Hypothalamic paraventricular nucleus, but not vasopressin, participates in chronic hyperactivity of the HPA axis in diabetic rats

Diabetes mellitus (DM), as chronic stress activates the hypothalamo-pituitary-adrenocortical axis. We examined whether arginine vasopressin (AVP) and the hypothalamic paraventricular nucleus (PVN) participate in DM-induced chronic stress symptoms. AVP-deficient Brattleboro or PVN-lesioned Wistar rats were used with heterozygous or sham-operated controls. The rats were studied 2 wk after a single injection of streptozotocin. The appearance of DM (enhanced water consumption and blood glucose elevation) and the chronic stress-like somatic changes (body weight decrease, thymus involution, adrenal gland hypertrophy) were not influenced by the lack of AVP. By contrast, PVN lesion significantly attenuated DM-induced thymus involution and adrenal gland hypertrophy as well as the increase in water consumption. The corticotropin-releasing hormone mRNA in PVN was diminished by DM and elevated by the lack of AVP without interaction. DM elevated the proopiomelanocortin (POMC) mRNA in the anterior lobe of the pituitary. The lack of AVP had no effect, whereas lesioning the PVN significantly diminished the elevation. The elevated basal corticosterone plasma levels detectable in DM were influenced neither by the lack of AVP nor by lesioning the PVN. Thus the lack of AVP had no influence on DM-induced chronic stress symptoms, but lesioning the PVN attenuated part of them. However, the lack of elevation in POMC mRNA after PVN lesion, together with the maintained corticosterone elevation, suggests that direct adrenal gland activation occurs in untreated DM.     

Effects of immobilization stress on tuberoinfundibular dopaminergic (TIDA) neuronal activity and prolactin levels in lactating and non-lactating female rats.

The effects of immobilization stress on the prolactin secretory response and on the activity of the tuberoinfundibular dopaminergic (TIDA) neurons were determined in intact, virgin female rats on the morning of diestrus or proestrus and in post-partum, lactating female rats. The virgin females exhibited a significant increase in circulating levels of prolactin which was evident by 1 minute and persisted during the immobilization (5 minutes). In contrast, the prolactin secretory response in lactating females was significantly attenuated compared to non-lactating animals. The activity of the TIDA neurons was not altered by the 5 minutes of stress. Even after 30 minutes of immobilization, TIDA neuronal activity was not affected in either the lactating or cycling females. These data suggest that the cycling female rat is capable of a prolactin secretory response to the stressor without inhibition of TIDA neuronal activity. It seems likely that prolactin releasing factors mediate this response. In contrast, stress did not produce a similar prolactin increase during lactation. It seems likely that, during lactation, the pituitary is not sensitive to releasing factors unless the TIDA neurons are inhibited. There appear to be differences in the sensitivity of the pituitary depending on the physiological state of the model employed.     

Lipolysis in rat adipocytes during pregnancy and lactation. The response to noradrenaline.

1. Lipolysis has been measured in parametrial adipocytes from virgin, pregnant and lactating rats. 2. The basal rate and the maximal rate of lipolysis, the latter measured in the presence of noradrenaline and theophylline, remained constant between the three experimental categories, with the exception of a significant transient increase in the basal rate at parturition. 3. The noradrenaline-stimulated lipolysis rate rose above the virgin rate during pregnancy and fell below it during lactation; inclusion of adenosine deaminase in incubations abolished these differences in response to noradrenaline. 4. Cyclic AMP phosphodiesterase activity was lower in adipocytes during pregnancy and lactation than in virgin animals.     

The effect of cholecystokinin, bombesin and calcitonin on food intake in virgin, lactating and postweaning female rats

During lactation food intake increases greatly without an accompanying large increase in body weight; therefore, this physiological state is an excellent example of non-obese hyperphagia. In the present study, we found that cholecystokinin (CCK-8) decreased food intake in lactating and virgin female rats. However, female rats were more resistant to the effect of CCK on eating following weaning of the pups. Bombesin (BB) suppressed food intake in virgin female rats and in lactating rats during early and mid lactation. Rats were resistant to its satiating effect during late lactation and during the postweaning period. Calcitonin potently suppressed food intake in virgin, lactating and postweaning rats. The present findings suggest that CCK and bombesin decrease food intake more effectively in virgin female rats and during earlier phases of lactation than during late lactation or postweaning.     

Absence of down-regulation of the insulin receptor by insulin. A possible mechanism of insulin resistance in the rat.

