Chronic hyperinsulinemia in the fetal rhesus monkey. Effects of physiologic hyperinsulinemia on fetal growth and composition.

One of the hallmarks of the hyperglycemic-hyperinsulinemic infant of the diabetic mother (IDM) is macrosomia and selective organomegaly. Primary hyperinsulinemia, with insulin levels similar to those observed in human IDMs at delivery, was produced in the fetal rhesus monkey during the last third of gestation. The effects of this physiologically relevant hyperinsulinemia, in the absence of hyperglycemia, on fetal growth were studied. Fetal macrosomia, with a 23% increase in total body weight, was observed in physiologically hyperinsulinemic fetuses. A similar 27% increase in weight was produced by fetal insulin levels that were 10 times higher. A logarithmic correlation was observed between fetal birth weight ratio and fetal plasma insulin concentration. In contrast to this increase in weight, skeletal growth, as measured by crown-heel length and head circumference, was not affected by hyperinsulinemia. Only cardiomegaly was found in the low-dose hyperinsulinemic fetuses, whereas cardiomegaly, hepatomegaly, and splenomegaly were produced by hyperinsulinemia in which insulin levels were in the highest range. Compositional analysis of heart and skeletal muscle indicated no differences in the protein, RNA and DNA concentration, or in the protein-to-DNA ratio in hyperinsulinemic fetuses. We interpret these data as indicating that fetal insulin plays the predominant role in controlling the normal, as well as the augmented, fetal weight characteristic of the human infant of the diabetic mother.

Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.

Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.

Feminizing Sertoli cell tumors in boys with Peutz-Jeghers syndrome.

We report the pathology findings in two cases of multicentric Sertoli cell testicular tumors in two young boys with probable Peutz-Jeghers syndrome. Four cases of such tumors occurring in boys with Peutz-Jeghers syndrome were previously reported. Each of the two boys reported in this paper had prominent gynecomastia, rapid growth, and advanced bone age. Serum levels of estradiol were markedly elevated. Anti-müllerian hormone was measured in the serum of one of the boys and was in the normal range for age. Bilateral orchiectomy was performed in each case because the neoplastic growth would most likely result in sterility, and curtailment of height potential was threatened from continued elevation of estradiol levels. Microscopically, greatly enlarged seminiferous tubules packed with ovoid Sertoli-like cells were present. Prominent eosinophilic basement membrane surrounded the tubules and intersected between the cells, forming hyalinized ovoid globules and microcalcifications. Ultrastructure revealed lamination of basement membranes surrounding adjacent cells, ovoid cells with abundant cytoplasm, and limited smooth endoplasmic reticulum. Studies of testicular tumor tissue from both cases revealed increased transcription of the aromatase cytochrome P450 gene using promoter II, the promoter directing aromatase expression in the normal ovary and testis. The levels of transcripts were comparable to corpus luteum, thus resulting in increased estrogen synthesis. Transcripts specific for placental-type aromatase promoters (I.1 and I.2) were not detected in significant levels in these tumors.

Anti-Müllerian hormone is a specific marker of sertoli- and granulosa-cell origin in gonadal tumors.

Sex cord stromal tumors are gonadal neoplasms containing Sertoli, granulosa, Leydig, or thecal cells, which originate from cells derived from either the sex cords (Sertoli and granulosa cell tumors) or the specific mesenchymal stroma (Leydig and thecal cell tumors) of the embryonic gonad. Only granulosa and Sertoli cells produce anti-Müllerian hormone (AMH). Our purpose was to investigate whether AMH can be used as a specific marker of human granulosa or Sertoli cell origin in gonadal tumors, to distinguish them from other primary or metastatic neoplasms, using immunohistochemistry. We studied 7 juvenile and 6 adult-type granulosa cell tumors of ovarian localization and 3 extraovarian metastases, 20 other ovarian tumors, 6 testicular Sertoli cell tumors, 2 gonadoblastomas, and 13 extragonadal tumors. Granulosa cell tumors, both juvenile- and adult-type of either ovarian or metastatic localization, showed an heterogeneous pattern of AMH immunoreactivity: Areas containing intensely or weakly AMH-positive cells were intermingled with AMH-negative areas. Although in most cases AMH-positive areas represented a minor proportion of tumor cells, we found a positive reaction in all the cases examined. In testes, although normal prepubertal Sertoli cells were intensely positive, testicular Sertoli cell tumors showed large areas of negative reaction, with few positive cells scattered throughout the tumor. AMH was also reactive in most of the cells of sex-cord origin in gonadoblastomas. No AMH immunoreaction was observed in other gonadal and extragonadal tumors. We conclude that AMH expression is conserved in only a small proportion of tumor cells of granulosa or Sertoli cell origin; however, a positive reaction in a few cells helps to distinguish between granulosa or Sertoli cell tumors or gonadoblastomas and other gonadal tumors of different origin.

