TRH Expression Research Articles

Abstract 091: Leptin-Thyroliberin Pathway in Left Ventricle Hypertrophy

Cardiac TRH (cTRH) induces LVH and fibrosis. The adiponectin Leptin induces TRH in CNS. We hypothesized that in obesity, the increase of cTRH induced by hyperleptinemia is responsible for the LVH, mostly attributed to pressure load. We studied obese hyperleptinemic with mild hypertension Agouti mice, and found LVH with higher (p<0.05) cTRH, fibrotic (I/III col,TGFβ) and hypertrophic (BNP,βMHC) marker genes vs lean (BL/6J). We normalized pressure in Agouti mice with hydroclorothiazide (20 mg/kg/day) since weaning (n=9) vs non treated mice. Still both developed (p<0.05) LVH, higher cTRH, fibrotic and hypertrophic marker genes expression, suggesting that LVH would not be due to hypertension. Opposite to Agouti model, we used obese normotensive ob/ob mice, lacking Leptin expression, and without LVH despite their obesity. We treated them with Leptin (80 ug/kg/day) or saline since weaning for 15 days. Only the treated group developed LVH (p< 0.05, n=7) demonstrating that it is Leptin dependant. Also we found increase (p<0.05) in cTRH, col III, TGFβ and BNP suggesting that leptin-TRH interaction is required in obesity-induced LVH. Finally, we evaluated whether Leptin administration, (n=6, 10 ug/kg/day) could induce hypertrophy in lean C57 mice with and withouth native cTRH system, by previous siRNA injection (5ug). Diuretic was given to Leptin groups, to avoid its hypertensive effect. Leptin induced cTRH expression, not observed in the groups with siRNA-TRH (p<0.05).This probably induce fibrotic and hypertrophic markers, demonstrating that the novel interaction is functional also in mildy hyperleptinemia at normal weight status. To confirm TRH's Leptin induction we used cardiomyocytes cell line H9C2 (n=6) stimulated with Leptin (10 and 100 ng/ml). TRH expression and precursor content increased (p<0.05) post Leptin treatment with both concentrations. Moreover in primary cardiomyocytes culture from neonate rats, Leptin stimulus (80 ng/ml, 24 hs) increased (p<0.05) TRH content vs controls, confirming the direct TRH-Leptin induction in heart cells. We described a novel Leptin-cTRH pathway which explains Leptin-induced LVH, highly likely since early stages.

Voluntary Exercise-Induced Activation of Thyroid Axis and Reduction of White Fat Depots Is Attenuated by Chronic Stress in a Sex Dimorphic Pattern in Adult Rats.

The activity of the hypothalamus-pituitary-thyroid (HPT) axis is inhibited by energy deficit, by acute or chronic stress, but activated by cold exposure or exercise. Because stress curtails acute cold induced activation of HPT, we evaluated the effect of chronic stress on HPT axis response to voluntary exercise, a persistent energy-demanding situation. Adult male and female Wistar rats were exposed to restraint stress, 30 min/day for 2 weeks, or to isolation (Iso) [post-natal day [PND] 30–63]. Exercise was performed (7 p.m.−7 a.m.) in a running wheel, sedentary controls stayed in individual cages (Sed); at 7 a.m. they were housed with their cage mate or individually (Iso); food intake by the exercised group was measured day and night to pair-fed Sed. At sacrifice, hormones, mRNA levels and tissue weights were quantified. Control or restrained adult rats had access to running wheel daily for 2 weeks. Compared to C, exercise decreased white adipose tissue (WAT) mass in females and males, increased hypothalamic paraventricular nucleus (PVN)-Trh expression in males proportionally to exercise performed, and increased TSH and T4 serum concentration in females. These changes were not detected in restrained groups. Starting at PND 63 control (2/cage) and isolated (1/cage) rats either exercised on 10 alternated nights or were sedentary. In control male animals, compared to Sed rats, exercise did not decrease WAT mass, nor changed HPT axis activity, but increased Pomc and deiodinase 2 (Dio2) expression in mediobasal hypothalamus (MBH), adrenergic receptor β3 and uncoupling protein-1 in brown adipose tissue. In control female animals, exercise decreased WAT mass, increased Pomc, Dio2, and Trhde expression in MBH, and TSH serum concentration. Iso females had lower TSH and T4 serum concentration, Dio2 and Trhde expression in MBH than controls. The stress response was higher in isolated males than females, but in males it did not alter the effects of exercise, in contrast to isolated females that had a blunted response to exercise compared to controls. In conclusion, chronic stress interferes with metabolic effects produced by exercise, such as loss of WAT mass, coincident with dampening of HPT activity.