Insulin resistance occurs in rat adipocytes during pregnancy and lactation despite increased or normal insulin binding respectively; this suggests that a post-receptor defect exists. The possibility has been examined that, although insulin binding occurs normally, internalization of insulin or its receptor may be impaired in these states. Insulin produced a dose-dependent reduction in the number of insulin receptors on adipocytes from virgin rats maintained in culture medium, probably due to internalization of the hormone-receptor complex. In contrast, adipocytes from pregnant and lactating rats did not exhibit this 'down-regulation' phenomenon. Down regulation was, however, apparent in all groups when the experiments were performed in Tris buffer (where receptor recycling is inhibited), suggesting that in pregnant and lactating rats insulin receptors are rapidly recycled back to the plasma membrane, whereas in virgin rats this recycling process is less effective. Internalization of insulin was also determined by using 125I-labelled insulin. Adipocytes from pregnant and lactating rats appeared to internalize similar amounts of insulin to virgin rats. In the presence of the lysosomal inhibitor chloroquine, adipocytes from pregnant rats internalized more insulin than virgin or lactating rats. These results suggest that adipocytes from pregnant and lactating rats internalize insulin and its receptor normally, whereas intracellular processing of the insulin receptor may differ from that in virgin rats. In addition the rate of lysosomal degradation of insulin may be altered in adipocytes from pregnant rats.     

Synergistic Interactions between Cytokines and AVP at the Blood-CSF Barrier Result in Increased Chemokine Production and Augmented Influx of Leukocytes after Brain Injury

Several lines of evidence indicate that the blood-cerebrospinal fluid barrier (BCSFB), which primarily resides in the choroid plexus (CP), plays a significant pathophysiological role not only in neuroinflammatory diseases, such as multiple sclerosis, but also in traumatic brain injury (TBI). Here we investigated how arginine vasopressin (AVP) regulates function of the BCSFB in the context of post-traumatic neuroinflammation. It has previously been shown that AVP exacerbates various forms of brain injury, but the mechanisms underlying this AVP action are poorly understood. Type 1A AVP receptor is highly expressed on the CP epithelium and the CP synthesizes AVP. Using the controlled cortical impact model of TBI, we demonstrated decreased post-traumatic production of proinflammatory mediators by the CP and reduced influx of inflammatory cells across the BCSFB in AVP-deficient Brattleboro rats when compared with Long-Evans rats, a parental strain for Brattleboro rats. Arginine vasopressin was also found to play an important role in post-traumatic activation of c-Jun N-terminal kinase (JNK) in the CP. In the CP epithelial cell cultures, AVP augmented the tumor necrosis factor-α– and interleukin-1β–dependent increase in synthesis of proinflammatory mediators, including neutrophil chemoattractants, an action largely dependent on the JNK signaling pathway. Under in vivo conditions, a selective JNK inhibitor decreased the post-traumatic production of neutrophil chemoattractants by the CP and reduced the influx of neutrophils across the BCSFB. These results provide evidence for the synergistic interactions between proinflammatory cytokines and AVP, a ligand for G protein-coupled receptors, and support a pathophysiological role of AVP in post-traumatic neuroinflammation.     

Brown adipose tissue activity in hypocaloric-diet fed lactating rats

Hypocaloric diet feeding reduced the mitochondrial protein content and whole tissue GDP-binding in interscapular brown adipose tissue from both virgin and lactating rats. A reduction in brown fat lipoprotein lipase activity was also detected in underfed virgin and lactating animals. These results indicate that lactation in the rat, even though it produces a reduction in brown fat activity, does not impair the capacity of the tissue to respond to a diminished caloric intake by lowering its activity further.     

Reduced noradrenaline turnover in brown adipose tissue of lactating rats

Brown adipose tissue properties as well as noradrenaline turnover in the tissue were determined in 15-day lactating rats and virgin controls. Brown adipose tissue thermogenic activity was reduced in lactating rats as shown by a decrease in weight, cytochrome oxidase activity and mitochondrial GDP-binding. The noradrenaline turnover rate was lower in brown adipose tissue from lactating rats. It is suggested that diminished sympathetic activity in brown adipose tissue may be a major cause of the reduced tissue thermogenic activity during lactation.     

