Differential metabolic response to 48 h food deprivation at different periods of pregnancy in the rat

Since during pregnancy the mother switches from an anabolic to a catabolic condition, the present study was addressed to determine the effect of 48 h food deprivation on days 7, 14 and 20 of pregnancy in the rat as compared to age matched virgin controls. Body weight, free of conceptus, decreased with food deprivation more in pregnant than in virgin rats, with fetal weight (day 20) also diminishing with maternal starvation. The decline of plasma glucose with food deprivation was greatest in 20 day pregnant rats. Insulin was highest in fed 14 day pregnant rats, and declined with food deprivation in all the groups, the effect being not significant in 7-day pregnant rats. Food deprivation increased plasma glycerol only in virgin and 20 day pregnant rats. Plasma NEFA and 3-hydroxybutyrate increased with food deprivation in all groups, the effect being highest in 20 day pregnant rats. Food deprivation decreased plasma triacylglycerols in 14 day pregnant rats but increased in 20 day pregnant rats. In 20-day fetuses, plasma levels of glucose, NEFA and triacylglycerols were lower than in their mothers when fed, and food deprivation caused a further decline in plasma glucose, whereas both NEFA and 3-hydroxybutyrate increased. Liver triacylglycerols concentration did not differ among the groups when fed, whereas food deprivation caused an increase in all pregnant rats and fetuses, the effect being highest in 20-day pregnant rats. Lipoprotein lipase (LPL) activity in adipose tissue was lower in 20 day pregnant rats than in any of the other groups when fed, and it decreased in all the groups with food deprivation, whereas in liver it was very low in all groups when fed and increased with food deprivation only in 20 day pregnant rats. A significant increase in liver LPL was found with food deprivation in 20 day fetuses, reaching higher values than their mothers. Thus, the response to food deprivation varies with the time of pregnancy, being lowest at mid pregnancy and greatest at late pregnancy, and although fetuses respond in the same direction as their mothers, they show a specific response in liver LPL activity.     

Effect of Acipimox on Plasma Lipids and Glucose/Insulin in Pregnant Rats

To determine how a reduction in maternal hypertriglyceridemia during late pregnancy may affect glucose/insulin relationships, pregnant and virgin rats were orally treated with acipimox, a potent antilipolytic agent. In 20-day pregnant rats receiving 80 mg of acipimox, plasma triglycerides (TG), free fatty acids (FFA), and glycerol decreased more than in virgin rats shortly after the drug (up to 7 hours), when compared with animals treated with distilled water, whereas plasma glucose level was unaffected by the treatment in either group of rats. When acipimox was given every 12 hours from day 17 to day 20 of pregnancy, plasma TG, FFA, and glycerol levels progressively increased, whereas they either decreased or did not change in virgin rats receiving the same treatment, with no effect in plasma glucose levels in either group. Fetal body weight was lower than in controls in 20-day pregnant rats that received acipimox for 3 days. On day 20 of pregnancy, 3 hours after receiving acipimox or distilled water, rats received a 2 g glucose/kg oral load and it was found that the change in plasma glucose was similar in both groups, whereas the increase in plasma insulin was greater in pregnant rats treated with acipimox. However, no difference was found in either variable after the oral glucose load in virgin rats receiving acipimox or distilled water. No differences in plasma glucose levels were found after intravenous (IV) administration of insulin in pregnant rats treated or not treated with acipimox. In conclusion, present results show that administration of acipimox during the last days of gestation inhibited lipolysis and decreased fetal weight. Over a short period of time, in pregnant rats, reductions of plasma FFA and TG after acipimox treatment improved the glucose-induced insulin release, but did not seem to have any effect in peripheral insulin resistance.     

Preservation of the effects of pregnancy on rat islets of Langerhans in tissue culture.

