John B. Susa

Chronic Hyperinsulinemia in the Fetal Rhesus Monkey: Effects on Fetal Growth and Composition

The delivery of 19 U of insulin a day for 21 days to the rhesus monkey fetus, coupled with the permeability properties of the placenta, has made it possible to produce fetal hyperinsulinemia in the presence of euglycemia. Fetal plasma insulin concentrations of 3525 μU/ml were attained with no apparent effects on the mother. In fetal macrosomia, a 34% increase in body weight was observed above the expected weight for gestational age (466 vs. 348 g). Relative organomegaly, matched for gestational age of fetal weight, was seen in the hyperinsulinemic fetuses with enlarged livers, placentas, hearts, and spleens. Liver composition in the fetuses was only slightly affected by hyperinsulinemia. Glycogen concentration was elevated, but not sufficiently, to explain the relative hepatomegaly produced. The lipid, protein, DNA, and RNA concentrations were not affected by hyperinsulinemia. Based on the similar DNA concentrations and protein/DNA ratios observed in hyperinsulinemic and control groups, the hepatomegaly appears to be the result of insulin-stimulated hyperplasia and not of hypertrophy. In the presence of normal plasma concentrations of growth substrates, insulin in the subhuman fetus has been shown to be a growth-promoting hormone that has specific growth-stimulating effects.

Temporal Response of Immunoreactive Erythropoietin to Acute Hypoxemia in Fetal Sheep

ABSTRACT. Acute hypoxemia was produced in chronically catheterized sheep fetuses to determine the response time necessary to increase plasma immunoreactive erythropoietin (Ep) concentration. Sodium nitrite (0.2 mM) was infused via a fetal vein to induce fetal hypoxemia. The resultant fetal methemoglobinemia was associated with a predictable, incremental decrease in arterial oxygen content. Twelve nitrite infusions were performed in eight fetal sheep preparations (gestational ages 115-146 days). Mean methemoglobin level increased to 33% of total Hb after 1- 2 h of NaNO2 infusion. These results were compared to those obtained in nine control studies in eight fetuses in which no change was observed for plasma Ep, arterial oxygen content, Pao2, pHa, or whole blood lactate. In the nitrite infused group, however, a significant and progressive increase in mean plasma Ep level over baseline levels was observed during the 4th and 5th h of hypoxemia (p<0.01). This change in Ep was significantly greater compared to the control group. These results, however, were confounded by the concomitant development of a lactic acidemia secondary to the fetal hypoxemia. To examine the theoretic possibility that lactic acidemia may primarily affect fetal Ep levels, an additional group of five fetuses was infused with L-lactic acid for the same time period. Although the decrements in pHa and whole blood lactate levels achieved in these fetuses were in excess of those observed during the nitrite infusions, this possibility was ruled out since no change in fetal plasma Ep levels occurred. We conclude that during the 4th h of acute fetal hypoxemia a predictable, progressive increase in plasma Ep level is observed. This response of plasma Ep to hypoxemia in late gestation fetal sheep is qualitatively similar to that observed in adult animals, thus demonstrating developmental maturity of the fetus.

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.

Effects of Hyperinsulinemia in the Primate Fetus

Nonhuman primate models of gestational diabetes have produced fetopathies most similar to those of the human infant of the mother with gestational diabetes (IGDM). Fetal hyperglycemia, hyperinsulinemia, macrosomia, selective organomegaly, intrauterine death, and placental hyperplasia are hallmarks of the fetopathy of the IGDM. The chronic infusion of insulin into the fetus of a normal pregnant rhesus monkey results in fetal hyperinsulinemia with normal to low plasma metabolic substrate concentrations. Under these conditions, fetal hyperinsulinemia is sufficient to cause fetal growth and hormone changes observed in the human IGDM. Our studies provide evidence that the soft tissue hyperplasia in the fetal macrosomia syndromes in humans and nonhuman primates in which fetal hyperinsulinemia is observed is the direct result of that chronic in utero hyperinsulinemia.

Glucose kinetics in infants of diabetic mothers.

