ABSTRACT
PURPOSE: Mitotane is the only approved treatment for metastatic adrenocortical carcinoma (ACC). Monitoring plasma levels is recommended, but its predictive value is insufficient. METHODS: This prospective study of the French ENDOCAN-COMETE network aimed to investigate the prognostic role of plasma mitotane levels pharmacokinetics and free or bound to lipoprotein fraction measurements during six consecutive months. Lipoprotein fractions were isolated by ultracentrifugation, and mitotane level was determined by HPLC-UV. Total, free, and lipoprotein fraction bound plasma mitotane were monitored every two months for six months with morphological assessment. The primary endpoint was overall survival (OS). RESULTS: 21 patients with metastatic ACC were included. Median overall survival was 23 months. The median free mitotane level per patient was 12% (± 7%), and the majority (88%) was bound to lipoprotein fractions. Several pharmacokinetics measures of total mitotane were related to OS: first level at one month (p = 0.026), mean level (p = 0.055), and area under the curve (AUC) (p = 0.048), with higher exposure associated to longer OS. Free mitotane (not bounded) and mitotane bounded to lipoprotein subfraction added no prognostic values. The relationship between the mitotane level and OS suggested a minimum "effective" threshold of 10-15 mg/L or an area under the curve above 100 mg/L/month with no individualized maximum value. CONCLUSION: This prospective study did not identify any added prognostic value of free mitotane level over the total level. Early total mitotane level measurements (before 3-6 months) were related to OS with a higher and faster exposure related to more prolonged survival.
ABSTRACT
We report a patient carrying a novel pathogenic variant p.(Tyr101Cys) in ISCA1 leading to MMDS type 5. He initially presented a psychomotor regression with loss of gait and language skills and a tetrapyramidal spastic syndrome. Biochemical analysis of patient fibroblasts revealed impaired lipoic acid synthesis and decreased activities of complex I and II of respiratory chain. While ISCA1 is involved in the mitochondrial machinery for iron-sulfur cluster biogenesis, these dysfunctions are secondary to impaired maturation of mitochondrial proteins containing the [4Fe-4S] clusters. Expression and purification of the human ISCA1 showed a decreased stability of the [2Fe-2S] cluster in the mutated protein.
Subject(s)
Amino Acid Substitution , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Mitochondrial Diseases/genetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Cells, Cultured , Child, Preschool , Humans , Male , Pedigree , Phenotype , Protein Domains , Protein StabilityABSTRACT
Congenital adrenal hyperplasia is an autosomal recessive disease due to functional abnormalities of adrenal steroid enzymes. The most common form of the disease is due to a 21-hydroxylase deficiency. The classical forms (most severe) are characterized by a deficiency in cortisol and sometimes in aldosterone, which may compromise the vital prognosis of neonates, and by an increase in androgen synthesis, leading to the virilization of girls' external genitalia at birth, followed by clinical signs of hyperandrogenism during childhood and adolescence. Neonatal screening has improved management and reduced morbidity and mortality in the neonatal period, but its performance could be broadly optimised by adjusting the assay techniques or the biomarkers used. The genetic diagnosis is difficult owing to the large genetic heterogeneity of the 6p21.3 region, which contains the CYP21A2 gene, especially with respect to the use of new-generation techniques of sequencing. Prenatal diagnosis is now possible as early as 6 weeks of gestation, but prenatal treatment remains controversial, awaiting results from prospective cohorts evaluating its long-term impact. Since conventional therapies have limitations, new therapies are currently being developed to allow better control of androgen synthesis and a substitutive treatment that respects the physiological rhythm of cortisol secretion, which would limit the development of long-term complications.