SAT-545 The Response of the Hypothalamic-Pituitary-Thyroid (HPT) Axis to a Palatable Diet or Exercise Depends on Sex and Housing Conditions in Adult Rats

Single housing induces behavioral, neuroendocrine and metabolic alterations. As thyroid hormones (TH) regulate several aspects of metabolism, and HPT axis is modulated by energy status and emotional stress, we evaluated the effect of isolation on HPT axis response to a palatable diet under basal conditions and after an acute stressor as restraint. Weaned female or male rat pups (Wistar) were caged alone (isolated, Iso; n=8) or in groups of 4 (group housed, Gh; n=12) at postnatal day (PND) 22. At adulthood (PND 63), Gh and Iso rats had access to chow or a palatable diet (Pd: peanuts, cookies, chow) for 1 or 3 months and exposed, in the latter, to 1 h of restraint stress (RES). Gh or Iso males and females increased their kcal intake of Pd, inducing white fat (WAT) accumulation (more in females), leptinemia and insulinemia. Pd for1 month increased Trh-degrading enzyme expression in both Gh sexes, and decreased TSH in Gh males. In females, T4 increased in Gh-Pd; TSH and T4 reduced in Iso-Pd rats. After 3 months of Pd, RES activated adrenal axis, but its response was curtailed by Pd in Gh-males, and to a lesser extent in Iso. In Gh-Pd females, corticosterone decreased but not in Iso. RES-induced inhibition of HPT axis was not diminished by Pd in Gh-males, as seen in the adrenal response, on the contrary, it decreased even further to a similar extent as did Iso in Trh expression, TSH and T4 levels; Iso reduced T3 levels independent of diet. In females, the only significant change was an increase in Trh mRNA levels in Iso-chow. Brown adipose tissue (BAT) gene expression was affected in Gh and Iso males by Pd (Adrb3 and Dio2) whereas in females, Dio2 decreased only in Iso-Pd. These results support a differential adrenal and HPT responses due to sex and housing. HPT axis activity increases by energy demands, we thus studied the effect of 2 weeks of voluntary wheel running (WR) in Gh and Iso adult rats. Controls were sedentary (Sed) pair-fed to the amount of food consumed by WR groups. Females ran more than males and, in either sex, Gh and Iso ran equivalently. WR reduced body weight gain in Iso and Gh males, without changes in fat mass. Gh females lost body weight, but not Iso; ovaric and subcutaneous fat decreased in Gh rats, Iso only lost subcutaneous fat. WR diminished PVN Trh mRNA levels and increased serum TSH in female Gh. Iso blunted several TH- and exercise-induced targets in BAT and WAT. WR stimulated BAT expression of Adrb3 and Ucp1 in Gh or Iso males whereas in Iso females, Dio2 and Ucp1. WR increased Adrb3 in WAT of Gh males or females and Pparg in Gh males and in Iso females. These results show the complex and multifactorial modulation of metabolism between males and females. In conclusion, single housing has long-term consequences in metabolism and HPT axis activity, and sex determines the hormonal responses to diet or exercise in adulthood. Grants: PAPIIT-IA200417 (LJH), IN204316 and CONACyT 284883 (PJB)

Differential regulation of thyrotropin-releasing hormone mRNA expression in the paraventricular nucleus and dorsomedial hypothalamus in OLETF rats

Thyrotropin-releasing hormone (TRH) plays an important role in the regulation of energy balance. While the regulation of TRH in the paraventricular nucleus (PVN) in response to changes of energy balance has been well studied, how TRH is regulated in the dorsomedial hypothalamus (DMH) in maintaining energy homeostasis remains unclear. Here, we assessed the effects of food restriction and exercise on hypothalamic Trh expression using Otsuka Long-Evens Tokushima Fatty (OLETF) rats. Sedentary ad lib fed OLETF rats (OLETF-SED) became hyperphagic and obese. These alterations were prevented in OLETF rats with running wheel access (OLETF-RW) or food restriction in which their food was pair-fed (OLETF-PF) to the intake of lean control rats (LETO-SED). Evaluation of hypothalamic gene expression revealed that Trh mRNA expression was increased in the PVN of OLETF-SED rats and normalized in OLETF-RW and OLETF-PF rats compared to LETO-SED rats. In contrast, the expression of Trh in the DMH was decreased in OLETF-SED rats relative to LETO-SED rats. This alteration was reversed in OLETF-RW rats as seen in LETO-SED rats, but food restriction resulted in a significant increase in DMH Trh expression in OLETF-PF rats compared to LETO-SED rats. Strikingly, while Trh mRNA expression was decreased in the PVN of intact rats in response to acute food deprivation, food deprivation resulted in increased expression of Trh in the DMH. Together, these results demonstrate the differential regulation of Trh expression in the PVN and DMH in OLETF rats and suggest that DMH TRH also contributes to hypothalamic regulation of energy balance.

