Brain fibronectin expression in prenatally irradiated mice.

Activation of gene transcription by radiation has been recently demonstrated in vitro. However, little is known on the specificity of these alterations on gene transcription. Prenatal irradiation is a known teratogen that affects the developing mammalian central nervous system (CNS). Altered neuronal migration has been suggested as a mechanism for abnormal development of prenatally irradiated brains. Fibronectin (FN), an extracellular glycoprotein, is essential for neural crest cell migration and neural cell growth. In addition, elevated levels of FN have been found in the extracellular matrix of irradiated lung. To test whether brain FN is affected by radiation, either FN level in insoluble matrix fraction or expression of FN mRNA was examined pre- and postnatally after irradiation. Mice (CD1), at 13 d of gestation (DG), served either as controls or were irradiated with gamma rays at 0.5 or 1 Gy. Control and irradiated animals were killed either at 13 DG, 14 DG, 17 DG, or 5, 6, or 14 d postnatal. Brain and liver were collected from offspring and analyzed for either total FN protein levels or relative mRNAs for FN and tubulin. Results of prenatal irradiation on reduction of postnatal brain weight relative to whole body weight and morphological reduction in cerebral cortex regions of postnatal brains are comparable to that reported by others. Insoluble matrix fraction (IMF) per gram of brain, liver, lung, and heart weight was not significantly different either between control and irradiated groups or between postnatal stages, suggesting that radiation did not affect the IMF. However, total amounts of FN in brain IMF at 17 DG were significantly different (p < .02) between normal (1.66 +/- 0.80 micrograms) and irradiated brains (0.58 +/- 0.22 microgram). FN mRNA was detectable at 13, 14, and 17 DG, but was not detectable at 6 and 14 d postnatal, indicating that FN mRNA is developmentally regulated. After 0.5 Gy of irradiation, expression of FN mRNA was reduced to 36% +/- 22% (1 h), 52% +/- 10% (1 d), and 76% +/- 10% (4 d) of the control level. After 1 Gy of irradiation, relative FN mRNA was 62% +/- 28% (1 h) and 75% +/- 3% (4 days) to the control level, respectively. This reduction was comparable to that reported by others for the cytoskeletal protein beta-actin. In contrast, mRNA for tubulin, another cytoskeletal protein, increased at 1 h after irradiation but then approached normal postnatally. The longer lasting alteration of FN may be more directly related to neural development, particularly if the reduction in FN is nonuniform.

Effects of glucose and insulin on fetal glucose oxidation and oxygen consumption.

Glucose and insulin clamp experiments were performed in late-gestation fetal lambs to quantify the separate and combined effects of physiological concentrations of fetal glucose (G; 7.3-62.6 mg/dl) and insulin (I; 2-119 uU/ml) on fetal glucose metabolism and O2 consumption. Fetal glucose utilization rate (GUR) varied from 2.82 to 15.12 mg.min-1.kg-1. Fetal CO2 production from fetal glucose carbon oxidation (CO2Pr) varied from 32 to 234 mumol.min-1.kg-1 and was directly related to G and I [CO2Pr = -0.00868 + 0.00578 (G) + 0.000901 (I) - 0.0000619 (G)2, r = 0.88] and to GUR (CO2Pr = 0.0159 GUR - 0.0130, r = 0.89). CO2Pr accounted for 54.7% of the mean GUR and for 35.9% of the mean umbilical O2 uptake (UO2U), ranging from 26.0% in the control studies to 36.5% in hyperinsulinemic-euglycemic studies and to 45.1% in hyperinsulinemic-hyperglycemic studies. UO2U varied from 0.200 to I [UO2U = 0.303 + [0.000813 (G)] + [0.0000461 (I)], r = 0.89] and to GUR (UO2U = 0.0098 GUR + 0.275, r = 0.91). These results define independent (additive) effects of G and I on glucose oxidation in the late gestation fetal lamb and demonstrate the necessity for considering the levels of both G and I when studying these aspects of fetal metabolism.

Effect of maternal glucose concentration on uteroplacental glucose consumption and transfer in pregnant sheep.

