Maria Vrontakis

Functional bitter taste receptors are expressed in brain cells

Humans are capable of sensing five basic tastes which are sweet, sour, salt, umami and bitter. Of these, bitter taste perception provides protection against ingestion of potentially toxic substances. Bitter taste is sensed by bitter taste receptors (T2Rs) that belong to the G-protein coupled receptors (GPCRs) superfamily. Humans have 25 T2Rs that are expressed in the oral cavity, gastrointestinal (GI) neuroendocrine cells and airway cells. Electrophysiological studies of the brain neurons show that the neurons are able to respond to different tastants. However, the presence of bitter taste receptors in brain cells has not been elucidated. In this report using RT-PCR, and immunohistochemistry analysis we show that T2Rs are expressed in multiple regions of the rat brain. RT-PCR analysis revealed the presence of T2R4, T2R107 and T2R38 transcripts in the brain stem, cerebellum, cortex and nucleus accumbens. The bitter receptor T2R4 was selected for further analysis at the transcript level by quantitative real time PCR and at the protein level by immunohistochemistry. To elucidate if the T2R4 expressed in these cells is functional, assays involving G-protein mediated calcium signaling were carried out. The functional assays showed an increase in intracellular calcium levels after the application of exogenous ligands for T2R4, denatonium benzoate and quinine to these cultured cells, suggesting that endogenous T2R4 expressed in these cells is functional. We discuss our results in terms of the physiological relevance of bitter receptor expression in the brain.

Estrogen induction of galanin synthesis in the rat anterior pituitary gland demonstrated by in situ hybridization and immunohistochemistry

The distribution of galanin messenger (mRNA) and galanin-like immunoreactivity (Gal-LI) in the anterior and posterior pituitaries of control and estrogen-implanted female rats was determined by in situ hybridization and immunohistochemical methods. In control ovariectomized animals galanin mRNA was undetectable in the posterior, intermediate and anterior pituitary. However, 4 days after implantation with 10 mg of diethylstilbestrol, galanin mRNA was clearly present in the anterior pituitary, but not in the posterior or intermediate lobe. By immunohistochemistry Gal-LI was readily visualized and detected in the posterior lobe, but clearly undetectable in cells of the intermediate and anterior pituitary lobes of control animals. After estrogen administration numerous cells exhibiting intense Gal-LI were evident in the anterior lobe, while Gal-LI remained unchanged in the intermediate and posterior lobes. These results indicate that in control animals galanin is stored, but not synthesized, in the posterior pituitary and that after estrogen administration galanin production is substantially increased in the anterior pituitary. We conclude that the expression of galanin in the anterior pituitary is regulated by estrogen and suggest that galanin may be a pituitary hormone.

Efficacy and safety of metformin during pregnancy in women with gestational diabetes mellitus or polycystic ovary syndrome: A systematic review

BACKGROUND Metformin is an effective oral anti-hyperglycemic agent that is widely used to manage diabetes mellitus type 2 in the general population and more recently, in pregnancy. However, as metformin crosses the placenta, its use during pregnancy raises concerns regarding potential adverse effects on the mother and fetus. OBJECTIVE (i) To provide background for the use of metformin during pregnancy through a narrative review and (ii) to critically appraise the published evidence on the efficacy and safety of using metformin during pregnancy through a systematic review. RESULTS Metformin appears to be effective and safe for the treatment of gestational diabetes mellitus (GDM), particularly for overweight or obese women. However, patients with multiple risk factors for insulin resistance may not meet their treatment goals with metformin alone and may require supplementary insulin. Evidence suggests that there are potential advantages for the use of metformin over insulin in GDM with respect to maternal weight gain and neonatal outcomes. Furthermore, patients are more accepting of metformin than insulin. The use of metformin throughout pregnancy in women with polycystic ovary syndrome reduces the rates of early pregnancy loss and preterm labor and protects against fetal growth restriction. There have been no demonstrable teratogenic effects, intra-uterine deaths or developmental delays with the use of metformin. CONCLUSIONS The publications reviewed in this paper support the efficacy and safety of metformin during pregnancy with respect to immediate pregnancy outcomes. Because there are no guidelines for the continuous use of metformin in pregnancy, the duration of treatment is based on clinical judgment and experience on a case-by-case basis.

