Showing papers in "Endocrinology in 1996"
TL;DR: This work presents the expression patterns of the PPAR subtypes in the adult rat, determined by in situ hybridization using specific probes for PPAR-alpha, -beta and -gamma, and by immunohistochemistry using a polyclonal antibody that recognizes the three rat PPar subtypes.
Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that can be activated by various xenobiotics and natural fatty acids. These transcription factors primarily regulate genes involved in lipid metabolism and also play a role in adipocyte differentiation. We present the expression patterns of the PPAR subtypes in the adult rat, determined by in situ hybridization using specific probes for PPAR-alpha, -beta and -gamma, and by immunohistochemistry using a polyclonal antibody that recognizes the three rat PPAR subtypes. In numerous cell types from either ectodermal, mesodermal, or endodermal origin, PPARs are coexpressed, with relative levels varying between them from one cell type to the other. PPAR-alpha is highly expressed in hepatocytes, cardiomyocytes, enterocytes, and the proximal tubule cells of kidney. PPAR-beta is expressed ubiquitously and often at higher levels than PPAR-alpha and -gamma. PPAR-gamma is expressed predominantly in adipose tissue and the immune system. Our results suggest new potential directions to investigate the functions of the different PPAR subtypes.
2,178 citations
TL;DR: It is demonstrated that leptin stimulates the reproductive endocrine system in both sexes of ob/ob mice and suggested that leptin may serve as a permissive signal to the reproductive system of normal animals.
Abstract: Leptin, a newly-discovered hormonal product of the obese (ob) gene, is expressed by adipocytes and thought to play a role in the regulation of food intake and metabolism. We tested the hypothesis that leptin signals metabolic information to the reproductive system by examining its effects on the reproductive system of ob/ob mice, which have a congenital deficiency in leptin and are infertile. We treated pair-fed males and females with leptin (50 microg twice daily, ip) or vehicle (n=10/group) for 14 days, after which the animals were bled and killed. Leptin-treated females had significantly elevated serum levels of LH, increased ovarian and uterine weights, and stimulated aspects of ovarian and uterine histology compared to controls. Leptin-treated males had significantly elevated serum levels of FSH, increased testicular and seminal vesicle weights, greater seminal vesicle epithelial cell height, and elevated sperm counts compared to controls. These results demonstrate that leptin stimulates the reproductive endocrine system in both sexes of ob/ob mice and suggest that leptin may serve as a permissive signal to the reproductive system of normal animals.
1,089 citations
TL;DR: It is suggested that estrogen action is required for fertility in male mice and that the mutation of the ER in ERKO males leads to reduced mating frequency, low sperm numbers, and defective sperm function.
Abstract: The reproductive system of male mice homozygous for a mutation in the estrogen receptor (ER) gene (ER knock-out; ERKO) appears normal at the anatomical level. However, these males are infertile, indicating an essential role for ER-mediated processes in the regulation of male reproduction. Adult ERKO male mice have significantly fewer epididymal sperm than heterozygous or wild-type males. Although spermatogenesis is occurring in some seminiferous tubules of 3- to 5-month-old ERKO males, other tubules either have a dilated lumen and a disorganized seminiferous epithelium with few spermatogenic cells or lack a lumen and contain mainly Sertoli cells. There are no obvious differences in seminiferous tubules at 10 days of age between wild-type and ERKO mice, but the lumen in ERKO males is dilated in all seminiferous tubules by 20 days. However, spermatogenesis progresses and similar numbers of sperm are present in the cauda epididymis of ERKO and wild-type males until 10 weeks of age. Disruption of spermatogenesis and degeneration of the seminiferous tubules become apparent after 10 weeks in the caudal pole of the testis and progresses in a wave to the cranial pole by 6 months. However, the seminal vesicles, coagulating glands, prostate, and epididymis do not appear to be altered morphologically in ERKO mice. Serum testosterone levels are somewhat elevated, but LH and FSH levels are not significantly different from those in wild-type males. Sperm from 8- to 16-week-old mice have reduced motility and are ineffective at fertilizing eggs in vitro. In addition, ERKO males housed overnight with hormone-primed wild-type females produce significantly fewer copulatory plugs than do heterozygous or wild-type males. These results suggest that estrogen action is required for fertility in male mice and that the mutation of the ER in ERKO males leads to reduced mating frequency, low sperm numbers, and defective sperm function.
835 citations
TL;DR: Results indicate that sequences corresponding to the cloned rat islet GLP-1 receptor are expressed in the pancreatic islets, lung, hypothalamus, stomach, heart, and kidney but not in adipose, liver, and skeletal muscle, suggesting that the observed extrapancreatic actions of GLp-1 may not be strictly confined to interactions with the defined receptor.