The present study was designed to measure the relationships between maternal arterial glucose concentration [( GI]A) and fetal arterial glucose concentration [( GI]a), uteroplacental glucose consumption (UPGC), and the rate of uteroplacental glucose transfer to the fetus (UPGT) in pregnant sheep in late gestation. [GI]A was controlled by a glucose clamp technique and the glucose flux rates of the uteroplacenta were quantified by the Fick principle. [GI]A varied from 1.81 to 154.7 mg/dl; [GI]a was directly related to [GI]A: [GI]a = 0.374 [GI]A + 1.81, r = 0.873, P less than 0.001. Fetal arterial blood oxygen content decreased with [GI]A (P less than 0.05) and fetal arterial blood lactate concentration increased with [GI]A (P less than 0.001). There was no significant effect of [GI]A on the rates of uteroplacental lactate production, uteroplacental oxygen consumption, fetal oxygen consumption, or uterine or umbilical blood flow. Both UPGC and UPGT were directly related to [GI]A: UPGC = -2.221 x 10(-3) chi 2 + 0.646 x -6.016, r = 0.80; UPGT = -1.208 x 10(-3) chi 2 + 0.405 x -2.416, r = 0.90. UPGC and UPGT were approximately parallel over the range of [GI]A studied (UPGC = 1.19 UPGT + 3.79, r = 0.764). These results demonstrate the importance of UPGC to maternal-fetal glucose homeostasis and indicate that factors regulating uteroplacental glucose consumption and transfer to the fetus become limiting at comparable levels of [GI]A and [GI]a. The estimated kinetic constants for UPGC represent the metabolism of glucose by the uteroplacental tissues, but the estimated kinetic constants for UPGT represent the metabolism of glucose by the fetus as well as the transfer of glucose by the uteroplacenta to the fetus.

The effects of streptozotocin on rates of glucose utilization, oxidation, and production in the sheep fetus.

Streptozotocin was injected into chronically catheterized, late gestation fetal sheep to produce hypoinsulinemia and to investigate the effects of hypoinsulinemia on the rates of utilization and production of glucose. Each fetus received two IV doses of streptozotocin (100 mg/kg estimated fetal weight per dose). Experiments were conducted before and five to six days after giving the streptozotocin. Experiments consisted of direct measurement of fetal glucose utilization rate (using [U-14C]glucose tracer) and umbilical glucose uptake rate (Fick principle) during basal and glucose infusion periods. Fetal endogenous production rate was calculated as the difference between rates of fetal glucose utilization and umbilical glucose uptake. Following streptozotocin injections the rate of fetal glucose utilization was reduced (5.50 +/- 0.34 to 4.13 +/- 0.32 mg/kg/min) as was the rate of fetal CO2 production from fetal glucose carbon oxidation (91.7 +/- 5.3 to 71.7 +/- 6.0 mumol/kg/min) and the rate of fetal glucose oxidation (2.75 +/- 0.16 to 2.15 +/- 0.18 mg/kg/min). At the same time the rate of fetal endogenous glucose production was enhanced (0.31 +/- 0.18 to 2.06 +/- 0.28 mg/kg/min). These changes in glucose metabolism were accompanied by hypoinsulinemia (16 +/- 2 to 8 +/- 1 muU/mL), an inhibition of insulin secretion in response to glucose infusion (16 +/- 2 to 34 +/- 2 muU/mL control, 8 +/- 1 to 9 +/- 1 muU/mL after streptozotocin), hyperglycemia (19.5 +/- 0.7 to 30.4 +/- 1.7 mg/dL), and a reduction in the rate of umbilical glucose uptake (5.19 +/- 0.34 to 2.07 +/- 0.40 mg/kg/min). The disturbances in glucose metabolism could be alleviated only in part by insulin infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of high levels of insulin on glucose utilization and glucose production in pregnant and nonpregnant sheep.

The present study was conducted to test the hypothesis that pregnancy in sheep alters the effects of insulin on glucose utilization and glucose production. Euglycemic, hyperinsulinemic glucose clamp experiments were performed in chronically catheterized, unstressed, fed or 24-hr fasted, nonpregnant sheep and fed, pregnant sheep. Endogenous glucose production rate for the whole sheep and glucose utilization rate of the uterine and nonuterine maternal tissues were measured in control and high-insulin periods by tracer technique using [6-3H]glucose. Control glucose utilization rate in the fed, nonpregnant sheep was significantly (P less than 0.05) greater than that in the fasted, nonpregnant sheep, 2.29 +/- 0.17 and 1.86 +/- 0.11 mg/min/kg, respectively, and also in the nonuterine maternal tissues of the pregnant sheep (1.71 +/- 0.18 mg/min/kg). Insulin stimulated glucose utilization 116.4 +/- 14.8% in the fed, nonpregnant sheep but only 82.8 +/- 11.0% in the fasted, nonpregnant sheep and 94.2 +/- 14.3% in the nonuterine tissues of the fed, pregnant sheep. Also, insulin suppressed endogenous glucose production to 53.2 +/- 5.6% in the fed, nonpregnant sheep, to 3.9 +/- 3.1% in the fasted, nonpregnant sheep, and to 9.0 +/- 3.7% in the fed, pregnant sheep. In the pregnant animals, uterine glucose uptake and uterine glucose utilization were not different and were not altered by changes in maternal insulin concentration. The results indicate that during late pregnancy glucose utilization is reduced and resistance to the effect of insulin to enhance glucose utilization is present in the nonuterine maternal tissues compared with nonpregnant, fed sheep. In contrast, the effectiveness of insulin to suppress glucose production in the pregnant sheep is greater than that in nonpregnant, fed sheep. These results also demonstrate that differential changes in the effect of insulin can exist simultaneously between peripheral (glucose consuming) and central (glucose producing) tissues. The changes in glucose utilization and in insulin effect in the pregnant sheep are both qualitatively and quantitatively similar to those of the nonpregnant sheep when fasted, suggesting that similar substrate and/or hormonal factors may be involved.