Subject(s)
Adrenal Hyperplasia, Congenital , Endocrinology/methods , Endocrinology/trends , Adrenal Hyperplasia, Congenital/classification , Adrenal Hyperplasia, Congenital/epidemiology , Adrenal Hyperplasia, Congenital/etiology , Adrenal Hyperplasia, Congenital/therapy , Child , Diagnosis, Differential , Female , Genetic Testing , Humans , Male , Pediatrics/methods , Pediatrics/trendsABSTRACT
Steroid resistance syndrome (mineralocorticoids, glucocorticoids, estrogens, androgens) is a rare clinical disorder, androgen insensitivity syndrome being the most commonly described. Resistance syndromes are characterized by elevated steroid hormone levels, secondary to an impaired signal transduction and a lack of negative feedback, without any specific clinical signs of steroid excess. In most cases, steroid hormone resistance is generally caused by steroid receptor mutations. Several nonsense and missense mutations or deletions have already been described for all steroid receptors, except for the progesterone receptor. The number of reported mutations is extremely variable, depending on the steroid receptor, ranging from 3 for the estrogen receptor to more than 500 for the androgen receptor. Loss of function of others factors implicated on the steroid hormone signaling pathway, such as molecular chaperones or coactivators, might also be involved in the pathogenesis of steroid resistance. Therapy is essentially symptomatic, there is currently no pharmacological strategy enabling restoration of the impaired hormone signaling pathway. Identification of steroid resistance syndrome and their characterization should allow a better understanding of underlying molecular defects, opening new avenues for the development of novel alternative options for the disease management.
Subject(s)
Endocrine System Diseases/physiopathology , Steroids/physiology , Animals , Endocrine System Diseases/genetics , Endocrine System Diseases/metabolism , Endocrine System Diseases/therapy , Humans , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Steroids/metabolismABSTRACT
Congenital lipodystrophies are heterogeneous genetic diseases, leading to the loss of adipose tissue. This loss of adipose tissue can be generalized or partial, thus defining different phenotypes. These lipodystrophies have a major metabolic impact, secondary to lipotoxicity. This lipotoxicity is responsible for insulin resistance, dyslipidemia and hepatic steatosis. The severity of the metabolic impact correlates with the severity of the loss of adipose tissue. Mutations in 15 predisposition genes are currently described; BSCL2 and AGPT2 genes are the major genes in the generalized forms. On the contrary, LMNA and PPARG gene mutations are recovered in partial lipodystrophies forms. These different genes encode for proteins involved in adipocyte physiology, altering adipocyte differentiation, triglycerides synthesis and lysis or playing a major role in the lipid droplet formation. Congenital lipodystrophies treatment is based on the management of metabolic comorbidities but recombinant leptin therapy appears to have promising results. These different points have been recently discussed during the 2015 Endocrine Society Congress, notably by S. O'Rahilly and are highlighted in this review.
Subject(s)
Lipodystrophy, Congenital Generalized/genetics , Lipodystrophy/congenital , Lipodystrophy/genetics , Adipocytes/physiology , Adipose Tissue/pathology , Adolescent , Adult , Angiopoietin-2/genetics , Female , GTP-Binding Protein gamma Subunits/genetics , Genetic Predisposition to Disease , Humans , Lamin Type A/genetics , Leptin/therapeutic use , Lipodystrophy/therapy , Lipodystrophy, Congenital Generalized/pathology , Lipodystrophy, Congenital Generalized/therapy , Mutation , PPAR gamma/genetics , Recombinant ProteinsABSTRACT
This review focuses on new aspects in craniopharyngiomas with emphasis on pathogenic mechanisms and treatment strategies that were presented at the joined Endocrine Society/International Society of Endocrinology meeting in Chicago in June 2014. Craniopharyngiomas are benign epithelial tumors arising from the pituitary stalk or gland. Two subtypes could be distinguished: an adamatinomatous form that is more common in children, and a papillary form that is observed almost exclusively in adults. Besides these histological differences, these two variants differ in some molecular features that have been recently identified and could have important therapeutic implications. Despite its histologically benign nature, the morbidities related to the tumor itself or its treatment raise many concerns and excess mortality rates up to nine times higher than in the general population has been reported. Among the potential sequelae of craniopharyngiomas, obesity seems the most frequent. The difficulty in the management of this obesity lies in its complex underlying pathophysiological mechanisms. Complete resection of localized tumors should be attempted while a limited hypothalamus-sparing surgery followed or not by radiotherapy should be adopted in tumors involving the hypothalamus. A multidisciplinary approach including, in particular, a dedicated neurosurgeon, and a therapeutic strategy tailored to the individual presentation of the craniopharyngioma in any patient should be initiated at diagnosis for improving the prognosis of these tumors.
