Effects of Pituitary Hormone Deficiency on Growth and Glucose ...

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Endocrinology 148(10):4812– 4820 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2007-0639

Effects of Pituitary Hormone Deficiency on Growth and Glucose Metabolism of the Sheep Fetus A. L. Fowden and A. J. Forhead Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, United Kingdom Pituitary hormones are essential for normal growth and metabolic responsiveness after birth, but their role before birth remains unclear. This study examined the effects of hypophysectomizing fetal sheep on their growth and glucose metabolism during the late normal and extended periods of gestation, and on their metabolic response to maternal fasting for 48 h near term. Fetal hypophysectomy reduced crown rump length (CRL), limb lengths, and body weight but increased ponderal index relative to controls near normal term. It also lowered the daily rate of crown rump length increment uniformly from 35 d before, to 20 d after normal term. Hypophysectomized (HX) fetuses had normal weight-specific rates of umbilical uptake, utilization, and oxidation of glucose but lower rates of umbilical oxygen uptake than controls near term. All these metabolic rates were significantly less in HX fetuses during the ex-

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ITUITARY HORMONES ARE essential for normal growth and metabolism after birth (1). They regulate the growth of bone and soft tissues, and have a key role in glucose homeostasis and energy balance (2). Postnatal deficiency of these hormones induced by hypophysectomy causes severe growth retardation in several species, including humans and sheep (3). Hypopituitarism also impairs the metabolic response to common physiological challenges, such as fasting, infection, and exercise, with adverse consequences for morbidity and mortality in the long term (2). In particular, hypophysectomy after birth limits endogenous glucose production and leads to severe hypoglycemia in conditions that increase the demand for glucose or reduce its supply (4). However, the role of pituitary hormones in growth and metabolism before birth is less well established (3). The initial studies of hypophysectomy in fetal sheep using five sets of twins showed that the hypophysectomized (HX) fetus was invariably smaller than its sham-operated sibling at normal term (5, 6). In subsequent experiments using larger numbers of single fetuses, the effects of hypophysectomy were more equivocal with significant reductions in fetal body weight at term in some studies (7, 8), but not in others (9 –12). Fetal hypophysectomy also has little, if any effect on fetal body weight near term in polytocous species, such as rats and First Published Online June 26, 2007 Abbreviations: CRL, Crown rump length; G6Pase, glucose-6-phosphatase; HX, hypophysectomized; PEPCK, phosphoenolpyruvate carboxykinase. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

tended period of gestation than in HX and intact fetuses near normal term. In contrast to controls, glucogenesis was negligible in HX fetuses during maternal fasting. Consequently, the rate of glucose utilization decreased significantly in fasted HX but not intact fetuses. Conversely, the rate of CO2 production from glucose carbon decreased in fasted intact but not HX fetuses. Fetal hypophysectomy also prevented the fasting-induced increases in plasma cortisol and norepinephrine concentrations seen in controls. These findings demonstrate that the pituitary hormones are important in regulating the growth rate and adaptive responses of glucose metabolism to undernutrition in fetal sheep. They also suggest that fetal metabolism is altered when gestational length is extended. (Endocrinology 148: 4812– 4820, 2007)

pigs (13, 14). In both single and twin sheep fetuses, hypophysectomy delayed bone ossification and reduced limb lengths, although its effects on crown rump length (CRL) appeared to be less pronounced (5–10). However, very little is known about the actual growth rate of HX fetuses in utero. In sheep, fetal hypophysectomy prevents the onset of labor and prolongs gestation indefinitely (15). The HX fetus continues to gain weight during the extended period of gestation but does not appear to elongate its limbs after normal term (5, 6, 9). It maintains normal circulating concentrations of glucose, lactate, and amino nitrogen for most of gestation but has lower rates of oxidative metabolism than intact fetuses close to normal term (6, 8). At delivery 25 d after normal term, HX sheep fetuses are hypoglycemic and have lower glycogen contents in several tissues, including the liver, than intact fetuses at term (6). However, nothing is known about the rates of metabolism and growth in HX sheep fetuses during the extended period of gestation. Nor is it clear whether deficiency of pituitary hormones in utero alters the fetal metabolic response to nutritional challenges. Therefore, this study investigated the effects of hypophysectomy on the growth and metabolism of the sheep fetus during the late normal and extended periods of gestation, and on its metabolic response to a short period of maternal food deprivation close to normal term. Materials and Methods Animals A total of 32 Welsh Mountain ewes carrying single fetuses were used in this study. During the experimental period, the ewes were housed individually and maintained on concentrate (200 g/d; Beart Ltd., Stowbridge, Suffolk, UK) and hay and water ad libitum. Half the daily ration

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Fowden and Forhead • Fetal Growth and Metabolism after Hypophysectomy

of concentrates was fed at 0800 h, whereas the remainder was given at 1700 h. Food but not water was withheld for 18 –24 h before surgery. All procedures were performed under the Animals (Scientific Procedures) Act 1986.

Surgical procedures Under general anesthesia (1.5% halothane in a 5:1 mixture of O2 and N2O2) at 105–110 d gestation (normal term 145 ⫾ 2 d), one of the following procedures was performed using the surgical techniques described previously (6, 16): 1) fetal hypophysectomy (HX, n ⫽ 17, nine females and eight males) with or without insertion of a device for measuring CRL (n ⫽ 7 and n ⫽ 10, respectively); 2) sham hypophysectomy alone (n ⫽ 3); or 3) insertion of a CRL measuring device alone (n ⫽ 7, three females, four males). Intravascular catheters were inserted into 13 of the HX fetuses at a second operation at either 125–129 (n ⫽ 7, four females, three males) or 153–158 d during the prolonged period of gestation (n ⫽ 6, three females, three males). The three sham-operated fetuses and five additional fetuses that had had no previous surgery were also catheterized intravascularly at 125–129 d gestation (five females and three males). The catheters were inserted into the umbilical vein, fetal dorsal aorta, and caudal vena cava, and into the maternal aorta via a femoral artery, as described previously (17). At the end of the experimental period, fetuses were either delivered by cesarean section under general anesthesia (20 mg/kg sodium pentobarbitone) or allowed to deliver naturally at term (intact control only, n ⫽ 7). The details of the experimental groups, their treatments, and gestational ages at surgery and delivery are shown in Table 1.