Use of fetal streptozotocin injection to determine the role of normal levels of fetal insulin in regulating uteroplacental and umbilical glucose exchange.

The present study was performed to determine the role of the normal fetal concentration of insulin in regulating placental-fetal glucose exchange. Fetal insulin deficiency was produced by streptozotocin injection into near term fetal sheep, and the effects of this insulin deficiency on net uteroplacental glucose uptake and net umbilical glucose uptake were measured. Each fetus received two or three doses of streptozotocin, 100 mg.kg-1.dose-1, given on separate days. This dosage of streptozotocin produced a 97.6% reduction in fetal pancreatic insulin content, a fall in fetal plasma insulin concentration (21 +/- 2 to 10 +/- 1 microU.ml-1), a rise in fetal plasma glucagon concentration (57 +/- 4 to 114 +/- 19 pg.ml-1), a rise in fetal blood glucose concentration (20.4 +/- 0.9 to 33.4 +/- 4.4 mg.dl-1), and a failure of insulin secretion in response to glucose infusion. Fetal blood oxygen content and umbilical oxygen uptake were normal and did not change during the entire study. Umbilical glucose uptake was reduced by 66% (5.98 +/- 0.38 to 2.02 +/- 1.31 mg.min-1.kg-1) after the streptozotocin-induced hypoinsulinemia and hyperglycemia but was returned to the control level by an insulin infusion into the fetus that reestablished the control maternal to fetal glucose concentration gradient. Net uteroplacental glucose uptake (consumption) did not change throughout the study. Because glucose concentration and umbilical glucose uptake could be normalized by an insulin infusion, it is unlikely that direct or toxic effects of streptozotocin on fetal or placental glucose metabolism were primarily responsible for the hyperglycemia and the reduced rate of umbilical glucose uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin and glucose concentrations on glucose utilization in fetal sheep.

Glucose and insulin clamp experiments were performed in vivo in chronically catheterized, late-gestation fetal lambs to quantify the effects of glucose and insulin on fetal glucose metabolism. Fetal glucose uptake from the placenta via the umbilical circulation (umbilical glucose uptake) was measured by application of the Fick principle, and fetal glucose utilization rate (GUR) was measured using [U-14C]glucose tracer. Fetal plasma insulin concentrations ranged from 2 to 119 microU.ml-1 and fetal blood glucose concentrations ranged from 7.3 to 62.6 mg.dl-1. GUR varied from 2.82 to 15.12 mg/min/kg and the exogenous glucose entry rate (umbilical glucose uptake + glucose infusion) varied from 2.46 to 13.95 mg/min/kg. The mean GUR [6.53 +/- 0.28 (SEM) mg/kg/min] was not different from the mean exogenous glucose entry rate [6.29 +/- 0.30 (SEM) mg/kg/min]. Multiple linear regression analysis on a glucose-by-insulin surface demonstrated a best-fit model of fetal glucose utilization following the quadratic equation: GUR = -0.322 + [0.289 (glucose)] + [0.108 (insulin)] - [0.00319 (glucose)2] - [0.000673 (insulin)2], r = 0.883 (all terms significant at p less than 0.02). This model predicted a GURmax of 10.56 mg/min/kg at blood glucose concentration = 45.3 mg/dl and plasma insulin concentration = 80 microU/ml and Km values for blood glucose concentration and plasma insulin concentration of 20.6 mg/dl and 10 microU/ml, respectively. According to this model, the glucose and insulin effects were additive. Furthermore, change in GUR was not proportionate to change in glucose concentration, accounting for a decreasing metabolic clearance rate at higher glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Fructose disposal and oxidation rates in the ovine fetus.

