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- Leptin resistance is not the primary cause of weight gain associated with reduced sex hormone levels in female mice. [JOURNAL ARTICLE]
- Endocrinology 2014 Aug 21.:en20141276.
Several studies have shown that estrogens mimic leptin's effects on energy balance regulation. However, the findings regarding the consequences of reduced sex hormone levels on leptin sensitivity are divergent. In the present study, we employed different experimental paradigms to elucidate the interaction between estrogens, leptin and energy balance regulation. We confirmed previous reports showing that ovariectomy caused a reduction in locomotor activity and energy expenditure leading mice to obesity and glucose intolerance. However, the acute and chronic anorexigenic effects of leptin were preserved in ovariectomized (OVX) mice despite their increased serum leptin levels. We studied hypothalamic gene expression at different time points after ovariectomy and observed that changes in the expression of genes involved in leptin resistance (suppressors of cytokine signaling and protein-tyrosine phosphatases) did not precede the early onset of obesity in OVX mice. On the contrary, reduced sex hormone levels caused an up-regulation of the long form of the leptin receptor (LepR), resulting in increased activation of leptin signaling pathways in OVX leptin-treated animals. The up-regulation of the LepR was observed in long-term OVX mice (30 days or 24 weeks after ovariectomy), but not 7 days after the surgery. In addition, we observed a progressive decrease in the co-expression of LepR and estrogen receptor-α in the hypothalamus after the ovariectomy resulting in a low percentage of dual-labeled cells in OVX mice. Taken together, our findings suggest that the weight gain caused by reduced sex hormone levels is not primarily caused by induction of a leptin resistance state.
- Changes in RFamide related peptide-1 (RFRP-1)-immunoreactivity during postnatal development and the estrous cycle. [JOURNAL ARTICLE]
- Endocrinology 2014 Aug 21.:en20141274.
Gonadotropin releasing hormone (GnRH) is a key player in the hypothalamic control of gonadotropin secretion from the anterior pituitary gland. It has been shown that the mammalian counterpart of the avian gonadotropin inhibitory hormone named RFamide-related peptide (RFRP) is expressed in hypothalamic neurons that innervate and inhibit GnRH neurons. The RFRP precursor is processed into two mature peptides RFRP-1 and RFRP-3. These are characterized by a conserved C-terminal motif Arg-Phe-NH2 but display highly different N-terminals. Even though the two peptides are equally potent in vitro, little is known about their relative distribution and their distinct roles in vivo. In this study, we raised an antiserum selective for RFRP-1 and defined the distribution of RFRP-1-immunoreactive (ir) neurons in the rat brain. Next, we analyzed the level of RFRP-1-immunoreactivity during postnatal development in males and females and investigated changes in RFRP-1-immunoreactivity during the estrous cycle. RFRP-1-ir neurons were distributed along the third ventricle from the caudal part of the medial anterior hypothalamus throughout the medial tuberal hypothalamus and were localized in, but mostly in between, the dorsomedial hypothalamic, ventromedial hypothalamic, and arcuate nuclei. The number of RFRP-1-ir neurons and the density of cellular immunoreactivity were unchanged from juvenile to adulthood in male rats during the postnatal development. However, both parameters were significantly increased in female rats from peri-puberty to adulthood, demonstrating prominent gender difference in the developmental control of RFRP-1 expression. The percentage of c-Fos positive RFRP-1-ir neurons was significantly higher in diestrus as compared to proestrus and estrus. In conclusion, we found that adult females, as compared to males, have significantly more RFRP-1-immunoreactivity pr cell, and these cells are regulated during the estrous cycle.
- LPXRFa, the Piscine Ortholog of GnIH, and LPXRF Receptor Positively Regulate Gonadotropin Secretion in Tilapia (Oreochromis niloticus). [JOURNAL ARTICLE]
- Endocrinology 2014 Aug 21.:en20132047.
