Lipolysis in rat adipocytes during recovery from lactation. Response to noradrenaline and adenosine.

The replenishment of lipid reserves of adipocytes following the removal of litters from lactating rats is associated with a 3-fold decrease in both the response of lipolysis to noradrenaline and the maximum rate of lipolysis (measured in the presence of noradrenaline plus adenosine deaminase); these adaptations do not appear to result from the changes in serum prolactin and insulin concentrations that occur on litter removal.     

Effect of chronic treatments with ghrelin on milk secretion in lactating rats

Ghrelin, a novel acylated peptide and endogenous ligand for growth hormone (GH) secretagogue receptor, was originally isolated from rat and human stomachs. In addition to its GH-releasing activity, ghrelin plays an important role in many physiological functions, including food intake, gastric acid secretion, neonatal development, and so on. In this study, the effect of daily treatment with ghrelin on milk production was investigated in lactating rats and the development of the pups was monitored. Daily subcutaneous injection of ghrelin into nursing dams for 8 days from parturition caused a significant increase in milk yield and litter weight gain. When litters nursed by ghrelin-treated and saline-treated dams were interchanged on day 4 of lactation, the growth curves were reversed. Daily injections of ghrelin also increased plasma GH levels. Northern blot analysis revealed that daily injection of ghrelin significantly increased mammary casein mRNA expression. In addition, RT-PCR analysis showed that a ghrelin receptor was present in the mammary glands of lactating rats. These results suggest that ghrelin may play an important role in milk production in lactating dams.     

Effects of serum on mammary epithelial proliferation in vitro during mammary gland development

The proliferative response of mammary gland epithelium from nonpregnant, pregnant, and lactating mice to mammary serum factor and insulin was studied in vitro. Mammary gland epiithelium from nonpregnant and lactating animals has a delayed proliferative response to mammary serum factor and insulin when compared to the response of epithelium from pregnant animals. The results show that as the animals go through pregnancy into lactation the mammary gland epithelium becomes less responsive to mammary serum factor while it retains its responsiveness to insulin. The concentration of mammary serum factor in sera from animals at various physiological stages is constant. Sera from hypophysectomized rats, on the other hand, show a 50% drop in mammary serum factor activity. This loss of activity cannot be reversed by injecting prolactin, 17-beta-estradiol, or growth hormone into the hypophysectomized animals. A hypothesis that the mammary gland is composed of two proliferative epithelial populations is developed, and the possible role of prolactin in stimulating DNA synthesis is discussed.     

Lactational [correction of Lacational] anovulation in rats and its dependency on progesterone

The relationship between prolactin (PRL) secretion and anovulation in lactating rats was studied. Normal lactating rats and lactating rats treated with antiserum against luteinizing hormone-releasing hormone at the time of postpartal ovulation were used. Normal lactating rats were treated with either a dopamine agonist (CB-154, 150 micrograms/rat) on Day 10 or 13, or pups removal on Day 7 or 10, and thereafter luteolysis and inhibition on PRL secretion were assessed. With the CB-154 treatment, the incidence of luteolysis increased as the lactational period advanced (42% vs 72%), whereas it decreased (73% vs 14%) with the pups removal. Thus, dopamine effectively inhibited PRL secretion during the later lactational stage, but could not do so during the earlier stage when there were mechanisms other than dopamine stimulating PRL secretion. Following luteal regression induced by CB-154, ovulation did not occur if the rats were treated with CB-154 on Day 10, whereas 50% of the rats ovulated within 4 days if treated on Day 13. Furthermore, in the lactating rats treated with anti-luteinizing hormone-releasing hormone serum during late pregnancy, ovulation was not observed until Day 10 of lactation. Since the serum progesterone levels were low in these rats due to the absence of ovulation and lactational corpora lutea, the blockade of ovulation was not due to elevated circulating progesterone during the early lactational period. The mechanism of ovulation blockade during lactation thus seems to shift from being progesterone independent to progesterone dependent at a similar period when the neuroendocrine control of PRL secretion shifts from dopamine independent to dependent.     

Alterations in response of rat white adipocytes to insulin, noradrenaline, corticotropin and glucagon after adrenalectomy. Correction of these changes by adenosine deaminase.