Tissue glucose transport and glucose transporters in suckling rats with endotoxic shock.

Hypoglycemia occurs without hyperinsulinemia in suckling rats with endotoxic shock. However, tissue glucose uptake during endotoxic shock is not well known in the newborn. GLUT1 is insulin insensitive and is the predominant glucose transporter in 10 day old rats. In the adult with endotoxic shock, noninsulin-mediated glucose uptake and GLUT1 gene expression increase. Therefore, we hypothesized that tissue glucose uptake and GLUT1 mRNA abundance increased in 10 day old rats with endotoxic shock. The present study showed that whole body glucose disposal increased 3 h after a Salmonella enteritidis lipopolysaccharide injection (LD90 at 72 h). Plasma insulin concentration was not altered. Tissue glucose uptake increased in liver (2.4-fold) and fat (2.6-fold). However, changes of GLUT1 protein concentration were not detected in liver. GLUT1 mRNA abundance increased in liver (9-fold) and fat (4-fold). GLUT1 mRNA abundance but not glucose uptake increased in muscle. Neither glucose uptake or GLUT1 mRNA abundance was altered in brain. The mRNA abundance of tissue-specific glucose transporters (GLUT2 and GLUT4) was not altered. Thus, tissue glucose uptake and GLUT1 mRNA abundance increased without hyperinsulinemia during endotoxic shock in 10 day old rats.

Perinatal nutrition enriched with omega-3 polyunsaturated fatty acids attenuates endotoxic shock in newborn rats.

Sepsis and septic shock continue to have a high mortality and morbidity in the newborn. Eicosanoids are important mediators in Gram negative septic shock. Omega-3 polyunsaturated fatty acids (omega-3) decrease production of biologically active 2-series eicosanoids. Therefore, we hypothesized that omega-3-enriched diet could decrease 2-series eicosanoids and attenuate endotoxic shock in newborn rats. Sprague-Dawley rat dams were fed with either omega-3 polyunsaturated fatty acid-enriched diet (omega-3 PURA) or omega-6 polyunsaturated fatty acid enriched diet (omega-6PUFA; controls) from the 16th day of gestation until 10 days after parturition. In 10 day old rats, shock was induced by an intraperitoneal injection of Salmonella enteritidis lipopolysaccharide. The omega-3PUFA decreased the mortality of endotoxic shock. In omega-6PUFA, lipopolysaccharide induced hyperglycemia at 2 h and hypoglycemia thereafter without an elevation in plasma insulin concentration, omega-3PUFA attenuated the hyperglycemia and hypoglycemia. omega-3PUFA attenuated the decrease of liver phosphoenolpyruvate ca-boxykinase mRNA abundance, suggesting preserved gluconeogenesis. Therefore, perinatal feeding with omega-3PUFA was beneficial in attenuating glucose dyshomeostasis in newborn rats with endotoxic shock and may be a novel approach to the prevention of endotoxic shock in the newborn.

Plasma endotoxin concentration after an intraperitoneal injection of endotoxin in fed and fasted suckling rats.

In the adult host response to endotoxin (lipopolysaccharide (LPS)) is dose-related. An intraperitoneal injection is commonly used for LPS administration in small animals. However, plasma endotoxin concentration following an intraperitoneal bolus injection of LPS is not well known. This study was performed to evaluate plasma endotoxin concentration following a bolus intraperitoneal injection of LPS in both fed and 24 h fasted 10 day old rats. Plasma endotoxin concentration increased in a dose-dependent manner after LPS injection (.03 or .1 mg/kg Salmonella enteritidis LPS) in both fed and fasted rats. Plasma endotoxin concentrations were higher (p < .05) in fed than fasted rats. A high dose of LPS (.1 mg/kg) induced 95 and 40% mortality in fed and fasted rats, respectively. A low dose of LPS (.03 mg/kg) induced 26.7% mortality in fed rats but no mortality in fasted rats. The hematocrit was significantly lower in fed than fasted rats. Plasma endotoxin inactivation was similar in fed and fasted rats. Host response appears to be related to plasma endotoxin concentration.

