Corticosterone treatment of pregnant low dose endotoxin-treated rats: inhibition of the inflammatory response.

PROBLEM: Can the endotoxin-induced inflammatory response, underlying experimental pre-eclampsia, in pregnant rats be inhibited by corticosterone? METHOD OF STUDY: On day 10 of pregnancy, rats were implanted with pellets containing 25% corticosterone and 75% cholesterol (n = 10) or with 100% cholesterol-pellets (n = 10). On day 14 of pregnancy, rats were infused with either endotoxin (1.0 microg/kg bw) or saline. Three days later, they were sacrificed. Cryostat kidney sections were immunohistologically stained for the presence of neutrophils (PMN) and monocytes (MO) and the expression of inflammation-associated adhesion molecules. RESULTS: In cholesterol-treated rats, endotoxin significantly increased glomerular numbers of PMN and MO, glomerular expression of ICAM-1 and VCAM-1 and glomerular numbers of LFA-1 and VLA-4-positive cells as compared with saline. Corticosterone treatment significantly inhibited glomerular infiltration of PMN, MO and LFA-1 positive cells after endotoxin infusion. It did not affect glomerular ICAM-1 or VCAM-1 expression or numbers of VLA-4 positive cells. CONCLUSIONS: It is concluded that pre-treatment with corticosterone inhibits the low dose endotoxin-induced glomerular inflammatory reaction in pregnant rats, most likely by inhibiting LFA-1 expression, thereby decreasing the adhesiveness of inflammatory cells for activated endothelial cells.

Interaction of late pregnancy and lactation in rats.

The effect of pregnancy on lactation was studied during the third week of lactational pregnancy in postpartum pregnant rats with a delay in implantation of only 1 day (1d-LP rats). In an experimental design in which the suckling litter was prevented from consuming solid food, lactational performance was estimated by weighing the ten-pup suckling litters on days 16-21 of lactation or by measuring maternal weight loss after a nursing spell on day 21. In 1d-LP rats, food consumption as well as lactational performance was lower than it was in nonpregnant lactating rats (L rats) and pregnant-lactating rats with a normal long delay of implantation of at least 6 days (LP rats). The time spent by the pups sucking at the nipples was not different among the three groups, but the number of milk ejections was diminished in 1d-LP dams. Restriction of daily food supply during days 16 to 21 of lactation diminished lactational performance more strongly in 1d-LP rats than it did in L rats; 1d-LP rats conserved protein stores and mobilized fewer minerals than did L rats. The weight and composition of the litter in vitro were not affected by the food restriction. In pregnant-lactating rats (LP and 1d-LP rats), the number of early resorptions was increased in comparison with pregnant rats, showing that lactation can affect the earlier stages of pregnancy. It was concluded that late pregnancy does not affect nursing behaviour, but suppresses lactation by restricting maternal food intake and mobilization of maternal stores. Measurements in serum indicate a causative role for oestradiol, but not for leptin.

FSH inhibits the augmentation by oestradiol of the pituitary responsiveness to GnRH in the female rat.

