Naomi D Neufeld

Ontogeny of the mammalian insulin receptor: Studies of human and rat fetal liver plasma membranes

Abstract Insulin binding to human fetal plasma liver membranes was studied in preparations segregated into three pools according to length of gestation: 15–18 weeks (Pool A), 19–25 weeks (Pool B), and 26–31 weeks (Pool C). Receptor numbers, calculated by extrapolation of Scatchard plots to the X axis, increased from 25 × 1010 sites per 100 μg protein in the youngest group (Pool A) to 46 × 1010 sites per 100 μg protein in Pool B. No further increase in receptor number was seen in Pool C. The affinity constant for insulin at tracer concentrations, K e (“empty site”), was 1.53 × 108 M−1 in Pool A and was only slightly higher than K f (“filled site”). K e was higher in Pool B, 1.75 × 108 M−1, and in Pool C reached a value of 5.63 × 108 M−1. In Pool C K f was 2.3 × 108 M−1. Insulin binding of liver plasma membranes from rat fetuses aged 14, 16, 18, and 21 (term) days and adults was also studied. Maximum binding capacity tended to increase with gestational age and was 130 × 1010 sites per 100 μg protein at term, which was in excess of that found in adult rats (89–90 × 1010). In addition, K e increased from 0.75 × 108 M−1 at 14 days to 3.02 × 108 M−1 at term, a value higher than that found in pregnant and nonpregnant adults. Dissociation of insulin in the presence of high concentrations of insulin was significantly enhanced in tissues from 18-day and term fetuses and adults, but not in membranes from fetal rats aged 14 and 16 days. These data appear to indicate that site-site interactions are not present in early fetal existence. These changes in insulin binding with increased length of gestation are not ascribable to changes in relative proportions of hematopoietic and parenchymal tissue. Human fetal plasma liver membranes demonstrated elevated insulin binding with increased gestational age, but comparison of fetal and adult liver could not be done. However, newborn human infants have been shown to have a higher capacity for binding insulin to circulating monocytes than adults. Also, human fetuses apparently lack the capability to diminish monocyte receptors in the presence of hyperinsulinemia. These experiments show that an increase in insulin receptor binding capacity and affinity also occurs in the liver of the rat fetus at term as compared to the adult rat. The reasons and mechanisms underlying enhanced capacity for insulin binding by fetal and newborn members of human and rodent species are not known.

Inhibition of Surfactant Production by Insulin in Fetal Rabbit Lung Slices

Summary: Incorporation of labeled glucose and fatty acid residues into saturated phosphatidylcholine was significantly reduced in lung slices from 27.5 days of gestation fetal rabbits during 90 min incubation in the presence of 100 μU/ml insulin. When 14C-glucose was used as substrate, incorporation into both phosphatidylcholine and saturated phosphatidylcholine was reduced by insulin. This occurred despite an increase in overall glucose utilization by the lung from 11.3 ± 3.9 to 16.3 ± 5.2 nmole/g tissue in the presence of insulin (P < 0.05). A decrease in incorporation of fatty acid residues into saturated phosphatidylcholine was also observed when 14C-paImitate was used as substrate, from 102 ± 4 to 90 ± 5 nmole palmitate/g tissue (P < 0.01). In the presence of insulin, there were significant reductions of both substrates appearing in lysophosphatidylcholine, a precursor of saturated phosphatidylcholine. There was no significant change in incorporation of glucose residues into glycogen or lactate under these conditions.Speculation: Hyperinsulinemia appears to be responsible for respiratory distress syndrome (RDS) in infants of diabetic mothers (IDM) and infants of gestational diabetic mothers (IGDM). Control of the maternal diabetic state to a degree which inhibits development of fetal hyperinsulinism reduces the incidence of RDS in IDM and IGDM by removing excess quantities of the factor, insulin, that is responsible for inhibition of surfactant production.

