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The numerous changes in metabolic pathways that accompany liver development entail associated changes in gene expression. Egr-1 is a zinc-finger transcription factor that regulates genes involved in cellular growth, differentiation, stress response, and apoptosis in many cell types. Egr-1 is induced in liver regeneration in rodents, but its role in normal hepatocyte function has not been characterized. We examined the developmental expression of Egr-1 in mouse liver and found that its expression increased during the suckling period. In screening the sequences of the genes involved in lactose assimilation, we found that the galactokinase gene Glk contains four potential Egr-1 binding sites in its proximal promoter. A minimal promoter of 155 nucleotides encompassing the four Egr-1 sites exhibited activity in hepatoma cell lines by transient transfection assays. Moreover, co-transfection of an Egr-1 expression plasmid increased promoter activity. Finally, mutations introduced into three of the four Egr-1 binding sites decreased activity, whereas mutation of the remaining site increased promoter activity. These data tie Egr-1 and galactokinase together in a developmentally regulated chain to prepare the neonate for suckling.

We examined the glycogenic response to glucose in cultured fetal and adult rat hepatocytes. After a 48-h culture in Dulbecco's modified Eagle's medium, 1 mM glucose, insulin, and cortisol, cells were cultured for 4 h in serum-free medium containing glucose (1-30 mM) and U-14C-glucose. Incorporation of 14C-glucose into glycogen was greater in fetal hepatocytes compared with adult hepatocytes at all glucose concentrations (p < 0.001). Net glycogenic rate in fetal cells was greatest between 1 and 8.3 mM (7.7- +/- 1.1-fold increase) compared with a 3.8- +/- 0.6-fold increase in adult cells. In contrast, there was a 19.4- +/- 2.7-fold increase in glycogen accumulated between 8.3 and 30 mM glucose in the adult and a 1.6 +/- 0.1-fold increase in the fetus. Total glycogen synthetase activity was higher in fetal than adult hepatocytes (p < 0.001), but the active a form was similar in fetal and adult hepatocytes. Glycogen synthase a/+b was stimulated at 8.3 mM or greater glucose in fetal hepatocytes, and 5.7 mM or greater in adult hepatocytes (p < 0.05). Total phosphorylase did not change with medium glucose, but glycogen phosphorylase a/a4+b decreased in adult hepatocytes incubated in 5.7 mM glucose or greater (p < 0.05). Fetal phosphorylase a/a+b was increased at 8.3 mM or greater glucose (p < 0.05). In contrast, both adult and fetal phosphorylase were activated by glycogen. A glucose-induced increase in active phosphorylase may induce the decrease in net glycogenic rate at high glucose in fetal hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

During the fetal period, hepatocytes are exposed to relatively low constant levels of glucose. The fetal to neonatal transition is characterized by remarkable changes in hepatic energy metabolism because of adaptation to intermittent oral feedings. Glucose transport, although not rate-limiting, is critical to the continuing supply of glucose to hepatocytes for storage and energy needs. Freshly isolated and cultured HFH and RFH express large amounts of both the low Km GLUT1 and the high Km GLUT2. Because cultured HFH, in contrast to RFH, do not downregulate GLUT1 and upregulate GLUT2 mRNA abundance in response to glucose, the reason for the change in GLUT abundance after birth in HFH is unclear. To define the mechanism by which hepatocytes change to the adult pattern of GLUT expression (largely GLUT2), we examined hormonal and substrate control of these transporters. RFH were isolated at 20 d gestation (n=4) and HFH were isolated at 13-20 wk (n=8), cultured for 24 h in 1 mM glucose DMEM with 10% FCS and then incubated for 4 h in stripped serum medium and 1 or 8.3 mM glucose with insulin (1μM), glucagon (1μM), or TGFβ (10 ng/mL). GLUT1 and GLUT2 mRNA was quantitated by densitometry and expressed relative to controls following Northern blot analysis of total RNA. In RFH and HFH, both insulin and glucagon increased GLUT1 mRNA abundance by 30-50% (p < 0.05), whereas TGFβ had no effect. In contrast, insulin enhanced GLUT2 mRNA abundance in RFH only in 8.3 mM glucose (p < 0.05) and in HFH in both 1 and 8.3 mM glucose (p < 0.05). Glucagon had no effect on GLUT2 mRNA abundance. TGFβ suppressed GLUT2 mRNA in RFH by 50% (p < 0.05), but not in HFH. We conclude that exogenous glucose and the capacity of the hepatocyte for endogenous glucose production (HFH are capable of gluconeogenesis) alter GLUT2 response to insulin. Glucose availability, as for other carbohydrate-regulated genes, is permissive for the action of insulin. The differential response to TGFβ remains to be explored. Table