The role of food intake on gastric mucosal growth and gastrin receptors during pregnancy and lactation

We examined the effects of pregnancy and lactation on mucosal growth and the numbers and affinity of gastrin receptors in the oxyntic gland mucosa in rats and compared these with changes in serum gastrin levels and food consumption. Gastric mucosal DNA, RNA, and protein contents were significantly increased during lactation. These changes were not observed in either pregnant or nonlactating rats which had given birth at the same time as the lactating animals. The gastrin-binding capacity of a membrane fraction of the oxyntic mucosa was also increased at the corresponding periods in lactating rats (Days 7, 15, 20). Scatchard plot analysis revealed that the number of gastrin receptors was significantly increased without any change in affinity. Food consumption and levels of serum gastrin remained unaltered in pregnant and non-lactating rats compared to virgin controls, but were significantly increased in lactating rats. Increased serum gastrin levels and gastrin binding capacities in lactating rats (Day 15) were abolished by preventing increased food consumption by means of pair feeding. The results demonstrate that the number of gastrin receptors in the oxyntic mucosa increases during lactation in rats. This increase is probably due to hypergastrinemia caused by increased food intake. The increased number of gastrin receptors may be involved in the mechanism of hypertrophic responses of the gastric mucosa in lactating rats.     

Enhanced glucose tolerance in the Brattleboro rat

[Arg⁸]-vasopressin (AVP) plays a crucial role in regulating body fluid retention, which is mediated through the vasopressin V₂ receptor in the kidney. In addition, AVP is involved in the regulation of glucose homeostasis via vasopressin V_1A and vasopressin V_1B receptors. Our previous studies demonstrated that vasopressin V_1A receptor-deficient (V_1AR−/−) and V_1B receptor-deficient (V_1BR−/−) mice exhibited hyperglycemia and hypoglycemia with hypoinsulinemia, respectively. These findings indicate that vasopressin V_1A receptor deficiency results in decreased insulin sensitivity whereas vasopressin V_1B receptor deficiency results in increased insulin sensitivity. In addition, vasopressin V_1A and vasopressin V_1B receptor double-deficient (V_1ABR−/−) mice exhibited impaired glucose tolerance, suggesting that the effects of vasopressin V_1B receptor deficiency do not influence the development of hyperglycemia promoted by vasopressin V_1A receptor deficiency, and that the blockage of both receptors could lead to impaired glucose tolerance. However, the contributions of the entire AVP/vasopressin receptors system to the regulation of blood glucose have not yet been clarified. In this study, to further understand the role of AVP/vasopressin receptors signaling in blood glucose regulation, we assessed the glucose tolerance of AVP-deficient homozygous Brattleboro (di/di) rats using an oral glucose tolerance test (GTT). Plasma glucose and insulin levels were consistently lower in homozygous di/di rats than in heterozygous di/+ rats during the GTT, suggesting that the blockage of all AVP/vasopressin receptors resulting from the AVP deficiency could lead to enhanced glucose tolerance.     

Amino acid metabolism and protein synthesis in lactating rats fed on a liquid diet.

1. Amino acid metabolism was studied in control virgin rats, lactating rats and virgin rats protein-pair-fed with the lactating rats (high-protein virgin rats). 2. Urinary excretion of nitrogen and urea was higher in lactating than in control virgin rats, and in high-protein virgin rats it was higher than in lactating rats. 3. The activities of urea-cycle enzymes (units/g) were higher in high-protein virgin than in lactating rats, except for arginase. In lactating rats the activities of carbamoyl-phosphate synthase, ornithine carbamoyltransferase and argininosuccinate synthase were lower than in control virgin rats. When the liver size is considered, the activities in lactating rats were similar to those in high-protein virgin rats, except for arginase. 4. N-Acetylglutamate content was higher in high-protein virgin rats than in the other two groups. 5. The rate of urea synthesis from precursors by isolated hepatocytes was higher in high-protein virgin rats than in the other two groups. 6. The flooding-dose method (L-[4-3H]phenylalanine) for measuring protein synthesis was used. The absolute synthesis rates of mammary gland, liver and small-intestinal mucosa were higher in lactating rats than in the other two groups, and in high-protein virgin rats than in control virgin rats 7. These results show that the increased needs for amino acids during lactation are met by hyperphagia and by a nitrogen-sparing mechanism.     

Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation

We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk. The observed changes indicate an active role of HSL in mammary gland lipid metabolism.     