Abstract: The incretin hormone glucagon-like peptide-1 (GLP-1) is an important regulator of postprandial insulin secretion. In addition to its insulinotropic actions on pancreatic beta-cells, GLP-1 enhances glucose disposal by insulin-independent mechanisms, suggesting that GLP-1 receptors are located on extrapancreatic tissues. In this study, we examined the tissue distribution of GLP-1 receptor (GLP-lR) messenger RNA (mRNA) in rat by RNAse protection, RT-PCR, and in situ hybridization. We identified GLP-1R mRNA in the lung, pancreatic islets, stomach, and kidney by the RNAse protection assay. RT-PCR analysis also detected GLP-1R mRNA in the hypothalamus and heart. In situ hybridization experiments identified receptor mRNA in the gastric pits of the stomach, large nucleated cells in the lung, crypts of the duodenum, and pancreatic islets. No localized specific grains were found in kidney, skeletal muscle, heart, liver, or adipocytes. These results indicate that sequences corresponding to the cloned rat islet GLP-1...
619 citations
TL;DR: The results indicate that maternal deprivation before weaning in male rats produces effects on CRF neural systems in both the central nervous system and pituitary that are apparent several months later and are probably associated with persistent alterations in behavioral response in adult rats.
Abstract: There is considerable evidence that CRF-containing neurons integrate the endocrine, autonomic, immune, and behavioral responses to stress. In this study we examined long term effects of early stress on developing hypothalamic and extrahypothalamic CRF neural systems in the rat brain and subsequent responses to stress in the adult. Specifically, we sought to determine whether adult male rats previously isolated for 6 h daily during postnatal days 2-20 react in a biochemically distinct manner to a mild foot shock stress compared to controls. Four treatment groups were examined: nondeprived (NDEP)/no shock, NDEP/shock, deprived (DEP)/no shock, and DEP/shock. Compared to the NDEP group, DEP rats exhibited an increase in both basal and stress-induced ACTH concentrations. Moreover, DEP rats exhibited a 125% increase in immunoreactive CRF concentrations in the median eminence and a reduction in the density of CRF receptor binding in the anterior pituitary compared to those in all NDEP rats. Alterations in extrahypothalamic CRF systems were also apparent in DEP vs. NDEP animals, with an observed 59% increase in the number of CRF receptor-binding sites in the raphe nucleus and an 86% increase in immunoreactive CRF concentrations in the parabrachial nucleus. These results indicate that maternal deprivation before weaning in male rats produces effects on CRF neural systems in both the central nervous system and pituitary that are apparent several months later and are probably associated with persistent alterations in behavioral response in adult rats.
512 citations
TL;DR: It is shown that prostaglandin (PG) E2 is the most potent factor which stimulates aromatase expression via cyclic AMP and promoter II, which could be the mechanism whereby estrogen biosynthesis is stimulated in breast sites adjacent to a tumor, leading in turn to increased growth and development of the tumor itself.
Abstract: In the present report, we show that prostaglandin (PG) E2 is the most potent factor which stimulates aromatase expression via cyclic AMP and promoter II. PGE2 acts via EP1 and EP2 receptor subtypes to stimulate both the PKC and PKA pathways. The combined stimulation of both of these pathways results in maximal expression of promoter II-specific CYP19 transcripts. Since PGE2 is a major secretory product both of breast tumor epithelial cells and fibroblasts, as well as of macrophages infiltrating the tumor site, then this could be the mechanism whereby estrogen biosynthesis is stimulated in breast sites adjacent to a tumor, leading in turn to increased growth and development of the tumor itself.
443 citations
TL;DR: Data support the conclusion that the antidiabetic actions of the thiazolidinediones are directly mediated through binding to PPAR gamma and the resulting active conformation of the receptor.
Abstract: The thiazolidinediones are novel insulin sensitizers that serve as orally active antidiabetic agents, in rodents, nonhuman primates, and man. We have examined the effects of 4-week oral administration of three thiazolidinediones (AD-5075, BRL 49653, and CS-045) on plasma glucose and triglyceride concentrations in obese hyperglycemic db/db mice. All three agents lower plasma glucose and triglyceride concentrations. Normal levels of glucose are achieved after treatment with AD-5075 (> 1.7 mg/kg) or BRL 49653 (> or = 30 mg/kg), whereas CS-045 (100 or 300 mg/kg) produces only modest reductions in either parameter. Although the thiazolidinediones have demonstrated insulin-sensitizing activities both in vivo and in vitro, their primary molecular target has been unclear. We have compared the in vivo antidiabetic actions described above with the in vitro activities on peroxisomal proliferator-activated receptor-gamma (PPAR gamma). Hamster PPAR gamma 1 was transiently expressed in COS-1 cells to study the binding ...
389 citations
TL;DR: Analysis of in vitro culture of early antral follicles revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis, and stage-dependent difference in the hormonal regulation of follicle apoptosis was demonstrated.
Abstract: Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.
368 citations
TL;DR: Results show that the human mononuclear osteoclast precursor circulates in the monocyte fraction and exhibits a monocyte phenotype, acquiring osteocline phenotypic features in the process of differentiation into mature functional osteoclasts.
