<p>Functioning as a “powerhouse”, the liver adapts to the metabolic needs of the body by maintaining a homeostatic balance. Prolactin receptor (PRLR) has been found to have a copious existence in the liver. Having established a well-defined role in both reproductive and endocrine systems, the role of this transmembrane protein in hepatocytes is yet to be elucidated. Due to its abundant nature, we hypothesized that PRLR is required for maintaining hepatic homeostasis and plays a role in liver diseases. To test this hypothesis, we defined two specific aims. The first was to explore whether PRLR loss-of-function affects liver structure and function in physiological conditions. The second was to determine whether PRLR is associated with liver pathology. We deleted the <i>Prlr</i> gene specifically in hepatocytes using a virus-based approach and evaluated liver function, transcriptome, and activities of downstream signaling molecules. Due to the absence of PRLR, we found that the urea cycle was disrupted, concomitant with excessive accumulation of urea in the blood; 133 genes exhibited differential expression, largely associated with hepatocyte structure, metabolism, and inflammation; and the activities of STAT3 and 5 were reduced. The results signify that PRLR indeed plays a homeostatic role in the liver. We also used <i>Prlr</i>^+/- mice to assess whether the loss of one allele of the <i>Prlr</i> gene alters maternal hepatic adaptations to pregnancy. As a result, in the pre-pregnancy state and during the first half of gestation, the expression of maternal hepatic PRLR protein was reduced approximately by half owing to <i>Prlr</i> insufficiency. However, during the second half of pregnancy, we observed compensatory upregulation of this molecule, leading to minimal interference in STAT 3 and 5 signaling and liver size. Contrary to a previous study in the breast and ovary, our results suggest that one allele of <i>Prlr</i> may be sufficient for the maternal liver to respond to this physiological stimulus (pregnancy). Furthermore, we examined the expression and activity of PRLR in fatty as well as cholestatic livers. Using a high fat diet, we induced non-alcoholic fatty liver disease (NAFLD). Strikingly and for the first time, we discovered that the short isoform of PRLR (PRLR-S) was completely inactivated in response to NAFLD, whereas the long isoform remained unchanged. This finding strongly suggests the involvement of PRLR-S in lipid metabolism. We also postulate that PRLR-L may be the major regulator of STAT signaling in the liver, consistent with other reports. Lastly, we induced extrahepatic cholestasis via bile duct ligation (BDL) in mice. As this liver disease progressed, the expression of both isoforms of PRLR generally declined and was surprisingly accompanied by increased STAT 3 and 5 activity. The data suggests that PRLR participates in this disease progression, with a disconnection between PRLR signaling and STAT proteins. Collectively, our preliminary studies suggest that PRLR signaling is required to maintain liver homeostasis and more prominently, is involved in liver diseases, especially NAFLD. These findings lay a foundation for our future studies.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Functioning as a “powerhouse”, the liver adapts to the metabolic needs of the body by maintaining a homeostatic balance. Prolactin receptor (PRLR) has been found to have a copious existence in the liver. Having established a well-defined role in both reproductive and endocrine systems, the role of this transmembrane protein in hepatocytes is yet to be elucidated. Due to its abundant nature, we hypothesized that PRLR is required for maintaining hepatic homeostasis and plays a role in liver diseases. To test this hypothesis, we defined two specific aims. The first was to explore whether PRLR loss-of-function affects liver structure and function in physiological conditions. The second was to determine whether PRLR is associated with liver pathology. We deleted the Prlr gene specifically in hepatocytes using a virus-based approach and evaluated liver function, transcriptome, and activities of downstream signaling molecules. Due to the absence of PRLR, we found that the urea cycle was disrupted, concomitant with excessive accumulation of urea in the blood; 133 genes exhibited differential expression, largely associated with hepatocyte structure, metabolism, and inflammation; and the activities of STAT3 and 5 were reduced. The results signify that PRLR indeed plays a homeostatic role in the liver. We also used Prlr+/- mice to assess whether the loss of one allele of the Prlr gene alters maternal hepatic adaptations to pregnancy. As a result, in the pre-pregnancy state and during the first half of gestation, the expression of maternal hepatic PRLR protein was reduced approximately by half owing to Prlr insufficiency. However, during the second half of pregnancy, we observed compensatory upregulation of this molecule, leading to minimal interference in STAT 3 and 5 signaling and liver size. Contrary to a previous study in the breast and ovary, our results suggest that one allele of Prlr may be sufficient for the maternal liver to respond to this physiological stimulus (pregnancy). Furthermore, we examined the expression and activity of PRLR in fatty as well as cholestatic livers. Using a high fat diet, we induced non-alcoholic fatty liver disease (NAFLD). Strikingly and for the first time, we discovered that the short isoform of PRLR (PRLR-S) was completely inactivated in response to NAFLD, whereas the long isoform remained unchanged. This finding strongly suggests the involvement of PRLR-S in lipid metabolism. We also postulate that PRLR-L may be the major regulator of STAT signaling in the liver, consistent with other reports. Lastly, we induced extrahepatic cholestasis via bile duct ligation (BDL) in mice. As this liver disease progressed, the expression of both isoforms of PRLR generally declined and was surprisingly accompanied by increased STAT 3 and 5 activity. The data suggests that PRLR participates in this disease progression, with a disconnection between PRLR signaling and STAT proteins. Collectively, our preliminary studies suggest that PRLR signaling is required to maintain liver homeostasis and more prominently, is involved in liver diseases, especially NAFLD. These findings lay a foundation for our future studies.