Effect of Growth Hormone (GH) Therapy on Growth, Body Composition, and Glucose Tolerance in Children with Prader Willi Syndrome (PWS). 377

Diagnostic ambiguity in classical CAH is unusual. We present two patients who were referred to us following longterm adrenal suppression in whom IV ACTH testing did not initially confirm the diagnosis. Pt#1 (7 y. o. F) was adopted at 4.5 y. Pregnancy was complicated by attempted termination using a synthetic progestin. Clitoromegaly at birth and dehydration at 10 d lead to a diagnosis of salt-losing 21-hydroxylase deficiency in Russia and she was treated with prednisone (2.5 mg b.i.d., 28 mg/M2 cortisol equiv.) and DOCA (3.5 mg q.d.). No laboratory data are available. At adoption, height was at -2.6 S.D. and weight was at 50th percentile for height. She was placed on 20 mg/M2 per day of cortisol (t.i.d. dosing) and 0.05 mg 9-alpha-fludrocortisone b.i.d. At 7 yr, height was at 15th%ile, but more than 6 morning 17OHP measurements were

Low Km glucose transporter GLUT-1 mRNA and protein is found in vivo in adult hepatocytes (AH) only with starvation and diabetes. In contrast to AH, hepatic glycogenesis during fetal life is dependent upon glucose entry into hepatocytes at low glucose concentration. We have quantitated low Km GLUT-1 mRNA as well as high Km GLUT-2 mRNA in cultured rat FH compared to male AH, as % of FH at isolation.

We examined the effects of maternal sodium beta-hydroxybutyrate (NaBOHB) on the primate fetus to investigate the impact of ketosis not associated with acidosis on fetal metabolism. After a loading dose (600 mg/kg), NaBOHB was infused for 70 min (300 mg/kg.hr) into the maternal femoral vein of eight pregnant baboons, and placental transfer and fetal and maternal metabolic changes were observed during an acute experimental protocol. Maternal arterial levels rose from 0.70 +/- 0.21 to 5.42 +/- 0.93 mM (p less than 0.001), and fetal arterial levels from 0.34 +/- 0.09 to 2.76 +/- 0.64 mM (p less than 0.01). A maternal-fetal gradient of approximately 2:1 was observed in both baseline and steady-state infusion conditions and is similar to the human maternal-fetal ketone gradient. This is in contrast to the sheep where significantly higher gradients have been described. The elevated lactate, from 1.90 +/- 0.34 to 2.88 +/- 0.54 mM (p less than 0.05) and somewhat decreased pO2 values in the fetus from 54.8 +/- 8.9 to 45.0 +/- 3.8 mm Hg (p greater than 0.05 less than 0.1), without change in oxygen consumption (2.00 +/- 0.28 versus 1.73 +/- 0.15 mM/min) are features common to conditions of increased levels of fetal energy substrate. NaBOHB does not appear to contribute to oxidative energy metabolism of the whole fetus but may contribute to lipid stores. The significance of higher levels of BOHB in the primate fetus compared to the sheep fetus remains to be elucidated.

