CART knock out mice have impaired insulin secretion and glucose intolerance, altered beta cell morphology and increased body weight

CART peptides are anorexigenic and are widely expressed in the central and peripheral nervous systems, as well as in endocrine cells in the pituitary, adrenal medulla and the pancreatic islets. To study the role of CART in islet function, we used CART null mutant mice (CART KO mice) and examined insulin secretion in vivo and in vitro, and expression of islet hormones and markers of beta-cell function using immunocytochemistry. We also studied CART expression in the normal pancreas. In addition, body weight development and food intake were documented. We found that in the normal mouse pancreas, CART was expressed in numerous pancreatic nerve fibers, both in the exocrine and endocrine portion of the gland. CART was also expressed in nerve cell bodies in the ganglia. Double immunostaining revealed expression in parasympathetic (vasoactive intestinal polypeptide (VIP)-containing) and in fewer sensory fibers (calcitonin gene-related peptide (CGRP)-containing). Although the expression of islet hormones appeared normal, CART KO islets displayed age dependent reduction of pancreatic duodenal homeobox 1 (PDX-1) and glucose transporter-2 (GLUT-2) immunoreactivity, indicating beta-cell dysfunction. Consistent with this, CART KO mice displayed impaired glucose-stimulated insulin secretion both in vivo after an intravenous glucose challenge and in vitro following incubation of isolated islets in the presence of glucose. The impaired insulin secretion in vivo was associated with impaired glucose elimination, and was apparent already in young mice with no difference in body weight. In addition, CART KO mice displayed increased body weight at the age of 40 weeks, without any difference in food intake. We conclude that CART is required for maintaining normal islet function in mice.     

Prolactin secretion after hypothalamic deafferentation in beef calves: response to haloperidol, alpha-methyl-rho-tyrosine, thyrotropin-releasing hormone and ovariectomy

In ruminant species photoperiod regulates prolactin (PRL) secretion. It is hypothesized that the inhibition of PRL secretion resides in dopaminergic neurons of the medial basal hypothalamus (MBH). To test this hypothesis, anterior (AHD), posterior (PHD) and complete (CHD) hypothalamic deafferentation and sham operation control (SOC) surgeries were carried out during May (long-day photoperiod) in beef heifer calves (6-8 mo old) to measure basal PRL secretion and PRL secretion as affected by intravenous secretagogues. On the day of surgery (day 0), PRL secretion reflected stress of anesthesia and surgery in all groups. Thyrotropin-releasing hormone (TRH), alpha-methyl-rho-tyrosine (alphaMrhoT), and haloperidol (HAL) was iv injected on days 11, 13 and 15, respectively. AHD, PHD, CHD, and SOC calves responded to TRH (100 microg) with an acute increase in PRL that peaked within 20 min. All heifers responded to alphaMrhoT (10 mg/kg BW) with an acute elevation in PRL within 10 min and remaining elevated for 3 h. HAL (0.1 mg/kg BW) induced an acute increase in PRL secretion in all groups, peaking within 15-30 min. Seven months later (December, short-day photoperiod) these heifers were ovariectomized. Basal plasma PRL levels were seasonally low, PRL secretion in AHD, PHD and CHD animals abruptly increased within 15 min to iv injection of 100 microg TRH to a greater amount than seen in SOC heifers. Although a biphasic effect on PRL secretion entrains under long-day and short-day photoperiods, hypothalamic deafferentation in cattle did not affect the pituitary gland's responsiveness to secretagogues.     

Involvement of hypothalamic dopamine in the regulation of prolactin secretion

The neuroendocrine control of prolactin (PRL) secretion is known to be a multifactorial process, but dopamine (DA) secreted by the tuberoinfundibular dopaminergic (TIDA) neurons of the hypothalamus is believed to exert a predominant inhibitory control on the secretion of PRL. The secretory activity of the TIDA neurons, including the rate of biosynthesis of DA and the rate of release of the neurohormone into hypophysial portal blood, can be readily evaluated in the rat. In most conditions in which an altered secretion of PRL has been documented, an altered secretory activity of the TIDA neurons has been found. When an acute reduction in the secretion of DA is observed, an increased secretion of PRL is associated, with an inverse relationship between DA and PRL concentrations in hypophysial portal and systemic blood, respectively. However, the secretion of PRL can be regulated by PRL itself through stimulation of the secretory activity of the TIDA neurons, and consequently hyperprolactinemia can be observed concomitantly with a sustained high secretion of DA, as seen after treatment with estrogen. The short loop feedback of PRL secretion seems to be impaired in the aging rat, since a sustained reduced hypothalamic secretion of DA is observed in spite of long-term hyperprolactinemia.     