Cardiovascular effects of neuropeptide Y in the nucleus tractus solitarius of rats: relationship with noradrenaline and vasopressin

The central haemodynamic effects of neuropeptide Y (NPY), both alone and together with either noradrenaline (NA) or vasopressin (AVP), have been investigated by microinjecting synthetic peptide into the nucleus tractus solitarius (NTS) of anaesthetized rats. NPY alone elicited dose-dependent changes in blood pressure (BP) and heart rate (HR); 470 fmol inducing a pressor response, and 4.7 pmol a fall in BP. The hypotensive response to 20 nmol NA was significantly modified by both simultaneous and prior injection of an ineffective dose (47 fmol) of NPY. Prior injection of a similar dose of NPY also modified the NTS pressor effect of 10 ng AVP. A relationship between the action of AVP and NPY in the NTS was further indicated by the finding that prior injection of an ineffective dose of AVP (1 ng) reduced the hypotensive response to 4.7 pmol NPY, and by the demonstration of contrasting effects of 4.7 pmol NPY in AVP-deficient Brattleboro rats compared to parent strain LE rats. These results, taken together with the recent localization of NPY-like immunoreactivity in the NTS, suggest a role for NPY in central cardiovascular control. In addition, NPY has been shown to exhibit functional interactions with both an amine neurotransmitter and a neuropeptide present in the NTS of rats.     

Impaired basal and noradrenaline-induced iodothyronine 5'-deiodinase activity in brown adipose tissue from pregnant and lactating rats

Basal iodothyronine 5'-deiodinase activity is lowered in brown fat from 20-day pregnant, 5 and 15-day lactating rats when compared with virgin controls. Acute noradrenaline treatment caused a seven fold increase in 5'-deiodinase activity in brown fat from virgin control rats. Late pregnant and lactating rats showed a reduction in noradrenaline-induced 5'-deiodinase activity in brown adipose tissue and the maximum impairment was observed in 15-day lactating rats. Lowered 5'-deiodinase activity in brown fat during late pregnancy and lactation correlates with the known reduction in the thermogenic activity of the tissue during these situations and agrees with the proposal that the rate of 3,5,3'-triiodothyronine generated in situ because of thyroxine 5'-deiodination could be an essential event related to thermogenesis in brown fat. Even though the relationship between 3,5,3'-triiodothyronine generation in the tissue and the specific thermogenic mechanisms of brown fat is unknown, present results indicate a close link between the thermogenic and 5'-deiodinase activities in physiological situations when brown adipose tissue needs to adapt to a low activity, such as that of the breeding cycle.     

Psychological stressors as a model of maternal adversity: diurnal modulation of corticosterone responses and changes in maternal behavior

Maternal adversity is associated with long-lasting consequences on cognitive development, behavior and physiological responses in rat offspring. Few studies have examined whether repeated maternal stress produces repeated activation of the hypothalamus-pituitary-adrenal (HPA) axis in mothers and whether it modifies maternal behavior. Here, we tested a novel model of perinatal stress using repeated exposure to "purely" psychological stressors throughout the gestation and lactation periods in rats. We first tested the diurnal influences of repeated 1-h strobe light exposure on maternal corticosterone secretion. Despite the hyporesponsiveness to stress documented in late pregnant and lactating mothers, we observed an enhanced response to strobe light in the afternoon compared to the morning in stressed mothers during lactation. Next, dams were exposed to 24-h forced foraging followed by 10-h wet bedding during the diurnal peak of corticosterone secretion. Although no corticosterone responses to forced foraging and wet bedding were observed, the combination of both stressors had a significant effect on maternal behavior. Mother-pup interactions were significantly altered during the first 8 days of lactation. Taken together, these findings suggest that lactating mothers maintain responsiveness to specific and repeated psychological stressors, in particular at the time of the diurnal peak in corticosterone secretion. Depending on the stressor applied, either neuroendocrine activation or changes in maternal behavior might be important determinants of the long-term consequences in the offspring. The combination of forced foraging, wet bedding and strobe light might represent a novel model of mild maternal adversity using "purely" psychological stressors.     