Islets of Langerhans, isolated from normal or 19-day pregnant rats, were cultured for 20 h at 37 degrees C in tissue culture medium 199. When islets were cultured in medium containing low glucose (5.5 mM), the higher adenylate cyclase activity and insulin secretory responses characteristic of islets from pregnant rats were maintained during the test period of 29 h. Islets from normal and pregnant rats were also cultured for 20 h in medium containing a very high glucose concentration (83.3 mM) in order to load the B cells with glycogen. It was found, after glycogen loading, that, while adenylate cyclase activity increased to a greater extent in islets from pregnant rats than controls, this activity was not increased in proportion to the striking changes in insulin release rate observed in pregnant rat islets. The results show that the difference in insulin secretory response between islets from normal and pregnant rats may be preserved when the islets are cultured for 20 h, and that these differences are enhanced for a variety of reasons after culture of islets in 83.3 mM glucose.     

Regulation of insulin release from isolated islets of Langerhans of the rat in pregnancy. The role of adenosine 3':5'-cyclic monophosphate

1. The concentrations of cyclic AMP were compared in islets of Langerhans isolated from the pancreases of normal female and pregnant rats and were higher in islets in pregnancy. 2. There was also a significant increase in adenylate cyclase activity in homogenates of islets from pregnant rats compared with those from normal rats. 3. Increased cyclic AMP concentration in islets from pregnant rats was reflected in increased protein kinase activity. When the cyclic AMP-dependent protein kinase activity was increased by 3-isobutyl-1-methylxanthine this stimulated activity was significantly greater in pregnancy. 4. Insulin-secretion studies with islets from normal and pregnant rats showed that theophylline or 3-isobutyl-1-methylxanthine, which raise intracellular cyclic AMP concentrations, caused a significantly greater insulin secretion in pregnancy. 5. It was also found that in the presence of a glucose concentration too low to stimulate insulin secretion, the latter could be induced if the cyclic AMP concentrations were raised sufficiently with 3-isobutyl-1-methylxanthine. 6. It is suggested that the higher cyclic AMP concentrations observed in islets in pregnancy mediate the greater insulin-secretory capacity, as well as the greater sensitivity of these islets to low glucose concentrations.     

Effect of starvation on nutrition and insulin secretion in pregnant rats and their fetuses

Pregnancy is thought to create a metabolic condition of accelerated starvation. To clarify this idea, the effect of fasting on pregnant rats (day 21 of gestation) and their fetuses was examined. Although pregnancy significantly increased plasma insulin, plasma ketone body concentrations in fed pregnant rats were higher than those of age-matched fed virgin rats. After 48 hr fasting (i.e., fasting during days 19-21 of gestation), plasma insulin was markedly decreased in virgin rats compared with term pregnant rats, while ketone bodies were significantly higher in pregnant rats than in virgin rats. Body weight was lower in fetuses from fasted mothers than those from fed mothers. Starvation also markedly diminished the insulin response to glucose in isolated, perfused pancreases in both virgin and pregnant rats. The amount of insulin released during glucose stimulation was greater in pregnancy, and the inhibitory effect of 48 hr fasting on insulin release was greater in virgin rats than in pregnant rats. It is possible, therefore, that in term pregnant rats a decrease in insulin release caused by fasting may cause more profound catabolism than in nongravid rats.     

Tryptophan Overload in the Pregnant Rat: Effect on Brain Amino Acid Levels and In Vitro Protein Synthesis

The concentration of most amino acids was higher in the brains of 19- and 21-day rat fetuses than in their respective mothers. After an intraperitoneal load of tryptophan to the mother, the intracerebral concentration of several amino acids (including leucine) decreased not only in the mothers, but also in their fetuses. The in vitro incorporation of pHJleucine into proteins in brain postmitochondrial supernatant fractions was enhanced in both the mothers and fetuses after tryptophan administration, but this effect disappeared when protein synthesis was calculated by using specific activities corrected for the amount of unlabeled leucine in the preparation. By this criterion, protein synthesis activity appeared similar in the brains of 19- and 21-day pregnant rats but was higher in their fetuses, especially in the 21-day subjects. Thus, protein synthesis in the brain was not altered by marked changes in the amino acid pool and more profound and prolonged metabolic disturbances must occur to cause permanent damage in the developing brain.     