Decreased gluconeogenesis and increased glucose disposal without hyperinsulinemia in 10-day-old rats with endotoxic shock.

Glucose dyshomeostasis is a common and life-threatening sign of endotoxic shock in the newborn. In this study, liver gluconeogenesis was evaluated in 10-day-old rats with endotoxic shock using the isolated perfused liver. Phosphoenolpyruvate carboxykinase (PEPCK) activity and PEPCK mRNA abundance were measured to confirm altered gluconeogenesis. Glucose disposal was also evaluated by a glucose tolerance test. Twenty-four-hour-fasted rats were studied to enhance gluconeogenesis and decrease glucose disposal. Rats received an intraperitoneal (IP) injection as follows: group 1 (fed-saline), 0.2 mL saline in fed rats; group 2 (fed-LPS), 0.1 mg/kg Salmonella enteritidis lipopolysaccharide (LPS) in fed rats; group 3 (fasted-saline), 0.2 mL saline in fasted rats; and group 4 (fasted-LPS), 0.1 mg/kg LPS in fasted rats. Isolated liver perfusion, determination of liver PEPCK activity and liver PEPCK mRNA abundance, and a glucose tolerance test were performed at 4 hours in fed rats and at 6 hours in fasted rats. LPS induced hypoglycemia (1.62 +/- 0.33 mmol/L, P < .05) at 6 hours in group 2 (fed-LPS), but not in group 4 (fasted-LPS). Hyperinsulinemia was not observed in either group 2 (fed-LPS) or group 4 (fasted-LPS). In group 2 (fed-LPS), liver gluconeogenesis decreased (3.0 +/- 0.3 mg/g liver, P < .01). PEPCK activity decreased from 0.65 +/- 0.07 (group 1) to 0.23 +/- 0.02 U (P < .01). PEPCK mRNA abundance also decreased from 100% +/- 10% to 40% +/- 10%. The glucose disappearance rate (t1/2) increased (P < .05) in group 2 (fed-LPS) and group 4 (fasted-LPS).(ABSTRACT TRUNCATED AT 250 WORDS)

Glycohemoglobin in postpartum women.

As women giving birth to large for gestational age (LGA) infants are at risk for glucose intolerance during pregnancy, fasting plasma glucose (FPG) and glycohemoglobin levels (Hb AIc) were studied in the immediate postpartum period (less than 10 days). These laboratory tests, in addition to infant birth weights and perinatal histories in a group of 146 women whose infants were above the 95th percentile for gestational age, were compared with those of a group of women whose infants were appropriate for gestational age (AGA: above the 25th percentile but below the mothers 75th percentile) as well as with those of a group of control mothers without diabetic risk factors. Mean Hb AIc and FPG were elevated in the mothers of the LGA infants. When either the LGA group alone or all 3 groups together were analyzed by linear regression, significant relationships were observed for maternal Hb AIc versus FPG (P less than .001); maternal Hb AIc versus birth weight corrected for gestational age (P less than .001); and maternal FPG versus birth weight corrected for gestational age (P less than .001). In addition, infant weight correlated with maternal prepregnancy weight (r = .36), maternal weight gain in pregnancy (r = .23), and maternal height (r = .17), but not with any of the paternal anthropometric features studied. Twenty-six women with LGA infants underwent postpartum oral glucose tolerance tests before discharge. Four had abnormal results and all had Hb AIc values above SD of the nonrisk control values (5.8% total Hb). Of the 16 mothers of LGA infants with FPG levels greater than 85 mg/dl (above SD of the nonrisk control values), 7 (44%) also had Hb AIc levels of more than 5.8%. Moreover, on retrospective analysis, the LGA infants manifested increased perinatal morbidity (P less than .05) compared to the combined control groups.

Endotoxin in newborn septic shock: significance, metabolic, and cardiovascular changes.