Plasma Membrane Insulin Receptors in Fetal Rabbit Lung

Summary: Previous studies have suggested that fetal hyperinsulinemia which occurs in offspring of diabetic mothers is responsible for diminished surfactant production and respiratory distress syndrome. Recognition of specific insulin effects on fetal lung tissue prompted us to characterize insulin receptors on plasma membranes of fetal rabbit lung tissue and to investigate the effects of maternal diabetes on such receptors. Six pairs of pregnant New Zealand rabbits were studied. One of each pair received alloxan (60 mg/kg) intravenously on day 14 of pregnancy, whereas the controls received saline. Animals were sacrificed on day 28 of gestation, and for each experiment, crude plasma membranes were prepared from maternal and pooled fetal lung tissue for 125I-insulin binding studies. Plasma glucose values were elevated for both maternal diabetic (246 ± 81 versus 98.5 ± 7.1 mg/dl; P < 0.001) and fetal diabetic offspring (160 ± 69 versus 55 ± 12 mg/dl; P < 0.02) in comparison to controls. Fetal diabetic offspring had plasma insulin values significantly higher than control fetuses (84.8 ± 25 versus 23.2 ± 3.7 μU/ml), (mean ± S.D.); P < 0.05). Insulin was undetectable in diabetic mothers.Lung membranes from fetuses of diabetic animals bound significantly more insulin than did those of control fetuses. Scatchard analysis yielded curvilinear plots of bound fractions versus total amount of insulin bound suggesting the presence of more than one class of receptors or negative cooperativity. Assuming two classes of receptors, one of high affinity and low capacity and another of low affinity and high capacity, we found that fetal membranes had a five-fold increase in binding capacity of high-affinity receptors as compared to adult membranes. In spite of marked hyperinsulinemia in the offspring of the diabetic animal, the fetal lung, far from experiencing a down-regulation of insulin receptor binding, showed increased insulin binding. This finding is consistent with observations made previously on circulating monocytes of infants of diabetic mothers.Speculation: Hyperinsulinemia occurring postnatally appears to lead to down-regulation of insulin binding. Although the mechanisms of down-regulation are not clearly understood, from a teleological standpoint the phenomenon may be viewed as partially protecting the organism from the effects of hyperinsdinism because less binding sites are available for insulin to exert its effects on target tissues. In the fetus, this protective mechanism does not appear to exist in at least two tissues examined thus far, circulating monocytes of the human and lung tissue of the rabbit. The fetus with hyperinsulinemia may therefore be subject to the double jeopardy of higher concentrations of insulin and higher binding of insulin to its target tissues.

Effect of Glyburide in Type II Diabetes Mellitus: Studies of Monocyte Membrane Fluidity, Lipid Composition, and Insulin Binding

Monocyte membrane lipid composition, fluidity, and insulin binding were studied in 10 previously untreated obese type II (non-insulin-dependent) diabetic subjects before and 2-7 days, 1 mo, and 3 mo after glyburide therapy. A significant reduction in fasting blood glucose levels occurred within 1 wk after initiation of therapy in all subjects. Serum insulin levels did not change significantly. After a transient increase at 2-7 days, insulin binding and receptor number decreased to less than pretreatment levels after 3 mo of therapy despite continued improved glycemic control. Molar cholesterol-phospholipid ratios increased significantly at 2-7 days and then reverted to pretreatment levels. A significant positive correlation between insulin binding and cholesterol-phospholipid ratios was seen at all durations of treatment. Membrane microviscosity as measured by fluorescence polarization of the probe diphenylhexatriene was significantly decreased after 3 mo of therapy. The results indicate that the glycemic effect of glyburide represents enhancement of insulin action and occurs independently of membrane insulin receptors.

204 INSULIN BINDING STUDIES IN NORMAL INFANTS (NI) AND INFANTS OF DIABETIC MOTHERS (IDM)

Insulin excess has been implicated in the greater perinatal morbidity and mortality in IDM. Since insulin acts by binding to cell surface receptors, we studied the receptors on cord blood monocytes of 22 NI and 8 IDM delivered by elective C-section at 36-38 wks. gestation. IDM had more receptor sites per monocyte (105,000) than NI (38,000) and 12 normal adults (25,000) studied similarly. The higher number of receptors in IDM occurred in the face of higher concentrations of insulin in their cord blood than in NI. Monocytes from both NI and IDM showed greater affinity for insulin than those from adults, (4.63 and 4.55 vs. 2.35×108 M−1 p<0.025). In NI of similar gestational age, a significant correlation was found between birthweight and insulin binding. Insulin binding to liver plasma membranes of fetal rats increased progressively from 14 d gestation through birth. At birth the maximum binding capacity was significantly greater per 100ug protein (25ng insulin) than in adult rat membranes, (17ng insulin, p<0.025). Scatchard analysis also showed that binding affinity constants were markedly greater for term fetuses than adults (Ke=3.94×108 vs. 2.85×108 M−1). Thus, in contrast to down-regulation of receptor number reported in adult hyperinsulinemia, IDM have an increase in receptor number which may be an exaggeration of the developmental process observed in normal fetuses. Greater binding of insulin to tissue of IDM may therefore expose these infants to greater hazards from the effects of insulin.

Relationship between Parenting Practices and Outcomes in the Treatment of Childhood Obesity (CO)

Relationship between Parenting Practices and Outcomes in the Treatment of Childhood Obesity (CO)

EFFECTS OF MATERAL DIABETES ON INSULIN RECEPTOR mRNA LEVELS IN FETAL RABBIT LIVER

Maternal diabetes is associated with marked increases in insulin receptor (IR) numbers on fetal tissue plasma membranes. We examined the effects of alloxan-induced diabetes in pregnant New Zealand rabbits on IR mRNA levels in fetal rabbit liver at 31 d. gestation.Total IR increased 4 fold in offspring of diabetics. Fetal rabbit liver Poly A+ RNA was subjected to gel electrophoresis, hydridization to a radiolabelled rat IR cDNA probe and autoradiographs analyzed by densitometry. Data were expressed as a percent of maximum intensity compared to a constitutive standard,(actin mRNA). Ratios of IR to actin mRNA were also calculated for each specimen from slot blot analyses. There was no change in IR m RNA levels in fetal liver from offspring of diabetic pregancies. CONCLUSIONS: Increases in IR number in fetal tissues of diabetic pregnancies is not due to an increase in receptor mRNA levels, but possibly may be explained by either post-transcriptional or translational changes, or by differences in membrane environment (i.e., fluidity) which alters receptor exposure.

