was fed ad libitum for a 12 week period of the feeding challenge
During the feeding challenge, lambs were housed in a single group in an open-front pole barn with free access to water and the pelleted feed available via an automated feeding behavior data acquisition system adapted for use in sheep (GrowSafe Systems Ltd, Airdrie, Alberta, Canada). Feed intake was continuously measured based on the weight difference of the feed bunk (accuracy to 0.01 kg) at the beginning and end of each feeding event for each individual animal as determined by a unique electronic ear tag worn by each lamb. The feed bunk had an opening, which permitted a single lamb’s head to enter, allowing only 1 animal to consume feed at a time and their ear tag to be scanned identifying the lamb consuming feed at each event. Feed was continuously available 24 hours per day.
Intake measurements were recorded throughout the entire 12 week period. Body weight (BW) was measured every 2 weeks when jugular blood was collected at 7:00 am into a heparinized blood collection tube (BD Vacutainer, Franklin Lakes, NJ; 143 USP units sodium heparin per 10 mL) prior to weighing. Blood was kept cold and centrifuged within 30 minutes at 4°C and 1500 × g for 15 minutes. Plasma was collected and aliquoted prior to storage at –20°C for analyses.
At the end of the 12 week feeding challenge, a catheter (Abbocath, 16ga; Abbott Laboratories, North Chicago, IL) was placed aseptically into the jugular vein 12 hours prior to the start of an intravenous glucose tolerance test (IVGTT). Catheters were sutured to the skin to secure them and an extension set (Seneca Medical, Tiffin, OH) attached for undisturbed infusion and sampling. The neck and shoulder area were covered with netting (Surgilast tubular elastic dressing retainer; Derma Science Inc, Princeton, NJ) to prevent catheter damage.
F2 lambs were maintained in neighboring individual pens with free access to water. No feed was provided for approximately 18 hours prior to and during the IVGTT, which has been described in detail. Jugular blood samples (approximately 6 mL) were obtained into chilled tubes (heparin plus NaF; 2.5 mg/mL; Sigma, St. Louis, MO) at –15 and 0 minutes relative to a 0.25 g/kg intravenous bolus infusion of 50% dextrose solution (Vedco) over 5 seconds. Blood samples were collected at 2, 5, 10, 15, 20, 30, 45, 60, 90, and 120 minutes after dextrose infusion. All blood samples were immediately placed on ice, centrifuged at 1500 × g and plasma was collected and stored at –80° C. Following the IVGTT, F2 lambs were subjected to a DEXA scan to determine body composition at the end of the feeding challenge.
Dual-energy x-ray absorptiometry
To accurately determine body composition (fat and lean tissue), DEXA (GE Lunar Prodigy 8743; GE Healthcare, Madison, WI) was utilized as previously described and validated for sheep. The whole-body scan mode was used for all animals and scan times were approximately 3 minutes, depending on the length of the animal. A single, experienced, blinded investigator performed all DEXA scans and quantified percentage body fat.
Biochemical and hormone assays
Glucose was measured colorimetrically in triplicate (liquid glucose hexokinase reagent; Pointe Scientific, Inc, Canton, MI) as previously described. The mean intraassay coefficient of variation (CV) was 1.6% and the interassay CV was 2.9%. Insulin was measured in duplicate by commercial radioimmunoassay (RIA) (Coat-A-Count insulin RIA; Siemens Medical Solutions Diagnostics, Los Angeles, CA) with an intra- and interassay CV of 8.4% and 11.1%, respectively, and a sensitivity of 2.6 μIU/mL. Plasma leptin was measured by radioimmunoassay (Multispecies leptin RIA; Linco Reseach, St. Charles, MO) as previously described with an intraassay CV of 4.1% and an interassay CV of 5.1%. Concentrations of cortisol were determined as described previously using Coat-A-Count cortisol RIA with a sensitivity of 5 ng/mL (Siemens Medical Solutions Diagnostics) with an intraassay CV of 6.1% and an interassay CV of 9.1%. Triiodothyronine was determined by RIA according to the manufacturer’s specifications (Coat-a-Count total T3) with intra- and interassay CVs of 3.4% and 4.4%, respectively, and a sensitivity of 16.2 ng/dL.
Statistical analysis
Postnatal hormone and metabolite data were analyzed as a repeated-measures analysis using the MIXED procedure of SAS (SAS Institute Inc, Cary, NC), with treatment, day, and their interaction in the model. Plasma hormone and metabolite concentrations during the feeding challenge were analyzed as repeated measures using the MIXED procedure (SAS Institute), with treatment, week, and their interaction in the model. The DEXA measurements, feed intakes, and BW gain during the feeding challenge were analyzed using the GLM procedure of SAS, with treatment in the model. GraphPad Prism (GraphPad Software Inc, La Jolla, CA) was used to calculate the area under the curve (AUC) for plasma glucose and insulin response curves during the IVGTT.