Increased gluconeogenesis in the rat at term gestation

In this study the contribution of maternal gluconeogenesis to the glucose homeostasis of the maternal-fetal unit has been studied in fed term pregnant rats. We have measured the activity of two gluconeogenic enzymes, the rates of lactate turnover and the rates of gluconeogenesis from lactate in fed term pregnant rats. A decrease in plasma glucose and liver glycogen concentrations, and an increase of plasma lactate and alanine concentrations were observed in fed 22-day pregnant rats compared to virgin controls. Also, liver and kidney phosphoenolpyruvate carboxykinase activities and liver lactate dehydrogenase and hexose bisphosphatase activities significantly increased in fed term pregnant rats compared to virgin rats. The lactate turnover rate and the rate of gluconeogenesis in vivo from L-[U14C] Lactate increased four- and two-fold respectively in fed pregnant rats compared to fed virgins.     

Initial permeability of the 19-day foetus to nickel

The administration of 63NiCl2 to 19-day pregnant rats produced a limited transfer of nickel to the foetus. An experimental model based on the incubation of the conceptuses still connected to the maternal organism resulted in an appreciable net transfer of nickel through the amniotic membranes. The placenta retained a significant amount of radioactive nickel as did the lining of the uterine wall. The circulation of nickel through the foetus following the direction maternal blood----placenta----foetus----amniotic membranes----endometrium----maternal venous blood, is postulated. This transfer could provide an additional protection to placental barrier against metal toxicity.     

Proposed mechanism for increased insulin-mediated glucose transport in adipose cells from young, obese Zucker rats. Large intracellular pool of glucose transporters

The mechanism for hyperresponsive insulin-mediated glucose transport in adipose cells from 30-day-old obese Zucker rats was examined. Glucose transport was assayed by measuring 3-O-methylglucose transport, and the concentration of glucose transporters was estimated by measuring specific D-glucose-inhibitable cytochalasin B binding. Insulin increased glucose transport activity by approximately 17 fmol/cell/min in cells from obese rats compared to 3 fmol/cell/min in lean littermates. Insulin increased the concentration of glucose transporters in the plasma membrane fraction by about 15 pmol/mg of membrane protein in both groups. The insulin-mediated decrease in the concentration of transporters in the low-density microsomal fraction was 30 pmol/mg of membrane protein for the obese rats compared to 15 pmol/mg of membrane protein for the lean controls. An estimated number of glucose transporters was calculated using membrane protein and enzyme recoveries for each group. Insulin increased the number of transporters in the plasma membrane by 3 X 10(6) sites/cell for the obese rats and only 0.6 X 10(6) sites/cell for the lean controls. In addition, insulin decreased the number of transporters/cell in the intracellular membrane pool by approximately 4 X 10(6) sites/cell for the obese rats and 0.9 X 10(6) sites/cells for the lean rats. The total number of transporters/cell was about 7 X 10(6) sites/cell for the obese animals and 1.6 X 10(6) sites/cell for the lean controls. In the basal state, more than 80% of these transporters were located in the intracellular pool for both the lean and obese rats. Thus, the marked hyperresponsive insulin-mediated glucose transport observed in adipose cells from 30-day-old obese Zucker rats may be the consequence of a marked increase in the number of glucose transporters in the intracellular pool.     

The Effect of Insulin on the Distribution of Glucose between the Blood Plasma and the Liver

In normal fasted rats whole liver tissue contains as much glucose as the blood plasma, i.e., the ratio of the concentrations is about unity. The concentration of glucose in hepatic intracellular water is about 1.2 times higher than in plasma water. In rats injected with insulin the concentration of glucose in the liver falls to a lesser extent than in the plasma: resulting in a ratio of concentrations higher than unity. If insulin hypoglycemia is prevented by the ingestion of glucose the concentration ratio is less than in hypoglycemic rats but still significantly above unity. In normal rats the specific activities of plasma and hepatic glucose do not differ significantly at 7.5, 15, and 30 minutes after the intravenous injection of C¹⁴-labeled glucose. In rats injected with insulin the specific activity of glucose is higher in the plasma than in the liver at 7.5 and 15 minutes, but not at 30 minutes following the injection of tracer. In insulin-treated hypoglycemic rats considerably higher concentrations of labeled glucose are found in hepatic intracellular than in plasma water. The penetration of C¹⁴-glucose from plasma into hepatic intracellular water is found to be fast. Excess insulin causes an accumulation of glucose within the liver cells by retaining newly formed glucose and by the taking up of glucose from the plasma against an existing concentration gradient.     