Experimental procedures In the animals with vascular catheters, blood samples (0.5–1.0 ml) were taken from the fetus and mother daily between 0900 and 1000 h to monitor well-being. The daily increment in CRL was also measured at this time in fetuses with a CRL measuring device. In fetuses with no vascular catheters (Table 1), the presence of a fetal heartbeat was confirmed on a weekly basis using Doppler ultrasound (UltraTec Pocket Doppler; Thames Medical, Goring by Sea, West Sussex, UK). Increments in CRL length were measured until birth in the seven intact fetuses and until delivery by cesarean section at 159- to 166-d gestation in the seven HX fetuses. Birth of the intact fetuses occurred at a mean gestational age of 143.7 ⫾ 0.7 d (Table 1). At least 6 d after vascular catheterization, measurements of fetal glucose and oxygen metabolism were made in the fed state in eight intact fetuses at 136 –139 d, including the three sham-operated fetuses, seven HX fetuses at 136 –139 d, and in six postmature HX fetuses at 158 – 163 d gestation. After this study, food but not water was withdrawn from the preterm ewes, and a second set of measurements were made in the fasted state in five intact and six HX fetuses at 139 –142 d gestation, when the animals had been without food for 48 h. In both fed and fasted animals, tritiated water (8 ␮Ci/ml; Amersham Intl., Buckinghamshire, UK) and universally labeled [14C]glucose (10 ␮Ci/ml in 0.09% NaCl weight/volume; ICN Biochemicals, High Wycombe, Buckinghamshire, UK) were infused together into the fetal caudal vena cava for 2– 4 h at known rates between 0.08 and 0.09 ml/min after an initial priming dose

Endocrinology, October 2007, 148(10):4812– 4820

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(3– 4 ml). Blood samples (3.5 ml) were taken simultaneously from the umbilical vein, fetal dorsal aorta, and maternal artery before (0 min), and, when steady state had been established, at known times approximately 120, 140, 160, and 180 min after beginning the infusion. The simultaneous blood samples were analyzed immediately for blood pH, gas tensions, packed cell volume, and O2 content (0.5 ml), and for labeled carbon dioxide (14CO2) where appropriate (1.0 ml). The remainder of the sample (2 ml) was added to a chilled tube containing EDTA for subsequent analyses. An aliquot (0.5 ml) of the EDTA-treated blood was deproteinized with zinc sulfate (0.3 m) and barium hydroxide (0.3 m), and the supernatant used for determination of both labeled and total concentrations of glucose. The remaining EDTA sample was centrifuged at 4 C, and the plasma stored at ⫺20 C until required for 3H2O and hormone measurements. An additional aliquot of fetal arterial blood (1 ml) was taken at 0 min, and placed in a chilled heparinized tube containing EGTA (5.0 ␮mol/ml blood) and glutathione (40 ␮mol/ml blood) for catecholamine assay. At the end of the experimental period, all HX and intact fetuses with vascular catheters and the uncatheterized, postmature HX fetuses with CRL measuring devices were delivered by cesarean section under general anesthesia (Table 1), and a blood sample was taken from the umbilical artery before administration of a lethal dose of anesthetic to the mother and fetus (200 mg/kg sodium pentobarbitone). The fetuses were then weighed and measured. A sample of liver (5–10 g) was collected from all intact and HX fetuses in the fasted state, and frozen immediately in liquid nitrogen before storage at ⫺80 C for the subsequent analyses of glycogen content and key gluconeogenic enzyme activities. The position of all catheters was also verified at autopsy. No pituitary remnants were found in the pituitary fossa of any of the HX fetuses. The hypothalamo-pituitary region of the HX fetuses was also examined using serial histological sections stained with hematoxylin and eosin. No pituitary cells were detected in any of the sections. The intact fetuses with CRL measuring devices that delivered spontaneously at term were weighed and measured immediately after birth (Table 1).

Biochemical analyses The blood gas tensions, packed cell volume, O2 content, and whole blood concentrations of glucose, [14C]glucose, 3H2O, and 14CO2, were measured in all five sets of simultaneous samples in the fed and fasted states. Blood O2 content was calculated from the percentage O2 saturation and the hemoglobin concentrations measured using an OSM2 Hemoximeter (Radiometer, Copenhagen, Denmark) that had been calibrated for ovine blood. Blood pH and partial pressures of O2 and CO2 were measured using an ABL5 Radiometer and corrected for a fetal body temperature of 39 C. Glucose concentrations were determined enzymatically in whole blood and plasma using a spectrophotometer (17) and an automated analyzer, respectively (2300 StatPlus; Yellow Springs Instruments Co. Inc., Yellow Springs, OH). Plasma 3H2O concentrations were measured using scintillation counting and converted to blood concentrations using the packed cell volume as described previously (8, 17). Labeled glucose and CO2 were determined using chemical methods published previously (17–20). Labeled glucose was separated from all other 14C-labeled products by anion exchange chromatography after preincubation with

TABLE 1. Numbers and gestational ages of the fetuses in the different treatment groups Gestational age (d) Treatment

Intact

HX

a

Additional surgical procedures

Growth measuring device Sham hypophysectomy and catheterization Catheterization alone Growth measuring device Growth measuring device and catheterization Catheterization alone

Natural delivery at term.