The energy-rich substrates available to the fasting stressed baboon neonate and infant are quantitatively similar to the metabolic fuels presented to the stressed low birth weight human newborn. Within a few hours after birth, fasting arterial plasma glucose levels in the baboon neonate approximate those of 4-6-week-old baboon infants after a 20-hr fast. Lactate levels are high and comparable for both age groups. In contrast, beta-hydroxybutyrate is quite low in the immediate neonatal period, but rises to significantly higher levels (P less than 0.001) after a fast at 4-6 weeks. In addition, glycerol levels are higher (P less than 0.02) in the fasted older infant compared with the fasting neonate. Computation of mean cerebral blood arteriovenous differences and oxygen equivalents for animals studied in the first 50 hr of life demonstrates that glucose uptake can account for 50% or less of cerebral oxygen consumption in the newborn period. In confirmation, the respiratory quotient in these animals is 0.52 +/- 0.06. Cerebral oxygen consumption in the immediate neonate is greater than can be explained by utilization of glucose and the small quantities of acetoacetate and beta-hydroxybutyrate available at this time. At birth, cerebral uptake of lactate is noted, but this phenomenon is not observed at 6 and 12 weeks of age.

Summary: The age-related changes in fasting arterial levels of energy substrates and insulin were studied at birth and/or 6 wk in eleven baboon infants. In addition, the renal contribution to glucose release in the primate infant was estimated. Arterial blood glucose levels were similar in six fasted newborns and in nine fasted 6-wk-old infants. Arterial blood lactate, alanine, pyruvate, glutamate, and glutamine levels were significantly higher (P < 0.01) in the new born animals, and β-hydroxybutyrate was significantly higher in the older animals (P < 0.001). Arterial plasma insulin levels were low in both groups. Levels of blood glucose in the inferior vena cava below the renal vein were significantly lower than levels in the aorta (P < 0.01). In contrast, levels of blood glucose in the inferior vena cava above the renal vein were significantly higher than in the aorta (P < 0.05). Computed renal vein glucose levels were higher than those in the aorta (P < 0.01). In the newborn infants, there was significant renal uptake of lactate, pyruvate, glycerol, and glutamine (P < 0.01), and release of β-hydroxybutyrate (P < 0.05). In the older animals, there was renal uptake of alanine, lactate, pyruvate, and glycerol (P 0.01). Mean cardiac output per kg body weight did not differ significantly in the newborn and 6-wk-old infants. Lactate uptake was potentially responsible for 59% of mean renal glucose output in the newborn and 76% of mean renal glucose output in the older infant. Net renal glucose release in eight 6-wk-old infants was estimated to be 3.5 ± 1.1 μM/min · kg (95% confidence limits, 0.7 < 3.5 < 6.2). Net renal glucose release in three newborn infants was 4.7, 5.4, and 19.8 μM/min · kg. There was a significant linear relationship between arterial lactate levels and renal glucose production in the older infants (P < 0.05). Extremely low arterial pH was associated with increased renal glucose release in the newborn, and high arterial pH with decreased or absent glucose release in the 6-wk-old animals. Speculation: The primate infant kidney has a capacity for gluconeogenesis which is apparently enhanced by acidosis and lactate availability. In the stressed human neonate, it is possible that renal gluconeogenesis could be a significant and uncontrolled source of new glucose, leading to hyperglycemia.

Fetal energy requirements can be met partially by protein catabolism during maternal substrate deprivation in sheep. The mechanism of this phenomenon is poorly understood. Hindlimb metabolism was studied in 7 chronically catheterized sheep pregnancies in the fed state. Catheters were placed in the fetal aorta and femoral vein in order to measure arteriovenous differences for substrate across the hindlimb. The hindlimb took up 2.9>5.8 10.8

Net cerebral uptake of energy substrates was measured by repetitive arteriovenous sampling in 9 baboon neonates in the first 3 days of life. Catheters were placed in the aorta, left ventricular outflow tract, and in the superior sagittal sinus. Cardiac output and cerebral cortex blood flow were determined by the radioactive microsphere method. Glucose (G) uptake was 9.4±2.1 μM/min/kg body weight or 11.2±2.6 μM/min/100 g cerebral cortex. Cerebral uptake of alanine was detected in 4/4 animals, and of lactate in 3/9 animals. There was no net lactate release and no net uptake or release of glycerol. Infusion of Na DL-βhydroxybutyrate (βOHB) to steady state levels of 2-6 mM total KB (acetoacetate + D-βOHB) did not alter G, lactate, or O2 uptake. However, KB uptake was correlated in a linear fashion with arterial KB to levels of 8 mM.