Recruitment of GLUT-4 glucose transporters by insulin in diabetic rat skeletal muscle

The cause of reduced insulin-stimulated glucose transport in skeletal muscle of diabetic rats was investigated. Basal and insulin-stimulated glucose uptake into hindquarter muscles of 7-day diabetic rats were 70% and 50% lower, respectively, than in nondiabetic controls. Subcellular fractionation of hindquarter muscles yielded total crude membranes, plasma membranes and intracellular membranes. The number of GLUT-4 glucose transporters was lower in crude membranes, plasma membranes and intracellular membranes, relative to non-diabetic rat muscles. These results were paralleled by reductions in D-glucose-protectable binding of cytochalasin B. Insulin caused a redistribution of GLUT-4 transporters from intracellular membranes to plasma membranes, in both control and diabetic rat muscles. This redistribution was also recorded using binding of cytochalasin B. The insulin-dependent decrement in glucose transporters in intracellular membranes was similar for both animal groups, but the gain and final amount of transporters in the plasma membrane were 50% lower in the diabetic group. The results suggest that insulin signalling and recruitment of GLUT-4 glucose transporters occur in diabetic rat muscle, and that the diminished insulin response may be due to fewer glucose transporters operating in the muscle plasma membrane.     

Cumulation of active metabolites of levo-alpha-acetylmethadol in the rat fetus and neonate

Levo-alpha-acetylmethadol (LAAM, 0.2 or 2.0 mg/kg/day) was orally administered to female Sprague-Dawley rats for one month prior to and throughout pregnancy. The rats were killed on the 18th day of gestation along with a group of 18-day pregnant females given a single oral 2.0 mg/kg dose of LAAM 24 hours earlier. Although cumulation of LAAM or its active metabolites was not seen in plasma or brain of pregnant rats given drug chronically, significant cumulation was observed in whole fetus and in fetal brain. In addition, a 2–3 fold elevation in the concentrations, and an even greater elevation of total content, was noted in the newborn pup. These data suggest that opiate intoxication soon after birth may be a factor responsible for the increased morbidity and mortality of rat pups prenatally exposed to LAAM.     

Gallium-67 distribution in pregnant mammals.

Studies on the distribution of 67Ga in pregnant rabbits showed a marked concentration of the isotope in uterine tissue and flushings of 5-day pregnant rabbits 24 hours after isotope injection. The isotope no longer localized throughout the uterine tissues and flushings on injection of 7- to 8-day pregnant rabbits but was specifically associated with the blastocysts and sites of implantation. As the gestation time progressed 67Ga increasingly concentrated in placental and mammary tissue while the concentration in serum and thymus tissue decreased. The marked concentration in placental tissue was readily discernible on total-body scans of pregnant rabbits. Column chromatography of uterine washings and placental extracts from pregnant rabbits and thymus extracts from estrous rabbits showed most of the isotope associated with moieties greater than or equal to 200,000 MW whereas the isotope in "milk" and extracts of mammary tissue was bound to components of 25,000-35,000 MW. Extracts of thymus from pregnant rats showed a marked, selective loss of 67Ga binding in two peaks of approximately 50,000 and 120,000 MW compared to the chromatographic profile of extracts from normal rat thymus.     

Estrogen effects on platelet-activating factor and platelet-activating factor acetylhydrolase activity in rat uterus during the late stages of pregnancy.