Galanin Transgenic Mice with Elevated Circulating Galanin Levels Alleviate Demyelination in a Cuprizone-Induced MS Mouse Model

Multiple Sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS) with a presumed autoimmune etiology. Approved treatments for MS are immunoregulatory and are able to reduce the inflammatory components of the disease. However, these treatments do not suppress progressive clinical disability. Approaches that directly protect myelin-producing oligodendrocytes and enhance remyelination are likely to improve long-term outcomes and reduce the rate of axonal damage. Galanin (GAL) is a bioactive neuropeptide that is widely distributed throughout the nervous system and has diverse neuromodulatory effects. In this study, using the cuprizone (CPZ) demyelination model of MS, we demonstrate that GAL has pronounced neuroprotective effects with respect to demyelination and remyelination. Using our GAL transgenic mouse (GAL-Tg), we identified a novel attenuation of OLs against CPZ induced demyelination, which was exerted independently of progenitor cells. Alleviation of myelin breakdown in the GAL-Tg mice was observed to be significant. Furthermore, we observed changes in the expression of the GAL receptor GalR1 during the demyelination and remyelination processes. Our data strongly indicate that GAL has the capacity to influence the outcome of primary insults that directly target OLs, as opposed to cases where immune activation is the primary pathogenic event. Taken together, these results suggest that GAL is a promising next-generation target for the treatment of MS.

Regulation of hepatic PPARγ2 and lipogenic gene expression by melanocortin

The central melanocortin system regulates hepatic lipid metabolism. Hepatic lipogenic gene expression is regulated by transcription factors including sterol regulatory element-binding protein 1c (SREBP-1c), carbohydrate responsive element-binding protein (ChREBP), and peroxisome proliferator-activated receptor gamma2 (PPARgamma2). However, it is unclear if central melanocortin signaling regulates hepatic lipogenic gene expression through the activation of these transcription factors. To delineate the molecular mechanisms by which the melanocortin system regulates hepatic lipid metabolism, we examined the effect of intracerebroventricular injection of SHU9119, a melanocortin receptor antagonist, on hepatic expression levels of genes involved in lipid metabolism in mice. SHU9119 treatment increased hepatic triglyceride content and mRNA levels of lipogenic genes, SREBP-1c, and PPARgamma2, whereas it did not cause any changes in hepatic ChREBP mRNA levels. These findings suggest that reduced central melanocortin signaling increases hepatic lipid deposition by stimulating hepatic lipogenic gene expression at least partly through the activation of SREBP-1c and PPARgamma2.

Galanin is highly expressed in bone marrow mesenchymal stem cells and facilitates migration of cells both in vitro and in vivo.

Galanin peptide has recently been found to be highly abundant in early embryonic mouse mesenchyme, while galanin and its receptors are expressed in embryonic mouse stem cells. Bone marrow mesenchymal stem cells (BMMSCs) represent the primary source for adult stem cell therapy. In this study we examined the abundance of galanin and its receptors in BMMSCs and evaluated its possible function. Galanin mRNA and protein were highly expressed in BMMSCs cultures up to four passages, while among the three galanin receptor subtypes (GalR1, GalR2, and GalR3) only GalR2 and to a lesser extent GalR3 were expressed. Using chemotaxis and wound assays we found that galanin protein increased the migration of BMMSCs. Furthermore, increased serum galanin levels in a galanin transgenic model enhanced the mobilization (homing) of injected BMMSCs in vivo. These data suggest a role for galanin in BMMSC migration, probably through activation of the GalR2 receptor.

Presence of Galanin-Like Immunoreactivity in Mesenchymal and Neural Crest Origin Tissues During Embryonic Development in the Mouse

Galanin is a highly conserved neuropeptide with a wide range of biological effects. Recently, through transcriptome analysis, galanin was identified in undifferentiated mouse embryonic stem cells as one of the most abundant transcripts. We have examined the developmental expression of galanin‐like immunoreactivity in mice from embryonic day 10 (E10) to embryonic day 15 (E15). At E10, galanin was readily detected in the undifferentiated head and trunk mesenchyme of both mesodermal and neural crest origin. There was also strong immunoreactivity in the mesenchymal spiral ridges of the outflow tract of the heart and the endocardial cushions. The highest level of galanin detected was at E13 in the craniofacial mesenchyme and proliferating chondrocytes in bones of both neural crest and mesoderm origin. Dorsal root ganglia and trigeminal ganglia contained galanin immunoreactive cells as well. These data indicate the presence of galanin peptide during periods of morphogenesis and thus a developmental role for the peptide in mesenchymal and neural crest origin tissues in the mouse embryo. Whether galanin has a growth and/or differentiating role, still remains to be demonstrated. Anat Rec, 2009.