403 STIMULATION OF NEONATAL HEPATIC UDP-GLUCURONYLTRANSPERASE ACTIVITY WITH PRENATAL THYROID ANALOG THERAPY

Unconjugated hyperbilirubinemia is often observed in congenital hypbthyroidism, due to impaired activity of the bilirubin-conjugating enzyme UDP-glucuironyl transferase (UDPGT-ase). This enzyme is tighly membrane bound and is dependent on membrane physical state, which varies with thyroid status and maturation. We examined the effects of maternal treatment with the non-halogenated thyroid analog, 3,5-dimethyl, 3′- isopropyl thyronine (DIMIT, 0.5 ug/100gm/d) to rats whose fetuses had been rendered hypothyroid (HYPO-Tx) by maternal propylthiouracil. Neonatal pups were sacrificed and liver microsomes prepared lor assessment of UDPGT-ase activity (nM/mg/min). Membrane fluidity was determined by fluorescence polarizaton (FP).Neonatal HYPO-TX was associated with reduction of UDPGT-ase activity, in comparison to euthyroid controls. Prenatal treatment with DIMIT stimulated the suppressed enzyme activity in HYPO-Tx. There was an inverse correlation between membrane fluidity (FP) and enzyme activity (r=-.51, n=23, p<0.05).Conclusion: Impaired conjugation of bilirubin in congenital hypothyroidism is the direct result of reduced membrane fluidity. Both of these detects can be reversed by prenatal treatment with DIMIT, a thyroid analog which readily crosses the placenta.

1239 DETECTION OF GESTATIONAL DIABETES IN THE FIRST TRIMESTER: ABNORMALITIES IN CELL MEMBRANE STRUCTURE AND FUNCTION

Detection of gestational diabetes (GD) is currently limited to the last trimester, whereas the associated letal abnormalities occur early in pregnancy due to disturbances in maternal metabolism. He have studied 39 pregnant subjects with varying degrees of carbohydrate tolerance at intervals during pregnancy. Heparinized blood samples (10-15cc) were obtained from all subjects after an overnight fast, for studies of [125]I-insulin binding, membrane phospholipid (PL) and membrane fluidity determinations using fluorescence polarization (FP).The data suggest that a detect in insulin binding and insulin action is present in women with GD, associated with disturbances in both membrane structure and biophysical behavior. This detect, similar to that seen in obese. Type II diabetic subjects is detectable betore 12 weeks gestation. Early detection ot GD, permitting the institution of appropriate measures of metabolic control in these subjects, should result in marked reduction of perinatal morbidity.

INSULIN RECEPTOR (IR) DEVELOPMENT IN NORMAL (N) AND DIABETIC (D) PREGNANCIES: DOES INSULIN (I) UP REGULATE THE RECEPTOR?

Offspring of D pregnancies have increased concentrations of IR on plasma membranes associated with differences in membrane lipid composition and physical structure. We have previously suggested that increased [I] occurring in human and animal offspring of D pregnancies caused up-regulation of IR. Since ambient [I] is the difference between its secretion by the pancreas and its degradation, we have studied these factors in rat fetuses from both N and D pregnancies. Twenty nine pregnant rats of timed gestation received either saline (N, n=13) or streptozotocin 60 mg/kg (D, n=16) on d 7 of pregnancy, and were sacrificed on d 21. Pooled fetal serum I levels were comparable (47 ± 8 vs. 34 ± 2μU/ml), whereas blood glucose levels were markedly elevated in offspring of D, (39 ± 13 vs. 321 ± 63 mg/dl, p < 0.001). Tracer I binding was also elevated on liver plasma membranes from D (5.3 ± 1.0 vs. 2.1 ± 0.3%/100μ g protein, p <0.02) due to increased high and low affinity receptors. I content of fetal pancreatic islets, quantitated by immunoperoxidase staining, was significantly lower in D offspring. I degradation was significantly higher in fetal liver homogenates from D offspring compared to N, (34 ± 1.7 vs. 26 ± 1.2% degradated/30 min/100μg protein, p<0.001).Conclusions: Elevation in IR concentrations on fetal liver membranes in D pregnancies appears to occur independently of I presented to the liver. The data support the concept that I degradation and IR regulation occur by separate pathways. A causal relationship between fetal hyperglycemia and/or hyperlipidemia and alterations of membrane structure increasing IR exposure cannot be excluded.