LPXRFamide (LPXRFa) peptides have been characterized for their ability to inhibit gonadotropin (GTH) release in birds and stimulate growth hormone (GH) release in frogs. However, their involvement in regulating the reproductive HPG axis in mammals and fish is inconclusive. To study the role of LPXRFa peptides in the regulation of GTH secretion, we cloned tilapia LPXRFa and LPXRF receptor (LPXRF-R). Processing of the tilapia preproLPXRFa liberated three mature LPXRFa peptides which varied in size and post-translational modifications. Phylogenetic analysis of LPXRFa and the closely related RFamide peptide PQRFa showed clear clustering of each peptide sequence with its orthologs from various vertebrates. Signal-transduction analysis of the tilapia LPXRF-R in COS-7 cells showed clear stimulation of CRE-dependent luciferase activity, while the human NPFFR1 showed suppression of forskolin-induced CRE-dependent activity in this system. Administration of the tilapia pyroglutaminated LPXRFa-2 peptide to primary cell culture of tilapia pituitaries, or to reproductive female tilapia by i.p. injection, positively regulated both LH and FSH release in vivo and in vitro. Using double-labeled fluorescent in-situ hybridization and immunofluorescence, βLH cells were found to co-express both tilapia LPXRFa and tilapia LPXRF-R mRNA, while some of the βFSH cells co-expressed only LPXRF-R mRNA. No co-expression of tilapia LPXRF-R was identified in GH-positive cells. These findings indicate that the LPXRFa system is a potent positive regulator of the reproductive neuroendocrine axis of tilapia.
- In Vivo Fluorescence Imaging and Urinary Monoamines as Surrogate Biomarkers of Disease Progression in a Mouse Model of Pheochromocytoma. [JOURNAL ARTICLE]
- Endocrinology 2014 Aug 19.:en20141431.
Pheochromocytoma is a rare but potentially lethal neuroendocrine tumor arising from catecholamine producing chromaffin cells. Especially for metastatic pheochromocytoma, the availability of animal models is essential for developing novel therapies. For evaluating therapeutic outcome in rodent pheochromocytoma models reliable quantification of multiple organ lesions depends on dedicated small animal in vivo imaging, which is still challenging and only available at specialized research facilities. Here, we investigated whether whole-body fluorescence imaging and monitoring of urinary free monoamines provide suitable parameters for measuring tumor progression in a murine allograft model of pheochromocytoma. We generated an mCherry-expressing mouse pheochromocytoma cell line by lentiviral gene transfer. These cells were injected subcutaneously into nude mice to perform whole-body fluorescence imaging of tumor development. Urinary free monoamines were measured by liquid chromatography with tandem mass spectrometry. Tumor fluorescence intensity and urinary outputs of monoamines showed tumor growth-dependent increases ( < 0.001) over the 30 days of monitoring post tumor engraftment. Concomitantly, systolic blood pressure was increased significantly during tumor growth. Tumor volume correlated significantly ( < 0.001) and strongly with tumor fluorescence intensity (= 0.946) and urinary outputs of dopamine ( = 0.952), methoxytyramine ( = 0.947), norepinephrine ( = 0.756) and normetanephrine ( = 0.949). Dopamine and methoxytyramine outputs allowed for detection of lesions at diameters below 2.3 mm. Our results demonstrate that MPC-mCherry cell tumors are functionally similar to human pheochromocytoma. Both tumor fluorescence intensity and urinary outputs of free monoamines provide precise parameters of tumor progression in this subcutaneous mouse model of pheochromocytoma. This animal model will allow for testing new treatment strategies for chromaffin cell tumors.
- Progesterone induces RhoA inactivation in male rat aortic smooth muscle cells through up-regulation of p27(kip1.) [JOURNAL ARTICLE]
- Endocrinology 2014 Aug 19.:en20141344.