No. of fetuses

At hypophysectomy or growth device surgery

7 3

105–109 106 –110

5 4 3

105–109 105–109

153–158

139 –141 159 –166 159 –163

7 3

105–109 105–109

125–129 153–158

139 –141 160 –163

At intravascular catheterization

125–129 126 –129

At delivery

141–146a 139 –141

and without glucose oxidase (19, 20). The mean recovery of [14C]glucose from the anion exchange column was 99.7 ⫾ 1.3% (n ⫽ 32). Therefore, no corrections for glucose recovery were made. In contrast, the mean recovery of 14CO2 was 71.9 ⫾ 0.6% (n ⫽ 22) and, therefore, all blood 14 CO2 values have been corrected for recovery. Plasma catecholamine concentrations were determined by high-pressure liquid chromatography using electrochemical detection (17). Recovery of isoprenaline added to the samples ranged from 63–97% and, therefore, all samples have been corrected for their respective recoveries. The limits of sensitivity of the method were 50 pg/ml for adrenaline and 30 pg/ml for noradrenaline. The interassay coefficients of variation for adrenaline and noradrenaline were 7.3 and 6.2%, respectively. Plasma concentrations of insulin, cortisol, and T4 were measured by RIA validated for use with ovine plasma (8, 21, 22). The interassay coefficients of variation for these three assays were 10.0, 13.7, and 10.0%, respectively, whereas the minimum detectable quantity of hormone was 1.5 ng/ml for cortisol, 5.0 ␮U/ml for insulin, and 7.0 ng/ml for T4. Hepatic glycogen content and the activities of glucose-6-phosphatase (G6Pase) [enzyme commission number (EC) 3.1.3.9] and phosphoenolpyruvate carboxykinase (PEPCK) (EC 4.1.1.32) were assayed using established methods described in detail elsewhere (23, 24). Hormone concentrations and enzyme activities were measured in duplicate, whereas all other biochemical analyses were measured in triplicate.

Calculations All calculations were made using equations derived for steady-state kinetics (8, 18). Umbilical blood flow was measured using the 3H2O steady-state diffusion technique (8). Net umbilical uptake of glucose and oxygen and net umbilical excretion rates of [14C]glucose and 14CO2 were calculated by Fick principle as the product of umbilical blood flow and the umbilical venous-arterial (uptake) or arteriovenous (excretion) concentration difference across the umbilical circulation. The fetal rates of utilization and production of glucose and of CO2 production from glucose carbon were measured using three pool tracer methodology as described previously (17, 18). These rates were used to calculate the glucose oxidation fraction and the fraction of fetal O2 consumption used to oxidize glucose carbon assuming that the amount of oxygen used in glucose carbon oxidation equals the amount of CO2 produced (18, 19). Endogenous glucose production by the fetus was calculated as the difference between the fetal rates of glucose use and umbilical glucose uptake. When studies were performed in the both fed and fasted states, the values for [14C]glucose and 14CO2 in the 0-min arterial and umbilical venous samples of the fasted study were subtracted from the subsequent samples before calculation of the glucose metabolic rates. All metabolic rates have been expressed per kg fetal body weight. No increase in fetal body weight was assumed to occur during the 48-h period of maternal food withdrawal. Fetal growth has been calculated in two ways: first, as an accumulated increment in CRL from the time of insertion of the CRL measuring device at 105–110 d gestation until delivery; and, second, as a rate of CRL increment over 5-d periods calculated retrospectively from the day of birth in the intact fetuses (Fig. 1). The accumulated increment in CRL was used to monitor daily changes in growth, whereas the rate of CRL increment was used to assess longitudinal changes in growth over longer periods of gestation with respect to normal term. In the HX fetuses, 144 d was defined as normal term for the retrospective assessment of CRL increment because this was the mean gestational age at which the intact fetuses were born. Therefore, the CRL of the lamb at delivery was obtained both by direct measurement (actual CRL) and by adding the accumulated increment in CRL over time to the actual CRL measured at insertion of the CRL measuring device (derived CRL). The mean discrepancy between the derived and actual measurements at delivery was 4.3 ⫾ 1.0% (n ⫽ 14) of the actual CRL or 11.1 ⫾ 2.4% when the discrepancy was expressed as a percentage of the increment in actual CRL that occurred between insertion of the CRL measuring device and delivery. The error in CRL measurement was similar in the intact and HX groups of fetuses. Ponderal index was calculated as body weight in kilograms divided by CRL in cubic meters.


Normal term

A CRL increment mm


200 180 160 140 120 100 80 60 40 20 0 0

5

10

15

20

25

30

35

40

45

50

55

Days after surgery

B CRL increment mm/day

4814

8

Normal term

a

a

a

6 4

ab a†

a†

a†

b a†

a

b a b a

a

a a

2 0 35-30 30-25 25-20 20-15 15-10 10-5

Days before term

5-0

0-5

5-10 10-15

Days after term

FIG. 1. Mean (⫾SEM) CRL increments calculated as accumulated increment from the day of surgery (A) and as a rate of daily increment over 5-d periods (B) assessed retrospectively and prospectively with respect to normal term (144 d) in intact (n ⫽ 7, open squares, open columns) and HX sheep fetuses (n ⫽ 7, filled diamonds and columns). Within a treatment group, columns with different letters as superscripts are significantly different from each other (P ⬍ 0.01, one way ANOVA with repeated measures). †, Significant difference between treatment groups (P ⬍ 0.02, two-way ANOVA).

Statistical analyses Steady state was defined as less than 10% variation of values around the mean for each sampling period with no consistent trend for the absolute values to increase or decrease with time. There were no significant differences between the intact fetuses that were and were not sham operated, and, therefore, these two groups have been combined for all further statistical analyses. Mean values ⫾ sem have been used throughout. Statistical analyses were made using Sigmastat (Jandel Scientific, Chicago, IL). Comparison of metabolic rates between treatments and nutritional states were made using one-way ANOVA, Student’s t test, and paired t tests, as appropriate. Comparison of CRL increments with time and treatment were made by one- or two-way ANOVA with repeated measures. For all statistical analyses, significance was accepted when P ⬍ 0.05.