Abstract: The osteoclast is known to be formed by fusion of circulating mononuclear precursor cells of haematopoietic origin. The precise nature of these circulating cells and, in particular, their relation to monocytes is unknown. We have developed an in vitro system of human osteoclast formation whereby human monocytes [CD14, CD11a, CD11b and HLA-DR positive, and tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), vitronectin receptor (VNR) negative] were isolated and cocultured for up to 21 days with UMR106 rat osteoblast-like cells or ST2 mouse preadipocytic bone marrow stromal cells in the presence of 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3) and macrophage colony stimulating factor (M-CSF). Numerous TRAP, VNR and CTR positive multinucleated cells, capable of extensive lacunar bone resorption, formed in these cocultures; the absolute requirements for this to occur were contact with the above bone stromal cells, 1,25(OH)2D3, and M-CSF. These results show that the human mononuclear osteocl...
365 citations
TL;DR: Synthetic human urocortin binds with high affinity to CRF receptor types 1, 2 alpha, and 2 beta, stimulates cAMP accumulation from cells stably transfected with these receptors, and acts in vitro to release ACTH from dispersed rat anterior pituitary cells.
Abstract: Urocortin, a new member of the CRF peptide family which also includes urotensin I and sauvagine, was recently cloned from the rat midbrain. The synthetic replicate of urocortin was found to bind with high affinity to type 1 and type 2 CRF receptors and, based upon its anatomic localization within the brain, was proposed to be a natural ligand for the type 2 CRF receptors. Using a genomic library, we have cloned the human counterpart of rat urocortin and localized it to human chromosome 2. Human and rat urocortin share 95% identity within the mature peptide region. Synthetic human urocortin binds with high affinity to CRF receptor types 1, 2 alpha, and 2 beta, stimulates cAMP accumulation from cells stably transfected with these receptors, and acts in vitro to release ACTH from dispersed rat anterior pituitary cells. In addition, the CRF-binding protein binds human urocortin with high affinity and can prevent urocortin-stimulated ACTH secretion in vitro. The inhibitory effect of the CRF-binding protein on ...
363 citations
TL;DR: Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase, suggesting a conserved function for this growth factor family in the regulation of growth and body size.
Abstract: Drosophila contain an insulin receptor homologue, encoded by the inr gene located at position 93E4-5 on the third chromosome. The receptor protein is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand- activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the inr gene and six independent mutations that lead to a loss of expression or function of the receptor protein were identified. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for inr in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size.
TL;DR: The results suggest that alpha- MSH, ACTH, and possibly beta-MSH, but not gamma-MSh, are capable of a physiological role in regulating human pigmentation, and that melanocytes in human skin are a specific target for these hormones.
Abstract: alpha-Melanocyte stimulating hormone (alpha-MSH) and ACTH increase the proliferation and melanogenesis of cultured human melanocytes. To further analyze how melanotropins produce these biological effects, we investigated the regulation of the melanocortin receptor MC1R expression by alpha-MSH and ACTH using Northern blot analysis and determine the relative affinity of the receptor for the structurally similar peptides alpha-MSH, ACTH, beta-MSH, and gamma-MSH. We also determined the relative potencies of these hormones to stimulate cAMP formation, tyrosinase activity, and melanocyte proliferation. The order of affinity and potency of the noted melanotropins in these assays were alpha-MSH = ACTH > beta-MSH > gamma-MSH. Because the binding affinity of each of these melanotropins for the MC1R correlated with its ability to stimulate human melanocyte proliferation and melanogenesis, we conclude that these effects are mediated specifically by binding to and activation of the MC1R. gamma-MSH stimulated cAMP formation without affecting proliferation or melanogenesis. However, we found that relative to alpha-MSH, the effect of gamma-MSH on cAMP formation was transient. Our results suggest that alpha-MSH, ACTH, and possibly beta-MSH, but not gamma-MSH, are capable of a physiological role in regulating human pigmentation, and that melanocytes in human skin are a specific target for these hormones.
TL;DR: It is discovered that expression of the AA-NAT gene in the rat pineal gland is essentially turned off during the day and turned on at night, resulting in a more than 150-fold rhythm.
Abstract: In vertebrates, the circadian rhythm in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] drives the daily rhythm in circulating melatonin. We have discovered that expression of the AA-NAT gene in the rat pineal gland is essentially turned off during the day and turned on at night, resulting in a more than 150-fold rhythm. Expression is regulated by a photoneural system that acts through an adrenergic-cAMP mechanism in pinealocytes, probably involving cAMP response element-binding protein phosphorylation. Turning off AA-NAT expression appears to involve de novo synthesis of a protein that attenuates transcription. A approximately 10-fold night/day rhythm in AA-NAT messenger RNA occurs in the retina, and AA-NAT messenger RNA is also detected at low levels in the brain.