Previously, we showed that progesterone (P4) at physiologic concentrations (5-500 nM) inhibits proliferation and migration of rat aortic smooth muscle cells (RASMC). The P4-induced migration inhibition in RASMC was resulted from Ras homolog gene family, member A (RhoA) inactivation induced by activating the cSrc/AKT/ERK 2/p38-mediated signaling pathway. We also demonstrated that up-regulation of p27(kip1) is involved in the P4-induced migration inhibition in RASMC. Since P4 can increase formation of the p27(kip1)-RhoA complex in RASMC, this finding led us to hypothesize that the P4-induced inactivation in RhoA might be caused by up-regulation of p27(kip1). Here, we showed that P4 increased phosphorylation of p27(kip1) at serine 10 (Ser10) in the nucleus, which in turn caused p27(kip1) translocation from the nucleus to the cytosol, subsequently increasing formation of the p27(kip1)-RhoA complex. These effects were blocked by knocking-down kinase interacting stathmin (KIS) using KIS siRNA. Knock-down of p27(kip1) abolished the P4-induced decreases in the level of RhoA protein in RASMC. However, pre-treatment of RASMC with the proteasome inhibitor, MG132, prevented the P4-induced degradation of p27(kip1) and RhoA. Taken together, our investigation of P4-induced migration inhibition in RASMC showed a sequence of associated intracellular events that included (a) increase in formation of the KIS-p27(kip1) complex in the nucleus; (b) phosphorylated nuclear p27(kip1) at Ser10; (c) increased cytosolic translocation of p27(kip1) and formation of the p27(kip1)-RhoA complex in the cytosol; and (d) degradation of p27(kip1) and RhoA through the ubiquitin-proteasome pathway. These findings highlight the molecular mechanisms underlying P4-induced migration inhibition in RASMC.
- Role of Melanocortin Signaling in Neuroendocrine and Metabolic Actions of Leptin in Male Rats with Uncontrolled Diabetes. [JOURNAL ARTICLE]
- Endocrinology 2014 Aug 19.:en20141169.
Although the antidiabetic effects of leptin require intact neuronal melanocortin signaling in rodents with uncontrolled diabetes (uDM), increased melanocortin signaling is not sufficient to mimic leptin's glucose-lowering effects. The current studies were undertaken to clarify the role of melanocortin signaling in leptin's ability to correct metabolic and neuroendocrine disturbances associated with uDM. To accomplish this, bilateral cannulae were implanted in the lateral ventricle of rats with streptozotocin-induced diabetes, and leptin was coinfused with varying doses of the melanocortin 3/4 receptor (MC3/4R) antagonist, SHU9119. An additional cohort of streptozotocin-induced diabetes rats received intracerebroventricular administration of either the MC3/4R agonist, melanotan-II, or its vehicle. Consistent with previous findings, leptin's glucose-lowering effects were blocked by intracerebroventricular SHU9119. In contrast, leptin-mediated suppression of hyperglucagonemia involves both melanocortin dependent and independent mechanisms, and the degree of glucagon inhibition was a associated with reduced plasma ketone body levels. Increased CNS melanocortin signaling alone fails to mimic leptin's ability to correct any of the metabolic or neuroendocrine disturbances associated with uDM. Moreover, the inability of increased melanocortin signaling to lower diabetic hyperglycemia does not appear to be secondary to release of the endogenous MC3/4R inverse agonist, Agouti-related peptide (AgRP), because AgRP knockout mice did not show increased susceptibility to the antidiabetic effects of increased MC3/4R signaling. Overall, these data suggest that 1) AgRP is not a major driver of diabetic hyperglycemia, 2) mechanisms independent of melanocortin signaling contribute to leptin's antidiabetic effects, and 3) melanocortin receptor blockade dissociates leptin's glucose-lowering effect from its action on other features of uDM, including reversal of hyperglucagonemia and ketosis, suggesting that brain control of ketosis, but not blood glucose levels, is glucagon dependent.
- Triiodothyronine Prevents Cardiac Ischemia/Reperfusion Mitochondrial Impairment and Cell Loss by Regulating miR30a/p53 Axis. [JOURNAL ARTICLE]
- Endocrinology 2014 Aug 19.:en20141106.
Mitochondrial dysfunctions critically affect cardiomyocyte survival during ischemia/reperfusion (I/R) injury. In this scenario p53 activates multiple signaling pathways that impair cardiac mitochondria and promote cell death. p53 is a validated target of miR-30 whose levels fall under ischemic conditions. Although triiodothyronine (T3) rescues post-ischemic mitochondrial activity and cell viability, no data are available on its role in the modulation of p53 signaling in I/R. Here we test the hypothesis that early T3 supplementation in rats inhibits the post I/R activation of p53 pro-death cascade through the maintenance of miRNA 30a expression. In our model, T3 infusion improves the recovery of post-ischemic cardiac performance. At the molecular level, the beneficial effect of T3 is associated with restored levels of miR-30a expression in the area at risk (AAR) that correspond to p53 mRNA downregulation. The concomitant decrease in p53 protein content reduces Bax expression and limits mitochondrial membrane depolarization resulting in preserved mitochondrial function and decreased apoptosis and necrosis extent in the AAR. Also in primary cardiomyocyte culture of neonatal rats, T3 prevents both miR-30a downregulation and p53 raise induced by hypoxia. The regulatory effect of T3 is greatly suppressed by miR-30a knockdown. Overall these data suggest a new mechanism of T3-mediated cardioprotection that is targeted to mitochondria and acts, at least in part, through the regulation of miR-30a/p53 axis.