Glucose kinetic studies were performed to define the glucose turnover rate with 78% enriched D-[U-13C] glucose by the prime constant infusion technique at less than or equal to 6 hours of age in nine infants of diabetic mothers (four insulin-dependent and five chemical diabetic patients) at term. Five normal infants were studied as control subjects. All infants received 0.9% saline intravenously during the study with the tracer. Fasting plasma glucose, insulin, and glucose13/12C ratios were measured during the steady state, and the glucose turnover rate was derived. The average plasma glucose concentration was similar during the steady state in the infants of the diabetic mothers and in the control infants, and the glucose turnover rate was not significantly different among the groups: 2.3 +/- 0.6 mg . kg-1 min-1 in infants of insulin-dependent diabetic patients; 2.4 +/- 0.4 mg . kg-1 min-1 in infants of chemical diabetic patients; and 3.2 +/- 0.3 mg . kg-1 min-1 in the control subjects. Good control of maternal diabetes evidenced by the normal maternal hemoglobin A1c and plasma glucose concentration at delivery and cord plasma glucose concentration resulted in glucose kinetic values in the infants of diabetic mothers that were indistinguishable from those of control subjects. The data further support the importance of good control of the diabetic state in the pregnant woman to minimize or prevent neonatal hypoglycemia.

Endogenous glucose production during constant glucose infusion in the newborn lamb.

Summary: Prompt diminution of endogenous hepatic glucose production is characteristic of the mature (adult) response to exogenous glucose infusion. We have tested the validity of this hypothesis in the neonatal period in 26 unanesthetized mixed breed term lambs and for comparison in eight 4− to 5-month-old mixed breed sheep. After a 7-hr fast, basal plasma glucose, insulin, and glucagon concentrations were determined following which the term lambs received either no glucose or 5.7, 11.7, or 21.7 mg glucose/kg/min over a period of 6 hr, and the 5-month-old sheep received either none or 5.7 mg glucose/kg/min. Glucose turnover was determined by the prime-constant infusion technique of Steele using 3H6 radiolabeled glucose during a 50-min turnover period which followed the 6-hr infusion of 0.45% saline or varying doses of glucose following the onset of fasting by 14 hr. Both newborn and adult animals maintained a constant plasma glucose concentration and glucose specific activity during the turnover period. Endogenous glucose production persisted in the term lamb until the exogenous glucose infusion reached the rate of 21.7 mg/kg/min. In contrast, the adult lambs reduced their endogenous glucose production with an exogenous glucose infusion rate of 5.7 mg/kg/min. At the time the endogenous glucose production rates were significantly reduced, the plasma insulin level in the newborn lamb was 5-fold greater than that of the adult sheep.Under steady state conditions of plasma glucose concentration and glucose specific activity, our data suggest that there is imprecise control of endogenous glucose production in the newborn lamb in contrast to the older sheep of 4–5 months of age. The absence of precise control may be due to decreased hepatic sensitivity for insulin.

Preserved Fetal Plasma Amino Acid Concentrations in the Presence of Maternal Hypoaminoacidemia

ABSTRACT: The effects on the conceptus of persistently decreased maternal plasma amino acid concentrations were studied in pregnant rats by the infusion of glucagon (0.21 mg/day) to the mother from day 14 to 20 of gestation with a subcutaneous, osmotically driven minipump. Controls received diluent. The experimental animals either had normal caloric intake and weight gain, or diminished caloric intake with no weight gain. Both experimental groups exhibited a decrease in plasma total amino acid concentration of approximately 50%. Maternal plasma glucose and insulin concentrations were unaffected except for slight decreases in the low weight gain group. At cesarean section on day 20, fetal weight was unaffected in the normal weight gain group, while the low weight gain animals exhibited intrauterine growth retardation. Fetal plasma glucose and insulin concentrations were unaffected. Despite the marked decrease in maternal plasma total amino acid concentration, fetal plasma total amino acid concentration was unaffected. Individual plasma amino acid concentrations in the normal weight gain mothers and fetuses revealed a spectrum of changes. Some maternal amino acids were decreased by more than 60% (α-aminobutyric acid, asparagine, threonine, glutamine, alanine) while others were unaffected (tyrosine, tryptophan, phenylalanine, histidine). In general, amino acids that were decreased in the mother exhibited no change or a lesser decrease in fetal plasma concentration, while those that were unaffected in the mothers showed increased fetal concentrations. Fetuses from the low weight gain mothers had plasma amino acid profiles that were similar to those of the normal weight gain mothers. We speculate that the preserved fetal plasma amino acid concentrations are due to enhanced placental transport of amino acid secondary to maternal substrate decrease.