Results Growth

Hypophysectomy of the sheep fetus at 105–109 d gestation reduced its body weight and actual CRL near to normal term compared with age-matched intact controls (Table 2). At 159 –166 d gestation, the body weight of the HX fetuses was significantly greater than the values found in HX fetuses near normal term and in newborn, intact lambs at birth (Table 2). The actual CRL of the postmature HX fetuses was also greater than that of the HX fetuses near normal term, and was



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TABLE 2. The mean (⫾SEM) values of body weight, CRL, ponderal index, limb lengths, and adrenal and placental weights in intact fetuses at 139 –141 d gestation and at birth, and in HX fetuses at 139 –141 and 159 –166 d gestation Treatment

Intact HX

Body wt (g)

CRL (cm)

Ponderal index (kg/m3)

Hind limb length (cm)

Forelimb length (cm)

3353 ⫾ 170 (8) 3139 ⫾ 244 (7)a 2759 ⫾ 107 (7)b,c 3926 ⫾ 200 (10)

50.5 ⫾ 0.5 (8) 49.0 ⫾ 0.7 (7) 44.2 ⫾ 0.6 (7)c,d 50.8 ⫾ 1.3 (10)

26.5 ⫾ 1.0 (8) 25.2 ⫾ 1.9 (7)a 32.9 ⫾ 1.9 (7)d 30.0 ⫾ 1.2 (10)b

32.6 ⫾ 0.4 (8) 32.2 ⫾ 1.1 (4)a 27.5 ⫾ 0.3 (7)c,d 29.6 ⫾ 0.5 (8)b

26.6 ⫾ 0.4 (8) 27.1 ⫾ 0.7 (4)a 22.5 ⫾ 0.4 (7)c,d 24.2 ⫾ 0.5 (8)b

Gestational age (d)

139–141 Birth 139–141 159–166

Placental wt (g)

Adrenal wt (mg)

268 ⫾ 18 (7)

425 ⫾ 33 (7)

282 ⫾ 15 (6) 247 ⫾ 29 (7)

249 ⫾ 17 (6)d 279 ⫾ 27 (7)b

Number of animals is shown in parentheses. wt, Weight. Significantly different from control at 139 –141 d: bP ⬍ 0.05 and dP ⬍ 0.01 (Student’s t test). Significantly different from HX fetus at 159 –166 d: aP ⬍ 0.05 and cP ⬍ 0.01 (Student’s t test).

similar to the values seen in intact fetuses near normal term and at birth (Table 2). The ponderal index of both groups of HX fetuses was significantly greater than that of the intact control fetuses in late gestation (Table 2). The lengths of both the forelimbs and hind limbs were shorter in the HX fetuses at 139 –141 d than in the age-matched intact controls (Table 2). At 159 –166 d, the limbs of the HX fetuses were longer than at 139 –141 d, but they were still shorter than those of intact fetuses near term or at birth. In common with previous findings (6, 8), adrenal weights were significantly lower in HX than intact fetuses, regardless of gestational age (Table 2). Fetal hypophysectomy had no apparent effect on placental weight; mean total placentome weight was similar in the intact and HX groups of fetuses. The actual CRL of the intact and HX fetuses at surgery did not differ significantly (intact, 31.6 ⫾ 0.6 cm, n ⫽ 7; HX, 33.0 ⫾ 0.6 cm, n ⫽ 7; P ⬎ 0.05). Compared with intact controls, fetal hypophysectomy reduced CRL increment from immediately after surgery throughout the rest of normal gestation until delivery by cesarean section 15–20 d after normal term (Fig. 1A). During the normal period of gestation, the mean daily increment in CRL of the HX fetuses was 3.0 ⫾ 0.2 mm (n ⫽ 7) compared with a mean value of 4.7 ⫾ 0.3 mm for the intact fetuses (n ⫽ 7, P ⬍ 0.01). In the HX fetuses, the mean daily increment in CRL remained at 3.0 ⫾ 0.2 mm (n ⫽ 7) during the prolonged period of gestation (Fig. 1A). When the rate of CRL increment was calculated over 5-d periods retrospectively and prospectively from normal term, the growth rate of the HX fetuses was uniform and did not vary significantly from 35 d before to 20 d after normal term (Fig. 1B). In contrast, the mean rate of CRL increment declined toward term in the intact fetuses (Fig. 1). In common with previous findings (16, 25), the growth rate of the intact fetuses was significantly lower in the last 15 d gestation than between 20 and 35 d before birth (Fig. 1B). Therefore, the mean rates of

CRL increment were significantly lower in HX than intact fetuses between 35 and 15 d before normal term but were similar in the two groups during the 15 d immediately before normal term (Fig. 1B). Metabolism

Fed state Basal values. Fetal hypophysectomy had no effect on the blood pH or gas status of the fetuses during either the normal or extended periods of gestation. The mean values of blood pO2, pCO2, O2 saturation, O2 content, or hemoglobin concentration were similar in the HX and intact groups of fetuses, and were within the range of values for those published previously for intact fetuses in late gestation (8, 17). Plasma concentrations of cortisol and T4 were significantly lower, whereas plasma levels of norepinephrine and total catecholamines were significantly higher in both groups of HX fetuses than in the intact fetuses at 136 –139 d gestation (Table 3). There were no significant differences in the plasma concentrations of insulin and epinephrine between intact and HX fetuses at either gestational age (Table 3). Fetal and maternal concentrations of blood glucose were also similar in the intact and HX groups of animals both before and after normal term (Table 3). In addition, there were no significant differences in blood glucose concentrations or plasma hormone concentrations between the two groups of HX fetuses (Table 3). Umbilical blood flow tended to be lower in HX than intact fetuses when expressed per kg fetal body weight, but this only reached statistical significance in the HX fetuses during the prolonged period of gestation (intact, 187 ⫾ 11 ml/min䡠kg, n ⫽ 8; HX, 136 –139 d, 158 ⫾ 19 ml/min䡠kg, n ⫽ 7, P ⬎ 0.05; HX, 158 –165 d, 130 ⫾ 19 ml/min䡠kg, n ⫽ 6, P ⬍ 0.02).