Gene expression of thyrotropin- and corticotrophin-releasing hormones is regulated by environmental salinity in the euryhaline teleost Sparus aurata.

In euryhaline teleosts, the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-interrenal axes (HPT and HPI, respectively) are regulated in response to environmental stimuli such as salinity changes. However, the molecular players participating in this physiological process in the gilthead seabream (Sparus aurata), a species of high value for aquaculture, are still not identified and/or fully characterized in terms of gene expression regulation. In this sense, this study identifies and isolates the thyrotropin-releasing hormone (trh) mRNA sequence from S. aurata, encoding prepro-Trh, the putative factor initiating the HPT cascade. In addition, the regulation of trh expression and of key brain genes in the HPI axis, i.e., corticotrophin-releasing hormone (crh) and corticotrophin-releasing hormone-binding protein (crhbp), was studied when the osmoregulatory status of S. aurata was challenged by exposure to different salinities. The deduced amino acid structure of trh showed 65-81% identity with its teleostean orthologs. Analysis of the tissue distribution of gene expression showed that trh mRNA is, though ubiquitously expressed, mainly found in brain. Subsequently, regulation of gene expression of trh, crh, and crhbp was characterized in fish acclimated to 5-, 15-, 40-, and 55-ppt salinities. In this regard, the brain gene expression pattern of trh mRNA was similar to that found for the crh gene, showing an upregulation of gene expression in seabream acclimated to the highest salinity tested. Conversely, crhbp did not change in any of the groups tested. Our results suggest that Trh and Crh play an important role in the acclimation of S. aurata to hypersaline environments.

Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A.

Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference. Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry. Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone. Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.

Abstract 027: Participation of Cardiac Thyrotroping Releasing Hormone (cTRH) in the Doxorrubicin (D) Induced Cardiotoxicityin in Mice

We have demonstrated TRH hyperactivity in the hypertrophied Left ventricle (LV) of SHR. Its specific inhibition attenuates hypertrophy development in spite of the significant higher pressure observed (Schuman M, Hypertension 2011). LV-TRH over-expression induces several features of the hypertrophied heart, including increases in the apoptotic index Bax/Bcl2, and the activated caspase 3 observed by immunohystochemistry (Schuman M, AJPH&C 2014). In addition, we have found that TRH expression was induced by D in primary cultures of cardiac cells. Based on these results we hypothesized that cTRH could participate in the D cardiotoxicity effects. Indeed, we used C57 adult males (n=10) with a single D or saline injection (200uL,10 mg/kg ip) which previously (24 h, under anesthesia) received an intracardiac injection of a specific TRH-siRNA to inhibit LV-TRH expression or scrambled Con-siRNA. Mice were sacrificed 2,4 and 7 days post D injection and body weight was measured. Genes expression was measured by real time PCR and protein by immunohystochemistry (ANOVA and Tukey test). Body weight showed a mild but not significant decrease in D treated animals. D significantly increased TRH gene expression and TRH protein content reaching the maximum at 7 days post injection (2d: 145%, 4d:190% and 7d: 250%) (p& 0.05), which were not observed in the groups with D+TRH-siRNA indicating the effectiveness of the specific TRH inhibition. Also at this time TRH inhibition attenuates (p&0.05) D-induced increase in the apoptotic index Bax/Bcl2 and the augmented activated caspase 3 content pointing out the participation of the cardiac TRH in the D-induced apoptosis. Similar results were observed with hypertrophic and fibrotic markers gene expression (BNP, BMHC and col III) which showed a significant increase (p& 0.05) only in the groups with D and the intact TRH system (D+Con-siRNA). Fibrosis results were confirmed by Sirius Red and Masson techniques. On the whole, we demonstrated for the first time that LV-TRH system is required for both, Doxorrubicin induction of apoptosis and consecutively hypertrophy and fibrosis in the mouse heart. Even more, we found that cTRH inhibition attenuates doxorrubicin induced damage suggesting a novel mechanisms in the cardiotoxicity injury.

Neonatal Maternal Separation Alters, in a Sex-Specific Manner, the Expression of TRH, of TRH-Degrading Ectoenzyme in the Rat Hypothalamus, and the Response of the Thyroid Axis to Starvation.