1. Adipocytes isolated from rats 6--9 days after adrenalectomy had significantly increased sensitivity to insulin action against noradrenaline-stimulated lipolysis. In the presence of adenosine deaminase there was no significant difference in insulin sensitivity between cells from adrenalectomized and sham-operated rats. 2. Adipocytes from adrenalectomized rats had decreased lipolytic responses to all concentrations of noradrenaline and glucagon tested and a decreased lipolytic response to low but not high concentrations of corticotropin. There was no difference in lipolytic response to theophylline after adrenalectomy. Adenosine deaminase corrected the differences in response to noradrenaline and glucagon resulting from adrenalectomy. 3. In the presence of adenosine deaminase rates of lipolysis, after stimulation by high concentrations of noradrenaline, glucagon, corticotropin or theophylline, were the same in cells from adrenalectomized or sham-operated rats. 4. These findings and previously reported effects of adenosine and adrenalectomy on adipocyte function are discussed. It is proposed that changes in adipocyte hormone responsiveness after adrenalectomy may result from changes in adenosine metabolism or release.     

氟中毒抑制大鼠泌鼠的实验研究

The effect of fluorosis on lactation, lactotroph function and ultrastructure were studied in lactating rats. The results were as follows: 1) Inhibition of lactation in lactating rats with chronic fluorosis was assessed by stunting growth of pups and decrease in the amount of milk suckled by pups in 30 min. Metoclopramide, a blocker of dopamine receptor, could improve lactation in these rats. 2) During chronic fluorosis serum PRL level was decreased, however, PRL content in pituitary was increased. Electronmicroscopic examination showed accumulation of large mature secretory granules and appearance of extremely large abnormal secretory granules in lactotroph cytoplasma. These findings indicate that hormone release of pituitary lactotrophs is obstructed in lactating rats with fluorosis, and the toxic effect of fluoride is mediated by an enhanced function of dopaminergic system in hypothalamus.     

Adenosine and the control of lipolysis in rat adipocytes during pregnancy and lactation.

The rate of noradrenaline-stimulated lipolysis is lower in fat-cells from lactating than from pregnant rats; this difference is eliminated by the addition of adenosine deaminase [Aitchison, Clegg & Vernon (1982) Biochem. J. 202, 243-247]. The activity of 5'-nucleotidase, and hence the capacity of the cells to synthesize adenosine, was the same in fat-cells and also stromal cells of adipose tissue from pregnant, lactating and male rats. The response and sensitivity of fat-cells to the anti-lipolytic effects of adenosine were measured by incubating cells in the presence of noradrenaline, adenosine deaminase (to remove endogenous adenosine) and various concentrations of the adenosine analogue N6-phenylisopropyladenosine (PIA). PIA caused a greater inhibition of the rate of noradrenaline-stimulated lipolysis in adipocytes from lactating than from pregnant rats. The concentration of PIA required to inhibit by 50% the rate of noradrenaline-stimulated lipolysis fell from over 100 nM for fat-cells from pregnant rats to 30 nM for fat-cells from lactating rats. The decreased rate of noradrenaline-stimulated lipolysis during lactation was not due to the smaller mean cell volume of adipocytes during this state.     

The ability of prolactin to change the sensitivity of the pituitary of the lactating rat to luteinising hormone releasing hormonein vitro

The ability of prolactin to influence the responsiveness of the lactating rat pituitary to luteinising hormone releasing hormone has been examinedin vitro. The pituitary responsivenessin vivo to luteinising hormone releasing hormone decreased as a function of increase in the lactational stimulus. Prolactin inhibited the spontaneousin vitro release of luteinising hormone and follicle stimulating hormone to a small extent, from the pituitary of lactating rats with the suckling stimulus. However, it significantly inhibited the release of these two hormones from luteinising hormone releasing hormone-stimulated pituitaries. The responsiveness of pituitaries of rats deprived of their litter 24 h earlier, to luteinising hormone releasing hormone was also inhibited by prolactin, although minimal. It was concluded that prolactin could be influencing the functioning of the pituitary of the lactating rat by (a) partially suppressing the spontaneous release of gonadotropin and (b) inhibiting the responsiveness of the pituitary to luteinising hormone releasing hormone.     