Neonatal hypoglycemia in the rhesus monkey: Effect on development and behavior☆

Abstract Neonatal hypoglycemia has been implicated as a cause of brain damage that may lead to cognitive, sensory, psychomotor, or behavioral deficits in children. Because nonhuman primates can serve as useful models of many aspects of human behavior, we have produced neonatal hypoglycemia in newborn rhesus monkeys to identify the specific nature of the deficits produced. Neonatal hypoglycemia was produced by the continuous subcutaneous delivery (begun in utero) of insulin for up to the first 4 hours after birth. A cognitive and behavioral testing program was begun under blind conditions when each animal reached 8 months of age. None of the measures of cognitive abilities or behavior distinguished experimental animals with 6.5 hours of hypoglycemia from controls. Ten hours of hypoglycemia resulted in motivational and adaptability problems that made it impossible for some animals to learn even the simplest tasks, but, when provided with additional attention and adequate motivation, these experimental animals performed as well as controls in tests designed to measure cognitive ability. We conclude that neonatal hypoglycemia of 10 hours duration results in adaptive difficulties in rhesus monkeys but, if special attention is devoted to these animals, there are no enduring cognitive or behavioral deficits.

Suppression of gluconeogenesis and endogenous glucose production by exogenous insulin administration in the newborn lamb.

Summary: Persistent endogenous hepatic glucose production is characteristic of the newborn lamb in response to exogenous glucose infusion. The hypothesis that decreased hepatic sensitivity for insulin may be a major mechanism accounting for the imprecision in glucose homeostasis was tested in 28 unanesthetized, mixed breed, term lambs. Either 0, 5.7, 6.2, or 12.5 mg glucose/kg/min was infused for a period of 2 hr after which simultaneous infusion of 0, 1.25, or 6.25 mU porcine insulin/kg/min was administered for 4 hr to produce a euglycemic, hyperinsulinemic steady state. Gluconeogenesis from lactate was measured by determining the ratio of 14C to 3H in plasma glucose during the simultaneous infusion of 14Cu-lactate and 3H6-glucose. Endogenous hepatic glucose production was determined by the prime-constant infusion technique of Steele using 3H6 radio-labeled glucose during a 50-min turnover period after the infusions. All animals maintained a constant plasma glucose concentration and glucose specific activity during the turnover period. Gluconeogenesis was significantly suppressed when insulin levels were in the 49–61 μU/ml range although endogenous glucose production was not affected. When plasma insulin concentrations were above 200 μU/ml, both gluconeogenesis and endogenous glucose production were significantly suppressed. These changes were observed irrespective of the steady state blood glucose concentration. Plasma glucagon concentrations were the same in all experimental groups and were not affected by glucose and insulin infusion nor were they responsive to differences in plasma glucose or insulin concentrations. Our data indicates that insulin may be the major hormone for the control of glucose homeostasis in the neonatal lamb and that there is a quantitative difference in the response of the various glucose homeostatic mechanisms to insulin.Speculation: Regardless of the plasma glucose concentration and the resultant counter-regulatory responses that may occur during prolonged continuous glucose infusion, endogenous glucose production in the neonatal lamb may be primarily controlled by insulin.

Phosphoenolpyruvate Carboxykinase in Experimental Intrauterine Growth Retardation in Rats

Summary: This study examines the role of impaired gluconeogenesis in the pathogenesis of neonatal hypoglycemia in intrauterine growth retardation (IUGR). IUGR was produced experimentally in eight pregnant rats by ligation of uterine arteries at the 17th day of gestation. Delivery occurred spontaneously at term. Sham operations were performed in five pregnant rats at the same gestational age and the fetuses delivered at term served as controls. The body weights of newborn rats with IUGR were significantly lower than the controls (5.32 ± 0.12 vs. 6.22 ± 0.06 g, mean ± SE, P < 0.001). The fetal liver weights were also significantly smaller in IUGR than in the control animals (0.224 ± 0.14 vs. 0.340 ± 0.12 g, P < 0.001). The activity of phosphoenolpyruvate carboxykinase (PEPCK) (the rate-limiting enzyme of gluconeogenesis) in liver cytosols was significantly lower in rats with IUGR (0.06 ± 0.01 vs. 0.11 ± 0.02 μM phosphoenolpyruvate/g liver/min when compared with controls (P < 0.05). A direct relationship between this enzyme and the brith weight was observed, suggesting a close relationship between intrauterine nutrition and the status of gluconeogenesis. The blood glucose level was also lower in growth-retarded fetuses (36.6 ± 4.7 vs. 69.6 ± 4.3 mg/dl, p < 0.001) when compared with controls. The data suggest that gluconeogenesis is impaired in IUGR and is partly responsible for the increase in the incidence of neonatal hypoglycemia in this group of subjects.Speculation: Neonatal hypoglycemia in IUGR is probably caused by a combination of rapid depletion of glycogen stores and impaired gluconeogenesis, the latter indicated by a decreased liver PEPCK activity. This may be related to a diminished enzyme synthesis on the basis of significant energy and oxygen deprivation.