Hypothalamic-pituitary-thyroid (HPT) axis activity is important for energy homeostasis, and is modified by stress. Maternal separation (MS) alters the stress response and predisposes to metabolic disturbances in the adult. We therefore studied the effect of MS on adult HPT axis activity. Wistar male and female pups were separated from their mothers 3 h/d during postnatal day (PND)2-PND21 (MS), or left nonhandled (NH). Open field and elevated plus maze tests revealed increased locomotion in MS males and anxiety-like behavior in MS females. At PND90, MS females had increased body weight gain, Trh expression in the hypothalamic paraventricular nucleus, and white adipose tissue mass. MS males had increased expression of TRH-degrading enzyme in tanycytes, reduced TSH and T3, and enhanced corticosterone serum concentrations. MS stimulated brown adipose tissue deiodinase 2 activity in either sex. Forty-eight hours of fasting (PND60) augmented serum corticosterone levels similarly in MS or NH females but more in MS than in NH male rats. MS reduced the fasting-induced drop in hypothalamic paraventricular nucleus-Trh expression of males but not of females and abolished the fasting-induced increase in Trh expression in both sexes. Fasting reduced serum concentrations of TSH, T4, and T3, less in MS than in NH males, whereas in females, TSH decreased in MS but not in NH rats, but T4 and T3 decreased similarly in NH and MS rats. In conclusion, MS produced long-term changes in the activity of the HPT axis that were sex specific; response to fasting was partially blunted in males, which could affect their adaptive response to negative energy balance.

Long-term exposure to triphenylphosphate alters hormone balance and HPG, HPI, and HPT gene expression in zebrafish (Danio rerio).

With the global decline in the use of polybrominated diphenyl ethers, the demand for alternative flame retardants, such as triphenylphosphate (TPP), has increased substantially. Triphenylphosphate is now detected in various environments including aquatic ecosystems worldwide. However, studies on the toxicological consequences of chronic TPP exposure on aquatic organisms are scarce. The zebrafish model was used to investigate the effects of long-term TPP exposure on the endocrine system. Zebrafish embryos were exposed to 5 µg/L, 50 µg/L, or 500 µg/L TPP for 120 d, and hormonal and transcriptional responses were measured along the hypothalamic-pituitary-gonad (HPG) axis, the hypothalamic-pituitary-interrenal (HPI) axis, and the hypothalamic-pituitary-thyroid (HPT) axis. Exposure to TPP significantly increased plasma 17β-estradiol, but decreased 11-ketotestosterone in both sexes. Gene expression data support these changes. In the HPI axis, plasma cortisol and proopiomelanocortin (pomc) and mineralocorticoid receptor transcripts increased in females, but in males cortisol decreased whereas pomc increased (p < 0.05). Thyroxine and triiodothyronine increased, and thyrotrophin-releasing hormone receptor 2 (trhr2) and trh expression were affected only in females (p < 0.05). In summary, long-term exposure to TPP enhanced estrogenicity in both males and females, potentially through influencing the HPG axis, but modulated the HPI, and HPT axes differently by sex, suggesting that both genomic and nongenomic responses might be involved. Environ Toxicol Chem 2016;35:2288-2296. © 2016 SETAC.

The Role of Hypothalamic NF-κB Signaling in the Response of the HPT-Axis to Acute Inflammation in Female Mice.

A large proportion of critically ill patients have alterations in the hypothalamus-pituitary-thyroid (HPT) axis, collectively known as the nonthyroidal illness syndrome. Nonthyroidal illness syndrome is characterized by low serum thyroid hormone (TH) concentrations accompanied by a suppressed central component of the HPT axis and persistent low serum TSH. In hypothalamic tanycytes, the expression of type 2 deiodinase (D2) is increased in several animal models of inflammation. Because D2 is a major source of T3 in the brain, this response is thought to suppress TRH expression in the paraventricular nucleus via increased local bioavailability of T3. The inflammatory pathway component RelA (the p65 subunit of nuclear factor-κB) can bind the Dio2 promoter and increases D2 expression after lipopolysaccharide (LPS) stimulation in vitro. We aimed to determine whether RelA signaling in tanycytes is essential for the LPS-induced D2 increase in vivo by conditional elimination of RelA in tanycytes of mice (RelA(ASTKO)). Dio2 and Trh mRNA expression were assessed by quantitative in situ hybridization 8 or 24 hours after saline or LPS injection. At the same time points, we measured pituitary Tshβ mRNA expression and serum T3 and T4 concentrations. In RelA(ASTKO) mice the LPS-induced increase in Dio2 and decrease in Trh mRNA levels in the hypothalamus were reduced compared with the wild-type littermates, whereas the drop in pituitary Tshβ expression and in serum TH concentrations persisted. In conclusion, RelA is essential for the LPS-induced hypothalamic D2 increase and TRH decrease. The central changes in the HPT axis are, however, not required for the down-regulation of Tshβ expression and serum TH concentrations.