The effect of follicle stimulating hormone (FSH) treatment on the pituitary response to gonadotrophin-releasing hormone (GnRH) was studied in rats in various reproductive conditions. A 3-day treatment of cycling rats with FSH (Metrodin; 10 IU/injection) lowered the spontaneous pre-ovulatory. LH-surge and suppressed the pituitary luteinizing hormone (LH) response to GnRH. FSH also suppressed the LH response of pseudopregnant (PSP) rats on day 8 of pseudopregnancy, but not that of day-8 PSP rats which had been ovariectomized on day 4 (OVX-PSP rats). GnRH induced self priming in cycling, PSP and OVX-PSP rats. Oestradiol strongly augmented the pituitary LH-response to GnRH injection in PSP and OVX-PSP rats, but not in cycling rats; probably because in these latter animals the LH response to GnRH was already augmented by endogenous oestradiol. FSH suppressed the LH response to GnRH in oestradiol-treated PSP and cycling rats; in these latter rats the suppression of the LH response was as strong as that in cycling rats not treated with oestradiol. FSH did not suppress the LH response of oestradiol-treated OVX-PSP rats. The effect of FSH was not associated with changes in plasma oestradiol and progesterone concentrations. Analysis of the data revealed that FSH specifically suppressed the augmentative effect of oestradiol, but did not affect the GnRH-self priming effect. It is concluded that under the influence of FSH, the ovaries produce a factor which suppresses the augmentative effect of oestradiol on the GnRH-induced LH response of the pituitary gland. It is suggested that this effect of FSH underlies the suppression of the spontaneous LH-surges of FSH-treated cycling rats. As the present putative 'oestrogen-antagonizing factor' did not suppress the GnRH-self priming effect, it is suggested that this factor is not identical to gonadotrophin surge inhibiting factor.

Progesterone stimulates pancreatic cell proliferation in vivo.

Treatment of cyclic and pregnant rats with progesterone stimulates cell proliferation within the islets of Langerhans. It was investigated whether this effect of progesterone depends on sex and/or the presence of the gonads or the presence of oestradiol. For this purpose, Silastic tubes containing progesterone were inserted s.c. in intact and gonadectomized male and female rats, and in gonadectomized female rats treated with oestradiol. After 6 days of progesterone treatment, rats were infused for 24 h with 5-bromo-2'-deoxyuridine (BrdU) and dividing cells were identified in pancreatic sections by immunostaining for BrdU. Progesterone treatment increased islet-cell proliferation in intact male and female rats (P < 0.05), but not in gonadectomized male and female rats or in gonadectomized female rats supplemented with oestradiol. Furthermore, in intact male and female rats, progesterone treatment also stimulated cell proliferation in extra-islet pancreatic tissue (P < 0.05). Identification of the proliferating cells, by double-immunocytochemistry, revealed that progesterone treatment stimulated proliferation of both alpha and beta cells within the pancreatic islets. In extra-islet pancreatic tissue, progesterone treatment stimulated proliferation in both duct (cytokeratin 20-immunoreactive) and non-duct cells. Progesterone treatment did not increase the number of single glucagon or insulin-containing cells outside the pancreatic islets, nor that of cytokeratin 20/insulin double-positive cells, suggesting that progesterone treatment did not stimulate differentiation of duct cells into endocrine cells. Progesterone treatment did not affect insulin responses to an i.v. glucose load (0.5 g/kg body weight). It is concluded that progesterone stimulates pancreatic cell proliferation indirectly; gonadal factor(s), not identical to oestradiol, is (are) probably involved.

Effects of food restriction on glucose tolerance, insulin secretion, and islet-cell proliferation in pregnant rats.

Pregnancy is associated with increased glucose-stimulated insulin secretion and increased pancreatic islet-cell proliferation. In the present study it was investigated whether increased food intake, as occurs during pregnancy, is involved in the regulation of these phenomena. From Day 0 of pregnancy, rats received each day the mean amount of food they consumed daily during the estrous cycle prior to conception. This food restriction regime resulted in lower maternal body weight, and in lower fetal weight on Day 20 of gestation, but did not affect fetal survival. Food-restricted rats showed decreased insulin responses to an i.v. glucose challenge on Day 13, and lower islet-cell replication rates on Day 14 of pregnancy than pregnant rats fed ad lib. Plasma lactogenic activity in food-restricted animals was increased on Days 11 and 13; plasma progesterone levels were unchanged, but plasma leptin concentrations declined progressively during food restriction. Glucose tolerance was normal, suggesting that food restriction improved insulin action. On Day 20 of pregnancy, insulin responses were similar in food restricted and ad lib-fed rats; glucose tolerance was still unchanged. It thus seems that the improved insulin action as present on Day 13 had disappeared on Day 20. Also on Day 20, lactogenic activity as well as progesterone concentrations were similar in food-restricted and ad lib-fed rats. It was concluded that increased food intake plays an important role in the stimulation of islet-cell proliferation and insulin secretion, as well as in the diminished insulin action during the second week of rat pregnancy.