The platelet-activating factor (PAF) concentration of the uterus spontaneously increased during pregnancy. When 17alpha-ethynylestradiol (0.25 mg/kg) was administered subcutaneously to pregnant rats for 3 days starting on Day 17 of pregnancy, some rats delivered prematurely on Day 20. However, none of the vehicle-treated (80% dimethylsulfoxide and 20% ethanol) pregnant rats delivered prematurely. The PAF concentration of the uterus in pregnant rats treated with 17alpha-ethynylestradiol was significantly higher than in those treated with vehicle on Days 19 and 20. On the other hand, the specific activity of uterine PAF-acetylhydrolase (PAF-AH) in pregnant rats treated with 17alpha-ethynylestradiol was significantly lower than in those treated with vehicle on Days 19 and 20, and the plasma PAF-AH activity in pregnant rats treated with estrogen was also significantly lower than in treated with vehicle on Days 18, 19, and 20. These findings indicate that estrogen increases PAF concentrations in the rat uterus, and this was correlated with a decrease in PAF-AH in the uterus and plasma. The increase in PAF concentrations in the uterus may be related to premature delivery and labor caused by PAF's known effect on myometrial contraction.     

Insulin release from the pancreas and fuel metabolism during late gestation in chemically diabetic rats

The effects of chemical diabetes and fasting on fuel metabolism and insulin secretory activity in late pregnancy were investigated. Female Wistar rats were made chemically diabetic (CD) by intravenous injection of streptozotocine (30 mg/kg) 2 weeks before conception. When CD pregnant rats were fed, plasma glucose and insulin levels were not significantly different from those of normal pregnant rats. Ketone body levels, however, were higher in CD pregnant rats than in normal pregnant rats, indicating insulin resistance in CD rats. Insulin secretion from the perfused pancreas caused by arginine or glucose was markedly decreased in CD pregnant rats. The pregnant rats were fasted for 2 days, from day 19 to 21 of gestation. Plasma glucose and insulin concentrations decreased similarly in the two groups, whereas ketone body concentrations in CD pregnant rats were significantly higher than those in normal pregnant rats. Glucose-induced insulin secretion by the perfused pancreas was markedly attenuated by fasting and was not significantly different in normal and CD pregnant rats. These observations suggest that diabetes mellitus accelerates starvation in late gestation, due to increased insulin resistance and poor insulin secretion, and that fasting in diabetic pregnancy amplifies ketogenesis.     

Effect of prenatal stress on the pituitary-adrenal axis in blue foxes

Handling is a source of stress for farm bred blue foxes. The influence of handling during the late gestation period on the pituitary--adrenal axis was studied in 10-day old male and female blue foxes. Cortisol and progesterone were measured by radioimmunoassay in the plasma, adrenal homogenates, and in vitro incubates from animals of both sexes. Adrenals were incubated in vitro in the absence or presence of ACTH. In addition, the adrenal weight and plasma concentration of ACTH were assessed. In cubs of both sexes, the adrenal weight was decreased after prenatal stress. The plasma concentration of progesterone and the adrenal cortisol in vitro production were elevated in the prenatally stressed female cubs, as compared to the control, along with the adrenal progesterone in vitro production in prenatally stressed male cubs. The adrenal cortisol and progesterone content and plasma ACTH and cortisol concentrations were not affected by prenatal stress. In conclusion, the results of this study suggest that the prenatal stress induced by handling pregnant vixens can affect the pituitary--adrenal axis in neonatal offspring, this effect being more pronounced in female cubs.     

Glucose metabolism "in vitro" in brown adipose tissue from pregnant and lactating rats

(U-14C)Glucose utilization has been studied "in vitro" in brown adipose tissue pieces from virgin, 20-day pregnant and 15-day lactating rats. Brown fat pieces from virgin rats increased their (U-14C)glucose utilization for (14C)CO2 production and for (14C)fatty acid and (14C)glycogen synthesis when insulin was present in the medium. Opposite changes were observed due to the presence of noradrenaline. Brown fat from late pregnant rats does not present any essential alteration in its capacity of metabolizing glucose and showed a pattern of responses to insulin and noradrenaline similar to that from virgins. Brown fat from mid lactating rats showed an intrinsic reduction in (U-14C)glucose utilization for oxidative pathways as well as for fatty acid synthesis, this reduction was present in all hormonal conditions. This data suggests a relationship between the lowered glucose metabolism and the known reduction in brown fat thermogenesis during mid lactation.     