Metabolome response to temperature-induced virulence gene expression in two genotypes of pathogenic Vibrio parahaemolyticus.

BackgroundVibrio parahaemolyticus is a main causative agent of serious human seafood-borne gastroenteritis disease. Many researchers have investigated its pathogenesis by observing the alteration of its virulence factors in different conditions. It was previously known that culture conditions will influence the gene expression and the metabolic profile of V. parahaemolyticus, but little attention has been paid on the relationship between them. In this study, for the first time, the metabolomics response in relation to the expression of two major virulence genes, tdh and trh, induced at three temperatures (4, 25 and 37 °C) was examined in two genotypes of pathogenic Vibrio parahaemolyticus (ATCC33846 (tdh+/trh−/tlh+) and ATCC17802 (tdh−/trh+/tlh+)).ResultsReverse transcription real-time PCR (RT-qPCR) analysis illustrated that the expression levels of tdh and trh induced at 25 °C in V. parahaemolyticus were significantly higher than those induced at 4 and 37 °C. Principal components analysis (PCA) based on the UPLC & Q-TOF MS data presented clearly distinct groups among the samples treated by different temperatures. Metabolic profiling demonstrated that 179 of 1,033 kinds of identified metabolites in ATCC33846 changed significantly (p <0.01) upon culturing at different temperatures, meanwhile 101 of 930 kinds of metabolites changed (p <0.01) in ATCC17802. Pearson’s correlation analysis highlighted the correlation between metabolites and virulence gene expression levels. At the threshold of | r | = 1, p <0.01, 12 kinds of metabolites showed extremely significant correlations with tdh expression, and 4 kinds of metabolites significantly correlated with trh expression. It is interesting that 3D, 7D, 11D-Phytanic acid showed the same trend with pyrophosphate, whose derivative could activate the degradation of phytanic acid. Several metabolites could be sorted into the same class by the method of chemical taxonomy, by assuming that they are involved in the same metabolic pathways.ConclusionsThis research can help to find biomarkers to monitor virulence gene expression, and can further help laboratory and clinical research of V. parahaemolyticus from the perspective of metabolomics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0688-5) contains supplementary material, which is available to authorized users.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Hypothalamo-Pituitary-Thyroid (HPT) Axis via Type 2 Deiodinase in Male Mice.

The hypothalamic activation of thyroid hormones by type 2 deiodinase (D2), catalyzing the conversion of thyroxine to T3, is critical for the proper function of the hypothalamo-pituitary-thyroid (HPT) axis. Regulation of D2 expression in tanycytes alters the activity of the HPT axis. However, signals that regulate D2 expression in tanycytes are poorly understood. The pituitary adenylate cyclase-activating polypeptide (PACAP) increases intracellular cAMP level, a second messenger known to stimulate the DIO2 gene; however, its importance in tanycytes is not completely characterized. Therefore, we tested whether this ubiquitously expressed neuropeptide regulates the HPT axis through stimulation of D2 in tanycytes. PACAP increased the activity of human DIO2 promoter in luciferase reporter assay that was abolished by mutation of cAMP-response element. Furthermore, PAC1R receptor immunoreactivity was identified in hypothalamic tanycytes, suggesting that these D2-expressing cells could be regulated by PACAP. Intracerebroventricular PACAP administration resulted in increased D2 activity in the mediobasal hypothalamus, suppressed Trh expression in the hypothalamic paraventricular nucleus, and decreased Tshb expression in the pituitary demonstrating that PACAP affects the D2-mediated control of the HPT axis. To understand the role of endogenous PACAP in the regulation of HPT axis, the effect of decreased PACAP expression was studied in heterozygous Adcyap1 (PACAP) knockout mice. These animals were hypothyroid that may be the consequence of altered hypothalamic T3 degradation during set-point formation of the HPT axis. In conclusion, PACAP is an endogenous regulator of the HPT axis by affecting T3-mediated negative feedback via cAMP-induced D2 expression of tanycytes.

Increased Thyroid Hormone Activation Accompanies the Formation of Thyroid Hormone-Dependent Negative Feedback in Developing Chicken Hypothalamus.