Subject(s)
Craniopharyngioma/etiology , Craniopharyngioma/therapy , Pituitary Neoplasms/etiology , Pituitary Neoplasms/therapy , Adult , Child , Craniopharyngioma/diagnosis , Drug Therapy , General Surgery , Humans , Obesity/etiology , Pituitary Neoplasms/diagnosis , RadiotherapyABSTRACT
A better understanding of molecular mechanisms responsible for tumorigenesis has allowed the development of targeted drugs designed to improve the outcome of cancer. In endocrine tumors, several molecules have demonstrated efficacy in terms of progression free survival during phase III trials such as vandetanib and cabozantinib in medullary thyroid carcinoma, sorafenib in differentiated thyroid carcinoma and everolimus or sunitinib for pancreatic neuroendocrine tumors. Rare cancer network has allowed ongoing phase III trials in malignant pheochromocytoma and adrenocortical carcinoma. However, to date no specific predictive biomarker has yet been identified for a personalized cancer medicine. We review recent advances in endocrine oncology concerning molecular targets identification, targeted therapies and predictive or prognostic markers.
Subject(s)
Endocrine Gland Neoplasms/drug therapy , Molecular Targeted Therapy , Antineoplastic Agents/therapeutic use , Carcinoma, Neuroendocrine , Clinical Trials, Phase III as Topic , Disease-Free Survival , Everolimus , Humans , Indoles/therapeutic use , Neuroendocrine Tumors/drug therapy , Niacinamide/analogs & derivatives , Niacinamide/therapeutic use , Pancreatic Neoplasms/drug therapy , Phenylurea Compounds/therapeutic use , Piperidines/therapeutic use , Prognosis , Pyrroles/therapeutic use , Quinazolines/therapeutic use , Sirolimus/analogs & derivatives , Sirolimus/therapeutic use , Sorafenib , Sunitinib , Thyroid Neoplasms/drug therapy , Treatment OutcomeABSTRACT
Three articles published in the NEJM in 2009 have renewed the interest for brown adipose tissue (BAT) in humans. This review reports interesting new findings on adipocyte cell types that have been presented at the last meeting of the Endocrine Society in Houston, TX, in June 2012. Many studies have focused on identifying factors involved in brown adipocyte lineage, the site of adaptive thermogenesis. Indeed, the role of the transcription factors, such as PRDM16, in brown adipocyte differentiation has been unambiguously established. Very recently, the concept of "beigeing", defined as the occurrence of thermogenic brown adipocytes in white adipose tissue, has emerged, leading to the identification, by Bruce Spiegelman's group, of a new muscular hormone, called irisin, which is able to stimulate the "beigeing". This finding should convey toward the discovery of new mutations involved in the pathogenesis of obesity and lipodystrophies, and should be translated into innovative therapeutic perspectives. Finally, the nature of BAT innervation has been clarified and the presence of an autoregulatory loop between BAT and notably hypothalamic paraventricular nuclei via the sensory and sympathetic nervous systems has been delineated. These feedback circuits appear to be crucial to control BAT thermogenic activity.
Subject(s)
Adipose Tissue, Brown , Adipose Tissue, White , Adipose Tissue , Obesity/therapy , Adipocytes , Adipocytes, Brown , Adipocytes, White , Adipose Tissue, Brown/innervation , Adipose Tissue, Brown/physiology , Animals , Cell Differentiation , DNA-Binding Proteins/physiology , Fibronectins/physiology , Humans , Thermogenesis , Transcription Factors/physiologyABSTRACT
This review focuses on a number of new data on biology and pathophysiology of the metabolic syndrome (MetS) and the involvement of nuclear receptors that have been presented during the last Endocrine Society meeting, held in Houston in June 2012. Several studies have reported beneficial effects of various orphan nuclear receptors, including SHP (Small Heterodimeric Partner, NR0B2) and LXR (Liver X Receptor, NR1H3 and NR1H2), on various components of MetS. By using an inactivation model of SHP, David Moore has shown that SHP exerts "antidiabetic" effects but associated with hepatic steatosis development. He also showed that DLPC (dilauroyl phosphatidylcholine), an unconventional phospholipid, exhibited anti-diabetic properties through its binding to LRH-1 (Liver Receptor Homolog-1, NR5A2), a molecular partner of SHP. Interestingly, Carolyn Cummins investigated LXR α and ß isoforms knock-out mice and provided experimental evidence for the detailed mechanisms involved in the deleterious metabolic effects of glucocorticoids, pointing out to the functional interaction between LXRß, and the glucocorticoid receptor. These new and original studies open new therapeutic opportunities for the management of metabolic disorders in humans by selective modulators of these receptors.