The effect of aging on prolactin secretion in rat pituitary monolayer culture.

A high basal rate of prolactin (PRL) secretion (.16±.03 μg/well/hr) was produced for over four weeks by pre-confluent male rat pituitary monolayer cell cultures. When the media was changed, a rapid release of microgram quantities of PRL occurred followed by a return to the basal PRL secretory rate by seven hours. Theophylline (3.8×10^?3M), but not dibutyrl cAMP (1×10^?3M), produced a significant (p<.02) increase in PRL secretion, and simultaneous addition of these agents potentiated the PRL secretory rate. TRH (2×10^?8M) had no effect on PRL release by six hours, whereas dopamine (4.9×10^?5M) produced a significant suppression (p<.002) of PRL secretion. In addition, the effects of theophylline, TRH, and dopamine on PRL secretion were similar in cultures of various ages. Ovine prolactin in concentrations up to 50 μg per ml produced no change in PRL secretion during 72 hours of incubation suggesting that PRL feedback control of its own secretion may be transmitted via the hypothalamus. These studies show that a high rate of PRL secretion can be maintained by pre-confluent monolayer cultures for extended periods of time, permitting repeated experimentation on the same wells.     

Transforming growth factor-beta and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system

Incubations of rat anterior pituitary cells with transforming growth factor (TGF)-beta 1 for 48 hr suppressed the secretion of basal prolactin (PRL) in a dose-dependent manner (ED50, 100 pg/ml). Activin, a gonadal hormone processing cysteine distribution similar to TGF beta, also suppressed basal PRL secretion, but it was less effective (ED50, 4 mg/ml). Treatment with TGF beta 1 significantly suppressed basal PRL secretion from the pituitary after 24 hr and up to 72 hr of incubation. TGF beta 1 also inhibited thyrotropin-releasing hormone-mediated PRL secretion and activin inhibited thyrotropin-releasing hormone-mediated PRL secretion slightly, but significantly. In addition, we also measured the secretion of growth hormone by cultured pituitary cells treated with TGF beta 1 or activin for 24 to 72 hr. TGF beta 1 and activin showed an opposite effect on growth hormone secretion; TGF beta stimulated and activin inhibited basal secretion of growth hormone. These results suggest that TGF beta 1 is a potent inhibitor of basal secretion of PRL by the pituitary, and both TGF beta 1 and activin play a multifunctional role in basal secretion of pituitary hormones.     

The physiology and mechanisms of the stress-induced changes in prolactin secretion in the rat

It is well known that stress in a number of forms induces the secretion of prolactin (PRL) in a number of species. What is not well known is that under certain conditions stress will also induce a decrease in PRL secretion. The conditions whereby stress decreases PRL are those where PRL secretion is elevated such as during the proestrous afternoon surge and during the nocturnal surge of pseudopregnancy. The physiologic significance of the stress-induced increase of PRL is suggested to be important in maintaining the competence of the immune system. The significance of the stress-induced decrease of PRL does not appear to have a major consequence on the physiology of reproduction in the rat and it is suggested that future studies be directed towards its significance in the immune system. The literature is reviewed dealing with the regulation of PRL secretion with emphasis on the factors that generate PRL surges in the rat. In addition the mechanism(s) of the stress-induced increase and decrease is (are) also examined. A hypothesis is presented suggesting an interaction between tuberoinfundibular dopamine secretion and a hypothalamic prolactin releasing factor in the generation of PRL surges and the differential effects of stress on PRL secretion.     

Prolactin after baclofen in healthy subjects and prolactinoma patients

Serum prolactin (PRL) levels in basal conditions (two samples) and 30, 60, 90, 120, 150 e 180 minutes after oral administration of baclofen (20 mg) were evaluated in 6 healthy subjects and in 10 patients with prolactinoma. The effect of baclofen (20 mg by mouth) on the PRL secretion cimetidine (400 mg i.v.) or domperidone (20 mg i.v.) induced were evaluated in 9 healthy women by administration of baclofen 60 minutes before cimetidine or domperidone. Baclofen was unable to significantly rise serum PRL levels in healthy subjects and in patients affected by prolactinoma and furthermore did not interfere with PRL rise domperidone induced. On the contrary baclofen decreased PRL rise cimetidine induced. It was concluded that: in basal condition, GABAb receptor don't play an obvious role in modulation of PRL secretion; when H2 istaminergic inhibition on PRL secretion is blocked (at an hypothalamic site), a GABA inhibition, b receptor mediated, on PRL secretion became more clear; the domperidone blockade of hypophysial dopaminergic receptors suggests that GABAb modulation of prolactin secretion don't obtain itself by dopaminergic pathways.     