TL;DR: Comparison with previous enzymology studies suggest the changing pattern of 11 beta-HSD2 mRNA is likely to be translated into enzyme activity and have significant parallels in human development.
Abstract: Glucocorticoids play important roles in development and 'fetal programming'. Fetal exposure to excess glucocorticoids reduces birth weight and causes later hypertension. To investigate these processes further we have determined the detailed category of 11 beta-hydroxysteroid dehydrogenase type2 (11 beta-HSD2, which potently inactivates glucocorticoids) and the mineralocorticoid receptor (MR) by in situ hybridisation from embryonic day 9.5 (E9.5, term = E19) until after birth in the mouse. Widespread abundant 11 beta-HSD2 mRNA expression from E9.5-E12.5 changes dramatically at approximately E13 to a limited tissue-specific pattern (kidney, hindgut, testis/bile ducts, lung and a few brain regions (later seen in cerebellum, thalamus, roof of midbrain, neuroepithelial regions in pons and near the subicular hippocampus)). Placenta (labyrinthine zone) and extra-embryonic membranes express abundant 11 beta-HSD2 mRNA until E15.5 but this ceases = E16.5. It is unclear to what extent rodent term placental 11 beta-HSD activity is due to persisting 11 beta-HSD2 protein. Convincing MR mRNA expression is seen from E13.5 and includes pituitary, heart, muscle and meninges with expression later in gut, kidney, thymus, discrete areas of lung and several brain regions (including hippocampus, rhinencephalon and hypothalamus). 11 beta-HSD2 and MR clearly co-localise = E18.5 in kidney and colon and might do so in discrete areas of lung (E14-15) and neuroepithelia near the subicular hippocampus. Probably elsewhere MR are non-selective and 11 beta-HSD2 is involved in protecting glucocorticoid receptors in fetal fetal tissues. Comparison with previous enzymology studies suggest the changing pattern of 11 beta-HSD2 mRNA is likely to be translated into enzyme activity and have significant parallels in human development.
TL;DR: In vivo antalarmnin (20 mg/kg body wt.) significantly inhibited CRH-stimulated ACTH release and carageenin-induced subcutaneous inflammation in rats, holding therapeutic promise in disorders with putative CRH hypersecretion, such as melancholic depression and inflammatory disorders.
Abstract: Corticotropin-releasing hormone (CRH) secreted from the hypothalamus is the major regulator of pituitary ACTH release and consequent glucocorticoid secretion. CRH secreted in the periphery also acts as a proinflammatory modulator. CRH receptors (CRH-R1, R2alpha, R2beta) exhibit a specific tissue distribution. Antalarmin, a novel pyrrolopyrimidine compound, displaced 12SI-oCRH binding in rat pituitary, frontal cortex and cerebellum, but not heart, consistent with antagonism at the CRHR1 receptor. In vivo antalarmnin (20 mg/kg body wt.) significantly inhibited CRH-stimulated ACTH release and carageenin-induced subcutaneous inflammation in rats. Antalarmin, or its analogs, hold therapeutic promise in disorders with putative CRH hypersecretion, such as melancholic depression and inflammatory disorders.
TL;DR: These findings indicate that ret/PTC2 is not only a biomarker associated with papillary thyroid carcinomas, but is also the only proven specific genetic event leading to the development of papilla thyroid carcinoma.
Abstract: The ret/PTC oncogene, a rearranged form of the ret proto-oncogene, has been found to be restricted to human papillary thyroid carcinomas. This report shows that transgenic mice with thyroid-targeted expression of the ret/PTC1 oncogene developed thyroid carcinomas with considerable similarities to human papillary thyroid carcinomas, particularly in the nuclear cytologic features and the presence of local invasion. Our findings indicate that ret/PTC2 is not only a biomarker associated with papillary thyroid carcinomas, but is also the only proven specific genetic event leading to the development of papillary thyroid carcinoma.
TL;DR: It is demonstrated that PTHrP acts principally as a paracrine factor, which promotes elongation of endochondral bone by restraining or delaying the pace of chondrocytic development and terminal differentiation of growth-plate chondsrocytes.
Abstract: To test the hypothesis that PTH-related peptide (PTHrP) is a paracrine regulator of endochondral bone development, we localized PTHrP and its cognate receptor during normal skeletal development at both messenger RNA (mRNA) and protein levels and compared the growth plate phenotypes of PTHrP-deficient [(PTHrP(-/-)] mice to those of normal littermates [PTHrP(+/+]. PTHrP mRNA was expressed adjacent to uncavitated joints, in the perichondrium of long bones and to a lower level in proliferating chondrocytes. In contrast, PTHrP protein was most evident at the interface of proliferating and hypertrophic zones, where it colocalized with PTH/PTHrP receptor mRNA and protein. Most strikingly, the proliferating zone was dramatically shorter in PTHrP(-/-) cartilage, although the percentage of cells in S-phase of the cell cycle in the proliferating zone was indistinguishable between PTHrP(+/+) and PTHrP(-/-) mice. Terminal differentiation of chondrocytes, which was characterized by cell hypertrophy, apoptosis (DNA frag...