Subject(s)
Metabolic Syndrome/physiopathology , Orphan Nuclear Receptors/physiology , Animals , Fatty Liver , Glucocorticoids/pharmacology , Humans , Hypoglycemic Agents , Liver X Receptors , Mice , Mice, Knockout , Phosphatidylcholines , Receptors, Cytoplasmic and Nuclear/physiologyABSTRACT
This review focuses on new aspects in growth hormone (GH) biology and pathophysiology presented at the Endocrine Society's meeting, in San Diego, in June 2010. First, we will describe recent advances in the understanding of cytokine hormone signaling via STAT5 in mammary gland development, highlighting the primary role of miR193b for differentiation of mammary stem cells into alveolar progenitor cells. We will examine the potential implication of endocrine and autocrine GH for mammary gland carcinogenesis. Three novel murine models bearing tissue-specific inactivation of GH receptor or JAK2 bring new insights into the large spectrum of GH effects on energy homeostasis. We will also report new data supporting a paracrine regulation of GH secretion in women by estrogen's action in the brain. Thereafter we will question the reasons for GH abuse for doping by assessing the hormonal impact on body composition and physical performance in recreational athletes. Finally, we will discuss the controversial issue of GH replacement in acromegalic patients presenting GH deficiency after treatment of acromegaly.
Subject(s)
Carrier Proteins/metabolism , Human Growth Hormone/metabolism , Human Growth Hormone/physiology , Acromegaly/therapy , Athletic Performance , Brain/metabolism , Breast/growth & development , Breast/metabolism , Breast Neoplasms/metabolism , Cell Transformation, Neoplastic/metabolism , Cytokines/metabolism , Energy Metabolism , Estrogens/metabolism , Female , Humans , MicroRNAs/metabolism , STAT5 Transcription Factor/metabolism , Stem Cells/metabolism , Substance-Related Disorders/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
Hypogonadotropic hypogonadism (HH) is defined by the absence of sex steroid synthesis associated with the lack of appropriate gonadotrophin secretion. This leads to a variable degree of impuberism, often diagnosed during childhood or adolescence. Genetics of HH involve many genes. However, molecular defects have been identified in only 30 % of patients. Kallmann syndrome (KS) is defined by the association of HH and anosmia. Six genes are involved in KS (KAL1, FGFR1, FGF8, PROK2, PROKR2 and CHD7). However, genetics of KS is complex, because of the variability of the phenotype for a similar molecular defect. Otherwise, heterozygous anomalies are frequently described. Identification in the same patient of several mutations in some of these genes (digenism) could account for this variability. Autosomal recessive transmission is frequently observed in familial cases of HH without anosmia. Molecular alterations have been identified for several neuropeptides or their corresponding receptors, which are involved in the physiology of the gonadotropic axis : GNRHR, KISS1R/GPR54, neurokinin B (TAC3), TACR3 and GNRH1 (and PROK2, PROKR2 and CHD7). Anomalies of leptin or its receptor are also involved in HH cases. A new negative regulating element has been recently identified in humans : RFRP3, which is ortholog of the avian GnIH (gonadotrophin inhibitory hormone). Recent progress about these neuropeptides leads to a new model of comprehension of the gonadotropic axis physiology, from a linear model to a network model, which regulates the central element of regulation of the gonadotropic axis, represented by the GnRH neurons.