Plasma concentrations of immunoreactive calcitonin in rats during lactation

A mild hypocalcemia (0.5 mg/dl) is observed in rats after 14 days of lactation, but plasma and thyroidal calcitonin (CT) levels are both increased on day 7 of lactation. Plasma CT levels are higher (x2) in lactating females than those found in virgin females from day 7 to the end of lactation (21 days). In vitro, the CT secretion rate after calcium stimulation(3 mM) is not different between lactating and virgin females. The rapid removal of pups after parturition in control females induces a rebound in plasma calcium (+1.4 mg/dl) and plasma phosphate (+1.6 mg/dl) associated with elevated plasma CT values. Our results suggest, that the transient mild hypocalcemia of lactation is preceded by increased plasma CT levels; and that it is not the cause of elevated plasma parathyroid hormone levels already reported by us.     

Increased glucagon receptors in chronically hypersomatotrophic and hyperglucagonemic rats

The effect of increased levels of growth hormone on glucagon binding by isolated hepatocytes and on the cellular cyclic AMP response to glucagon was evaluated in rats bearing growth hormone-secreting tumor (Mt-T-W15) and in rats treated with rat growth hormone. An increased binding, due to an increased number of receptors, was observed in both groups of animals. Glucagon binding did not correlate with plasma glucagon levels, suggesting a failure of down regulation, possibly due to an effect of growth hormone and insulin on the number of receptors. Tumor-bearing and growth hormone-treated rats had larger hepatocytes so that, when hormone binding was expressed in terms of square micrometer of membrane surface, it appeared decreased. When the tumor was removed the increase in the number of glucagon receptors per cell persisted, even though the average cell size returned toward normal. It is suggested that this retention of the receptors may have been the result of continuing hyperinsulinism. Basal cAMP levels were elevated in hepatocytes of tumor-bearing and growth hormone-treated animals, possibly due to cell hypertrophy. On the other hand, the maximum cAMP response to glucagon was not altered by the experimental procedures. A negative effect of insulin on cAMP accumulation may explain this apparent paradox. Indeed, hepatocytes isolated from rats following tumor removal, but with continuing hyperinsulinemia, had a lower maximum cAMP response, even though the glucagon binding per cell or per unit of cell surface was increased.     

Reduction of litter size during lactation in rats greatly influences fatty acid profiles in dams

Journal of Physiology and Biochemistry - This study aimed to determine in lactating rats how fatty acid profiles are affected by litter size. On day 2 after parturition, litters of lactating rats...     

Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro.

1. The turnover rate of L-[1-14C]leucine was increased by 35% in lactating rats compared with virgin rats. Starvation or removal of pups (24 h) returned the value to that of the virgin rat. 2. Incorporation of L-[U-14C]leucine into lipid and protein of mammary glands of lactating rats in vivo increased 7-fold and 6-fold respectively compared with glands of virgin rats. Lactation caused no change in the incorporation of L-[U-14C]leucine into hepatic lipid and protein. 3. The production of 14CO2 from L[l-14C]leucine (in the presence of glucose) was similar in isolated acini from glands of fed (chow) and starved lactating rats. Feeding with a 'cafeteria' diet caused a slight decrease, and removal of pups a large decrease, in the oxidative decarboxylation of leucine. 4. Oxidation of L-[2-14C]leucine to 14CO2 was increased about 3-fold in acini from starved lactating rats or lactating rats fed on a 'cafeteria' diet compared with rats fed on a chow diet. Insulin decreased the formation of 14CO2 in all three situations. 5. Incorporation of L-[U-14C]- and [2-14C]-leucine into lipid was decreased in acini from starved lactating rats and lactating rats fed on a 'cafeteria' diet. Insulin tended to increase the conversion of [2-14C]leucine into lipid, but this was significant only in the case of the acini from 'cafeteria'-fed rats. 6. Experiments with (-)-hydroxycitrate indicate that the major route for conversion of leucine carbon into lipid in acini is via citrate translocation from the mitochondria. 7. The physiological implications of these findings are discussed.     