TL;DR: IGF-I of peripheral origin contributes to the initiation of female puberty by stimulating LHRH release from the hypothalamus, an effect that appears to be amplified by the increased synthesis of IGF-I receptors in the ME during first proestrus.
Abstract: In several species, including humans, circulating insulin-like growth factor I (IGF-I) levels increase during the onset of puberty, suggesting that this peptide contributes to attaining sexual maturity. Because IGF-I elicits LHRH release from the median eminence (ME) of immature female rats in vitro, we hypothesized that it may represent one of the peripheral signals suspected to link somatic development to the LHRH-releasing system at puberty. We now present evidence in support of this concept. Quantitation of IGF-I messenger RNA (mRNA) levels by ribonuclease protection assay revealed that expression of the IGF-I gene did not change in the medial basal hypothalamus or preoptic area of female rats during peripubertal development. In contrast, the contents of both IGF-Ia and IGF-Ib mRNA, the two alternatively spliced forms of the IGF-I gene, increased significantly in the liver during the early proestrous phase of puberty. This change was followed by an elevation in serum IGF-I levels during the late proestrous phase of puberty along with a concomitant increase is serum gonadotropin levels. The proestrous change in serum IGF-I levels was accompanied by a selective increase in IGF-I receptor (IGF-IR) mRNA in the ME. Small doses of IGF-I (2-200 ng), administered intraventricularly, effectively induced LH release in both juvenile and peripubertal female rats, an increase prevented by prior immunoneutralization of LHRH actions. Importantly, intraventricular injections of IGF-I (20 ng), administered twice daily in the afternoon to immature animals, significantly advanced puberty. Thus, these results suggest that IGF-I of peripheral origin contributes to the initiation of female puberty by stimulating LHRH release from the hypothalamus, an effect that appears to be amplified by the increased synthesis of IGF-I receptors in the ME during first proestrus.
TL;DR: These data provide evidence for non-genomic effects of steroid hormones involving protein kinase associated signal transduction pathways traditionally associated with effects at the cell membrane and provide a possible explanation for estrogen's effects on neuronal genes lacking estrogen response elements but which contain cAMP response elements.
Abstract: Estrogen treatment of ovariectomized rats rapidly increases immunoreactivity for the phosphorylated form of the cAMP response element binding protein (CREB)in neurons of the preoptic area and the bed nucleus of the stria terminalis. These effects were detected within 15 minutes after estrogen exposure. Since the antisera used for these studies detect CREB phosphorylation at ser133, which is important for transcriptional activation these data provide a possible explanation for estrogen's effects on neuronal genes lacking estrogen response elements (EREs) but which contain cAMP response elements (CREs). These data also provide evidence for non-genomic effects of steroid hormones involving protein kinase associated signal transduction pathways traditionally associated with effects at the cell membrane.
TL;DR: Osteoblasts aside from their role of mediating osteoclastic resorption promoters are also involved in inhibiting bone resorptive activity through the synthesis of an osteoclast resor adaptation inhibitor.
Abstract: Current knowledge indicates that osteoblasts play an integral role in osteoclastic bone resorption through an osteoclast-stimulating activity produced by osteoblasts in response to resorption-promoting osteotropic factors. Previously, we have shown that the inhibitory action of the bisphosphonates on bone resorption in part is mediated by osteoblasts. The aim of the present study was to investigate whether the bisphosphonate-generated inhibition is due to these compounds decreasing the synthesis of the osteoclast-stimulating activity or is the result of osteoblasts synthesizing an osteoclast resorption inhibitor. Using the osteoblastic cell line CRP 10/30, which produces osteoclast- stimulating activity, constitutively and employing isolated rat osteoclasts cultured on ivory, evidence was obtained indicating that the bisphosphonates ibandronate and alendronate at a concentration of 10(-7) M induce osteoblasts to synthesize an osteoclast inhibitor that reduces pit formation by more than 50%. The inhibitor ...
TL;DR: The results indicate that there are unique features of D2 that distinguish it from the two other selenodeiodinases, and suggests that it could play a role in peripheral, as well as intracellular, T3 production.