Glucose consumption by various tissues in pregnant rats: effects of a 6-day euglycaemic hyperinsulinaemic clamp.

In the course of pregnancy maternal tissues become increasingly more insensitive to insulin. As 6 days of euglycaemic hyperinsulinaemic clamping, from day 8 until 14 of gestation, ameliorates total glucose consumption, we analysed the contribution of individual tissues in this phenomenon. We measured not only glucose consumption, but also concentrations of the glucose transporter protein GLUT4 in selected tissues. On day 15 of pregnancy in saline-infused (pregnant control) rats, 18F-fluoro-2-deoxy-D-glucose (FDG) consumption, as measured by positron emission tomography, as well as GLUT4 content were diminished in heart (P < 0.05), and in brown (P < 0.05) and white adipose tissues (P < 0.05) when compared with non-pregnant controls. During clamping, on day 13 of the experiments, both in pregnant and non-pregnant rats FDG consumption was increased in liver (P < 0.05), skeletal muscle (P < 0.05), brown (P < 0.05) and white adipose tissues (P < 0.05) when compared with saline-infused controls. In both the reproductive conditions, only in white adipose tissue was this increased FDG consumption associated with increased GLUT4 levels (P < 0.05); GLUT4 content of m. gastrocnemius, m. soleus, heart and brown adipose tissue was unaffected by clamping. In non-pregnant rats, 24 h after clamping ceased, FDG consumption was diminished in heart compared with control non-pregnant rats (P < 0.05). In pregnant rats, however, 24 h after clamping (i.e. day 15 of pregnancy) both FDG consumption (P < 0.05) and GLUT4 content (P < 0.05) were still increased in white adipose tissue only, when compared with saline-infused pregnant rats on the same day of gestation. It is suggested that hyperinsulinaemic euglycaemic clamping ameliorates insulin action halfway pregnancy, in particular by stimulation of glucose consumption and GLUT4 protein content in white adipose tissue.

Proliferation of pancreatic islet-cells in cyclic and pregnant rats after treatment with progesterone.

The effect of progesterone (P) on pancreatic islet-cell proliferation and function of cyclic and pregnant rats was investigated in vivo. Silastic tubes containing P were inserted s.c. in cyclic rats for 7 or 14 days and in pregnant rats from day 7 to 14, from day 14 to 21 or from day 7 to 21 of pregnancy. 5-Bromo-2-deoxyuridine (BrdU) was infused during the last 24h of the treatment; the proportion of dividing islet-cells was determined in pancreatic sections, which were immunostained for BrdU. Islet-cell function was determined by measuring glucose and insulin response to a standard intravenous glucose challenge. P treatment increased P and 20alpha-dihydroprogesterone (20alpha-OHP) levels in cyclic rats; in pregnant rats, only the plasma levels of 20alpha-OHP were elevated. Both 7 and 14 days of P treatment stimulated islet-cell proliferation in cyclic rats. In pregnant rats, P treatment increased islet-cell proliferation on day 14, but not on day 21 after either 7 or 14 days of P treatment. P did not affect plasma lactogenic activity in pregnant rats; plasma concentrations of prolactin were decreased after 14 days of P treatment in cyclic rats, but were not affected in pregnant rats. P treatment had no effect on glucose tolerance and glucose-stimulated insulin secretion in any of the groups. It was concluded that: 1. in vivo P stimulates islet-cell proliferation, but does not affect islet-cell function, 2. the stimulatory effects of P are indirect and possibly mediated by the P metabolite 20alpha-OHP and 3. at the end of gestation, stimulation of islet-cell proliferation is inhibited by some factor, which is not identical to P.