Regulation of galanin by dexamethasone in the rat anterior pituitary and the uterus

Galanin is a 29 amino acid neuropeptide widely distributed throughout the mammalian nervous and endocrine system. We have previously reported that estrogen dramatically increases galanin gene expression and protein synthesis in the anterior pituitary (AP), while the expression in the uterus (UT) of the same animals is transient and similar to the induction of protooncogenes (c-fos, c-jun, c-myc). In order to examine if this pattern of induction is specific to estrogen administration, we investigated the effect of glucocorticoids, another steroid, on the gene expression of galanin in the AP and in the UT of ovariectomized female rats and in the AP of male rats. Using Northern blot analysis, the AP and the UT showed almost undetectable levels of galanin mRNA, but in vivo treatment of female rats with 1 mg/kg body weight of dexamethasone (DEX) led to a significant increase of galanin mRNA levels in both AP and UT. Similarly, DEX (0.1-5 mg/kg i.p.) significantly stimulated galanin mRNA levels in the AP of the male rats. In both males and females the peak of induction was at 9 h after injection that is different from the 3-hour peak after estrogen administration. Daily injection of DEX for up to 7 days sustained the levels of galanin mRNA in both the AP and the UT, in contrast to the transient induction of galanin in the UT after estrogen administration. No change was noted in the galanin protein content of AP (control = 30 +/- 3.5 ng/mg protein; DEX treated = 38 +/- 4.2 ng/mg protein). Interestingly, in the UT of ovariectomized rats the combination of DEX and DES (diethylstilbestrol) treatment for 2 days resulted in a synergistic stimulation of galanin mRNA. In summary, these data demonstrate a tissue- and steroid-specific regulation of the galanin gene in AP and UT and suggest that DEX regulates the galanin gene possibly through a pathway different from estrogen.

An elevated level of circulating galanin promotes developmental expression of myelin basic protein in the mouse brain

Myelinogenesis is a scheduled process that is regulated by the intrinsic properties of the cell and extracellular signals. Galanin (GAL) is a bioactive neuropeptide that is widely distributed throughout the nervous system. Chronic increase in circulating GAL levels protects the demyelination processes. Furthermore, GAL is synthesized in myelin-producing glial cells, such as oligodendrocytes and its expression level is at its highest between postnatal days 10 and 40. In the present study, we use our GAL transgenic mouse model to examine the effects of GAL on postnatal myelinogenesis in the CNS. Although we observed no difference in the proliferation of oligodendrocyte precursor cells, we found that GAL has a strong pro-myelinating effect. The transgenic mice at postnatal day 10 appeared to undergo myelinogenesis at an accelerated rate, as demonstrated by the increase in myelin basic protein (MBP) synthesis. The immunohistochemical results are consistent with our preliminary findings that suggest that GAL is a regulator of myelination and may be one of the myelination promoters. This finding is especially important for studies focusing on endogenous molecules for treating myelin-related diseases, such as multiple sclerosis and other leukodystrophies.

Bromocriptine inhibits galanin gene expression in the rat pituitary gland.

The chronic effects of diethylstilbestrol (DES) and bromocriptine on the expression of galanin in the rat anterior pituitary were examined and compared with the expression of prolactin and vasoactive intestinal peptide (VIP) after the same type of hormonal manipulation. Total pituitary RNA was subjected to Northern blot analysis 2, 7, and 28 days after rats were implanted sc with a 10-mg DES pellet with or without concurrent treatment with bromocriptine (5 mg/kg per day). Estrogen treatment resulted in a rise in galanin, prolactin, and VIP messenger RNA (mRNA) that was particularly evident for galanin. Coadministration of bromocriptine with DES suppressed the response to estrogen with galanin, prolactin, and VIP mRNA levels being in general lower than when treatment with estrogen alone was used. In the combined treatment group there was an increase in the levels for the three mRNA species over the control group. Pituitary content of galanin and prolactin measured by radioimmunoassay exhibited a temporal pattern similar to that of corresponding mRNA levels after administration of estrogen alone or in combination with bromocriptine. The rise in galanin content (up to 78-fold) was higher than the increase in prolactin immunoreactivity (up to 6.5-fold compared to control). Bromocriptine partially suppressed the estrogen-induced increase in the content of these peptides. The changes in galanin and prolactin peptide levels in the plasma paralleled those found in the pituitary. This study demonstrates that the neuropeptides galanin and VIP are coregulated with prolactin after estrogen and bromocriptine administration. Since galanin and VIP are known to stimulate prolactin secretion it is possible that they mediate, at least in part, the effects of estrogen and bromocriptine on pituitary size and/or prolactin release.