Fructose disposal and oxidation rates were measured in fetal lambs receiving a constant intravenous infusion of D-[U-14C]fructose. Approximately 60% of the infused tracer entered the placenta, but loss of fructose into the maternal circulation was negligible. Fructose was metabolized to lactate and CO2 in both the placenta and fetus, whereas there was no detectable conversion to glucose. In well-fed ewes the fetal disposal and utilization rates of fructose were 2.4 +/- 0.17 and 0.97 +/- 0.09 mg/min. kg, respectively. The umbilical excretion rate of CO2 originating from the oxidation of fetal fructose was 18.1 +/- 1.3 mumol/min. kg or 5.3% of total fetal CO2 production. This excretion rate is one-fifth of the CO2 excretion rate from fetal glucose carbon. In four ewes comparison of fructose metabolism in the fed and fasted states showed a significant decrease of fructose production and oxidation with fasting. Although fructose is present in high concentrations in the fetal blood of ungulates, its contribution to fetal oxidative metabolism is relatively small in comparison to glucose.

The effect of hyperinsulinaemia on glucose utilization and oxidation and on oxygen consumption in the fetal lamb.

In order to measure the effect of hyperinsulinaemia on fetal glucose metabolism and oxygen consumption, we applied the glucose-clamp technique to experiments in fifteen late-gestation, unstressed, chronically catheterized fetal lambs. In a control period, and immediately thereafter following 2 h of hyperinsulinaemia, we measured fetal glucose utilization and oxidation rates (radioactive tracer methodology) and net fetal uptake rates of exogenous glucose and oxygen uptake rates (Fick principle). During the period of hyperinsulinaemia, fetal glucose concentration was maintained at the average control period value by a variable rate of glucose infusion into the fetus in response to serial 10 min measurements of fetal arterial blood glucose concentration. Hyperinsulinaemia in the fetus (12.12 +/- 1.92 ng X ml-1 (mean +/- S.E.M.) arterial plasma) resulted in a 13% increase in net fetal oxygen uptake rate (0.310 +/- 0.011 to 0.349 +/- 0.012 mmol. min-1 X kg-1), a 106% increase of fetal glucose uptake rate (4.54 +/- 0.43 to 9.35 +/- 0.50 mg X min-1 X kg-1) and an 83% increase of fetal glucose utilization rate (4.94 +/- 0.43 to 9.05 +/- 0.83 mg X min-1 X kg-1). Fetal glucose uptake and utilization rates were not different from each other during the control and the hyperinsulinaemia periods. The fraction of glucose that was oxidized (0.58 +/- 0.05, control; 0.54 +/- 0.05, hyperinsulinaemia) did not change significantly; thus the glucose oxidation rate, the rate of entry of glucose into non-oxidative pathways, and the amount of oxygen used for glucose oxidation all increased in proportion to glucose utilization. These results suggest that insulin promotes the entry of glucose into fetal tissues, thereby increasing fetal glucose utilization and oxidation rates and substituting glucose oxidation for that of other substrates. The insulin-enhanced glucose utilization rate also increases slightly fetal metabolic rate.

Effect of insulin on glucose uptake in near-term fetal lambs.

Glucose clamp experiments were performed in 27 chronically catheterized, late-gestation fetal lambs in order to measure the effect of fetal insulin concentration on fetal glucose uptake at a constant glucose concentration. Fetal arterial blood glucose concentration was measured over a 30-min control period and then maintained at the control value by a variable glucose infusion into the fetus while insulin was infused at a constant rate into the fetus. Plasma insulin concentration increased from 21 +/- 10 (SD) to 294 +/- 179 (SD) microU X ml-1. The exogenous glucose infusion rate necessary to maintain constant glycemia during the plateau hyperinsulinemia averaged 4.3 +/- 1.6 (SD) mg X min-1 X kg-1. In a subset of 13 animals, total fetal exogenous glucose uptake (FGU; sum of glucose uptake from the placenta via the umbilical circulation plus the steady-state exogenous glucose infusion rate) was measured during the control and hyperinsulinemia period. FGU was directly related to insulin concentration (y = 4.24 + 0.07x) at insulin levels less than 100 microU/ml and increased 132% above control at insulin levels above 100 microU/ml. Hyperinsulinemia did not affect fetal glucose uptake from the placenta via the umbilical circulation. These studies demonstrate that insulin concentration is a major factor controlling glucose uptake in the near-term fetal lamb, and that an increase of fetal insulin does not affect the transport of glucose to the fetus from the placenta.