Role of increased insulin demand in the adaptation of the endocrine pancreas to pregnancy.

During gestation the demand for insulin increases due to a decrease in insulin sensitivity of the maternal tissues. Simultaneously, pancreatic islet-cell proliferation, as well as insulin production and secretion increase. Both phenomena appear to be caused by the actions of pregnancy hormones. We studied the relationship between the two phenomena by investigating whether the supply of exogenous insulin affects the secretion of pregnancy hormones and islet function during gestation. For that purpose rats were treated with high doses of insulin (4.8 IU day-1 by sub-cutaneous osmotic mini pumps) so that the endogenous demand for insulin was fully satisfied from day 8-14 of gestation. Euglycaemia (5.0 mM) was maintained by intra venous infusion of glucose. The treatment suppressed insulin synthesis, as measured by in situ hybridization, in both pregnant and cyclic rats. In addition, in pregnant rats the increments in insulin secretion and in islet-cell proliferation were partly prevented. Furthermore, the data also suggest that in pregnant rats the treatment partly prevented the decrease in insulin sensitivity. Finally, the treatment did not affect the plasma concentrations of progesterone, prolactin and placental lactogen, but prevented the rise in growth hormone concentrations in pregnant rats. The present data suggest that, next to direct effects of pregnancy hormones and growth hormone on the pancreatic islets, a decreased insulin sensitivity in the maternal tissues, induced by actions of the same hormones, is involved in the regulation of islet function during gestation.

Role of gonadotrophin releasing hormone baseline concentrations in the control of pituitary gonadotrophin and ovarian steroid secretion in the pseudopregnant rat.

To study the effect of moderately elevated gonadotrophin releasing hormone (GnRH) baseline concentrations during the luteal and the follicular phase, pseudopregnant rats were infused s.c. with GnRH at several doses for 5 days. These rats were also treated with oestradiol or sham-treated during the last 3 days of GnRH treatment. GnRH infusions started on day 7 or day 3 of the luteal phase of the ovulatory cycle; in the rat, the luteal phase or pseudopregnancy lasts about 10 days. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) responses were induced by i.v. injection of GnRH on days 12 (after expected luteolysis) or on day 8 (before expected luteolysis). In normal rats the LH and FSH responses induced by GnRH on day 12 were higher than on day 8 (approximately 160 and approximately 50% respectively). In GnRH-infused rats the LH and FSH responses were not increased. In these rats the luteal phase was extended (the plasma progesterone concentrations remained high) and the onset of the follicular phase was postponed (plasma oestrogen concentrations did not increase). Oestradiol increased the day 12 LH and FSH responses; this effect of oestradiol was suppressed by GnRH infusion. On day 8, exogenous oestradiol also increased the LH and FSH responses, but again the effect of oestradiol was suppressed when the animals were concomitantly infused with GnRH. These data may suggest that in the rat, GnRH baseline concentrations participate in the neuroendocrine system controlling gonadotrophin secretion and hence the ovulatory cycle.

Effect of insulin on endocrine pancreas function during late pregnancy in the rat.

To partly or completely satisfy the increasing demand for insulin, pregnant rats were infused SC with human insulin (2.4 or 4.8 IU/day) from day 14 to day 20 of gestation. Cyclic control rats underwent the same procedure of 6 days of insulin-treatment. During the treatment all groups of rats were hypoglycaemic, but foetal survival was not affected. The low dose treatment prevented the characteristic rise of the insulin response to a glucose challenge during pregnancy, both in vivo and in vitro, while the high dose treatment suppressed the insulin response, as well as the pancreatic insulin content. The insulin responses and insulin contents of pregnant rats were higher than those of the corresponding cyclic control rats. These results support the hypothesis that during gestation the increased insulin demand, due to the actions of placental hormones, is the cause of the increased insulin secretion. However, it cannot be excluded that direct effects of placental hormones on the islets of Langerhans are also involved.