Resting echocardiographic abnormalities have been documented in healthy young diabetics. In order to evaluate the physiologic relevance of this finding and to determine LV responses to the stress of exercise in young diabetics, 28 normal children and 30 otherwise healthy type I diabetics had M-mode echocardiograms of the LV and aorta (Ao) at rest and immediately following maximal exercise on a supine bicycle ergometer. Adequate studies were obtained in 26 controls (aged 10.7 to 17.7 yrs) and 25 diabetics (aged 9.2 to 18.3 yrs). Duration of exercise was less for diabetics (8.5±5 vs. 7.0±5 MIN, p

1121 ECHOCARDIOGRAPHIC EVIDENCE FOR IMPAIRED MYOCARDIAL PERFORMANCE IN CHILDREN WITH TYPE I DIABETES MELLITUS

The assessment of energy expenditure (EE) in humans in a normal environment has recently become possible through measurement of the differential excretion of doubly labeled heavy water (2H218O) following oral ingestion. After 2H218O distribution, CO2 production and EE may be calculated from differential 2H and 18O enrichment in urine samples at 1 and 7 days. Further, isotope dilution at 4 hours may be used to calculate total body water and fat-free mass (FFM). We studied 10 individuals with Prader-Willi syndrome (PWS) (8-24 years) and 5 normal obese children (NO) (7-16 years). None had other chemical abnormalities, and both groups were similarly obese (PWS 157±9.7%, NO 152±11.7% ideal body weight). PWS were 47.9±2.3% fat comp. to NO 45.6±1.9% (N.S.). However, % fat linearly correlated with body mass index (kg/m2) only in PWS (r=0.93). Two normal-weight PWS had 40% and 37% body fat. EE in PWS was 2103±203 kcal vs. 3478±407 kcal, NO (p

Glucose (Glu) levels are exceedingly low in the ovine fetus and the ungulate liver has little measurable glucokinase. Nonetheless, at term, the fetal (Fet) sheep liver contains significant glycogen stores. Recent suggestions that glycogenesis in the adult may proceed through the gluconeogenic pathway led us to examine the capacity of lactate (Lac) to serve as a glycogenic precursor in the chronically catheterized Fet sheep. Six ovine fetuses were prepared with aortic (Ao) and inferior vena cava (IVC) catheters. On day 5 post surgery, after a maternal (Mat) 48 hour fast, a steady state Mat venous infusion of Glu was begun sufficient to raise Mat blood Glu from 19.41.6 mg/dl to 48.6±6.2 mg/dl. A simultaneous infusion of 300 uC (U-14C) Lac was administered to the Fet IVC over a 5 hour period to steady state. Six Fet Ao and Mat art. samples were obtained q 10 min during the last 60 min of the infusion. The fetus and ewe were then sacrificed and liver removed for determination of glycogen and 14C glycogen enrichment. Minimal radiolabel was found in Mat blood or liver glycogen. Fet blood levels were Lac 13.6±1.3 mg/dl (SA 42±9 uc/mg×103), fructose 70.2±10.4 mg/dl (SA 1.0±.2 uc/mg×103), Glu 23.2±4.3 mg/dl. No Lac-derived labelled Fet Glu could be reliably detected. Fet hepatic glycogen was 40.0±6.5 mg/g liver (SA 1.4±.5 uc/mg×103). The SA of glycogen and absent Glu labelling suggests that Lac is a precursor of Fet glycogen through the gluconeogenic path. This is teleologically important, since placental conversion of Mat Glu to Lac maintains higher levels of Lac in the Fet circulation and seems to be a means for Fet conservation of energy stores.

The contribution of renal gluconeogenesis to glucose homeostasis in the newborn has been neglected despite its well-established role in fasting glucose homeostasis. We have studied the activity of the key gluconeogenic enzyme PEPCK (forward spectro-photometric reaction) in normal and IUGR rat neonates (uterine vessel ligation) fasted for 4 hours postpartum (34°C).