Subject(s)
Gonads/metabolism , Hypogonadism/metabolism , Hypothalamus/metabolism , Pituitary Gland/metabolism , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Extracellular Matrix Proteins/genetics , Female , Fibroblast Growth Factor 8/genetics , Gastrointestinal Hormones/genetics , Humans , Hypogonadism/genetics , Kallmann Syndrome/genetics , Kallmann Syndrome/metabolism , Leptin/genetics , Male , Nerve Tissue Proteins/genetics , Neuropeptides/genetics , Olfaction Disorders/genetics , Pituitary Hormone Release Inhibiting Hormones/metabolism , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, Leptin/genetics , Receptors, Peptide/geneticsABSTRACT
Steroidogenic factor 1 (SF-1) gene, identified by Keith Parker in 1992, encodes for an orphan nuclear receptor, NR5A1, whose expression is detected during fetal life in adrenal and gonadal steroidogenic tissues, but also in the developing hypothalamus and in pituitary gonadotropic cells. SF-1 knock-out mouse models exhibit complete adrenal and gonadal agenesis. Human mutations of this transcription factor, were initially associated with primary adrenal failure and male gonadal dysgenesis with various degrees of under androgenization. More recently, identification of novel SF-1 mutations responsible for isolated 46, XY gonadal dysgenesis or 46, XX primary ovarian insufficiency, underscores its central role in the control and maintenance of adrenal and reproductive functions. A better understanding in the regulatory mechanisms of SF-1 signaling pathway, will open new avenues for diagnostic and therapeutic managements of sex differentiation disorders and infertilities.
Subject(s)
Adrenal Glands/growth & development , Gonadal Dysgenesis/genetics , Gonadal Dysgenesis/pathology , Gonads/growth & development , Ovarian Diseases/genetics , Ovarian Diseases/pathology , Steroidogenic Factor 1/genetics , Steroidogenic Factor 1/physiology , Animals , Female , Humans , Male , Mice , Phenotype , Steroidogenic Factor 1/biosynthesisABSTRACT
The pituitary hormone prolactin (PRL) exerts pleiotropic effects, which are mediated by a membrane receptor (PRLR) present in numerous cell types including adipocytes. Brown adipose tissue (BAT) expresses uncoupling proteins (UCPs), involved in thermogenesis, but also secretes leptin, a key hormone involved in the control of body weight. To investigate PRL effects on BAT, we used the T37i brown adipose cell line, and demonstrated that PRLRs are expressed as a function of cell differentiation. Addition of PRL leads to activation of the JAK/STAT and MAP kinase signaling pathways, demonstrating that PRLRs are functional in these cells. Basal and catecholamine-induced UCP1 expression were not affected by PRL. However, PRL combined with insulin significantly increases leptin expression and release, indicating that PRL potentiates the stimulatory effect of insulin as revealed by the recruitment of insulin receptor substrates and the activation of phosphatidylinositol 3-kinase. To explore the in vivo physiological relevance of PRL action in BAT, we showed that leptin content was significantly increased in BAT of PRLR-null mice compared with wild-type mice, highlighting the involvement of PRL in the leptin secretion process. This study provides the first evidence for a functional link between PRL and energy balance via a cross-talk between insulin and PRL signaling pathways in brown adipocytes.
Subject(s)
Adipocytes/drug effects , Adipocytes/metabolism , Adipose Tissue, Brown/drug effects , Gene Expression Regulation/drug effects , Insulin/pharmacology , Leptin/metabolism , Prolactin/pharmacology , Adipocytes/cytology , Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Animals , Carrier Proteins/metabolism , Cell Differentiation , Cell Line , DNA-Binding Proteins/metabolism , Ion Channels , Janus Kinase 2 , Membrane Proteins/metabolism , Mice , Mice, Knockout , Milk Proteins/metabolism , Mitochondrial Proteins , Mitogen-Activated Protein Kinases/metabolism , Protein Binding , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, Leptin , Receptors, Prolactin/deficiency , Receptors, Prolactin/genetics , Receptors, Prolactin/metabolism , STAT3 Transcription Factor , STAT5 Transcription Factor , Signal Transduction/drug effects , Trans-Activators/metabolism , Transcription, Genetic/genetics , Uncoupling Protein 1ABSTRACT
Type I pseudohypoaldosteronism (PHA1) is a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Typical biochemical features include high levels of plasma aldosterone and renin, hyponatremia and hyperkalemia. Different mutations of the human mineralocorticoid receptor (hMR) gene have been identified in subjects affected by the autosomal dominant or sporadic form of the disease. Our laboratory has investigated a large number of subjects with familial and sporadic PHA1. Several different mutations have been detected, which are localized in different coding exons of the hMR gene. These mutations either create truncated proteins, either affect specific amino acids involved in receptor function. In this paper, we review hMR mutations described to date in PHA1 and their functional characterization. We discuss the absence of mutations in some kindreds and the role of precise phenotypic and biological examination of patients to allow for identification of other genes potentially involved in the disease.