The effect of d-fenfluramine on anterior pituitary hormone release in the rat: in vivo and in vitro studies

Administration of d-fenfluramine, a serotonin-releasing drug, to male rats induced a dose-dependent increase in both serum prolactin and corticosterone concentrations. Serum growth hormone levels increased, but not significantly, at a dose of 1.25 mg/kg i.p. and decreased significantly at higher doses. When rats were pretreated with the serotonin uptake inhibitor fluoxetine (10 mg/kg i.p.) 30 min prior to injection of d-fenfluramine (5 mg/kg i.p.), the serum prolactin response to d-fenfluramine was partially inhibited, whereas the growth hormone response was not significantly modified. Fluoxetine pretreatment increased the serum corticosterone to the same level as did d-fenfluramine. d-Fenfluramine's effect on prolactin and growth hormone release was further tested in a hypothalamic-pituitary in vitro system. The addition of d-fenfluramine (5-500 ng/mL) for 30 min to rat hypothalami resulted in an enhancement of prolactin and growth hormone-releasing activities. These were expressed as the ability of the media in which the hypothalami had been incubated to stimulate prolactin and growth hormone release by cultured pituitary cells. The data suggest that the effect of d-fenfluramine on prolactin secretion is exerted through the hypothalamus and is probably mediated, at least partially, by a serotoninergic mechanism. The mechanism of d-fenfluramine's effect on corticosterone and growth hormone release needs further evaluation.     

Brain angiotensinergic mediation of enhanced water consumption in lactating rats

The mechanism by which lactating rats increase fluid consumption to meet the demands of milk production is unknown. Because ANG II is the most potent dipsogenic stimulus known, this study examined whether angiotensinergic signaling plays a role in enhanced drinking in lactating rats. ANG II administered intracerebroventricularly caused a significantly greater dipsogenic response in lactating rats than in control rats, suggesting that dipsogenic responsivity to ANG II is enhanced in the brains of lactating rats. The angiotensin type 1 (AT1) ANG II receptor subtype antagonist SKF-108566, also given intracerebroventricularly, caused a significant reduction in water consumption in lactating rats, whereas it did not significantly affect water intake in control rats. In contrast, stimulation of drinking by the muscarinic agonist carbachol, also administered intracerebroventricularly, did not differ between lactating and control rats. Inhibition of drinking by the muscarinic antagonist atropine also did not differ significantly between lactating and control rats. These results suggest that the increased drinking in lactating rats involves an increased responsivity to ANG II in neurons that mediate dipsogenesis, as well as an enhancement in the amount of angiotensinergic input to these ANG II-responsive neurons.     

Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling

Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.     

Dynamic of the GRF-induced GH response in genetically obese Zucker rats: influence of central and peripheral factors

To determine the time onset of the growth hormone (GH) alteration in the genetically obese rat, we studied the in vivo and in vitro rat growth hormone releasing factor (rGRF(1-29)NH2)-induced GH secretion in 6- and 8-week-old lean and obese male Zucker rats. Under sodium pentobarbital anesthesia, rGRF(1-29)NH2 (GRF) was injected intravenously at two doses: 0.8 and 4.0 micrograms/kg b.w. Basal serum GH concentrations were similar in lean and obese age-matched animals. The GH response to both GRF doses tested was unchanged in 6-week-old obese rats as compared to their lean litter mates. In contrast, a significant decrease of the GH secretion in response to 4.0 micrograms/kg b.w. GRF was observed in the 8-week-old obese rats. The effect of GRF (1.56, 6.25 and 12.5 pM) was further studied in vitro, in a perifusion system of freshly dispersed anterior pituitary cells of lean and obese Zucker rats. Basal GH release was similar in the 6-week-old animal group. In contrast, it was significantly decreased in 8-week-old obese rats as compared to their lean litter mates. Stimulated GH response to 1.56 and 6.25 pM GRF was significantly greater in the 6-week-old obese group than in the age-matched control group. In contrast, the GH response to all GRF concentrations tested was significantly decreased in the 8-week-old obese rats as compared to their respective lean siblings. In 8-week-old obese rats, a decrease of GH pituitary content and an increase of hypothalamic somatostatin (SRIF) concentration were observed. Insulin and free fatty acid serum were significantly increased in 8-week-old obese rats. In contrast, lower insulin-like growth factor I serum levels were observed in the obese animals as compared to their lean litter mates. Finally, to further clarify the role of the periphery in the inhibition of GH secretion observed in the 8-week-old fatty rats, we exposed cultured pituitary cells of 8-week-old lean animals to 17% serum of their obese litter mates. A significant decrease of GRF-stimulated GH secretion of lean rat pituitary cells exposed to the obese serum was noted (P less than 0.05). This study demonstrates that, in the obese Zucker rat, an alteration of the GH response to GRF is evident by the 8th week of life. This defective GH secretion could be related to peripheral and central abnormalities.     