The role of endotoxin in newborn septic shock is reviewed. Metabolic and cardiovascular changes, as known to us, are described with special emphasis that the newborn is not a "small adult." Developmental or maturational changes are hypothesized to be the major cause of these differences between age groups. Finally, it is our belief that newborn septic shock is an important topic for further investigation.

Testing for thyroid function recovery in children and adolescents with Hashimoto thyroiditis.

Thyroid function in children and adolescents with primary hypothyroidism owing to Hashimoto thyroiditis was studied to evaluate criteria for the discontinuation of thyroxine therapy. A cohort of 29 children and adolescents was prospectively studied for one year. A thyrotropin-releasing hormone stimulation test with measurements of basal and stimulated thyrotropin and thyroxine was performed at the beginning of the study and 6 and 12 months later. In 59 percent of patients, persistent biochemical evidence of hypothyroidism was observed. Interindividual variations and variations among measurements in individual patients were much greater than those reported for healthy individuals. In one patient, all measured values were consistently normal, and the therapy was successfully discontinued. No single measurement or test could predict the natural course of disease. In summary, children and adolescents with hypothyroidism owing to Hashimoto thyroiditis can not have discontinued replacement thyroxine therapy on the basis of any single evaluation of thyroid function, as proposed for adults. If the decision to discontinue the therapy in this age group is made on the basis of previous follow-up, a substantial possibility of relapse remains and continuous follow up is necessary.

Concurrent clinical and metabolic derangements in the newborn rat: a late phase sepsis model.

Gram negative sepsis is a leading cause of human newborn morbidity and mortality. The clinical signs and glucose and lactate concentration during the late phase of newborn rat endotoxicosis has not been well characterized. In order to define a late phase model of sepsis, simultaneous clinical signs (loss of response to pain [RP] and/or righting reflex [RR]), and metabolic (glucose, lactate) concentrations were studied in the 10 day old Sprague-Dawley rat. The rats were fasted for four hours and then injected with either saline (control) or 0.1 mg per kg Salmonella enteritidis (LD90 @ 24) endotoxin intraperitoneally. Rats were then examined every 15 minutes beginning two hours post injection for the presence or absence of loss of RR and/or loss of RP. Central blood samples were collected for determination of glucose and lactate at the moment of loss of the predetermined clinical parameter. Metabolic parameters were also determined on saline treated controls time matched to the experimental groups. Rats were grouped as follows: GrI, (n = 10), saline three hours post injection; GrIII, (n = 10), saline five hours post injection; GrII, (n = 45), endotoxin at the moment of loss of RR; GrVI, (n = 14), endotoxin at 8.5 hrs post injection (termination of experiment, all with loss of RR); GrIV, (n = 11) endotoxin at loss of RP; and GrV, (n = 10) saline at 8.5 hr post injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Cord blood thyrotropin screening for primary hypothyroidism on Caribbean island: a rapid communication.

Screening programs using determinations of serum thyroxine have demonstrated that congenital hypothyroidism occurs in one in 4,000 live births in North America. More than 90 percent of affected infants have primary hypothyroidism with elevated plasma thyrotropin (TSH) levels. Since the feasibility of newborn screening and incidence of congenital hypothyroidism in other less well developed areas of the world is not well defined, a study was undertaken of neonatal primary hypothyroid screening infants born on the Caribbean island of St. Lucia in the Lesser Antilles. Three hundred thirteen cord blood samples were collected on filter paper and transported 3,000 miles to Loyola University of Chicago, Stritch School of Medicine (LUMC). From LUMC, the samples were transported to the Illinois State Metabolic Screening Laboratory for determination of TSH by radioimmunoassay (RIA). In this group of newborns, the mean TSH level in cord blood was 10.23 +/- 0.29 microIU per ml (SEM). It is concluded that screening programs for neonatal primary hypothyroidism can be performed using reference laboratories far removed from the population under observation.

Cord blood thyrotropin screening for congenital hypothyroidism. Three years' experience on the Island of Saint Lucia.