The hypothalamic-pituitary-thyroid axis is governed by hypophysiotropic TRH-synthesizing neurons located in the hypothalamic paraventricular nucleus under control of the negative feedback of thyroid hormones. The mechanisms underlying the ontogeny of this phenomenon are poorly understood. We aimed to determine the onset of thyroid hormone-mediated hypothalamic-negative feedback and studied how local hypothalamic metabolism of thyroid hormones could contribute to this process in developing chicken. In situ hybridization revealed that whereas exogenous T4 did not induce a statistically significant inhibition of TRH expression in the paraventricular nucleus at embryonic day (E)19, T4 treatment was effective at 2 days after hatching (P2). In contrast, TRH expression responded to T3 treatment in both age groups. TSHβ mRNA expression in the pituitary responded to T4 in a similar age-dependent manner. Type 2 deiodinase (D2) was expressed from E13 in tanycytes of the mediobasal hypothalamus, and its activity increased between E15 and P2 both in the mediobasal hypothalamus and in tanycyte-lacking hypothalamic regions. Nkx2.1 was coexpressed with D2 in E13 and P2 tanycytes and transcription of the cdio2 gene responded to Nkx2.1 in U87 glioma cells, indicating its potential role in the developmental regulation of D2 activity. The T3-degrading D3 enzyme was also detected in tanycytes, but its level was not markedly changed before and after the period of negative feedback acquisition. These findings suggest that increasing the D2-mediated T3 generation during E18-P2 could provide the sufficient local T3 concentration required for the onset of T3-dependent negative feedback in the developing chicken hypothalamus.

Abstract P141: Cardiac TRH Partly Mediates Angiotensin II-induced Fibrotic and Hypertrophy Effects in "in vivo" and "in vitro" Models

Cardiac TRH (cTRH) is overexpressed in the hypertrophied ventricle (LV) of the SHR. Additionally in vivo siRNA-TRH treatment induced downregulation of LV-TRH preventing cardiac hypertrophy and fibrosis demonstrating that TRH is involved in hypertrophic and fibrotic processes. Moreover, in a normal heart, the increase of LV TRH expression alone could induce structural changes where fibrosis and hypertrophy could be involved, independently of any other system alterations. Is well-known the cardiac hypertrophy/ fibrotic effects induced by AII, raising the question of whether specific LV cTRH inhibition might attenuates AII induced cardiac hypertrophy and fibrosis in mice. We challenged C57 mice with AII (osmotic pumps,14 days; 2 mg/kg) to induce cardiac hypertrophy vs saline. Groups were divided and , simultaneously to pump surgery, injected intracardiac with siRNA-TRH and siRNA-Con as its control. Body weight, water consume and SABP were measured daily. As expected, AII significantly increased SABP (p&lt;0.05) in both groups treated , although cardiac hypertrophy (heart weight/body weight) was only evident in the group with the cardiac TRH system undamaged, suggesting that the cardiac TRH system function as a necessary mediator of the AII-induced hypertrophic effect. As hypothesized, we found an AII-induced increase of TRH (p&lt;0.05) gene expression (real-t PCR) confirmed by immunofluorescence that was not observed in the group AII+siRNA-TRH demonstrating the specific siRNA treatment efficiency. Furthermore, AII significantly increase (p&lt;0.05) BNP (hypertrophic marker), III collagen and TGFB (fibrosis markers) expressions only in the group with AII with the cardiac TRH system intact. On the contrary, the group with AII and the cTRH system inhibited, shows genes expressions similar to the saline control group. We confirmed these results by immunofluorescence. Similar fibrotic results were observed with NIH3T3 cell culture where we demonstrated that AII induced TRH gene expression (p&lt;0.05) and its inhibition impedes AII-induced increase of TGFB and III/I collagens expressions telling us about the role of the cTRH in the AII fibrosis effects. Our results point out that the cardiac TRH is involved in the AII-induced hypertrophic and fibrotic effects.

Fasting Enhances Pyroglutamyl Peptidase II Activity in Tanycytes of the Mediobasal Hypothalamus of Male Adult Rats.