Subject(s)
Exons/genetics , Mutation , Pseudohypoaldosteronism/genetics , Receptors, Mineralocorticoid/genetics , Aldosterone/blood , Genes, Dominant/genetics , Humans , Hypokalemia/genetics , Hypokalemia/physiopathology , Hyponatremia/genetics , Hyponatremia/physiopathology , Kidney/physiopathology , Mineralocorticoids/metabolism , Pedigree , Predictive Value of Tests , Pseudohypoaldosteronism/blood , Pseudohypoaldosteronism/congenital , Pseudohypoaldosteronism/physiopathology , Receptors, Mineralocorticoid/metabolism , Renin/blood , Salts/metabolismABSTRACT
The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.
Subject(s)
Cell Nucleus/metabolism , Osteosarcoma/metabolism , Parathyroid Hormone-Related Protein/metabolism , Receptors, Parathyroid Hormone/metabolism , Transcription Factors/metabolism , Cell Nucleus/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Genes, Reporter , Humans , Osteosarcoma/genetics , Parathyroid Hormone/metabolism , Parathyroid Hormone-Related Protein/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Receptors, Parathyroid Hormone/genetics , Transcription Factors/genetics , Tumor Cells, CulturedABSTRACT
Aldosterone effects are mediated by the MR, which possesses the same affinity for mineralocorticoids and glucocorticoids. In addition to the existence of mechanisms regulating intracellular hormone availability, we searched for human MR splice variants involved in tissue-specific corticosteroid function. We have identified a new human MR isoform, hMRDelta5,6, resulting from an alternative splicing event skipping exons 5 and 6 of the human MR gene. hMRDelta5,6 mRNAs are expressed in several human tissues at different levels compared with wild-type human MR, as shown by real time PCR. Introduction of a premature stop codon results in a 75-kDa protein lacking the entire hinge region and ligand binding domain. Interestingly, hMRDelta5,6 is still capable of binding to DNA and acts as a ligand-independent transactivator, with maximal transcriptional induction corresponding to approximately 30-40% of aldosterone-activated wild-type human MR. Coexpression of hMRDelta5,6 with human MR or human GR increases their transactivation potential at high doses of hormone. Finally, hMRDelta5,6 is able to recruit the coactivators, steroid receptor coactivator 1, receptor interacting protein 140, and transcription intermediary factor 1alpha, which enhance its transcriptional activity. Ligand-independent transactivation and enhancement of both wild-type MR and GR activities by hMRDelta5,6 suggests that this new variant might play a role in modulating corticosteroid effects in target tissues.
Subject(s)
Adrenal Cortex Hormones/metabolism , Alternative Splicing/genetics , Receptors, Mineralocorticoid/genetics , Trans-Activators/genetics , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Humans , Molecular Sequence Data , Protein Isoforms , Rabbits , Receptors, Mineralocorticoid/metabolism , Trans-Activators/metabolism , Transcription, Genetic/geneticsABSTRACT
The mineralocorticoid receptor (MR), a ligand-dependent transcription factor, mediates aldosterone actions in a large variety of tissues. To explore the functional implication of MR in pathophysiology, transgenic mouse models were generated using the proximal human MR (hMR) promoter to drive expression of hMR in aldosterone target tissues. Tissue-specific analysis of transgene expression in two independent transgenic animal (TG) lines by ribonuclease protection assays revealed that hMR is expressed in all mineralocorticoid-sensitive tissues, most notably in the kidney and the heart. TG exhibit both renal and cardiac abnormalities. Enlarged kidneys were histologically associated with renal tubular dilation and cellular vacuolization whose prevalence increased with aging. Renal clearance studies also disclosed a significant decrease in urinary potassium excretion rate in TG. hMR-expressing animals had normal blood pressure but developed mild dilated cardiomyopathy (increased left ventricle diameters and decreased shortening fraction), which was accompanied by a significant increase in heart rate. Differential gene expression analysis revealed a 2- to 5-fold increase in cardiac expression of atrial natriuretic peptide, serum- and glucocorticoid-induced kinase, and early growth response gene 1 as detected by microarrays; renal serum- and glucocorticoid-induced kinase was also induced significantly. Altogether, TG exhibited specific alteration of renal and cardiac functions, thus providing useful pathophysiological models to gain new insights into the tissue-specific mineralocorticoid signaling pathways.