TABLE 3. The mean (⫾SEM) concentrations of fetal and maternal blood glucose, and of fetal plasma cortisol, insulin, T4, epinephrine, norepinephrine, and total catecholamines in intact and HX fetuses in the fed and fasted state at different gestational ages Treatment

Intact HX

a b

Nutritional Gestational No. of state age (d) animals

Fed Fasted Fed Fasted Fed

136–139 139–141 136–139 139–141 159–166

8 5 7 5 6

Blood glucose (mmol/liter) Fetus

0.80 ⫾ 0.06 0.53 ⫾ 0.04a 0.74 ⫾ 0.07 0.40 ⫾ 0.05a 0.70 ⫾ 0.10

Mother

Cortisol (ng/ml)

2.34 ⫾ 0.08 13.7 ⫾ 1.7 1.18 ⫾ 0.11a 41.0 ⫾ 10.0a 2.40 ⫾ 0.09 7.7 ⫾ 0.9b 1.42 ⫾ 0.17a 7.8 ⫾ 1.1c 2.29 ⫾ 0.20 7.5 ⫾ 0.7b

Insulin (␮U/ml)

T4 (ng/ml)

21.8 ⫾ 2.0 133 ⫾ 11 14.0 ⫾ 1.6a 116 ⫾ 14 21.4 ⫾ 2.4 23 ⫾ 6c 16.8 ⫾ 1.0a 19 ⫾ 4c 15.1 ⫾ 2.8 24 ⫾ 9c

Total Epinephrine Norepinephrine catecholamines (pg/ml) (pg/ml) (pg/ml)

65 ⫾ 28 65 ⫾ 41 62 ⫾ 26 108 ⫾ 40 22 ⫾ 10

215 ⫾ 38 880 ⫾ 268a 560 ⫾ 145b 610 ⫾ 149 693 ⫾ 265b

280 ⫾ 62 945 ⫾ 234a 615 ⫾ 141b 718 ⫾ 119 715 ⫾ 262b

Significantly different from the value in the fed state for the corresponding treatment group at 136 –139 d (aP ⬍ 0.05, Student’s t test). and c Significantly different from the value in intact fetuses in the same nutritional state (bP ⬍ 0.05, cP ⬍ 0.01 Student’s t test).

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Metabolic rates. In common with previous findings (8), fetal hypophysectomy had no apparent effect on the weight-specific rates of umbilical uptake and utilization of glucose close to normal term. The mean values of these rates did not differ significantly between the intact and HX fetuses at 136 –139 d gestation (Table 4). Neither was there any significant difference in the rate of CO2 production from glucose carbon between these two groups of fetuses when values were expressed on a weight-specific basis (Table 4). The rate of endogenous glucose production was negligible in both the intact and HX fetuses at 136 –139 d gestation (Table 4). During the prolonged period of gestation, the weight-specific rates of umbilical glucose uptake, glucose utilization, and CO2 production from glucose carbon in the HX fetuses were significantly lower than those observed in both the HX and intact groups of fetuses at 136 –139 d (Table 4). The mean rate of endogenous glucose production remained insignificant in the HX fetuses during the extended period of gestation (Table 4). There was no significant difference in the transplacental gradient in plasma glucose between the postmature HX fetuses at 159 –165 d (2.56 ⫾ 0.23 mmol/liter, n ⫽ 6) and either the HX or intact groups of fetuses at 136 –139 d gestation (HX, 2.43 ⫾ 0.12 mmol/liter, n ⫽ 7; intact, 2.20 ⫾ 0.36 mmol/liter, n ⫽ 8; P ⬎ 0.05, all cases). Compared with intact controls near term, the weight-specific rates of umbilical O2 uptake were significantly lower in HX fetuses at both 136- to 139- and 159- to 165-d gestation (P ⬍ 0.02; Table 4). The amount of O2 used to oxidize glucose carbon was significantly less in the postmature HX fetuses than in either the younger HX fetuses or the intact controls near term (P ⬍ 0.05). The glucose oxidation fraction and the fraction of umbilical O2 uptake used to oxidize glucose carbon did not differ significantly between the intact and HX fetuses at either gestational age (Table 4). Fasted state Basal values. Maternal fasting for 48 h reduced maternal and fetal concentrations of blood glucose by 35–50% in both the intact and HX groups of animals (Table 3). The degree of hypoglycemia and the mean decrements in fetal and maternal blood glucose concentrations were similar in the two groups at 139 –141 d gestation (P ⬎ 0.05 all cases). There was a significant increment in the fetal plasma cortisol concen-

tration during fasting in the intact fetuses (⫹28.2 ⫾ 10.1 ng/ml, n ⫽ 5; P ⬍ 0.05 paired t test), but not in the HX fetuses (HX, ⫺0.2 ⫾ 0.8 ng/ml, P ⬎ 0.05 paired t test; P ⬍ 0.02). Consequently, the absolute cortisol concentration in the fasted state was significantly greater in the intact than HX fetuses (P ⬍ 0.02; Table 3). The increases in the fetal concentrations of plasma norepinephrine and epinephrine in response to maternal fasting did not reach statistical significance in either group (Table 3). However, the increment in total catecholamine concentrations was significant in the intact (⫹680 ⫾ 264 pg/ml, n ⫽ 5; P ⬍ 0.05, paired t test) but not in the HX fetuses at 139 –141 d gestation (⫹103 ⫾ 176 pg/ml, n ⫽ 6; P ⬎ 0.05, paired t test). Plasma insulin concentrations decreased significantly during maternal fasting in both intact and HX fetuses; mean decreases were similar in the two groups (Table 3). No changes in plasma T4 concentration occurred in response to maternal fasting in either the intact or HX fetuses (Table 3). There were also no significant changes in blood pH, gas status, or hemoglobin concentration during maternal fasting; all values remained within the range of values published previously for animals in the fed state (8). Umbilical blood flow increased during the 48-h period of maternal fasting in the HX fetuses (⫹33 ⫾ 13 ml/min䡠kg, n ⫽ 6; P ⬍ 0.05, paired t test), but not in the intact animals (⫹4 ⫾ 20 ml/min䡠kg, n ⫽ 5; P ⬎ 0.05, paired t test). Metabolic rates. Maternal fasting for 48 h significantly reduced the rate of umbilical glucose uptake in both the intact and HX fetuses at 139 –141 d; mean decrements were similar in the two groups (Fig. 2). The transplacental plasma glucose concentration gradient also decreased to a similar extent in the HX and intact fetuses (intact, ⫺0.98 ⫾ 0.25 mmol/liter, n ⫽ 5; HX, ⫺0.89 ⫾ 0.21 mmol/liter, n ⫽ 6; P ⬍ 0.02, paired t test, both cases). In the HX fetuses, there was also a significant decrease in the rate of fetal glucose utilization, which was of a similar magnitude to the decrease in umbilical glucose uptake (Fig. 2). There was also a tendency for the rate of fetal glucose use to decrease during fasting in the intact fetuses, but the mean decrement was not significant. Therefore, endogenous glucose production remained negligible in the HX fetuses but occurred at a significant rate in the intact fetuses after maternal fasting for 48 h (Fig. 2). No significant change in umbilical O2 uptake was ob-