Abstract: Type 2 deiodinase (D2) is a low K(m) iodothyronine deiodinase that catalyzes the removal of a single iodine from the phenolic ring of T4 or rT3. We sequenced and subcloned the open reading frame from a partial complementary DNA (cDNA) clone (2.1 kilobases) prepared by Genethon (Z44085) from a human infant brain cDNA library. The open reading frame encodes a putative 273-amino acid protein of 31 kDa with greater than 70% similarity to the Rana catesbeiana D2 protein. Transient expression of the cDNA produces a low K(m) (5 nM for T4; 8 nM for rT3) propylthiouracil- and gold thioglucose-resistant 5'-deiodinase in 293-HEK cells. Human D2, like human type 1 (D1) and type 3 (D3) deiodinases, is a selenoenzyme, as evidenced by 1) the presence of two in-frame UGA codons (positions 133 and 266), 2) the synthesis of a 31-kDa 75Selabeled protein in D2 cDNA-transfected cells, and 3) the requirement for a 3'-selenocysteine incorporation sequence element for its translation. Unlike D1 and D3, we were not able to covalently label overexpressed D2 with N-bromoacetyl [125I]T3 or -T4. We found that the human D2 messenger RNA is 7-8 kilobases and is expressed in brain, placenta, and, surprisingly, cardiac and skeletal muscle. Type 2 deiodinase activity was also present in human skeletal muscle. These results indicate that there are unique features of D2 that distinguish it from the two other selenodeiodinases. The expression of D2 in muscle suggests that it could play a role in peripheral, as well as intracellular, T3 production.
TL;DR: It is proposed that the beta 3-adrenergic receptor plays a central role in regulating the release of leptin from the adipocyte and that insulin-stimulated leptin release is blocked by simultaneous activation of cAMP-dependent protein kinase.
Abstract: Various model systems have been used to study the expression of the recently cloned ob gene, leptin. Here we report that freshly isolated rat white adipocytes incubated with insulin release leptin in a rapid and concentration-dependent manner (EC50 of 0.221 +/- .075 nM). Insulin-stimulated leptin release could be detected as early as 30 min and a maximal 2-3 fold effect was produced by 10 nM insulin. The effect of insulin was completely blocked by simultaneous activation of cAMP-dependent protein kinase. Using the activation of lipolysis as an index of cAMP-dependent protein kinase activity, we show that inhibition of leptin release by norepinephrine or the selective beta 3-adrenergic receptor agonist, CL316,243, occurred in parallel to activation of cAMP-dependent protein kinase. In addition, beta 1- and beta 2-adrenergic receptor antagonists did not impair the ability of norepinephrine or CL316,243 to inhibit leptin release from the adipocytes. These findings suggest that the beta 3-adrenergic receptor ...
TL;DR: In this article, the effects of EGF, TGF alpha, and insulin-like growth factor I (IGF-I) on transcription from consensus estrogen response elements (ERE) in human ovarian adenocarcinoma calls were assessed.
Abstract: Modulation of steroid receptor-dependent transcription by extra- cellular ligands represents a novel mechanism of steroid receptor regulation. We have assessed the effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulin-like growth factor I (IGF-I) on transcription from consensus estrogen response elements (ERE) in estrogen receptor (ER)-positive BG-1 human ovarian adenocarcinoma calls. EGF, TGF alpha, IGF-I, and estradiol (E2) enhanced transcription in a dose-dependent manner using either a strong or a minimal promoter, and ICI 164,384, a specific ER antagonist, inhibited these responses. Combinations of E2 with TGF alpha or IGF-I induced synergistic activation of transcription from an ERE, whereas as additive response was observed with combinations of IGF-I and TGF alpha of EGF. Tetradecanoyl 12-phorbol 13-acetate (TPA), a protein kinase C (PKC) activator, stimulated ERE-mediated transcription, and this effect was inhibited by ICI 164,384. Bisindolylmaleimide,...
TL;DR: Combined replacement therapy with T4 and T3 (in proportions similar to those secreted by the normal rat thyroid) completely restored euthyroidism in thyroidectomized rats at much lower doses of T4 than those needed to normalize T3 in most tissues when T4 alone was used.
Abstract: We have recently shown that it is not possible to restore euthyroidism completely in all tissues of thyroidectomized rats infused with T4 alone. The present study was undertaken to determine whether this is achieved when T3 is added to the continuous sc infusion of T4. Thyroidectomized rats were infused with placebo or T4 (0.80 and 0.90 microgram/100 g BW.day), alone or in combination with T3 (0.10, 0.15, or 0.20 microgram/100 g BW.day). Placebo-infused intact rats served as euthyroid controls. Plasma and 12 tissues were obtained after 12 days of infusion. Plasma TSH and plasma and tissue T4 and T3 were determined by RIA. Iodothyronine deiodinase activities were assayed using cerebral cortex, pituitary, brown adipose tissue, liver, and lung. Circulating and tissue T4 levels were normal in all the groups infused with thyroid hormones. On the contrary, T3 in plasma and most tissues and plasma TSH only reached normal levels when T3 was added to the T4 infusion. The combination of 0.9 microgram T4 and 0.15 microgram T3/100 g BW.day resulted in normal T4 and T3 concentrations in plasma and all tissues as well as normal circulating TSH and normal or near-normal 5'-deiodinase activities. Combined replacement therapy with T4 and T3 (in proportions similar to those secreted by the normal rat thyroid) completely restored euthyroidism in thyroidectomized rats at much lower doses of T4 than those needed to normalize T3 in most tissues when T4 alone was used. If pertinent to man, these results might well justify a change in the current therapy for hypothyroidism.