Uptake of glucose and release of gluconeogenic precursors by the hindlimb of the fasting baboon infant was evaluated in 6 baboon neonates after delivery by cesarean section at term and 8 6-week-old baboon infants. Arteriovenous (AV) differences across the hindlimb were determined by measuring substrate levels in the aorta and the inferior vena cava below the level of the renal veins and above a unilaterally occluded femoral vein. There were no significant AV differences for pyruvate, beta-hydroxybutyrate, glutamate or glutamine. Production and uptake was calculated using hindlimb blood flows determined by the radioactive microsphere method. Glucose consumption by the hindlimb in the neonate was 2.4 μM/min. Uptake by the entire carcass was estimated to be 9.6 μM/min, almost twice the previously estimated cerebral cortex glucose consumption (5.6 μM/min). Estimated splanchnic and renal glucose production (15.7 μM/min) could account for all (15.2 μM/min) of estimated cerebral and carcass glucose uptake. The fasting neonate has a substantial glucose utilization rate exclusive of the requirement of the central nervous system.

29 DECREASE IN CYTOCHROME P-450 DEPENDENT 3-N-DEMETHYLATION OF CAFFEINE MEASURED BY THE CAFFEINE 13 CO 2 BREATH TEST (CET) FOLLOWING GROWTH HORMONE THERAPY IN GROWTH HORMONE (GH) DEFICIENT CHILDREN

1 ECHOCARDIOGRAPHIC ASSESSMENT OF LEFT VENTRICULAR (LV) FUNCTION IN HEALTHY ADOLESCENTS FOLLOWING MAXIMAL SUPINE EXERCISE

In order to assess the contribution of the direct [phosphorylation(P) of glucose] vs indirect [P of other precursors) pathway of glycogenesis in the fetus(fet), ten baboon fet of 140 d gestation were studied after placement of femoral artery (FA) and umbilical vein(UV) catheters. On day 4 post surgery, after a 24h fast, maternal(mat) baboons were sedated and mat plasma glucose(G) was raised to 68±3 mg/dl with 10%G to mat vein. A primed dose of 50 uc/h U-14C L and 2-3H G (n=5), or U-14C F and 3-3H G(n=5) was infused to steady state into the UV over 3h. Six FA samples were obtained 90-180 min, the fet was sacrificed under mat-fet general anesthesia, and kidney and liver removed for determination of glycogen (gly) and 14C, 3H gly enrichment. Fetal blood 14C G derived from L SA was 4.0±.5, 14C G from F SA was 11.0±1.5, 2-3H G SA was 42.0+2.5, and 3-3H G SA, was 35.0±5.(uc/mg(103). Organ gly SA was calculated as uc/mg(103): SA organ gly/SA G reflects the fraction of gly derived from label. 14C SA kidney gly/SA G was greater than 2-3H or 3-3H ratio for F and L fet(p

Regulation of gluconeogenesis has been studied in 4 baboon fetuses (135-140 d.) with catheters chronically placed in the inferior vena cava and aorta, using a single isotope-labeled precursor technique. A primed infusion of [u-14C] lactate (Lac) was administered to the fetus to steady state over 150 min. Net Lac to glucose (Glu) conversion was evaluated by comparing fetal (Fet) and Maternal (Mat) dpm and specific activity (S.A.) of Lac and Glu during Mat fed (4), fasting (4), and Glu infusion (1) (Iglu). Mat/Fet Glu dpm were 36.0±1.9% fed, 51.4±3.1% fasting (p

TRANSSPHENOIDAL REMOVAL OF PITUITARY MICROADENOMAS IN THE TREATMENT OF CHILDHOOD CUSHING'S DISEASE

In order to evaluate the physiological adaptations to feeding in low birth weight for gestation infants and the infants or gestational diabetic mothers (IGDM), blood glucose (G), plasma insulin (PI) and free fatty acid (FFA) responses to the ingestion of formula by 42 normal newborns were studied in the first three days of life. Thses measurements were made at fasting, and 30 and 60 min after feeding. Blood glucose values rose significantly over fasting at 30 min on all 3 days, and the values at 30 min on day 3 were significantly higher than those on day 1. The only significant change in the FFA values was a lower fasting value on day 3 than on day 2. Fasting PI values on day 1 were significantly higher than those on day 3. The decrease in PI values and relatively stable FFA values after formula feeding are in contrast to changes that occur with the administration of a large quantity of glucose either orally or intravenously. This differences in metabolic homeostasis was apparent not only in the normal infant but in the IGDM and low birth weight for gestation infants as well.