TABLE 4. The mean (⫾SEM) rates of umbilical uptake, utilization, and production of glucose, CO2 production from glucose carbon and of umbilical oxygen uptake, and the glucose oxidation fraction and the fraction and amount of umbilical oxygen uptake used for oxidation of glucose carbon in the fed state in intact (n ⫽ 8) and HX (n ⫽ 7) fetuses at 136 –139 d and in HX fetuses at 159 –165 d (n ⫽ 6) Treatment

Intact HX

Intact HX a b

Fetal rates of glucose metabolism (␮mol/min䡠kg)

Gestational age (d)

Umbilical uptake

Utilization

136 –139 136 –139 159 –165

29.0 ⫾ 2.1 28.8 ⫾ 1.3 15.7 ⫾ 2.5a,b

28.9 ⫾ 2.1 30.6 ⫾ 1.3 20.9 ⫾ 2.2a,b

136 –139 136 –139 159 –165

Umbilical O2 uptake ␮mol/min䡠kg

294 ⫾ 17 222 ⫾ 13a 183 ⫾ 15a,b

Production

1.1 ⫾ 2.4 1.6 ⫾ 1.7 5.2 ⫾ 2.6

O2 used for glucose oxidation

Fraction

Amount ␮mol/min䡠kg

0.283 ⫾ 0.018 0.343 ⫾ 0.037 0.231 ⫾ 0.051

84.4 ⫾ 7.5 76.1 ⫾ 7.1 42.3 ⫾ 11.0a,b

Significantly different from the values in the intact fetuses at 136 –139 d (P ⬍ 0.05). Significantly different from the values in the HX fetuses at 136 –139 d (P ⬍ 0.05).

CO2 production from fetal glucose carbon

83.4 ⫾ 7.8 74.5 ⫾ 6.9 49.2 ⫾ 10.5a,b

Glucose oxidation fraction

0.497 ⫾ 0.049 0.402 ⫾ 0.059 0.374 ⫾ 0.063


Glucose utilization

Control

40

HX

30

30

20

20

10

10

0 -10

- 10

-20

†

†

-30

D

Glucose production

30

Control

20

*†

10

†

HX

- 20 - 30

CO2 production

Control

HX

100 75 50 25

5 0

0

-5

- 25

Glucogenic capacity. The hepatic glycogen content and hepatic activities of G6Pase and PEPCK were significantly lower in HX than intact fetuses in the fasted state at 139 –141 d gestation (Fig. 3). Renal activity of G6Pase but not PEPCK was also significantly less in HX than intact fetuses after maternal fasting (Fig. 3). Discussion

This study demonstrates for the first time that hypophysectomy of the sheep fetus reduces the fetal growth rate uniformly from the time of surgery until delivery 15–25 d after normal term. The rate of CRL increment in the HX fetuses was only 50% of that seen in the intact controls in the period between 35 and 15 d before normal term. This reduced growth rate was accompanied by reductions in the CRL, limb

lengths, and body weight of HX fetuses close to the end of the normal period of gestation. Fetal hypophysectomy also decreased the rate of fetal oxygen consumption both before and after normal term compared with intact controls. In addition, during the prolonged period of gestation, HX fetuses had lower rates of glucose uptake, glucose use, and CO2 production from glucose carbon than HX fetuses and intact controls close to normal term. Furthermore, HX fetuses were unable to produce glucose endogenously in response to a decrease in the umbilical glucose supply induced either by prolonged gestation or by maternal fasting near to normal term. These findings demonstrate that the pituitary hormones are essential for normal growth in utero and have an important role in the metabolic response to undernutrition in the sheep fetus. In the current study, the reductions in CRL increment, actual CRL, limb lengths, and body weight observed after hypophysectomy were accompanied by an increase in pon-

Liver 60

Glycogen content mg/g

served in response to maternal fasting in either the HX or intact fetuses; mean rates of O2 uptake in the fasted state were similar to those in the fed state (Table 4). Therefore, the rate of umbilical O2 uptake remained significantly lower in the HX fetuses (212 ⫾ 9 ␮mol/min䡠kg, n ⫽ 6) than in the intact controls (275 ⫾ 12 ␮mol/min䡠kg, n ⫽ 5; P ⬍ 0.01) after maternal fasting for 48 h. During fasting, the glucose oxidation fraction tended to increase in the HX fetuses (⫹0.139 ⫾ 0.070, n ⫽ 6), whereas it tended to decrease in the intact fetuses (⫺0.084 ⫾ 0.039), although neither of these changes reached statistical significance (P ⬎ 0.05, paired t test, both cases). The rate of CO2 production from glucose carbon decreased significantly in response to maternal fasting in the intact but not in the HX fetuses (Fig. 2). There were also significant reductions in the fraction and amount of the umbilical O2 uptake used to oxidize glucose carbon with maternal fasting in the intact fetuses (fraction, ⫺0.111 ⫾ 0.021; amount, ⫺30.6 ⫾ 8.2 ␮mol/min䡠kg, n ⫽ 5; P ⬍ 0.05, paired t test, both cases), but not in the HX fetuses (fraction, ⫺0.072 ⫾ 0.065; amount, ⫺10.4 ⫾ 12.3 ␮mol/min䡠kg, n ⫽ 6; P ⬎ 0.05, paired t test, both cases).