Prolactin-releasing peptide (PrRP) is a member of the family of RF-amide peptides. These peptides have typical C-terminal sequence -Arg-Phe-NH2 and similar biological effects. PrRP was discovered as an endogenous ligand of an orphan receptor GPR10 while searching for a factor responsible for a prolactin secretion. This effect was not later confirmed and nowadays, PrRP is mainly considered as an anorexigenic peptide. This is supported by a fact that PrRP and GPR10 deficient mice suffer from hyperphagia and late-onset obesity. Besides GPR10, PrRP is bound to NPFF2 receptor whose endogenous ligand is neuropeptide FF (NPFF). In this study, the PrRP's analogues modified at the N-terminus with fatty acids of different lenghts were tested in vitro on binding and activation MAPK/ERK1/2 signalling pathway. In in vivo experiments on food intake, the central anorexigenic effects of lipidized PrRP-analogues were tested provided their crossing blood brain barrier. Binding studies showed that all analogues bound to rat pituitary RC-4B/C cells with high affinity, analogues containing fatty acid with Ki of one order of magnitude lower than native PrRP. High affinity was also confirmed for binding to cells overexpressing GPR10 receptor and cell membranes with overexpressed NPFF2 receptor. All tested analogues...

Background: Interactions between the neuroendocrine and immune system play an importatnt role in maintaining homeostasis. This communication is mediated by cytokines, neurotransmiters and hormones through endocrine, paracrine and autocrine signaling. Prolactin (PRL), hormone of anterior pituitary, is produced by a number of other tissues and cells of immune system. On periphery, PRL is cytokine. Sepsis is an inflamatory response of the organism to severe infection, Th1 immune response is activated and PRL could participate in it. Toll-like receptors (TLR) play a key role in a recognition of bacteial components and mediate a systemic response (with PRL secretion) during infection. It is supposed that activated immune system leads to increasing of PRL, TLR2 and TLR4 gene expression. We detected PRL, TLR2 a TLR4 mRNA levels in monocytes from patiens with system inflammation. We studied influence of single nucleotide polymorphism (SNP -1149 G/T) in PRL gene promotor, it supposed that G allele increases PRL expression. Materials and Methods: For the pilot study 30 patients diagnose with severe infectious event. Collectoin of patiens blood samples was performed consequently three times. Control group comprised 40 healthy individuals. One blood sample was taken from each healthy subject. For testing of...

Introduction: Diabetes mellitus is a polygene disease and on its manifestation have influence also enviromental factors. We have studied the role of extrapituitary prolactin (PRL) and toll-like receptors (TLR) 2 and 4 in the etiopathogenesis of autoimmune diabetes. PRL is mainly produced by hypophysis, but in small concentrations also in the periphery, where it participates in the immune reactions. Therefore, we investigated the influence of the levels of monocytic PRL mRNA on the development of diabetes, and also the influence of G allele of the -1149 G/T polymorphism in the extrapituitary promotor, which has already been associated with other autoimmune diseases. TLRs are receptors of the immune cells that recognize patogenes entering into the body. They play an important role in the iniciation of the immune response. We aimed to find out their function in the pathogenesis of the autoimmune diabetes by the detection of their mRNA levels and protein levels expressed on the cell surface of the monocytes. Material and methods: In this study we included 30 T1D and 21 LADA patients. Three control groups consisted of 23 T2D patients, 23 patients with a nondiabetic disease (neDM) and 60 healthy blood donors (TO). Blood samples have been taken from the individuals. From these blood samples we isolated...