Neuroendocrine regulation of prolactin secretion in adult female rhesus monkeys during different phases of the menstrual cycle: role of neuroexcitatory amino acid (NMA)

The present study attempts to examine the role of N-methyl-D, L-aspartate (NMDA) receptors in the central regulation of prolactin (PRL) secretion, which may be involved in ovarian function and its alteration by glutamate in various phases of the menstrual cycle of female rhesus monkeys (Macaca mulatta). The results suggest that the glutaminergic component of the control system, which governs PRL secretion by utilizing NMDA receptors, may have an important role in regulating changes in PRL secretion. The response of PRL during the luteal phase of the cycle was different from that observed in follicular and menstrual phases. Steroids may influence the NMDA-dependent drive to release PRL. N-methyl-D-aspartic acid (NMA) involvement in the regulation of PRL secretion may occur through activation of the PRL-stimulating system depending on the physiological state or steroidal milieu. It is possible, therefore, that the NMA-induced release of PRL-releasing factors (PRF) and PRL are enhanced in the presence of ovarian feedback.     

Stimulation of prolactin secretion from turkey anterior pituitary cells in culture

Prolactin (PRL) secretion by monolayer cultures of turkey anterior pituitary cells was significantly increased (up to 44-fold) by vasoactive intestinal peptide (VIP), arginine vasotocin (AVT), and by an extract of turkey hypothalami (HE). Several other neuropeptides (including thyrotropin-releasing hormone) and neurotransmitters were ineffective in influencing PRL secretion at doses up to 10(-6) M. The dynamic PRL response to HE and VIP was studied using superfused pituitary cells attached to microcarrier beads. HE, administered in 30-min pulses, resulted in a significant, dose-related increase in PRL secretion from a basal secretion rate of 2.32 ng/min/10(7) cells to a peak secretion rate of 127.13 ng/min/10(7) cells at the highest dose of HE tested (1 mg tissue-equivalent weight/ml). VIP significantly increased PRL secretion at all doses studied (from 10(-10) to 10(-6) M), with 10(-8) M VIP producing a response similar to that observed with 1 mg/ml HE. A highly significant (P less than 0.001) linear relationship was demonstrated between the log-dose of VIP administered and peak PRL secretion rate. These studies suggest that VIP, but not TRH, may be a physiological stimulus for PRL release in the turkey.     

Prolactin-releasing peptides

Physiologic control of prolactin (PRL) secretion is largely dependent upon levels of dopamine accessing the adenohypophysis via the hypophysial portal vessels. However, it is clear that other factors of hypothalamic origin can modulate hormone secretion in the absence or presence of dopamine. Several neuropeptides have been identified as PRL releasing factors (PRFs) but none of these peptides appears to be a major determinant of PRL secretion in vivo. There remain uncharacterized activities in hypothalamic extracts that can alter secretion and production of the hormone. In addition, there exist a wide variety of substances (neurotransmitters, neuromodulators, neuropeptides) that can act within the hypothalamus to modify the neuroendocrine regulation of PRL secretion. These factors may not be considered true PRFs because their actions are not exerted directly at the level of the lactotroph; however, they can act in brain to stimulate PRL release in vivo and therefore might be considered PRL releasing peptides (PRPs).     

In vivo and in vitro effect of naloxone on prolactin response to TRH in rat

In adult male Wistar rats submitted to a standardized noise stress, intravenous TRH induced a prolactin (PRL) secretory response. Prior IV naloxone administration not only lowered plasma PRL levels in those stressed rats but abolished also the stimulatory action of TRH. This effect was further studied by superfusion experiments on enriched PRL cell suspensions (70% lactotrophs) from female adult Wistar rats. Naloxone kept unaffected the basal PRL secretion but lowered significantly that induced by TRH. These experiments suggest a dual effect of naloxone on rat PRL secretion, one exerted on central opioid receptors lowering stress-related increased basal PRL levels, the other inhibiting the TRH-dependent PRL secretion exerted at the lactotroph level itself.     

Prolactin secretion and intracellular Ca(2+) change in rat lactotroph subpopulations stimulated by thyrotropin-releasing hormone

Thyrotropin-releasing hormone (TRH) may stimulate lactotrophs to increase intracellular Ca(2+) and to secrete prolactin (PRL). In this study, PRL contents in lactotrophs were determined by the sequential cell immunoblot assay (SCIBA) and their changes in intracellular Ca(2+) was analyzed by confocal microscopy. Significant correlations were found in the corresponding parameters between TRH treatments with a recovery interval of 2 h. Measuring the PRL contents after the first TRH treatment and then determining the intracellular Ca(2+) changes after the second TRH treatment revealed four lactotroph subpopulations. Type I cells (51%) showed significant responses of both PRL secretion and intracellular Ca(2+) concentration. Type II cells (22%) increased in PRL secretion, but without changes in intracellular Ca(2+). Type III cells (17%) have increased in intracellular Ca(2+), but without changes in PRL secretion. Type IV cells (10%) did not show changes in PRL secretion and intracellular Ca(2+).     