- 50

†

-10

Kidney

Glycogen G6Pase PEPCK

G6Pase PEPCK

12

50

10

40

8

30

6

20 10 0

*

4

* *

*

2

Enzyme activity U/g

C Rate of glucose metabolism mol/min/kg

HX

Rate of glucose metabolism mol/min/kg

Control

40

B

Glucose uptake

0

FIG. 2. Mean (⫾SEM) rates of umbilical glucose uptake (A), glucose utilization (B), glucose production (C), and CO2 production from glucose carbon (D) in intact control (n ⫽ 5) and HX fetuses (n ⫽ 6) in the fed state at 136 –139 d (open columns) and fasted state at 139 –141 d (striped columns), and the mean change in value during the 48-h period of maternal fasting (filled columns). †, Significant change from zero during fasting within a treatment group (P ⬍ 0.02, paired t test). *, Significant rate of glucose production (P ⬍ 0.02, paired t test).

Rate of glucose metabolism mol/min/kg

A

4817

Rate of CO2 production mol/min/kg


0

FIG. 3. Mean (⫾SEM) values of glycogen content, and activities of G6Pase and PEPCK in the liver and kidneys of intact (n ⫽ 5, open columns) and HX fetuses (n ⫽ 6, striped columns) at 139 –141 d after maternal fasting for 48 h. *, Significantly different from the corresponding value in the intact fetuses (P ⬍ 0.01, Student’s t test).

4818


deral index. This is consistent with previous findings of increased sc fat deposition in HX fetuses at term (5, 12). Therefore, skeletal growth appears to be more adversely affected than somatic tissue growth by hypophysectomy of the sheep fetus. This poor growth may be due to fetal deficiency of GH, thyroid hormones, glucocorticoids, or a combination of these hormones. The skeletal defects observed in HX fetuses closely resemble those seen after fetal thyroidectomy (26, 27), which suggests that hypothyroidism may be an important contributory factor to the retarded growth after hypophysectomy. However, treatment of the HX sheep fetus with either T4 or GH alone does not restore the CRL or limb lengths to normal values, although GH administration prevents the excessive accumulation of sc fat (8, 9, 12). Administration of GH also has little, if any, effect on the growth of the normal, intact sheep fetus during late gestation (28). Similarly, the low cortisol concentrations in the HX fetuses during late gestation are unlikely to account for their reduced growth rate because cortisol inhibits rather than enhances fetal growth during late gestation (25). Circulating concentrations of IGF-I are low in the HX sheep fetus (29), which may contribute to their retarded growth because plasma IGF-I levels are positively correlated with body weight in intact sheep fetuses during late gestation (30). Together, these observations suggest that the reduced growth of the HX fetus is due to multiple endocrine deficiencies rather than the lack of a specific pituitary hormone. Although fetal growth slowed after hypophysectomy, it did not stop. Even during the extended period of gestation, the CRL of the HX fetuses continued to increase at a steady rate similar to that seen before normal term. In contrast to previous findings (29), the actual CRL, body weight, and limb lengths of the postmature HX fetuses in the current study were greater than those seen in HX fetuses 20 d earlier in gestation. Indeed, the rate of CRL increment in the HX fetuses was greater than that seen in sheep fetuses made insulin deficient during the last 20 –30 d gestation by fetal pancreatectomy (16). Insulin concentrations were normal in HX fetuses even in the prolonged period of gestation, and, therefore, insulin may have been responsible for the continued growth of these fetuses, albeit at a lower rate than in intact fetuses with normal insulin and pituitary hormone levels. Because GH antagonizes the actions of insulin in utero (31), the unopposed action of insulin in the HX fetus may also account for its increased fat content and higher ponderal index because insulin normally stimulates fetal lipogenesis during late gestation (32). Hypophysectomy has little effect on the fetal rates of glucose metabolism in fed conditions but prevented some of the metabolic and endocrine responses to maternal fasting during late gestation. HX fetuses were unable to produce glucose endogenously, and, as a consequence, their rate of glucose use decreased significantly in response to the decrease in umbilical glucose supply induced by maternal fasting. They were also unable to reduce their rate of CO2 production from glucose carbon when glucose availability decreased during fasting. In contrast, a similar decrease in glucose availability in the intact fetuses reduced glucose carbon oxidation and activated glucogenesis, which ameliorated the decrease in fetal glucose use. There were no changes in fetal O2 con-