Lack of effect of oxytocin on the numbers of “synaptic” ribbons,cyclic guanosine monophosphate and serotonin N-acetyltransferase activity in organ-cultured pineals of three strains of rats

In addition to the stimulating influence of the sympathetic system on the function of the mammalian pineal gland, neuropeptides such as neuropeptide Y, vasoactive intestinal polypeptide and arginine-vasopressin (AVP) are thought to function as modulators. Since AVP has been shown to influence pineal melatonin synthesis, the aim of the present study was to investigate the possible effects of the second hypothalamic nonapeptide oxytocin (OT), which likewise has been detected in the pineal gland. We therefore studied synaptic ribbon (SR) numbers, N-acetyltransferase (NAT) activity and the intracellular concentration of cyclic guanosine monophosphate (cGMP) following in vitro incubation of rat pineals in media containing OT (10^-5 M), noradrenaline (NA, 10^-5 M) or both NA and OT. Pineal glands were derived from rats of three different strains (Sprague-Dawley, Long-Evans and the AVP-deficient strain Brattleboro). Neither morphological nor biochemical analyses showed a difference between control and OT-incubated organs in any of the strains tested. In Brattleboro rats, but not in the other strains, noradrenaline slightly increased the number of SR which was not observed when NA and OT were combined. The addition of NA resulted in distinct augmentation of NAT activity and cGMP content, which were not affected by additional OT application. These results suggest that oxytocin is not crucially involved in the regulation of pineal gland function.     

Hormonal regulation of retinoic acid-binding proteins in the mammary gland.

A cytosolic retinoic acid-binding (RAB) protein that sediments specifically as a 2S component on sucrose density gradients was detected in the mammary glands of virgin, pregnant and lactating rats. Mammary cytosol from pregnant rats contained significantly higher concentrations of cytosolic RAB protein than did cytosol from either virgin or lactating rats. The glands of pregnant animals exhibited increased concentration of cytosolic RAB protein during the first 5 days of pregnancy, and a steady decline was observed thereafter. The concentration of cytosolic RAB protein dropped to the value observed during lactation on the day 20 of pregnancy. Moreover, throughout lactation, low concentrations of cytosolic RAB protein were maintained. Daily treatment of virgin and lactating animals with 5 micrograms of oestradiol-17 beta for 1 week increased cytosolic RAB protein to concentrations comparable with those seen in pregnant rats. Progesterone, however, did not affect the mammary cytosolic RAB protein content of virgin rats. These results suggest hormonal involvement in the regulation of cytosolic RAB protein concentration of mammary gland during differentiation.     

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.     

Food restriction selectively increases hypothalamic orexin-B levels in lactating rats

Orexins are hypothalamic peptides implicated in the regulation of ingestive and other behaviours. Here we investigated prepro-orexin expression and hypothalamic orexin-A and -B levels in lactating rats, which display marked hyperphagia, with or without food restriction for 2 days or treatment with bromocriptine, which inhibits milk production and thus reduces the energy losses of lactation. Neither prepro-orexin gene expression nor hypothalamic orexin-A peptide levels were changed in any of these lactating groups compared with age-matched virgin controls. However, hypothalamic orexin-B levels were significantly higher in lactating rats that were food-restricted for 2 days (P<0.05) compared with non-lactating controls and with lactating rats that were either freely-fed or bromocriptine-treated. Thus, food restriction superimposed on lactation selectively increases hypothalamic orexin-B levels, suggesting that orexin-A and -B may be differentially released or cleared. Changes in orexin-B availability may influence physiological activities other than energy homeostasis, perhaps inducing arousal.     

Maternal condition reduces fear behaviors but not the endocrine response to an emotional threat in virgin female rats

Lactating dams and maternal virgin females are less fearful in behavioral tests compared with non-maternal animals, suggesting that maternal condition per se reduces the negative value of threatening stimuli. In addition, lactating females exhibit a diminished hypothalamic-pituitary-adrenal response to potential environmental threats. Can the maternal condition, independently of the endocrine profile of lactation, promote a reduction in the behavioral as well as in the endocrine response to an emotional stressor? To answer this question, anxiety-related and fear behaviors as well as the levels of corticosterone were evaluated in response to a bright-lit open field-loud noise model in maternal and non-maternal non-ovariectomized virgin females and lactating dams in the presence of the pups. Maternal animals, both lactating and virgin, presented an increased exploration of the bright-lit open field and a significant reduction of fear behaviors, indicated by the decreased flight and immobility responses to the subsequent activation of a loud noise, in comparison to non-maternal virgins. Interestingly, maternal virgin females, as non-maternal rats, showed high corticosterone plasma levels, in contrast to the lower endocrine response exhibited by lactating dams when confronted to this threat. Present results suggest that maternal condition allows females to take risks when caring for their young, a behavioral strategy that is independent of the reduced hypothalamic-pituitary-adrenal axis response characteristic of lactation. This evidence points towards a clear dissociation in the mechanisms regulating behavioral and endocrine responses to emotional stressors during motherhood.