- Somatotropinomas, But Not Nonfunctioning Pituitary Adenomas, Maintain a Functional Apoptotic RET/Pit1/ARF/p53 Pathway That Is Blocked by Excess GDNF. [JOURNAL ARTICLE]
- Endocrinology 2014 Aug 19.:en20141034.
Acromegaly is caused by somatotroph cell adenomas (somatotropinomas [ACROs]), which secrete GH. Human and rodent somatotroph cells express the RET receptor. In rodents, when normal somatotrophs are deprived of the RET ligand, GDNF, RET is processed intracellularly to induce overexpression of Pit1, which in turn leads to p19Arf/p53-dependent apoptosis. Our purpose was to ascertain whether human ACROs maintain the RET/Pit1/p14ARF/p53/apoptosis pathway, relative to nonfunctioning pituitary adenomas (NFPAs). Apoptosis in the absence and presence of GDNF was studied in primary cultures of 7 ACROs and 3 NFPAs. Parallel protein extracts were analyzed for expression of RET, Pit1, p14Arf, p53, and phospho-Akt. When GDNF deprived, ACRO cells, but not NFPAs, presented marked level of apoptosis that was prevented in the presence of GDNF. Apoptosis was accompanied by RET processing, Pit1 accumulation, and p14ARF and p53 induction. GDNF prevented all these effects via activation of phospho-AKT. Overexpression of human Pit1 (hPit1) directly induced p14Arf/p53 and apoptosis in a pituitary cell line. Using in silico studies, 2 cEBPα consensus-binding sites were found to be 100% conserved in mouse, rat, and hPit1 promoters. Deletion of 1 cEBPα site prevented the RET-induced increase in hPit1 promoter expression. TaqMan qRT-PCR for RET, Pit1, Arf, TP53, GDNF, SF-1, and GH was performed in RNA from whole ACRO and NFPA tumors. ACRO but not NFPA adenomas express RET and Pit1. GDNF expression in the tumors was positively correlated with RET and negatively correlated with p53. In conclusion, ACROs maintain an active RET/Pit1/p14Arf/p53/apoptosis pathway that is inhibited by GDNF. Disruption of GDNF's survival function might constitute a new therapeutic route in acromegaly.
- Thyroid hormone receptors and resistance to thyroid hormone disorders. [JOURNAL ARTICLE]
- Nat Rev Endocrinol 2014 Aug 19.
Thyroid hormone action is predominantly mediated by thyroid hormone receptors (THRs), which are encoded by the thyroid hormone receptor α (THRA) and thyroid hormone receptor β (THRB) genes. Patients with mutations in THRB present with resistance to thyroid hormone β (RTHβ), which is a disorder characterized by elevated levels of thyroid hormone, normal or elevated levels of TSH and goitre. Mechanistic insights about the contributions of THRβ to various processes, including colour vision, development of the cochlea and the cerebellum, and normal functioning of the adult liver and heart, have been obtained by either introducing human THRB mutations into mice or by deletion of the mouse Thrb gene. The introduction of the same mutations that mimic human THRβ alterations into the mouse Thra and Thrb genes resulted in distinct phenotypes, which suggests that THRA and THRB might have non-overlapping functions in human physiology. These studies also suggested that THRA mutations might not be lethal. Seven patients with mutations in THRα have since been described. These patients have RTHα and presented with major abnormalities in growth and gastrointestinal function. The hypothalamic-pituitary-thyroid axis in these individuals is minimally affected, which suggests that the central T3 feedback loop is not impaired in patients with RTHα, in stark contrast to patients with RTHβ.
- Thyroid function: Subclinical hypothyroidism and thyroid autoimmunity increase the risk of miscarriage. [JOURNAL ARTICLE]
- Nat Rev Endocrinol 2014 Aug 19.