Differential involvement of protein kinase C in basal versus acetylcholine-regulated prolactin secretion in rat anterior pituitary cells during aging

Although it is well known that plasma concentration of prolactin (PRL) increases during aging in rats, how the anterior pituitary (AP) aging per se affects PRL secretion remains obscure. The objectives of this study were to determine if changes in the pituitary PRL responsiveness to acetylcholine (ACh; a paracrine factor in the AP), as compared with that to other PRL stimulators or inhibitors, contribute to the known age-related increase in PRL secretion, and if protein kinase C (PKC) is involved. We also determined if replenishment with aging-declined hormones such as estrogen/thyroid hormone influences the aging-caused effects on pituitary PRL responses. AP cells were prepared from old (23-24-month-old) as well as young (2-3-month-old) ovariectomized rats. Cells were pretreated for 5 days with diluent or 17beta-estradiol (E(2); 0.6 nM) in combination with or without triiodothyronine (T(3); 10 nM). Then, cells were incubated for 20 min with thyrotropin-releasing hormone (TRH; 100 nM), angiotensin II (AII; 0.2-20 nM), vasoactive intestinal peptide (VIP; 10(-9)-10(-5) M), dopamine (DA; 10(-9)-10(-5) M), or ACh (10(-7)-10(-3) M). Cells were also challenged with ACh, TRH, or phorbol 12-myristate 13-acetate (PMA; 10(-6) M) following PKC depletion by prolonged PMA (10(-6) M for 24 h) pretreatment. We found that estrogen priming of AP cells could reverse the aging-caused effects on pituitary PRL responses to AII and DA. In hormone-replenished cells aging enhanced the stimulation of PRL secretion by TRH and PMA, but not by AII and VIP. Aging also reduced the responsiveness of cells to ACh and DA in suppressing basal PRL secretion, and attenuated ACh inhibition of TRH-induced PRL secretion. Furthermore, ACh suppressed TRH-induced PRL secretion mainly via the PMA-sensitive PKC in the old AP cells, but via additional mechanisms in young AP cells. On the contrary, basal PRL secretion was PKC (PMA-sensitive)-independent in the old AP cells, but dependent in the young AP cells. Taken together, these results suggest differential roles of PMA-sensitive PKC in regulating basal and ACh-regulated PRL responses in old versus young AP cells. The persistent aging-induced differences in AP cell responsiveness to ACh, DA, TRH, and PMA following hormone (E(2)/T(3)) replenishment suggest an intrinsic pituitary change that may contribute, in part, to the elevated in vivo PRL secretion observed in aged rats.     

Significant qualitative differences exist between thyrotropin and prolactin secretory dynamics induced by pituitary cell swelling

Cell swelling produced by a variety of techniques is a potent stimulus intensity-related inducer of an immediate secretory burst of thyroid-stimulating hormone (TSH) and prolaction (PRL) secretion from anterior pituitary cells. A 2-min "square wave" exposure to either hyposmolarity or isotonic urea induced stimulus intensity-correlated TSH and PRL secretory bursts peaking within 3 min, but the PRL zenith occurred 1 min later than that of TSH. With continuous exposure to these stimuli, TSH secretion rapidly decreased and remained only slightly above the unstimulated rate after 5 min. PRL secretion fell to and remained below the unstimulated level after 10 min. After stopping the stimulus, another secretory burst ("off" response) occurred with PRL, but not with TSH. A progressive "ramp" increase in stimulus intensity over 18 min induced a corresponding gradual increase in TSH secretion; there was a progressive depression, rather than increase, in PRL secretion during the stimulus ramp, with an off response secretory burst when the stimulus was discontinued. Removal of extracellular Ca2+ or addition of verapamil to the medium did not alter the dynamics of hyposmolarity-induced TSH secretion, but markedly altered those of PRL secretion; there was no off response PRL secretion and a hyposmolar ramp induced a corresponding gradual increase in PRL secretion, with a return to baseline after removing the stimulus. The dramatic qualitative differences in the response of the thyrotroph and lactotroph may reflect differences between the cell types in the size of secretory vesicles, membrane potential, the mechanism of exocytosis, and/or the role of Ca2+ influx across the plasmalemma.