Fasting down-regulates the hypothalamus-pituitary-thyroid (HPT) axis activity through a reduction of TRH synthesis in neurons of the parvocellular paraventricular nucleus of the hypothalamus (PVN). These TRH neurons project to the median eminence (ME), where TRH terminals are close to the cytoplasmic extensions of β2 tanycytes. Tanycytes express pyroglutamyl peptidase II (PPII), the TRH-degrading ectoenzyme that controls the amount of TRH that reaches the anterior pituitary. We tested the hypothesis that regulation of ME PPII activity is another mechanism by which fasting affects the activity of the HPT axis. Semiquantitative in situ hybridization histochemistry data indicated that PPII and deiodinase 2 mRNA levels increased in tanycytes after 48 hours of fasting. This increase was transitory, followed by an increase of PPII activity in the ME, and a partial reversion of the reduction in PVN pro-TRH mRNA levels and the number of TRH neurons detected by immunohistochemistry. In fed animals, adrenalectomy and corticosterone treatment did not change ME PPII activity 72 hours later. Methimazole-induced hypothyroidism produced a profound drop in tanycytes PPII mRNA levels, which was reverted by 3 days of treatment with T4. The activity of thyroliberinase, the serum isoform of PPII, was increased at most fasting time points studied. We conclude that delayed increases in both the ME PPII as well as the thyroliberinase activities in fasted male rats may facilitate the maintenance of the deep down-regulation of the HPT axis function, despite a partial reactivation of TRH expression in the PVN.

Flatfish metamorphosis: A hypothalamic independent process?

Anuran and flatfish metamorphosis are tightly regulated by thyroid hormones that are the necessary and sufficient factors that drive this developmental event. In the present study whole mount in situ hybridization (WISH) and quantitative PCR in sole are used to explore the central regulation of flatfish metamorphosis. Central regulation of the thyroid in vertebrates is mediated by the hypothalamus–pituitary–thyroid (HPT) axis. Teleosts diverge from other vertebrates as hypothalamic regulation in the HPT axis is proposed to be through hypothalamic inhibition although the regulatory factor remains enigmatic. The dynamics of the HPT axis during sole metamorphosis revealed integration between the activity of the thyrotrophes in the pituitary and the thyroid follicles. No evidence was found supporting a role for thyroid releasing hormone (trh) or corticotrophin releasing hormone (crh) in hypothalamic control of TH production during sole metamorphosis. Intriguingly the results of the present study suggest that neither hypothalamic trh nor crh expression changes during sole metamorphosis and raises questions about the role of these factors and the hypothalamus in regulation of thyrotrophs.

TGFβ2 regulates hypothalamic Trh expression through the TGFβ inducible early gene-1 (TIEG1) during fetal development

The hypothalamus regulates the homeostasis of the organism by controlling hormone secretion from the pituitary. The molecular mechanisms that regulate the differentiation of the hypothalamic thyrotropin-releasing hormone (TRH) phenotype are poorly understood. We have previously shown that Klf10 or TGFβ inducible early gene-1 (TIEG1) is enriched in fetal hypothalamic TRH neurons. Here, we show that expression of TGFβ isoforms (1-3) and both TGFβ receptors (TβRI and II) occurs in the hypothalamus concomitantly with the establishment of TRH neurons during late embryonic development. TGFβ2 induces Trh expression via a TIEG1 dependent mechanism. TIEG1 regulates Trh expression through an evolutionary conserved GC rich sequence on the Trh promoter. Finally, in mice deficient in TIEG1, Trh expression is lower than in wild type animals at embryonic day 17. These results indicate that TGFβ signaling, through the upregulation of TIEG1, plays an important role in the establishment of Trh expression in the embryonic hypothalamus.

Inhibition of angiotensin II increases hypothalamic thyroid hormone and stimulates orexigenic NPY/AgRP neurons (LB437)

We have demonstrated that mice lacking renin (Ren1c ‐/‐) are lean and resistant to diet‐induced obesity, which is associated with increased energy expenditure and uncoupling proteins (UCPs) expression. Surprisingly, plasma catecholamine levels of Ren1c‐/‐ mice were similar to wild type (WT) mice, and their plasma total T3 and T4 were lower than those of WT, although they showed no obvious signs of hypothyroidism. We here investigate whether mice lacking renin display true hypothyroidism, and if so, the specific mechanism by which they develop it. The Ren1c‐/‐ mice have approximately 50% levels of free T3, freeT4 and TSH in the plasma, as well as reduced TRH expression in the hypothalamus compared to WT. Administration of TRH or angiotensin II (Ang II) both restored plasma TSH and thyroid hormones in the Ren1c‐/‐ mice, while these hormones were not affected by human renin, which stimulates mouse MAP kinases but does not cleave mouse angiotensinogen to Ang I. Moreover, the Ang II receptor blocker losartan decreased TRH, TSH, and thyroid hormones of WT mice to the levels similar to those in mice lacking renin. Furthermore, mice lacking renin and WT mice treated with losartan have significantly increased hypothalamic expression of type 2 deiodinase (Dio2), which converts T4 to the more potent T3, and have increased thyroid hormones in the hypothalamus, which likely inhibits TRH through feedback. Both mice lacking renin and WT mice treated with losartan have 3‐fold higher uncoupling protein‐2 (UCP2), orexigenic neuropeptide Y (NPY) and agouti‐related protein (AgRP), as well as 5‐fold lower anorexigenic proopiomelanocortin (POMC) gene expression in the hypothalamus relative to untreated WT mice. In contrast, Ren1c‐/‐ mice treated with human renin had similar expression levels of these genes. These results suggest that the inhibition of Ang II not of Ang‐independent effects of renin causes hypothyroidism and enhances food intake, which likely decrease energy expenditure. We hypothesize that changes in gene expression of the hypothalamus do not contribute to increased energy expenditure of the Ren1c‐/‐ mice, and that hypothalamic hyperthyroidism feedback inhibits TRH, leading to systemic hypothyroidism. Caution should be paid to hypothyroidism when using high dose of inhibitors of the renin angiotensin system.Grant Funding Source: National Science Council: 102‐2320‐B‐009 ‐002 ‐MY3