sumption in response to maternal fasting in either group of fetuses, although the weight-specific rate of umbilical O2 uptake was lower in HX than intact fetuses in both fed and fasted states. These differences in basal and fasting-induced changes in fetal metabolism are due, in part, to the different endocrine environments of the HX and intact fetuses. Previous studies have shown that the low rate of O2 consumption in the HX fetus is due to hypothyroidism and can be restored to normal values by T4 treatment (8). Similarly, the rate of fetal glucose production in response to maternal fasting and other stressful stimuli is known to be dependent on both cortisol and catecholamine concentrations in the sheep fetus (17, 33). Cortisol enhances the fetal glucogenic capacity by increasing glycogen deposition, gluconeogenic enzyme activities, and the abundance of hepatic adrenoreceptors, whereas catecholamines actually activate the glucogenic pathways once they are functionally competent (23, 33–35). Although catecholamine concentrations were high in HX fetuses relative to controls in fed conditions, there were no increases in catecholamine or cortisol concentrations in the HX fetuses in response to maternal fasting in the current study, in contrast to the findings in the intact fetuses. This may account, in part, for the inability of the HX fetus to produce glucose endogenously in response to maternal fasting during late gestation. Certainly, hepatic glycogen content and hepatic activities of the key gluconeogenic enzymes, G6Pase and PEPCK, were low in HX fetuses compared with controls at the end of the 48-h period of maternal fasting. When glucose availability decreases in intact fetuses during maternal fasting, the primary oxidative substrate switches from glucose to amino acid carbon, which leads to decreases in the rate of CO2 production from glucose carbon, and in the fraction and amount of umbilical O2 uptake used to oxidize glucose (19, 36). These changes are associated with protein catabolism and an increased release of amino acids from skeletal muscle in fetuses of fasted ewes (37). Because cortisol increases fetal protein catabolism and amino acid oxidation (38, 39), the fasting-induced increase in plasma cortisol observed in the intact fetuses in the current study is likely to facilitate the switch in oxidative metabolism and, therefore, the decrease in glucose carbon oxidation. Therefore, the low cortisol levels in the HX fetuses during maternal fasting may limit amino acid availability for oxidation and explain the sustained rate of glucose carbon oxidation, even though the degree of hypoglycemia was similar to that seen in the intact fetuses. The decrease in glucose availability during fasting may also pose less of a metabolic challenge to the HX fetuses because they had been growing more slowly and were smaller at the time of fasting than the intact fetuses. The lower weight-specific rates of umbilical glucose and O2 uptake in the HX fetus during the extended period of gestation suggest that the placental capacity to supply nutrients does not keep pace with the growth of these fetuses beyond normal term. In intact fetuses, the increasing nutrient demands for growth between mid and late gestation are met by altering placental nutrient consumption and by increasing placental blood flow, the transplacental glucose concentration gradient, and the placental abundance of nutrient transporters, such as the glucose transporters (36). Umbilical blood flow was lower in the postmature HX fetuses than in


HX and intact fetuses near normal term, which may have reduced the delivery of substances, like oxygen, which are flow limited (40). However, there were no changes in placental weight or the transplacental glucose concentration gradient that could have accounted for the reduced umbilical supply of glucose. To date, nothing is known about placental glucose transporters or the rate of uteroplacental glucose consumption in the postmature HX fetus, although there are changes in placental ultrastructure in these animals, which may influence nutrient transfer (41). Whatever their cause, the reduced rates of umbilical uptake of glucose and O2 in the postmature HX fetus do not appear to be too detrimental because HX fetuses maintain their preterm CRL increment during the extended period of gestation and survive in utero for up to 50 d beyond normal term (5, 6, 15). Because insulin regulates glucose uptake into fetal tissues (32), the reduced rates of glucose carbon use and oxidation in the postmature HX fetuses may have been due, in part, to the tendency for lower insulin levels during the extended period of gestation. Alternatively, the reductions in weight, type I fiber number, and in capillary density seen in specific fetal muscles after hypophysectomy (7, 42) may reduce muscle glucose consumption with implications for the rate of glucose utilization by the fetus as a whole, particularly as the abnormalities in fat and muscle composition become more pronounced with advancing gestational age (5, 12). Although the mean rate of fetal glucose production was not significantly different from zero during the prolonged period of gestation, the rate of glucose use was higher than the rate of umbilical glucose uptake in four of the six postmature HX fetuses. Therefore, endogenous glucose production may occur in some fetuses during prolonged gestation, although only at a low rate. Norepinephrine concentrations were high in the postmature HX fetuses, but whether these can stimulate glucogenesis when the hepatic glycogen content and gluconeogenic enzyme activities are low remains unknown (33, 35). Certainly, high norepinephrine concentrations were not associated with endogenous glucose production in HX fetuses close to term in either fed or fasted conditions. These observations are consistent with previous findings that catecholamines do not readily activate glucogenesis unless fetal cortisol levels have reached a threshold value of 15 ng/ml (17, 33). The effects of pituitary hormone deficiency on the fetal metabolic and endocrine responses to undernutrition observed in the present study have more widespread implications for the fetal responses to other adverse intrauterine conditions. Increases in the circulating concentrations of cortisol and the catecholamines occur in the normal fetus in response to hypoxemia, acidemia, anesthesia, umbilical cord compression, and reduced uterine blood flow. These endocrine responses aid fetal survival not only through their actions on glucose and other metabolites but also via cardiovascular effects that redirect fetal cardiac output away from peripheral tissues of the fetus toward more essential tissues, such as the adrenals, brain, and placenta (43). The absence of these endocrine changes in fetuses with hypopituitarism may increase their vulnerability to the normal range of challenges that occur in utero during late gestation. Similarly, the lower rates of umbilical uptake of glucose and


4819

oxygen observed during the extended period of gestation in the present study may explain, in part, the increased mortality and morbidity of human infants born after 41-wk gestation (44). In summary, the pituitary hormones have an important role in fetal growth, particularly of the axial and appendicular skeleton. They also regulate the basal rate of oxygen metabolism and the adaptive changes in glucose metabolism that occur in response to undernutrition in the sheep fetus. The results of the present study also suggest that fetal metabolism is altered when gestation is extended beyond normal term, although further studies are required to determine whether these changes are the consequence of an aging placenta or of prolonged endocrine deficiencies. Acknowledgments We thank Mrs. S. Nicholls and Mr. S. Gentle for their care of the animals and help during surgery, and Mrs. N. Daw for her assistance with the biochemical analyses. Received May 14, 2007. Accepted June 21, 2007. Address all correspondence and requests for reprints to: A. L. Fowden, Department of Physiology, Development and Neuroscience, University of Cambridge, Physiology Building, Downing Street, Cambridge CB2 3EG, United Kingdom. E-mail: [email protected]. This work was supported by the Wellcome Trust and the Biotechnology and Biological Sciences Research Council. Disclosure Summary: The authors have nothing to disclose.

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