Objectives: Hormones and cytokines are known to act as regulatory messengers between the neuroendocrine and immune systems. The innate immune system identifies infectious agents by means of pattern-recognition receptors. These receptors recognize pathogen-specific macromolecules called pathogen-associated molecular patterns. Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity via interaction with membrane receptors on immune-competent cells. Glucans are also released into the systemic circulation of patients with fungal infections. Recent evidence confirms the existence of glucan-specific receptors on cells outside the immune system. We hypothesized that glucans may directly interact with pituitary cells as an early signaling event in fungal infections. Methods: We characterized the receptor-mediated interaction of glucan derived from Candida albicans with pituitary cells using surface plasmon resonance. Prolactin levels were assayed by commercial ELISA. TLR2, TLR4 and CD14 mRNA levels were assessed by RT-PCR. Results: A single glucan-specific binding site was identified on rodent somatomammotroph (KD = 3.9) μM) and human folliculostellate cell (KD = 3.6) μM) membranes. Coincubation of glucan with somatomammotroph cells for 72 h significantly (p < 0.01) increased prolactin accumulation by 56-62% over that observed in cells treated with media alone. Glucan also increased TLR4 and CD14 gene expression in human folliculostellate cells. Conclusions: Pituitary cells directly recognize and respond to fungal cell wall glucans resulting in stimulation of pituitary cell TLR4 and CD14 gene expression. In addition, glucan stimulates secretion of prolactin, a hormone that plays an important role in the response to infection.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.

One of the oldest, extant, lineages of vertebrates, the sea lamprey, was used to clarify the evolutionary origin and divergence of the growth hormone receptor (GHR) family. A single, full-length, cDNA, and a second, partial, cDNA were identified and shown to encode proteins that share amino acid identity with GHRs and prolactin receptors (PRLR s) previously identified. The complexity of the dynamic signaling system, with special emphasis on this system in fish and in the context of the evolution of this system, is discussed in the first chapter. The second chapter integrates the new insights gained by these studies. Included is a newly proposed phylogenetic analysis and revised nomenclature-system for vertebrate GHRs that better represents the evolutionary history of the receptor family. The molecular evolution of the receptors is, furthermore, highlighted as the backdrop for the continued discussion regarding how the GH-family of hormones exhibit such coordinated and pleiotropic actions.

The role of endogenous dopamine, utilizing a dopamine antagonist (fluphenazine; FLU), in modulation of gonadotropin, growth hormone (GH) and prolactin (PRL) secretion during the early postpartum period and various stages of the estrous cycle was investigated in Holstein cows. Experiment 1 was conducted in anovulatory early postpartum cows. Fluphenazine caused a decrease (P < .05) in mean serum LH concentration and LH pulse frequency. Likewise, FLU caused a (P < .05) decrease in mean GH concentration. These results suggest that endogenous dopamine, at least in part, is responsible for regulation of LH and GH secretion in anovulatory Holstein cows. Experiment 2 was conducted in cyclic lactating Holstein cows during the mid-luteal phase of the estrous cycle. Mean serum LH and FSH concentrations, pulse frequencies, and peak amplitudes did not change in response to FLU. FLU did not affect mean serum GH concentration. These results suggest that a dopamine-mediated mechanism for modulation of gonadotropin and GH secretion is absent or perhaps overridden by high progesterone concentration during the luteal phase of the estrous cycle in lactating dairy cows. Experiment 3 was conducted during the early follicular phase of the estrous cycle in Holstein cows. During the follicular phase, FLU caused a decrease (P < .05) in mean serum LH concentration and LH pulse frequency. However, FLU had no effect on mean serum FSH concentration or pulse frequency. Further, FLU increased (P < .05) GH concentrations during the follicular phase. Experiment 4 was conducted during the early metestrus phase of the estrous cycle. During the metestrus phase, FLU tended to decrease (P < .1) mean LH concentration and suppressed (P < .05) LH pulse frequency but had no effect on FSH secretion. Fluphenazine caused a transient increase (P < .05) in mean serum GH concentration. The results of the third and fourth experiments suggest that, during the early follicular and metestrus phases of the estrous cycle, when progesterone concentration is low, modulation of LH and GH secretion, at least in part, is modulated by endogenous dopamine. However, a dopamine mediated mechanism for FSH secretion is absent during both phases of the estrous cycle in lactating Holstein cows. In all experiments FLU increased (P < .01) PRL secretion indicating that endogenous dopamine suppresses PRL secretion in cattle regardless of ovarian status. It is concluded that: 1) endogenous dopamine plays a stimulatory role in LH secretion during the anovulatory postpartum period and during the estrous cycle only when serum progesterone is low. 2) FLU decreased GH secretion in anovulatory postpartum Holstein cows but it increased GH secretion during the follicular and metestrus phases of the estrous cycle. However FLU had no effect on GH secretion during the luteal phase of the estrous cycle. Thus it appears that, modulation of GH secretion is dependent upon reproductive status and ovarian hormones secretion. / Ph. D.

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