Effects of estradiol benzoate and progesterone on superoxide dismutase activity in the thymus of rats

The activity of mitochondrial superoxide dismutase (MnSOD) and cytosol superoxide dismutase (CuZnSOD) was measured in corresponding subcellular fractions prepared from the thymi of intact and chronically gonadectomized (GX) rats of both sexes, as well as of GX male and female rats injected subcutaneously with a single dose of 5 microg estradiol benzoate (EB) and/or 2 mg progesterone (P). Animals were sacrificed 2 h or 24 h following hormone treatment. In the females, the activity of MnSOD in the thymus was stable during the estrous cycle and did not change after ovariectomy. Treatment of GX females with estradiol benzoate resulted 2 h later in a significant elevation of MnSOD activity, whereas 24 h later the activity returned back to control values. On the other hand, treatment of GX females with progesterone had no effect on the MnSOD activity. However, combined hormone treatment, in which EB injection preceded progesterone injection by one hour, enhanced the effect on MnSOD activity similar to that of estradiol benzoate alone. The activity of CuZnSOD in cycling rats was increased in proestrus, whereas removal of the ovaries kept the values at low diestrus and estrus levels. Contrary to MnSOD, CuZnSOD activity did not change after EB treatment of GX females, while progesterone increased the enzyme activity at 2 h and 24 h after hormone treatment. However, combined EB+P treatment proved to be ineffective. In the males, neither MnSOD nor CuZnSOD activity was affected by the removal of testes or by progesterone treatment of GX animals. Only EB injection to GX rats significantly increased CuZnSOD activity 24 h later.     

Modulation of long-term memory and extinction responses induced by growth hormone (GH) and growth hormone releasing hormone (GHRH) in rats

The purpose of this work is to study the participation of growth hormone (GH) and growth hormone releasing hormone (GHRH) in the modulation of long-term memory and the extinction response of a passive avoidance task in rats. However, the effect on memory vary according to the age of the animals due to plasma levels of either hormone being modified during the aging process. Male Wistar rats were divided according to age into two experimental blocks (young rats 3 months old and aged rats 24 months old at the start of the experiment) where each block received the same treatment. Each experimental block was then divided randomly into three groups where two were experimental and the other served as control. The animals were then submitted to a one-trial passive avoidance conditioning and tested for memory retention 24 hrs after as well as twice a week until the extinction response occurred. The control group received an isotonic saline solution and the other two groups received 0.8 U.I. Of GH or 4 mcg of GHRH respectively. All substances were in a 0.08 ml volume and applied 24 hrs before training as well as 24 hrs before each retention session. The results indicate that GH and GHRH modulate longterm memory as well as the extinction response and in either case the response seems to vary with age. GH and GHRH facilitates long-term memory in young rats but not in aged rats. Finally, whereas GH delays the extinction response in both groups, GHRH retards the extinction only in aged rats.     

Can a mouse be the object of maternal aggression on the part of a rat?

It has been found that mice do not evoke interspecies maternal aggression in lactating female rats even if the latter kill little rats of their litter. Putting mice in cages with rats bearing for the first time, and their progeny for 24 hours after labor, increases the frequency of infanticide and progeny leaving by female rats. The infanticide by lactating rats towards their own litter does not depend on whether these females exhibit muricid or not. Putting mice in cages with rats 3-6 days before bearing for the first time or during 24 hours after labor by repeatedly bearing rats does not influence the frequency of progeny leaving and infanticide reactions of female rats.     

Hormonal and Metabolic Studies in Pheochromocytoma

One patient with benign and another with malignant pheochromocytoma have been studied in an attempt to elucidate the effect of increased catecholamines on the response of blood sugar, unesterified fatty acids, insulin and growth hormone to a glucose load. The presence of increased catecholamines in both patients appeared to produce increased fasting plasma unesterified fatty acid levels, carbohydrate intolerance and an unusual plasma growth hormone response to glucose. There was no interference with the normal decrease in plasma unesterified fatty acids after glucose ingestion. The malignant tumour, but not the benign one, was associated with low plasma insulin levels.After removal of the benign tumour the fasting unesterified fatty acid levels became normal. In addition, following glucose ingestion there appeared to be a more normal plasma insulin and growth hormone response and improved glucose tolerance. After removal of the primary malignant tumour it is noteworthy that the distant metastases secreted abnormal amounts of both adrenaline and noradrenaline.