TL;DR: Testing the hypothesis that insulin-like growth factor I (IGF-I), a known osteogenic factor, modulates VEGF expression in osteoblasts concludes that IGF-I enhances osteoblast synthesis of V EGF, which may then act locally on endothelium to stimulate angiogenesis, an essential component of bone growth and remodeling.
Abstract: Formation of new capillaries, a critical component of tissue growth and repair, is a recognized process in the development, formation, and remodeling of bone. Vascular endothelial growth factor (VEGF), a potent angiogenic factor with specific mitogenic actions on endothelial cells, is produced in a regulated manner by many cell types, including osteoblasts. The aim of the present investigation was to test the hypothesis that insulin-like growth factor I (IGF-I), a known osteogenic factor, modulates VEGF expression in osteoblasts. In human SaOS-2 osteoblast-like cells, 10 nM IGF-I increased the abundance of VEGF messenger RNA (mRNA) by 4-fold above the control value at 2h, and the elevated levels of mRNA returned to near basal by 8 h. IGF-I stimulated VEGF mRNA levels at IGF-I concentrations as low as 1-2 nM. The stability of VEGF mRNA was not increased after IGF-I treatment, and actinomycin D abrogated the enhanced expression of VEGF mRNA by IGF-I, indicating that the action of IGF-I was probably mediated...
TL;DR: The genomic structure and the corresponding complementary DNA (cDNA) sequence of the human CRF2 receptor is reported, and there is no evidence for the existence of aCRF2 beta receptor homolog in humans.
Abstract: Two CRF receptor subtypes (CRF1 and CRF2 receptors) with distinct brain localizations and pharmacological profiles have recently been cloned and characterized. For the CRF2 receptor subtype, at least 2 splice forms with different 5'-coding sequences (CRF2 alpha and CRF2 beta) have been identified in rat. In this article, we report the genomic structure and the corresponding complementary DNA (cDNA) sequence of the human CRF2 receptor. The gene coding for human CRF2 receptor consists of at least 12 exons and spans approximately 30 kilobases. The cDNA sequence in the protein-coding region is 94% identical to that of the reported rat CRF2 alpha receptor. At present, there is no evidence for the existence of a CRF2 beta receptor homolog in humans. The encoded receptor is 411 amino acids in length and is 70% identical to the human CRF1 receptor, with least sequence homology in the N-terminal extracellular domain (47% identical). Cells transfected with the full-length human CRF2 receptor cDNA responded to rat/human CRF and sauvagine by increasing the intracellular cAMP level, with EC50 values of approximately 20 and 1 nM, respectively. The CRF- and sauvagine-induced accumulation of intracellular cAMP could be competitively inhibited by the CRF receptor antagonist D-Phe-CRF. This pharmacological profile was comparable to that of the rat CRF2 alpha receptor. The relative abundance of the CRF2 receptor messenger RNA appears to be lower in humans than in rats for the tissues studied thus far.
TL;DR: Results show an inhibitory effect of NO donors on Leydig cell steroidogenesis, and suggest that NO can be directly inhibiting cholesterol side-chain cleavage enzyme (cytochrome P450scc) as it does with other heme proteins, including different cytochromes P450.
Abstract: Testicular macrophages as well as endothelial cells, which are intimately associated with Leydig cells, constitute a potential source of paracrine nitric oxide (NO) in the testis. In the present study, we investigated the effect of NO donors on MA-10 murine Leydig tumor cell line and rat Leydig cell steroidogenesis. We show that NO donors inhibit human CG-induced steroidogenesis in both type of cells. We also studied NO mechanism of action. Contrary to what is observed in many other systems, NO inhibitory effect on Leydig cell steroidogenesis is not mediated by cyclic GMP (cGMP) because NO fails to increase cGMP production, and cGMP analogs do not reproduce NO effect. NO does not modify the production of cAMP, the main second messenger that mediates gonadotropin action. When we studied NO effect over the steroidogenic pathway in MA-10 cells, we found that NO was inhibiting the conversion of cholesterol to pregnenolone. Taken together these results show an inhibitory effect of NO donors on Leydig cell ster...
TL;DR: The existence of a synergistic interaction between decreased BAT and Western diet to cause marked obesity and its accompanying disorders, such as insulin resistance and hyperlipidemia, is demonstrated and gives further support for the view that an important function of BAT is protection from diet-induced obesity, diabetes, and insulin resistance.