Pancreatic beta-cell function and islet-cell proliferation: effect of hyperinsulinaemia.

Pancreatic beta-cell function was studied in adult female rats, in which endogenous insulin demand was fully met by SC infusion of human insulin (4.8 IU/24 h) for 6 days, resulting in hyperinsulinaemia and severe hypoglycaemia. The amount of pancreatic endocrine tissue declined by 40%, (pro)insulin mRNA, as determined by in situ hybridisation by 95%, and the amount of stored insulin by 90%. Islet-cell proliferation as determined by 24 h of BrdU infusion declined by 60%. Basal glucose levels normalized within 2 days after the insulin treatment was ended, whereas about 1 week was needed to restore the amount of pancreatic insulin, glucose-induced insulin release, and glucose tolerance to normal values. The amount of endocrine tissue recovered within 48 h and mRNA abundance within 96 h after discontinuation of the insulin infusion, whereas at that time islet-cell proliferation still showed a sixfold increase, before returning to control levels after 1 week. These results show that after a period of suppression of beta-cell function, recovery of insulin synthetic capacity does not immediately result in normalization of insulin stores and insulin release. Under these conditions, episodes of hyperglycaemia may occur, which may act as a stimulus for islet-cell proliferation.

Arginine stimulated glucagon and insulin secretion by islets of Langerhans of pregnant and lactating rats.

Glucagon secretion by isolated pancreatic rat islets was not affected by an increase of the glucose concentration from 2.5 to 5.0 mM, but was stimulated by 25 mM arginine. This stimulation was only slightly increased by pregnancy and lactation. Insulin secretion increased, when the glucose concentration was raised from 2.5 to 5.0 mM, especially in pregnant islets and was also stimulated by arginine. The arginine stimulated insulin release was greatly augmented during late pregnancy and lactation. Islet content did not reflect the changes in secretion: glucagon content increased in the course of pregnancy and lactation, while insulin content was increased during late pregnancy, but not during lactation. It is concluded that, unlike insulin, glucagon is only marginally involved in the increase in anabolism during pregnancy and lactation.

Effect of the TRH analogue, CG 3703, on prolactin secretion in the pseudopregnant rat.

The effect of the TRH analogue, CG 3703 (AG), on secretion of prolactin (Prl) was studied in pseudopregnant rats. AG stimulated Prl secretion of incubated pituitary glands in the presence of 5.10(-8) M dopamine (DA) but not in the presence of 0 or 10(-6) M DA. AG did not inhibit the in vitro secretion of Prl. AG postponed the peaks of Prl which were generated between 0100 and 1200 h (delay after 500 micrograms AG: 3-4 h); the height of these nocturnal Prl peaks was not affected by AG. AG did not induce Prl secretion during the phase of low Prl secretion (the interphase) between 2000 and 0100 h. The DA-release blocking drug, HA 966, induced peaks of Prl during the interphase. These peaks were as high as spontaneous nocturnal Prl peaks and were blocked by AG. The results show that AG has two opposite effects on Prl secretion: a direct, stimulating effect, which is modulated by DA, and an indirect inhibitory effect, which is probably executed by DA, released by AG from the tuberoinfundibular DA neurons.

Dopamine and TRH-induced prolactin secretion in pseudopregnant rats.