Cord blood thyrotropin (TSH) screening for congenital primary hypothyroidism has been in effect on the island of St. Lucia for the past three years. Umbilical cord blood samples are obtained on Guthrie filter paper and then transported 3,000 miles to Loyola University of Chicago and delivered to the Illinois State Metabolic Screening Laboratory. There TSH is measured by radioimmunoassay (RIA). After three years, 1,789 newborns have been screened, and the mean value is 6.23 +/- 0.13 microIU per ml. This mean value is less than previously reported by us in 1986 (10.23 +/- 0.29 microIU per ml).13 It is concluded that this screening service continues to be possible far removed from the population under observation. No case of primary hypothyroidism has been detected. Our decreased mean TSH value is due to the new method currently used by the Illinois State Metabolic Screening Laboratory. Congenital hypothyroidism will not be missed provided internal controls are established and rigidly observed.

Fast hemoglobins and red blood cell metabolites in citrate phosphate dextrose adenine stored blood.

Blood was drawn from 10 fasted, healthy volunteers and stored under standard blood bank conditions in citrate-phosphate-dextrose-adenine (CPDA-1). Blood was sampled before storage (Day 0) and on Days 5, 12, 19, 26, and 35. Laboratory testing for glucose, HbAla + b, HbAlc, pyruvic acid, lactic acid, adenosine triphosphate (ATP), 2, 3-diphosphoglycerate (2,3DPG), plasma free hemoglobin (Hb) and pH (blood gases) were performed. In addition, P50 was also serially measured in two of the individuals and in their stored blood. Significant elevations of HbAla + b and HbAlc (fast hemoglobins) were found on Days 12 and 19 of storage (p less than 0.05). These elevations of fast hemoglobins are due to hypoxia, acidosis, and hyperglycemia. Following the initial elevation of the fast hemoglobins (Hbs), there was a decline in their concentration, from Day 12, which could partly be explained by cell death.

Cepharanthine (biscoclaurine alkaloid) treatment in endotoxic shock of suckling rats.

Gram-negative sepsis/septic shock causes significant mortality in newborns. However, there has been no established method for newborn endotoxic shock treatment. Prostaglandins play a role in endotoxic shock. Cepharanthine is a biscoclaurine alkaloid that primarily inhibits phospholipase A2. Therefore, the effects of cepharanthine have been studied on endotoxic shock in newborn rats. Cepharanthine decreased the 24 h mortality of endotoxic shock in a dose-related manner. At the dose of 0.2 mg kg-1 it effectively reduced the mortality from 90 to 21% in newborn rats. It also induced hyperglycaemia in control rats and blunted the hypoglycaemia of endotoxic shock. Cepharanthine did not suppress body weight gain nor did it delay death as seen with glucocorticoid treatment. We conclude that cepharanthine is beneficial in the treatment of newborn endotoxic shock.

Accidental carbon monoxide poisoning.

In this case report of an accidental automobile carbon monoxide poisoning, we identify the following risk factors: freezing temperature, young passenger age, location in the rear of the auto, smaller patient mass, and auto disrepair. The pathogenesis of carbon monoxide poisoning is reviewed. Emergency treatment and suggested criteria for hyperbaric oxygen use in pediatric patients are discussed.

Endotoxemia following enteral refeeding in children.

Plasma endotoxin-like activity, tumor necrosis factor alpha (TNFalpha) concentrations, core body temperature, and liver functions were measured before and after enteral feeding in children who had been deprived of enteral feeding for 5 days because of their illness. Transient endotoxemia and elevations in plasma TNFalpha concentrations occurred. Core body temperature, aspartate aminotransferase, alamine aminotransferase, and bilirubin concentrations were normal in patients who had elevated plasma endotoxin-like activity. Transient endotoxemia following enteral feeding may be due to the translocation from the gastrointestinal (GI) tract as a result of increased mesenteric circulation and peristalsis. No clinical consequences were noted despite transient endotoxemia. The transient endotoxemia is not due to the immature GI tract; instead, it results from enteral feeding following the deprivation of enteral feeds.

Glycosuria and maternal licking in rats.

Maternal anogenital licking (MAGL) has been studied to understand the mechanism of maternal behaviour. The present study showed that rats had glycosuria at the concentration of 18-20 mg/dl and glucose was the preference of postpartum rats. MAGL increased on suckling rats separated for 24 h. However, wiping anogenital region attenuated the increase of MAGL. Therefore, glucose preference of postpartum rats may be involved in MAGL.