Plasma amino acid concentrations in pregnant rats and in 21-day foetuses.

Plasma amino acid concentrations were determined in virgin female rats, in pregnant rats (12 and 21 days after impregnation) and in 21-day foetuses. The total amino acid concentration in plasma decreases significantly with pregnancy, being lower at 12 than at 21 days. Alanine, glutamine+glutamate and other 'gluconeogenic' amino acids decrease dramatically by mid-term, but regain their original concentrations at the end of the pregnancy. With most other amino acids, mainly the essential ones, the trend is towards lower concentrations which are maintained throughout pregnancy. These data agree with known nitrogen-conservation schemes in pregnancy and with the important demands on amino acids provoked by foetal growth. In the 21-day foetuses, concentrations of individual amino acids are considerably higher than in their mothers, with high plasma foetal/maternal concentration ratios, especially for lysine, phenylalanine and hydroxy-proline, suggesting active protein biosynthesis and turnover. All other amino acids also have high concentration ratios, presumably owing to their requirement by the foetuses for growth. Alanine, glutamine+glutamate, asparagine+aspartate, glycine, serine and threonine form a lower proportion of the total amino acids in foetuses than in the virgin controls or pregnant rats, probably owing to their role primarily in energy metabolism in the adults. The results indicate that at this phase of foetal growth, the placental amino acid uptake is considerable and seems to be higher than immediately before birth.     

Response to acute nickel toxicity in rats as a function of sex

Summary The effects of different nickel chloride doses upon blood and plasma glucose and essential metal homeostasis were studied in male and female rats. A definite sex-dependent response to injections of nickel has been observed for both the increase in plasma and blood glucose levels and the time at which these levels peak. Males showed a fast recovery from the rise in glucose levels and were much less affected by changes in the other parameters studied. In females, an extended rise in glucose levels was observed. All these effects are clearly nickel dose-dependent. Plasma, liver and kidney copper levels rose significantly in females while only a small decrease was observed in male kidneys. Zinc levels rose in all organs studied but males recovered to basal levels after the study period, whereas females maintained maximum levels at the end of the same period. An increase in urinary excretion of iron was observed. The present results show that the sex differences to acute nickel toxicity can be a helpful way to study metal interaction and discriminate between specific toxicity due to nickel or that induced by the associated hyperglucagonemia.     

Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity

The effects of insulin therapy in streptozotocin diabetic rats on the glucose transport response to insulin in adipose cells have been examined. At sequential intervals during subcutaneous insulin infusion, isolated cells were prepared and incubated with or without insulin, and 3-O-methylglucose transport was measured. Insulin treatment not only reversed the insulin-resistant glucose transport associated with diabetes, but resulted in a progressive hyperresponsiveness, peaking with a 3-fold overshoot at 7-8 days (12.1 +/- 0.3 versus 3.4 +/- 0.1 fmol/cell/min, mean +/- S.E.) and remaining elevated for more than 3 weeks. During the peak overshoot, glucose transporters in subcellular membrane fractions were assessed by cytochalasin B binding. Insulin therapy restored glucose transporter concentration in the plasma membranes of insulin-stimulated cells from a 40% depleted level previously reported in the diabetic state to approximately 35% greater than control (38 +/- 4 versus 28 +/- 2 pmol/mg of membrane protein). Glucose transporter concentration in the low-density microsomes from basal cells was also restored from an approximately 45% depleted level back to normal (50 +/- 4 versus 50 +/- 6 pmol/mg of membrane protein), whereas total intracellular glucose transporters were further increased due to an approximately 2-fold increase in low-density microsomal membrane protein. However, these increases remained markedly less than the enhancement of insulin-stimulated glucose transport activity in the intact cell. Thus, insulin treatment of diabetic rats produces a marked and sustained hyperresponsive insulin-stimulated glucose transport activity in the adipose cell with little more than a restoration to the non-diabetic control level of glucose transporter translocation. Because this enhanced glucose transport activity occurs through an increase in Vmax, insulin therapy appears to be associated with a marked increase in glucose transporter intrinsic activity.