Tissue-Specific Alterations in Thyroid Hormone Homeostasis in Combined Mct10 and Mct8 Deficiency

The monocarboxylate transporter Mct10 (Slc16a10; T-type amino acid transporter) facilitates the cellular transport of thyroid hormone (TH) and shows an overlapping expression with the well-established TH transporter Mct8. Because Mct8 deficiency is associated with distinct tissue-specific alterations in TH transport and metabolism, we speculated that Mct10 inactivation may compromise the tissue-specific TH homeostasis as well. However, analysis of Mct10 knockout (ko) mice revealed normal serum TH levels and tissue TH content in contrast to Mct8 ko mice that are characterized by high serum T3, low serum T4, decreased brain TH content, and increased tissue TH concentrations in the liver, kidneys, and thyroid gland. Surprisingly, mice deficient in both TH transporters (Mct10/Mct8 double knockout [dko] mice) showed normal serum T4 levels in the presence of elevated serum T3, indicating that the additional inactivation of Mct10 partially rescues the phenotype of Mct8 ko mice. As a consequence of the normal serum T4, brain T4 content and hypothalamic TRH expression were found to be normalized in the Mct10/Mct8 dko mice. In contrast, the hyperthyroid situation in liver, kidneys, and thyroid gland of Mct8 ko mice was even more severe in Mct10/Mct8 dko animals, suggesting that in these organs, both transporters contribute to the TH efflux. In summary, our data indicate that Mct10 indeed participates in tissue-specific TH transport and also contributes to the generation of the unusual serum TH profile characteristic for Mct8 deficiency.

Interleukin 6 Deficiency Modulates the Hypothalamic Expression of Energy Balance Regulating Peptides during Pregnancy in Mice

Pregnancy is associated with hyperphagia, increased adiposity and multiple neuroendocrine adaptations. Maternal adipose tissue secretes rising amounts of interleukin 6 (IL6), which acts peripherally modulating metabolic function and centrally increasing energy expenditure and reducing body fat. To explore the role of IL6 in the central mechanisms governing dam's energy homeostasis, early, mid and late pregnant (gestational days 7, 13 and 18) wild-type (WT) and Il6 knockout mice (Il6-KO) were compared with virgin controls at diestrus. Food intake, body weight and composition as well as indirect calorimetry measurements were performed in vivo. Anabolic and orexigenic peptides: neuropeptide Y (Npy) and agouti-related peptide (Agrp); and catabolic and anorectic neuropeptides: proopiomelanocortin (Pomc), corticotrophin and thyrotropin-releasing hormone (Crh and Trh) mRNA levels were determined by in situ hybridization. Real time-PCR and western-blot were used for additional tissue gene expression and protein studies. Non-pregnant Il6-KO mice were leaner than WT mice due to a decrease in fat but not in lean body mass. Pregnant Il6-KO mice had higher fat accretion despite similar body weight gain than WT controls. A decreased fat utilization in absence of Il6 might explain this effect, as shown by increased respiratory exchange ratio (RER) in virgin Il6-KO mice. Il6 mRNA levels were markedly enhanced in adipose tissue but reduced in hypothalamus of mid and late pregnant WT mice. Trh expression was also stimulated at gestational day 13 and lack of Il6 blunted this effect. Conversely, in late pregnant mice lessened hypothalamic Il6 receptor alpha (Il6ra), Pomc and Crh mRNA were observed. Il6 deficiency during this stage up-regulated Npy and Agrp expression, while restoring Pomc mRNA levels to virgin values. Together these results demonstrate that IL6/IL6Ra system modulates Npy/Agrp, Pomc and Trh expression during mouse pregnancy, supporting a role of IL6 in the central regulation of body fat in this physiological state.