Subject(s)
Heart/physiology , Immediate-Early Proteins , Kidney/physiology , Mice, Transgenic , Nuclear Proteins , Receptors, Mineralocorticoid/biosynthesis , Animals , Blotting, Northern , DNA, Complementary/metabolism , DNA-Binding Proteins/biosynthesis , Early Growth Response Protein 1 , Humans , Kidney/metabolism , Male , Mice , Models, Genetic , Myocardium/metabolism , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/biosynthesis , Receptors, Mineralocorticoid/genetics , Recombinant Proteins/metabolism , Signal Transduction , Sodium-Potassium-Exchanging ATPase/biosynthesis , Time Factors , Tissue Distribution , Transcription Factors/biosynthesisABSTRACT
Leptin, the ob gene product, is produced by adipose tissue and is submitted to a complex hormonal and metabolic regulation. Leptin plays a critical role in the balance of body weight. Here we report on secretion and hormonal regulation of leptin by brown adipocytes. Using the recently established T37i cell line, we show that leptin expression and secretion occurred as a function of cell differentiation. In differentiated T37i cells, insulin induced leptin release ( approximately 0.25 ng/10(6) cells/h) in a concentration-dependent manner (EC50=0.1 nM), and this was totally suppressed by beta3-adrenergic ligand, thiazolidinedione, cycloheximide, or actinomycin D. Insulin induced a strong, rapid (within 2 h) but transient fivefold increase in leptin mRNA levels. This transcriptional control of ob gene expression by insulin involved both phosphatidylinositol 3-kinase- and MAP kinase-dependent pathways. Glucocorticoids inhibited both insulin-stimulated leptin secretion and ob gene expression without affecting leptin mRNA stability (t(1/2)=3h05). Altogether, our results demonstrate that brown adipocytes express and secrete leptin, whose hormonal regulation clearly differs from that described in white adipose tissue. These findings point to tissue-specific molecular mechanisms and suggest that leptin might exert direct effects on energy homeostasis through an autocrine mechanism.
Subject(s)
Adipocytes/drug effects , Adipose Tissue, Brown/drug effects , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Insulin/pharmacology , Leptin/metabolism , Adipocytes/cytology , Adipose Tissue, Brown/cytology , Animals , Dose-Response Relationship, Drug , Leptin/genetics , Mice , Transcription, Genetic/drug effects , Tumor Cells, CulturedABSTRACT
Uncoupling proteins (UCP), specific mitochondrial proton transporters that function by uncoupling oxidative metabolism from ATP synthesis, are involved in thermoregulation and control of energy expenditure. The hibernoma-derived T37i cells, which possess functional endogenous mineralocorticoid receptors (MR), can undergo differentiation into brown adipocytes. In differentiated T37i cells, UCP1 mRNA levels increased 10- to 20-fold after retinoic acid or beta-adrenergic treatment. Interestingly, UCP2 and UCP3 mRNA was also detected. Aldosterone treatment induced a drastic decrease in isoproterenol- and retinoic acid-stimulated UCP1 mRNA levels in a time- and dose-dependent manner (IC(50) approximately 1 nM aldosterone). This inhibition was unaffected by cycloheximide and did not modify UCP1 mRNA stability (half-life time = 5 h), indicating that it occurs at the transcriptional level. It involves both the MR and/or the glucocorticoid receptor (GR), depending on the retinoic or catecholamine induction pathway. Basal UCP3 expression was also significantly reduced by aldosterone, whereas UCP2 mRNA levels were not modified. Finally, as demonstrated by JC1 aggregate formation in living cells, aldosterone restored mitochondrial membrane potential abolished by isoproterenol or retinoic acid. Our results demonstrate that MR and GR inhibit expression of UCP1 and UCP3, thus participating in the control of energy expenditure.