The stimulatory effect of TRH on prolactin (Pr1) secretion by the anterior pituitary gland (APG) of the pseudopregnant (PSP) rat was studied in vivo and in vitro. TRH, 500 micrograms, did not increase Pr1 release during the Pr1 peaks which are generated daily between 01.00 and 12.00 for about 10 days (mean height of the peaks: 302 +/- 99 ng mL-1), nor did TRH induce Pr1 secretion during the phase of low (12 +/- 1 ng Pr1 mL-1) secretion (the "interphase", between 20.00 and 01.00 h). 25 mg/kg b.w. of the dopamine- (DA) release blocking drug, 1-hydroxy-3-amino-pyrrolidone-2 (HA 966) did not itself induce peaks of Pr1 during the interphase, but in 1 animal out of 6 which were treated with this dose of HA 966, TRH (500 micrograms) induced a peak of Pr1 with a height of 264 ng mL-1 x 100 mg HA 966/kg b.w. induced during the interphase peaks of Pr1 which were as high as spontaneous Pr1 peaks (343 +/- 95 ng mL-1). TRH increased these Pr1 peaks to 844 +/- 48 ng mL-1. Apparently, HA 966 not only lowers DA concentrations but also somehow increases the TRH-responsiveness of the lactotrophs. In vitro, the secretion of Pr1 by incubated pituitary glands was suppressed to the same extent by 5 x 10(-8) and 10(-6) M DA. TRH stimulated Pr1 secretion in the presence of 5 x 10(-8) M DA, but not in the presence of 10(-6) M DA or in the absence of DA. It is concluded that in vitro TRH acts as a physiological antagonist of DA. It is suggested that in vivo the effectivity of TRH as a Pr1-releasing factor may be modulated by DA and that this effect of DA is independent from its Pr1-release suppressing effect.

Metabolism of pregnant-lactating rats is adapted to pregnancy rather than to lactation.

In pregnant-lactating rats implantation was induced on day 4 of lactation so that, as an exception, lactation coincided with the period of high fetal growth. The already present suckling litters of these animals lagged behind in growth, but the "second" litters were at birth normal in size and weight. Such pregnant-lactating rats were tested in vivo with intravenous glucose loads and compared with cyclic and lactating rats. Glucose tolerance was unaffected by the reproductive state. Pregnant-lactating rats showed, just as during their first pregnancy, low basal glucose levels. Their basal insulin levels and insulin responses, however, were decreased in comparison with the first pregnancy and resembled those of lactating rats. This may be due to an increased insulin turnover, because in vitro insulin responsiveness and insulin content of both "pregnant-lactating" and "pregnant" islets were increased in comparison with "cyclic" and "lactating" islets. It was concluded that the metabolism of pregnant-lactating rats is adapted to the pregnant rather than to the lactational state.

Regulation of peripheral glucagon concentrations in cyclic, pregnant, and lactating rats.

In the rat, peripheral glucagon concentrations were studied throughout pregnancy and lactation. Basal glucose concentrations were decreased during late pregnancy and during lactation, but basal glucagon concentrations were not affected. Infusion of glucose (7.4 mg/min) caused an elevation of the glucose concentrations, which became lower in the course of lactation, and a suppression of the glucagon concentrations which was the same throughout pregnancy and lactation. Ingestion of 336 mg of glucose or 1 g of rat chow throughout pregnancy and lactation induced a transient increase of the glucose concentrations and a biphasic glucagon response: following a short-lasting elevation, the glucagon concentrations became suppressed. The glucagon responses to these tests did not change during pregnancy and lactation. It is concluded that the regulation of the peripheral glucagon concentration is not affected by pregnancy or lactation, and that the response of the glucagon concentration to a metabolic challenge varies with the kind of test (oral or intravenous) used.

Effect of in vivo pre-treatment with oestradiol and either GnRH, GnRH agonistic analog or GnRH antagonistic analog on GnRH-stimulated secretion of LH in vitro.

In vivo treatment with GnRH or with GnRH agonistic analog (AG), but not with GnRH antagonistic analog (ANT), depleted the LH stores of the rat pituitary gland. This depletion was potentiated by oestradiol. Oestradiol augmented the in vitro LH response of the pituitary gland to GnRH. This augmenting effect of oestradiol became smaller with increasing rates of in vivo administration of GnRH or AG, but not with ANT. With respect to both depletion of the LH stores and suppression of the augmenting effect of oestradiol, AG ist about 20 times as potent as GnRH.