Baseline concentrations of glucose and insulin in all samples before infusion were averaged to give baseline concentrations. Plasma glucose and insulin during the IVGTT were analyzed as repeated measures using MIXED procedure of SAS (SAS Institute), with treatment and time and their interaction in the model. The AUC and fasting concentrations of glucose and insulin were analyzed using the GLM procedure of SAS with treatment in the model. Birth type (twin vs single) was initially included in all models but was found to be nonsignificant ( P < .39) and was therefore removed. Data are provided as mean ± SEM throughout with P ≤ .05 was considered significant.
Results
Birthweights and measures together with growth patterns up to 8 months of the age of F2 female offspring have been published. Briefly, the birthweight of DEX F2 female lambs was less ( P = .03) than control F2 female lambs (5.72 ± 0.26 vs 6.62 ± 0.29 kg). There was no difference in birthweight between lambs born as singles or twin ( P = .39; single 6.28 ± 0.46 vs twin 5.75 ± 0.55). In control F2 lambs, plasma leptin increased ( P < .05) from postnatal day 2 to 3 and remained higher than values of DEX F2 female lambs on day 4 ( P < .05), returning to levels seen in DEX F2 lambs from day 5 to 11 ( Figure 1 , A). Plasma cortisol was increased at birth and on days 1, 3, 6, and 7 of age in DEX F2 lambs compared with control F2 lambs ( Figure 1 , B). There was no difference in plasma insulin, glucose, and triiodothyronine during the first 11 days of age between DEX and control F2 lambs ( Figure 1 , C, D, and E, respectively).
At 10 months of age, when F2 ewes were transitioned onto the experimental pelleted ration, there was no difference in control and DEX F2 female offspring body weights ( Table ). There was also no difference in any of the DEXA measurements between control and DEX F2 female lambs before the feeding challenge ( Table ). During the feeding challenge, DEX F2 female offspring put on more weight both in absolute terms and as a percent of initial weight ( Figure 2 , A and B) and consumed more feed ( Figure 2 , C and D) than control F2 female offspring. At the end of the feeding challenge, the body weight of DEX F2 was 2.5 kg heavier than controls but because of the fact that controls were 1.5 kg heavier (not significant) at the beginning of the challenge, the final weights of the 2 groups were similar between control and DEX F2 offspring ( Table ). However, the percentage body fat and grams of adipose tissue was increased 28.4% and 37.6% of the initial measurements, respectively, in DEX F2 offspring compared with control F2 female offspring ( Table ).
| Variable | Control (n = 8) | DEX (n = 9) | P value |
|---|---|---|---|
| Prefeeding challenge | |||
| Body weight, kg | 62.4 ± 3.7 | 60.9 ± 3.1 | .39 |
| Crown rump length, cm | 117.4 ± 2.7 | 115.9 ± 2.4 | .35 |
| Percent fat | 10.1 ± 1.3 | 11.8 ± 0.9 | .16 |
| Bone mineral density | 1.12 ± 0.03 | 1.12 ± 0.02 | .5 |
| Fat tissue, g | 6224 ± 1105 | 7161 ± 786 | .26 |
| Lean tissue, g | 54,046 ± 2624 | 51,055 ± 2587 | .22 |
| Postfeeding challenge | |||
| Body weight, kg | 82.5 ± 4.3 | 85.0 ± 3.3 | .33 |
| Crown rump length, cm | 125.9 ± 1.8 | 124.0 ± 1.6 | .24 |
| Percent fat | 17.6 ± 1.6 | 23.9 ± 1.3 | .003 |
| Bone mineral density | 1.16 ± 0.02 | 1.19 ± 0.02 | .22 |
| Fat tissue, g | 13,300 ± 1705 | 17,996 ± 1575 | .04 |
| Lean tissue, g | 61,273 ± 2492 | 57,565 ± 2261 | .15 |
| Percent change from initial to final | |||
| Body weight (% of initial) | 32.2 ± 2.9 | 40.0 ± 2.6 | .05 |
| Percent fat (% of initial) | 74.1 ± 6.9 | 102.5 ± 6.1 | .01 |
| Fat tissue (% of initial) | 113.7 ± 5.6 | 151.3 ± 4.9 | .02 |
| Lean tissue (% of initial) | 13.4 ± 1.9 | 12.8 ± 1.7 | .56 |
Plasma leptin concentrations were similar at the start of the feeding challenge in the 2 groups, but at day 42, halfway through the feeding challenge, and at the end of the feeding challenge, plasma leptin was increased in DEX F2 offspring compared with control F2 offspring ( Figure 3 , A). Plasma cortisol concentrations were similar between F2 offspring at the start and for the first 14 days of the feeding challenge, but from day 28 to the end of the feeding challenge, plasma cortisol was higher in DEX F2 female offspring than controls ( Figure 3 , B).