Abstract: Previous studies have indicated that rodents are relatively resistant to diet-induced obesity and that this resistance may be mediated in part by the capacity for diet-induced thermogenesis in brown adipose tissue (BAT). To test this hypothesis, we fed UCP-DTA transgenic with toxigene-mediated ablation of BAT and their control littermates a "Western diet" [21% (wt/wt) fat] or normal mouse chow [6.5% (wt/wt) fat]. The diets were begun at weaning (19 days old). At the age of 12 weeks, transgenic mice receiving the Western diet were markedly obese. The increased body weight and total body lipid content were significantly greater in transgenic mice receiving the Western diet than were the additive individual effects of Western diet (in control mice) and decreased BAT (in chow-fed mice), suggesting a synergistic interaction between diminished BAT and diet. A synergistic effect of Western diet and BAT ablation was also observed for morbid metabolic complications, such as insulin resistance, hyperglycemia, and hyperlipidemia. These metabolic changes were accompanied by increased expression of tumor necrosis factor-alpha and decreased expression of GLUT4 and beta 3-adrenergic receptor messenger RNA levels in white adipose tissue of UCP-DTA transgenic mice receiving the Western diet compared to those in the other experimental groups. As previously described, transgenic mice with diminished brown fat are hyperphagic. Of note, the degree of hyperphagia in transgenics compared to controls was similar whether the animals were fed chow or a Western diet. Thus, the synergistic effect of Western diet on obesity in transgenic mice was not mediated by a further stimulation of food intake. Overall, this study demonstrates the existence of a synergistic interaction between decreased BAT and Western diet to cause marked obesity and its accompanying disorders, such as insulin resistance and hyperlipidemia, and gives further support for the view that an important function of BAT is protection from diet-induced obesity, diabetes, and insulin resistance.
TL;DR: Activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system.
Abstract: Current studies have shown that atresia of ovarian follicles is induced through apoptosis in granulosa cells. Several articles have been devoted to study of the molecular mechanisms responsible for APO-1/CD95 (Fas) is a cell surface protein that can mediate apoptosis in lymphoid cells, and Fas ligand was recently identified in a cytotoxic T cell line. To clarify the involvement of the Fas-Fas ligand system in granulosa cell apoptosis, we investigated the expression of Fas and Fas ligand at an individual cell level. For this purpose, we raised specific polyclonal antibodies against Fas and Fas ligand. Western blotting confirmed that our anti-Fas antibodies (anti-P2 and anti-P4) detect a specific band with a mol wt of 45 kDa in the lysate of ovaries from immature PMSG-treated rats or adult cyclic rats. In immature PMSG-treated rats, immunohistochemical analysis with these antibodies revealed specific staining of granulosa cells in secondary and tertiary follicles at an early stage of atresia, but not in healthy follicles. Fas messenger RNA was also found in granulosa cells of early atretic follicles using in situ hybridization. On the other hand, the anti-Fas ligand antibody (anti-P5) detected a specific 31-kDa band on a Western blot of the oocytes lysate, and the staining with the serum was localized to oocytes in most of developing follicles. Colocalization of Fas and Fas ligand in certain follicles intimately correlated with granulosa cell apoptosis, which was revealed by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling staining of DNA strand breaks. Finally, we found that interferon-gamma increased Fas expression on granulosa cells in vitro. Coculturing interferon-gamma-pretreated granulosa cells with zona-free oocytes induced granulosa cell apoptosis, which was confirmed by Hoechst 33342 dye staining and terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling, and the killing effect of oocytes was abolished by the addition of anti-P2, which was expected to interrupt the interaction between Fas and Fas ligand. These results demonstrate that activation between Fas and Fas ligand. These results demonstrate that activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system.
TL;DR: A diet supplemented with fiber is able to significantly alter proglucagon gene expression and modulate GLP-1 and insulin secretion and deepen the understanding of the beneficial role of fiber in improving glucose homeostasis.
Abstract: Intestinal hormones stimulate more than 50% of the insulin response after oral glucose administration. Short chain fatty acids stimulate mucosal adaptation and may alter proglucagon messenger RNA and release of the insulin secretagogue, glucagon-like peptide-1 (GLP-1). Sprague-Dawley rats ingested a fiber-free elemetnal diet or an elemental diet supplemented with 30% fiber providing similar energy and nutrients for 14 days. The cecal and colonic short chain fatty acids contents were significantly higher in the 30% fiber group. Ileal proglucagon messenger RNA levels were significantly higher in the 30% group vs. the 0% group (11.47 +/- 0.87 vs. 6.52 +/- 0.87 densitometer units), respectively. Similar trends were seen in the colon (13.36 +/- 1.0 vs. 10.90 +/- 0.77 densitometer units; P = 0.07). Plasma GLP-1, insulin, and C peptide levels 30 min postoral glucose were significantly higher in the 30% fiber group vs. the 0% group (19.8 +/- 1.2 vs. 15.4 +/- 1.2 pg/ml, 2.67 +/- 0.4 vs. 1.29 +/- 0.5 ng/ml, and 964.4 +/- 94.4 vs. 530.2 +/- 120.4 pM, respectively). Plasma glucose and glucagon did not differ between groups. A diet supplemented with fiber is able to significantly alter proglucagon gene expression and modulate GLP-1 and insulin secretion. These novel findings deepen our understanding of the beneficial role of fiber in improving glucose homeostasis.